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During manufacturing of an exemplary CD19-directed CAR-T cell product, patient T cells were transduced with a viral vector encoding the CD19-directed, 41BBζ endodomain-containing chimeric antigen receptor (CAR) construct, enabling permanent genetic modification of the target T cells. Lentiviral transduction with a self-inactivating (SIN) vector can offer certain advantages in safety and consistency over other DNA delivery methods. Although the integration patterns of wildtype HIV-1 have been well-characterized and shown to have a low risk of insertional transformation, the addition of an artificial construct may have the potential to affect integration patterns. Existing techniques for viral integration mapping, such as ligation-mediated polymerase chain reaction (LM-PCR), have limitations precluding comprehensive, unbiased, high-throughput insertion site mapping. To characterize insertional events in engineered CAR-T cells, we developed an unbiased NGS assay and computational pipeline capable of characterizing viral integration events genome-wide. The method leverages Tn5 transposase, coupled with a long terminal repeat-specific PCR, to map integration events down to nulceotide resolution. This assay identified over 150,000 high-confidence integration events across 23 patient-derived CAR-T cell products. Analysis of these sites revealed a similar integration preference to lentiviruses described in the literature: driven by GC content, local nucleotide content, chromatin status, and transcriptional activity, with no indication of preferential integration at oncogenic loci. In conclusion, in this assay a CD19-directed, CAR-containing lentivirus exhibited a low risk integration profile with no significant evidence of construct-specific risk factors. Furthermore, the findings suggest that the NGS assay may be useful for more efficient, cost-effective, and sensitive measurement of CAR-T integration events for a range of constructs. Citation Format: Timothy G. Johnstone, Rajagopal Chari, David Koppstein, Ronald J. Hause, Rafael Ponce, Adrian W. Briggs, Francois Vigneault. Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1541.

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells that have metastasized. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative viral replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. Recently, in a retrospective analysis of a Phase III melanoma trial investigators found that about two-thirds of the lesions injected with talimogene laherparepvec shrank 50% or more. And the same effect was seen in about a third of all uninjected tumors in the skin and lymph nodes and about a sixth of uninjected visceral lesions providing an indication that the treatment is triggering the desired immune system effect. Ongoing clinical trials are investigating T-VEC in combination with the immune checkpoint inhibiting antibodies ipilimumab and pembrolizumab in advanced melanoma. We sought to determine if the combination of intratumoral injection of talimogene laherparepvec and the systemic delivery of the checkpoint anti-PD1 antibody could increase the anti-tumor efficacy in a preclinical syngeneic contralateral tumor model. Using established MC-38 colon carcinoma tumors in C57Bl/6 mice, we delivered three intratumoral injections three days apart of the talimogene laherparepvec surrogate OncoVEXmuGM-CSF and twice weekly systemic injections of an antagonistic anti-PD-1 antibody. Either OncoVEXmuGM-CSF or anti-PD-1 alone induced modest tumor growth inhibition/tumor regressions of contralateral tumors. In combination, the OncoVEXmuGM-CSF injected tumor displayed 8/10 complete regressions and the distant tumors had 2/10 complete regressions. Peripheral blood was analyzed at 4 days and 10 days post the initial OncoVEXmuGM-CSF and anti-PD-1 antibody injections. OncoVEXmuGM-CSF increased the percent of PD-L1+ CD4 and CD8 T cells and the combination increased the percent of activated CD8+ T cells. Our findings demonstrate that localized therapy with talimogene laherparepvec augments the systemic anti-tumor immune response seen with anti-PD-1 therapy, providing a strong rationale for continued investigation of such combinations in the clinic. Citation Format: Julia Piasecki, Tiep le, Rafael Ponce, Courtney Beers. Talilmogene laherparepvec increases the anti-tumor efficacy of the anti-PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 258. doi:10.1158/1538-7445.AM2015-258

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) immunotherapy designed to selectively replicate in tumors and to initiate an anti-tumor immune response. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. The talimogene laherparepvec-induced immune mediated mechanism of action in both the virus-injected and distant tumors have yet to be fully understood therefore, we evaluated viral replication, tumor cell lysis, and the changes in immune cell populations in both the injected and distant tumors using syngeneic contralateral tumor models. Animals received a single intratumoral dose of the murine surrogate of talimogene laherparepvec (OncoVEXmuGM-CSF), which induced regression in the majority of virus-injected tumors and tumor growth inhibition/regression in the contralateral (uninjected) tumors. HSV-1 antigen was only detected by IHC in the virus-injected tumor and not the contralateral tumor (and no other tissues). Virally-mediated tumor destruction (oncolysis) was also localized to only the virus-injected tumor. In preliminary experiments, morphometric analysis of the tumor tissue revealed that the percent area of CD3+ and CD8+ lymphocytes were significantly increased in both the virus-injected and contralateral tumors compared to the formulation control mice. In addition, the percent area occupied by CD103+ cells (a marker found on potent cytotoxic T cell stimulating dendritic cells) was increased in the virus-injected tumor compared to the contralateral tumor and the tumors in the control animals. Although cellular infiltration was increased in both virus-injected and contralateral tumors and was inversely correlated with tumor volume, the decreased volume of injected tumors was attributed to viral oncolysis. Taken together these data supports that talimogene laheparepvec activates a systemic T cell mediated anti-tumor immune response. Citation Format: Julia Piasecki, Jim Rottman, Tiep Le, Rafael Ponce, Courtney Beers. Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2015-4287