Chimeric Antigen Receptor (CAR) T-cell Therapy Platform

Abstract

This application provides methods and compositions for treating cancer using CAR T cell therapy platforms. Methods and uses of CAR T cells for treating diseases and conditions such as cancer, particularly any disease and condition associated with elevated adenosine or other related markers, are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63 / 238927, filed August 31, 2021, and U.S. Provisional Patent Application No. 63 / 355396, filed June 24, 2022, the entire disclosures of which are incorporated by reference.

[0002] CAR T-cell therapy has shown exciting breakthroughs in the treatment of patients with hematopoietic malignancies, leading to FDA approval of CAR T-cell therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM). However, the antitumor activity of CAR T-cell therapy in solid tumors has been modest so far in clinical studies. The suboptimal effects of current CAR T-cell therapy on solid tumors are likely due to i) the immunosuppressive solid tumor microenvironment, ii) suboptimal T cell persistence in vivo after cell infusion, and iii) suboptimal CAR T cell trafficking in vivo. Therefore, it is desirable to develop novel CAR-T cell therapy technology platforms that exhibit improved cytotoxicity against tumors, especially solid tumors. [Background technology]

[0003] An important but largely overlooked issue in CAR T cell therapy for tumors, especially solid tumors, is nutrient competition between tumor cells and T cells in the nutrient-poor tumor microenvironment (TME). The TME represents a dramatic example of metabolic burden, where the high metabolic demands of cancer cells can limit CAR T cell function through competition for nutrients (i.e., glucose) and by producing immunosuppressive metabolites (e.g., adenosine). Thus, to be effective, CAR T cell therapy requires a reliable source of nutrients.

[0004] Inosine is a common component of food, and studies have shown that inosine has neuroprotective, cardioprotective, and immunomodulatory effects. Although inosine has a weak binding affinity with adenosine receptors, early studies have shown that inosine produces anti-inflammatory effects associated with activation of adenosine receptors, mainly A2a and A3 receptors, whose activation may contribute to the reduction of proinflammatory cytokines, as well as tissue protective effects against endotoxin-induced and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced inflammation. However, some of the literature has shown opposite findings, that inosine analogs can be proinflammatory and that A2AR signaling can maintain Th1 and antitumor immunity in mice. Recent studies have shown that inosine is an important bacterial metabolite that acts to promote Th1 cell activation in a context-dependent manner via T cell-specific A2AR signaling. Specifically, in the presence of IFN-gamma, inosine strongly boosted Th1 differentiation of naive T cells, whereas in the absence of IFN-gamma, inosine inhibited Th differentiation. Because adoptive T cell transfer and immune checkpoint inhibitors have shown the potential to transform a suppressive TME into a supportive TME, it is reasonable that inosine could amplify the antitumor effects of T cell therapy or immune checkpoint inhibitors. In addition, an independent study showed that inosine could strongly sensitize tumor cells to the cytolytic effects of immune cells by improving tumor immunogenicity.

[0005] Adenosine deaminase (ADA) is known to have antitumor activity. Several groups have developed ADA as a cancer therapy. For example, PEG-ADA2 (adenosine deaminase 2) was developed and showed antitumor activity in mouse models. The half-life of ADA is only a few hours long, and PEG conjugation did not significantly increase the half-life of the protein. Therefore, for ADA to serve as an effective cancer therapy, the half-life must be improved.

[0006] ADA reduces the levels of the immunosuppressive metabolite adenosine by irreversibly converting it to inosine. ADA is a key enzyme in purine metabolism, required for the degradation of adenosine from food and for the turnover of nucleic acids in tissues. ADA1 and ADA2 are the two ADA isoenzymes in humans. ADA1 is primarily responsible for the intracellular activity of most somatic cells, especially lymphocytes. ADA1 deficiency in humans induces severe combined immunodeficiency (SCID). ADA2 is the predominant form present in human plasma. ADA1 and ADA2 may play different roles in regulating immune responses, independent of their ADA activity. Studies have shown that ADA1 has a costimulatory effect in T cell-mediated immunity by binding to Teff and CTLs that express CD26. In contrast, ADA2 binds to neutrophils, CD16+ monocytes, NK cells, B cells, and CD39+ Tregs, which do not express CD26. A recent study showed that PEGylated ADA2 inhibited tumor growth in mice by targeting adenosine in an enzymatic activity-dependent manner and modulating immune responses. In addition, ADA1 has 100-fold higher affinity for adenosine than ADA2.

[0007] Adenosine signaling has emerged as a key immunometabolic checkpoint in tumors. ATP or NAD+ can accumulate in the TME and generate adenosine by ectonucleotidases, including CD39, CD73, CD38, CD203a, ALP, and PAP53-61. Studies have shown that adenosine can allow tumor cells to escape immune surveillance by suppressing the function of multiple potential defensive immune cells, including T cells, DCs, NK cells, macrophages, and neutrophils, while enhancing the activity of immunosuppressive cells, such as MDSCs and Tregs. In addition, adenosine activates cancer-associated fibroblasts and induces the formation of new blood vessels.

[0008] Several drugs, such as small molecules and mAbs, have been developed that target CD73 and CD38 to limit their production, or A2AR and A2BR to limit their binding to immune cells, and preclinical studies have shown their antitumor activity alone and in combination with other immunotherapies, including immune checkpoint inhibitors and adoptive cell transfer. However, early clinical studies show that their antitumor effects are not optimal. This can be explained by the fact that multiple ectonucleotidases contribute to the production of extracellular adenosine, and adenosine binds to multiple receptors, such as A2AR, A2BR, and A3R, suppressing antitumor immunity.

[0009] Although cure rates for some malignancies have improved significantly, outcomes for patients with advanced solid tumors remain grim in recent decades, highlighting the need for new therapeutic approaches. Oncolytic vaccinia viruses are an attractive addition to current treatment options for solid tumors due to their safety and potential to infect, replicate, and digest tumor cells. Clinical studies have shown that intratumoral or intravenous injection of oncolytic vaccinia viruses is safe and can induce tumor lysis, but the antitumor efficacy of oncolytic vaccinia viruses is suboptimal, and most tumors developed. This is most likely due to the limited activation of antitumor T cell responses in tumors and in the immunosuppressive tumor environment. Thus, an oncolytic vaccinia virus that 1) subverts the immunosuppressive tumor microenvironment, and 2) effectively supports T cell function, would overcome the current limitations of oncolytic vaccinia viruses.

[0010] There is growing evidence that T cells can control tumor growth and survival in cancer patients with both early and late stages of disease. For example, adoptive transfer of T cells has been shown to effectively treat sporadic tumors, including Hodgkin's lymphoma, nasopharyngeal carcinoma, neuroblastoma, and malignant melanoma. However, tumor-specific T cell responses are difficult to incorporate and maintain in cancer patients and are limited by the numerous immune evasion mechanisms of tumor cells selected during immunoediting. Therefore, it would be desirable to develop alternative strategies that utilize T cells in cancer therapy that have the ability to overcome tumor immune evasion mechanisms.

[0011] This application addresses the disadvantages that exist in current cancer treatment methods. Methods and compositions are provided for treating cancer using engineered immune cells (e.g., CAR-T cells, CAR-NK cells, CAR-NKT cells, macrophages) or recombinant oncolytic viruses that encode adenosine deaminase. The engineered immune cells or recombinant oncolytic viruses encode one or more other heterologous proteins. This application also provides methods and uses of the engineered immune cells or oncolytic viruses for treating diseases and conditions such as cancer, particularly any disease and condition associated with elevated adenosine or other related markers. Summary of the Invention

[0012] Embodiments of the claimed invention are directed to engineered cells that contain a nucleotide sequence encoding adenosine deaminase (ADA). In certain embodiments, the cells are T cells, natural killer cells, natural killer T cells, dendritic cells, macrophages, mesenchymal stem cells, and derivatives thereof.

[0013] Embodiments of the claimed invention are directed to pharmaceutical compositions comprising the engineered cells.

[0014] Further embodiments of the claimed invention are directed to methods of administering to a patient a pharmaceutical composition comprising the engineered cells in an amount effective to treat cancer.

[0015] Another embodiment of the present invention is directed to a recombinant oncolytic virus comprising a nucleotide sequence encoding ADA, wherein the nucleotide sequence encoding ADA is operably linked to a promoter.

[0016] An embodiment of the present invention is directed to a recombinant protein comprising a nucleotide sequence encoding ADA. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows a conjugated ADA design used in an embodiment of the invention. [Diagram 2] Figure 2 shows the principle of CD26 / ADA-CD3-scFv expressing CAR T cell therapy. CAR T cells are engineered to express tumor-specific CAR, membrane CD26 and secreted bispecific ADA-CD3-scFv. These CAR T cells 1) target and kill specific tumor cells; 2) stimulate CAR T cells with ADA-CD3-scFv (the binding affinity of CD3 and CD3-scFv is higher than that of ADA-CD26, so CD3-scFv tethers ADA to T cells, while CD3-scFv itself can activate T cells), and induce CAR T cell activation and migration; 3) release CAR T cells from adenosine-mediated immune suppression; and 4) provide inosine as an alternative carbon source for CAR T cells and improve the antitumor activity of CAR T cell therapy. [Diagram 3] FIG. 3 shows the structures of a CD26 / ADA-CD3-scFv-expressing CAR retroviral vector and a CD26 / ADA-CD3-scFv-expressing retroviral vector targeting HER2. [Figure 4] Figure 4 shows that Rv-CD26-transduced CAR T cells can resist TGF-beta suppression of CD26 expression, whereas CD26 expression on CAR T cells not transduced with Rv-CD26 was suppressed by TGF-beta. [Figure 5A-B]Figure 5A and Figure 5B show that Rv-CD26-transduced CAR T cells exhibited enhanced migration ability in fluorescent migration assays (Figure 5A) and transwell migration assays (Figure 5B). [Figure 6] FIG. 6 shows the mechanism of action of ADA-CD3-scFv on metabolically reprogrammed CAR T cells: (A) CD3 and CD3-scFv have higher binding affinity than that of CD26-ADA, so CD3-scFv tethers ADA to T cells, while CD3-scFv can directly activate T cells via CD3; (B) ADA engages CD26 and co-stimulates CAR T cells; and (C) ADA converts ADO into INO, overcoming ADO-mediated immune suppression in CAR T cells, while providing INO as an alternative energy source for CAR T cell survival and proliferation. [Figure 7A] Expression of ADA-CD3 (ADA-CD3-scFv) by retroviral vector-transduced HEK293T or Jurkat T cells: (FIG. 7A) Western blotting of ADA-CD3-scFv using monoclonal Ab against ADA in HEK293T cells transduced with a retroviral vector expressing ADA-CD3-scFv with or without the human IL2 signal peptide compared to non-transduced HEK293T cells. [Figure 7B] (FIG. 7B) Western blotting of ADA-CD3-scFv using monoclonal Ab against ADA in Jurkat T cells transduced with a retroviral vector expressing ADA-CD3-scFv with human IL2 signal peptide or a lentiviral vector expressing the CRISPR ADA activation element compared to non-transduced Jurkat T cells. [Figure 8A-B]Expression of ADA-CD3-scFv (ADA-CD3) is shown: (FIG. 8A) ELISA measurement of ADA in supernatants or cell lysates of HEK293T cells transduced with Rv-ADA-CD3-IL2sp (signal peptide), transduced with Rv-ADA-CD3 (no IL2sp), or not transduced; (FIG. 8B) ELISA measurement of ADA in supernatants on days 1 and 4 of co-culture of Jurkat T cells with HEK293T cells transduced with Rv-ADA-CD3-IL2sp (signal peptide), transduced with Rv-ADA-CD3 (no IL2sp), or not transduced. [Figure 8C] (FIG. 8C) Flow analysis of ADA-CD3 on Jurkat T cells or Rv-CD26-transduced Jurkat T cells after coculture with Rv-ADA-CD3-IL2sp (signal peptide), Rv-ADA-CD3 (no IL2sp), or untransduced HEK293T cells. [Figure 9A] Showing activity of ADA (ADA1) by ADA enzyme activity assay: (Figure 9A) ADA enzyme activity assay of co-cultures of Jurkat T cells and HEK293T cells transduced with Rv-ADA-CD3 (with IL2sp) or not (top: original reading; bottom: background reading of non-transduced HEK293T cells subtracted); [Figure 9B-C] (Figure 9B) ADA binding assay of Jurkat-Dual or Jurkat-Dual-CD26 T cell lines; and (Figure 9C) ADA binding assay of Jurkat-NEFA or Jurkat-NFAT-CD26 T cell lines. [Figure 10A] Using the Jurkat NFAT reporter T cell line, we show T cell activation: ( Fig. 10A ) Jurkat NFAT reporter T cells were supplemented with supernatants from Rv-ADA or Rv-ADA-CD3-transduced HEK293T cells for 6 h and subjected to luciferase assay; [Figure 10B]( Fig. 10B ) Jurkat NFAT reporter T cells were added with supernatants from HEK293T cells transduced with Rv-ADA or Rv-ADA-CD3 for 24 h and subjected to luciferase assay; [Figure 10C] ( Fig. 10C ) Jurkat NFAT reporter T cells were added with supernatants from HEK293T cells transduced with Rv-ADA-CD3 or Rv-ADA-CD3(IL2sp) for 6 or 24 h and subjected to luciferase assay; [Figure 10D] (FIG. 10D) The expression levels of ADA-CD3(IL2sp) were calculated. [Figure 11A] Showing the antitumor activity of ADA-CAR-T cells using in vitro LDH assay: (Figure 11A) Expression levels of GPC3 CAR in different groups of cells by flow analysis; [Figure 11B] LDH assay of CAR T cell groups against GPC-positive HepG2 or Huh7 liver tumor cell lines. [Figure 11C] LDH assay of CAR T cell groups against GPC-positive HepG2 or Huh7 liver tumor cell lines. [Figure 11D] LDH assay of CAR T cell groups against GPC-positive HepG2 or Huh7 liver tumor cell lines. [Figure 11E] LDH assay of CAR T cell groups against GPC-positive HepG2 or Huh7 liver tumor cell lines. [Figure 12A] The results of antitumor efficacy and toxicity studies carried out in mice are shown. [Figure 12B] The results of antitumor efficacy and toxicity studies carried out in mice are shown. [Figure 12C] The results of antitumor efficacy and toxicity studies carried out in mice are shown. [Figure 12D] The results of antitumor efficacy and toxicity studies carried out in mice are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] An embodiment of the present invention relates to engineered CAR T cells that express a tumor-specific CAR and express ADA (Figures 1 and 3). The engineered cells 1) target and kill tumor antigen-specific tumor cells; 2) CAR T cells are costimulated by ADA via C26 signaling; 3) CAR T cells are relieved from adenosine-mediated immune suppression; and 4) provide inosine as an alternative carbon source for CAR T cells, improving the antitumor activity of CAR T cell therapy.

[0019] Research has shown that extracellular adenosine in the TME has a significant inhibitory effect on immune responses, suppressing T cell function and stabilizing immune suppressive regulatory cells. Thus, ADA, which irreversibly converts adenosine to inosine, provides an energy source for CAR T cells while relieving them from adenosine-driven immune suppression. In addition, ADA mediates an effective co-stimulatory signal and promotes T-cell proliferation independently of its enzymatic activity. Figure 1 illustrates an example of ADA-CAR T cell conjugation.

[0020] An overview of the ADA-CD3-scFv / CD26 overexpressing metabolically reprogrammed (MR)CAR T cell therapy platform is shown in Figure 2. ADA-CD3-scFv / CD26-MRCAR T cells expressing tumor Ag-specific CAR and ADA-CD3-scFv / CD26 complex improve the antitumor activity of CAR T cell therapy through a series of mechanisms: i) the CAR molecule targets and directs T cells to kill tumor Ag-positive tumor cells, ii) the ADA / CD26 complex relieves adenosine-mediated immune suppression by providing inosine as a carbon source for CAR T cells by converting adenosine to inosine in the TME, and iii) CD26 mediates CAR T cell trafficking and remains bound to the T cell surface via ADA. Such ADA-CD3-scFv / CD26-MRCAR T cells have the potential to overcome the current limitations of CAR T cell therapy for solid tumors.

[0021] The ADA-CD3-scFv / CD26-MRCAR in Figure 3 includes the following components: a tumor antigen (e.g., HER2, GPC3)-specific scFv (small chain variable fragment), a chimeric antigen receptor including a spacer, hinge, transmembrane domain (e.g., CD28 and / or 4-1BBL), CD3 zeta domain; an ADA-CD3-scFv bispecific fusion protein, and CD26. These components were linked with a 2A sequence or expressed in separate viruses (e.g., retroviral vector, lentiviral vector). Ecto-ADA is important for protecting T cells from adenosine-mediated immune suppression, whereas cell-free ADA is not. Therefore, the development of an effective strategy to tether ADA on the surface of T cells is desirable. Rv-ADA T cells can express ~ng ADA / ml per 24 hours, and the expression of ADA by Rv-ADA T cells is only increased by 20% compared to that of unmodified T cells. However, the ADA concentration in human breast tumor tissue, normal tissue, or serum is 16.4-47.7ug / g, 11.5ug / g, or 160-242ng / ml, respectively. Therefore, T cell therapy with ADA is significantly limited by the ADA expression level of T cells. Tethering ADA to the cell surface by engaging CD26 on the T cell surface overcomes this limitation.

[0022] In one embodiment, the CAR comprises an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 18, 20, 22, and 24, 10, 12, 14, and 16, 2, 4, 6, 8, 9, 19, 21, 23 and 25, 11, 13, 15, and 17.

[0023] As shown in Figure 2, CD3-scFv engages with CD3 on infused CAR-T cells or endogenous T cells by tethering ADA on the T cell surface and promoting ADA-CD26 engagement and activating T cells via CD3 signals. When ADA engages with CD26, it activates infused CAR T cells or endogenous T cells. ADA converts adenosine to inosine to overcome adenosine-mediated immune suppression in CAR T cells and provide inosine as an alternative energy source for CAR T cells.

[0024] Figure 3 also shows a vector containing only the ADA-CD3-scFv bispecific fusion protein and CD26. This vector could potentially be used together with a vector expressing a CAR against any type of tumor antigen to transduce human T cells to produce MRCAR T cells.

[0025] The retroviral vector system is used to express the HER2-specific CAR, membrane CD26 and secreted ADA-CD3-scFv (Figure 3). The retroviral vector system was also used to express only membrane CD26 and secreted ADA-CD3-scFv (Figure 3). T cell codon optimization was used to improve transgene expression of ADA in human T cells. An optimized human IL2 signal peptide was added to express ADA by human T cells. Human peripheral blood monocytes were activated by plate-bound CD3 and CD28 antibodies for 2 days, followed by viral transduction. Expression and secretion of ADA1 by T cells was confirmed by Western blotting (Figure 7) and ELISA (Figure 8). ADA expression effectively converted adenosine to inosine (Figure 9). As shown in Figure 10, ADA-CD3 expression effectively activated human T cells (Figure 10). In vitro LDH cytotoxicity assays demonstrated that GPC3-specific MR-CAR exhibited enhanced cytotoxicity against GPC3-positive liver cancer cell lines, such as HepG2 and Huh7 (Figure 11).

[0026] Overexpression of CD26 in CAR T cells is important for CAR T cell therapy. As shown in Figure 4, in the presence of TGF-beta, the expression of CD26 on CAR T cells decreased within 48 hours, suggesting that the expression of CD26 may be suppressed by the tumor microenvironment. Rv-CD26 can restore CD26 expression in CAR T cells. As shown in Figure 4, T cells transduced with Rv-CD26 expressed CD26 at higher levels compared to unmodified T cells. In addition, T cells transduced with Rv-CD26 resisted TGF-beta-mediated immune suppression and maintained CD26 expression at high levels. In addition, T cells transduced with Rv-CD26 had improved migration ability as demonstrated by in vitro fluorescent migration assay and transwell migration assay (Figure 5).

[0027] A further embodiment of the invention relates to ADA-CAR T cell therapy in a transplantable mouse tumor model. The results show the efficacy and antitumor properties of the CAR T cell therapy model. MR-CAR T cells showed improved antitumor efficacy in two mouse models. In the first model, NSG mice were inoculated with 2x10 e6 Huh7 HCC tumor cells were inoculated into the right flank. On day 7, Huh7 tumor-bearing mice were inoculated with PBS, 2x10 e6 GPC3-CAR T cells, or 2x10 e6 In the second model, NSG mice were treated with GPC3-ADA1-CD3-CD26-CAR (referred to as GPC3-MR-CAR) and subsequently monitored for tumor size by caliper (Figure 12A) and body weight measurement (Figure 12B). e6 A549 NSCLC tumor cells were inoculated into the right flank. On day 7, A549 tumor-bearing mice were inoculated with PBS, 2x10 e6 HER2-CAR T cells, or 2x10 e6The mice were treated with HER2-ADA1-CD3-CD26-CAR (referred to as HER2-MR-CAR) and subsequently monitored for tumor growth by caliper (Figure 12C) and body weight measurement (Figure 12D). In both models, MR-CAR T cells showed improved antitumor activity. MR-CAR T cells significantly inhibited tumor growth in both Huh7 HCC and A549 NSCLC mouse models, whereas conventional CAR T cells only moderately inhibited the growth of Huh7 or A549 tumors. In addition, no significant difference in body weight was observed in either Huh7 HCC or A549 NSCLC mouse models, suggesting that MR-CAR T cells did not induce toxicity in the mouse models. The promising results in the mouse model provide a good prediction of the efficacy of the CAR T cell platform in human subjects.

[0028] Another embodiment of the present invention relates to an optimized ADA that exhibits improved ADA activity compared to conventional ADA. In one embodiment, the optimized ADA is an ADA-Fc fusion protein that significantly extends the half-life of ADA and improves its anti-tumor activity (Figure 1). In another embodiment, the optimized ADA is an ADA conjugated to an antibody (Figure 1).

[0029] Another embodiment of the present invention relates to an engineered oncolytic virus expressing ADA or an ADA derivative such as ADA-Fc (Figure 1). The engineered oncolytic virus expressing ADA will express ADA or ADA-Fc that can infect and replicate in tumor cells and convert ADO to INO in tumor tissues, improving the anti-tumor effect of the oncolytic virus.

[0030] In one embodiment, the engineered oncolytic virus comprises an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 22, and 24. EXAMPLES

[0031] CAR T cell generation material : OKT3 (ortho Biotech) - 1mg / ml CD28 (Azide-free (NA) / Low Endotoxin (NE) (Pharmingen #33740D) - 1mg / ml Plates: Non-tissue culture treated 24-well plates (Falcon #1147)

[0032] Protocol : Day 0 : 1. Dilute antibodies to a final concentration of 1ug / ml in sterile water (e.g., to coat 6 wells, add 3uL of OKT3 and 3uL of CD28 to 3ml of sterile water) 2. Add 0.5ml (=0.5ug of antibody) of antibody solution per well to a 24-well plate; seal with parafilm. 3. Incubate overnight at +4°C. Day 1 : 1. 5x10 6 PBMCs are resuspended in 10 ml of complete RPMI (10% FCS). 2. Aspirate the antibody solution and wash wells once with RPMI or DMEM complete. 3. Aspirate the medium and add 1x10 cells per well. 6 PBMCs are plated in a final volume of 2 ml complete medium. Day 2: 1. Aspirate and discard approximately 1 ml from each well and replace with 1 ml of complete RPMI medium containing 200 U / ml IL-2. 2. Coat non-tissue culture treated 24-well plates with Retronectin; seal with parafilm. 3. Place the Retronectin coated plates at +4° C. overnight. Day 3 : 1. Remove the RetroNectin solution and wash the wells once with RPMI or DMEM complete. (Store RetroNectin in a 15cc tube at +4°C; can be reused once within one month) 2. Add 500-700ul of viral supernatant to the wells. Place the plate in the incubator for 30 minutes. 3. Remove the viral supernatant; repeat step 2. 4. During the incubation period, incubate the blast cells with 400 U / cc of IL-2. 5x10 5 Prepare cells / cc. 5. Remove the viral supernatant. Add 1.5cc of viral supernatant. 6. Add 500ul of blast cells (2.5x10 5 cell) 7. Centrifuge the plate at 1000g for 30 minutes and then return the plate to the incubator (optional) Day 5 / 6 : 1. Remove 1 cc of medium from the well; transfer (by scraping) the cells from the Retronectin coated plate to a new well; add 1 cc of complete medium containing 100 U / ml IL-2. 2. Give IL-2 (100 U / ml) every 2-3 days. Refreeze or use within 7 to 21 days.

[0033] MR-CAR resisted TGF-beta-mediated suppression of CD26 expression on CAR T cells and exhibited greater migratory capacity. Human PBMCs were activated with CD3 / CD28 antibodies and subsequently transduced with Rv-CD26 as described above. Rv-CD26 transduced T cells (Rv-CD26) or non-transduced T cells (NT) were cultured for 48 hours in the presence or absence of 5ng / ml, 10ng / ml or 20ng / ml TGF-beta. T cells were harvested and stained with FITC-conjugated human CD26 specific antibody. CD26 expression on T cells was then detected by flow analysis and presented as MFI (median fluorescence intensity). In the absence of TGF-beta, Rv-CD26 transduced human T cells expressed higher levels of CD26 compared to non-transduced T cells (Figure 4, Rv-CD26 vs. NT = MFI 1420 vs. MFI 801). When NT T cells were cultured for 48 hours in the presence of TGF-beta (5ng / ml, 10ng / ml, or 20ng / ml), CD26 expression decreased from MFI 801 to MFI 603, MFI 599, or MFI 626, respectively, while CD26 expression on Rv-CD26-transduced T cells remained unchanged after 48 hours of culture in the presence of TGF-beta (5ng / ml, 10ng / ml, or 20ng / ml). These results indicated that Rv-CD26-transduced CAR T cells could resist TGF-beta inhibition of CD26 expression, while CD26 expression of CAR T cells not transduced with Rv-CD26 was suppressed by TGF-beta.

[0034] To investigate the migration ability of human T cells, in vitro transwell migration assay and fluorescent migration assay were performed. A. Transwell Cell Migration Assay Protocol (by Cell Counting) 1. Grow enough cells (Rv-CD26-transduced HER2 CAR T cells or control HER2 CAR T cells) to perform a cell migration assay in the desired medium and culture conditions. 2. Harvest the cells, resuspend the cells in serum-free medium, and count the number of cells using a hemocytometer. Resuspend the cells in serum-free medium at a concentration of 1.5x10 6 Resuspend in cells / mL or to your desired concentration of cells. 3. Lower chamber: Add 235 μL of HER2 positive A549 cell supernatant (chemoattractant) and 365 μL of serum free medium to the lower chamber for both NT (non-transduced) and CD26-transduced HER2-CAR T cells to 600 μL. 4. Carefully place the top chamber (Transwell, 5 mm) into the bottom chamber, making sure no air bubbles are trapped between the top and bottom chambers. 5. Upper chamber: Add 300 μL (5 million cells) of cell suspension (both NT and Rv-CD26 transduced) to each well of the upper chamber. 6. Carefully replace the plate cover and incubate the cell migration chamber in a CO2 incubator at 37°C for 2-24 hours. 7. At different time points (2, 4, 22 / 24 hours) cells are counted using a hemocytometer.

[0035] B. Fluorescence cell migration assay protocol (by fluorescence intensity using the Abcam transwell migration assay kit ab235696) standard curve : 1. Each cell type requires a separate standard curve. 50 μL of cell suspension (1.8x10 6 Cells / mL, 90,000 cells or 1x10 6 Prepare a standard curve by adding 50,000 cells / mL of 100 mM NaCl. 2. Serially dilute the cells 1:1 into wash buffer to generate a cell standard curve (90000, 45000, 22500, 11250, 5625, 2812, 1406 and 703) in a 96-well white plate, total volume 100 μL. 3. As a blank, use 100 μL of wash buffer. 4. Add 10 μL of cell dye to each well. 5. Incubate at 37℃ for 1 hour. 6. Read fluorescence at Ex / Em=530 / 590 nm. 7. Plot a standard curve of cell number vs. RFU obtained. 8. Use a linear trend line to fit the data points.

[0036] Counting migrated cells : 1. Make the desired cell staining solution depending on the number of wells. Add 100 μL of cell stain to 1 mL of cell dissociation solution. Mix well. 2. Add 550 μL of the mixture to each well of the lower chamber (after 22 hours, step A6). Depending on the experimental design, this can also be after 2 hours or 4 hours. 3. Incubate in a CO2 incubator at 37°C for 60 minutes. 4. After incubation, transfer 110 μL of the mixture from the lower chamber to a 96-well white plate. Read the plate at Ex / Em=530 / 590 nm. Multiply the reading by 5 to account for the 5-fold higher volume in each well of the 24-well plate.

[0037] Data analysis : The number of migrated cells was calculated using the linear equation obtained from the standard curve.

[0038] As shown in Figure 5, the results of the fluorescent migration assay and the transwell migration assay showed that Rv-CD26-transduced HER2 CAR T cells exhibited enhanced migration ability compared to HER2 CAR T cells.

[0039] ADA-CD3-scFv expression by MR-CAR T cells is biologically functional Western blotting was used to measure the expression of ADA-CD3(scFv) in Figure 7. Rv-ADA-CD3(scFv) or Rv-ADA-CD3(scFv)-IL2sp (human IL2 signal peptide was inserted at the 5´ end of ADA-CD3(scFv) to increase the expression of ADA-CD3(scFv)) was transduced into 293T cells, and the cell lysates were subjected to Western blotting with anti-ADA antibody (Figure 7A). The results showed that Rv-ADA-CD3-IL2sp expressed ADA-CD3 at a higher level compared to RV-ADA-CD3 (without IL2sp). Then, Rv-ADA-CD3-IL2sp was compared with a lentiviral vector expressing the ADA CRISPR activation element. Lv-ADA(CRISPR activation) virus or Rv-ADA-IL2sp was transduced into Jurkat T cells. Jurkat T cells transduced with Lv-ADA (CRISPR activated) were selected by puromycin. ADA expression was measured by Western blotting with anti-ADA mAb on days 7, 14 or 18 of T cell culture. As shown in Figure 7B, Rv-ADA1-IL2sp expressed ADA at higher levels compared to lenti-ADA activation. These results supported that Rv-ADA-CD3-IL2sp is an optimized strategy for expressing ADA-CD3(scFv) in CAR T cells.

[0040] Secretion of ADA-CD3 (scFv) by MRCAR T cells was measured by ELISA against human ADA. As shown in Figure 8A, ADA in the supernatant or cell lysate of HEK293T cells transduced with Rv-ADA-CD3-IL2sp (signal peptide), transduced with Rv-ADA-CD3 (no IL2sp), or not transduced (NT) was measured by ELISA. The results showed that Rv-ADA-CD3-scFv-IL2sp (ADA1-CD3-IL2sp) or Rv-ADA-CD3-scFv (no IL2sp, ADA1-CD3) expressed ADA-CD3 in both the supernatant or cell lysate. And Rv-ADA-CD3-IL2sp expressed higher levels of ADA-CD3 than Rv-ADA-CD3.

[0041] Then, HEK293T cells transduced with Rv-ADA-CD3-IL2sp or Rv-ADA-CD3 were co-cultured with Jurkat T cells to mimic stress conditions (increased cell density and cell contact) for cell culture to confirm ADA-CD3 expression. On the first or fourth day of co-culture, cell culture medium was collected and subjected to ELISA measurement of ADA. The results showed that Rv-ADA-CD3-IL2sp induced higher ADA expression than NT or Rv-ADA-CD3. We observed that the NT group had ADA expression (Figure 8B). This may be explained by the background expression of ADA by Jurkat T cells or HEK293T cells.

[0042] Next, we examined whether ADA-CD3 engaged CD3-expressing Jurkat T cells or CD3 / CD26 double-positive CD26- Jurkat T cells transduced with Rv-CD26. Jurkat T cells transduced with Rv-ADA-CD3-IL2sp or CD26- Jurkat T cells were cultured for 24 h, stained with PE-conjugated mAb against ADA, and subjected to flow analysis of ADA expression. As shown in Figure 8C, the ADA level on Jurkat T cells transduced with Rv-ADA-CD3 was higher than that on Jurkat T cells, suggesting that CD3-scFv tethered ADA-CD3 on the surface of Jurkat T cells. Compared with the ADA level on Jurkat T cells transduced with Rv-ADA-CD3, the ADA level on CD26-Jurkat T cells transduced with Rv-ADA-CD3 was further increased, suggesting that the engagement of ADA and CD26 further enhanced the binding of ADA-CD3 to CD26-Jurkat T cells. Therefore, these data suggested that the ADA-CD3 we designed could be secreted from T cells and engage with T cells.

[0043] Next, the enzymatic activity of secreted ADA-CD3 was examined. Because ADA-CD3 has high binding affinity with T cells and would engage on the surface of T cells, T cells were directly used to measure ADA-CD3 enzymatic activity. First, HEK293T cells were transduced with Rv-ADA-CD3 and seeded overnight in 96-well plates at different densities. After 24 hours, cells were counted and the cell densities were shown in Figure 9A. The same amount of Jurkat T cells was added to the cell culture for 6 hours. The cell culture medium was removed and the cells were subjected to direct ADA activity assay. As shown in Figure 9A, at a cell culture density of 10e5 / well or 5x10e5 / well, Rv-ADA-CD3 resulted in higher enzymatic activity of converting ADO to INO compared with the control group, suggesting that ADA secretion was stress condition mediated (left: original reading; right: background reading of untransduced HEK293T cells was subtracted).

[0044] Next, we investigated the binding efficiency of ADA-CD3 on T cells. Jurkat-Dual (NF-kB and IRF / IFN), Jurkat-Dual transduced with Rv-CD26 (Jurkat-Dual-CD26), Jurkat-NFAT, or Jurkat-NFAT transduced with Rv-CD26 (Jurkat-NFAT-CD26) reporter cell lines were co-cultured with 293T cells transduced with Rv-ADA or Rv-ADA-CD3 (scFv) for 24 h. The cell culture medium was then used to measure ADA by ADA activity assay. ADA membrane binding efficiency was calculated using Rv-ADA-CD3-293T / CD26-Jurkat T as the maximum value and 293T / JurkatT W / O CD26 as the background. The results showed that secreted ADA-CD3 fully engaged with both Jurkat-Dual and CD26-Jurkat-Dual (Figure 9B), or Jurkat-NFAT and CD26-Jurkat-NFAT (Figure 9C), while secreted ADA (without CD3-scFv) only engaged with CD26-Jurkat-Dual (Figure 9B) or CD26-Jurkat-NFAT (Figure 9C) with 40% efficiency of secreted ADA-CD3. Thus, these results indicated that ADA-CD3 improved the engagement of ADA.

[0045] Since either CD3-scFv or ADA could provide stimulatory or costimulatory signals to human T cells, we next examined whether secreted ADA-CD3 (scFv) could activate human T cells. As shown in Figures 10A and 10B, Rv-ADA (ADA1) or Rv-ADA-CD3 (ADA1-CD3) were transduced into 293T cells. 293T cell culture medium (supernatant) or 293T cells were added to the culture of NFAT-luciferase Jurkat reporter cell line. The cells were cultured for 6 hours (Figure 10A) or 24 hours (Figure 10B), and then the cell culture medium was collected and subjected to luciferase assay. Only 293T cells transduced with Rv-ADA-CD3 induced T cell activation at 24 hours, suggesting that the secretion of ADA-CD3 was mediated by stress conditions (cell culture at high cell density for 24 hours). We also compared Rv-ADA-CD3 and Rv-ADA-CD3-IL2sp. Jurkat NFAT reporter T cells were added with the supernatant of HEK293T cells transduced with Rv-ADA-CD3 or Rv-ADA-CD3(IL2sp) for 6 or 24 hours and subjected to luciferase assay. The results showed that Rv-ADA-CD3-IL2sp induced T cell activation more effectively than Rv-ADA-CD3. ADA-CD3(IL2sp) expression level was calculated as shown in Figure 10D. Rv-ADA-CD3 or Rv-ADA-CD3-IL2sp could induce ADA-CD3 expression of 0.3ug / ml or >0.5ug / ml at 24 hours, respectively.

[0046] LDH cytotoxicity assay of MR-CAR T cells LDH Assay Protocol Day 1 In 96-well U-bottom cell culture plates, tumor cells (1x10e4) were co-cultured with effector cells CAR-T and NT-T (untransduced T cells as negative control) in 200ul DMEM medium for 4 hours with gradient E:T ratios as follows: Maximum loading (positive control) wells were added with 20ul cell lysis buffer and incubated for 30 minutes. TIFF2024533109000002.tif49170 After 4 hours: Detection of lactate dehydrogenase (LDH) was performed using the Enzo LDH cytotoxicity EST assay (Cat♯ ENZ-KIT157) as specified by the manufacturer. 1. To pellet the cells, the plates will be centrifuged at 250 x g for 2 minutes. 2. Transfer 100 ul of cell supernatant to each well of a new flat bottom optically clear 96 well plate. 3. Add 100 ul of working solution to each well. 4. Protect the plate from light and incubate at room temperature for 30 minutes. 5. Add 50 ul of Stop Solution to each well. 6. Measure the absorbance at 490 nm using a microplate reader. Specific lysis was calculated using the following formula: (Experimental-Spontaneous Release) / (Maximum Load-Spontaneous Release) x 100%

[0047] As shown in Figure 11, CAR T cells were co-cultured with HepG2 or Huh7 liver cancer cells and subjected to LDH assay as described above. Figure 11B and Figure 11C showed that at 4 hours of co-culture, MR-CAR (GPC3-ADA1-CD3-CD26) T cells showed improved cytotoxicity against HepG2 (Figure 11B) or Huh7 (Figure 11C) at an E:T ratio of 20:1 compared to GPC3-CAR T cells, while no significant difference was observed at an E:T ratio of 1:1. This is likely due to the time required for expression and secretion of ADA-CD3 (scFv). As a result, at an E:T ratio of 1:1, MR-CAR showed significantly improved cytotoxicity against HepG2 (Figure 11D) or Huh7 (Figure 11E) at either 18 hours or 24 hours of co-culture of MR-CAR T cells and tumor cells. These results suggested that MR-CAR T cells exhibited enhanced cytotoxicity against specific tumor cells in vitro.

[0048] In vivo mouse study of MR-CAR T cell therapy Six-week-old NSG mice were purchased from Jax laboratory, with 5 mice per group. To investigate the effect of MR-CAR T-cell therapy in liver cancer, groups of NSG mice were first inoculated subcutaneously on the right flank with 2x10e6 Huh7 HCC tumor cells in 200ul of medium on day 0. On day 7, mice bearing Huh7 tumors were treated with 200ul of PBS, 2x10e6 GPC3-CAR T cells in 200ul of medium, or 2x10e6 GPC3-ADA1-CD3-CD26-CAR T cells in 200ul of medium (referred to as GPC3-MR-CAR) via the tail vein. Mice were monitored every 2-3 days. Tumor size was measured with a caliper, and tumor size was calculated as tumor size=LxWxW / 2 (L: tumor length, W: tumor width). Mouse body weight was also measured to monitor the toxicity of MR-CAR T-cell therapy.

[0049] To investigate the effect of MR-CAR T cell therapy in lung cancer, groups of NSG mice were first inoculated subcutaneously on the right flank with 2x10e6 A549 NSCLC tumor cells in 200ul of medium on day 0. On day 7, A549 tumor-bearing mice were treated with 2x10e6 HER2-CAR T cells, or 2x10e6 HER2-ADA1-CD3-CD26-CAR T cells (represented as HER2-MR-CAR) in 200ul of medium, via the tail vein, or no treatment (NT) as control. Tumor size and body weight were measured as described above.

[0050] In both models, MR-CAR T cells showed improved antitumor activity. MR-CAR T cells significantly inhibited tumor growth in both Huh7 HCC or A549 NSCLC mouse models, while the original CAR T cells only moderately inhibited the growth of Huh7 or A549 tumors. In addition, no significant difference in body weight was observed in both Huh7 HCC and A549 NSCLC mouse models, suggesting that MR-CAR T cells did not induce toxicity in these mouse models.

[0051] SEQUENCE LISTING: The following sequence listing is part of this disclosure.

[0052] HER2-scFv-CD28TM-CD3zeta-ADA1-CD26 (SEQ ID NO: 1)

[0053] HER2-scFv-CD28TM-CD3zeta-ADA2-CD26 (SEQ ID NO: 2)

[0054] HER2-scFv-CD28TM-CD3zeta-ADA1 (SEQ ID NO: 3)

[0055] HER2-scFv-CD28TM-CD3zeta-ADA2 (SEQ ID NO: 4)

[0056] IL2SP-ADA1-CD26 (SEQ ID NO: 5)

[0057] ADA2-CD26 (SEQ ID NO: 6)

[0058] IL2SP-ADA1 (SEQ ID NO: 7) MRRMQLLLLIALSLALVTNSMAQTPAFDKPKVELHVHLDGSIKPETILYYGRRRGIALPANTAEGLLNVIGMDKPLTLPDFLAKFDYYMPAIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVEPIPWNQAEGDLTPDEVVALVGQGLQEGERDFGVKARSILCCMRHQPNWSPKVVELCKKYQQQTVVAIDLAGDETIPGSSLLPGHVQAYQEAVKSGIHRTVHAGEVGSAEVVKEAVDILKTERLGHGYHTLEDQALYNRLRQENMHFEICPWSSYLTGAWKPDTEHAVIRLKNDQANYSLNTDDPLIFKSTLDTDYQMTKRDMGFTEEEFKRLNINAAKSSFLPEDEKRELLDLLYKA YGMPPSASAGQNL

[0059] ADA2 (SEQ ID NO:8) MLVDGPSERPALCFLLAVAMSFFGSALSIDETRAHLLLKEKMMRLGGRLVNTKEELANERLMTLKIAEMKEAMRTLIFPPSMHFFQAKHLIERSQVFNILRMMPKGAALHLHDIGIVTMDWLVRNVTYRPHCHICFTPRGIMQFRFAHPTPRPSEKCSKWILLEDYRKRVQNVTEFDDSLLRNFTLVTQHPEVIYTNQNVVWSKFETIFFTISGLIHYAPVFRDYVFRSMQEFYEDNVLYMEIRARLLPVYELSGEHHDEEWSVKTYQEVAQKFVETHPEFIGIKIIYSDHRSKDVAVIAESIRMAMGLRIKFPTVVAGFDLVGHEDTGHSLHDYKEALMIPAKDGVKLPYFFHAGETDWQGTSIDRNILDALMLNTTRIGHGFALSKHPAVRTYSWKKDIPIEVCPISNQVLKLVSDLRNHPVATLMATGHPMVISSDDPAMFGAKGLSYDFYEVFMGIGGMKADLRTLKQLAMNSIKYSTLLESEKNTFMEIWKKRWDKFIADVATK

[0060] CD26 (SEQ ID NO: 9) MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSRKTYTLTDYLKNTYRLKLYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPDGQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEERIPNNTQWVTWSPVGHKLAYVWNNDIYVKIEPNLPSYRITWTGKEDIIYNGITDWVYEEEVFSAYSALWWSPNGTFLAYAQFNDTEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVNPTVKFFVVNTDSLSSVTNATSIQITAPASMLIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICDYDESSGRWNCLVARQHIEMSTTGWVGRFRPSEPHFTLDGNSFYKIISNEEGYRHICYFQIDKKDCTFITKGTWEVIGIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYTKVTCLSCELNPERCQYYSVSFSKEAKYYQLRCSGPGLPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPSKKLDFIILNETKFWYQMILPPHFDKSKKYPLLLDVYAGPCSQKADTVFRLNWATYLASTENIIVASFDGRGSGYQGDKIMHAINRRLGTFEVEDQIEAARQFSKMGFVDNKRIAIWGWSYGGYVTSMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSRAENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDH GIASSTAHQHIYTHMSHFIKQCFSLP

[0061] HER2-scFv-ADA1-CD28TM-CD3zeta-CD26 (SEQ ID NO: 10)

[0062] HER2-scFv-ADA2-CD28TM-CD3zeta-CD26 (SEQ ID NO: 11)

[0063] HER2-scFv-CD28TM-CD3zeta-ADA1-CD28TM-CD3zeta-CD26 (SEQ ID NO: 12)

[0064] HER2-scFv-CD28TM-CD3zeta-ADA2-CD28TM-CD3zeta-CD26 (SEQ ID NO: 13)

[0065] ADA1-CD28TM-CD3zeta-CD26 (SEQ ID NO: 14)

[0066] ADA2-CD28TM-CD3zeta-CD26 (SEQ ID NO: 15)

[0067] ADA1-CD28TM-CD3zeta (SEQ ID NO: 16) MRRMQLLLLIALSLALVTNSMAQTPAFDKPKVELHVHLDGSIKPETILYYGRRRGIALPANTAEGLLNVIGMDKPLTLPDFLAKFDYYMPAIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVEPIPWNQAEGDLTPDEVVALVGQGLQEGERDFGVKARSILCCMRHQPNWSPKVVELCKKYQQQTVVAIDLAGDETIPGSSLLPGHVQAYQEAVKSGIHRTVHAGEVGSAEVVKEAVDILKTERLGHGYHTLEDQALYNRLRQENMHFEICPWSSYLTGAWKPDTEHAVIRLKNDQANYSLNTDDPLIFKSTLDTDYQMTKRDMGFTEEEFKRLNINAAKSSFLPEDEKRELLDLLYKAYGMPPSASAGQNLSSDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0068] ADA2-CD28TM-CD3zeta (SEQ ID NO: 17) MLVDGPSERPALCFLLAVAMSFFGSALSIDETRAHLLLKEKMMRLGGRLVLNTKEELANERLMTLKIAEMKEAMRTLIFPPSMHFFQAKHLIERSQVFNILRMMPKGAALHLHDIGIVTMDWLVRNVTYRPHCHICFTPRGIMQFRFAHPTPRPSEKCSKWILLE DYRKRVQNVTEFDDSLLRNFTLVTQHPEVIYTNQNVVWSKFETIFFTISGLIHYAPVFRDYVFRSMQEFYEDNVLYMEIRARLLPVYELSGEHHDEEWSVKTYQEVAQKFVETHPEFIGIKIIYSDHRSKDVAVIAESIRMAMGLRIKFPTVVAGFDLVGHEDTGH SLHDYKEALMIPAKDGVKLPYFFHAGETDWQGTSIDRNILDALMLNTTRIGHGFALSKHPAVRTYSWKKDIPIEVCPISNQVLKLVSDLRNHPVATLMATGHPMVISSDDPAMFGAKGLSYDFYEVFMGIGGGMKADLRTLKQLAMNSIKYSTLLESEKNTFMEIWKKRWDKFIADVATKSSDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQQNQLYNELNLRREEYDVLDKRRGRDRPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0069] HER2-scFv-CD28TM-CD3zeta-ADA1-CD3-scFv-CD26 (SEQ ID NO: 18)

[0070] HER2-scFv-CD28TM-CD3zeta-ADA2-CD3-scFv-CD26 (SEQ ID NO: 19)

[0071] HER2-scFv-CD28TM-CD3zeta-ADA1-CD3-scFv (SEQ ID NO: 20)

[0072] HER2-scFv-CD28TM-CD3zeta-ADA2-CD3-scFv (SEQ ID NO: 21)

[0073] ADA1-CD3-scFv-CD26 (SEQ ID NO: 22)

[0074] ADA2-CD3-scFv-CD26 (SEQ ID NO: 23)

[0075] ADA1-CD3-scFv (SEQ ID NO: 24) MRRMQLLLLIALSLALVTNSMAQTPAFDKPKVELHVHLDGSIKPETILYYGRRRGIALPANTAEGLLNVIGMDKPLTLPDFLAKFDYYMPAIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVEPIPWNQAEGDLTPDEVVALVGQGLQEGERDFGVKARSILCCMRHQPNWSPKVVELCKKYQQQTVVAIDLAGDETIPGSSLLPGHVQAYQEAVKSGIHRTVHAGEVGSAEVVKEAVDILKTERLGHGYHTLEDQALYNRLRQENMHFEICPWSSYLTGAWKPDTEHAVIRLKNDQANYSLNTDDPLIFKSTLDTDYQMTKRDMGFTEEEFKRLNINAAKSSFLPEDEKRELLDLLYKAYGMPPSASAGQNLSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS

[0076] ADA2-CD3-scFv (SEQ ID NO: 25) MLVDGPSERPALCFLLAVAMSFFGSALSIDETRAHLLLKEKMMRLGGRLVLNTKEELANERLMTLKIAEMKEAMRTLIFPPSMHFFQAKHLIERSQVFNILRMMPKGAALHLHDIGIVTMDWLVRNVTYRPHCHICFTPRGIMQFRFAHPTPRPSEKCSKWILLEDYRKRVQNVTEFDDSLLRNFTLVTQHPEVIYTNQNVVWSKFETIFFTISGLIHYAPVFRDYVFRSMQEFYEDNVLYMEIRARLLPVYELSGEHHDEEWSVKTYQEVAQKFVETHPEFIGIKIIYSDHRSKDVAVIAESIRMAMGLRIKFPTVVAGFDLVGHEDTGHSLHDYKEALMIPAKDGVKLPYFFHAGETDWQGTSIDRNILDALMLNT TRIGHGFALSKHPAVRTYSWKKDIPIEVCPISNQVLKLVSDLRNHPVATLMATGHPMVISSDDPAMFGAKGLSYDFYEVFMGIGGGMKADLRTLKQLAMNSIKYSTLLESEKNTFMEIWKKRWDKFIADVATKSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGGSGGGGGSGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS

[0077] CD28-TM (SEQ ID NO: 26) DPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0078] CD3-zeta (SEQ ID NO:27) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0079] G4S linker (SEQ ID NO: 28) GGGGSGGGGSGGGGS

[0080] CD3-scFv (SEQ ID NO: 29) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSG GGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS

[0081] T2A sequence (SEQ ID NO:30) GSGEGRGSLLTCGDVEENPGP

[0082] P2A sequence (SEQ ID NO:31) GSGATNFSLLKQAGDVEENPGP

[0083] IL2 leadersequence (SEQ ID NO:32) MRRMQLLLLIALSLALVTNS

[0084] The term "substantially" is defined as being understood by one of ordinary skill in the art to be largely, but not necessarily completely, what is specified. In any disclosed embodiment, "substantially," "about," "generally," and "approximately" may be substituted with "within [a percent]" of what is specified, where the percent includes 0.1, 1, 5, and 10 percent.

[0085] The foregoing outlines features of some embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they can readily use the present disclosure as a basis for designing or modifying other processes or structures to accomplish the same purposes and / or achieve the same advantages as the embodiments presented herein. Those skilled in the art should also appreciate that such equivalent structures do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. The scope of the present invention should be determined solely by the language of the following claims. In the claims, "comprising" is intended to mean "including at least" such that the list of recited elements in the claim is an open group. The terms "a," "an," and other singular forms are intended to include their plural forms unless specifically excluded.

Claims

1. Engineered cells that express a tumor-specific chimeric antigen receptor (CAR) and adenosine deaminase (ADA).

2. The cell of claim 1 , wherein the ADA is ADA1 or ADA2.

3. The cell of claim 1 , wherein the ADA is a secreted protein or a membrane-bound protein.

4. The cell of claim 1 , wherein the ADA is operably linked to one or more of a linker sequence, a human CD3-specific ligand, an antibody, an scFv, or a derivative thereof.

5. The cell described in claim 4, further comprising an IL2 signal peptide operably linked to ADA.

6. The cell of claim 1 , further comprising human CD26 or a derivative thereof.

7. 10. A pharmaceutical composition comprising the engineered cells of claim 1 and a pharmaceutically acceptable carrier.

8. A recombinant protein comprising an amino acid sequence encoding adenosine deaminase (ADA) operably linked to one or more of a linker sequence, a human CD3-specific ligand, an antibody, an scFv, or a derivative thereof.

9. The recombinant protein of claim 8, wherein the ADA is ADA1 or ADA2.

10. A pharmaceutical composition comprising the recombinant protein of claim 8 and a pharmaceutically acceptable carrier.

11. Use of the engineered cells of claim 1 and / or the recombinant protein of claim 8, said use comprising administering the engineered cells and / or recombinant protein to a patient in an effective amount for the treatment of a disease.

12. The use of claim 11, wherein the treatment includes immunomodulation of T cells.

13. The use described in claim 11, wherein the disease is cancer.

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