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  • br with the CD z containing

    2020-07-04

    
    with the CD3z-containing NKG2D CAR, NK Pertussis Toxin expressing the DAP12-containing NKG2D CAR provided an enhanced in vitro cyto-toxicity and increased IFNg release. Thus, CAR design with DAP12 signaling domain offers new opportunities to enhance the antitumor activity and specificity of engineered NK cells.
    Almost all the previous studies using NKG2D CARs were pursued with immune effector cells transduced with integrating viral vectors. When applied clinically, permanent DNA CAR expression mediated by integrating viral vectors cannot be simply shut off when severe toxicity associated with cytokine storm or on-target and off-tumor toxicity occurs. VanSeggelen et al.37 have reported a strain- and pre-conditioning-dependent lethal toxicity in mice treated with T cells transduced with retroviral vectors encoding NKG2D DNA CARs, especially when a second-generation NKG2D CAR (NKG2D28z) and a NKG2D CAR co-expressed with DAP10 (NKG2Dz10) were used.38 CARs can also be expressed by transfection through mRNA electroporation. Using short-lived CAR-expressing cells modified with this non-integrating technology, the duration and potency of CAR effects can be controlled by different dosing and infusion schemes. Understandably, the transient expression of CARs on im-mune cells requires multiple infusions to achieve antitumor effects. It, however, provides good opportunity to test CAR clinical safety. Through discontinuing the infusion of mRNA CAR-modified im-mune effector cells, an excessive response caused by the toxicity related to recognition of normal tissues and/or cytokine storms can be stopped. Moreover, transfection of mRNA encoding a CAR is more economical in testing new CARs, particularly when performing an initial clinical trial of a novel CAR. There were two previous studies
    that assessed in vitro effects of NKG2D CAR mRNA transfected ab T cells and NK cells, respectively,27,29 but the impact of these RNA
    CARs has not been evaluated previously in in vivo models. Expression of the NKG2Dp RNA CAR in the current study significantly augmented the tumoricidal activity of NK cells against several solid tumor cell lines in vitro and displayed significant anti-tumor effects in a mouse model with established tumors.
    After confirming the effectiveness of NKG2D ligand-targeting mRNA CAR NK cells in vitro and in animal studies, we initiated a pilot clin-ical trial (NCT03415100) based on the hypothesis that the local treat-ment of metastatic colorectal cancer by intraperitoneal infusion and intratumoral injection of the CAR-NK cells Pertussis Toxin would be safe and effec-tive. This clinical trial was performed with three vital safety consider-ations: (1) human primary NK cells were used as immune effector cells for CAR therapy. Given the limited lifespan of the cytotoxic cells, especially when haploidentical NK cells were used in patients,5 there will be little concern about persistent CAR-associated side effects; (2) using a non-integrating mRNA electroporation technology for CAR gene transfection further ensures the transient nature of CAR expres-sion; and (3) local treatment instead of systemic delivery of CAR NK cells was performed to limit systemic exposure to CAR cells.
    In the current study, clinical-grade CAR-NK cell products that met release criteria were successfully manufactured for all three patients.
    Molecular Therapy
    Table 3. Summary of Clinical Characteristics of Three Patients Treated with NKG2Dp CAR-NK Cells
    CT/PET in
    Subject ID
    Age/Sex
    Disease
    Prior Therapies
    CAR-NK Treatment
    GVHD
    Target Sites
    Status
    sigmoid colon cancer + extensive
    2017: 3 cycles of oxaliplatin and capecitabine (XELOX)
    liver metastases, bone and lung
    SD
    DOD
    metastasis + peritoneal
    carcinomatosis + malignant ascites
    2017: paclitaxel liposome chemotherapy
    2014: 8 cycles of irinotecan +fluorouracil+cetuximab
    sigmoid colon cancer + extensive
    2014: surgical resection of liver metastases
    liver metastases + peritoneal
    negative
    SD
    DOD
    2015: 4 cycles of irinotecan+fluorouracil +cetuximab
    carcinomatosis + malignant ascites
    2017: surgical resection of metastases in right testicle
    and sigmoid colostomy
    rectal cancer + subcapsular
    2017: 2 cycles of oxaliplatin+capecitabine
    negative
    CMR/SD
    DOD
    liver metastases
    local injections
    2017: 1 cycle of irinotecan+fluorouracil
    2018: 3 cycles of irinotecan+fluorouracil +bevacizumab
    M, male; F, female; i.p., intraperitoneal; SD, stable disease in target site; CMR, complete metabolic response; DOD, death of disease.
    This technical implantation success rate is the most important feasi-bility endpoint. However, due to transient CAR expression feature, we were unable to evaluate the in vivo fate of adoptively transferred CAR-NK cells. All three patients met the safety endpoint, as the CAR-NK cells were well tolerated with no serious adverse events, including high-grade cytokine release syndrome. Multiple dose levels of CAR-NK cells were tested, but no dose-limiting toxicities occurred.