CN114634580B - Development of membrane anchored IL-15 super complex and application thereof in tumor immune cell treatment - Google Patents

Abstract

The invention relates to the field of tumor immunotherapy, in particular to development of a membrane anchored IL-15 super complex and application thereof in the field of tumor immunotherapy, mainly comprising carrier construction of the IL-15 super complex, wherein the carrier comprises a signal peptide, a Sushi domain of IL-15-N-72D, IL-15Rα, a connecting peptide, a CD4 molecular bracket and a suicide gene R structure, and is connected with an exogenous inserted gene through a P2A connecting peptide. The invention mainly enables immune cells to continuously express IL-15, improve the quantity of TSCM, increase the function of the immune cells, enable the immune cells to keep continuous killing function, induce apoptosis when necessary, ensure the safety of treatment and have great significance in the field of clinical treatment of the immune cells through the molecular design of transmembrane and suicide gene R structures.

Description

Development of a membrane-anchored IL-15 supercomplex and its application in tumor immune cells therapeutic applications

Technical field

The invention relates to the field of immune cell tumor treatment, and specifically relates to the development of a membrane-anchored IL-15 super complex and its application in tumor immunotherapy.

Background technique

Adoptive immune cell therapy is a new type of anti-tumor treatment in the field of biomedicine following surgery, chemotherapy, radiotherapy and targeted therapy. This method mainly collects the patient's own immune cells, and after in vitro modification, activation, culture and amplification, enhances the targeted killing function of the cells, and then infuses them back into the patient's body to achieve killing by enhancing the body's immune system function. and the purpose of destroying tumor cells. At present, adoptive immune cell therapy mainly includes CAR-T, LAK, CIK, DC, NK and TCR, among which CAR-T cell immunotherapy is the most popular. CAR-T (Chimericantigen receptor T cell, chimeric antigen receptor T cell), which uses genetic modification technology to transfer genetic material with specific antigen recognition domains and T cell activation signals into T cells. TAA can activate the killing effect of T cells, continuously kill and produce cytokines. After the development in recent years, tumor immune cell therapy has achieved clinical breakthroughs, especially CAR-T cells targeting the CD19 antigen, which have remarkable efficacy in hematological tumors and lymphomas, and have achieved complete success in some patients. ease. This persistence is directly related to long-term immune surveillance by CAR T cells. Once the antigen is cleared in the immune response, the immune cells' role is lost. Therefore, there are still many clinical bottlenecks in current tumor immune cell therapy technology. A large number of studies have shown that the relapse rate within 1 year of treated patients after achieving complete remission is as high as 46%. Analysis of the patient's immune cell subpopulations and the reinfused CAR-T cells after reinfusion showed that the ratio of Tcm and Tscm in the product and the ratio of Tcm and Tscm in the subjects after reinfusion were related to the remission time and CAR-T The maintenance of anti-tumor activity showed a positive correlation. The higher the ratio of Tcm and Tscm among CAR-positive T cells in a patient's infused CAR-T cells, the greater the likelihood that the patient will achieve long-term remission. Preclinical animal experiments also show that infusion of CAR-T cells, in which Tcm is the main subpopulation, can significantly extend the survival time of mice. The currently known Tcm preparation method is to isolate primary naive T cells from peripheral blood, while TSCM only accounts for a small part (2-3%) of peripheral blood mononuclear cells (PBMC), even through in vitro expansion, It is difficult to meet clinical needs.

IL-15 is a pleiotropic cytokine with similar biological functions to IL-2. It can not only activate T cells, B cells and NK cells, and mediate the proliferation and survival of these cells, but also activate and maintain and expand CD8+ memory T cells and maintain the stemness of T cells. It will not promote apoptosis of activated T cells or promote Treg proliferation, and CD8+ T cells are extremely sensitive to IL-15. Therefore, IL-15 is an excellent choice for anti-tumor treatment.

IL-15Rα is a high-affinity receptor for IL-15. It first binds to IL-15 on the cell surface. The IL-15/IL-15Rα complex must eventually bind to the other two receptor subunits, IL-15Rβ/IL-2R. Only the combination of subunit and γC subunit can stimulate the proliferation of T lymphocytes. The IL-15/IL-15Rα complex interacts with the IL-15Rβ/IL-2R subunit and γC subunit in two main ways: one is the IL-15/IL-15Rα complex formed on the surface of activated monocytes and other cells It presents IL-15 to nearby target cells expressing IL-15Rβ/IL-2R and γC receptors through "trans" presentation, that is, IL-15 is "presented" from one cell to another. Another way is that IL-15Rα presents IL-15 in "cis" to the same cell that expresses IL-15Rβ/IL-2R and γC receptors, performing signal transduction in the same cell. Among them, trans presentation is the most important. However, the trans-presentation mechanism requires one cell to present its membrane-bound IL-15/IL-15Rα complex to another target cell, which is actually a "cell" expressing IL-15Rβ/IL-2R and γC receptors. The interaction with "cells" requires that one cell must come into contact with another cell to produce an effect. This is not only required for the regulation of the body's immune system, but also restricts the use of IL-15/IL- to a certain extent. The 15Rα complex carries out immunotherapy for related diseases (such as tumors and infections).

Therefore, how to use genetic engineering technology to allow cells to produce membrane-anchored IL-15 supercomplexes is a question of scientific value and worthy of in-depth discussion.

Contents of the invention

The present invention provides membrane-anchored IL-15 supercomplexes for immune cell therapy. This super complex can not only be continuously expressed on the cell membrane surface, but also contains the "suicide gene" R structure with a small molecular weight, which greatly promotes the proliferation and survival of immune cells, improves the stemness and vitality of immune cells, and ensures the safety of the product.

In order to solve the above problems, the present invention provides the following technical solutions:

Specifically, the IL-15 super complex includes signal peptide, "suicide gene" R structure, IL-15 mutant IL-15-M-N72D, "Sushi" domain of IL-15Rα, and CD4 molecules. Scaffold, the complex is connected to the exogenous inserted gene fragment through the 2A connecting peptide.

The R structure is a CD20 epitope polypeptide, which is expressed on the cell surface and can be cleared by rituximab in the form of CDC and ADCC in vivo.

The IL-15 mutant IL-15-M-N72D in the IL-15 super complex and the "Sushi" domain of IL-15Rα are connected through a linker peptide (Linker);

The IL-15 super complex uses the CD-4 transmembrane region as a scaffold to anchor it on the cell membrane;

The 2A connecting peptide used to connect the IL-15 super complex to the exogenous inserted gene fragment is P2A or F2A;

Specifically, the IL-15 super complex includes: "suicide gene" R structure, Linker1, interleukin-15 mutant IL-15-M-N72D, Linker2, "Sushi" domain of IL-15Rα, CD-4 transmembrane region and connected sequentially;

The signal peptide is CD8, GM-CSF, CD4, CD28, CD137, or mutations/modifications thereof, or combinations thereof.

Specifically, the signal peptide is a signal peptide derived from CD8, and its amino acid sequence is the sequence shown in SEQ ID NO: 1 (DMWTWILFLVAAATRVHS);

Specifically, the amino acid sequence of the R structure of the "suicide gene" is the sequence shown in SEQ ID NO: 2 (ACPNSNPSLC);

Specifically, the amino acid sequence of the IL-15 mutant IL-15-M-N72D is the sequence shown in SEQ ID NO: 3 (NWVNVISDLKKIEDLIQSMHIDATLYTASDVHPSCKVTAMKCFLLELQVISLESGDASIHDDVENLIILANDSLSSNGNVTESGCAECEELEEKNIKEFLWSFVHIVQMFINTS);

Specifically, the "Sushi" domain of IL-15Rα is the first cysteine amino acid residue (C1) after the signal peptide of IL-15Rα, and terminates at the fourth cysteine amino acid residue after the signal peptide. residue (C4). The amino acid sequence of this "Sushi" domain is the sequence shown in SEQ ID NO: 4 (ITCPPPMSVEHADIWAKSYSLYSRERYICNSAFKRKAGTSSLTECVTNKATNVAHWTTPSLKCIRD);

Specifically, the amino acid sequence of the CD-4 transmembrane region molecular scaffold is the sequence shown in SEQ ID NO: 5 (VNVVMRATKNTCVWGTSKMSKNKAKVSKRKAVWVNAGMWCSDSGVSNIKVTWSTVMAIVGGVAGI GGI):

Specifically, the linker peptide Linker can be (GxSy)n, where: G is glycine, S is serine, x is 1, 2, 3 or 4, y is 1, 2, 3 or 4, and n is 1, 2 ,3,4,5 or 6.

Specifically, the amino acid sequence of the Linker is selected from SEQ ID NO: 6 (SGGGGSGGGGSGGGGSGGGGSGGGSLQ), SEQ ID NO: 7 (GGGGS), SEQ ID NO: 8 (GGGGSGGGGSGGGGSGGGGS), SEQ ID NO: 9 (EAAAK), SEQ ID NO : The group consisting of 10 (EAAAKEAAAKEAAAK), SEQ ID NO: 11 (GSADDAKKDAAKKDGKS), SEQ ID NO: 12 (SSADDAKKDAAKKDDAKKDDAKKDA);

Preferably, the amino acid sequence of "Linker1" is SEQ ID NO: 6, and the amino acid sequence of "Linker2" is SEQ ID NO: 7; Preferably, the 2A connecting peptide is P2A, and its amino acid sequence is SEQ ID NO: 13 (GSGATNFSLLKQAGDVEENPGP );

On the other hand, the present invention provides a biological molecule vector and a host cell. The biological molecule vector and host cell include nucleic acid molecules encoding the above-mentioned IL-15 super complex.

In another aspect, the present invention provides an application method of the above-mentioned IL-15 supercomplex in tumor immune cell therapy.

Specifically, the cells used in the tumor immune cell therapy include but are not limited to NK cells, CAR-T cells, TCR-T, DC and other cells.

Description of the drawings

Figure 1 Schematic diagram of IL-15 supercomplex

Figure 2 SDS-PAGE detection results of recombinant IL-15 supercomplex

Figure 3 Flow cytometry detection of IL-15-CD19-CAR expression on T cell surface

Figure 4 Amplification curves of different types of CAR-T cells

Figure 5 Comparison of killing efficiency of different types of CAR-T

Figure 6 Detection of expression levels of TSCM-like cells by flow cytometry

Figure 7 Detection of suicide function of IL-15 supercomplex

beneficial effects

Compared with the existing technology, the present invention has the following advantages:

(1) This complex can be continuously expressed in immune cells and anchored to the cell membrane through the shorter CD-4 structural element. It does not require the addition of exogenous cytokine IL-15, allowing immune cells to continue to proliferate and survive. Exert anti-tumor activity;

(2) When abnormal conditions occur, it can activate the expression of the "suicide gene" R structure, induce cell apoptosis, and ensure safety;

(2) Each fragment is the smallest structural element necessary to function and has a small molecular weight;

(3) By increasing the number of TSCM, the stemness and vitality of immune cells are enhanced, making the function of immune cells more durable and effective, and preventing tumor recurrence caused by immune cell therapy.

Detailed ways

The present invention will be further described in detail below with reference to specific examples. CD19-CAR-T cells are selected in the following examples. However, this example is not used to limit the present invention, but is only used to illustrate the present invention.

Example 1 Gene synthesis and vector construction of IL-15 super complex

Commissioned by General Biosystems (Anhui) Co., Ltd. to build it. According to the signal peptide (SEQ ID NO: 1), "suicide gene" R structure (SEQ ID NO: 2), Linker1 (SEQ ID NO: 6), IL-15 mutant IL-15-M-N72D (SEQ ID NO :3), Linker2 (SEQ ID NO: 7), the “Sushi” domain of IL-15Rα (SEQ ID NO: 4), and the CD-4 transmembrane region (SEQ ID NO: 5) to assemble the sequence. After optimization, the genes were routinely synthesized and cloned into plasmid vectors. The IL-15 supercomplex model is shown in Figure 1.

Example 2 Expression and Purification of IL-15 Super Complex

Transfer the expression vector into the expression host strain (E.coli DH5α), wait for the host strain to amplify for about 8-10 hours, then add 0.8mM IPTG (isopropyl-B-D-thiogalactose) to induce the expression of the plasmid. After induction for about 6 hours, collect the bacterial cells, add disruption buffer (pH 7.5, 20mM Tris-HCl, 50mM NaCl, 1% Triton-100, 20% glycerol) for ultrasonic disruption, and pass the supernatant through a nickel column. After the sample loading is completed, use the eluent (pH 7.5, 20mM Tris-HCl, 50mM NaCl, 0.1% TritonX-100, 500mM imidazole, 20% glycerol) to elute the nickel column and collect the eluate. Dialyze into 20mM Tris, 50mM NaCl pH7.5, 20% glycerol, and concentrate by ultrafiltration after completion of dialysis. The purified IL-15 super complex was identified by SDS-PAGE, and the results are shown in Figure 2. The molecular weight in Figure 2 is approximately 27KD, which is consistent with the theoretical molecular weight.

Example 3 Construction of CAR plasmid

Commissioned by General Biosystems (Anhui) Co., Ltd. to build it.

The plasmid of CAR-CD19 is PST#1;

The sequence was assembled according to the order of IL-15 mutant IL-15-M-N72D, the "Sushi" domain of IL-15Rα, Fc fusion protein, P2A and CAR genes, and it was PST#2;

According to the signal peptide, "suicide gene" R structure, Linker1, IL-15 mutant IL-15-M-N72D, Linker2, "Sushi" domain of IL-15Rα, CD-4 transmembrane region, P2A and CAR genes The plasmid in which the sequence is assembled is PST#3.

Example 4 Packaging of lentiviral vectors

① Preparation of 293T cells: 24 hours before transfection, spread out a 10cm culture dish with 9×10 ⁶ cells/dish (10ml); when the cell confluence reaches 80-90%, it can be used for transfection;

②Virus packaging: A total of 22.5ug of expression plasmid and helper plasmid are added for transfection according to the proportion in Table 1.

Table 1. Plasmid addition ratio

③After changing the medium of 293T cells, mix the plasmid solution prepared above with Lipo8000 and transfer it to the culture medium of 293T cells, mix gently, and culture in a 37°C, 5% CO 2 cell culture incubator. 8 hours after infection, replace the DMEM culture medium with new serum; collect the supernatant 48 hours after infection and store it at 4°C. The same volume of serum-containing DMEM culture medium was added, and the supernatant was collected again 72 hours after infection. The virus supernatant collected twice was centrifuged at 3000 rpm and 4°C for 10 min to remove cell debris, and the supernatant was filtered through a 0.45 μm filter. . Add the pre-prepared virus extract into a 50ml centrifuge tube, and then slowly drop the lentivirus supernatant onto the virus extract to form a layer (the volume ratio of virus extract to virus supernatant is 1:4). Centrifuge for 4 hours at 4°C and resuspend the pellet in a 1.5 mL centrifuge tube. Take a small amount to detect the virus titer using the QPCR method, and store the virus at -80°C for a long time.

Analysis of the results shows that the titer of the virus packaged by the scheme designed in this patent is significantly higher than that of the virus packaged by the CAR-CD19 plasmid. Compared with the Fc transmembrane region, the viral expression of IL15 supercomplex with CD4 as the transmembrane region is also significantly increased.

Table 2. Virus titer test results

Group Virus titer TU/ml A 2.1*10 ⁸ B 5.9*10 ⁹ C 9.2*10 ⁹

Example 5 Isolation of immune cells to be modified from human peripheral blood

① Human peripheral blood comes from healthy donors. After collecting the peripheral blood, first add 20ml of lymphocyte separation solution into a 50ml centrifuge tube, and then carefully add 20ml of whole blood along the tube wall to ensure that the stratification is obvious. After slowly rising and falling, centrifuge at 650g for 20 minutes, absorb the buffy coat layer into a 50ml centrifuge tube, add PBS to wash, discard the supernatant, resuspend in RPMI 1640 culture medium, and use CD3 antibody beads to isolate CD3-positive T cells. , Group A added cell stimulating factors CD3, CD28, and IL-2, with final concentrations of 200ng/ml, 200ng/ml, and 40ng/ml respectively for culture. Group B and Group C were added with CD3, CD28, IL-7, and IL-21, and the final concentrations were 200ng/ml, 200ng/ml, 10ng/mL, and 20ng/ml respectively.

②After 24 hours, use the prepackaged three groups of viruses A, B, and C according to the MOI value of 100, that is, lentivirus: T cells = 100:1, and culture it in a 37°C, 5% CO 2 incubator to obtain the virus. Infection with different types of CAR-T cells.

Example 6 Detection of IL-15 expression on T cell surface after infection by flow cytometry

The green fluorescent protein (GFP) carried by the lentiviral vector itself was used as a screening marker, and flow cytometry was used to detect the expression of IL-15 on the surface of CAR-T cells after infection with A, B, and C viruses. Centrifuge the CAT-T cells at 2000 rpm for 5 minutes and resuspend them in PBS. Take 100ul of cell resuspension and run it on the machine for detection. The test results are shown in Figure 3. The expression of IL-15 in group C was higher than that in groups A and B.

Example 7 In vitro proliferation and maintenance ability of CD19 CAR-T cells capable of secreting IL-15

After infecting T cells with lentiviruses from groups A, B, and C, the number of CAR-T cells was counted by microscopy at time points of 2, 4, 6, 8, 10, 12, 14, and 16 days respectively. Flow cytometry was used, using CD3 molecules as a gate, and using an antibody-coupled fluorescent molecule that specifically recognized anti-CD19scFv for flow labeling to count the number of CAR-positive cells. The expansion and proliferation curves of CAR-T cells prepared by lentivirus infection in groups A, B, and C are shown in Figure 4. As can be seen from the figure, the expansion level of CAR-T cells in group C was significantly increased.

Example 8 Evaluation of Cell Killing Experimental Effects

(1) Cultivate K562 cells expressing CD19 and T cells isolated from peripheral blood respectively;

(2) Three days before the start of the experiment, T cells isolated from peripheral blood were infected with viruses from groups A, B, and C at an MOI of 100, and the infected cells were placed at 37°C and 5% CO ₂ After 96 h of culture, the cell killing experiment was performed;

(3) Collect 1.0×10 ⁶ cells of target cells (CD19+K562) and 1.5×10 ⁶ cells of effector cells (T cells infected in step 2). After centrifugation at 2000 rpm for 6 min, discard the supernatant and use 1 mL of 1 ×Resuspend in PBS solution.

(4) Centrifuge again at 2000rpm for 6min and discard the supernatant.

(5) Effector cells were resuspended in 700 μL medium (1640 medium + 10% FBS), and target cells were resuspended in 1 mL medium (1640 medium + 10% FBS);

(6) Set up experimental wells with effect-to-target ratios of 1:1 and 10:1, and set up a control group with 3 duplicate holes in each group;

(7) Centrifuge the plate at 250 × g for 5 minutes, place it in an incubator at 37°C and 5% CO2 for 24 hours, and then centrifuge the plate again at 250 × g for 5 minutes;

(8) Take 50 μL of the supernatant into a new 96-well plate, and add 50 μL of substrate solution to each well (the operation should be protected from light);

(9) Incubate in the dark for 25-30 minutes;

(10) Add 50 μL stop solution to each well;

(11) Detect the absorbance at 490nm with a microplate reader;

(12) Take the average of 3 duplicate wells; subtract the average background absorbance value of the culture medium from the absorbance values of all experimental wells, target cell wells and effector cell wells; subtract the volume correction control from the maximum absorbance value of the target cells. The average absorbance value;

(13) Put the corrected value obtained in step 12 into the following formula to calculate the percentage of cytotoxicity produced by each effect-to-target ratio, where killing efficiency = (experimental well-effector cell well-target cell well)/(target cell well) Maximum cell hole - target cell hole) × 100%. The results are shown in Figure 5. T cells are in the control group. Compared with groups A, B, and C, the CAR-T cells in group C have a better killing effect on target cells, indicating that the CAR-T cells containing IL15-CAR-CD19 super complex Cells can enhance the killing ability of effector cells.

Expression of TSCM-like cells in CAR-T cells in Group C of Example 9

After infecting T cells with lentiviruses from groups A, B, and C respectively, flow cytometry was used to analyze the number of TSCM-like cells on the 12th day and screen CD45RA+CD45RO-CCR7+CD95+ cells. The results are shown in Figure 6. It can be seen from the results that the T cells infected with lentivirus in group C have a higher number of TSCM-like cells. CD19-specific CAR-T cells generated from membrane-bound IL-15 exhibit long-term persistence and excellent anti-tumor activity in vivo.

Example 10 Detection of suicide function of IL-15 super complex

1. Verify that CAR-T cells in groups A, B, and C achieve "suicide" through the CDC pathway. After incubating CAR-T cells in groups A, B, and C with 25% baby rabbit complement and different concentrations of rituximab (100ug/ml and 200ug/ml) for 4 hours, the samples were stained with AnnexinV/PI and passed through flow Cytology analysis was used to evaluate the apoptosis of CAR-T cells in groups B and C.

2. Verify that the CAR-T cells in groups B and C achieve "suicide" through the ADCC pathway. NK cells from the same donor were used as effector cells. The effector-target ratio was 8:1 and 16:1. After incubating the effector cells, target cells and 100ug/ml rituximab for 48 hours, AnnexinV/PI was used. Stained samples were used to evaluate apoptosis in CAR-T cells in groups B and C through flow cytometry analysis.

The results in Figure 7 show that the CAR-T cells in group C can achieve "suicide" through the CDC pathway and ADCC pathway, while groups A and B have no suicide function.

sequence list

<110> The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College)

<120> Development of a membrane-anchored IL-15 supercomplex and its application in tumor immune cell therapy

<141> 2022-03-06

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Claims (7)

1. A membrane anchored IL-15 super complex characterized by: the IL-15 super complex is a signal peptide, a suicide gene R structure, a connecting peptide 1, a Sushi domain of IL-15-N72D, IL-15 Ralpha of IL-15 mutant, a connecting peptide 2 and a CD4 transmembrane region molecular scaffold which are sequentially connected, and the IL-15 complex is anchored on a cell membrane through the CD4 scaffold; wherein the signal peptide is a signal peptide derived from CD8, and the amino acid sequence is shown in SEQ ID NO:1 is shown in the specification; the suicide gene R structure is a CD20 epitope polypeptide, the expression of the suicide gene R structure on the surface of a cell can be cleared by rituximab in vivo through CDC and ADCC forms, and the amino acid sequence of the R structure is shown as SEQ ID NO:2 is shown in the figure; the amino acid sequence of the IL-15 mutant IL-15-N72D is shown in SEQ ID NO:3 is shown in the figure; the amino acid sequence of the "Sushi" domain of IL-15Rα is shown in SEQ ID NO:4 is shown in the figure; the amino acid sequence of the CD4 transmembrane domain scaffold is shown in SEQ ID NO:5 is shown in the figure; the amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO:6 is shown in the figure; the amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: shown at 7.

2. A lentiviral expression vector, characterized in that: a nucleic acid molecule comprising a nucleic acid encoding the membrane-anchored IL-15 super complex of claim 1.

3. An immune cell capable of secreting an IL-15 complex, characterized by: infecting the immune cells with a virus comprising a nucleic acid molecule encoding the IL-15 complex of claim 1 and an exogenous insertion gene sequence; the immune cells are selected from CAR-T, NK, TCR-T, DC cells.

4. A pharmaceutical composition for immunotherapy comprising the membrane-anchored IL-15 super complex of claim 1, or the vector of claim 2, or the immune cell of claim 3.

5. Use of a membrane-anchored IL-15 super complex of claim 1, or a vector of claim 2, or an immune cell of claim 3, in the manufacture of a medicament for inducing immune cell proliferation and/or tumor treatment; the tumor is blood or solid tumor; the cells are selected from the group consisting of CAR-T, NK, TCR-T, DC cells.

6. The use according to claim 5, wherein the nucleic acid molecule encoding the membrane-anchored IL-15 super complex is linked to the foreign insert gene sequence by P2A; the amino acid sequence of P2A is shown in SEQ ID NO: shown at 13.

7. The construction method of the membrane anchored IL-15 super complex expression vector is characterized by comprising the following steps:

(1) Constructing a nucleic acid molecule encoding the membrane-anchored IL-15 super complex of claim 1;

(2) Connecting the nucleic acid molecule obtained in the step (1) with CAR-CD19 through P2A to construct a lentiviral vector, wherein the amino acid sequence of P2A is SEQ ID NO: 13;

(3) And (3) carrying out virus packaging on the obtained lentiviral vector by using 293T cells, respectively after 48h and 96h, collecting virus supernatant, and concentrating to obtain the virus vector for expressing the IL-15 complex and the CAR-CD 19.