METHODS & RESULTS PiggyBac-manufactured anti-BCMA Centyrin-based CAR-T therapeutic exhibits improved potency and durability David Hermanson, Burton E. Barnett, Srinivas Rengarajan, Rebecca Codde, Xinxin Wang, Yening Tan, Christopher E. Martin, Jenessa B. Smith, Jin He § , Rohit Mathur § , Jing Yan § , Sattva Neelapu § , Eric M. Ostertag, Devon J. Shedlock ABSTRACT INTRODUCTION CONCLUSIONS Chimeric antigen receptor (CAR) T cells have been extremely effective in treating acute lymphoblastic leukemia and have shown promise against other malignancies including multiple myeloma (MM). However, relatively poor potency and durability continue to affect efficacy. Addressing these limitations, we have developed a novel CAR-T cell therapeutic with enhanced stem cell memory phenotype, reduced immunogenicity, and no evidence of CAR-mediated tonic activity. P-BCMA-101 employs a BCMA-specific Centyrin™ rather than a single chain variable fragment (scFv) for antigen detection and is engineered using the piggyBac™ (PB) DNA modification system. Centyrins™ are fully human and have similar binding affinities but are smaller, more thermostable and predicted to be less immunogenic than a scFv. Furthermore, PB modification of human T cells requires only in vitro transcribed mRNA and plasmid DNA, eliminating the need for lentivirus or γ–retrovirus, resulting in significant time and cost savings. Additionally, the increased cargo capacity of PB permits the incorporation of a safety switch and a selectable gene into the product. The former is incorporated for optional depletion in vivo in case of adverse events and the latter allows enrichment of CARTyrin+ cells using a non-genotoxic drug, leading to greater consistency in patient product material. Characterization of P-BCMA-101 revealed > 70% of CD8 + cells possessed a stem-cell memory phenotype (i.e. CD45RA+ CCR7+ CD62L+ CD95+) and >95% of the cells expressed anti-BCMA CARTyrin. In addition, no tonic signaling or T cell exhaustion was observed, highlighted by low levels of PD-1, Lag3, and Tim-3. Cells exhibited specific and robust in vitro target-cell killing, cytokine production, and proliferation in response to BCMA+ tumor cells. In vivo anti-tumor efficacy of P-BCMA-101 was evaluated in NSG mice bearing luciferase+ MM.1S cells, an aggressive human MM-derived cell line, and tumor growth was monitored by bioluminescent imaging (BLI). Following tumor implantation, animals received a single IV administration of either 4 x 10 6 or 12 x 10 6 P-BCMA-101 cells. All untreated control animals succumbed to disease within four weeks of the treatment date. Conversely, tumor burden was reduced to the limit of detection by BLI within 7 days of P- BCMA-101 treatment. As opposed to lentivirus-based products in the same animal model, P-BCMA-101 persists and expands in the animals, eliminates tumors from relapse and prolongs survival, with most animals surviving 100 days post-tumor implant without re- administration of the product. Lastly, the effectiveness of the safety switch has been demonstrated both in vitro and in vivo. P-BCMA-101 is the first-in-class of Centyrin™-based CAR-T therapeutics modified using PB and is predicted to have improved potency and durability given the phenotype and non-immunogenic properties of Centyrins™. We plan to initiate a phase I clinical trial of P- BCMA-101 for the treatment of patients with relapsed and/or refractory MM. Poseida Therapeutics, Inc. 4242 Campus Point Court Suite 700, San Diego, CA, 92121. § MD Anderson Cancer Center, Houston, TX • P-BCMA-101 is a novel CAR-T cell product that uses a smaller and potentially less immunogenic Centyrin™ binder to target BCMA for the treatment of MM • Manufacturing using the PB DNA Modification System requires only mRNA (transposase) and a single plasmid (transposon) • The process does not require virus, cytokines or magnetic beads, and is easily scalable to generate patient doses yielding >95% CARTyrin- expressing cells • P-BCMA-101 predominantly exhibits a stem cell memory phenotype and no significant expression of inhibitory/exhaustion markers • In vitro studies demonstrate specific cell lysis and secondary proliferation against BCMA + target cells • In vivo studies using a NSG™ mouse model demonstrate potency and unprecedented durability of P-BCMA-101 • Rapid initial eradication of tumor • Prolonged elimination of tumor relapse • Protection is dose-dependent Figure 1: P-BCMA-101 Phenotype: P-BCMA-101 cells were evaluated by flow cytometry for typical T cell markers following the manufacturing process. (A) Expression of T cell phenotypic markers on CD4+ and CD8+ T cells. (B) Expression of commonly associated exhaustion/activation markers. (C) Expression of the CARTyrin following secondary activation. Figure 4: Expanded in vivo Study Using P-BCMA-101: P-BCMA-101 cells were evaluated in a NSG™ mouse model using MM.1S/luciferase + cells. Female mice aged 6-8 weeks were injected with 7.5x10 5 MM.1S/luciferase + cells that were allowed to grow for 21 days. Once tumor burden had been confirmed, mice were injected IV with vehicle, 4x10 6 , or 12x10 6 P-BCMA-101 cells. Tumor burden was monitored using BLI. (A) Graph represents the Mean + SEM for each treatment group. (B) Survival graph. BLI revealed no sign of tumor in the two deaths in the 4x10 6 dose. (C) Individual mice are plotted as a single line for low (left) and high (right) dose groups. All animals from the treatment group were euthanized and submitted for pathology. scFv Centyrin™ Size: ~250 aa ~90 aa Components: Heavy & Light Chain Variable Regions, Flexible Linker Fibronectin type III domain Derived: Mouse/RodentAbs Human Stability: Not as stable as original IgG format Stable Figure 2: In vitro P-BCMA-101 Activity: P-BCMA-101 cells were evaluated for their (A) in vitro cytotoxicity and (B) cytokine production in response to K562 (BCMA - ) and K562/BCMA (BCMA + ) tumor lines. (A) 24-hour killing of Luciferase positive K562 or K562/BCMA cells mixed at the indicated effector:target ratio. All samples were run in triplicate. (B) IFN-γ was measured from the supernatant of the 24-hour killing assay using an ELISA. (C) CFSE labeled P-BCMA-101 cells were co-cultured in the presence of H929 (BCMA + ) or K562 cells for 4 days, harvested and evaluated by flow. All samples were run in duplicate. Figure 3: In vivo P-BCMA-101 Activity: P-BCMA-101 product cells were evaluated in an NSG™ mouse model. Male mice were injected with 1.5x10 6 MM.1S/Luciferase + Cells that were allowed to implant over a 21 day period. Following tumor implant, mice were dosed with 5x10 6 P-BCMA-101 cells via IV administration. Tumor burden was monitored using (A) BLI as well as through (B) M-Protein measurements in blood. A B 1. This poster is available on the Poseida website at poseida.com/publications/ 2. Centyrin™ is a registered trademark of Janssen Pharmaceuticals, Inc. Poseida has licensed certain rights to the Centyrin™ technology platform from Janssen Pharmaceuticals, Inc. for use in autologous T cell therapeutics Abstract #3759 In Vivo Results
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METHODS & RESULTS
PiggyBac-manufactured anti-BCMA Centyrin-based CAR-T therapeutic exhibits improved potency and durabilityDavid Hermanson, Burton E. Barnett, Srinivas Rengarajan, Rebecca Codde, Xinxin Wang, Yening Tan, Christopher E. Martin, Jenessa B. Smith, Jin He§, Rohit Mathur§, Jing Yan§, Sattva Neelapu§, Eric M. Ostertag, Devon J. Shedlock
ABSTRACT
INTRODUCTION
CONCLUSIONS
Chimeric antigen receptor (CAR) T cells have been extremely effective in treating acutelymphoblastic leukemia and have shown promise against other malignancies includingmultiple myeloma (MM). However, relatively poor potency and durability continue toaffect efficacy. Addressing these limitations, we have developed a novel CAR-T celltherapeutic with enhanced stem cell memory phenotype, reduced immunogenicity, andno evidence of CAR-mediated tonic activity.
P-BCMA-101 employs a BCMA-specific Centyrin™ rather than a single chain variablefragment (scFv) for antigen detection and is engineered using the piggyBac™ (PB) DNAmodification system. Centyrins™ are fully human and have similar binding affinities butare smaller, more thermostable and predicted to be less immunogenic than a scFv.Furthermore, PB modification of human T cells requires only in vitro transcribed mRNAand plasmid DNA, eliminating the need for lentivirus or γ–retrovirus, resulting insignificant time and cost savings. Additionally, the increased cargo capacity of PB permitsthe incorporation of a safety switch and a selectable gene into the product. The former isincorporated for optional depletion in vivo in case of adverse events and the latter allowsenrichment of CARTyrin+ cells using a non-genotoxic drug, leading to greater consistencyin patient product material.
Characterization of P-BCMA-101 revealed > 70% of CD8+ cells possessed a stem-cellmemory phenotype (i.e. CD45RA+ CCR7+ CD62L+ CD95+) and >95% of the cells expressedanti-BCMA CARTyrin. In addition, no tonic signaling or T cell exhaustion was observed,highlighted by low levels of PD-1, Lag3, and Tim-3. Cells exhibited specific and robust invitro target-cell killing, cytokine production, and proliferation in response to BCMA+tumor cells. In vivo anti-tumor efficacy of P-BCMA-101 was evaluated in NSG mice bearingluciferase+ MM.1S cells, an aggressive human MM-derived cell line, and tumor growthwas monitored by bioluminescent imaging (BLI). Following tumor implantation, animalsreceived a single IV administration of either 4 x 106 or 12 x 106 P-BCMA-101 cells. Alluntreated control animals succumbed to disease within four weeks of the treatment date.Conversely, tumor burden was reduced to the limit of detection by BLI within 7 days of P-BCMA-101 treatment. As opposed to lentivirus-based products in the same animal model,P-BCMA-101 persists and expands in the animals, eliminates tumors from relapse andprolongs survival, with most animals surviving 100 days post-tumor implant without re-administration of the product. Lastly, the effectiveness of the safety switch has beendemonstrated both in vitro and in vivo.
P-BCMA-101 is the first-in-class of Centyrin™-based CAR-T therapeutics modified usingPB and is predicted to have improved potency and durability given the phenotype andnon-immunogenic properties of Centyrins™. We plan to initiate a phase I clinical trial of P-BCMA-101 for the treatment of patients with relapsed and/or refractory MM.
Poseida Therapeutics, Inc. 4242 Campus Point Court Suite 700, San Diego, CA, 92121. §MD Anderson Cancer Center, Houston, TX
• P-BCMA-101 is a novel CAR-T cell product that uses a smaller andpotentially less immunogenic Centyrin™ binder to target BCMA forthe treatment of MM
• Manufacturing using the PB DNA Modification System requires onlymRNA (transposase) and a single plasmid (transposon)
• The process does not require virus, cytokines or magnetic beads, andis easily scalable to generate patient doses yielding >95% CARTyrin-expressing cells
• P-BCMA-101 predominantly exhibits a stem cell memory phenotypeand no significant expression of inhibitory/exhaustion markers
• In vitro studies demonstrate specific cell lysis and secondaryproliferation against BCMA+ target cells
• In vivo studies using a NSG™ mouse model demonstrate potency andunprecedented durability of P-BCMA-101
• Rapid initial eradication of tumor• Prolonged elimination of tumor relapse• Protection is dose-dependent
Figure 1: P-BCMA-101 Phenotype: P-BCMA-101 cells were evaluated by flow cytometry for typical T cell markers following themanufacturing process. (A) Expression of T cell phenotypic markers on CD4+ and CD8+ T cells. (B) Expression of commonly associatedexhaustion/activationmarkers. (C) Expression of the CARTyrin following secondary activation.
Figure 4: Expanded in vivo Study Using P-BCMA-101: P-BCMA-101 cells were evaluated in aNSG™ mouse model using MM.1S/luciferase+ cells. Female mice aged 6-8 weeks were injected with7.5x105 MM.1S/luciferase+ cells that were allowed to grow for 21 days. Once tumor burden had beenconfirmed, mice were injected IV with vehicle, 4x106, or 12x106 P-BCMA-101 cells. Tumor burdenwas monitored using BLI. (A) Graph represents the Mean + SEM for each treatment group. (B)Survival graph. BLI revealed no sign of tumor in the two deaths in the 4x106 dose. (C) Individual miceare plotted as a single line for low (left) and high (right) dose groups. All animals from the treatmentgroup were euthanized and submitted for pathology.
scFv Centyrin™
Size: ~250 aa ~90 aa
Components: Heavy & Light ChainVariable Regions,
Flexible Linker
Fibronectin type III domain
Derived: Mouse/RodentAbs Human
Stability: Not as stable as original IgG format
Stable
Figure 2: In vitro P-BCMA-101 Activity: P-BCMA-101 cells were evaluated for their (A) in vitro cytotoxicity and (B) cytokineproduction in response to K562 (BCMA-) and K562/BCMA (BCMA+) tumor lines. (A) 24-hour killing of Luciferase positive K562 orK562/BCMA cells mixed at the indicated effector:target ratio. All samples were run in triplicate. (B) IFN-γ was measured from thesupernatant of the 24-hour killing assay using an ELISA. (C) CFSE labeled P-BCMA-101 cells were co-cultured in the presence of H929(BCMA+) or K562 cells for 4 days, harvested and evaluated by flow. All samples were run in duplicate.
Figure 3: In vivo P-BCMA-101 Activity: P-BCMA-101 product cells were evaluated in an NSG™ mouse model. Male micewere injected with 1.5x106 MM.1S/Luciferase+ Cells that were allowed to implant over a 21 day period. Following tumor implant,mice were dosed with 5x106 P-BCMA-101 cells via IV administration. Tumor burden was monitored using (A) BLI as well as through(B) M-Protein measurements in blood.
A
B
1. This poster is available on the Poseida website at poseida.com/publications/2. Centyrin™ is a registered trademark of Janssen Pharmaceuticals, Inc. Poseida has licensed certain rights to the
Centyrin™ technology platform from Janssen Pharmaceuticals, Inc. for use in autologous T cell therapeutics
Abstract #3759
In Vivo Results
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