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Introduction Methods Results Conclusions Engineering T cells for adoptive cell therapies (ACT) typically rely on lenti-, retro-, or adeno-associated virus to deliver the payload sequences. However, for personalized therapies, such as the generation of neoepitope-specific TCR T cell therapies, relying on viral vectors is not reasonable for just-in-time manufacturing timelines. PACT Pharma has developed a highly efficient, DNA-mediated (non-viral) proprietary precision genome engineering approach that simultaneously abolishes endogenous TCR expression while introducing the sequences encoding the tumor-targeted neoTCR to yield ‘natural’ patient-specific tumor mutation-targeted T cells for testing and infusion back to patients. PACT’s precision genome engineering technology has been translated into highly efficient, single-step, just-in-time GMP manufacturing of bespoke neoTCR T cells for clinical testing of personalized adoptive cell therapy for patients with solid tumors. Furthermore, the PACT precision genome engineering technology has also been applied successfully to other primary human cell types, including natural killer and hematopoietic stem cells. Highly Efficient, Non-Viral Precision Genome Engineering for the Generation of Personalized NeoEpitope-Specific Adoptive T cell Therapies Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam Litterman, Fabrizia Urbinati, Susan Foy, Theresa Hunter, Albert Tai, Michael Bethune, Songming Peng, Olivier Dalmas, Alex Franzusoff, Stefanie Mandl PACT Pharma, 2 Corporate Drive, South San Francisco, CA 94080, USA. Abstract: 4858 • PACT has developed a highly efficient, site-specific, DNA-mediated non-viral precision genome engineering platform - applicable to multiple primary human cell types, including T cells, HSCs and NKs. • PACT engineered T cells display patient-specific neoTCRs on the cell surface at native levels under the endogenous promoter, and of native sequence. The expression does not diminish over time. • Using PACT’s approach, patient T cells are engineered to target neoepitopes using neoTCR sequences identified by PACT’s imPACT isolation technology. Engineered T cells show functional activity against their respective targets. • PACT’s precision genome engineering technology has been translated for just-in-time GMP manufacturing of bespoke NeoTCR-T cells for clinical testing of personalized adoptive cell therapy for patients with solid tumors. Figure 7. neoTCR T cells expressing neo12 TCR or F5 (MART1 TCR) showed functional activity as measured by antigen-specific (A) IFNγ cytokine secretion, (B) target cell killing and, (C) proliferation. Engineered T cells from healthy and patient donors were found to both be of the “Younger” Tscm/Tcm phenotype (see poster 3758). 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 % kill M N 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 % diving cells M N Neo12 T cells F5 T cells Peptide (nM) Peptide (nM) Target cell killing T cell proliferation 0.01 0.1 1 10 100 1000 0 4000 8000 12000 16000 IFN Peptide (nM) IFN (pg/mL) M N A B C → 1 2 (+) control (-) control (-) H2O control Brightfield Fluorescence BFU-E (erythroid) CFU-GM (myeloid) Knock-out (KO) of endogenous TCR (Endo-TCR) Knock-in (KI) of PACT neoTCR at native TCR genome locus L 1 (-) DNA Only → MND ZsGreen ZsGreen CD56 34% 68 Hrs 20 Hrs 0 Hrs NeoE-specific TCR sequences (see poster 3714) are cloned into homologous recombination (HR) DNA templates. These HR templates are used with site- specific nucleases to engineer primary human T cells. The single-step (non-viral) precision genome engineering results in the seamless replacement of the endogenous TCR with the patient’s neoE- specific TCR (of native sequence), whose expression is under endogenous regulation. Figure 5. Cells modified to express mCherry with the neoTCR were monitored using time- lapse fluorescence microscopy. (A) Cells showed high levels of mCherry expression 2-3 days post-modification. (B) T cells were cultured with tumor cells expressing ZsGreen and the specific neoantigen (neo12) and HLA-A02 complex. At baseline, edited (red) and non-edited T cells (grey) were round and smaller in size than tumor cells (green). After encountering antigen-expressing tumor cells, neoTCR T cells became elongated, formed immunological synapses and killed the target tumor cell. The non-edited T cells did not show any cytotoxic activity. Images were taken at 1 hour intervals. Figure 4. (A) The intentional disruption of endoTCR expression removes potential for CD3 subunit competition in the intracellular TCR assembly process, thus reconstituting surface neoTCR expression at native levels. (B) Consistent expression of engineered TCRs was observed regardless of TCR identity. (C) T cells assayed for NeoTCR expression by dextramer staining showed similar rates of editing & TCR expression levels 10d or 27d post engineering. Figure 2. Crosslinking, ligation, and use of primers specific to the NeoTCR insert were used to obtain sequences around the site(s) of integration by Targeted Locus Amplification (TLA). The reads mapped to the genome are binned in 10 kb intervals. Significant read depths were obtained only around the targeted integration site (on chromosome 14), with no significant evidence of off-target genomic insertion by this analysis. Figure 8. (A) Hematopoietic Stem Cells (HSCs) were engineered using a ZsGreen cassette driven by the MND promoter. (B) A schematic of in-out PCR is shown, (C) which was used to check for site- specific, precise integration of the cassette. (D) Engineered cells (green) demonstrated proliferative and multi-lineage capacity in a methylcellulose colony forming cell assay. Figure 9. Natural Killer (NK) cells were engineered using the same ZsGreen expression cassette shown in Figure 8a. (A) In-out PCR, schematized in Figure 8b, confirmed site-specific, precise integration of the cassette. Engineered T cells were used for the positive and negative controls using TCR-specific and ZsGreen-specific primers. (B) High levels of ZsGreen expression was observed in a significant fraction of the CD3-/CD5-/CD56+ engineered cell population 11 days post-modification,. # TCR cells in population (normalized) Neo12 MART1 NY-ESO 0 10000 20000 30000 TCR expression/cell (MFI) Endo TCR PACT TCR CD3 expression levels NeoTCR+ NeoTCR+ Day 10 Day 27 NeoTCR peptide-HLA % of Live Cells A B C Figure 3. (A) Antibody staining for endogenous TCR and peptide-HLA staining for neoTCR reveals that the engineering results in high frequency knock-in of the NeoTCR, with some TCR- cells and few WT T cells remaining. Knock-in is evidenced by neoTCR expression in the absence of an exogenous promoter. (B) Engineering was carried out multiple times using the same neoTCR with similar results. T cells from cancer patients showed similar efficiencies to healthy donors (data not shown). 24 75 neoTCR (peptide-HLA) Endo TCR signal TCR– PACT NeoE TCR Mock EndoTCR KO + PACT NEOTCR-P1 Cassette EndoTCR 0 15 30 45 60 75 % of CD8 + Live Cells NeoTCR A B EndoTCR % of CD8+ Live Cells C B Precision genome engineering is highly efficient and consistent TLA confirms targeted integration at only the intended locus Engineered neoTCRs are stably expressed at endogenous levels Figure 1. Genomes of individual primary human CD8 and CD4 T cells are engineered with site-specific nucleases in a single-step transfection process to yield efficient, targeted replacement of the endogenous TCR with the therapeutic neoTCR sequences. In this way, the expression of the endogenous TCR is abolished, ensuring natural expression and regulation of the inserted neoTCR. A A D Engineered T cells rapidly express neoTCR and perform effector function A B Engineered NeoTCR-P1 T cells display potent antigen-specific activity Non-Viral precision genome engineering replaces the endogenous TCR with a therapeutic neoTCR B PACT NeoTCR Knock-in Engineered HSCs maintain multi-lineage potential Natural killer cells can be efficiently engineered
