introduction methods conclusions - pact pharma, inc....abstract: 4858 pact pharma, 2 corporate...

Introduction Methods

Results

ConclusionsEngineering T cells for adoptive cell therapies (ACT) typically rely on lenti-, retro-, or adeno-associated virus to deliverthe payload sequences. However, for personalized therapies, such as the generation of neoepitope-specific TCR T celltherapies, relying on viral vectors is not reasonable for just-in-time manufacturing timelines. PACT Pharma hasdeveloped a highly efficient, DNA-mediated (non-viral) proprietary precision genome engineering approach thatsimultaneously abolishes endogenous TCR expression while introducing the sequences encoding the tumor-targetedneoTCR 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 GMPmanufacturing of bespoke neoTCR T cells for clinical testing of personalized adoptive cell therapy for patients with solidtumors. Furthermore, the PACT precision genome engineering technology has also been applied successfully to otherprimary 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 TherapiesKyle 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 engineeringplatform - 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 theendogenous 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 sequencesidentified by PACT’s imPACT isolation technology. Engineered T cells show functional activity against theirrespective targets.

• PACT’s precision genome engineering technology has been translated for just-in-time GMP manufacturing ofbespoke NeoTCR-T cells for clinical testing of personalized adoptive cell therapy for patients with solidtumors.

Figure 7. neoTCR T cells expressing neo12 TCR or F5 (MART1 TCR) showed functionalactivity as measured by antigen-specific (A) IFNγ cytokine secretion, (B) target cell killingand, (C) proliferation. Engineered T cells from healthy and patient donors were found toboth be of the “Younger” Tscm/Tcm phenotype (see poster 3758).

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Knock-in (KI) of PACT neoTCR at native TCR genome locus

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NeoE-specific TCR sequences (see poster 3714) arecloned into homologous recombination (HR) DNAtemplates. These HR templates are used with site-specific nucleases to engineer primary human Tcells. The single-step (non-viral) precision genomeengineering results in the seamless replacement ofthe endogenous TCR with the patient’s neoE-specific TCR (of native sequence), whose expressionis 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 dayspost-modification. (B) T cells were cultured with tumor cells expressing ZsGreen and thespecific neoantigen (neo12) and HLA-A02 complex. At baseline, edited (red) and non-editedT cells (grey) were round and smaller in size than tumor cells (green). After encounteringantigen-expressing tumor cells, neoTCR T cells became elongated, formed immunologicalsynapses and killed the target tumor cell. The non-edited T cells did not show any cytotoxicactivity. Images were taken at 1 hour intervals.

Figure 4. (A) The intentional disruption of endoTCR expression removes potential for CD3 subunitcompetition in the intracellular TCR assembly process, thus reconstituting surface neoTCR expression atnative levels. (B) Consistent expression of engineered TCRs was observed regardless of TCR identity. (C) Tcells assayed for NeoTCR expression by dextramer staining showed similar rates of editing & TCRexpression levels 10d or 27d post engineering.

Figure 2. Crosslinking, ligation, and use of primers specific to the NeoTCR insertwere used to obtain sequences around the site(s) of integration by TargetedLocus Amplification (TLA). The reads mapped to the genome are binned in 10 kbintervals. Significant read depths were obtained only around the targetedintegration site (on chromosome 14), with no significant evidence of off-targetgenomic insertion by this analysis.

Figure 8. (A) Hematopoietic Stem Cells (HSCs) were engineered using a ZsGreen cassette driven bythe 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 proliferativeand multi-lineage capacity in a methylcellulose colony forming cell assay.

Figure 9. Natural Killer (NK) cells were engineered using the same ZsGreen expression cassetteshown in Figure 8a. (A) In-out PCR, schematized in Figure 8b, confirmed site-specific, preciseintegration of the cassette. Engineered T cells were used for the positive and negative controls usingTCR-specific and ZsGreen-specific primers. (B) High levels of ZsGreen expression was observed in asignificant fraction of the CD3-/CD5-/CD56+ engineered cell population 11 days post-modification,.

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Figure 3. (A) Antibody staining for endogenous TCR and peptide-HLA staining for neoTCR reveals that theengineering results in high frequency knock-in of the NeoTCR, with some TCR- cells and few WT T cellsremaining. 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 fromcancer patients showed similar efficiencies to healthy donors (data not shown).

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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 areengineered with site-specific nucleases in a single-step transfection process toyield efficient, targeted replacement of the endogenous TCR with thetherapeutic neoTCR sequences. In this way, the expression of the endogenousTCR is abolished, ensuring natural expression and regulation of the insertedneoTCR.

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Engineered NeoTCR-P1 T cells display potent antigen-specific activity

Non-Viral precision genome engineering replaces the endogenous TCR with a therapeutic neoTCR

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Engineered HSCs maintain multi-lineage potential

Natural killer cells can be efficiently engineered