Chimeric antigen receptor and carrier and immune cell modified by chimeric antigen receptor

Abstract

The invention discloses a chimeric antigen receptor as well as a carrier and an immune cell modified by the chimeric antigen receptor, the chimeric antigen receptor is IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv, and the chimeric antigen receptor is combined with another chimeric antigen receptor CD8a signal-GPC3 scFv-CD8a hinge-CD8a transmembrane-CD28-CD3 delta for use. The CAR modified immune cells express membrane type IL-15 and secrete and express'immune checkpoint molecule 'monoclonal antibodies. The co-expression of the cell factor IL-15 and the'immune checkpoint molecule 'monoclonal antibody not only prolongs the life cycle of the immune cells, but also prevents the inhibition signal channel of the immune checkpoint molecule by secreting and expressing the'immune checkpoint molecule' monoclonal antibody, so that the tumor cell killing effect of the immune cells is improved.

Description

Technical Field

[0001] The present invention belongs to the field of cell immunotherapy, and specifically relates to a chimeric antigen receptor and a modified carrier and immune cell thereof. Background Art

[0002] In addition to surgery, chemotherapy, and radiotherapy, clinical cancer treatment methods have seen an increasing use of biological treatment technology in recent years, including monoclonal antibody technology, cytokines, tumor vaccines, immune cells, etc., especially immune cell tumor treatment technology, which has received widespread attention and attention with the success of CAR-T in the treatment of large B-cell lymphocytic leukemia. Cell therapy for cancer has gradually come into people's view.

[0003] At present, there are five CAR-T drugs on the market in China: Fosun Kite's Akirensel Injection, Innovent Biologics' Ruiqiorensel Injection, Innovent Biologics' Ikiorensel Injection jointly developed by Innovent and Reindeer Biopharmaceuticals, Heyuan Biopharmaceuticals' Nakiorensel Injection, and Coherus Pharmaceuticals' Zewokiorensel. Chongqing Precision Biopharmaceuticals' Pukirensel Injection is applying for listing. These drugs are mainly used to treat large B-cell lymphoma, multiple myeloma, B-cell acute lymphoblastic leukemia and other blood system tumors.

[0004] Cell drugs for solid tumors have not yet appeared. One of the main problems is that when immune cells interact with tumor cells, immune cells are stimulated to express immune checkpoint molecule ligands, and tumor cells express immune checkpoint molecule receptors. After the ligand binds to the receptor, the signal pathway is activated, the activity of immune cells is inhibited, and the function of immune cells to kill tumor cells is greatly reduced. At present, monoclonal antibodies of immune checkpoint molecules or their receptor antibody drugs are often prescribed for clinical treatment of solid tumors in combination or alone. There are 7 immune checkpoint monoclonal antibody drugs that have been launched abroad: Lpilimumab and Tremelimumab for CTLA-4; Nivolumab and Pembrolizumab for PD-1; Atezolizumab, Durvalumab, and Avelumab for PD-L1. Two monoclonal antibody drugs for PD-1, Daboshu and BeiGene, have also been launched in China. Although the application of immune checkpoint monoclonal antibodies has effectively improved the efficacy of immune cells, such as T cells and NK cells, in killing tumor cells, these monoclonal antibody drugs are expensive and require multiple infusions to control tumors. For the families of tumor patients, this is a heavy economic burden, resulting in the limitation of the clinical application of monoclonal drugs. Therefore, if immune checkpoint monoclonal antibodies are constructed into immune cells for secretory expression, while improving the activity of immune cells, the limitations of in vitro medication are avoided, the economic burden of tumor patients is reduced, tumors are well treated, and the national tumor incidence and mortality rate are reduced. Secondly, after the currently constructed CAR-T or CAR-NK cells are infused back into tumor patients, although some T cells differentiate into memory T cells, most cells die within a certain period of time, thereby greatly shortening the immune cell treatment cycle and failing to achieve a good therapeutic effect. A research team reported that in the presence of IL-15 cytokines, the life cycle of immune cells such as T cells, NK cells, and iNKT cells will be greatly extended. Therefore, when designing CAR, domestic and foreign research teams will consider simultaneously constructing IL-15 into CAR molecules for secretory expression. After the modified CAR is loaded into NK cells, the life cycle of CAR-NK cells is greatly extended. There are also literature reports that the expression of IL-15 cytokine on NK cell membrane not only significantly improves NK activity, but also significantly prolongs the life cycle of NK cells compared to secretory expression. Therefore, the IL-15 membrane-localized expression gene fragment and the immune checkpoint molecule monoclonal antibody scFv gene are constructed into CAR at the same time, which can enhance the activity of immune cells and prolong the life cycle of immune cells, and effectively kill and control the occurrence and development of tumors until the tumor is eliminated. Summary of the invention

[0005] The purpose of the present invention is to provide an enhanced CAR design structure that can be applied to the preparation of immune cells modified with a variety of chimeric antigens for clinical tumor treatment.

[0006] According to the first aspect of the present invention, a chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv is provided, which comprises IL-2 signal peptide, hIL-15, CD8a transmembrane region, P2A connecting peptide, CD8a signal peptide, scFv, preferably connected in sequence. The chimeric antigen receptor is used in combination with another chimeric antigen receptor CD8a signal-GPC3 scFv-CD8a hinge-CD8a transmembrane-CD28-CD3δ, wherein the other chimeric antigen receptor comprises CD8a signal peptide, GPC3scFv, CD8a hinge region, CD8a transmembrane region, CD28 intracellular region and CD3δ, preferably connected in sequence, and the other chimeric antigen receptor is controlled by the promoter EF1a.

[0007] Preferably, the chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv is controlled by the promoter CMV.

[0008] Preferably, the GPC3 scFv has an amino acid sequence as shown in SEQ ID NO:2, and its encoding base sequence is as shown in SEQ ID NO:1.

[0009] Preferably, the amino acid sequence of the CD8a signal peptide is shown as SEQ ID NO:4, and the encoding base sequence thereof is shown as SEQ ID NO:3.

[0010] Preferably, the amino acid sequence of the hinge region of CD8a is shown as SEQ ID NO:6, and its encoding base sequence is shown as SEQ ID NO:5.

[0011] Preferably, the amino acid sequence of the CD8a transmembrane region is shown as SEQ ID NO:8, and its encoding base sequence is shown as SEQ ID NO:7.

[0012] Preferably, the amino acid sequence of the intracellular region of CD28 is shown as SEQ ID NO:10, and the encoding base sequence thereof is shown as SEQ ID NO:9.

[0013] Preferably, the amino acid sequence of CD3δ is shown as SEQ ID NO:12, and the encoding base sequence thereof is shown as SEQ ID NO:11.

[0014] Preferably, the amino acid sequence of the IL-2 signal peptide is shown as SEQ ID NO:14, and the encoding base sequence thereof is shown as SEQ ID NO:13.

[0015] Preferably, the amino acid sequence of hIL-15 is shown as SEQ ID NO:16, and the encoding base sequence is shown as SEQ ID NO:15.

[0016] Preferably, the amino acid sequence of P2A is shown as SEQ ID NO:20, and its encoding base sequence is shown as SEQ ID NO:19.

[0017] Preferably, the amino acid sequence of scFv is as shown in SEQ ID NO:22 (ie, PD-1 scFv), and its encoding base sequence is as shown in SEQ ID NO:21.

[0018] The present invention further provides a vector comprising a polynucleotide encoding the above chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv and another chimeric antigen receptor CD8a signal-GPC3 scFv-CD8a hinge-CD8a transmembrane-CD28-CD3δ.

[0019] The present invention also provides an engineered immune cell, which is transduced with the above-mentioned chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv and CD8a signal-GPC3 scFv-CD8a hinge-CD8a transmembrane-CD28-CD3δ.

[0020] Furthermore, the engineered immune cells express hIL-15 in a membrane-localized manner.

[0021] The hIL-15 disclosed in the present invention is expressed in a membrane-localized manner.

[0022] Furthermore, the immune cell membrane secretes and expresses PD-1 scFv.

[0023] Furthermore, the immune cell membrane simultaneously secretes and expresses hIL-15 and PD-1 scFv.

[0024] Furthermore, the above-mentioned anti-immune checkpoint ligand and receptor molecule scFv can be selected from one or more (e.g., a combination of two or three) of PD-1, PD-L1, CTLA-4, TIM-3, and LAG-3, preferably PD1 scFv.

[0025] Preferably, the immune cells disclosed in the present invention are T cells, iNKT cells, NK cells, or γδT cells.

[0026] Preferably, the immune cells are iNKT cells.

[0027] The present invention further relates to the use of the chimeric antigen receptor, a vector comprising a polynucleotide encoding the chimeric antigen receptor, or an engineered immune cell in the preparation of a drug for treating cancer.

[0028] The chimeric antigen modified immune cells disclosed in the present invention not only express hIL-15 cytokines by membrane localization, but also secrete PD-1 scFv. The expression of hIL-15 by membrane localization of immune cells can significantly prolong their life cycle; the secretion of PD-1 scFv can selectively inhibit the PD-1 / PD-L1 signaling pathway, weaken the inhibitory effect of the signal on the activity of immune cells, thereby improving the tumor cell killing effect of immune cells. The iNKT cells modified with chimeric antigen receptors co-localize the expression of hIL-15 and secrete the expression of PD-1 scFv, which have two advantages: first, natural iNKT cells themselves have an immunomodulatory effect, which can regulate the activity of immune cells in the body's blood; second, the co-membrane localization of hIL-15 and secretion of PD-1 scFv by CAR-iNKT cells can significantly improve their killing activity and targeting. The two molecules expressed by CAR-iNKT cells not only regulate their own activity, but also the secreted PD-1 scFv antibodies also regulate the activity of the patient's own T lymphocytes, which can maintain the functions of T cells and iNKT cells for a long time.

[0029] The chimeric antigen receptor structure designed and constructed in the present invention can be used as a molecular platform to design different chimeric antigen receptors with strong specific targeting according to different tumor targets, and is widely used in clinical treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Design a structural representation for the CAR sequence.

[0031] Figure 2A -C is the construction and identification diagram of CAR-iNKT cells, where A: CAR-iNKT cell transfection efficiency flow cytometer analysis results; B: CAR-iNKT cell membrane localization expression of IL-15 flow cytometer analysis results; C: CAR-iNKT cell secretion expression of PD-1 monoclonal antibody ELISA test results.

[0032] Figure 3A-G is the in vitro efficacy analysis diagram of CAR-iNKT cells, where A: flow cytometric analysis results of liver cancer cell GPC3 antigen; B: flow cytometric analysis results of liver cancer cell CD1d antigen; C: ELISA determination of LDH method to analyze the in vitro killing activity of CAR-iNKT cells against HepG2 target cells; D: ELISA determination of LDH method to analyze the in vitro killing activity of CAR-iNKT cells against SK-Hep-1 target cells; E: ELISA detection results of TNF-a secretion and expression by CAR-iNKT cells; F: ELISA detection results of IFN-γ secretion and expression by CAR-iNKT cells; G: ELISA detection results of IL-6 secretion and expression by CAR-iNKT cells. DETAILED DESCRIPTION

[0033] The present invention is further described below through specific implementation cases, in which the immune cells are iNKT cells as an example.

[0034] Example 1: CAR structure design and virus packaging Three CARs were designed at the same time, and the extracellular domain recognition structure was GC3 scFv, targeting liver cancer cells or tissues, of which GC328z and 1928z15PD were control CARs. The specific CAR design scheme is as follows ( Figure 1 ): GC328z: EF1a-CD8a signal-GPC3 scFv–CD8a hinge–CD8a transmembrane region-CD28–CD3δ (linked to the promoter EF1a in the viral vector) GC328z15PD: EF1a-CD8a signal-GPC3 scFv–CD8a hinge–CD8a transmembrane-CD28–CD3δ / / CMV-IL2 signal-IL15- CD8a transmembrane-P2A-CD8a signal-PD1 scFv (attached to the promoters EF1a and CMV of the viral vector, respectively) 1928z15PD: EF1a-CD8a signal-CD19 scFv–CD8a hinge–CD8a transmembrane-CD28–CD3δ / / CMV-IL2 signal-IL15- CD8a transmembrane-P2A-CD8a signal-PD1 scFv (connected to the promoters EF1a and CMV of the viral vector, respectively), wherein the gene sequence of CD19 scFv is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18.

[0035] All sequences were artificially synthesized and packaged by a third party, Shandong Weizhen Biotechnology Co., Ltd., according to conventional methods, with a titer of more than 10^8 PFU / ml.

[0036] The synthetic sequences are shown in the table below.

[0037]

[0038] Example 2: iNKT cell culture and expansion 1. Use lymphocyte separation solution to separate and purify peripheral blood PBMC, and use high-pressure purified water to break red blood cells and count them; 2. Prepare NKT complete medium by adding 100 IU / ml IL-2, 5 ng / ml IL-21, and 100 ng / ml a-Galcer to X-Vivo 15 basal medium; 3. Add the PBMC cell suspension to a vessel pre-coated with 100 ng / ml TCR V beta11 monoclonal antibody at a concentration of 2.5×10^6 / ml. Place in a 37°C, 5% CO2 incubator; 4. Cultivate for 9 days, replenishing fluid every 2-3 days; 5. Collect cells on Day 10, centrifuge at 1000 rpm for 10 min, remove supernatant, add 2 ml RPMI1640 medium to resuspend cells into single cell suspension; 6. Add reagents according to the instructions of anti-iNKT microbeads to purify iNKT cells and count them; 7. Prepare NKT expansion medium by adding 100 IU / ml IL-2, 10 ng / ml IL-7, and 2.5% UltraGRO to X-Vivo 15 basal medium; 8. Add the iNKT cell suspension purified in step 6 to the vessel pre-coated with 3 μg / ml CD3 and 3 μg / ml CD28 monoclonal antibodies on Day 9, add the culture medium prepared in step 7 at a concentration of 1×10^6 / ml, and place in a 37°C, 5% CO2 incubator, which is counted as Day 0; 9. Cultivate for 9 days, replenishing fluid every 2-3 days; 10. Collect cells on Day 9 and count them.

[0039] Example 3: Construction and identification of CAR-iNKT cells 1. iNKT stimulation culture: add 50ng / ml anti-CD3 monoclonal antibody, 50ng / ml anti-CD28 monoclonal antibody, and 150IU / ml IL-15 to the culture medium and stimulate the culture for 48h; 2. Add the empty lentivirus (Shandong Weizhen Biotechnology Co., Ltd.) and CAR-packaged lentivirus to a 24-well plate coated with Retronectin, set the MOI value to 5, then add the corresponding volume of iNKT cells, centrifuge at 1000g, 32°C for 40 minutes, transfer the culture plate to an incubator, and continue culturing for 5 days; 3. Take cells and culture supernatant separately. CAR and IL-15 expression were detected by flow cytometry. PD-1 scFv expression was detected by ELISA. The specific results are shown in Figure 2A -C.

[0040] The results in Figure 2 show that the transfection efficiency of CAR-iNKT cells is as high as over 70%, while the efficiency of IL-15 membrane localization expression is over 15%, and the secretion of PD-1 scFv antibody into the supernatant can reach over 700pg / ml. The successful construction of CAR-iNKT cells can express IL-15 membrane-localized and PD-1 scFv secreted, achieving the expected results.

[0041] Example 4: Detection of CAR-iNKT cells killing liver tumor cells in vitro and cytokine expression 1. HepG2 and SK-Hep-1 cells were revived and the expression of GPC3 and CD1d antigens was detected by flow cytometry. The specific results are shown in Figure 3A -B; 2. Take appropriate amount of HepG2 and SK-Hep-1 cells, inoculate into 96-well plates according to different gradients, and culture overnight; 3. Design a 96-well plate killing scheme according to the CK-12 manual. Set the effector-target ratio to 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, and set high control, high control Blank, low control, and low control Blank at the same time. Make 2 duplicate wells and culture for 24 hours; 4. Detect the OD value of each well according to the CK-12 manual. The specific results are shown in Figure 3C -D; 5. Detect the expression of TNF-a, IFN-γ, and IL-6 cytokines according to the ELISA instructions. For specific results, see Figure 3E -G.

[0042] The kill rate is calculated using the following formula: A = Sample well OD value - Sample well Blank OD value B = high control OD value - high control Blank OD value C = low control OD value - low control Blank OD value Kill rate (%) = (AC) ÷ (BC) × 100.

[0043] The results in Figure 3 show that HepG2 highly expresses GPC3 antigen, SK-Hep-1 lowly expresses or hardly expresses GPC3 antigen, and both cancer cells express CD1d very low. From the killing effect, GC328z-CARiNKT and GC328z15PD-iNKT can efficiently kill HepG2 cells, but have almost no killing effect on SK-Hep-1 cells. However, natural iNKT cells and 1928z15PD-iNKT have low killing effects on HepG2 and SK-Hep-1 cells. The experimental results fully demonstrate that GC3-CARiNKT can specifically target liver cancer cells. In the process of CAR-iNKT cells killing tumor cells, some cytokines can be secreted, such as IFN-γ, TNF-a, etc.

[0044] Example 5: Detection of tumor inhibition activity in CAR-iNKT cell model animals 1. The HepG2 liver cancer model was established subcutaneously in SCID mice. The mice were divided into 5 groups, with 30 mice in each group; 2. Expand the culture of CAR-iNKT cells, transfect iNKT cells with empty lentiviral vector (iNKT control), and count; 3. The first group of model mice were injected with normal saline via tail vein, 200 μl per mouse, and marked as control group; The second group of model mice were injected with iNKT control cells via tail vein, 200 μl / 2×10^6 / mouse, and were marked as iNKT control group; The third group of model mice were injected with 28z cells via tail vein, 200 μl / 2×10^6 / mouse, and were marked as GC328z group; The fourth group of model mice were injected with 28z15PD cells via tail vein, 200 μl / 2×10^6 / mouse, and were marked as GC328z15PD group; The fifth group of model mice were injected with 19z15PD cells via tail vein, 200 μl / 2×10^6 / mouse, and were marked as 1928z15PD group; 4. Daily observation indicators: mental state, dietary status, after the model mice died, tumor tissues were dissected, tumor lesion diameters were measured, and the number and time of death of model mice in each group were recorded; 5. After the injection of immune cells, the mice were observed for a total of 60 days. At the end of the experiment, tumor tissues were dissected from the surviving model mice and the diameter of the tumor lesions was measured. The specific results are shown in Table 1.

[0045] Table 1 Results of drug efficacy test on inhibition of HepG2 tumor foci in CAR-iNKT model mice

[0046] The results of killing tumor lesions by injecting CAR-iNKT and iNKT cells into model animals showed that the diameter of tumor lesions in model mice injected with GC328z-CARiNKT and GC328z15PD-iNKT was significantly reduced compared with natural iNKT cells and 1928z15PD-iNKT, and the effect of GC328z15PD-iNKT was significantly better than that of GC328z-CARiNKT, indicating that the activity of GC328z15PD-iNKT cells that secrete and express PD-1 scFv is much stronger than that of GC328z-CARiNKT, and that secretion and expression of PD-1 scFv plays an important role in improving the activity of CAR-iNKT cells.

[0047] Example 6: Detection of the survival cycle of CAR-iNKT cells in Balb / c mice 1. Purchase 50 Balb / c mice and randomly divide them into 5 groups, 10 mice in each group; 2. The first group of mice were injected with normal saline via tail vein, 200 μl per mouse, and marked as control group; The second group of mice were injected with iNKT control cells via tail vein, 200 μl / 2×10^6 / mouse, and marked as iNKT control group; The third group of mice were injected with 28z cells via tail vein, 200 μl / 2×10^6 / mouse, and was marked as GC328z group; The fourth group of mice were injected with 28z15PD cells via tail vein, 200 μl / 2×10^6 / mouse, and was marked as GC328z15PD group; The fifth group of mice were injected with 19z15PD cells via tail vein, 200 μl / 2×10^6 / mouse, and were labeled as 1928z15PD group; 3. On Day 1, 3, 7, 10, 15, 20, 25, 30, 40, 50, and 60 after cell injection, tail vein blood was collected and anti-human CD3 monoclonal antibody and anti-iNKT monoclonal antibody were added to detect the survival of CAR-iNKT cells. The specific results are shown in Table 2.

[0048] Table 2 Results of survival cycle detection of CAR-iNKT cells in Balb / c mice

[0049] The results showed that GC328z15PD-iNKT and 1928z15PD-iNKT cells expressing IL-15 in membrane-localized mice survived significantly longer than natural iNKT cells and GC328z-CARiNKT, indicating that IL-15 cytokine can prolong the life cycle of iNKT cells.

[0050] Finally, it should be noted that the above content is only for illustrative purposes and not for limiting the scope of protection of the present invention. A person skilled in the art may modify or substitute equivalents for the technical solution of the present invention without departing from the essence of the present invention, and the modifications or equivalent substitutions shall be included in the scope of protection of the claims of the present invention.

Claims

1. A chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv, characterized in that: It includes IL-2 signal peptide, hIL-15, CD8a transmembrane region, P2A connecting peptide, CD8a signal peptide and scFv.

2. The chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8asignal-scFv according to claim 1, characterized in that The chimeric antigen receptor is controlled by the CMV promoter.

3. The chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8asignal-scFv according to claim 1, characterized in that The amino acid sequence of the IL-2 signal peptide is shown in SEQ ID NO:

14. The amino acid sequence of hIL-15 is shown in SEQ ID NO: 16, The amino acid sequence of the CD8a transmembrane region is shown in SEQ ID NO:

8. The amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO:

4. The amino acid sequence of P2A is shown in SEQ ID NO: 20, The amino acid sequence of PD-1 scFv is shown in SEQ ID NO:

22.

4. The chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8asignal-scFv according to claim 1, characterized in that The anti-immune checkpoint ligand and receptor molecule scFv are selected from one or more of PD-1, PD-L1, CTLA-4, TIM-3, and LAG-3, for example, a combination of two or three, preferably PD1 scFv.

5. A vector comprising a polynucleotide encoding the chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv according to any one of claims 1 to 4 and another chimeric antigen receptor CD8a signal-GPC3 scFv-CD8a hinge-CD8a transmembrane-CD28-CD3δ, wherein the other chimeric antigen receptor comprises a CD8a signal peptide, a GPC3 scFv, a CD8a hinge region, a CD8a transmembrane region, a CD28 intracellular region and CD3δ, and the other chimeric antigen receptor is controlled by the promoter EF1a.

6. An engineered immune cell, characterized in that: The immune cell is transduced with a vector of the chimeric antigen receptor IL2 signal-IL15-CD8a transmembrane-P2A-CD8a signal-scFv and another chimeric antigen receptor CD8asignal-GPC3 scFv-CD8a hinge-CD8a transmembrane-CD28-CD3δ polynucleotide as described in claim 1, wherein the other chimeric antigen receptor includes a CD8a signal peptide, a GPC3 scFv, a CD8a hinge region, a CD8a transmembrane region, a CD28 intracellular region and CD3δ, and the other chimeric antigen receptor is controlled by the promoter EF1a.

7. The immune cell according to claim 6, characterized in that The immune cells are iNKT cells, NK cells, and γδT cells.

8. Use of the chimeric antigen receptor according to claim 1 in the preparation of a drug for treating cancer.

9. Use of the carrier according to claim 5 in preparing drugs for treating cancer.

10. Use of the engineered immune cells according to claim 6 in the preparation of drugs for treating cancer.

Images