A single-chain antibody targeting cd70 and uses thereof

Abstract

The application belongs to the technical field of immunotherapy, and particularly relates to a ScFv targeting CD70, a humanized single-chain antibody capable of recognizing CD70 and application thereof. The amino acid sequence of the light chain variable region of the ScFv targeting CD70 is shown in SEQ ID NO: 8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 or a functional variant thereof. The ScFv after humanization of the ScFv targeting CD70, and a vector, a CAR and an immune cell containing the humanized ScFv can effectively eliminate tumor target cells expressing CD70 antigens.

Description

[0001] This application is a divisional application of the application filed on June 18, 2020, entitled "A Single-Chain Antibody Targeting CD70 and Its Application Thereof," with application number 2020105593668. Technical Field

[0002] This invention belongs to the field of immunotherapy technology, specifically relating to a CD70-targeting ScFv, a humanized single-chain antibody that can recognize CD70, and their applications. Background Technology

[0003] CD70 is a member of the tumor necrosis factor receptor (TNFR) superfamily. It is a type II transmembrane protein that is mainly expressed in activated T cells, B cells, natural killer cells, and dendritic cells. CD70 is highly expressed in various tumor tissues, including renal cell carcinoma, hematopoietic malignancies (including AML, NHL, HL, etc.), thymic tumors, ovarian cancer, glioma, and nasopharyngeal carcinoma.

[0004] Chimeric antigen receptors (CARs) are artificial receptors that mimic the function of tumor receptors (TCRs). They consist of a tumor-associated antigen-binding domain, an extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain is typically the CD3ζ chain or FcRγ, or linked to one or more co-stimulatory molecules, such as 4-1BB (CD137), CD28, or ICOS (CD278). CD70 is highly expressed in various tumors, but is only expressed in normal activated T cells, B cells, natural killer cells, and dendritic cells. Designing CARs targeting CD70 allows for a wider range of tumor applications and offers higher safety. Summary of the Invention

[0005] One of the objectives of this invention is to provide an isolated antibody or antigen-binding fragment, characterized in that it can recognize CD70.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The light chain CDR region of the ScFv contains amino acid sequences such as CDR1, CDR2 as shown in SEQ ID NO:1 and CDR3 as shown in SEQ ID NO:3; wherein, the amino acid sequence of CDR2 in the light chain CDR region of the ScFv is AAS.

[0007] Furthermore, the heavy chain CDR region of the ScFv contains amino acid sequences such as CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0008] Furthermore, the amino acid sequence of the light chain variable region of the CD70-targeting ScFv is as shown in SEQ ID NO:8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 or a functional variant thereof.

[0009] The second objective of this invention is to provide a humanized single-chain antibody that can recognize CD70.

[0010] Although humanization techniques have been reported extensively, especially after site-directed mutagenesis for stability and affinity optimization, the resulting humanized single-chain antibodies require extensive validation to confirm their effectiveness. Theoretically, single-chain antibodies can be used to prepare chimeric antigen receptors, but in practice, not every single-chain antibody can be used for this purpose. This requires inventors to engage in creative work to find products with unexpected effects among the numerous modified single-chain antibodies available.

[0011] The method for modifying single-chain antibodies involves searching a human framework library to obtain a framework sequence with high similarity to a murine framework. A murine CDR (variable region) is then inserted into the variable region of the human framework to form a humanized antibody. Finally, the humanized FR (constant region) is mutated using multiple or unit site-directed mutagenesis to restore the antibody's stability and antigen recognition activity. The results of this method are random; only under proper configuration can the humanized antibody structure remain stable, and its affinity be suitable for recognizing target antigens on the tumor surface, thereby activating CAR-T cells.

[0012] To achieve the above objectives, the technical solution of the present invention is as follows: The CD70-targeting ScFv was humanized, and the amino acid sequence of the light chain variable region of the humanized single-chain antibody is shown in any one of SEQ ID NO:9-12, and the amino acid sequence of the heavy chain variable region of the humanized single-chain antibody is shown in any one of SEQ ID NO:13-16.

[0013] Furthermore, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment that recognizes CD70 are connected by a linker. In some embodiments, the linker amino acid sequence is as shown in SEQ ID NO:17; in some embodiments, the linker amino acid sequence is as shown in SEQ ID NO:18, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33; in some embodiments, the heavy chain variable region and light chain variable region are connected in a VH-linker-VL manner, and in some embodiments, the heavy chain variable region and light chain variable region are connected in a VL-linker-VH manner.

[0014] A third objective of this invention is to provide a CAR structure comprising the CD70-recognizing ScFv and a CD70-recognizing humanized single-chain antibody, an expression vector, and immune cells.

[0015] To achieve the above objectives, the technical solution of the present invention is as follows: The CAR structure containing the humanized single-chain antibody that recognizes CD70 includes an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain that recognizes the CD70 antigen.

[0016] Furthermore, the antigen recognition region for recognizing the CD70 antigen is selected from ScFv, characterized in that the light chain CDR region contains amino acid sequences as shown in SEQ ID NO:1 (CDR1, CDR2) and SEQ ID NO:3 (CDR3), and the heavy chain CDR region contains amino acid sequences as shown in SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3); wherein, the amino acid sequence of CDR2 in the light chain CDR region of the ScFv is AAS.

[0017] Furthermore, the antigen recognition region for recognizing the CD70 antigen includes a heavy chain variable region as shown in SEQ ID NO:7, a light chain variable region as shown in SEQ ID NO:8, or a heavy chain variable region as shown in SEQ ID NO:13, a light chain variable region as shown in SEQ ID NO:9, or a heavy chain variable region as shown in SEQ ID NO:14, a light chain variable region as shown in SEQ ID NO:10, or a heavy chain variable region as shown in SEQ ID NO:15, a light chain variable region as shown in SEQ ID NO:11, or a heavy chain variable region as shown in SEQ ID NO:16, and a light chain variable region as shown in SEQ ID NO:12 (ScFv).

[0018] Preferably, the single-chain antibody recognizing the CD70 antigen comprises a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:10, or a heavy chain variable region as shown in SEQ ID NO:16 and a light chain variable region as shown in SEQ ID NO:12.

[0019] In some embodiments, the hinge region of the chimeric antigen receptor for the anti-CD70 antigen may be derived from CD8, IgG4, and CD7, etc.

[0020] Furthermore, the amino acid sequence of the hinge region is as shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, the hinge region may also originate from the IgG4 hinge region; the transmembrane region originates from the transmembrane region of the CD8 molecule or CD28 molecule; the intracellular signal originates from the intracellular signal region of CD28 or CD137 and the intracellular sequence of CD3.

[0021] Furthermore, the amino acid sequence of the transmembrane region is shown in SEQ ID NO:26 or SEQ ID NO:27.

[0022] Furthermore, the intracellular signal amino acid sequence is shown in SEQ ID NO:28 or SEQ ID NO:29.

[0023] Furthermore, the amino acid sequence of the CD3 activation region is shown in SEQ ID NO:30.

[0024] The inventors designed four different humanized ScFvs and three different CAR structures for random combination. Through evaluation in five aspects—ScFv affinity, stability, CAR expression stability, CAR-T cell efficacy, and safety—the inventors verified that the antibodies or antigen-binding fragments that recognize CD70 and the modified humanized single-chain antibodies described by the inventors can function in different CAR structures and are applicable to all CAR combinations targeting CD70.

[0025] Preferably, the chimeric antigen receptor amino acid sequence is SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25.

[0026] Furthermore, the expression vector containing the CAR structure is any one of lentiviral expression vector, retroviral expression vector, adenovirus expression vector, adeno-associated virus expression vector, DNA vector, RNA vector, or plasmid.

[0027] Furthermore, the expression vector promoter containing the CAR gene uses five repeated HRE regulatory elements in combination with a weakly activated CMV mini promoter to form the 5HRE-CMV mini promoter.

[0028] Preferably, the vector is a lentiviral vector, characterized in that the vector contains an RRE element and an oPRE element.

[0029] Furthermore, the vector is characterized in that it comprises a prokaryotic replicon pUCOri sequence for plasmid replication; a kanamycin or ampicillin resistance gene sequence for large-scale amplification of the target strain; a viral replicon SV40Ori sequence for enhancing replication in eukaryotic cells; a lentiviral packaging cis-element for lentiviral packaging; a promoter for eukaryotic transcription of the chimeric antigen receptor gene; and a CAR-encoding gene for integrating target recognition, signal transduction, and signal initiation.

[0030] In some embodiments, the vector comprises a left (5') retroviral LTR, a Psi(Ψ) packaging signal, a central polypurine segment / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operatively linked to a polynucleotide encoding a CAR covered herein, and a right (3') retroviral LTR.

[0031] In some implementations, the CAR vector contains a hepatitis B virus post-transcriptional regulatory element (HPRE) or a prairie dog post-transcriptional regulatory element (WPRE); the promoter of the 5'LTR is replaced by a heterologous promoter, which is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter.

[0032] When the lentiviral vector is used for T-cell transduction, it exhibits high CAR-positive expression, remains stable during patient cell culture, and does not decrease in positivity over time. Therefore, cells infected with the lentiviral vector possess the ability to kill target cells.

[0033] The fourth objective of this invention is to provide a pharmaceutical composition and its use in the preparation of a drug for malignant tumors.

[0034] To achieve the above objectives, the technical solution of the present invention is as follows: The pharmaceutical composition comprises the CD70-recognizing ScFv, the humanized single-chain antibody, the CAR structure, or the immune cells.

[0035] In some embodiments, CAR-expressing cells may also express or be used in combination with other active agents. These active agents may be immune checkpoint inhibitors, such as PD1 / PDL1, TIM3, CTLA4, LAG3, and TGFRβ inhibitors. In other embodiments, these active agents may be peptides or fusion proteins, such as fusion proteins formed by PD-1-CD137-CD3 molecules. In other embodiments, these active agents may be antibody drugs, such as monoclonal antibody drugs or bispecific antibody drugs. In other embodiments, these active agents may be kinase inhibitors, such as the tyrosine kinase inhibitor dasatinib.

[0036] Furthermore, the malignant tumor is a malignant tumor that expresses CD70, including but not limited to renal cell carcinoma, esophageal cancer, mesothelioma, gastric cancer, adenocystic carcinoma, ovarian cancer, endometrial cancer, breast cancer, head and neck squamous cell carcinoma, glioma, lung cancer, osteosarcoma, thyroid cancer, melanoma, pancreatic cancer, lymphoma, acute myeloid leukemia, or multiple myeloma.

[0037] In some embodiments, the cells are activated and stimulated in the presence of a PI3K pathway inhibitor.

[0038] In some embodiments, the composition is applied via a composition comprising the cells, and the routes of administration include oral, intravenous, peritoneal infusion, subcutaneous injection, and local administration to a tumor or organ. Preferably, the composition is administered via intravenous, intraperitoneal, or subcutaneous injection.

[0039] Furthermore, this application also relates to: 1. A CD70-targeting ScFv, characterized in that the light chain CDR region of the ScFv comprises amino acid sequences as shown in SEQ ID NO:1 (CDR1, CDR2) and SEQ ID NO:3 (CDR3); the heavy chain CDR region of the ScFv comprises amino acid sequences as shown in SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3); wherein the amino acid sequence of CDR2 in the light chain CDR region of the ScFv is AAS.

[0040] 2. The CD70-targeting ScFv according to claim 1, characterized in that the amino acid sequence of the light chain variable region of the CD70-targeting ScFv is as shown in SEQ ID NO:8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 or a functional variant thereof.

[0041] 3. A humanized single-chain antibody capable of recognizing CD70, characterized in that the ScFv targeting CD70 described in any one of items 1-2 is humanized, wherein the amino acid sequence of the light chain variable region of the humanized single-chain antibody is as shown in any one of SEQ ID NO: 9-12, and the amino acid sequence of the heavy chain variable region of the humanized single-chain antibody is as shown in any one of SEQ ID NO: 13-16.

[0042] 4. The humanized single-chain antibody capable of recognizing CD70 according to item 3, characterized in that the linker amino acid sequence of the antibody is as shown in SEQ ID NO:17 or SEQ ID NO:18 or SEQ ID NO:31 or SEQ ID NO:32 or SEQ ID NO:33.

[0043] 5. A CAR structure comprising the ScFv that can identify CD70 as described in any one of items 1-2.

[0044] 6. A CAR structure comprising a humanized single-chain antibody that recognizes CD70 as described in any one of items 3-4.

[0045] 7. The CAR structure according to item 6, characterized in that the amino acid sequence of the hinge region of the CAR structure is as shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0046] 8. The CAR structure according to item 6, characterized in that the amino acid sequence of the transmembrane region of the CAR structure is as shown in SEQ ID NO:26 or SEQ ID NO:27.

[0047] 9. The CAR structure according to item 6, characterized in that the intracellular signal amino acid sequence of the CAR structure is as shown in SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0048] 10. The CAR structure according to claim 6, characterized in that the amino acid sequence of the CAR structure is as shown in SEQ ID NO:22 or SEQ ID NO:23 or SEQ ID NO:24 or SEQ ID NO:25 or a functional variant thereof.

[0049] 11. An expression vector comprising the CAR structure described in any one of items 6-10.

[0050] 12. The expression vector according to item 11, characterized in that the expression vector is any one of lentiviral expression vector, retroviral expression vector, adenovirus expression vector, adeno-associated virus expression vector, DNA vector, RNA vector, and plasmid.

[0051] 13. The expression vector according to item 12, wherein the expression vector is a lentiviral expression vector.

[0052] 14. Immune cells comprising the expression vector described in any one of items 11-13.

[0053] 15. The immune cell according to item 14, characterized in that the immune cell is a stem cell, T cell, NK cell, monocyte or macrophage.

[0054] 16. The immune cell according to claim 15, characterized in that the immune cell is a T cell.

[0055] 17. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the CD70-recognizing ScFv of any one of claims 1-2, or the CD70-recognizing humanized single-chain antibody of any one of claims 3-4, or the CAR structure of any one of claims 6-10, or the immune cells of any one of claims 14-16.

[0056] 18. Use of the pharmaceutical composition described in item 17 in the preparation of a drug for malignant tumors.

[0057] 19. The application according to item 18, characterized in that the malignant tumor is a CD70-expressing malignant tumor, including but not limited to renal cell carcinoma, esophageal cancer, mesothelioma, gastric cancer, adenocystic carcinoma, ovarian cancer, endometrial cancer, breast cancer, head and neck squamous cell carcinoma, glioma, lung cancer, osteosarcoma, thyroid cancer, melanoma, pancreatic cancer, lymphoma, acute myeloid leukemia, or multiple myeloma.

[0058] In summary, the CD70-targeting CAR (chimeric antigen receptor) described in this invention, after being expressed in immune cells, can not only maintain the positive rate of CD70-targeting chimeric antigen receptor (CAR) in patient cell culture, but also enhance the proliferation and tumor-killing ability of CAR-T cells, and can be used for targeted cancer therapy.

[0059] The beneficial effects of this invention are as follows: 1) The antibody or antigen-binding fragment for recognizing CD70 provided by this invention can efficiently recognize the CD70 antigen; 2) The antibody or antigen-binding fragment for recognizing CD70 provided by this invention can recognize CD70-positive tissue, cell and blood samples.

[0060] 3) The chimeric antigen receptor against CD70 antigen constructed from the antibody or antigen-binding fragment that recognizes CD70 provided by this invention can effectively clear tumor target cells expressing CD70 antigen after being expressed in immune cells, and can maintain the positive rate of the chimeric antigen receptor (CAR) targeting CD70 in the patient cell culture process, and can be used for targeted therapy of tumors. Attached Figure Description

[0061] Figure 1 Detection of CD70 expression in tissues.

[0062] Figure 2 Designed for CD70 murine antibody and humanization.

[0063] Figure 3 For CD70 antibody affinity detection.

[0064] Figure 4 To validate the ability of CD70 antibodies to recognize CD70 on the cell surface.

[0065] Figure 5 CAR structure constructed for CD70 antibody ScFv.

[0066] Figure 6 To validate the expression of CD70 CAR.

[0067] Figure 7 To target CD70 CART for effective in vitro killing.

[0068] Figure 8 To target CD70 CART for effective in vivo killing. Detailed Implementation

[0069] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Experimental methods in the preferred embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd Edition, J. Sambrook et al.), or as recommended by the manufacturer. The examples provided are for better illustration of the invention, but are not intended to limit the scope of the invention to the examples given. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0070] Example 1: Detection of CD70 expression in normal tissues Singleton PCR was used (one gene amplified per plate, one plate for the internal control gene, and one plate for the target gene). Skov3 cell cDNA was used as the template for the positive control; the negative control was a reaction mixture without cDNA template, using dH2O as the template. This experiment used Skov3 cell cDNA synthesized from the same reverse transcription to amplify GAPDH with the same primer pair, to demonstrate the comparability between the experimental and internal control plates. Peripheral blood lymphocytes were used as the control group. The relative expression level of CD70 in each tissue was calculated using the classical method of 2-ΔΔCt. The results are as follows: Figure 1 As shown: using peripheral blood lymphocytes as a control, CD70 expression in lymph nodes and tonsils was slightly higher than in control samples, while it was slightly lower in the small intestine, lungs, spleen, stomach, thymus, prostate, and trachea. Other tissue samples showed significantly lower expression than control samples. This indicates that CD70 is almost not expressed in normal tissues except for those rich in lymphocytes.

[0071] Example 2: CD70 murine antibody and humanization design The structure of mouse antibody CDR is as follows Figure 2 As shown, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0072] Through molecular docking simulation, the mouse antibody backbone sequence was modified to humanize it, resulting in two humanized ScFvs. Based on this, these two ScFvs were subjected to reverse mutation and immunogenicity optimization to obtain ScFvs with good activity and lower immunogenicity, resulting in a total of four optimized humanized ScFvs.

[0073] Design humanized ScFv sequences, wherein the amino acid sequences of the light chain variable region are shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; and the amino acid sequences of the heavy chain variable region are shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0074] Example 3: CD70 antibody affinity detection The interaction kinetics between hCD70-hFc and CD70-ScFv were analyzed using an HIS1K sensor to immobilize the target CD70. Analyte concentration gradients were set at 200 nM, 100 nM, 50 nM, and 25 nM. Specific experimental procedures are detailed in the instrument operating instructions. Results are as follows: Figure 3 As shown in Table 1, ScFv has a good affinity for CD70, with affinity constants KD = 16.4 nM and 4.65 nM.

[0075] Table 1. CD70 antibody affinity assay

[0076] Example 4: Verification of the ability of CD70 molecules to recognize CD70 on the cell surface ScFv targeting CD70 was used to perform flow cytometry staining on CD70-positive cells 786-O, exogenously constructed K562-CD70 cells, and CD70-negative K562 cells. Results are as follows: Figure 4 As shown, ScFv targeting CD70 can recognize CD70 on the surface of CD70-positive 786-O cells and exogenously constructed K562-CD70 cells. Therefore, the murine and humanized anti-CD70 antibodies or peptide fragments containing the CDR regions of SEQ ID NO: 1, AAS, and SEQ ID NO: 3~SEQ ID NO: 6 disclosed can be used as CD70 detection reagents.

[0077] Example 5: Preparation of lentiviruses expressing chimeric antigen receptors targeting human CD70 antigen (1) Construction of chimeric antigen receptor targeting human CD70 antigen A chimeric antigen receptor sequence containing a single-chain antibody ScFv against human CD70 antigen was synthesized, comprising the hinge region, transmembrane region, and intracellular signaling segment of the hFc region. The humanized single-chain antibody against human CD70 antigen includes the following amino acid sequences: a heavy chain variable region as shown in SEQ ID NO:7; a light chain variable region as shown in SEQ ID NO:8; a heavy chain variable region as shown in SEQ ID NO:13; a light chain variable region as shown in SEQ ID NO:9; a heavy chain variable region as shown in SEQ ID NO:14; a light chain variable region as shown in SEQ ID NO:10; a heavy chain variable region as shown in SEQ ID NO:15; a light chain variable region as shown in SEQ ID NO:11; a heavy chain variable region as shown in SEQ ID NO:16; and a light chain variable region as shown in SEQ ID NO:12. The transmembrane region is derived from the CD8 or CD28 sequence; and the intracellular signaling segment is derived from the CD28 or CD137 and CD3 sequences. Twelve groups of humanized anti-CD70 chimeric antigen receptors and one mouse control group were finally synthesized. The structure is shown below. Figure 5 As shown.

[0078] Enzyme digestion was performed according to the manufacturer's instructions. The digestion products were isolated and recovered. The target fragment and vector fragment were then ligated using T4 ligase (Promega) to obtain a lentiviral vector expressing a chimeric antigen receptor. Plasmids were extracted using a plasmid extraction kit (Invitrogen), following the manufacturer's instructions.

[0079] (2) Packaging of lentiviruses In this embodiment, lentivirus packaging was performed using the calcium phosphate method. The specific steps are as follows: 293T cells were cultured in DMEM medium containing 10% FBS (w / v) to optimal condition. ddH2O, lentiviral vector, and 2.5mM CaCl2 were added to a 15mL centrifuge tube in a specific ratio and mixed thoroughly. 2× HBS was then added dropwise to the mixture, and after mixing, the mixture was allowed to stand at room temperature for 15 minutes. The mixture was then added dropwise to the prepared 293T cells, and the cells were cultured overnight. The culture medium was then replaced with fresh DMEM medium containing 10% FBS (w / v). Cell supernatants were collected after 48h and 72h of culture, and then purified and aliquoted.

[0080] Example 6: Preparation of T cells modified with chimeric antigen receptors of CD70 antigen (1) Lentiviral infection of T cells 1) Isolation of human peripheral blood mononuclear cells Lymphocytes were isolated using gradient centrifugation. After centrifugation, the second white lymphocyte layer was taken, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human peripheral blood mononuclear cells.

[0081] 2) Lentiviral vector infection of T lymphocytes PBMCs were activated with anti-CD3 and CD28 monoclonal antibodies and then infected with lentiviruses. After infection, the cells were cultured for approximately 10 days to verify their in vivo and in vitro biological characteristics. The obtained chimeric antigen receptor T cells were named according to their corresponding lentiviral vectors.

[0082] 3) Detection of chimeric antigen receptor (CAR) expression targeting human CD70 antigen During the culture process, the CAR positivity rate of virus-infected T cells cultured to day 6 and day 9 was detected. The detection method was flow cytometry, and the antibody used was Protein-L-PE. Protein-L recognizes the antibody light chain, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L; therefore, Protein-L can be used to detect the CAR positivity rate. Results are as follows... Figure 6 As shown, CAR can be normally expressed on the surface of T cells after infection with different viruses.

[0083] Example 7: Validation of the anti-tumor effect of T lymphocytes expressing a chimeric antigen receptor targeting CD70 CD70-positive 786-O (abbreviated as 786-O-luc) and A549-Luc (negative) cells, which stably express firefly luciferase, were used as target cells.

[0084] CAR-T cells were prepared using peripheral blood mononuclear cells from healthy donors, with an effector-to-target ratio of 8:1 / 4:1 / 2:1. The killing effect was assessed using the standard method provided with the Steady-Glo® Luciferase Assay System (Promega Cat. #E2520) kit, and the killing rate was calculated using the following formula: The result of the killing is as follows Figure 7 As shown, the constructed CARTs all exhibit good in vitro killing effects.

[0085] Example 8: Validation of the antitumor effect of T lymphocytes expressing CD70 chimeric antigen receptor in an animal model A mouse xenograft model of human CD70-positive tumor cell line was established to verify the anti-tumor effect of T lymphocytes expressing chimeric antigen receptors targeting CD70 in the animal model.

[0086] The NOG mice used for in vivo validation were NOD.Cg-PrkdcscidII2rgtm1Sug / JicCrl, bred by Mamoru Ito of the China Institute of Laboratory Animal Science (CIEA) in Japan. This is the most common strain used internationally for CAR-T-related tumorigenesis experiments. The tumorigenesis target cells used for in vivo validation were 786-O cells. After tumor formation, CAR-T cells and virus-free PBMC cells were injected via the tail vein into the mice. Tumor volume was measured every 7 days after CAR-T cell injection, and mouse survival was observed and recorded daily. Results are as follows: Figure 8 As shown, the constructed CD70 CAR-T cells significantly reduced tumor volume in mice treated with the CAR-T therapy, demonstrating good in vivo killing activity.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of a CD70-targeting ScFv in the preparation of a CAR-structured drug for treating malignant tumors expressing CD70, characterized in that, The light chain variable region of the ScFv contains amino acid sequences such as CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1 and SEQ ID NO:3; the heavy chain variable region of the ScFv contains amino acid sequences such as CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5 and CDR3 as shown in SEQ ID NO:6; wherein, the amino acid sequence of CDR2 in the light chain variable region of the ScFv is AAS; The malignant tumors mentioned are clear cell adenocarcinoma of the kidney, breast cancer, squamous cell carcinoma of the head and neck, glioma, lung cancer, melanoma, or acute myeloid leukemia.

2. The use according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the CD70-targeting ScFv is shown in SEQ ID NO:8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7 or a functional variant thereof.

3. The use according to claim 1 or 2, characterized in that, The ScFv is a humanized single-chain antibody that can recognize CD70, and the amino acid sequence of the light chain variable region of the humanized single-chain antibody is as shown in any one of SEQ ID NO:9-12, and the amino acid sequence of the heavy chain variable region of the humanized single-chain antibody is as shown in any one of SEQ ID NO:13-16.

4. The use according to claim 3, characterized in that, The linker amino acid sequence of the antibody is shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:

33.

5. The use of a CAR structure containing a CD70-recognizing ScFv for preparing CD70-targeting immune cells, characterized in that, The light chain variable region of the ScFv contains amino acid sequences such as CDR1, CDR2 as shown in SEQ ID NO:1 and CDR3 as shown in SEQ ID NO:3; the heavy chain variable region of the ScFv contains amino acid sequences such as CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5 and CDR3 as shown in SEQ ID NO:6; wherein, the amino acid sequence of CDR2 in the light chain variable region of the ScFv is AAS.

6. The use according to claim 5, characterized in that, The ScFv is a humanized single-chain antibody that can recognize CD70, and the amino acid sequence of the light chain variable region of the humanized single-chain antibody is as shown in any one of SEQ ID NO:9-12, and the amino acid sequence of the heavy chain variable region of the humanized single-chain antibody is as shown in any one of SEQ ID NO:13-16.

7. The use according to claim 6, characterized in that, The amino acid sequence of the hinge region of the CAR structure is shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:

21.

8. The use according to claim 6, characterized in that, The amino acid sequence of the transmembrane region of the CAR structure is shown in SEQ ID NO:26 or SEQ ID NO:

27.

9. The use according to claim 6, characterized in that, The intracellular signal amino acid sequence of the CAR structure is shown in SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:

30.

10. The use according to claim 6, characterized in that, The amino acid sequence of the CAR structure is shown in SEQ ID NO:22 or SEQ ID NO:23 or SEQ ID NO:24 or SEQ ID NO:25 or a functional variant thereof.

11. The use according to any one of claims 5-10, characterized in that, The CAR structure is contained in the expression vector.

12. The use according to claim 11, characterized in that, The expression vector is any one of a retroviral expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, or a plasmid.

13. The use according to claim 12, characterized in that, The retroviral expression vector is a lentiviral expression vector.

14. The use according to claim 11, characterized in that, The expression vector is a DNA vector or an RNA vector.

15. The use according to any one of claims 5-10, 12-14, characterized in that, The immune cells are T cells, NK cells, monocytes, or macrophages.

16. The use according to claim 15, characterized in that, The immune cells mentioned are T cells.

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