<p>All growth curves of first implantation and subsequent expansions with and without continuous BRAF inhibitor (PLX4720) diet. Error bars are SEM.</p>
Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups.
CAR-T cell therapies have proven safe and efficacious for hematologic malignancies, but there remains a significant unmet need for effective cell therapy options for solid tumors. CAR-engineered induced pluripotent stem cell (iPSC)-derived effector cells allow for the treatment of cancer as an off-the-shelf allogeneic cell therapy. Gamma delta (γδ) T cells exhibit the cytolytic features of conventional alpha beta (αβ) CD8+ T cells with additional capabilities for innate recognition of tumors. For example, expression of CD16 on γδ T cells can mediate antibody-dependent cellular cytotoxicity (ADCC) against tumors. Here we describe development of an iPSC-derived CAR γδ T cell platform which can target solid tumors through both CAR-mediated recognition and ADCC when combined with a therapeutic antibody.
Methods
Primary γδ T cells were enriched and expanded in culture to enable reprogramming to iPSCs by delivery of pluripotency genes. These T cell derived iPSCs (TiPSCs) were used to produce γδ T cells using a proprietary differentiation process. The TiPSC line was engineered with a CAR targeting EGFR and a membrane bound form of IL-15 to enhance T cell persistence. Tumor spheroids were generated from EGFR+Her-2+ SKOV-3 ovarian tumor cells. Cytolysis of spheroids was evaluated using CAR-T cells alone or in combination with anti-HER2 antibody (trastuzumab).
Results
Batches of CAR-T cells were generated using a proprietary differentiation process yielding >90% pure CAR+ γδ T cells. The TiPSCs contained the rearranged γδ TCR gene and upon differentiation to T cells, uniformly expressed a Vγ9Vδ2 TCR and expressed high levels of CD16. CAR γδ T cells were effective in killing SKOV-3 spheroids. When cultured with SKOV-3 spheroids in an ADCC assay, CAR γδ T cells exhibited enhanced cytotoxicity in the presence of trastuzumab but not isotype control antibody. Activity of the γδ T cells was not reliant on additional exogenous cytokine due to the engineered form of membrane-associated IL-15.
Conclusions
We have demonstrated that iPSC-derived γδ T cells mediate anti-tumor activity in human solid tumor models through multiple pathways. The combination of two modes of tumor recognition (CAR and CD16/antibody) enabled more potent killing of solid tumor spheroids. The ability to manufacture large batches of iPSC derived CAR γδ T cells will enable a true off-the-shelf allogenic cell therapy for solid tumors.
To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models.We established 12 PDXs from BRAF inhibitor-progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches.We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals.Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. See related commentary by Hartsough and Aplin, p. 1550.
<p>A. heat map of complete RPPA data median centered, all tumor grafts untreated. Data are mean of 3 biological replicates, red is up, green is down. B. Principal component analysis (PCA) of median centered data C. heat map of fold change between untreated tumor grafts and on chronic BRAF inhibitor therapy. D. Principal component analysis (PCA) of fold change data.</p>
