CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Fibroblasts Impair Migration and Antitumor Activity of NK-92 Lymphocytes in a Melanoma-on-Chip Model.

Adoptive cell therapy in solid tumors, such as melanoma, is impaired, but little is known about the role that the fibroblasts present in the tumor microenvironment could exert. However, the mechanism at play is not well understood, partly due to the lack of relevant pre-clinical models. Three-dimensional culture and microfluidic chips are used to recapitulate the dynamic interactions among different types of cells in the tumor microenvironment in controlled and physiological settings. In this brief report, we propose a reductionist melanoma-on-a-chip model for evaluating the essential role of fibroblasts in the antitumor activity of lymphocytes. To this end, 3D melanoma spheroids were monocultured and co-cultured with human dermal fibroblasts and the NK-92 cell migration towards the tumor compartment was tested in a commercially available microfluidic device. Utilizing confocal microscopy, we observed the different recruitment of NK-92 cells in the presence and absence of fibroblasts. Our results show that fibroblasts' presence inhibits immune effector recruiting by exploiting a 3D pre-clinical tumor model.

Recruitment, Infiltration, and Cytotoxicity of HLA-Independent Killer Lymphocytes in Three-Dimensional Melanoma Models.

Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma.

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae.

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

PURPOSE: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. EXPERIMENTAL DESIGN: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. RESULTS: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. CONCLUSIONS: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.