WO2024087267A1 - 一种靶向TGFβRII的嵌合抗原受体及其用途 - Google Patents

一种靶向TGFβRII的嵌合抗原受体及其用途 Download PDF

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WO2024087267A1
WO2024087267A1 PCT/CN2022/132043 CN2022132043W WO2024087267A1 WO 2024087267 A1 WO2024087267 A1 WO 2024087267A1 CN 2022132043 W CN2022132043 W CN 2022132043W WO 2024087267 A1 WO2024087267 A1 WO 2024087267A1
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chimeric antigen
antigen receptor
domain
seq
amino acid
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French (fr)
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王永生
陈玥
李丹
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四川大学华西医院
成都华西精准医学产业技术研究院有限公司
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Definitions

  • the present invention belongs to the field of tumor treatment, and specifically relates to a chimeric antigen receptor targeting TGF ⁇ RII and a use thereof.
  • Tumor immunotherapy is a treatment method that controls and eliminates tumors by restoring the body's normal anti-tumor immune response.
  • Tumor immunotherapy includes monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, etc.
  • tumor immunotherapy was rated as the most important scientific breakthrough of the year by Science magazine in 2013 due to its excellent efficacy and innovation.
  • chimeric antigen receptor modified T cells (CAR-T) therapy and CTLA-4, PD-1/PD-L1 antibody therapy are considered to be the three major advances in tumor immunotherapy.
  • CAR-T therapy uses antigen-antibody fragments, combined with T cell intracellular activation and proliferation signals, to construct CAR-T, which enables T cells to directly obtain the specific recognition ability of antibodies and become effector T cells that are not dependent on HLA (human leukocyte antigen) restrictions.
  • Designed CAR-T cells can be cultured and grown in the laboratory, reaching billions.
  • the amplified CAR-T cells are injected into the patient's body. After injection, the T cells will also proliferate in the patient's body and kill tumor cells with corresponding specific antigens, survive for a long time and form immune memory.
  • chimeric antigen receptors on immune cells other than T cells such as monocytes, natural killer cells, neutrophils, etc., so that these immune cells can also obtain specific recognition of tumor cells and play the role of immunotherapy.
  • CAR chimeric antigen receptors
  • CAR chimeric antigen receptors
  • the extracellular domain is a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to antigens, or scFv and a hinge region (Hinge) that acts as a connector.
  • the intracellular domain includes a co-stimulatory domain and a signal transduction domain.
  • the transmembrane domain connects the extracellular domain of CAR to the intracellular domain and anchors the receptor to the T cell membrane.
  • scFv is the key to antigen recognition and targeting, and is an important factor affecting the therapeutic effect.
  • ROR1 is a transmembrane protein in the RTK (receptor tyrosine kinase) family. It exists during embryonic development and is related to the development of the central nervous system, heart, lung and bone tissue. Postnatally, except for a small amount of expression in adipose tissue, pancreas, lung and developing B cells (pre-B cells, immature B cells), it is not expressed in most normal tissues. However, it is expressed in many blood and solid malignancies.
  • RTK receptor tyrosine kinase
  • ROR1 is highly expressed to varying degrees in malignant tumors such as chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, melanoma, etc. And in ovarian cancer and triple-negative breast cancer, the degree of ROR1 expression is associated with poor prognosis. ROR1 tends to be expressed in poorly differentiated tumors. Cancer cells expressing ROR1 show stronger invasion, metastasis and recurrence capabilities, and express marker genes related to epithelial-mesenchymal transition (EMT). Therefore, ROR1 CAR-T developed based on the ROR1 target has been widely studied and has shown effective targeted killing of tumor cells.
  • EMT epithelial-mesenchymal transition
  • TGF- ⁇ transforming growth factor ⁇
  • TGF- ⁇ tumor cells, regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), and other cells. It is highly expressed in most solid tumor lesions such as ovarian cancer, lung cancer, and colorectal cancer.
  • TGF- ⁇ directly inhibits T cell activity by binding to TGF- ⁇ receptors TGF ⁇ RI and TGF ⁇ RII. After binding to dimerized TGF ⁇ RII, TGF- ⁇ recruits TGF ⁇ RI to form heterotetramers, which causes phosphorylation of intracellular SMAD2 and SMAD3.
  • TGF- ⁇ can promote T cells to differentiate into Tregs, which in turn can produce TGF- ⁇ and further promote immunosuppression and tumor tolerance. Therefore, the immunosuppression of TGF- ⁇ is considered to be one of the main reasons for the failure of anti-tumor activity.
  • the object of the present invention is to provide a chimeric antigen receptor targeting TGF ⁇ RII, and a dual-target chimeric antigen receptor formed by combining the chimeric antigen receptor with a traditional tumor-targeting chimeric antigen receptor.
  • the present invention provides a chimeric antigen receptor, which is a chimeric antigen receptor capable of recognizing and binding to TGF ⁇ RII.
  • the chimeric antigen receptor comprises an antigen recognition domain, a transmembrane domain, and an intracellular domain; the antigen recognition domain is a single-chain antibody of TGF ⁇ RII.
  • amino acid sequence of the single-chain antibody of the above-mentioned TGF ⁇ RII is shown in SEQ ID NO.19.
  • transmembrane domain is any one or more of CD28 transmembrane segment, CD8, CD3 ⁇ , CD134, CD137, ICOS, DAP10, and CD27;
  • the intracellular domain includes a co-stimulatory signal domain, and the co-stimulatory signal domain is any one or more of the intracellular segment of CD28, 4-1BB, ICOS, CD27, OX40, MyD88, and CD40.
  • transmembrane domain is CD8, and the amino acid sequence is shown in SEQ ID NO.21; the intracellular domain is 4-1BB, and the amino acid sequence is shown in SEQ ID NO.23.
  • the above-mentioned chimeric antigen receptor also includes a signal peptide 2.
  • the amino acid sequence of the signal peptide 2 is as shown in SEQ ID NO.15.
  • the above-mentioned chimeric antigen receptor also includes a protein tag sequence, preferably a MYC tag sequence, and the amino acid sequence is shown in SEQ ID NO.17.
  • the chimeric antigen receptor comprises the following fragments connected in sequence: signal peptide 2, protein tag sequence, single-chain antibody of TGF ⁇ RII, CD8 transmembrane domain, 4-1BB co-stimulatory signal domain;
  • the chimeric antigen receptor amino acid sequence is as shown in SEQ ID NO.30.
  • the present invention also provides a dual-target chimeric antigen receptor, comprising a chimeric antigen receptor 1 and a chimeric antigen receptor 2;
  • chimeric antigen receptor 1 is a chimeric antigen receptor capable of recognizing and binding to tumor-specific antigens or tumor-associated antigens;
  • chimeric antigen receptor 2 is the chimeric antigen receptor described in any one of claims 1 to 7.
  • the dual-target chimeric antigen receptor is a combination of chimeric antigen receptor 1 and chimeric antigen receptor 2, or a combination of chimeric antigen receptor 1 and chimeric antigen receptor 2.
  • the above-mentioned dual-target chimeric antigen receptor is formed by connecting chimeric antigen receptor 1 and chimeric antigen receptor 2 through a polypeptide fragment; preferably, the polypeptide fragment is a self-cleaving peptide fragment, more preferably a self-cleaving peptide P2A fragment, and the amino acid sequence is shown in SEQ ID NO.13.
  • the chimeric antigen receptor 1 comprises an antigen recognition domain, a transmembrane domain, and an intracellular domain;
  • the antigen recognition domain is a single-chain antibody capable of recognizing and binding to a tumor-specific antigen or a tumor-associated antigen;
  • the tumor-specific antigen or tumor-associated antigen is any one or more of CD19, CD20, MUC1, ROR1, EGFR, EGFRvIII, HER2, ERBB3, ERBB4, VEGFR1, VEGFR2, EpCAM, CD44, and IGFR.
  • the above-mentioned single-chain antibody is a single-chain antibody of ROR1; preferably, the amino acid sequence of the single-chain antibody of ROR1 is as shown in SEQ ID NO.3.
  • transmembrane domain is any one or more of CD28 transmembrane segment, CD8, CD3 ⁇ , CD134, CD137, ICOS, DAP10, and CD27;
  • the intracellular domain includes a co-stimulatory signal domain and a signal transduction domain, wherein the co-stimulatory signal domain is any one or more of the intracellular segment of CD28, 4-1BB, ICOS, CD27, OX40, MyD88, and CD40; and the signal transduction domain is CD3 ⁇ or Fc ⁇ RI.
  • transmembrane domain is the CD28 transmembrane segment, and the amino acid sequence is shown in SEQ ID NO.7;
  • co-stimulatory signal domain is the CD28 intracellular segment, and the amino acid sequence is shown in SEQ ID NO.9;
  • signal transduction domain is CD3 ⁇ , and the amino acid sequence is shown in SEQ ID NO.11.
  • the above-mentioned chimeric antigen receptor 1 also includes a signal peptide 1 and a hinge region; preferably, the amino acid sequence of the signal peptide 1 is as shown in SEQ ID NO.1, and the amino acid sequence of the hinge region is as shown in SEQ ID NO.5.
  • the chimeric antigen receptor 1 comprises the following fragments connected in sequence: signal peptide 1, single chain antibody of ROR1, hinge region, CD28 transmembrane domain, CD28 intracellular segment co-stimulatory signal domain, CD3 ⁇ signal transduction domain;
  • amino acid sequence of the chimeric antigen receptor 1 is as shown in SEQ ID NO.29.
  • amino acid sequence of the above-mentioned dual-target chimeric antigen receptor is shown in SEQ ID NO.26.
  • the present invention also provides a gene, which is a gene encoding the above-mentioned chimeric antigen receptor or the above-mentioned dual-target chimeric antigen receptor; preferably, the nucleotide sequence of the gene encoding the above-mentioned chimeric antigen receptor is as shown in SEQ ID NO.27, and the nucleotide sequence of the gene encoding the above-mentioned dual-target chimeric antigen receptor is as shown in SEQ ID NO.25.
  • the present invention also provides a vector, which is a vector containing the above gene; the vector is a plasmid or a virus.
  • the present invention also provides a host cell, which is a host cell expressing the above-mentioned chimeric antigen receptor or the above-mentioned dual-target chimeric antigen receptor, preferably a host cell containing the above-mentioned vector.
  • the host cell is an immune response cell, preferably at least one of a T cell, a monocyte, a natural killer cell or a neutrophil, and more preferably a T cell.
  • the present invention also provides the use of the chimeric antigen receptor, dual-target chimeric antigen receptor, gene, vector or host cell in preparing a drug for preventing and/or treating tumors.
  • the present invention provides a chimeric antigen receptor targeting TGF ⁇ RII.
  • TGF ⁇ RII TGF ⁇ RII
  • it can bind to TGF ⁇ RII of tumor cells to enhance the recognition and targeting effect of immune cells on tumors.
  • TGF ⁇ RII TGF ⁇ RII of immune cells themselves to achieve the blocking of TGF ⁇ RII of immune cells themselves, hindering the binding of TGF ⁇ to TGF ⁇ receptor of immune cells, thereby reducing the inhibitory effect of TGF ⁇ on immune cells.
  • the chimeric antigen receptor sequence targeting TGF- ⁇ RII does not contain CD3 ⁇ activation signal, and will not activate T cells to perform cytotoxic effects.
  • the chimeric antigen receptor targeting TGF ⁇ RII is used in combination with the traditional tumor-targeted chimeric antigen receptor to construct a dual-target chimeric antigen receptor-modified immune cell, which can not only achieve tumor-specific targeting and kill the tumor through the tumor-targeted chimeric antigen receptor, but also reduce the inhibitory effect of the inhibitory cytokine TGF ⁇ on immune cells through the action of the chimeric antigen receptor targeting TGF ⁇ RII, while enhancing the tumor recognition and targeting effect, thereby achieving a synergistic anti-tumor effect.
  • Figure 1 is a diagram showing the structural composition of the chimeric antigen receptor functional unit in the viral vector of the present invention, wherein: A is a chimeric antigen receptor that single-targets ROR1, and B is a dual-target chimeric antigen receptor.
  • FIG2 shows the expression of CAR of the present invention on T cells.
  • Figure 3 shows the expression of tumor cell surface antigens.
  • Figure 4 shows the level of IFN- ⁇ release after co-culture of CAR-T and tumor cells.
  • Figure 5 shows the level of IL-2 release after co-culture of CAR-T and tumor cells.
  • Figure 6 shows the level of TNF- ⁇ release after co-culture of CAR-T and tumor cells.
  • Figure 7 shows the detection of cells secreting IFN- ⁇ after co-culture of CAR-T and tumor cells.
  • Figure 8 is the detection of T cell granzyme B release after CAR-T cells kill tumor cells in vitro.
  • Figure 9 shows the proliferation of T cells after co-culture of CAR-T cells and tumor cells.
  • Figure 10 shows the detection of pSMAD2/3 signaling pathway in T cells after CAR-T cells kill tumor cells in vitro.
  • FIG. 11 shows the growth inhibitory effect of CAR-T cells on the SKOV3 ovarian cancer peritoneal dissemination model in vivo.
  • the raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.
  • Example 1 Synthesis of the full-length gene of the dual-target chimeric antigen receptor and completion of the construction of the recombinant plasmid vector
  • the expression framework from the 5 end to the 3 end is: signal peptide 1-Anti-ROR1 scFv-hinge-CD28 transmembrane segment-CD28 intracellular segment-CD3 ⁇ -P2A-signal peptide 2-MYC protein tag-Anti-TGF- ⁇ RII scFv-CD8 transmembrane domain-4-1BB intracellular segment.
  • Coding nucleotide sequence (SEQ ID NO.2):
  • Coding nucleotide sequence (SEQ ID NO.4):
  • Hinge amino acid sequence (SEQ ID NO.5): ESKYGPPCPPCP
  • Coding nucleotide sequence SEQ ID NO.6
  • Coding nucleotide sequence (SEQ ID NO.8):
  • Coding nucleotide sequence (SEQ ID NO.10):
  • Coding nucleotide sequence (SEQ ID NO.12):
  • Coding nucleotide sequence (SEQ ID NO.14):
  • CD8 signal peptide 2 (signal peptide 2) (SEQ ID NO.15):
  • Coding nucleotide sequence (SEQ ID NO.16):
  • Amino acid sequence of MYC tag (SEQ ID NO.17): EQKLISEEDL encoding nucleotide sequence (SEQ ID NO.18):
  • Coding nucleotide sequence (SEQ ID NO.20):
  • Coding nucleotide sequence (SEQ ID NO.22):
  • Coding nucleotide sequence (SEQ ID NO.24):
  • the italic part is the nucleotide sequence encoding chimeric antigen receptor 1 (SEQ ID NO.26), the underlined part is the nucleotide sequence encoding the polypeptide connecting chimeric antigen receptor 1 and chimeric antigen receptor 2 (SEQ ID NO.14), and the remaining part is the nucleotide sequence encoding chimeric antigen receptor 2 (SEQ ID NO.27)
  • the italic part is the amino acid sequence of chimeric antigen receptor 1 (SEQ ID NO.29)
  • the underlined part is the amino acid sequence of the polypeptide connecting chimeric antigen receptor 1 and chimeric antigen receptor 2 (SEQ ID NO.13)
  • the remaining part is the amino acid sequence of chimeric antigen receptor 2 (SEQ ID NO.30).
  • a nucleotide sequence encoding a complete dual-target chimeric antigen receptor (SEQ ID NO. 25) was synthesized and inserted into the EcoRI-BamH1 site of the lentiviral pWPXLd vector, which is preceded by an EF-1 ⁇ promoter, and transformed into competent Escherichia coli cells. After correct sequencing, the plasmid was extracted and purified using a plasmid purification kit from Qiagen. The purification steps were referred to the instructions of the kit to obtain a high-quality plasmid of the recombinant expression vector. The structure of the inserted target fragment is shown in Figure 1B.
  • Day 1 Plate. Digest 293T cells with good density at 90%, subculture at 1:3, about 4 x 10 6 cells/10ml/10cm plate, culture overnight at 5% CO 2 and 37°C. After 24 hours, the cell density is about 70-90% (not more than 90%).
  • the transfection steps are as follows:
  • psPAX2 packetaging plasmid
  • pMD.2G envelope plasmid
  • pWPXLd lentivirus vector, prepared in Example 1
  • Day 4 48 hours after transfection, collect cell supernatant, add 10 ml preheated DMEM complete medium, culture at 37°C with 5% CO 2 ; filter viral supernatant with 0.45 ⁇ m filter and store at 4°C (up to 1 week).
  • Day 5 72 hours after transfection, collect the viral supernatant, filter with a 0.45 ⁇ m filter, and store at 4°C.
  • Centrifugation conditions are: 20°C, 70000g, 2h. Wait until the centrifuge speed rises to 70000g before leaving. After centrifugation, pour out the culture medium and invert the centrifuge tube on the sterilized filter paper to absorb the remaining culture medium. Resuspend the virus precipitate with PBS. Divide the resuspended virus into 1.5ml EP tubes and store in a -80°C refrigerator for use.
  • Peripheral blood was collected using sodium heparin anticoagulation tubes. The ratio of lymphocyte separation solution volume to blood volume was 1:1. The peripheral blood was slowly added to the lymphocyte separation solution, and the mixture was centrifuged. The conditions were 1000g, 30min, 18°C, and acceleration/deceleration of 1. After centrifugation, the blood was divided into 4 layers, and the layer where PBMC was located was the middle white cloud-like layer. The tip of the gun slowly aspirated the middle white cloud-like layer. The aspirated cells were added to 10ml X VIVO medium and centrifuged at 300g for 10min. The supernatant was discarded, and the red cells were lysed with 10ml lysate, mixed and allowed to stand for 5min.
  • Retronectin recombinant human fibronectin
  • BSA bovine serum albumin
  • ROR1 CAR-T expressed the ROR1 CAR gene
  • ROR1-TGF ⁇ RII CAR-T expressed the ROR1 CAR gene and anti-TGF ⁇ RII scFv, indicating that the dual CAR was successfully constructed.
  • a nucleotide sequence (SEQ ID NO. 26) encoding chimeric antigen receptor 1 (signal peptide 1-Anti-ROR1 scFv-hinge-CD28 transmembrane segment-CD28 intracellular segment-CD3 ⁇ , see Figure 1A) was prepared by referring to the method of Example 1, and then a CAR-T containing only chimeric antigen receptor 1, i.e., ROR1 CAR-T, was prepared by referring to the method of Example 2.
  • Flow cytometry was used to detect the expression levels of ROR1 and TGF ⁇ RII in different tumor cell lines.
  • Ovarian cancer cells SKOV3, breast cancer cells MDA-MB-231, lung cancer cells A549 and ovarian cancer cells A2780 were incubated with PE-labeled antibodies that specifically recognize human ROR1 and APC-labeled antibodies that specifically recognize human TGF ⁇ RII.
  • T cells (ROR1 CAR-T (Comparative Example 1) group, ROR1-TGF ⁇ RII CAR-T (Example 2) group, control T cell group) were co-cultured with 1 ⁇ 10 4 ROR1-positive target cells: ovarian cancer cell line SKOV3, breast cancer cell line MDA-MB-231, lung cancer cell line A549, or ROR1-negative target cells: ovarian cancer cell line A2780, with the ratio of effector cells to target cells (effect-target ratio) being 1:1 or 2:1, and cultured for 24 hours in the absence of TGF- ⁇ or in the presence of 5 ng/ml TGF- ⁇ .
  • the levels of IFN- ⁇ (Figure 4), IL-2 ( Figure 5) and TNF- ⁇ ( Figure 6) in the cell culture supernatant were detected by ELISA.
  • the ROR1-TGF ⁇ RII CAR-T group released more IFN- ⁇ , IL-2 and TNF- ⁇ cytokines after co-culture with ROR1-positive tumor cells, and had stronger killing ability.
  • the dual-targeted CAR-T also released almost no cytokines and had no off-target toxicity.
  • T cells 1 ⁇ 10 6 T cells (ROR1 CAR-T group, ROR1-TGF ⁇ RII CAR-T group, control T cell group) were co-cultured with breast cancer cell line MDA-MB-231 and ovarian cancer cell line SKOV3 at an effector-target ratio of 3:1 for 24 hours. Flow cytometry was used to detect the level of intracellular IFN- ⁇ released by CD3-positive T cells (Figure 7).
  • T cells 1 ⁇ 10 6 T cells (ROR1 CAR-T group, ROR1-TGF ⁇ RII CAR-T group, and control T cell group) were co-cultured with lung cancer cell line A549 at an effector-target ratio of 3:1 for 4 hours. Flow cytometry was used to detect the level of intracellular granzyme B released by CD8-positive T cells (Figure 8).
  • T cells were labeled with FAR RED cell tracing fluorescent dye.
  • the cell tracing fluorescent dye is attached to the cell membrane. If the cell divides, the membrane dye on one cell will be distributed to the membranes of two new cells as the cell divides, so that the fluorescence on the cell will be weakened by half. Therefore, the weaker the fluorescence intensity, the less fluorescent dye there is on the cell membrane, and the more the cell divides and proliferates.
  • the two CAR-T cells were co-cultured with breast cancer cells MDA-MB-231, with an effector-target ratio of 3 to 1; after 24 hours, the fluorescence of the T cells was detected.
  • the horizontal axis represents the intensity of FAR RED fluorescence signals
  • the vertical axis represents the number of cells in a certain intensity range. The closer the peak of the curve is to the origin, the weaker the fluorescence signal.
  • the degree of division of ROR1-TGF ⁇ RII CAR-T is greater than that of the ROR1 CAR-T group, that is, the cell proliferation ability of this group is stronger.
  • T cells 1 ⁇ 10 6 T cells (ROR1 CAR-T group, ROR1-TGF ⁇ RII CAR-T group, control T cell group) were co-cultured with ovarian cancer cell line SKOV3 at an effector-target ratio of 3:1, and cultured for 24 hours in the absence of TGF- ⁇ or in the presence of 5ng/ml, 10ng/ml, and 20ng/ml TGF- ⁇ .
  • Flow cytometry was used to detect the expression level of pSMAD2/3, a downstream signaling pathway of TGF- ⁇ , in CAR-positive or CAR-negative T cells (Figure 10).
  • TGF- ⁇ RII In the presence of TGF- ⁇ , after CAR-T cells were co-incubated with tumor cells, TGF- ⁇ RII on the surface of T cells dimerized and bound to TGF- ⁇ , and then formed a heterotetramer with TGF- ⁇ RI, thereby activating downstream signaling pathways and phosphorylating intracellular SMAD proteins.
  • the dual-targeted ROR1-TGF ⁇ RII CAR-T group because it expresses anti-TGF ⁇ RII scfv, can block TGF ⁇ RII on its own surface, thereby preventing TGF ⁇ RII from binding to TGF- ⁇ , blocking intracellular SMAD protein phosphorylation, and thus resisting the inhibitory effect of TGF- ⁇ on T cells.
  • a model of peritoneal dissemination of human ovarian cancer was established using NSG immunodeficient mice and SKOV3 (SKOV3-luc) cells stably expressing luciferase. T cells were intraperitoneally injected and transfused to evaluate the inhibitory effect of T cells on tumor growth.
  • SKOV3 SKOV3-luc
  • mice Purchase 5-week-old NSG mice, keep them in the animal room for 1 week to adapt to the environment, and inject 5 ⁇ 10 5 SKOV3-luc cells intraperitoneally to establish a model.
  • bioluminescence imaging was performed to detect the tumor burden in vivo.
  • the mice were randomly divided into four groups on average, namely ROR1 CAR-T group, ROR1-TGF ⁇ RII CAR-T group, unmodified control T cells and PBS group, with 5 experimental mice in each group.
  • the preparation process of CAR-T cells was carried out according to Example 2.
  • each CAR-T group 2 ⁇ 10 6 CAR-positive T cells were intraperitoneally injected back into the mice.
  • the number of unmodified control T cells was the same as the total amount of the CAR-T group.
  • the cells were resuspended in 100 ⁇ l PBS and injected back into the mice intraperitoneally.
  • Tumor burden was assessed by intraperitoneal injection of luciferin into mice every week and imaging using a bioluminescence imaging system.
  • the dual-target CAR-T compared with the unmodified ROR1 CAR-T, the dual-target CAR-T has stronger killing activity against tumor cells, which is reflected in the release of more cytokines and stronger release of granzyme B; at the same time, it has stronger proliferation activity, which is conducive to the long-term survival of CAR-T. It also showed a stronger anti-tumor effect in the ovarian cancer peritoneal metastasis model. This shows that the dual-target CAR-T designed in the present invention can significantly increase the efficacy of T cells against solid tumors when TGF- ⁇ is inhibited.
  • the principle of the present invention is to design a chimeric antigen receptor targeting TGF- ⁇ RII.
  • it can bind to the TGF ⁇ RII of tumor cells to enhance the recognition and targeting effect of immune cells on tumors.
  • it can also target the TGF ⁇ RII of immune cells themselves to achieve the blocking of TGF ⁇ RII of immune cells themselves, hinder the binding of TGF ⁇ to the TGF ⁇ receptor of immune cells, thereby reducing the inhibitory effect of TGF ⁇ on immune cells.
  • the chimeric antigen receptor sequence targeting TGF- ⁇ RII does not contain CD3 ⁇ activation signal, and will not activate T cells to perform cytotoxic effects.
  • the chimeric antigen receptor targeting TGF ⁇ RII is used in combination with the traditional tumor-targeted chimeric antigen receptor to construct a dual-target chimeric antigen receptor-modified immune cell, which can not only achieve tumor-specific targeting and kill the tumor through the tumor-targeted chimeric antigen receptor, but also reduce the inhibitory effect of the inhibitory cytokine TGF ⁇ on immune cells through the action of the chimeric antigen receptor targeting TGF ⁇ RII, while enhancing the tumor recognition and targeting effect, thereby achieving a synergistic anti-tumor effect.
  • the method provided in the embodiment of the present invention can be adopted, in which the chimeric antigen receptor targeting TGF- ⁇ RII of the present invention is connected to the chimeric antigen receptor targeting the tumor (such as the chimeric antigen receptor of ROR1) through a self-cleaving peptide, and the immune cells (such as T cells) are modified to make CAR-T.
  • the combination form can also be reasonably changed.
  • the plasmid expressing the chimeric antigen receptor targeting TGF- ⁇ RII and the plasmid expressing the chimeric antigen receptor targeting the tumor are respectively transferred into the immune cells (such as T cells), so that the surface of the immune cells is modified with two chimeric antigen receptors at the same time, and the two chimeric antigen receptors are not connected, which can also constitute a combination form to achieve the same synergistic anti-tumor effect as the embodiment of the present invention.

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Abstract

提供了一种能够识别和结合TGFβRII的嵌合抗原受体，所述嵌合抗原受体与能够识别和结合肿瘤特异性抗原或肿瘤相关抗原的嵌合抗原受体联用构建双靶点嵌合抗原受体修饰的免疫细胞，具有优异的抗肿瘤效果。

Description

一种靶向TGFβRII的嵌合抗原受体及其用途 技术领域
本发明属于肿瘤治疗领域，具体涉及一种靶向TGFβRII的嵌合抗原受体及其用途。
背景技术
正常情况下，免疫系统可以识别并清除肿瘤微环境中的肿瘤细胞，但为了生存和生长，肿瘤细胞能够采用不同策略，使人体的免疫系统受到抑制而不能正常的杀伤肿瘤细胞，从而在抗肿瘤免疫应答的各阶段得以幸存。肿瘤免疫治疗就是通过恢复机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法。肿瘤免疫疗法包括单克隆抗体类免疫检查点抑制剂、治疗性抗体、癌症疫苗、细胞治疗等。近几年，肿瘤免疫治疗由于其卓越的疗效和创新性，在2013年被《科学》杂志评为年度最重要的科学突破，其中嵌合抗原受体修饰的T细胞(Chimeric antigen receptor T cells，CAR-T)治疗与CTLA-4、PD-1/PD-L1抗体治疗被认为肿瘤免疫治疗三大进展。
CAR-T疗法就是利用抗原抗体片段，联合T细胞胞内激活、增殖信号，构建CAR-T，可使T细胞直接获得抗体的特异识别能力、成为不依赖HLA(人类白细胞抗原)限制的效应T细胞。经过设计的CAR-T细胞可在实验室培养生长，达到数十亿之多将扩增后的CAR-T细胞注入到患者体内，注入之后的T细胞也会在患者体内增殖，并杀死具有相应特异性抗原的肿瘤细胞，长期存活并形成免疫记忆。也有研究将嵌合抗原受体表达于T细胞以外的免疫细胞，诸如单核细胞、自然杀伤细胞、中性粒细胞等，使这些免疫细胞也获得特异识别肿瘤细胞，发挥免疫治疗的功能。
CAR修饰的免疫细胞对肿瘤实现免疫治疗的疗法核心在于嵌合抗原受体(CAR)的设计。CAR主要由三个功能域构成，分别是胞外结构域、跨膜结构域和胞内结构域。一般来说，胞外结构域为负责识别并结合抗原的单克隆抗体的单链可变片段(scFv)，或者scFv与一段起连接作用的铰链区(Hinge)构成。胞内结构域包括共刺激结构域和信号转导结构域。跨膜结构域则将CAR的细胞外结构域与胞内结构域连接，并将受体锚定到T细胞膜上。其中，scFv是进行抗原识别靶向的关键，是影响治疗效果的重要因素。
至今为止，已有诸多靶向肿瘤特异性抗原或肿瘤相关抗原的CAR-T报道，例如，ROR1是RTK(receptor tyrosine kinase)家族中的一个跨膜蛋白。在胚胎发育过程中存在，与中枢神经系统、心、肺和骨组织的发育有关。产后，除了在脂肪组织、胰腺、肺和发育中的B细胞(前B细胞、未成熟B细胞)上少量表达，在绝大部分正常组织中不表达。然而在许多血液和实体恶性肿瘤中有表达。目前研究表明，ROR1在慢性淋巴细胞白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤、卵巢癌、乳腺癌、结肠癌、肺癌、胰腺癌、 黑色素瘤等恶性肿瘤中有不同程度的高表达。并且在卵巢癌、三阴乳腺癌中，ROR1表达的程度与不良预后有关。ROR1倾向于表达在低分化肿瘤中，表达ROR1的癌细胞显示出更强的侵袭转移和复发的能力，并表达与上皮间质转化(Epithelial-Mesenchymal Transition，EMT)相关的标志基因。因此，基于ROR1靶点开发的ROR1 CAR-T，目前已有较多研究，表现出有效的对肿瘤细胞的靶向杀伤作用。
遗憾的是，嵌合抗原受体以及表达嵌合抗原受体的免疫细胞至今在实体瘤临床应用进展缓慢，仅取得有限的治疗应答。主要原因之一是肿瘤微环境中存在大量的抑制免疫细胞的细胞因子，这些细胞因子使得表达嵌合抗原受体的免疫细胞无法达到并维持一个较高的数量水平。
在众多抑制免疫细胞的细胞因子中，转化生长因子β(TGF-β)是由肿瘤细胞、调节性T细胞(regulatory T cells,Treg)、骨髓来源抑制性细胞(myeloid-derived suppressor cells,MDSC)等细胞产生的，在卵巢癌、肺癌、结直肠癌等大多数实体瘤病灶内都有高水平表达。TGF-β通过结合TGF-β受体TGFβRI和TGFβRII直接抑制T细胞活性。TGF-β与二聚化的TGFβRII结合后招募TGFβRI形成异源四聚体，从而引起胞内的SMAD2和SMAD3的磷酸化。磷酸化SMADs诱导抑制性转录程序，导致细胞因子产生减少、细胞杀伤毒性降低和抑制抗原结合时T细胞扩增。此外，TGF-β可促使T细胞分化为Treg，而Treg又可产生TGF-β，并进一步促进免疫抑制和肿瘤耐受，因此TGF-β的免疫抑制被认为是抗肿瘤活性失败的主要原因之一。
因此，在已有的靶向肿瘤的CAR-T基础上，为进一步提升其疗效，如何采用合理手段有效规避TGF-β等抑制免疫细胞的细胞因子的影响，还有待进一步探究。
发明内容
本发明的目的在于提供一种靶向TGFβRII的嵌合抗原受体，以及该嵌合抗原受体与传统的肿瘤靶向的嵌合抗原受体联合形成的双靶点嵌合抗原受体。
本发明提供了一种嵌合抗原受体，它是能够识别和结合TGFβRII的嵌合抗原受体。
进一步地，上述的嵌合抗原受体包括抗原识别结构域、跨膜结构域、胞内结构域；所述抗原识别结构域是TGFβRII的单链抗体。
更进一步地，上述TGFβRII的单链抗体氨基酸序列如SEQ ID NO.19所示。
进一步地，上述跨膜结构域是CD28跨膜段、CD8、CD3ζ、CD134、CD137、ICOS、DAP10、CD27中的任意一种或多种；
所述胞内结构域包括共刺激信号域，所述共刺激信号域为CD28胞内段、4-1BB、ICOS、CD27、OX40、MyD88、CD40中的任意一种或多种。
更进一步地，上述跨膜结构域是CD8，氨基酸序列如SEQ ID NO.21所示；所述胞内结构域是4-1BB，氨基酸序列如SEQ ID NO.23所示。
进一步地，上述的嵌合抗原受体，还包括信号肽2，优选地，所述信号肽2的氨基酸序列如SEQ ID NO.15所示。
更进一步地，上述的嵌合抗原受体，还包括蛋白标签序列，优选为MYC标签序列，氨基酸序列如SEQ ID NO.17所示。
更进一步地，上述嵌合抗原受体包括顺序连接的如下片段：信号肽2、蛋白标签序列、TGFβRII的单链抗体、CD8跨膜结构域、4-1BB共刺激信号域；
优选地，所述嵌合抗原受体氨基酸序列如SEQ ID NO.30所示。
本发明还提供了一种双靶点嵌合抗原受体，包括嵌合抗原受体1和嵌合抗原受体2；
其中，嵌合抗原受体1是能够识别和结合肿瘤特异性抗原或肿瘤相关抗原的嵌合抗原受体；嵌合抗原受体2是权利要求1～7任一项所述的嵌合抗原受体。
进一步地，上述的双靶点嵌合抗原受体是嵌合抗原受体1和嵌合抗原受体2的组合，或是嵌合抗原受体1与嵌合抗原受体2连接而成。
更进一步地，上述的双靶点嵌合抗原受体，它是嵌合抗原受体1与嵌合抗原受体2通过多肽片段连接而成；优选地，所述多肽片段是自剪切肽片段，更优选为自剪切肽P2A片段，氨基酸序列如SEQ ID NO.13所示。
进一步地，上述嵌合抗原受体1包括抗原识别结构域、跨膜结构域、胞内结构域；所述抗原识别结构域是能够识别和结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体；
优选地，所述肿瘤特异性抗原或肿瘤相关抗原是CD19、CD20、MUC1、ROR1、EGFR、EGFRvIII、HER2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、CD44、IGFR中的任意一种或多种。
更进一步地，上述单链抗体是ROR1的单链抗体；优选地，所述ROR1的单链抗体的氨基酸序列如SEQ ID NO.3所示。
更进一步地，上述跨膜结构域是CD28跨膜段、CD8、CD3ζ、CD134、CD137、ICOS、DAP10、CD27中的任意一种或多种；
所述胞内结构域包括共刺激信号域和信号转导域，所述共刺激信号域为CD28胞内段、4-1BB、ICOS、CD27、OX40、MyD88、CD40中的任意一种或多种；所述信号转导域为CD3ζ或FcεRI。
更进一步地，上述跨膜结构域是CD28跨膜段，氨基酸序列如SEQ ID NO.7所示；所述共刺激信号域为CD28胞内段，氨基酸序列如SEQ ID NO.9所示；所述信号转导域为CD3ζ，氨基酸序列如SEQ ID NO.11所示。
进一步地，上述嵌合抗原受体1还包括信号肽1、铰链区；优选地，所述信号肽1的氨基酸序列如SEQ ID NO.1所示，所述铰链区的氨基酸序列如SEQ ID NO.5所示。
更进一步地，上述嵌合抗原受体1包括顺序连接的如下片段：信号肽1、ROR1的单链抗体、铰链区、CD28跨膜结构域、CD28胞内段共刺激信号域、 CD3ζ信号转导域；
优选地，所述嵌合抗原受体1的氨基酸序列如SEQ ID NO.29所示。
进一步地，上述的双靶点嵌合抗原受体氨基酸序列如SEQ ID NO.26所示。
本发明还提供了一种基因，它是编码上述的嵌合抗原受体或编码上述双靶点嵌合抗原受体的基因；优选的，所述编码上述的嵌合抗原受体基因核苷酸序列如SEQ ID NO.27所示，编码上述双靶点嵌合抗原受体的基因核苷酸序列如SEQ ID NO.25所示。
本发明还提供了一种载体，它是含有上述的基因的载体；所述载体是质粒或病毒。
本发明还提供了一种宿主细胞，它是表达上述的嵌合抗原受体或表达上述双靶点嵌合抗原受体的宿主细胞，优选为含有上述的载体的宿主细胞。
进一步地，上述宿主细胞为免疫反应细胞，优选为T细胞、单核细胞、自然杀伤细胞或中性粒细胞中的至少一种，更优选为T细胞。
本发明还提供了上述的嵌合抗原受体、双靶点嵌合抗原受体、基因、载体或宿主细胞在制备预防和/或治疗肿瘤的药物中的用途。
本发明的有益效果：本发明提供过了一种靶向TGFβRII的嵌合抗原受体。一方面，其可以结合肿瘤细胞的TGFβRII，增强免疫细胞对肿瘤的识别靶向作用，另一方面，其还可以靶向免疫细胞自身的TGFβRII，实现对免疫细胞自身TGFβRII的封闭，阻碍TGFβ与免疫细胞的TGFβ受体结合从而减轻TGFβ对免疫细胞的抑制作用，同时，靶向TGF-βRII的嵌合抗原受体序列中不包含CD3ζ激活信号，不会激活T细胞执行细胞毒性作用。
进一步地，将靶向TGFβRII的嵌合抗原受体与传统的肿瘤靶向的嵌合抗原受体联合使用，构建双靶点嵌合抗原受体修饰的免疫细胞，既能够通过肿瘤靶向的嵌合抗原受体实现肿瘤特异性靶向，对肿瘤产生杀伤，又能够借由靶向TGFβRII的嵌合抗原受体的作用，在增强肿瘤识别靶向效果的同时，减轻抑制细胞因子TGFβ对免疫细胞的抑制作用，从而实现协同抗肿瘤的效果。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明病毒载体中嵌合抗原受体功能单元结构组成图。其中：A为单靶向ROR1的嵌合抗原受体，B为双靶点嵌合抗原受体。
图2为本发明CAR在T细胞上的表达情况。
图3为肿瘤细胞表面抗原表达情况。
图4为CAR-T与肿瘤细胞共培养后IFN-γ释放水平。
图5为CAR-T与肿瘤细胞共培养后IL-2释放水平。
图6为CAR-T与肿瘤细胞共培养后TNF-α释放水平。
图7为CAR-T与肿瘤细胞共培养后分泌IFN-γ的细胞检测。
图8为CAR-T细胞体外杀伤肿瘤细胞后T细胞颗粒酶B释放检测。
图9为CAR-T细胞与肿瘤细胞共培养后T细胞增殖情况。
图10为CAR-T细胞体外杀伤肿瘤细胞后T细胞胞内pSMAD2/3信号通路检测。
图11为CAR-T细胞在体内对SKOV3卵巢癌腹腔播散模型的生长抑制作用。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
实施例1、合成双靶点嵌合抗原受体全长基因，完成重组质粒载体的构建
先准备本实施例中制备的双靶点嵌合抗原受体的表达框架。表达框架从5端到3端依次为：信号肽1-Anti-ROR1 scFv-hinge-CD28跨膜段-CD28胞内段-CD3ζ-P2A-信号肽2-MYC蛋白标签-Anti-TGF-βRII scFv-CD8跨膜结构域-4-1BB胞内段。
其中各段的序列为：
嵌合抗原受体1的信号肽1的氨基酸序列(SEQ ID NO.1)：
Figure PCTCN2022132043-appb-000001
编码核苷酸序列(SEQ ID NO.2)：
Figure PCTCN2022132043-appb-000002
ROR1的单链抗体(Anti-ROR1 scFv)的氨基酸序列(SEQ ID NO.3)：
Figure PCTCN2022132043-appb-000003
编码核苷酸序列(SEQ ID NO.4)：
Figure PCTCN2022132043-appb-000004
Figure PCTCN2022132043-appb-000005
铰链区(Hinge)的氨基酸序列(SEQ ID NO.5)：ESKYGPPCPPCP
编码核苷酸序列(SEQ ID NO.6)：
Figure PCTCN2022132043-appb-000006
CD28跨膜段的氨基酸序列(SEQ ID NO.7)：
Figure PCTCN2022132043-appb-000007
编码核苷酸序列(SEQ ID NO.8)：
Figure PCTCN2022132043-appb-000008
CD28胞内段的氨基酸序列(SEQ ID NO.9)：RSKRSRGCHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS
编码核苷酸序列(SEQ ID NO.10)：
Figure PCTCN2022132043-appb-000009
CD3ζ的氨基酸序列(SEQ ID NO.11)：
Figure PCTCN2022132043-appb-000010
编码核苷酸序列(SEQ ID NO.12)：
Figure PCTCN2022132043-appb-000011
P2A的氨基酸序列(SEQ ID NO.13)：GSGATNFSLLKQAGDVEENPGP
编码核苷酸序列(SEQ ID NO.14)：
Figure PCTCN2022132043-appb-000012
Figure PCTCN2022132043-appb-000013
CD8信号肽(信号肽2)的氨基酸序列(SEQ ID NO.15)：
Figure PCTCN2022132043-appb-000014
编码核苷酸序列(SEQ ID NO.16)：
Figure PCTCN2022132043-appb-000015
MYC标签的氨基酸序列(SEQ ID NO.17)：EQKLISEEDL编码核苷酸序列(SEQ ID NO.18)：
Figure PCTCN2022132043-appb-000016
Anti-TGFβRII-scFv的氨基酸序列(SEQ ID NO.19)：
Figure PCTCN2022132043-appb-000017
编码核苷酸序列(SEQ ID NO.20)：
Figure PCTCN2022132043-appb-000018
CD8跨膜结构域的氨基酸序列(SEQ ID NO.21)：
Figure PCTCN2022132043-appb-000019
编码核苷酸序列(SEQ ID NO.22)：
Figure PCTCN2022132043-appb-000020
Figure PCTCN2022132043-appb-000021
4-1BB胞内段的氨基酸序列(SEQ ID NO.23)：
Figure PCTCN2022132043-appb-000022
编码核苷酸序列(SEQ ID NO.24)：
Figure PCTCN2022132043-appb-000023
完整的编码双靶点嵌合抗原受体的核苷酸序列(SEQ ID NO.25)：
Figure PCTCN2022132043-appb-000024
Figure PCTCN2022132043-appb-000025
其中，斜体部分为编码嵌合抗原受体1的核苷酸序列(SEQ ID NO.26)，下划线部分为编码连接嵌合抗原受体1和嵌合抗原受体2的多肽的核苷酸序列(SEQ ID NO.14)，剩余部分为编码嵌合抗原受体2的核苷酸序列(SEQ ID NO.27)
完整的双靶点嵌合抗原受体的氨基酸序列(SEQ ID NO.28)：
Figure PCTCN2022132043-appb-000026
Figure PCTCN2022132043-appb-000027
其中，斜体部分为嵌合抗原受体1的氨基酸序列(SEQ ID NO.29)，下划线部分为连接嵌合抗原受体1和嵌合抗原受体2的多肽的氨基酸序列(SEQ ID NO.13)，剩余部分为嵌合抗原受体2的氨基酸序列(SEQ ID NO.30)。
合成编码完整的双靶点嵌合抗原受体的核苷酸序列(SEQ ID NO.25),并将其插入到慢病毒pWPXLd载体EcoRI-BamH1位点，该位点前面有EF-1α启动子，转化到大肠杆菌感受态细胞，经测序正确后，使用Qiagen公司的质粒纯化试剂盒提取并纯化质粒，纯化步骤参照试剂盒说明书，获得重组表达载体的高品质质粒，插入的目的片段结构参见图1B。
实施例2、重组载体转化细胞
1、293T细胞的培养和传代：
打开生物安全柜，用75％的酒精棉擦拭台面，并将移液器、移液枪、枪尖盒、15ml离心管、离心管架、10cm2新的细胞培养皿放于生物安全柜中，关闭柜门，开启生物安全柜的紫外开关，照射半小时以消毒灭菌。将含10％的胎牛血清和100U/ml青霉素链霉素的DMEM及胰酶放于37℃水浴锅中预热。打开生物安全柜，开启通风开关，将已长到80％-90％的293T细胞培养皿从37℃、5％的CO 2的培养箱中取出，放于生物安全柜。用75％的酒精消毒双手、培养基瓶瓶口、移液管筒口等。用无菌移液管吸尽培养皿中的培养基，弃于废液缸中。向培养皿中滴入2ml胰酶，镜下观察细胞，直到细胞变圆分离，吸除胰酶。向培养皿中加入6-8ml新鲜完全培养基，轻柔吹打下细胞。将细胞悬液分于其他培养皿，并添加培养基以达到每皿10ml。十字式晃动培养皿数次，摇匀细胞，镜下观察后放入37℃培养箱。24小时后观察细胞状态，待细胞长到80％-90％时进行下一次传代培养。
2、慢病毒原液的获取：
Day 1:铺板。90％密度状态良好的293T细胞消化，1:3传代,约4 x 10 6cells/10ml/10cm平皿，5％CO 2、37℃培养过夜。24h细胞密度约70-90％(不超过90％)。
Day 2：转染。转染前所有试剂平衡至室温。
转染步骤如下：
a.在10ml BD管中配制以下DNA混合物(每10cm平皿)，psPAX2(packaging plasmid)3μg；pMD.2G(envelope plasmid)1.5μg；pWPXLd(lentivirus vector，由实施例1制备得到)6μg。
b.加入DMEM培养基定容至980μl，混匀。
c.Highgene转染试剂(爱博泰克公司)20μl缓慢滴入DNA溶液，轻轻吹打至混匀。
d.室温静置15min。
e.将1ml转染混合物滴入平皿，“十”字式(各10次)轻轻摇晃混匀后放回5％CO 2，37℃培养箱。8-12h后吸去培养基，使用预热的DMEM完全培养基进行换液，放回5％CO 2，37℃培养箱至48h。
Day 4：转染后48h。收细胞上清，加预热的DMEM完全培养基10ml，5％CO 2，37℃培养；病毒上清0.45μm滤器过滤，4℃保存(最多1周)。
Day 5：转染后72h，收病毒上清，0.45μm滤器过滤，4℃保存。
3、慢病毒的浓缩：
仪器：超高速离心仪，配套的转子与套筒，超速离心管，配平用天平。将套筒及天平至生物安全柜紫外仪下消毒。确保每个套筒内无液滴后，将合适的离心管放至套筒里。将用0.45μm的滤器过滤的病毒悬液加至离心管内。每个装了病毒悬液的离心管严格配平，使用精度为0.001g及以上的天平,盖上套筒盖后再次用天平验证是否完全配平。将配平好的各套筒装至离心仪转子里，准备离心。离心：离心条件为：20℃，70000g,2h。待离心仪速度上升至70000g再离开。离心完毕后，将培养基倒掉，离心管倒置在灭菌滤纸上吸干剩余培养基。使用PBS重悬病毒沉淀。将重悬的病毒分装至1.5ml EP管里，-80℃冰箱保存，待使用。
4、人外周血T淋巴细胞的分离：
采用肝素钠抗凝管收集外周血。淋巴细胞分离液体积与血液体积比例为1:1，将外周血缓慢加入淋巴细胞分离液上，并将此混合液离心。条件是1000g，30min，18℃，加/减速度为1。离心过后可以看到血液分为4层，PBMC所在层为中间白色云絮状层。枪尖缓慢吸取中间白色云絮状层。吸取的细胞加入到10ml X VIVO培养基中离心，300g，10min。弃上清，用10ml裂红液进行裂红，混匀后静置5min。离心300g，10min。弃上清，用10ml X VIVO培养基重悬，并计数。离心300g，10min。弃上清，用5％人血清，100IU/ml IL-2的X VIVO培养基重悬淋巴细胞，根据细胞数量加入对应数量的CD3/CD28刺激磁珠。5％CO 2，37℃培养。
5、病毒感染T细胞
病毒感染T细胞前一天，用Retronectin(重组人纤连蛋白)稀释液包被十二孔板(浓度为50μg/ml)，每孔1ml。孔板封口后放入4℃过夜备用。感染当天，吸去Retronectin稀释液，使用2％BSA(牛血清白蛋白)溶液封闭30min。吸去BSA液体，使用PBS润洗一次。各孔加入1×10 6个细胞，以及病毒浓缩液，放入离心机进行离心感染，条件为32℃，1000g，2h。离心完成后取出孔板，放回37℃，5％CO 2细胞培养箱培养。24h后使用完全培养基换液。
6、检测T细胞表面CAR分子表达：
ROR1 CAR病毒以及ROR1-TGFβRII双CAR病毒感染T细胞72h后进 行流式细胞术检测CAR的表达(图2)。
如图2所示，ROR1 CAR-T表达ROR1 CAR基因，而ROR1-TGFβRII CAR-T表达ROR1 CAR基因以及抗TGFβRII的scFv，表明双CAR构建成功。
对比例1、ROR1 CAR-T的制备
参照实施例1的方法制备编码嵌合抗原受体1(信号肽1-Anti-ROR1 scFv-hinge-CD28跨膜段-CD28胞内段-CD3ζ，参见图1A)的核苷酸序列(SEQ ID NO.26)，然后参照实施例2的方法制备得到仅有嵌合抗原受体1的CAR-T，即ROR1 CAR-T。
以下通过实验例证明本发明的有益效果。
实验例1、检测肿瘤细胞表面抗原表达
采用流式细胞仪检测不同肿瘤细胞株ROR1和TGFβRII的表达水平。
方法：利用PE标记的特异识别人ROR1的抗体以及APC标记的特异识别人TGFβRII的抗体孵育卵巢癌细胞SKOV3、乳腺癌细胞MDA-MB-231和肺癌细胞A549和卵巢癌细胞A2780。
结果：经流式细胞仪检测，最终发现不同的肿瘤细胞ROR1及TGFβRII的表达水平不同。其中MDA-MB-231细胞的ROR1及TGFβRII表达水平最高；SKOV3中度表达ROR1及TGFβRII；A549中度表达ROR1，几乎不表达TGFβRII；A2780细胞ROR1表达为阴性，中度表达TGFβRII(见图3)。
实验例2、双靶向CAR-T的功能检测
1、CAR-T细胞体外杀伤肿瘤细胞后细胞因子释放量检测
方法：将T细胞(ROR1 CAR-T(对比例1)组、ROR1-TGFβRII CAR-T(实施例2)组、对照T细胞组)与1×10 4的ROR1表达阳性的靶细胞：卵巢癌细胞株SKOV3，乳腺癌细胞株MDA-MB-231，肺癌细胞株A549，或者ROR1表达阴性的靶细胞：卵巢癌细胞株A2780，共培养，效应细胞与靶细胞的比例(效靶比)为1:1或2:1，在无TGF-β或存在5ng/ml TGF-β的情况下，培养24h。使用ELISA检测细胞培养上清中IFN-γ(图4)、IL-2(图5)和TNF-α(图6)的水平。
结果：与单纯的ROR1 CAR-T组相比，ROR1-TGFβRII CAR-T组与ROR1表达阳性的肿瘤细胞共培养后，能释放更多的IFN-γ、IL-2和TNF-α细胞因子，具有更强的杀伤能力。而与ROR1表达阴性的肿瘤细胞共培养后，双靶向的CAR-T也几乎不释放细胞因子，没有脱靶毒性。
2、CAR-T细胞体外杀伤肿瘤细胞后T细胞胞内INF-γ释放检测
方法：将1×10 6个T细胞(ROR1 CAR-T组、ROR1-TGFβRII CAR-T组、对照T细胞组)与乳腺癌细胞株MDA-MB-231、卵巢癌细胞株SKOV3共培养，效靶比3:1，培养24h。使用流式细胞术检测CD3阳性的T细胞释放胞内IFN-γ的水平(图7)。
结果：与单纯的ROR1 CAR-T组相比，ROR1-TGFβRII CAR-T组与ROR1表达阳性的肿瘤细胞共培养后，分泌胞内IFN-γ的细胞数量更多，具有更强 的杀伤能力。
3、CAR-T细胞体外杀伤肿瘤细胞后T细胞颗粒酶B释放检测
方法：将1×10 6个T细胞(ROR1 CAR-T组、ROR1-TGFβRII CAR-T组、对照T细胞组)与肺癌细胞株A549共培养，效靶比3:1，培养4h。使用流式细胞术检测CD8阳性的T细胞释放胞内颗粒酶B的水平(图8)。
结果：与单纯的ROR1 CAR-T组相比，ROR1-TGFβRII CAR-T组与ROR1表达阳性的肿瘤细胞共培养后，能更快地释放颗粒酶B，具有更强的细胞毒性。
4、CAR-T细胞与肿瘤细胞共培养后T细胞增殖情况
方法：使用FAR RED细胞示踪荧光染料标记T细胞；该细胞示踪荧光染料附着于细胞膜上，若细胞分裂，一个细胞上的膜染料会随分裂而分布到两个新的细胞的膜上，从而细胞上的荧光会减弱一半；所以荧光强度越弱，表明细胞膜上的荧光染料更少，则细胞分裂越多，增殖越多。
将两种CAR-T细胞与乳腺癌细胞MDA-MB-231共培养，效靶比为3比1；24h后，检测T细胞的荧光。
结果：如图9所示，横轴代表FAR RED荧光信号强度，纵轴代表某一强度区间内细胞数量，曲线形成的峰越靠近原点，则荧光信号越弱。由图可知，在CD8、CD4阳性细胞群中，ROR1-TGFβRII CAR-T分裂程度都大于ROR1 CAR-T组，即该组细胞增殖能力更强。
4、CAR-T细胞体外杀伤肿瘤细胞后T细胞胞内pSMAD2/3信号通路检测
方法：将1×10 6个T细胞(ROR1 CAR-T组、ROR1-TGFβRII CAR-T组、对照T细胞组)与卵巢癌细胞株SKOV3共培养，效靶比3:1，在无TGF-β或存在5ng/ml、10ng/ml、20ng/ml TGF-β的情况下，培养24h。使用流式细胞术检测CAR阳性或者CAR阴性的T细胞胞内TGF-β下游信号通路pSMAD2/3的表达水平(图10)。
结果：在TGF-β存在的情况下，CAR-T与肿瘤细胞共孵育后，T细胞表面的TGF-βRII二聚化与TGF-β结合，进而与TGF-βRI形成异四聚体，从而激活下游的信号通路，磷酸化胞内的SMAD蛋白。而双靶向的ROR1-TGFβRII CAR-T组，因为自身表达抗TGFβRII的scfv，可以封闭自身表面的TGFβRII，从而阻止TGFβRII与TGF-β结合，阻断胞内SMAD蛋白磷酸化，从而抵制TGF-β对T细胞的抑制作用。实验结果显示双靶向的ROR1-TGFβRII CAR-T组，在TGF-β存在的情况下，CAR阳性部分的T细胞胞内pSMAD2/3表达低于单纯ROR1 CAR-T组。
5、CAR-T细胞在体内对SKOV3卵巢癌腹腔播散模型的生长抑制作用
方法：利用NSG免疫缺陷小鼠，采用稳定表达荧光素酶的SKOV3(SKOV3-luc)细胞，建立人卵巢癌腹腔播散模型，腹腔注射回输T细胞，评估T细胞对肿瘤的生长抑制作用。
①购买5周龄的NSG小鼠，于动物房饲养1周适应环境，予以5×10 5 个SKOV3-luc细胞腹腔注射以建立模型。一周后予以生物发光成像以检测体内肿瘤负荷。根据肿瘤负荷将小鼠平均随机分成四组，分为ROR1 CAR-T组、ROR1-TGFβRII CAR-T组、未修饰的对照T细胞以及PBS组，每组实验鼠数目为5只。CAR-T细胞的制备过程按照实施例2进行。
②CAR-T组每组腹腔注射回输2×10 6个CAR阳性的T细胞，未修饰的对照T细胞回输数量与CAR-T组总量相同，用100μl PBS重悬细胞，腹腔注射回输入小鼠体内。
③每周向小鼠腹腔注射荧光素并使用生物发光成像系统进行成像，从而评估肿瘤负荷。
结果：与PBS组、未修饰的对照T细胞组相比，两种CAR-T均能抑制SKOV3肿瘤细胞的生长，并且ROR1-TGFβRII CAR-T在体内具有更强的肿瘤抑制作用(图11)。
综上，与未改造的ROR1 CAR-T相比，双靶点CAR-T对肿瘤细胞的杀伤活性更强，体现在可以释放更多的细胞因子，具有更强的颗粒酶B释放；同时具有更强的增殖活性，有利于CAR-T长期存活。在卵巢癌腹腔转移模型中也展现出更强的抗肿瘤效应。这表明本发明中设计的双靶点CAR-T可以显著增加T细胞在TGF-β抑制时对实体瘤的疗效。
本发明的原理在于，设计了一种靶向TGF-βRII的嵌合抗原受体，一方面，其可以结合肿瘤细胞的TGFβRII，增强免疫细胞对肿瘤的识别靶向作用，另一方面，其还可以靶向免疫细胞自身的TGFβRII，实现对免疫细胞自身TGFβRII的封闭，阻碍TGFβ与免疫细胞的TGFβ受体结合从而减轻TGFβ对免疫细胞的抑制作用，同时，靶向TGF-βRII的嵌合抗原受体序列中不包含CD3ζ激活信号，不会激活T细胞执行细胞毒性作用。
进一步地，将靶向TGFβRII的嵌合抗原受体与传统的肿瘤靶向的嵌合抗原受体联合使用，构建双靶点嵌合抗原受体修饰的免疫细胞，既能够通过肿瘤靶向的嵌合抗原受体实现肿瘤特异性靶向，对肿瘤产生杀伤，又能够借由靶向TGFβRII的嵌合抗原受体的作用，在增强肿瘤识别靶向效果的同时，减轻抑制细胞因子TGFβ对免疫细胞的抑制作用，从而实现协同抗肿瘤的效果。
基于这一原理，可以采用本发明实施例提供的方式，以本发明靶向TGF-βRII的嵌合抗原受体通过自剪切肽与肿瘤靶向的嵌合抗原受体(如ROR1的嵌合抗原受体)连接的形式联用，修饰免疫细胞(如T细胞)制成CAR-T，但同时，根据本领域的基本常识，也可以对其联用形式进行合理的改变，例如分别将表达靶向TGF-βRII的嵌合抗原受体的质粒和表达肿瘤靶向的嵌合抗原受体(如ROR1的嵌合抗原受体)的质粒转入免疫细胞(如T细胞)，使得免疫细胞表面同时修饰有两种嵌合抗原受体，且这两种嵌合抗原受体不相连接，同样可以构成一种联用形式，实现与本发明实施例同样的协同抗肿瘤的效果。

Claims (23)

  1. 一种嵌合抗原受体，其特征在于，它是能够识别和结合TGFβRII的嵌合抗原受体。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，包括抗原识别结构域、跨膜结构域、胞内结构域；所述抗原识别结构域是TGFβRII的单链抗体。
  3. 如权利要求2所述的嵌合抗原受体，其特征在于，所述TGFβRII的单链抗体氨基酸序列如SEQ ID NO.19所示。
  4. 如权利要求2所述的嵌合抗原受体，其特征在于，所述跨膜结构域是CD28跨膜段、CD8、CD3ζ、CD134、CD137、ICOS、DAP10、CD27中的任意一种或多种；
    所述胞内结构域包括共刺激信号域，所述共刺激信号域为CD28胞内段、4-1BB、ICOS、CD27、OX40、MyD88、CD40中的任意一种或多种。
  5. 如权利要求4所述的嵌合抗原受体，其特征在于，所述跨膜结构域是CD8，氨基酸序列如SEQ ID NO.21所示；所述胞内结构域是4-1BB，氨基酸序列如SEQ ID NO.23所示。
  6. 如权利要求1～5任一项所述的嵌合抗原受体，其特征在于，还包括信号肽2，优选地，所述信号肽2的氨基酸序列如SEQ ID NO.15所示。
  7. 如权利要求6所述的嵌合抗原受体，其特征在于，还包括蛋白标签序列，优选为MYC标签序列，氨基酸序列如SEQ ID NO.17所示。
  8. 如权利要求7所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体包括顺序连接的如下片段：信号肽2、蛋白标签序列、TGFβRII的单链抗体、CD8跨膜结构域、4-1BB共刺激信号域；
    优选地，所述嵌合抗原受体氨基酸序列如SEQ ID NO.30所示。
  9. 一种双靶点嵌合抗原受体，其特征在于，包括嵌合抗原受体1和嵌合抗原受体2；
    其中，嵌合抗原受体1是能够识别和结合肿瘤特异性抗原或肿瘤相关抗原的嵌合抗原受体；嵌合抗原受体2是权利要求1～7任一项所述的嵌合抗原受体。
  10. 如权利要求9所述的双靶点嵌合抗原受体，其特征在于，它是嵌合抗原受体1和嵌合抗原受体2的组合，或是嵌合抗原受体1与嵌合抗原受体2连接而成。
  11. 如权利要求10所述的双靶点嵌合抗原受体，其特征在于，它是嵌合抗原受体1与嵌合抗原受体2通过多肽片段连接而成；优选地，所述多肽片段是自剪切肽片段，更优选为自剪切肽P2A片段，氨基酸序列如SEQ ID NO.13所示。
  12. 如权利要求9所述的双靶点嵌合抗原受体，其特征在于，所述嵌合抗原受体1包括抗原识别结构域、跨膜结构域、胞内结构域；所述抗原识别结构域是能够识别和结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体；
    优选地，所述肿瘤特异性抗原或肿瘤相关抗原是CD19、CD20、MUC1、 ROR1、EGFR、EGFRvIII、HER2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、CD44、IGFR中的任意一种或多种。
  13. 如权利要求12所述的双靶点嵌合抗原受体，其特征在于，所述单链抗体是ROR1的单链抗体；优选地，所述ROR1的单链抗体的氨基酸序列如SEQ ID NO.3所示。
  14. 如权利要求12所述的双靶点嵌合抗原受体，其特征在于，所述跨膜结构域是CD28跨膜段、CD8、CD3ζ、CD134、CD137、ICOS、DAP10、CD27中的任意一种或多种；
    所述胞内结构域包括共刺激信号域和信号转导域，所述共刺激信号域为CD28胞内段、4-1BB、ICOS、CD27、OX40、MyD88、CD40中的任意一种或多种；所述信号转导域为CD3ζ或FcεRI。
  15. 如权利要求14所述的双靶点嵌合抗原受体，其特征在于，所述跨膜结构域是CD28跨膜段，氨基酸序列如SEQ ID NO.7所示；所述共刺激信号域为CD28胞内段，氨基酸序列如SEQ ID NO.9所示；所述信号转导域为CD3ζ，氨基酸序列如SEQ ID NO.11所示。
  16. 如权利要求12～15任一项所述的双靶点嵌合抗原受体，其特征在于，所述嵌合抗原受体1还包括信号肽1、铰链区；优选地，所述信号肽1的氨基酸序列如SEQ ID NO.1所示，所述铰链区的氨基酸序列如SEQ ID NO.5所示。
  17. 如权利要求16所述的双靶点嵌合抗原受体，其特征在于，所述嵌合抗原受体1包括顺序连接的如下片段：信号肽1、ROR1的单链抗体、铰链区、CD28跨膜结构域、CD28胞内段共刺激信号域、CD3ζ信号转导域；
    优选地，所述嵌合抗原受体1的氨基酸序列如SEQ ID NO.29所示。
  18. 如权利要求9～17任一项所述的双靶点嵌合抗原受体，其特征在于，氨基酸序列如SEQ ID NO.26所示。
  19. 一种基因，其特征在于，它是编码权利要求1～8任一项所述的嵌合抗原受体或编码权利要求9～18任一项所述双靶点嵌合抗原受体的基因；优选的，所述编码权利要求1～8任一项所述的嵌合抗原受体基因核苷酸序列如SEQ ID NO.27所示，编码权利要求9～18任一项所述双靶点嵌合抗原受体的基因核苷酸序列如SEQ ID NO.25所示。
  20. 一种载体，其特征在于，它是含有权利要求19所述的基因的载体；所述载体是质粒或病毒。
  21. 一种宿主细胞，其特征在于，它是表达权利要求1～8任一项所述的嵌合抗原受体或表达权利要求9～18任一项所述双靶点嵌合抗原受体的宿主细胞，优选为含有权利要求20所述的载体的宿主细胞。
  22. 如权利要求21所述的宿主细胞，其特征在于，所述宿主细胞为免疫反应细胞，优选为T细胞、单核细胞、自然杀伤细胞或中性粒细胞中的至少一种，更优选为T细胞。
  23. 权利要求1～8任一项所述的嵌合抗原受体、权利要求9～18任一项 所述的双靶点嵌合抗原受体、权利要求19所述的基因、权利要求20所述的载体或权利要求21～22任一项所述的宿主细胞在制备预防和/或治疗肿瘤的药物中的用途。
PCT/CN2022/132043 2022-10-28 2022-11-15 一种靶向TGFβRII的嵌合抗原受体及其用途 WO2024087267A1 (zh)

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