Construction method of CAR-T cell co-expressing cytokine IL15 and lymph node homing signal CCR7

Abstract

The invention relates to a construction method of a CAR-T cell co-expressing a cell factor IL15 and a lymph node homing signal CCR7, and the method can promote T cell proliferation, activation, apoptosis resistance and depletion, maintain low differentiation phenotype and other excellent characteristics. The CAR-T optimization strategy and application have obvious effects in the aspects of breaking through immune barrier inhibition, increasing tumor infiltration, improving T cell activity and the like.

Description

Technical Field

[0001] The present invention relates to the field of CAR-T immune cell technology, and specifically to a method for constructing CAR-T cells that co-express the cytokine IL15 and the lymph node homing signal CCR7, and to a CAR-T optimization strategy and application that optimizes the CAR-T cells to enhance proliferation, activation, anti-apoptosis and anti-exhaustion, maintain a low-differentiation phenotype and other excellent properties, and exhibit significant effects in breaking through immune barrier inhibition, increasing tumor infiltration and improving T cell activity. Background Art

[0002] CAR-T therapy has the characteristics of strong specificity and high selectivity. It has become the most popular treatment for hematological malignancies and has shown significant response rates and breakthrough therapeutic effects. Even some patients with relapsed and refractory large B-cell lymphoma (LBCL) and B-cell acute lymphoblastic leukemia (B-All) have achieved lasting complete remission after CD19 CAR-T treatment. However, the high cost of treatment, safety and recurrence after treatment limit the further clinical application of CAR-T therapy. Studies have shown that CAR-T cell exhaustion is the main reason for disease recurrence in patients after cell therapy, which is mainly manifested in poor survival, proliferation, homing and tumor infiltration of CAR-T cells in vivo. Usually, CAR-T cells will disappear three weeks after reinfusion. Therefore, prolonging the survival time of CAR-T cells in vivo and improving the proliferation and homing ability of CAR-T cells in vivo are crucial for the clinical application of CAR-T in the treatment of malignant tumors.

[0003] The proper combination of CAR and immunomodulatory factors can enhance the anti-tumor ability of CAR-T cells, while synergizing the activation of tumor-reactive receptor T cells and the formation of their memory. Cytokine IL15 has a significant effect in reshaping the memory-like immune phenotype of T cells, prolonging survival time, reducing the expression of T cell exhaustion markers to increase anti-apoptosis characteristics, and promoting the preservation of stem cell memory-like CD8+T cells (TSCM) phenotype. In addition to the damage and differentiation status of T cells affecting the efficacy of CAR-T cell therapy, TE cell "homing defect" is another major factor leading to ineffective immunotherapy. The lymph node homing and recirculation marker CCR7 can promote the lymph node homing immune phenotype of T cells, T cell homing and extravasation can improve the persistence of CAR-T cells in the tumor microenvironment, and the lymphocyte recirculation accompanied by T cell homing makes the distribution of lymphocytes in the peripheral immune organs and tissues in the body more reasonable, which is expected to break through the inhibition of the immune barrier.

[0004] Therefore, the addition of elements that improve the immune phenotypes of CAR-T cells such as proliferation / persistence / homing / chemotaxis in vivo will hopefully increase the response rate and durability of CAR-T cell therapy in the treatment of malignant tumors in patients. Summary of the invention

[0005] In response to the above problems, the present invention provides a method for constructing CAR-T cells that co-express the cytokine IL15 and the lymph node homing signal CCR7, and simultaneously co-express CAR, cytokine IL15 and the lymph node homing signal CCR7, thereby improving the immune phenotypes such as proliferation, survival, and exhaustion of CAR-T cells, and improving the efficacy of CAR-T cells in tumor treatment.

[0006] A method for constructing CAR-T cells that co-express cytokine IL15 and lymph node homing signal CCR7, wherein the IL15 and CCR7 genes replace the coGFP and Puro genes in the pCDH-CMV-CAR-EF1-coGFP-T2A-puro plasmid to obtain the target plasmid pCDH-CMV-CAR-EF1-IL15-T2A-CCR7, and the virus is packaged and transfected. The nucleotide sequence of IL15 is shown in SEQ ID NO.2, and the nucleotide sequence of CCR7 is shown in SEQ ID NO.3.

[0007] Furthermore, after 15x7-CD19-CAR-Jurkat cells were co-incubated with target cells Raji in gradient numbers, the expression of CD69 protein activation in CAR-Jurkat cells was detected; after 15x7-CD19-CAR-T cells were co-incubated with target cells Raji, the target cell killing effect was detected by LDH.

[0008] Furthermore, CD19-CAR-T and 15x7-CD19-CAR-T cells were cultured in basal culture medium with autologous plasma and ampicillin / streptomycin antibiotics alone, without the addition of cytokines such as IL2, IL15, and IL7, and the proliferation of CAR-T cells was measured by a cell counter for 1, 2, 4, 6, and 10 days in vitro; CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with target cells Raji at different effector-target ratios, and the secretion of cytokines IL-2 and INF-γ in CAR-T cells was detected by Elisa.

[0009] Furthermore, the basal culture medium was simply supplemented with autologous plasma and ampicillin / streptomycin antibiotics without the addition of cytokines such as IL2, IL15, and IL7. RNA was extracted on the 6th day of culture, and the expression of stem cell-like / memory-like marker IL-7R on the surface of CD19-CAR-T and 15x7-CD19-CAR-T cells and the expression of inhibitory receptors PD-1, TIM-3, LAG3, and CTLA4 on the surface of T cells were detected by qPCR to evaluate the immune phenotype of CAR-T cells.

[0010] The present invention provides a method for constructing CAR-T cells that co-express the cytokine IL15 and the lymph node homing signal CCR7, which promotes T cell proliferation, activation, anti-apoptosis and exhaustion, maintains low-differentiation phenotype and other excellent characteristics, and exhibits significant effects in breaking through immune barrier inhibition, increasing tumor infiltration and improving T cell activity. CAR-T optimization strategy and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Design and transcriptional level detection of CD19-CAR-Jurkat and 15x7-CD19-CAR-Jurkat cells.

[0012] Figure 2 It is the expression of all genes (CD19, CCR7 and IL-15) of CD19-CAR-T and 15x7-CD19-CAR-T cells in T cells after lentivirus infection of T cells.

[0013] Figure 3 This is the activation of Jurkat cells after CD19-CAR-Jurkar and 15x7-CD19-CAR-Jurkat cells were co-incubated with Raji target cells.

[0014] Figure 4 It is the killing efficiency of CD19-CAR-T and 15x7-CD19-CAR-T on target cells Raji under different effector-target ratios.

[0015] Figure 5 It is the secretion of cytokines IL-2 and INF-γ after CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with target cells Raji.

[0016] Figure 6 It is the proliferation of T cells after CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with target cells Raji.

[0017] Figure 7 It is the measurement of T cell immune phenotype indicators after CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with target cells Raji. DETAILED DESCRIPTION

[0018] The following is a further detailed description of the method for constructing CAR-T cells that co-express the cytokine IL15 and the lymph node homing signal CCR7 of the present invention in conjunction with the accompanying drawings and specific examples.

[0019] The present invention firstly uses pCDH-CMV-MCS-EF1-coGFP-T2A-Puro plasmid (generation 0) as a basis to construct the second-generation CAR of the single-target CD19 specific scFv-CD8 hinge region, transmembrane region + co-stimulatory signal (CD28 or 4-1BB) in the multiple cloning site (MCS) NheⅠ and EcoRⅠ in the form of enzyme ligation to construct CD19-CAR (generation 1, number 51#); then, using the CAR plasmid in the first generation as a template, homologous recombination is used to replace the coGFP and Puro genes in the plasmid with the IL15 and CCR7 genes respectively (CAR plasmid co-expressing IL15 and CCR7 (generation 2: referred to as 15x7-CAR, number 57#). After the above plasmids are packaged by lentivirus and transduced with T cells, the expression levels of CAR-T, IL15 and CCR7 genes are detected by qPCR, and the protein expression levels are detected by flow cytometry. Example

[0020] Plasmid design and construction The CAR expression plasmids were all digested and ligated by enzyme digestion. The target CAR gene was synthesized by whole gene synthesis. Then, the CAR gene product and the pCDH-CMV-MCS-EF1-coGFP-T2A-Puro basic plasmid were digested by NheⅠ and EcoRⅠ, so that both had the same sticky ends and were used for subsequent T4 DNA ligase ligation experiments, and finally the target plasmid pCDH-CMV-CAR-EF1-coGFP-T2A-puro plasmid was obtained.

[0021] The CAR plasmid co-expressing IL15 and CCR7 was used by homologous recombination (ClonExpressTM II One Step Cloning Kit) to replace the coGFP and Puro genes in the plasmid with the IL15 and CCR7 genes, respectively, and finally the target plasmid pCDH-CMV-CAR-EF1-IL15-T2A-CCR7 (pCDH-15x7-CAR) was obtained.

[0022] Virus packaging specific process The mixed packaging plasmids were configured in a ratio of target gene plasmid: pSPAX2: pMD2G (4:3:1), and the transfection mixture was obtained in the manner of 1ug plasmid corresponding to 2ul PEI25K linearized polyethyleneimine transfection reagent, and 293T cells were transfected. The viral supernatants were collected 24h, 48h, and 72h after the transfection solution was changed, and the viruses were concentrated by centrifugation at 20000xg for 2h. The virus titers were determined by Lenti-Pac™ Lentivirus Titer Detection Kit, and then aliquoted and frozen at -80℃ for later use.

[0023] Jurkat cells and T cells were infected at MOI=10 and MOI=100, respectively, to construct effector cells: CAR-Jurkat cells and CAR-T cells.

[0024] Detection of gene expression levels of CAR-T cell products qPCR quantitative analysis: Real-time quantitative PCR (qPCR) was used to measure the mRNA level of CAR-T cells. RNA was extracted from WT-T cells, CAR-T cells, and 15x7-CAR-T cells using the RNAEasy Fast Animal Tissue / Cell Total RNA Extraction Kit (Tiangen: DP451). Subsequently, the HiScript® II One Step qRT-PCR SYBR Green Kit from Weizan was used, and qPCR amplification was performed based on specific primers. Finally, the 2-∆∆ct value was used to determine the mRNA expression levels of CAR, IL15, and CCR7 genes in WT-T cells, CAR-T cells, and 15x7-CAR-T cells. (The qPCR detection of the expression of the stem cell-like / memory-like marker IL-7R on the surface of T cells and the expression of the inhibitory receptors PD-1, TIM-3, LAG3, and CTLA4 on the surface of T cells also adopted the same protocol.) Flow cytometry quantitative analysis: Flow cytometry was used to measure the protein levels of CAR-T cells. CAR, IL15, and CCR7 protein levels were detected using CD19-CAR, IL15, and CCR7 specific antibodies.

[0025] like Figure 1 The design and transcription level detection of CD19-CAR-Jurkat and 15x7-CD19-CAR-Jurkat cells are shown. CD19-CAR is normally expressed in CD19-CAR-Jurkat cells; CD19-CAR, IL15, and CCR7 are all normally expressed in 15x7-CD19-CAR-Jurkat cells. Figure 2 As shown, after lentivirus infected T cells, all genes (CD19, CCR7 and IL-15) of CD19-CAR-T and 15x7-CD19-CAR-T cells were expressed on T cells, proving the success of the plasmid design and construction of the present invention.

[0026] CAR-Jurkat activation assay A high-throughput method for identifying novel chimeric antigen receptors of Jurkat cells was used to evaluate the sensitivity of CAR and the specific recognition efficiency of target cells. CAR-Jurkat cells and target cells were co-cultured: CAR-Jurkat = 1x10 5 Target cells are 0, 1x10 3, 5x10 3 , 1x10 4 , 5x10 4 , 1x10 5 The cells were plated in 96-well plates. After 24 hours, the expression of CD69 in CAR-Jurkat cells was detected by flow cytometry and compared with the original NC-Jurkat cells to analyze the activation efficiency of CAR-Jurkat cells.

[0027] CAR-T cell killing experiment 96-well plate with opaque flat bottom, 5 x10 luciferase stably transfected target cells 3 cells / well, 4 replicate wells. Effector cells were cultured at four ratios of 1:1, 5:1, 10:1, and 20:1. After 24 hours of culture, the supernatant was removed by centrifugation, and then the cells were added with lysis buffer and firefly luciferin substrate. After incubation for 15-20 minutes, the chemiluminescence value was detected in a chemiluminometer or an ELISA reader with a chemiluminescence detection module (380-780nm can be measured). The obtained values ​​were obtained using the formula (1-(experimental group / maximum value of target cells)) x 100% to obtain the effector cell killing efficiency values ​​at four ratios of 1:1, 5:1, 10:1, and 20:1.

[0028] like Figure 3 As shown, 1x106 cells of CD19-CAR-Jurkar and 15x7-CD19-CAR-Jurkat cells were compared with 1x10 6 , 5x10 5 , 1x10 5 , 5x10 4 , 1x10 4 , 5x10 3 , 0, Raji target cells with different cell number gradients were co-incubated for 24 hours, and the expression of CD69 activation index in CD19-CAR-Jurkar and 15x7-CD19-CAR-Jurkat cells was measured. The results showed that the activation of CD69 index increased with the increase of the number of target cells Raji in CD19-CAR-Jurkar and 15x7-CD19-CAR-Jurkat cells, while there was no change in NC-Jurkat cells. Tip: Both CD19-CAR-Jurkar and 15x7-CD19-CAR-Jurkat cells can effectively recognize target cells through CD19-CAR and be activated by antigens, and the activation is higher with the increase of antigen density. It is worth noting that compared with CD19-CAR-Jurkar, 15x7-CD19-CAR-Jurkat cells have a stronger activation effect under the same antigen density conditions.

[0029] like Figure 4 As shown, CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with Raji target cells at effector-target ratios of 1:1, 5:1, 10:1, and 20:1 for 24 hours, and the killing efficiency of CD19-CAR-T and 15x7-CD19-CAR-T cells on target cells Raji (CD19+) and K562 (CD19-) was measured. The results showed that the killing efficiency of CD19-CAR-T and 15x7-CD19-CAR-T cells on target cells Raji (CD19+) increased with the increase of effector-target ratio, while there was no change for K562 (CD19-). Tip: Both CD19-CAR-T and 15x7-CD19-CAR-T cells can effectively and specifically kill target cells through CD19-CAR. It is worth noting that compared with CD19-CAR-T, 15x7-CD19-CAR-T cells have higher killing efficiency under the conditions of effector-target ratios of 5:1 and 10:1.

[0030] Detection of CAR-T cell proliferation ability in vitro WT-T cells, CD19-CAR-T cells, and 15x7-CD19-CAR-T cells were transfected and cultured. According to the instructions of the CCK-8 cell proliferation-toxicity detection kit, the absorbance (A) value at 450 nm was detected by an ELISA instrument on the 1st, 2nd, 4th, 6th, and 10th days of culture, and the proliferation capacity of WT-T cells, CD19-CAR-T cells, and 15x7-CD19-CAR-T cells was calculated.

[0031] like Figure 5 As shown, NC-T, CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with Raji target cells at an effector-target ratio of 10:1 for 24 hours, and the secretion of cytokines IL-2 and INF-γ was measured. The results showed that the secretion of cytokines IL-2 and INF-γ by 15x7-CD19-CAR-T cells was more significant than that of CD19-CAR-T and NC-T cell groups. It suggests that 15x7-CD19-CAR-T cells have stronger proliferation and tumoricidal activity.

[0032] like Figure 6As shown, NC-T, CD19-CAR-T and 15x7-CD19-CAR-T cells were cultured in basal medium with autologous plasma and ampicillin / streptomycin antibiotics (without adding cytokines such as IL2, IL15, and IL7) for 1, 2, 4, 6, and 10 days in vitro, and the proliferation of CAR-T cells was measured by a cell counter. The results showed that on days 6 and 10, the cell proliferation effect of 15x7-CD19-CAR-T cells was more significant than that of CD19-CAR-T and NC-T cell groups.

[0033] like Figure 7 As shown, NC-T, CD19-CAR-T and 15x7-CD19-CAR-T cells were cultured in basal medium with autologous plasma and ampicillin / streptomycin antibiotics alone (without adding cytokines such as IL2, IL15, and IL7). After culture for 6 days, qPCR was used to detect the expression of stem cell-like / memory-like marker IL-7R on the surface of T cells and the expression of inhibitory receptors PD-1, TIM-3, LAG3, and CTLA4 on the surface of T cells. The results showed that compared with CD19-CAR-T and NC-T cell groups, the expression of stem cell-like / memory-like marker IL-7R in 15x7-CD19-CAR-T cells was significantly increased, while the expression of inhibitory receptors PD-1, TIM-3, and LAG3 on the surface of T cells was significantly decreased. This suggests that 15x7-CD19-CAR-T cells have a better memory-like phenotype and anti-aging effect.

[0034] The above is only a preferred embodiment of the present invention for improving T cell immune phenotype and enhancing tumor killing effect by CAR-T cells co-expressing cytokine IL15 and lymph node homing signal CCR7. It should be pointed out that for ordinary technicians in this technical field, without departing from the precursor of the method of the present invention, the construction strategy for co-expressing CAR and improving the immune phenotype state of T cells including cytokines, lymph node homing signals, initial T cells / memory T cells or chemokine receptors, etc. and their application in tumor treatment, as well as corresponding improvements and supplements, should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing CAR-T cells that co-express cytokine IL15 and lymph node homing signal CCR7, characterized in that: The IL15 and CCR7 genes replaced the coGFP and Puro genes in the pCDH-CMV-CAR-EF1-coGFP-T2A-puro plasmid to obtain the target plasmid pCDH-CMV-CAR-EF1-IL15-T2A-CCR7. The virus was packaged and transfected. The nucleotide sequence of IL15 is shown in SEQ ID NO.2, and the nucleotide sequence of CCR7 is shown in SEQ ID NO.

3.

2. The method for constructing CAR-T cells co-expressing cytokine IL15 and lymph node homing signal CCR7 according to claim 1, characterized in that: After 15x7-CD19-CAR-Jurkat cells were co-incubated with target cells Raji in gradient numbers, the expression of CD69 protein activation in CAR-Jurkat cells was detected; after 15x7-CD19-CAR-T cells were co-incubated with target cells Raji, the target cell killing effect was detected by LDH.

3. The method for constructing CAR-T cells co-expressing cytokine IL15 and lymph node homing signal CCR7 according to claim 1, characterized in that: CD19-CAR-T and 15x7-CD19-CAR-T cells were cultured in basal culture medium with autologous plasma and ampicillin / streptomycin antibiotics alone, without the addition of cytokines IL2, IL15, and IL7, and the proliferation of CAR-T cells was measured by cell counter for 1, 2, 4, 6, and 10 days in vitro. CD19-CAR-T and 15x7-CD19-CAR-T cells were co-incubated with target cells Raji at different effector-target ratios, and the secretion of cytokines IL-2 and INF-γ in CAR-T cells was detected by Elisa.

4. The method for constructing CAR-T cells co-expressing cytokine IL15 and lymph node homing signal CCR7 according to claim 1, characterized in that: The basal culture medium was simply supplemented with autologous plasma and ampicillin / streptomycin antibiotics without the addition of cytokines IL2, IL15, and IL7. RNA was extracted on the 6th day of culture, and the expression of stem cell-like / memory-like marker IL-7R on the surface of CD19-CAR-T and 15x7-CD19-CAR-T cells and the expression of inhibitory receptors PD-1, TIM-3, LAG3, and CTLA4 on the surface of T cells were detected by qPCR to evaluate the immune phenotype of CAR-T cells.

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