CN106117367B

CN106117367B - HER-3 specific chimeric antigen receptor and application thereof

Info

Publication number: CN106117367B
Application number: CN201610483185.5A
Authority: CN
Inventors: 冯振卿; 唐奇; 许国贞; 刘振云; 朱进; 唐小军; 熊四平
Original assignee: Sinobioway Cell Therapy Co Ltd
Current assignee: Qinhuangdao Weiming Jianchangxing Medical Health Technology Co ltd
Priority date: 2016-06-24
Filing date: 2016-06-24
Publication date: 2020-02-11
Anticipated expiration: 2036-06-24
Also published as: CN106117367A

Abstract

The invention discloses a HER-3 specific chimeric antigen receptor which is formed by connecting intracellular signal structures of a human anti-HER-3 single-chain antibody, human antibodies CD8TM, CD137 and CD3 zeta in series. The invention also discloses an amino acid sequence and a nucleic acid sequence of the HER-3 specific chimeric antigen receptor. The chimeric antigen receptor is used for modifying T lymphocytes, and the modified lymphocytes can be used for treating HER-3 gene expression-related tumors.

Description

HER-3 specific chimeric antigen receptor and application thereof

Technical Field

The invention relates to the technical field of cellular immunity, in particular to a HER-3 specific chimeric antigen receptor and application thereof.

Background

Adoptive Cell Therapy (ACT) for tumor is to induce, activate and amplify the precursor cells of autologous or allogeneic anti-tumor effector cells in vitro by using activators such as IL-2, anti-CD 3 monoclonal antibodies, specific polypeptides and the like, and then transfuse the cells to tumor patients to improve the anti-tumor immunity of the patients, so as to achieve the purpose of treating and preventing relapse. Adoptive immune effector cell therapy receives attention from people because of its unique advantages, and has achieved remarkable results in antitumor therapy in the very active research field in recent decades of tumor immunotherapy. ACT is developed through NK, gamma delta T, CD3AK, DC-CIK, LAK, TIL, CIK, EAAL and other stages, and its curative effect, specificity, overall effective rate, side effect and other aspects are improved gradually. Whereas activation of T cells requires stimulation by two signals. The TCR-CD3 complex on the surface of the T cell is combined with the antigen peptide-MHC to provide a first signal for the activation of the T cell; the costimulatory molecules on the surface of the T cells bind to the corresponding ligands, providing a second signal for T cell activation. T cells should therefore be engineered. Mainly adopts a gene modification method to carry out T Cell Receptor (TCR) and CAR gene modification on the prepared cells in vitro. Although both are the mainstream direction of tumor ACI, there is still a great difference, advantage and disadvantage between Chimeric Antigen Receptor (CAR) modified T cells and TCR modified T cell (TCR-T) therapy, wherein CAR-T has the greatest advantage that its anti-tumor effect is exerted independently of HLA molecules and the choice of tumor antigen target is not limited to proteinaceous substances.

In recent years, adoptive immunotherapy of tumors based on T cell modification by Chimeric Antigen Receptors (CARs) has been a major advance. The modified T cell not only has the killing capacity of targeting tumor cells, but also can overcome the local immunosuppression microenvironment of the tumor and break the immune tolerance state of a host. CARs have now evolved to the fourth generation through constant improvement. The first generation CARs consisted primarily of scFv and ITAMs (usually CD3 ζ) that recognize tumor associated antigens, but T cells were unable to proliferate sufficiently due to the CD3 ζ activation signal alone, and the activated T cells survived for a short time in vivo. Second generation CARs add a costimulatory molecule such as CD28, CD134, CD137, etc., based on the first generation CARs, enhancing T cell activation, proliferation, in vivo maintenance time, and thus enhancing anti-tumor effects. The third generation CAR introduces more than one costimulatory molecule on the basis of the second generation CAR. The fourth generation CAR (TRUCKS, T cells redirected for systemic cytokine killing) mainly introduces cytokines such as IL-12, IL-23, IL-27, etc., recruits innate immune cells such as macrophages to kill TAA negative tumor cells.

Depending on the T cell location in which the CAR is located, CARs are largely divided into three parts: extracellular antigen binding region, transmembrane region, intracellular signal transduction region. Wherein the extracellular antigen-binding region is generally composed of a single chain fragment variable (scFv) sequence of a monoclonal antibody against a Tumor Associated Antigen (TAA) (the light and heavy chain variable regions are formed by a hinge region linkage). This part functions to recognize tumor associated antigens, binding CAR-T cells to the tumor. The transmembrane region is usually composed of dimeric proteins such as CD3, CD28, and CD 8. The intracellular signal transduction domain is mainly an immunoreceptor tyrosine-based activation motif (ITAM, usually CD3 ζ or fcepsilonraj). Its main function is to transduce signal to T cell and raise the capacity of resisting tumor and secreting cell factor.

CAR-T cell immunotherapy has great advantages in surgery, radiotherapy and chemotherapy in traditional tumor treatment. The tumor killing precision is high, the CAR-T cell treatment adopts the antigen-antibody specific combination technology, only tumor cells expressed by tumor surface antigens are killed, and the damage to normal cells is small; the killing tumor range is wide, the CAR-T cells can be eliminated as long as tumor-associated antigens are expressed, and the killing tumor-associated antigens are effective on metastatic tumor recurrent tumors; the pain of radiotherapy and chemotherapy is avoided for the patient, and the health is rapidly recovered.

HER-3(human epidermal growth factor receptor3) is also called ErbB3, is a type I transmembrane tyrosine kinase receptor and is a unique member of ErbB family of tyrosine kinase receptors. Its function, regulatory activity and sensitivity to tumor-targeted therapies are closely related to the dimerization of other members of the ErbB family. HER-3 is highly expressed by squamous cell carcinoma of head and neck (squamous carcinoma), uveal melanoma, gastric, ovarian, prostate and bladder cancers which are common to various malignant tumors such as breast cancer and colorectal cancer. Because of its widespread expression in cancer tissues and important functions in cancer progression, attempts to target cancer therapy for ErbB family members have become the focus of extensive research. The preparation of CAR-T targeting HER-3 is of great significance for treating tumors.

Disclosure of Invention

Based on the technical problems of the background art, the present invention aims to provide a HER-3 specific chimeric antigen receptor.

The present invention also aims to provide the amino acid sequence and the nucleic acid sequence of the chimeric antigen receptor.

The invention also aims to provide application of the chimeric antigen receptor.

In order to achieve the above object, the present invention provides a HER-3 specific chimeric antigen receptor comprising a human anti-HER-3 single-chain antibody, human antibodies CD8TM, CD137, and CD3 zeta, wherein the intracellular signaling structures are connected in series.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1.

Preferably, the nucleic acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3.

Preferably, the amino acid sequence of the human anti-HER-3 single-chain antibody is shown in SEQ ID NO. 4.

Preferably, the nucleic acid sequence of the human anti-HER-3 single-chain antibody is shown in SEQ ID NO. 5.

Preferably, the human anti-HER-3 single-chain antibody comprises a light chain variable region and a heavy chain variable region, wherein the nucleic acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

Preferably, the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO. 10.

Preferably, a connecting peptide is arranged between the heavy chain molecule and the light chain molecule of the human anti-HER-3 single-chain antibody, and the amino acid sequence of the connecting peptide is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.

Preferably, the amino terminal of the chimeric antigen receptor contains a signal peptide, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 6.

Preferably, the amino acid sequence of CD137 is shown as SEQ ID NO. 7.

Preferably, the nucleic acid sequence of CD137 is shown as SEQ ID NO. 8.

Preferably, the intracellular signaling structures of human antibodies CD8TM, CD137, and CD3 ζ are connected in series to form a T cell costimulatory signaling structure, and the amino acid sequence thereof is shown in SEQ ID NO. 9.

The invention also provides application of the HER-3 specific chimeric antigen receptor in preparing a medicament for treating HER-3 related tumors.

The HER-3 related tumor comprises breast cancer, colon cancer, rectal cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, uveal melanoma and the like.

According to the invention, HER-3 antibody Fab is screened from a fully human antibody library according to a laboratory, an anti-HER-3 ScFv sequence is synthesized through codon optimization and gene synthesis, and the synthesized ScFv is cloned into pSFG-CD8TM-CD137-CD3 zeta, so that a HER-3-scFv-CD8TM-CD137-CD3 zeta chimeric antigen receptor is constructed, and the chimeric antigen receptor HER-3-scFv-CD8TM-CD137-CD3 zeta is obtained.

The chimeric antigen receptor and lentivirus packaging plasmids PeqPam3 and RD114env are used for packaging the lentivirus in 293T cells, and the T lymphocytes are infected by the lentivirus. Elisa is used for detecting the secretion of IFN-gamma of CAR-T cells under antigen stimulation and the killing effect of CCK8 method on HER-3 positive tumor cells.

The invention discloses a structure of a HER-3 specific chimeric antigen receptor, which kills tumor cells by infecting and modifying T lymphocytes in vitro and releasing IFN-gamma and the like from the modified T lymphocytes. The HER-3 specific chimeric antigen receptor can be used for the targeted therapy of HER-3 related tumors.

Drawings

FIG. 1 is an electrophoretogram of a fragment of HER-3scFv of interest obtained in example 1 of the present invention, wherein M is a standard marker.

FIG. 2 is an electrophoretogram of a fragment of the order CD8TM-CD137-CD3 ζ obtained in example 1 of the present invention, wherein M is a standard marker.

FIG. 3 shows the expression of the HER-3 specific chimeric antigen receptor obtained by the present invention after transfection of 293T cells by Western Blotting in example 2 of the present invention.

FIG. 4 shows the expression of HER-3 in tumor cells of different logarithmic growth phases as measured by Western Blotting.

FIG. 5 shows the detection of the killing of tumor cells by HER-3 specific chimeric antigen receptor modified T cells obtained by the present invention.

FIG. 6 shows IFN- γ expression of HER-3 specific chimeric antigen receptor modified T cells of the invention upon stimulation with HER-3 antigen.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to specific examples.

Example 1: construction of HER-3 specific chimeric antigen receptor lentiviral vector

1. The Fab sequence of HER-3 resistance screened by the laboratory is utilized to select the sequences of VH and VL for designing scFv sequence in CAR vector, adopting OptimumGene (TM) gene design software of Kingsler biotechnology company to further optimize codon, adding NcoI and BamHI enzyme cutting sites at two ends of the optimized sequence respectively, completing gene synthesis of the optimized sequence by Nanjing Kingsler biotechnology company, cloning the synthesized sequence in pUC57 vector, and naming the plasmid as HER-3-scFv-pUC 57.

HER-3-scFv-pUC57 was digested with restriction enzymes NcoI and BamH I as follows: mu.g of HER-3-scFv-pUC57, 1. mu.L of NcoI, 1. mu.L of BamH I, and 2. mu.L of 10 Xdigestion buffer, 20. mu.L of which was supplemented with water, left overnight in a water bath at 37 ℃, electrophoresed the digested product on 1% agarose gel, excised the desired band under ultraviolet, and recovered the desired fragment using a DNA gel recovery kit (AXYGEN Co.); as a result, as shown in FIG. 1, the pUC57-HER-3-ScFv vector was cleaved with NcoI and BamH I to release the desired band.

The pSFG-CD8TM-CD137-CD3 zeta vector was digested with NcoI and BamH I in the same manner, and the target fragment was recovered by agarose gel electrophoresis. As a result, the pSFG-CD8TM-CD137-CD3 zeta vector cleaved with NcoI and BamH I released the desired band, as shown in FIG. 2.

Connecting the recovered HER-3-ScFv with an enzyme digestion vector through T4DNA ligase, wherein the reaction system is as follows: mu.L of LHER-3-ScFv, 2. mu.L of vector digestion product, 1. mu.L of 10 Xligation buffer, 1. mu.L of ligase were supplemented with water to 10. mu.L, and incubated overnight in a water bath at 16 ℃. The ligation product was added to DH5TM competent E.coli competent preparation (CaCl) according to molecular cloning, third edition ₂Method)]The cells were cultured overnight in a37 ℃ bacterial incubator. A single colony is picked up and,and (3) carrying out amplification culture, extracting a plasmid of a positive clone, carrying out enzyme digestion and sequencing, and naming the correct vector as HER-3-scFv-CD8TM-CD137-CD3 zeta.

Example 2: HER-3 specific chimeric antigen receptor expression identification

The retrovirus vector HER-3-scFv-CD8TM-CD137-CD3 zeta was extracted with reference to the instructions in the endotoxin-free plasmid Profibration kit (Tiangen), the extracted plasmid was transfected into human embryonic kidney cells 293T with PI transfection reagent, after 48h, washed once with PBS, the cells were lysed with cell protein extraction Reagent (RIPA), the proteins of the transfected 293T cells were separated by 10% SDS-PAGE, transferred at constant current (300mA, 1h) to PVDF membrane, incubated with anti-CD 3 zeta (1: 1000) antibody, and incubated overnight at 4 ℃. After 3 washes with PBST, the cells were incubated with secondary HRP goat anti-mouse antibody (1: 5000) for 1h at room temperature. After color development by adding ECL, imaging was performed by using a ChemiDoc XRS System from Bio-Rad, and the results are shown in FIG. 3.

As can be seen from fig. 3: the recombinant plasmid constructed by the invention can detect the expression of a target band, the size of the target band is consistent with that of the male ginseng CD19, and the untransfected 293T cell has no band.

Example 3: preparation of HER-3 specific chimeric antigen receptor modified T lymphocytes

1. Package containing anti-HER-3 chimeric antigen receptor lentivirus

The retrovirus packaging vector RD114 and the HER-3-scFv-CD8TM-CD137-CD3 zeta retrovirus vector are co-transfected into GP2-293T cells by using an operation instruction in an endotoxin-free plasmid big extract kit (Tiangen organisms), cell supernatant is collected 48h after transfection, the cell supernatant is centrifuged at 4000rpm for 10min, and cells and cell debris in the supernatant are removed. Filtering with 0.45 μm filter membrane, packaging, and freezing.

Preparation of T lymphocytes

Fresh anticoagulated blood from 20mL healthy volunteers was collected and Peripheral Blood Mononuclear Cells (PBMC) were isolated using lymph separation medium (GE). The separated cells were stimulated with CD3 and CD28 plates for 48h, and induced with T lymphocyte culture medium GT-T551 (TAKARA) plus 3% autologous plasma to obtain T lymphocytes.

Preparation of CAR-T cells

Non-tissue culture plates 24-well plates were coated with 50. mu.g/mL of RetroNectin (TAKARA), 500. mu.L per well, overnight at 4 ℃. mu.L of virus supernatant was added to each well and incubated at 37 ℃ for 30 min. The viral supernatant was removed, 500. mu.L of viral supernatant was added, incubation was carried out at 37 ℃ for 30min, and 1.5mL of viral supernatant and 0.5mL of diluted T-lymphocytes were added to each well to remove viral supernatant. Thus, HER-3-scFv-CD8TM-CD137-CD3 zeta T cells, namely HER-3 specific CAR-T cells, were obtained.

Example 4: killing effect of HER-3 specific CAR-T cell on HER-3 related tumor

Western Blotting detection of HER-3 expression in tumor cells

Selecting human melanoma cells A375, human breast cancer cells MDA-MB-231 and human breast cancer cells MCF-7 in logarithmic growth phase, cracking the cells by using a cell protein extraction Reagent (RIPA), extracting cell proteins, and carrying out Western Blotting detection, wherein the detection result is shown in figure 4: HER-3 expression was detected in human breast cancer cells MDA-MB-231 and MCF-7, whereas HER-3 expression was not detected in human melanoma cells A375.

Killing detection of tumors by CAR-T cells

Adjusting the tumor cell culture medium to 5X 10 ⁶mL, 50 μ L per well, as per E: t (effector cell to target cell ratio) is 16: 1. 8: 1. 4: 1. 2: 1, tumor cells are added in 2.5X 10 ⁶1.25X 10 ⁶6.25X 10 pieces ⁵3.125X 10 ⁵A plurality of; collecting T cells and CAR-T cells after the cells are completely attached to the wall, adjusting the cell concentration to 1 × 10 ⁶50. mu.L/well for 12 h. And (3) discarding the supernatant, adding 100 mu L of diluted CCK8, incubating for 4-6 hours, and detecting the light absorption value of OD450 by using an enzyme-labeling instrument.

The tumor cells are human melanoma cell A375, human breast cancer cell MDA-MB-231 and human breast cancer cell MCF-7.

The detection results are shown in fig. 5: the killing rate of CAR-T cells on HER-3 expressing tumor cells is higher than that of HER-3 non-expressing tumor cells; the killing effect of the CAR-T cells on tumor cells is higher than that of T cells and higher than that of a pure culture medium.

ELISPOT detection of expression of IFN-gamma in CAR-T cells after stimulation with HER-3 antigen

IFN-. gamma.antibody was diluted with sterile coating and added to an ELISPOT (Millipore, Cat. No. MAIPS4510) plate at 100. mu.L/well and left overnight at 4 ℃. The next day the plates were washed 2 times with sterile coating solution. Add 200. mu.L of complete medium to each well and block for 1h at room temperature. The serum-containing cell culture solution RPMI-1640 was washed off and washed three times with PBS. The HER-3 extracellular domain polypeptide was prepared and diluted in serum-containing cell culture medium RPMI-1640 to a final concentration of 1. mu.g/mL, 100. mu.L per well. Adjusting the concentration of CAR-T cells to be detected to 1X 10 ⁶Per mL, 100. mu.L of 5% CO at 37 ℃ per well ₂After 24h, the cells and medium were discarded and washed 3 times with ELISPOT wash. Biotin-labeled detection antibody was added to each well at 100. mu.L/well and left overnight at 4 ℃. The plate was washed 4 times with ELISPOT wash. HRP-labeled avidin was added to each well at 100. mu.L/well and left at room temperature for 45 min. After washing 3 times with ELISPOT wash, the cells were washed 2 times with PBS. Adding 100 mu L of ACE chromogenic substrate into each hole, and developing for 20-60 min at room temperature. After the appearance of a distinct colony point, the reaction was terminated by 3 washes with sterile water. The plates were air dried and the number of spots formed was counted using an ELISPOT plate reader, the results of which are shown in FIG. 6. As can be seen from fig. 6: CAR-T cells are capable of secreting IFN- γ under stimulation by specific HER-3 antigens.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A HER-3 specific chimeric antigen receptor is characterized in that the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1.

2. A gene encoding the HER-3 specific chimeric antigen receptor of claim 1, having the nucleic acid sequence shown in SEQ ID No. 3.

3. Use of the HER-3 specific chimeric antigen receptor of claim 1 in the manufacture of a medicament for the treatment of HER-3 related tumors.