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Your Name Oct, 6 th 2015 Janssen F2F Meeting Springhouse, PA PSMA-specific CAR-T Memory Stem Cell Therapy Jenessa B. Smith, Rebecca Codde, Yening Tan, Chris E. Martin, Burton E. Barnett, Srinivas Rengarajan, Eric M. Ostertag, Devon J. Shedlock Poseida Therapeutics, Inc., 4242 Campus Point Court, Suite 700, San Diego, CA, 92121 ABSTRACT INTRODUCTION METHODS & RESULTS CONCLUSIONS Eliminates Solid Tumors in Multiple Prostate Cancer Models Figure 2: Initial screen of RNA-electroporated CARTyrin cells. (a) Surface expression of human PSMA (hPSMA) on tumor cells. K562.hPSMA and PC3.hPSMA were engineered to stably express hPSMA. (b) Surface expression of PSMA on RNA-electroporated K562 cells. PSMA RNA amounts per reaction (ug). (c/d) Degranulation of indicated RNA-electroporated CAR T cells against K562 cells transiently expressing varying levels of hPSMA (c) or tumor cells (d). Figure 1: Construction, delivery, specificity, and phenotype of P-PSMA-101 with piggyBac transposition and Poseida manufacture process. Figure 3: In vitro activity of piggyBac-engineered CARTyrin cells against PSMA+ tumor cells. (a) Binding of histidine (His)-tagged, recombinant hPSMA antigen (rhPSMA) with an anti-His tag antibody. Mock transposed T cells in gray. The CAR indicated above each histogram is shown in blue. PSMA-specific J591 antibody- based scFv (J591) transposed CAR T cells were used to test comparability. (b) IFN-γ secretion against indicated target tumor cells at 1:1 effector: target ratio. (c/d) Cytolytic activity against the indicated tumor cells in 24-hr assays. Mock P-PSMA-101 Day 41 Pre- Infusion Day 55 No tumor by caliper (Day 20-25) Day 27 Day 20 Day 69 CD62L CD45RA a) b) c) Figure 4: In vivo activity of piggyBac-engineered CARTyrin cells against established LNCaP tumor. (a) Experimental outline. Study 1: dose study at 1, 5, or 10e6 P-PSMA-101 cells. Study 2: efficacy study at 4e6 P-PSMA-101 cells, Mock-transposed and P-BCMA-101 cells as specificity controls, and J591 CAR-T cells. (b/c) Bioluminescence (BLI) imagining (b) and caliper measurement (c) of Study 1 over time. Figure 5: In vivo efficacy of P-PSMA-101 at suboptimal “stress test” dose (Study 2). (a/b) Caliper measurements (a) and quantification of BLI (b) of NSG mice bearing established LNCaP tumor. Mice were treated with 4e6 of the indicated CARTyrin cells at Day0. (c) Survival curves of Study 2. Median survival in days indicated by number. (d) Quantification of CD8+ cells/μl of peripheral blood at the indicated time points. Cells were gated on size, human CD45+CD3+CD8+ and quantified with beads. (e) Flow cytometric analysis of CD45RA versus CD62L in a representative P-PSMA-101 CARTyrin-treated mouse (top) or Mock-transposed T cells (bottom) over time. Cells were gated on size, mouse CD45(-), human CD3+, CD8+ T cells. Figure 6: Efficacy of P-PSMA-101 in a model of bone metastasis. (a) Schematic of bone metastasis study. PC3luc.hPSMA tumor cells were injected into the peri-tibial region in NSG mice. Cohorts were treated 4 days post tumor implant with the indicated CAR and dose. (b) Quantification of bioluminescent imaging (c) Quantification of CD8+ cells/μl of peripheral blood at the indicated time points. Cells were gated on size, human CD45+CD3+CD8+ and quantified with beads. a) b) c) d) e) 20:1 10:1 5:1 2.5:1 1:1 0.1:1 0.01:1 0 20 40 60 80 100 24hr Killing Assay LNCaP n=2 independent donors Specific Lysis (%) (normalized to mock) K562.BCMA K562.hPSMA LNCaP 0 10,000 20,000 30,000 50,000 24hr 1:1 co-culture n=3 independent donors IFN- (pg/mL) Mock P-BCMA-101 J591-based scFv P-PSMA-101 K562.BCMA K562.PSMA LNCaP 0 10 20 30 40 50 60 70 80 24hr 10:1 Killing Assay n=4 indepedent donors Specific Lysis (%) (normalized to mock) P-BCMA-101 J591-based scFv P-PSMA-101 P-BCMA-101 P-PSMA-101 J591-based scFv a) c) d) b) rhPSMA; anti-His tag Centryin TM is a registered trademark of Janssen Pharmaceuticals, Inc. Poseida has licensed certain rights to the CARTyrintechnology platform from Janssen Pharmaceuticals, Inc. for use in autologous T cell therapeutics a) b) PSMA RNA 20 10 5 2.5 1.25 0.625 0.3125 0.1 0 20 40 60 80 4hr Degranulation K562 cells electroporated with hPSMA (ug) CD107a+ (%) PSMA isotype c.) 20 10 5 2.5 1.25 0.625 0.3125 0 PSMA Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein overexpressed on the surface >80% of primary and metastatic prostate cancers. PSMA is a promising therapeutic target, but early first-generation chimeric antigen receptor (CAR) T-cell therapies have lacked clinical efficacy. Here we developed a novel CAR T-cell product (P-PSMA-101) via the non-viral piggyBac™ DNA Modification System for transposition of a tri-cistronic transgene encoding a safety switch, a PSMA-specific Centyrin- based CAR (CARTyrin), and a selection gene- features that may improve safety and therapeutic efficacy. We identified a lead anti-PSMA CARTyrin (P-PSMA-101) that displayed exquisite specificity for PSMA isoform 1 in a safety screen testing >5,600 surface proteins. Using piggyBac we obtained >95% CAR+ T- cells after selection and expansion. Importantly, our unique production methodology leads to exceptionally high levels of T-stem cell memory (Tscm) cells, an early memory population that has recently been reported to correlate with clinical responses in several CAR T-cell clinical trials. In vitro, P- PSMA-101 specifically proliferated, lysed, and secreted IFN-γ and IL-2 against PSMA+ LNCaP or PSMA- engineered K562s. P-PSMA-101 eliminated established solid tumors in two different in vivo prostate cancer models, one subcutaneous and the other intratibial to model bone metastasis. In the first study, P-PSMA-101 eliminated established LNCaP subcutaneous tumor in 100% of immune-compromised mice for the duration of the study at a 12e6 dose (42 days post-treatment), while 2/3 of the low-dose (5e6) animals remained tumor-free. In the second study, we tested the previously clinically-applied anti-PSMA J591 scFv-based CAR for comparability. P-PSMA-101 demonstrated enhanced anti-tumor efficacy and survival (>70 days, end of study) in comparison to J591 CAR T-cell-treated mice (41 days) at a “stress test” dose of 4e6 T-cells against subcutaneous LNCaP solid tumors in NSG mice. The >60% Tscm P- PSMA-101 expanded in vivo and gave rise to differentiated effector CAR+ T-cells that were detected in the peripheral blood concomitant with a decrease in tumor burden below detectable caliper and BLI imaging limits. P-PSMA-101 then contracted yet persisted in the peripheral blood with >70% of T-cells retaining a Tscm phenotype. Additional in vivo studies utilizing a PSMA-engineered PC3 bone metastasis prostate cancer model demonstrated potent P-PSMA-101 anti-tumor efficacy at a 4e6 dose. In comparison, the J591 CAR showed significant anti-tumor activity at a 12e6, but not at 4e6. P-PSMA-101 is a first-in-class Centyrin-based CAR T-cell therapeutic that exhibits an exceptionally high, persistent frequency of Tscm and mediates durable anti-solid tumor efficacy that surpasses previously established anti-PSMA CAR T-cell therapy in these in vivo models. Current efforts will continue towards clinical application in patients with metastatic castrate resistance prostate cancer. • P-PSMA-101 is Poseida’s CAR-T memory stem cell product for mCRPC • P-PSMA-101 eliminated solid tumors and significantly prolonged survival in an established subQ LNCaP model • P-PSMA-101 gave rise to differentiated CAR-T populations, but following solid tumor elimination, persisted as CD45RA + CD62L + memory CARTyrin + cells • P-PSMA-101 reduced tumor burden at 3x lower dose than a clinical PSMA binder in an peri-tibial bone metastasis model 0 10 20 30 40 50 60 70 0 500 1000 1500 2000 2500 3000 Caliper Measurements Day post T cell injection Tumor Volume (mm3) Mock P-BCMA-101 J591-based scFv P-PSMA-101 0 5 10 15 20 25 30 35 40 45 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 Caliper Measurements Tumor Volume (mm3) Vehicle n=4 1e7 n=3 5e6 n=3 1e6 n=4 Days post T cell injection 0 5 10 15 20 25 30 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 BLI Days post T cell injection Luminescence (p/sec/m2) Mock P-BCMA-101 J591 J591 12e6 P-PSMA-101 Mock P-BCMA-101 J591 P-PSMA101 J591 12e6 0 10 20 30 40 50 60 100 150 CD8 + CAR-T cells in blood Cells/uL Day 7 Day 14 Day 21 Day 28 b) c) Adapted from Restifo et al Blood 2014 Tscm Tcm Tem Teff 0 20 40 60 80 100 After Poseida Process Tcm Tem Teff 0 20 40 60 80 Before Poseida Process CD8+ (%) Naive + Tscm CD45RA Teff Tem Tcm P-PSMA-101 CD62L Tscm Naive/Tscm Tcm Tem Teff 0 20 40 60 % Transposed piggyBac TM a) Confirmatory Screen of Centryin Binder Adapted from Maus et al Blood 2014 Overview of Poseida Manufacturing Process PSMA-101 binder Binder control CTLA-4 Positive Control Isotype control 5,647 human plasma membrane proteins Phenotype of Poseida T cell Products K562.BCMA K562.hPSMA LNCap PC3.hPSMA PC3 K562 media 0 20 40 60 80 100 4hr Degranulation n=2-7 indepdendent experiments CD107a+ (%) No RNA P-BCMA-101 J591-based scFv P-PSMA-101 0 10 20 30 40 50 60 70 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 BLI Day post T cell injection Luminescence (p/sec/m2) Mock P-BCMA-101 J591-based scFv P-PSMA-101 1 10 100 1,000 10,000 100,000 CD8+ Peripheral Blood Cells/uL Day 6 Day 13 Day 20 Day 27 Day 42 Day 55 Day 69 0 10 20 30 40 50 60 70 0 25 50 75 100 Survival Percent survival 34 37 41 >70 d.)
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