Evolution by innovation as a driving force to improve TCR-T therapies

Adoptive cell therapies continually evolve through science-based innovation. Specialized innovations for TCR-T therapies are described here that are embedded in an End-to-End Platform for TCR-T Therapy Development which aims to provide solutions for key unmet patient needs by addressing challenges of TCR-T therapy, including selection of target antigens and suitable T cell receptors, generation of TCR-T therapies that provide long term, durable efficacy and safety and development of efficient and scalable production of patient-specific (personalized) TCR-T therapy for solid tumors. Multiple, combinable, innovative technologies are used in a systematic and sequential manner in the development of TCR-T therapies. One group of technologies encompasses product enhancements that enable TCR-T therapies to be safer, more specific and more effective. The second group of technologies addresses development optimization that supports discovery and development processes for TCR-T therapies to be performed more quickly, with higher quality and greater efficiency. Each module incorporates innovations layered onto basic technologies common to the field of immunology. An active approach of “evolution by innovation” supports the overall goal to develop best-in-class TCR-T therapies for treatment of patients with solid cancer

GENETIC CONTROL OF CELL-MEDIATED LYMPHOLYSIS IN MOUSE

H-2 congenic mouse strains were tested in vitro to investigate the genetic control of cell-mediated lympholysis (CML). Combinations were selected such that differences in various segments of H-2 could be examined for their ability to stimulate production of effector cells and to serve as targets for lysis. Particular emphasis was directed towards understanding the roles of LD and SD. SD-region differences are important in the sensitization of effector cells and they also function as strong targets for lysis, or as markers for the CML targets. LD differences are also important for sensitization of cytotoxic effector cells, but they serve only as very weak targets for lysis. Collaboration occurs between LD and SD in generation of CML. The nature of this interaction can be of two types: together LD and SD can produce CML which neither difference alone can stimulate; LD can enhance a CML response stimulated by SD-region differences alone

NOD/scid IL-2Rgnull mice: a preclinical model system to evaluate human dendritic cell-based vaccine strategies in vivo

<p>Abstract</p> <p>Background</p> <p>To date very few systems have been described for preclinical investigations of human cellular therapeutics <it>in vivo</it>. However, the ability to carry out comparisons of new cellular vaccines <it>in vivo </it>would be of substantial interest for design of clinical studies. Here we describe a humanized mouse model to assess the efficacy of various human dendritic cell (DC) preparations. Two reconstitution regimes of NOD/scid IL2Rg^null(NSG) mice with adult human peripheral blood mononuclear cells (PBMC) were evaluated for engraftment using 4-week and 9-week schedules. This led to selection of a simple and rapid protocol for engraftment and vaccine evaluation that encompassed 4 weeks.</p> <p>Methods</p> <p>NSG recipients of human PBMC were engrafted over 14 days and then vaccinated twice with autologous DC via intravenous injection. Three DC vaccine formulations were compared that varied generation time <it>in vitro </it>(3 days versus 7 days) and signals for maturation (with or without Toll-like receptor (TLR)3 and TLR7/8 agonists) using MART-1 as a surrogate antigen, by electroporating mature DC with <it>in vitro </it>transcribed RNA encoding full length protein. After two weekly vaccinations, the splenocyte populations containing human lymphocytes were recovered 7 days later and assessed for MART-1-specific immune responses using MHC-multimer-binding assays and functional assessment of specific killing of melanoma tumor cell lines.</p> <p>Results</p> <p>Human monocyte-derived DC generated <it>in vitro </it>in 3 days induced better MART-1-specific immune responses in the autologous donor T cells present in the humanized NSG mice. Moreover, consistent with our <it>in vitro </it>observations, vaccination using mature DC activated with TLR3 and TLR7/8 agonists resulted in enhanced immune responses <it>in vivo</it>. These findings led to a ranking of the DC vaccine effects <it>in vivo </it>that reflected the hierarchy previously found for these mature DC variations <it>in vitro</it>.</p> <p>Conclusions</p> <p>This humanized mouse model system enables comparisons among different DC vaccine types to be rapidly assessed <it>in vivo</it>. In addition, <it>ex vivo </it>analyses of human CD3⁺T cells recovered from the spleens of these mice are also possible, including studies on lymphocyte subsets, Th1/Th2 polarization, presence of regulatory T cells and the impact of DC vaccination on their functions.</p

A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma

We present a generic dendritic cell (DC) vaccine strategy for patients with renal cell carcinoma (RCC) based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs. Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs) specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naïve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP)-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease

Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

<p>Abstract</p> <p>Background</p> <p>Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) <it>in vitro</it>. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.</p> <p>Methods</p> <p>In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.</p> <p>Results</p> <p>Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of <it>in vitro </it>transcribed (<it>ivt</it>)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of <it>ivt</it>RNA.</p> <p>Conclusions</p> <p>This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or <it>ivt</it>RNA as sources of specific antigen.</p

Selective Bispecific T Cell Recruiting Antibody and Antitumor Activity of Adoptive T Cell Transfer

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. Methods: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40+ and EpCAM+). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met+ human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase+ melanoma cells by TCR-, as well as of CEA+ colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of AC