Abstract 2557: Assessing the therapeutic efficacy of disease-specific T-cell biofactories

Abstract

Current protein-based cancer therapies have several disadvantages, which include general toxicity, lack of response at the administered dose and differing responses from patients to patient. To circumvent these issues, we propose a platform in which engineered T-cells specifically recognize tumors and secrete therapeutic peptides directly at the disease site leading to targeted cancer cell killing. This approach will increase the specificity of the therapy and decrease toxicity to healthy cells. We transformed acute T-cell lymphoma cells into biofactories for site-specific synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. The effector T-cell line was engineered by stably introducing a chimeric T-cell receptor to recognize Folate-Receptor alpha (FRα) or Mesothelin (MSLN) protein. Upon binding of the effector T-cells to the receptor, an intracellular cascade directed expression of non-human proteins is induced. Specific T-cell binding to human ovarian cancer cell lines and signaling was measured by in vitro co-culture using luciferase production as a surrogate for therapeutic peptide secretion. We demonstrated that T-cells can be genetically programed to synthesize and secrete proportionate amounts of engineered proteins upon engaging the tumor-associated antigens (FRα or MSLN) on a human ovarian cancer cell line, OVCAR3 (FRα+MSLN+). A FRα-MSLN- ovarian cancer cell line, A2780cis, was used as the non-targeted negative control. The difference in protein secretion following stimulation by the two cell lines, as measured by luciferase activity, was statistically significant within 1 hour. It reached ~35-fold within 1 to 3 days, and we observed stable expression for at least 10 days. The luminescent signal was proportionate to the number of OVCAR3 cells. To further validate the specificity of target engagement, we generated A2780cis-FRα positive and A2780cis-MSLN positive cell lines and demonstrated selective binding and activation of the corresponding effector cells in co-culture assays. No binding was detected to the A2780cis-vector control cells. In vivo results for T-cell biofactories targeting OVCAR3 tumors 24 hours post-stimulation validated the in vitro results. Currently, we are engineering T-cell biofactories to release cytotoxic peptides and are assessing their therapeutic efficacy against cancer cells in vitro using co-culture assays and supernatant transfer. Our results show that T-cells can be genetically reprogrammed to serve as biofactories for the synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. Importantly, these studies demonstrate the feasibility of developing the next generation of adoptively transferred T-cell therapies to target tumors that express FRα (e.g., ovarian, breast, lung) and/or MSLN (e.g. ovarian, lung, pancreatic) on their cell surfaces for cancer therapy. Citation Format: Claire E. Repellin, Puja Patel, Lucia Beviglia, Harold Javitz, Lidia C. Sambucetti, Parijat Bhatnagar. Assessing the therapeutic efficacy of disease-specific T-cell biofactories [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 2557.

Lentivirus Manufacturing Process for Primary T‐Cell Biofactory Production

“…A unified protocol was finally generated and the results are presented in Figure 2K. A ≈4.8‐fold increase in the titer over a generally reported protocol, also used in our previous work, [ 6 ] was observed. To demonstrate the scalability of the process, which will also be relevant in phase I/II trials, we translated the lentivirus production process to T175 flask.…”

“…As such, options for many cell‐based diseases (e.g., cancers, viral infections, autoimmune disorders) that evade the immune system are limited. To address this challenge, we recently reported [ 6 ] on a cell‐based living vector that, upon interacting with the antigen‐presenting target cells, synthesizes proportionate amounts of desired proteins in situ. While the transformation and subsequent stabilization is realizable with immortalized cell lines, this is a challenge with primary cells that have limited lifespan.…”

“…Effect of A) producer cell seeding densities; B) types of producer cell lines; C) DNA quantities used for the transfection; D) types of transfection reagents; E) types of cell growth surfaces; F) types of supernatant clarification processes; G) supernatant harvesting times; H) sodium butyrate (HDAC inhibitor); I) 2AP (protein kinase R inhibitor); and J) pAdV and its combination with 2AP. A unified protocol, based on the favorable values for the above factors, was generated and compared (K) with the generally reported protocol, that has also been used by us (see Repellin et al [ 6 ] ). L) The process was scaled‐up from 100 mm plate (55 cm 2 ) to 175 cm 2 flask.…”

“…Figure 3 illustrates the application of two different lentivirus vectors (Figure 3A) for generating two different effector cells, FRα‐CAR T cells and FRα‐specific T‐cell biofactory cells, both of which can also be redirected to other cell‐surface antigens and has been shown in our previous work. [ 6,29 ] The lentivirus vectors, shown in Figure 3A(i), were first used to understand transduction in immortalized cell lines (HEK293T cells, Jurkat clone E6‐1 cells). This has been presented in Figure 3B and was instrumental in developing the transduction procedure for primary T cells.…”

“…The S/N was found to be favorable at lower effector‐to‐target cell ratios. Based on the detailed experiments conducted with A2780cis and described previously, [ 6,33 ] we are able to rule out allorecognition as the major contributor of the engineered T‐cell activities.…”