Dual CAR-T cells to treat cancers co-expressing NKG2D and PD1 ligands in xenograft models of peritoneal metastasis.

While the expression of either NKG2D ligands or PD-1 ligands has been reported in various types of cancers, the co-expression of the two sets of ligands in the same tumour tissues is still un-investigated. After examining 68 primary ovarian cancer samples, we observed around 80% of the co-expression in low grade serous and endometrioid ovarian cancer samples. We then constructed a dual CAR system that splits the conventional single-input of a 2nd generation CAR into two independent chimeric receptors, one composed of the NKG2D extracellular domain linked with DAP12 for T cell activation and another using the PD-1 extracellular domain linked with 4-1BB for costimulatory signal 2 input. Given the limitation of the low-affinity PD-1 receptor in recognizing cancer cells with low levels of PD-1 ligands, we also used a high-affinity scFv specific to PD-L1 in our combinatorial approach to expand the range of target cancer cells with different expression levels of PD-L1. The two types of dual CAR-T cells were generated through electroporation of non-viral piggyBac transposon plasmids and were effective in eliminating the target cancer cells. Especially, the dual CAR-T cells with anti-PD-L1 scFv were capable of eradicating established tumors in mouse models of peritoneal metastasis of colorectal cancer and ovarian cancer. Since both NKG2D ligands and PD-1 ligands have been marked as favourable cancer therapeutic targets, the new dual CAR-T cells developed in this study hold attractive application potential in treating metastatic peritoneal carcinoma.

T Cells Expressing NKG2D CAR with a DAP12 Signaling Domain Stimulate Lower Cytokine Production While Effective in Tumor Eradication.

Cytokine-related toxicity associated with the use of highly active chimeric antigen receptor T cells (CAR-T cells) is a significant clinical problem. By fusing the natural killer group 2D (NKG2D) ectodomain to 4-1BB and the DAP12 cytoplasmic domain containing only one immunoreceptor tyrosine-based activation motif, we have developed a 2nd-generation (2nd-Gen) NKG2D CAR for stable expression in human T cells. When compared to T cells modified with NKG2D CAR containing the commonly used CD3ζ activation domain, T cells expressing the NKG2D-DAP12 CAR stimulated lower level release of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-2 during tumor cell lysis and their proliferative activity was lower upon repeated antigen stimulation, although no difference between the two CARs was observed in mediating in vitro tumor cell lysis. In tumor-bearing NSG mice, both types of CAR-T cells displayed similar anti-tumor activity, being able to completely eradicate established solid tumor xenografts. However, treatment with the NKG2D-CD3ζ CAR-T cells led to the death of most mice from xenogeneic graft versus host disease starting 30 days post-CAR-T cell injection, which was associated with a higher level of cytokine release, whereas all the mice treated with the NKG2D-DAP12 CAR-T cells survived well. Thus, the incorporation of the DAP12 activation domain in a CAR design may possibly provide a potential clinical advantage in mitigating the risk of cytokine release syndrome (CRS).

T cells with split CARs specific for NKG2D ligands and PD-L1 exhibit improved selectivity towards monocyte-derived cells while effective in eliminating acute myeloid leukaemia in vivo.

PURPOSE: The expression of NKG2D ligands and PD-L1 has been detected on acute myeloid leukaemia (AML) cells, as well as normal cells of the myeloid lineage. To target leukemic cells while minimizing collateral damage to normal cells, we constructed a split dual CAR system based on the AND-gate logic. METHODS: The NKG2D extracellular domain linked with DAP12 without a co-stimulatory signal was used for the basal activation of T cells, and used together with the PD-L1-specific chimeric costimulatory receptor containing the 4-1BB activating domain for co-stimulatory signal 2 input. This dual CAR displayed cell-type specificity and activity similar as a 2nd generation NKG2D ligand-specific CAR. RESULTS: When compared to CD64 and PD-L1-specific 2nd generation CARs, we observed that the split dual CAR offered an improved myeloid cell type selectivity. For example, PD-L1-specific CAR-T cells lysed all tested myeloid cell types that expressed PD-L1, including M0 macrophages (Mø0), LPS-polarized Mø1, IFN-γ polarized Mø1, IL-4 polarized Mø2, monocytes, immature dendritic cells (imDCs), mature DCs, as well as KG-1 AML cells, while the dual CAR-T cells displaying killing activity only towards LPS polarized Mø1, mature DCs and KG-1 cells that expressed both NKG2D ligands and PD-L1. In a mouse liquid tumor model, the dual CAR-T cells were effective in eradicating established KG-1 AML xenografts. CONCLUSION: The improved cell type specificity offered by our split dual CAR-T cell system targeting paired antigens would favour the reduction of the on-target off-tumor toxicity towards normal myeloid cells during the treatment of myeloid leukaemia.

CXCR4 and anti-BCMA CAR co-modified natural killer cells suppress multiple myeloma progression in a xenograft mouse model.

The highly restricted expression of B-cell maturation antigen (BCMA) on plasma cells makes it an ideal target for chimeric antigen receptor (CAR) immune cell therapy against multiple myeloma (MM), a bone marrow cancer. To improve the infiltration of ex vivo expanded human natural killer (NK) cells into the bone marrow, we electroporated these cells with mRNA encoding the chemokine receptor CXCR4. The CXCR4-modified NK cells displayed increased in vitro migration toward the bone marrow niche-expressing chemokine CXCL12/SDF-1α and augmented infiltration into the bone marrow compartments in mice. We further modified the CXCR4-NK cells by electroporation of mRNA encoding a CAR targeting BCMA. After the intravenous injection of the double-modified NK cells into a xenograft mouse model of MM, we observed significantly reduced tumor burden in the femur region of the living mice and the extended survival of the tumor-bearing mice. Collectively, this study provides the experimental evidence that the co-expression of CXCR4 and anti-BCMA CAR on NK cells is a possible effective way to control MM progression.

Vγ9Vδ2 T cells expressing a BCMA-Specific chimeric antigen receptor inhibit multiple myeloma xenograft growth.

Vγ9Vδ2 T cells are immune effector cells capable of killing multiple myeloma (MM) cells and have been tested in clinical trials to treat MM patients. To enhance the MM cell killing function of Vγ9Vδ2 T cells, we introduced a BCMA-specific CAR into ex vivo expanded Vγ9Vδ2 T cells through electroporation of the CAR-encoding mRNA. The modified Vγ9Vδ2 T cells displayed a high cytolytic activity against BCMA-expressing MM cell lines in vitro, while sparing BCMA-negative cells, including normal B cells and monocytes. Subsequently, we intravenously injected KMS-11 human MM cells to generate a xenograft mouse model. The treatment of the tumor-bearing mice with Zometa and anti-BCMA CAR- Vγ9Vδ2 T cells resulted in a significant reduction of tumor burden in the femur region, as well as the overall tumor burden. In association with the decrease in tumor burden, the survival of the MM cell-inoculated mice was markedly prolonged. Considering the potential of Vγ9Vδ2 T cells to be used as off-the-shelf products, the modification of these cells with a BCMA-specific CAR could be an attractive option for cancer immunotherapy against bone marrow cancer MM.

A CD123-specific chimeric antigen receptor augments anti-acute myeloid leukemia activity of Vγ9Vδ2 T cells.

Aim: To investigate whether anti-CD123 chimeric antigen receptor (CAR)-expressing Vγ9Vδ2 T cells could be an alternative for acute myeloid leukemia (AML) treatment. Materials & methods:Ex vivo expanded Vγ9Vδ2 T cells were electroporated with anti-CD123 CAR-encoding mRNA. The effector function and specificity of the modified Vγ9Vδ2 T cells were examined by in vitro cytotoxicity, degranulation and cytokine release level. The in vivo function was analyzed using the xenograft KG1-luc model with NOD-SCID-γc-/- mice. Results: The modified Vγ9Vδ2 T cells exhibited significantly improved effector activities against both AML cell lines and primary AML cells in vitro. In the xenograft mouse model, the modified Vγ9Vδ2 cells displayed an enhanced tumor control potency. Conclusion: Anti-CD123 CAR-expressing Vγ9Vδ2 T cells may serve as an alternative way to target AML.

piggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells.

Promising progress has been made in adoptive transfer of allogeneic natural killer (NK) cells to treat relapsed or refractory acute myeloid leukemia (AML). In this regard, chimeric antigen receptor (CAR)-modification of NK cells is considered as a compelling approach to augment the specificity and cytotoxicity of NK cells against AML. Using a non-viral piggyBac transposon technology and human peripheral blood-derived primary NK cells, we generated CAR-NK cells to target NKG2D ligands and demonstrated their in vitro activity in lysing cancer cells expressing the ligands and in vivo efficacy in inhibiting tumor growth in a xenograft KG-1 AML model. We further generated CAR-NK cells co-expressing transgenes for the NKG2D CAR and interleukin-15 (IL-15). The ectopic expression of IL-15 improved the in vitro and in vivo persistence of NKG2D CAR-NK cells, leading to enhanced in vivo tumor control and significant prolongation of mouse survival in the KG-1 AML model. Collectively, our findings demonstrate the ectopic expression of IL-15 as an important means to improve the antileukemic activity of NKG2D CAR-NK cells. Our study further illustrates the feasibility of using the piggyBac non-viral platform as an efficient and cost-effective way for CAR-NK cell manufacturing.

Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells.

Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the in vitro anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions.

CXCR1 Expression to Improve Anti-Cancer Efficacy of Intravenously Injected CAR-NK Cells in Mice with Peritoneal Xenografts.

One reason underlying the failure of current chimeric antigen receptor (CAR) immune therapy to treat solid tumors adequately is insufficient tumor infiltration of CAR immune cells. To address the issue, we electroporated natural killer (NK) cells with two mRNA constructs encoding the chemokine receptor CXCR1 and a CAR targeting tumor-associated NKG2D ligands. The CXCR1-modified NK cells displayed increased migration toward tumor supernatants in vitro and augmented infiltration into human tumors in vivo in subcutaneous and intraperitoneal xenograft models. Most importantly, the cytotoxicity of the CAR-NK cells was not affected by CXCR1 transgene expression, and the enhanced tumor trafficking following intravenous injection resulted in significantly increased antitumor responses in mice carrying established peritoneal ovarian cancer xenografts. Collectively, our findings suggest that the coexpression of CXCR1 and a CAR may provide a novel strategy to enhance therapeutic efficacy of NK cells against solid cancers.

Experimental treatment of colorectal cancer in mice with human T cells electroporated with NKG2D RNA CAR.

Aim: Peritoneal metastasis is often present in end-stage neoplastic diseases, including recurrent colorectal cancer and is associated with decreased overall survival. Novel methods are needed. Materials & methods: We constructed first-, second- and third-generation chimeric antigen receptors (CARs) specific for NKG2D ligands and modified human T cells with mRNA electroporation. Results: NKG2D CAR expression was detectable for at least 6 days postelectroporation and mediated efficient cytotoxicity against NKG2DL+ tumor cells, but not NKG2DL-cells. Multiple infusions of the first-generation CAR-T cells into immunodeficient mice bearing established peritoneal colorectal xenografts led to significantly reduced tumor burden. Conclusion: mRNA CAR is an economical way to test new CARs and potentiates controlling on-target/off-tumor toxicity and cytokine storms. The use of NKG2D RNA CARs to treat colorectal peritoneal metastasis warrants further investigation.

Electroporation of NKG2D RNA CAR Improves Vγ9Vδ2 T Cell Responses against Human Solid Tumor Xenografts.

Vγ9Vδ2 T cell-based anticancer immunotherapy has shown some promise in early-phase clinical trials but there is still large room for improvement. Using the extracellular domain of the human NKG2D, a stimulatory receptor expressed by Vγ9Vδ2 T cells, we constructed NKG2D ligand-specific chimeric antigen receptors (CARs). We adopted a non-viral CAR approach via mRNA electroporation to modify Vγ9Vδ2 T cells and demonstrated that, upon interaction with the NKG2D ligand-positive cancer cells, the CARs substantially enhanced the cytotoxic activity of the modified cells toward multiple cultured solid tumor cell lines, including those resistant to Zometa treatment. Repeated doses of the CAR-expressing cells resulted in tumor regression in mice with established tumors, extending median survival time by up to 132% as compared to the PBS control group. The findings suggest clinical potential for RNA CAR-modified Vγ9Vδ2 T cells to treat a wide variety of NKG2D ligand-expressing cancers.