Figure S1, S2, S3, S4, S5, S6, S7, S8, S9, Table S1

Abstract Purpose: Chimeric antigen receptor (CAR) T-cell therapies have shown potential in solid tumors. A higher proportion of stem cell–like memory T cells (TSCM) in CAR T-cell products could enhance engraftment, persistence, and prolong immune activity. This phase I trial (NCT04249947) evaluated the safety and efficacy of P-PSMA-101, an autologous TSCM-rich, bone-tropic CAR T-cell therapy targeting prostate-specific membrane antigen (PSMA), in patients with metastatic castrate-resistant prostate carcinoma (mCRPC). Secondary endpoints included objective response rate, prostate-specific antigen (PSA) response, and radiographic progression-free survival. Patients and Methods: The final P-PSMA-101 product was produced from leukapheresis using the piggyBac DNA transposon–based platform, which integrates a multicistronic transgene encoding an inducible caspase 9 (iCasp9) safety switch in addition to the CAR, generating TSCM-rich CAR T cells. Results: Among 33 treated patients, 18% (n = 6) had dose-limiting toxicities. Cytokine release syndrome (CRS) occurred in 61% (n = 20), with grade ≥3 CRS seen in 9% (n = 3). Activation of the iCasp9-based safety switch was required in 24% (n = 8) of cases, including one toxicity that was ultimately fatal and successful resolution of symptoms in the other seven. P-PSMA-101 demonstrated antitumor activity, with 21% (n = 7) of patients achieving a ≥50% PSA decline (PSA50 response). Among 13 RECIST-evaluable patients, one partial response was observed. Stable disease was observed in 61% (n = 20) of patients, with 21% (n = 7) maintaining disease stability for ≥3 months. Two patients experienced sustained remissions exceeding 12 months, characterized by PSA declines of more than 90%, corroborated by pharmacokinetic, biomarker, and PSMA-PET imaging data. Conclusions: Robust expansion of P-PSMA-101 CAR T cells resulted in toxicity but also durable responses in patients with mCRPC. Future trials of CAR T therapy may be informed by the results of this nonviral engineering, TSCM cell–enriched approach. See related commentary by Lee et al., p. 3417

Autologous CAR-T therapies targeting B-cell maturation antigen (BCMA) in relapsed/refractory multiple myeloma (RRMM) have demonstrated therapeutic clinical responses. Here, we present the characterization and interim Phase I data for P-BCMA-ALLO1, a TSCM-predominant allogeneic CAR-T therapy targeting BCMA in heavily pretreated relapsed/refractory multiple myeloma. Preclinical analyses reveal a strong correlation between CD8+ TSCM phenotype and in vivo potency in mouse xenograft models. In early clinical data (NCT04960579), among the 11 of 33 evaluable patients who received enhanced lymphodepletion, 82% (9/11) responded, with 63.6% (7/11) achieving very good partial response (VGPR) or better. All patients started therapy a median of 1 day after enrollment, with every patient receiving P-BCMA-ALLO1 infusion, and resulting in a 100% intent-to-treat (ITT) rate with no use of bridging therapy. CRS was reported in 21.2% (7/33) across all cohorts, all grade ≤2. The median time to peak CAR-T cell expansion (Tmax) was 10 days post-infusion. Consistent with preclinical findings, CAR-T cell expansion is accompanied by differentiation from a predominantly TSCM phenotype to a TEM/TEFF phenotype, with trafficking and persistence observed in bone marrow. These data suggest that a TSCM cellular phenotype may offer significant advantages in efficacy, safety, and cellular persistence in the context of allogeneic CAR-T therapy. Clinical trial: NCT04960579.

B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapy has shown high initial response rates in relapsed/refractory multiple myeloma (RRMM), but most patients eventually relapse with progressive disease. Previous studies have identified a less differentiated, CD19-positive progenitor subpopulation of MM cells that contributes to resistance and poor survival. Furthermore, anti-CD19 CAR-T cells given after high-dose melphalan and stem cell transplantation have shown to improve patient survival and can eliminate MM progenitor cells that are resistant to BCMA-directed CAR-T cells. P-BCMACD19-ALLO1 is a fully allogeneic CAR-T therapy designed to target BCMA and CD19. Additionally, P-BCMACD19-ALLO1 is a T stem cell memory (TSCM)-rich product derived from healthy donor T cells using a nonviral, transposon-based system for transgene delivery, with knockout of TRBC1/2 and B2M to prevent graft-versus-host disease and reduce host T-cell mediated rejection, respectively. TSCM -rich CAR-Ts, including our BCMA-targeting allogeneic P-BCMA-ALLO1 CAR-T for RRMM (NCT04960579), have shown favorable efficacy and safety in the clinic. To develop P-BCMACD19-ALLO1, we constructed the BCMA CAR using two fully human, single-domain, heavy-chain variable domains (VHs) that independently bind BCMA and are joined together by a G4S linker in tandem. The tandem anti-BCMA VHs binds to all clinical escape mutants, including teclistamab-resistant R27P mutation. The anti-CD19 CAR binder comprises of a fully human VH against CD19. Both CARs are expressed from a single, multi-cistronic transgene that also contains an inducible Caspase-9 safety switch and a dihydrofolate reductase mutein. Herein, we show enhanced in vivo potency of P-BCMACD19-ALLO1against BCMA/CD19-double positive RPMI-8226 MM xenograft model using an optimized ICD combination that incorporates a novel co-stimulatory domain derived from the tumor necrosis factor receptor family member transmembrane activator and CAML interactor (TACI) in the CD19 CAR and the standard 4-1BB in the BCMA CAR. P-BCMACD19-ALLO1 demonstrates target-specific, in vitro cytotoxicity against tumor cells expressing BCMA, CD19, or both antigens but lacks activity against antigen-negative tumor cells. P-BCMACD19-ALLO1 also demonstrates potent, in vitro killing of tumor cells expressing R27P, P33Del, P33S and S30Del escape mutants. Lastly, P-BCMACD19-ALLO1 effectively eliminates primary bone marrow CD81+CD19+ progenitor cells as well as colony-forming cells derived from MM patient samples, suggesting that P-BCMACD19-ALLO1 may increase the depth and the durability of response. Taken together, our data underscore the therapeutic potential of P-BCMACD19-ALLO1 and support its application for treating RRMM to be further evaluated in clinical trials. Steven Wang, Iris Pang, Claudia Chang, Michelle Burrascano, Nick DeMarco, Danny Mendoza-Reyes, Sonia Reyes, Arturo Barcenas, Pastor Nieto, Jose Diaz, Garret Arauz, Chris Lynn, Tony Nguyen, Connor Reed, Mona Connerney, Quy Le, Julia Coronella, Devon J. Shedlock. Enhanced potency of BCMA/CD19 dual-targeting allogeneic CAR-T cells for relapsed/ refractory multiple myeloma with 4-1BB/TACI intracellular domains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4811.

Introduction: P-BCMA-ALLO1 is an allogeneic BCMA targeting CAR-T for the treatment of RRMM. It is manufactured at an in-house GMP facility from healthy donor T cells using Cas-CLOVER™ gene editing and non-viral transposon-based integration (piggyBac® DNA Delivery System) to express a human anti-BCMA VH-based CAR. These technologies enable P-BCMA-ALLO1 to be a T stem cell memory (TSCM)-rich product with high purity characterized by near-100% CAR+ cells. Methods: The P-BCMA-ALLO1-001 study (NCT04960579) is enrolling RRMM patients (pts) refractory to or who have relapsed following IMID, PI and CD38 mAb. Pts previously treated with BCMA-targeted therapy were allowed. The study employs a 3 + 3 dose escalation design to test multiple P-BCMA-ALLO1 doses from multiple product lots manufactured from different donors. Several lymphodepletion (LD) regimens are being investigated to enable optimal CAR-T expansion. The primary objective is to assess the safety and determine maximum tolerated dose of P-BCMA-ALLO1. The key secondary objective is to evaluate its anti-myeloma effect. Here, we present data on pts treated with a dose of approximately 2 x 106 cells/kg, after completing a 3-day LD regimen (cyclophosphamide 750 mg/m2/day and fludarabine 30 mg/m2/day). Results: At the time of data cutoff, 21 pts with 4+ weeks of follow up had been treated in this LD arm. None required bridging therapy and the median time from enrollment to start of study treatment was just 1 day (range: 0-9 days), highlighting the rapid availability of P-BCMA-ALLO1, being an off-the-shelf CAR-T. 100% of the intent to treat (ITT) population received P-BCMA-ALLO1. The median pt age was 61 (39-76) years, 53% were female and 42% were minorities. The median time since multiple myeloma diagnosis was 7.0 (1.0-15.1) years. Pts were heavily pretreated, with a median of six (2-14) prior lines of therapy. 62% had undergone prior BCMA-targeted CAR-T and/or T cell engager (TCE) therapy, 29% had previously received both a BCMA-targeted CAR-T and/or TCE and Talquetamab. Thirteen (62%) pts had high-risk FISH anomalies. P-BCMA-ALLO1 was well tolerated with no DLTs or GvHD. The most common treatment-emergent adverse events (TEAEs) were neutropenia (71%), leukopenia (67%), anemia (52%), thrombocytopenia (48%), and CRS (43%). The most common ≥ G3 TEAEs were neutropenia (67%), leukopenia (67%) anemia (43%), and thrombocytopenia (33%). Most AEs were considered unrelated to P-BCMA-ALLO1. Of nine (43%) pts with CRS, seven (33%) had G1 CRS and two (10%) had G2 CRS; no pt had > G3 CRS. Three (14%) pts experienced ICANS, all were G1. P-BCMA-ALLO1 demonstrated an encouraging overall response rate (ORR) in all relevant pt subsets. The ORR in the ITT population was 90%, including 6 CR/sCR, 5 VGPRs, and 8 PRs. The ORR was 100% in BCMA naïve pts and 85% in pts who previously received BCMA-targeted therapy. The ORR was 83% in the subset of 6 pts who received both prior BCMA-targeted agents, and Talquetamab. To assess whether BCMA expression impacted clinical activity, baseline BCMA levels (BCMA MESF on CD138+CD38+ bone marrow cells) were evaluated for 12 pts, showing a median MESF of 4,685 (2,125-23,537). The median BCMA MESF for the 6 BCMA-naïve pts tested was 8,859 (4,098-23,537), while the median for the 6 pts with prior BCMA therapy was 2744 (2125-5912). P-BCMA-ALLO1 cellular kinetics (CK) were assessed (n=15) using ddPCR demonstrating a median Cmax of 49,430 (66-501,678) cp/µg of the CAR transcript at a median Tmax of 10 (7-21) days. The median CK for the BCMA naïve pts (n=7) was 72.077 (10,904 - 501,678) cp/µg, while the median CK for BCMA exposed pts (n=8) was 27,878 (66 - 310,209) cp/µg. There was no statistical difference between these groups. Conclusions: P-BCMA-ALLO1 is a non-viral, TSCM-rich, allogeneic BCMA-targeting CAR-T quickly delivered to all eligible pts without the need for bridging therapy. P-BCMA-ALLO1 demonstrates a promising ORR and favorable safety profile when administered with optimized LD. It is highly active, regardless of prior exposure to BCMA-targeted autologous CAR-T or TCE therapy. As an allogeneic therapy, it also offers significant practical advantages over autologous CAR-T, which require apheresis, prolonged, often uncertain manufacturing, and bridging therapy; and TCE, which require chronic administration. The P-BCMA-ALLO1-001 clinical trial is actively enrolling pts. Updated safety and efficacy data will be presented at ASH 2024.

Abstract Introduction: Poseida is developing innovative allogeneic T stem cell memory rich CAR-T both for hematologic malignancies (HM) and solid tumors (ST) including P-BCMA-ALLO1 which targets BCMA for MM, and P-MUC1C-ALLO1 targeting MUC1-C for epithelial-derived ST. Optimal LD for allogeneic cell therapies remains to be established. Most research has focused on HM, where patients (pts) have likely undergone HSCT and are, therefore, LD experienced in contrast to ST pts. Consequently, ST pts may require higher doses of LD chemotherapy to achieve LD depth comparable to HM pts. This study sought to compare LD with higher doses of cy across our early phase 1 trials (NCT04960579/NCT05239143) which are enrolling ST and MM pts, respectively. Methods: P-BCMA-ALLO1 and P-MUC1C-ALLO1 are 3+3 phase 1 dose escalation studies enrolling heavily pre-treated pts. LD arm S used cy 300 mg/m2 + fludarabine (flu) 30 mg/m2 × 3 days, followed by CAR-T. Alternative arms tested higher cy doses: P1 (500 mg/m2) and P2 (1,000 mg/m2), both with flu. We assessed LD depth and cellular kinetics (CK) by measuring WBC, serum IL-15, and CAR-T transcripts (qPCR). Results: By Jan 4, 2024, 42 pts received P-BCMA-ALLO1 (22 in S, 13 in P1, 7 in P2), and 30 pts received P-MUC1C-ALLO1 (19 in S, 9 in P1, 2 in P2). ST pts presented with higher baseline WBC compared to MM pts with median counts of 5.7×103 vs. 4.4×103/µL. LD was less pronounced in ST pts with a median WBC nadir of 1.1×103/µL for both S and P1. In MM pts median WBC nadir was 0.6×103 for S vs. 0.2×103 for P1. For P-BCMA-ALLO1, CK at 2×106 cells/kg dose showed a mean Cmax of 130,811 copies (cp)/µg in P2 and 6,191 in P1 compared to 170 in S. Median WBC nadir was significantly (p=0.0002) lower for P2 (0.1×103/µL) compared to S (0.6×103), with corresponding AUC0-13d values of 3.5×103/µL for P2 vs. 10×103 for S (p=0.0013). Peak serum IL-15 in P2 was 66.1 pg/mL, significantly higher than 29.7 in S (p=0.0110). In P1, there was a trend towards improved LD with median WBC nadir for P1 of 0.3×103/µL and AUC0-13d of 6.4×103/µL.P1 LD for P-MUC1C-ALLO1 did not improve CK, with WBC nadir and AUC 0-13d values comparable to S. P-MUC1C-ALLO1 CK at 2×106 cells/kg showed a mean Cmax of 692 cp/µg DNA in S, 23 in P1, and 4,200 in P2. P-MUC1C-ALLO1 pts receiving P2 achieved the deepest LD (WBC nadir: 0.1×103/µL). Peak IL-15 in ST pts was increased and delayed to day 8 in P2, compared to day 0 - 1 in S or P1. Conclusion: Increasing cy dose to 500 or 1,000 mg/m2 improves CK for P-BCMA-ALLO1 pts due to enhanced LD depth. For P-MUC1C-ALLO1 pts, increasing cy to 500 does not improve LD depth or CK compared to 300. This difference suggests the need for higher LD doses in ST pts than MM pts to achieve optimal CK. This differential need for LD chemo for these two studies is unlikely due to cellular characteristics because of similar manufacturing technology with the only difference being the binder. Citation Format: Sabrina Haag, Jeff D. Eskew, Katherine McArthur, Joanne McCaigue, Sepideh Vaziri, Samuel DePrimo, Christopher E. Martin, Catherine Gregovics, Ann Murphy, Hamid Namini, Ellen Christie, Marcela Marinez-Prieto, Rajesh Belani, Stacey Cranert, Julia Coronella, Devon Shedlock. Solid tumor patients require higher cyclophosphamide (cy) dose than multiple myeloma (MM) patients to achieve adequate lymphodepletion (LD) necessary to enable allogeneic CAR-T expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT070.

Abstract Introduction: Despite therapeutic advances, multiple myeloma remains incurable. BCMA targeting immunotherapies, such as bispecific T-cell engagers (TCE) and autologous CAR-T provide high response rates, but relapses are common. Autologous CAR-T are logistically challenging due to the need for apheresis, prolonged manufacturing and occasional manufacturing failures. Importantly, pts who have progressed after a prior BCMA targeting immunotherapy are an emerging area of high unmet need. Emerging data indicate that autologous CAR-T have lower clinical activity in pts ho have progressed on TCE. Methods: P-BCMA-ALLO1 is an allogeneic CAR-T therapy manufactured from healthy donor T-cells available “off-the-shelf” and being evaluated in a phase 1 clinical trial (P-BCMA-ALLO1-001) in RRMM pts. This primary objective is to determine the maximum tolerated dose of P-BCMA-ALLO1, and the key secondary objective is to investigate the anti-myeloma activity. The pts must have progressed on a prior proteasome inhibitor, immunomodulatory drug and anti-CD38 monoclonal antibody. The study allows enrollment of pts who have received prior BCMA targeting therapy. The study is exploring escalating P-BCMA-ALLO1 doses and several different lymphodepletion chemotherapy (LD) regimens. Here we report the safety and early efficacy results for the 5 pts who were treated with P-BCMA-ALLO1 after having progressed on BCMA targeting CAR-T, TCE or both. These pts were treated in arms P1 (LD: cyclophosphamide (cy) 500 mg/m2 + fludarabine (flu) 30 mg/m2 X 3 days) or arm P2 (LD: cy 1000 mg/m2 + flu 30 mg/m2 X 3 days) at a P-BCMA-ALLO1 dose of > 2 X 106 to <6 X 106 cells/kg. Results: The median pt age was 62 years and median prior lines of therapy was 10. Three pts were treated in arm P2 and 2 in arm P1. Two pts had received prior teclistamab, 2 had received prior CAR-T and 1 had received prior teclistamab and CAR-T. P-BCMA-ALLO1 was well tolerated with no dose limiting toxicities or graft vs. host disease. Three of the five pts developed cytokine release syndrome (all grade (G) 2) and one developed G2 immune effector cell neurotoxicity syndrome. Three of the five pts (60%) achieved clinical responses with all three achieving the best response of very good partial response. The two non-responders had previously received and failed to achieve clinical response with teclistamab. One pt who had previously received both teclistamab and CAR-T achieved VGPR. Conclusion: In conclusion, P-BCMA-ALLO1 is an allogeneic CAR-T that is available “on-demand” with activity in RRMM pts who have progressed following prior BCMA targeted CAR-T and TCE. We believe this is the first such report of an allogeneic CAR-T showing clinical activity in such a pt population with high unmet need. Enrollment is continuing and updated data will be presented at the meeting. Citation Format: Bhagirathbhai Dholaria, Leyla Shune, Andrew Kin, Katherine McArthur, Jeff D. Eskew, Christopher E. Martin, Sabrina Haag, Joanne McCaigue, Hamid Namini, Sam DePrimo, Stacey Cranert, Julia Coronella, Devon Shedlock, Rajesh Belani. Clinical activity of P-BCMA-ALLO1, a B-cell maturation antigen (BCMA) targeted allogeneic chimeric antigen receptor T-cell (CAR-T) therapy, in relapsed refractory multiple myeloma (RRMM) patients (pts) following progression on prior BCMA targeting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT071.

Autologous Chimeric Antigen Receptor (CAR) T cells targeting CD19 have revolutionized the treatment of relapsed or chemotherapy-refractory B cell malignancies. However, many patients experience disease progression due to the loss or reduced expression of CD19. Autologous therapies also pose challenges for access, as each CAR-T dose requires patient apheresis and individual manufacture, leading to associated wait times and the need for bridging therapy. Here, we describe P-CD19CD20-ALLO1, a fully allogeneic CAR-T product expressing two full-length CARs targeting CD19 and CD20, respectively. P-CD19CD20-ALLO1 is currently being investigated in an open-label, multicenter Phase 1 study in subjects with relapsed/refractory B cell malignancies (NCT06014762) and is the most advanced allogeneic dual-targeting CAR-T in clinical development. P-CD19CD20-ALLO1 is a T stem cell (TSCM)-rich product derived from healthy donor pan-T cells using a nonviral transposon-based system for transgene delivery, with knockout of TRBC1/2 and B2M. Clinical data from Poseida and others have demonstrated safety and efficacy advantages associated with TSCM CAR-T, including for P-BCMA-ALLO1, our BCMA-targeting allogeneic CAR-T for Relapsed/Refractory Multiple Myeloma (NCT04960579). Both the CD19 and CD20-targeting CARs employ novel fully human single-domain VH binders. In nonclinical studies, P-CD19CD20-ALLO1 demonstrated potent antigen-specific anti-tumor activity against multiple tumor models in vivo, across multiple healthy donors and dose levels. Beyond expected advantages of targeting two antigens to prevent antigen escape, we also observed potency advantages for P-CD19CD20-ALLO1 compared to its CD19- and CD20-single targeting counterparts. In vitro potency was evaluated using serial restimulation against the CD19- and CD20-positive RAJI cell line, as well as RAJI cells engineered to express only CD19 or CD20. In these assays, P-CD19CD20-ALLO1 CAR-T cells showed superior potency against WT (CD19+CD20+) RAJI cells compared to the CD19- or CD20-single targeting products. Interestingly, this superiority was also observed against Raji cells expressing only one of the two target antigens, CD19 or CD20, suggesting that the increased potency of P-CD19CD20-ALLO1 is not solely due to the dual binding of CAR molecules to more antigens on the same target cell. Mechanistically, we observed that over three restimulations with WT (CD19+CD20+) RAJI cells, P-CD19CD20-ALLO1 CAR-T cells expressed and/or sustained higher expression of effector cytokines such as IFNγ, FasL, Granzyme A, and Granulysin than either CD19 or CD20 single-antigen targeting CAR-T cells. In addition to improved in vitro potency, we assessed the in vivo efficacy of the dual-targeting product in comparison with the single-targeting products in a stress xenograft model of WT (CD19+CD20+) RAJI. P-CD19CD20-ALLO1 CAR-T cells were superior to the CD19-targeting CAR-T product. P-CD19CD20-ALLO1, a dual-targeting, fully allogeneic TSCM-rich CAR-T product for CD19 and CD20-positive B-cell malignancies, demonstrates robust antigen-specific activity against DLBCL and CLL models and outperforms its single-targeting counterparts even in the presence of only a single antigen on target cells.

Potency assays are required for cellular and gene therapy (CGT) final product (FP) lot release, stability testing, and manufacturing updates, as per the FDA draft guidance on Potency Tests for CGT Products (2011), and Manufacturing Changes and Comparability for Human CGT Products (2023). These assays by definition measure the ability of a product to effect a desired result, and the FDA allows for “considerable flexibility” in determining the appropriate potency assay. However, standard in vitro potency assays are limited in their capacity to meaningfully assess manufacturing comparability and consistency, or product quality to provide predictive value for clinical performance. For chimeric antigen receptor (CAR)-T cell products, certificates of analysis typically report key FP attributes such as CAR expression, cell concentration and viability, impurities and target-specific in vitro potency, among others. However, clinical effectiveness of CAR-T is dependent on complex pharmacokinetics (PK)/ pharmacodynamics (PD) including in vivo engraftment, trafficking, drug exposure kinetics, tumor antigen-driven T cell expansion in tumor microenvironment, survival and persistence, and other factors that are not easily recapitulated in short-term in vitro assays. We developed a bioassay for assessing CAR-T FP potency using a tumor-bearing murine NSG xenograft model to evaluate in vivo performance of GMP-manufactured autologous (Auto) or allogeneic (Allo) CAR-T FPs. We hypothesized that this complex in vivo system may better model PK/PD and provide a more meaningful way to assess CAR-T FP potency. Preclinical dose-finding data from both RUO and GMP anti-BCMA CAR-T lots were used to establish a potency specification for RPMI-8226 tumor control at a “stress dose” of 5e6 CAR-T cells at <60% of the tumor volume compared to the untreated control group at 21 days after CAR-T cell infusion. Using these specifications, we evaluated the in vivo potency performance of GMP-manufactured Auto FP samples (P-BCMA-101; NCT03288493) generated from multiple myeloma (MM) patients and administered in the clinic with known responses ranging from poor (no response or stable disease) to significant (very good partial response [VGPR] or stringent complete response [sCR]). In addition, GMP-manufactured Allo FP samples generated from healthy donors (P-BMCA-ALLO1; NCT04960579) were also tested. We report that half of the P-BCMA-101 products (50%) previously demonstrating favorable clinical outcomes in MM patients were also able to control RPMI-8226 tumor in vivo, while none of the P-BCMA-101 products (0%) that resulted in poor clinical responses were capable of tumor control. These data show translational relevance considering the known clinical outcomes in MM patients, albeit at a high level of stringency at current specifications. Interestingly, all the Allo products (100%) performed at least as well as or better than the Auto products capable of controlling tumor, demonstrating a greater potency of Allo CAR-T cells manufactured from healthy donors. There is great demand for potency assays that can reliably assess manufacturing consistency and product quality, and that might provide some level of predictive value for clinical performance, especially considering the current limitations of standard in vitro potency assays. In vivo potency bioassays have been proposed or utilized for other products with complex biological mechanisms of action including vaccines, gene therapies, and other cell therapy products. The potency bioassay described herein demonstrated a level of positive predictive value for clinical performance using Auto FPs of known clinical outcome in MM patients. In the Auto CAR-T setting, this assay could be utilized for correlative studies including possible biomarker identification. In the Allo CAR-T setting, this potency bioassay may inform FP manufacturing consistency and quality for lot release to ensure patients receive only the highest quality products.

P-BCMA-ALLO1 is an allogeneic CAR-T that targets B-cell Maturation Antigen (BCMA) and is currently being investigated for the treatment of RRMM. P-BCMA-ALLO1 cells are manufactured from healthy donor T-cells using non-viral transposon-based integration (piggyBac® DNA Delivery System) that introduces a human anti-BCMA V H-based CAR and an iCas9 safety switch. The piggyBac® DNA delivery system produces a highly enriched T stem cell memory product. The Cas-CLOVER™ Site-Specific Gene Editing System eliminates endogenous T cell receptor (TCR) expression via knockout of the TCR beta chain 1 gene to prevent graft-vs-host disease (GvHD), and the beta-2 microglobulin gene to reduce Major Histocompatibility Complex (MHC) class I expression to eliminate host-vs-graft responses. P-BCMA-ALLO1 demonstrated compelling activity in MM xenografts, providing rationale for this first-in-human phase I study. The primary objective is to assess the safety and maximum tolerated dose (MTD) of P-BCMA-ALLO1 based on dose limiting toxicity (DLT) in RRMM patients with measurable disease who have received a proteasome inhibitor (PI), immunomodulatory agent (IMiD) and anti-CD38 monoclonal antibody therapy. Key secondary objective will assess the anti-myeloma effect of P-BCMA-ALLO1. Exploratory objectives will assess CAR-T related cytokines, serum BCMA levels and cellular kinetics. The trial utilizes a standard 3 + 3 dose escalation design to test seven planned dose levels of intravenous P-BCMA-ALLO1 ranging from 0.0625 × 10 6 to 15 × 10 6 cells/kg (NCT04960579). Multiple lymphodepletion (LD) strategies and cyclic administration of P-BCMA-ALLO1 are being investigated. Repeat P-BCMA-ALLO1 administration is allowed for patients who do not achieve at least a partial response with the first dose after 4 weeks of follow up. We are currently dosing patients in arm S (Cyclophosphamide (Cy) 300 mg/m 2 + Fludarabine (Flu) 30 mg/m 2 X 3 days), arm P1 (Cy 500 mg/m 2 + Flu 30 mg/m 2 X 3 days), arm P2 (Cy 1,000 mg/m 2 + Flu 30 mg/m 2 X 3 days) and arm C (multiple P-BCMA-ALLO1 doses following Cy 300 mg/m 2 + Flu 30 mg/m 2 X 3 days). The median patient age was 73 (33, 85) years, 61% were female, and median time since MM diagnosis was 6.27 (1.48,18.95) years. Eight (35%) patients had high risk disease by cytogenetic criteria. The enrolled patients were heavily pretreated, having received median 7 (2,16) prior lines of therapy, with 30% having undergone prior BCMA targeting therapy. As of 10 Jul 2023, 24 patients received P-BCMA-ALLO1 at 4 dose levels. Twenty-two were treated in arm S, 1 each in arms P1 and P2. One patient received 0.25 X 10 6 cells/kg (cohort -1, arm S), 7 received 0.75 X 10 6 cells/kg (cohort 1, arm S), 12 received 2 X 10 6 cells/kg (cohort 2, 10 in arm S, 1 in arm P1 and 1 in arm P2), and 4 received 6 X 10 6 cells/kg (cohort 3). One of the cohort 2 patients received a second P-BCMA-ALLO1 infusion at the cohort 2 dose level following disease progression. The median time from enrollment to start of LD chemotherapy was 2 (1-8) days and from enrollment to CAR-T infusion was 7 (6-13) days. No patient needed bridging therapy between enrollment and the start of LD. Twenty-two (all in arm S) of the 24 treated patients completed DLT evaluation by data cutoff. None of the patients treated thus far had DLTs. Most common treatment emergent adverse events (TEAEs) were anemia (36%), neutropenia (36%), constipation (36%) and leukopenia (32%). Most common ≥ grade (G) 3 TEAEs were neutropenia (36%), leukopenia (32%) and anemia (23%). Most of the AEs were considered unrelated to P-BCMA-ALLO1. Three (14%) patients developed grade 1 Cytokine Release Syndrome (CRS). One (4%) patient experienced grade 1 Immune Effector Cell Associated Neurotoxicity Syndrome (ICANS), which resolved with one dose of corticosteroids. None of the patients experienced GvHD by the last follow up. P-BCMA-ALLO1 is a non-viral transposon-generated allogeneic “off-the-shelf” CAR-T that is rapidly available for administration and well tolerated in a heavily pretreated RRMM patient population with minimal CRS/ICANS risk. Updated results including safety, preliminary efficacy, and selected biomarkers will be presented at the American Society of Hematology 2023 meeting.

We would like to respond to a recent commentary by Frederic D. Bushman in the March 2023 issue of Molecular Therapy, speculating on the possible role of the piggyBac transposon in a CAR-T trial involving two lymphomas emerging from engineered CAR-T cells. 1 Bushman F.D. DNA transposon mechanisms and pathways of genotoxicity. Mol. Ther. 2023; 31: 613-615 Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar DNA transposon mechanisms and pathways of genotoxicityFrederic D. BushmanMolecular TherapyFebruary 6, 2023In BriefRecently two lymphomas were reported in a chimeric antigen receptor (CAR) T cancer therapy trial (the CARTELL study)1,2 in which the piggyBac DNA transposon was used to deliver an engineered receptor gene to human T cells. The transformed cells were found to contain multiple integrated transposon DNA copies (4–24 per cell) and harbor multiple rearrangements in cellular chromosomal DNA. A variety of factors may have contributed to transformation, including aspects of the vector design and cell processing. Full-Text PDF

Devon J Shedlock, PhD, is Chief Scientific Officer, Cell Therapies at Poseida Therapeutics, Inc., a clinical-stage biopharmaceutical company utilizing proprietary genetic engineering platform technologies to create cell and gene therapeutics with the capacity to cure. He joined Poseida as its first employee in 2015 and most recently served as Senior Vice President of Research and Development. Dr Shedlock is a key scientific contributor in the application of the Company’s proprietary gene engineering platform technologies to develop novel cell therapy programs. Before joining Poseida, he held positions as an adjunct assistant professor of pathology and laboratory medicine at the Perelman School of Medicine and associate director of the T-Cell Engineering Laboratory that is part of Carl June’s group, both at the University of Pennsylvania. Dr Shedlock received his PhD in cell and molecular biology from the University of Pennsylvania, and a BS with Honors in biology from Ursinus College in Collegeville, Pennsylvania.

98 Background: P-PSMA-101 is an autologous CAR-T therapy targeting PSMA, with a high percentage of stem cell memory T cells (T SCM ) associated with efficacy, safety, and bone homing (particularly relevant to prostate cancer). It is manufactured using a novel non-viral transposon system (piggyBac) that creates high T SCM products. Genes are inserted encoding a PSMA-targeted Centyrin CAR, iCasp9-based safety switch, and DHFR to purify CAR-T cells. P-PSMA-101 completely eliminated tumors in intractable murine models of prostate cancer, providing rationale for this phase 1 trial (NCT04249947). Methods: Patients with mCRPC treated with or not eligible for a CYP17 inhibitor or second-generation antiandrogen, and a taxane were enrolled. P-PSMA-101 was manufactured from apheresed T cells and administered IV following a standard 3-day cy/flu lymphodepletion regimen. Dose escalation from 0.25-15 x 10 6 cells/kg is planned. Results: As of September 30, 2021, P-PSMA-101 had been administered to 10 heavily pretreated patients (median 7 prior regimens; range 3-15). Single infusions of 0.25 (n=5) to 0.75 (n=5) x 10 6 cells/kg have been assessed, with dose escalation continuing. P-PSMA-101 cells were shown to expand in blood via qPCR assay, peaking 2-3 weeks after infusion, consistent with the high percentage of T SCM . Significant antitumor responses were seen in this preliminary data set. Declines in PSA were seen in 7 patients (>50% in 3 and >99% in 1). Of 4 patients who had pre- and post-treatment FDG and PSMA-PET imaging, 3 demonstrated marked to complete resolution of abnormal uptake at known metastatic disease sites, with concordance in bone and CT scans, and/or circulating tumor cells (CTC). In 1 case, post-treatment tumor biopsy demonstrated infiltration by P-PSMA-101 CAR-T cells and elimination of tumor cells (pathologic complete response). Safety was consistent with expectations for a CAR-T product. CRS was seen in 60% (10% Gr ≥3) of patients. DLT was seen in 1 patient with macrophage activation syndrome/uveitis, and was the only Gr ≥3 CRS event. Immune effector cell-associated neurotoxicity syndrome (ICANS) has not occurred. CRS marker elevations were modest (max IL-6: 642.6 pg/mL). The most common AEs were cytopenias, infections, and constitutional symptoms (Gr ≥3 60%, 10%, and 0%), as expected with lymphodepletion. Treatable related ocular AEs were noted in 3 patients. Conclusions: These results parallel preclinical findings that P-PSMA-101 can produce marked efficacy in mCRPC, and very low doses are highly efficacious, consistent with unique product attributes such as the T SCM phenotype and bone tropism. This is the first report demonstrating profound antitumor effects of a novel PSMA-directed CAR-T-cell platform with concordant biochemical, radiographic, and pathologic parameters, demonstrating that therapeutic benefit of unarmored CAR-T cells in a major solid tumor is possible. Clinical trial information: NCT04249947.

[This corrects the article DOI: 10.1016/j.omtn.2022.06.003.].