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The Application of ‘Drug-Reversible’ CAR-T Cells Directed Against Recipient Hematopoietic Cells as a Selective Conditioning Strategy for Stem Cell Transplantation Nina Timberlake 1 , Maximilian Richter 1 , Xinxin Wang 1 , Claire Koechlein 1 , Armenuhi Avanesyan 1 , Nicholas Cam 1 , Yening Tan 1 , Natalie Chen 1 , Kip Hermann 2 , Bruce E. Torbett 2 , Eric M. Ostertag 1 , Devon J. Shedlock 1 and Julian D. Down 1 CONCLUSIONS 1 Poseida Therapeutics Inc., 4242 Campus Point Court, San Diego, CA 92121 2 The Scripps Research Institute, 10550 N. Torrey Pines Rd., San Diego, CA 92037 • PB CAR-T cells targeted against c-kit or CD133 deplete hematopoietic progenitor cells from human and non-human primate bone marrow, and primitive CAFCs from human CD34 + cells. • PB CAR-T cells exhibit a stem cell memory phenotype and naturally express CXCR4, although expression can be increased by 24h culture with added factors. • PB CAR-T cells successfully home to bone marrow within 16 hours after injection. • These data support the use of PB CAR-T cells to target endogenous HSCs in the bone marrow as a minimal, non-genotoxic HSC transplant regimen. Figure 5: Activity of anti-c-kit and CD133 CAR-T cells against long-term cobblestone area forming cells (CAFCs): Following co-culture of the CAR-T cells with human mobilized peripheral blood CD34+ cells for 48h (effector to target cell ratio of 3:1), the cells were plated on MS-5 stromal cells over serial dilutions for the generation of CAFCs over 2 months. At 5 weeks post-plating, both CAR-T cells significantly reduced the absolute number of CAFCs suggesting these CAR-T cells successfully target very primitive cells. Depletion of More Primitive Hematopoietic Cells in Long-Term Stromal Cultures by Both Anti-c-kit and CD133 CAR-Ts The transplant of autologous or allogeneic hematopoietic stem cells (HSCs) has the proven ability to treat a wide array of malignant and non-malignant hematological diseases. The preparative regimen, however, routinely entails aggressive and genotoxic treatment with total body irradiation and/or chemotherapy, which brings severe and even life-threatening complications that limit its broader application. Previous experimental studies have established that depletion of recipient HSCs is an essential requirement of these conditioning regimens in allowing successful engraftment of the composite donor HSCs. Animal and clinical studies have also indicated that alloreactive anti-HSC donor T cells additionally facilitate stem cell engraftment, but this is often accompanied by the risks of graft versus host disease. This has prompted the consideration of alternative conditioning methods for the depletion of HSCs with less toxic side-effects, such as anti-c-kit and anti-CD45 antibody-directed treatments. In this way, more precise HSC targeting may also be achieved by the application of short-lived, genetically engineered chimeric antigen receptor (CAR)-T cells for stem cell transplantation conditioning. We developed a novel and controllable CAR-T approach for recipient HSC targeting via genetic modification using the non-viral piggyBac™ (PB) transposon system. As opposed to viral vector delivery systems, the relatively large carrying capacity of PB allows the stable introduction of at least three separate genes encoded within the same tri-cistronic transgene cassette. This includes a second-generation CAR that targets either human c-kit (CD117) or prominin-1 (CD133), markers known to be antigenically expressed on the surface of HSCs. In addition, a drug resistance element serves as a selection gene that, in combination with a non-genotoxic drug, provides an effective method of CAR-T cell purification during manufacture. Importantly, a small molecule drug-inducible safety switch gene is also included to facilitate rapid in vivo clearance of the CAR-T cells after depletion of recipient HSCs and prior to donor HSC transplant. Lastly, as a result of the manufacturing process, the majority of the CAR-T cells express chemokine receptors such as CXCR4 that can allow more selective trafficking to the bone marrow (BM) for eradication of resident HSCs. To select a lead candidate from a panel of anti-HSC CAR constructs, CD3/CD28-stimulated T cells from human peripheral blood were first electroporated with mRNA encoding each of the CAR candidates directed against either c-kit or CD133. CAR surface expression was confirmed in transfected T cells by flow cytometry. In vitro functional assays were performed by co-culturing mRNA-transfected CAR-T cells with mouse or human cell lines (EML-C1, TF-1 and K562), expressing either c-kit or CD133, as well as mouse and human primary BM cells. Lead CAR candidates were identified from their specific activation of the CAR-T cells through degranulation, measured by CD107a expression and secretion of IFNγ. Furthermore, those CARs were also capable of selectively depleting c-kit or CD133 positive cells. Interestingly, some mRNA-transfected CAR-T cells retained effector activity against target c-kit+ TF-1 cells even in the presence of its soluble ligand, stem cell factor. Next, lead CAR candidates were co-expressed with the selection and drug-inducible safety switch genes in the same tri-cistronic transgene and then stably delivered to T cells using PB. The manufacturing process yielded CAR-T cells that were mainly of the T memory stem cell (Tscm) phenotype, as determined by positive expression of CD62L and CD45RA, and also expressed high levels of the CXCR4 chemokine receptor. Similar to the mRNA-transfected CAR-T cells, these stably-transposed cells were capable of extensive effector capabilities including specific depletion of c-kit or CD133 expressing target cells. Future studies will evaluate PB-produced lead anti-HSC CAR-T cells in immune-deficient NSG mice with pre-established xenogeneic human hematopoietic chimerism, along with standard busulfan or radiation conditioning controls. This approach constitutes a novel targeted biological therapy that is envisaged to lead the way towards minimally toxic transplant regimens for depletion of endogenous HSCs in the BM and to procure their replacement with engrafted allogeneic or gene-corrected stem cells. INTRODUCTION Need for Alternative Conditioning Therapies prior to HSC Transplants • More than 5,000 patients per year in the U.S. are treated with myeloablative conditioning regimens prior to HSC transplants. Most of these conditioning regimens consist of high doses of genotoxic radiation or busulfan. The use of stem cell transplants is limited by the major life-threatening complications associated with these regimens. • Antibodies directed against antigens expressed on HSCs, such as c-kit and CD45, have been considered as alternatives. • CAR-T cells may provide more effective, selective, and safer depletion of HSCs residing in the bone marrow. • PiggyBac™ (PB) is a non-viral delivery system with a large cargo capacity that allows introduction of multiple genes, including a selection marker and a safety switch that can clear CAR-T cells prior to donor HSC transplant. • PB-produced CAR-T cells exhibit a stem cell memory (SCM) phenotype for enhanced in vivo potency, and may better home to bone marrow than other HSC- directed pre-conditioning alternatives. ABSTRACT A Figure 4: Activity of anti-c-kit or CD133 CAR-T cells against the CD34 + CD38 - progenitor population and CFUs from mobilized peripheral blood CD34 + cells: CD34 + cells isolated from human mobilized peripheral blood were incubated with anti-c-kit and CD133 CAR-T cells for 48h followed by FACS phenotyping of remaining cells (A) and CFU survival assay (B). The anti-c-kit CAR-T depleted >95% of ckit + and CD133 + cells from the primitive CD34 + CD38 - population, while the anti-CD133 CAR-T depleted >90% of CD133+ cells from this population (A). Both the anti-c-kit and CD133 CAR-T cells also reduced colony formation at all E:T ratios tested (B). Depletion of Hematopoietic Progenitors from Human Mobilized Peripheral Blood CD34 + Cells A B B Figure 1: PB vector construct and manufacturing process: (A) A constitutive promoter is used to drive the tri- cistronic cassette consisting of a safety switch, the chimeric antigen receptor (CAR), and a selection gene with flanking chromatin insulators; (B) Pan T cells are isolated from an apheresis product, and then electroporated with anti-c-kit or anti-CD133 CAR piggyBac™ transposon plasmid DNA and in vitro-transcribed piggyBac™ transposase mRNA. The electroporated cells are then activated, expanded, and selected prior to freezing. The process yields >1 x 10 9 cells with >95% CAR expression. Figure 2: PB CAR-T phenotype: PB CAR-T cells directed against c-kit and CD133 antigens were evaluated by flow cytometry for typical T-cell markers following the manufacturing process. (A) Expression of CD4, CD8 and memory markers demonstrating the stem cell memory phenotype of PB CAR-T cells; (B) PB CAR-T cells express CXCR4, a marker commonly associated with bone marrow homing. Figure 3: Activity of anti-c-kit and CD133 CAR-T cells against bone marrow colony forming cells: Following 48h co-culture of CAR-T cells with human or non-human primate (Rhesus macaque) bone marrow cells (effector to target cell ratio of 3:1), remaining cells were scored for survival of hematopoietic colony forming units (CFUs). CFU survival was determined by the number of colonies per plate compared to cells incubated with mock transfected CAR-T cells. 3 of the 8 anti-c- kit CAR-T cell candidates resulted in a >70% decrease in the number of surviving human bone marrow CFUs. The CAR-T cells were species cross-reactive and depleted hematopoietic progenitors from non-human primate bone marrow as well. Phenotype of PB CAR-T Cells Depletion of Hematopoietic Progenitors from Human and Non-Human Primate Bone Marrow METHODS & RESULTS piggyBac anti-c-kit & CD133 CAR Vector Design and CAR-T Cell Production B CD8+ A CD8 CD4 CD4+ 34.2 CD8+ 62.4 CD45RA CD62L Q2 83.7 Q3 11.9 CCR7 CD45RA Q1 3.72 Q4 0.65 Q2 82.1 Q3 12.4 Q4 0.45 Q1 5.06 CXCR4 CD133 CAR (1) c-kit CAR (1) Unstained 82.1% 83.7% Migration of PB CAR–T Cells to the Bone Marrow of NSG Mice Figure 6: Bone marrow homing of PB CAR-T cells: PB CAR-T cells were cultured with (mod. culture) or without (std. culture) factors to increase CXCR4 expression. Cells from each treatment group were labeled separately, mixed, and injected IV into 4-week old, irradiated NSG mice. (A) CXCR4 expression increases after 24h in modified culture; (B) input cell ratio; (C) 16h after cell injection, CAR-T cells, regardless of treatment, were found at equal ratios to input cells in the blood and bone marrow. A Std. Culture (CFSE) Unstained Mod. Culture (CTV) CXCR4 hCD45 SSC CFSE CTV B CFSE CTV 69% Mod. culture cells 30% Std. culture cells C hCD45 SSC CFSE CTV 10% Human cells 67.4% Mod. culture cells 32.2% Std. culture cells 66.8% Mod. culture cells 32.3% Std. culture cells 0.62% Human cells M o ck c-kit CAR (1) CD133 CAR ( 2) %Survival CD34+CD38-CD133+ cells M o c k c- ki t CAR (1) CD133 CAR (2) %Survival CD34+CD38-cKit+ cells Mock c - ki t CAR (2 ) C D1 3 3 CAR (3) Fraction Surviving CAFCs (norm. to Mock; 95% CI) Moc k c-kit CAR (2) CD1 3 3 CAR ( 3 ) Blood Bone marrow 2 weeks 5 weeks TTAA ITR Insulator Poly(A) (sv40) T2A TTAA ITR Insulator T2A Selection Selection Safety Switch Safety Switch CAR CAR Promoter Promoter Selection Safety Switch CAR Promoter Human bone marrow Non-human primate bone marrow Apheresis Product Isolated T- Cells Nucleoporation and Gene Transposition Selection Expansion PB-CAR Transposon pDNA Transposase mRNA + Cryostored CAR-T Cell Product
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