Summary of treatment-related adverse events with acapatamab monotherapy occurring in ≥ 5% of patients

Representativeness of study participants

Radiographic progression-free survival in patients according to baseline tumor PSMA expression

List of worst grade 4 treatment-emergent adverse events in dose exploration and dose expansion

Summary of dose-limiting toxicities

Acapatamab-induced PSA response in patients who received prior lutetium-PSMA therapy

List of prior therapies in patients who received acapatamab monotherapy

Duration of PSA response in dose expansion

Key medications used for the management of cytokine release syndrome

AbstractPurpose: Safety and efficacy of acapatamab, a prostate-specific membrane antigen (PSMA) x CD3 bispecific T-cell engager were evaluated in a first-in-human study in metastatic castration-resistant prostate cancer (mCRPC). Patients and Methods: Patients with mCRPC refractory to androgen receptor pathway inhibitor therapy and taxane-based chemotherapy received target acapatamab doses ranging from 0.003 to 0.9 mg in dose exploration (seven dose levels) and 0.3 mg (recommended phase II dose) in dose expansion intravenously every 2 weeks. Safety (primary objective), pharmacokinetics, and antitumor activity (secondary objectives) were assessed. Results: In all, 133 patients (dose exploration, n = 77; dose expansion, n = 56) received acapatamab. Cytokine release syndrome (CRS) was the most common treatment-emergent adverse event seen in 97.4% and 98.2% of patients in dose exploration and dose expansion, respectively; grade ≥ 3 was seen in 23.4% and 16.1%, respectively. Most CRS events were seen in treatment cycle 1; incidence and severity decreased at/beyond cycle 2. In dose expansion, confirmed prostate-specific antigen (PSA) responses (PSA50) were seen in 30.4% of patients and radiographic partial responses in 7.4% (Response Evaluation Criteria in Solid Tumors 1.1). Median PSA progression-free survival (PFS) was 3.3 months [95% confidence interval (CI): 3.0–4.9], radiographic PFS per Prostate Cancer Clinical Trials Working Group 3 was 3.7 months (95% CI: 2.0–5.4). Acapatamab induced T-cell activation and increased cytokine production several-fold within 24 hours of initiation. Treatment-emergent antidrug antibodies were detected in 55% and impacted serum exposures in 36% of patients in dose expansion. Conclusions: Acapatamab was safe and tolerated and had a manageable CRS profile. Preliminary signs of efficacy with limited durable antitumor activity were observed. Acapatamab demonstrated pharmacokinetic and pharmacodynamic activity.

Best (unconfirmed) prostate-specific antigen response in dose exploration

Pharmacokinetics of acapatamab

List of dose-limiting toxicities

ARDENT is a phase 1 study evaluating safety and tolerability of SC291, a hypoimmune (HIP), allogeneic, CD19-directed CAR T cell therapy in subjects with Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia (CLL). SC291 is derived from healthy donor CD4+ and CD8+ T cells that are genetically engineered to disrupt function of CD3 and HLA class I/II, overexpress CD47, and express a CD19-directed CAR. A primary obstacle for allogeneic CAR T cell approaches remains their immune recognition and rejection. Patient immune responses prevent durable allogeneic CAR T clinical responses despite the use of enhanced lymphodepletion (LD) regimens. We therefore developed hypoimmune CAR T cells that avoid immune recognition and have the potential to deliver clinical efficacy while using a standard LD chemotherapy regimen (as defined by approved autologous CAR T products). The HIP engineering approach involves the depletion of HLA class I/II to prevent adaptive immune rejection and the overexpression of CD47 to prevent innate immune rejection, which has previously demonstrated alloimmune protection in preclinical models ( Nat Biotechnol 2019;37(3):252-258; Proc Natl Acad Sci U S A 2021;118(28):e2022091118; J Exp Med. 2021;218(3):e20200839; Nat Biotechnol. 2023; doi: 10.1038/s41587-023-01784-x, and Nat Commun. 2023;14(1):2020). The initial patient, a 74-year-old male with CLL (unmutated IGHV, del(11q), BTK C481S mutation) and 3 prior lines of therapy (FCR, ibrutinib, venetoclax + rituximab), received a LD regimen of cyclophosphamide 500 mg/m 2 and fludarabine 24 mg/m 2 (daily for 3 days) followed by a starting dose of 60 million CAR+ SC291 cells (of which approximately 80% are fully HIP engineered cells). SC291 was well tolerated with no observed CRS or ICANS. The patient developed non-neutropenic fever after LD (prior to SC291 infusion). At the Day 28 visit post-SC291 infusion, the patient had a partial response (per iwCLL 2018 criteria), along with significant (99%) B-cell reduction and improvements in platelet levels. Analysis of cellular kinetics is ongoing. SC291 is a mixture of T cells, in which portions are partially or fully HIP-engineered. As such, we evaluated the initial patient's immune response against SC291 subpopulations using a panel of previously described in vitro assays. Blood samples from the patient were taken pre-treatment (Day -5) and at the Day 13 and Day 28 visits following SC291 treatment and were assessed for immune evasion. The patient's PBMCs were sorted into CD3+ T cells and CD3-CD56+ NK cells and incubated with the following subpopulations sorted from SC291 drug product (DP): (i) fully HIP engineered (CD19 HIP CAR T cells), (ii) HLA I/II negative T cells without CD47 overexpression (DKO), and (iii) HLA I/II positive CAR T cells overexpressing CD47 (WT CD19 CAR T cells). Patient serum from Day -5 and the Day 28 visit were used for donor-specific antibody analyses, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) against the DP subpopulations. At Day -5, T cell EliSpot assays showed no immune responses against any of the SC291 sorted subpopulations, but by Days 13 and 28, these assays produced high and increasing IFN-g spot frequencies against the WT CD19 CAR T cells. Similar results were shown with T cell cytotoxicity assays (Fig. 1B). In contrast, no T cell activation and no T cell killing was observed with DKO and CD19 HIP CAR T cells at any time point. As expected, patient NK cells vigorously killed DKO T cells at all time points but spared WT CD19 CAR T and CD19 HIP CAR T cells (Fig. 1B). No antibodies against any sorted subpopulation were detected at Day -5, but IgG antibody binding of the WT CD19 CAR T population was observed at Day 28. These antibodies did not bind to DKO or CD19 HIP CAR T cells, and there was no CDC or ADCC response against these subpopulations. Interestingly, with the LD regimen used, we observed a 95% and 99% reduction of CD19+ cells on Days 0 and 28, respectively, while T cells rebounded from an 88% reduction on day 0 to pretreatment levels on Day 28 (Fig. 1A). These data demonstrate the preliminary safety and tolerability of SC291 in the initial treated patient. Immune assays demonstrated that the CD19 HIP CAR T cell subpopulation effectively evades the host adaptive and innate immune responses and could overcome the allogeneic barrier in humans. Additional data from the ARDENT study will be presented at the time of the conference.

PDF file - 12MB, Movie of SCp2 cells infected with vector (left movie) or Znf217 (right movie) following a scratch with a pipette tip. This movie ran for 20.25 hours

PDF file - 117K, Microarray target genes from MCF7 siRNA. Target genes identified by microarray expression analysis from MCF7 cells Znf217 siRNA

PDF file - 37K, Drug list identified in silico. List of drugs identified in silico to inhibit breast cancer cell lines that overexpress ZNF217. Correlation of ZNF217 expression in the cell line panel with the drug panel of ~50,000 drugs generated by the NCI Developmental Therapeutics Program (dtp.nci.nih.gov) identified several drugs that selectively inhibited growth of cells, assessed by GI50, expressing high levels of ZNF217 with a low drug concentration

Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering a CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate in vivo CD19-directed chimeric antigen receptor (CAR) T cells in patients. For an in vivo gene delivery platform there are several approaches for administration that could be considered including direct intravenous (IV) administration or extracorporeal delivery (ECD). With ECD, patients would first undergo apheresis, then fusosomes would be retained with patient peripheral blood mononuclear cells (PBMCs) in the apheresis bag for a brief period of time before the entire contents of the bag is IV delivered into the same patient. Here, we demonstrate the feasibility of this approach to generate in vivo CAR T cells in a clinical setting with a mock infusion and discuss the translation of these findings to direct IV administration. A healthy donor was apheresed to collect PBMCs using standard apheresis equipment. No further cell enrichment, separation, or washing was performed. CD8-targeted fusosome formulated in an isotonic cryoprotective buffer was administered into the apheresis bag by gravity via an infusion set at a dose of ~0.5 IU/cell, gently mixed and retained for 30 min. The contents of the bag were mock infused into sample collection bags at a controlled flow rate using an infusion pump. Samples were collected before and after fusosome addition, after 30 min retention, and every 30 min during the mock infusion. Cell counts and viability were measured by complete blood count (CBC) and flow cytometry analyses. Fusosome binding to CD8 + T cells was measured by flow cytometry. Cytokines and complement components in supernatants were measured by ELISA or MSD. Residual fusosome in the supernatant was measured by ddPCR. Transduction of CD8 + T cells was measured by flow cytometry and vector copy number (VCN) analysis after PBMCs were washed, activated with CD3/CD28 beads, and cultured for 8 days. Early transcription events were measured by ddPCR using specific amplicons targeting different DNA species generated during reverse transcription. The CD8-targeted fusosome was successfully deliveredusing aseptic techniques to a freshly collected apheresis bag.PBMC viability stayed high (>%95) throughout the process. There were no major changes in white blood cell, platelet, or red blood cell levels as measured by CBC analysis. There were no changes in CD8 + T cell phenotype throughout the time course. Fusosome binding to CD8 + T cells was detected as early as fusosome addition to the apheresis bag (~5 min) and increased over the course of the process. Analysis of vector genomes in the supernatants indicated presence of unbound residual fusosome. Exposure of PBMC to fusosome did not induce cytokine secretion or complement activation throughout the process. Early events of transduction of cells could be detected by analysis of reverse transcription intermediates. Analysis of cultured cells via both flow cytometry and VCN analysis indicated generation of CD8 + CAR T cells upon short-term exposure to fusosome. In vivo delivery ofCD8-targeted fusosome encoding a CD19CAR transgene enables a novel CAR T cell approach. This study demonstrated that administration of fusosomes via ECD is operationally feasible in a clinical setting and scale as a novel method of administration to generate CD8 + CD19CAR T cells in vivo. ECD setup also served as an in vitro pharmacokinetic model for direct IV infusion. In scenarios where in vivo exposure is limited due to very rapid clearance (e.g. few minutes), ECD has the benefit of reducing the volume of distribution and bringing T cells and fusosomes in close proximity for efficient gene delivery. The findings of study suggest that with rapid binding of fusosomes to CD8 + T cell leading to early transcription events, and an in vivo half-life of more than 30 min, fusosome may generate CAR T cells successfully via direct IV administration.

Introduction: Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate CD19-directed chimeric antigen receptor (CAR) T cells in patients after direct intravenous delivery or extracorporeal delivery (ECD), i.e., a short-term exposure of apheresis product to fusosome before re-infusion into the patient. Here, we demonstrate that an improved manufacturing process resulted in higher fusosome titer and quality, increasing the potency of CAR T generation from resting CD8 T cells. This potency is also observed when fusosomes are evaluated in a clinically relevant mock ECD approach. Methods: Fusosome titer was quantified by ddPCR directly for physical titer (vector genomes, GQA) or after infections of SupT1 cells for functional titer (IU/mL). Transduction of CD8 T cells was measured by flow cytometry and vector copy number analysis after short-term (1-4 hour) incubation of PBMCs with fusosome followed by wash and culture in resting or CD3/CD28-activated conditions. To assess CD19CAR T cell generation at a mock clinical setting and scale, a healthy donor was apheresed to collect PBMCs, fusosome was administered into the apheresis bag and PBMCs were sampled from the bag over a 2.5-hour time course for further analysis. In vivo anti-tumor efficacy of the fusosome was measured in immune-deficient NSG mice engrafted intravenously with Nalm6, then PBMCs followed by fusosome one day later. Tumor growth was monitored by bioluminescence imaging. Results: Fusosomes produced by the improved process had both a higher functional concentration (>14-fold higher IU/mL) and better infectivity (>10-fold higher IU/GQA) in comparison to fusosomes produced by the original process. Exposure of PBMCs to fusosomes produced by the improved process led to increased fusosome binding to CD8 T cells at high IU/PBMC doses and higher transduction efficiency in CD8 T cells at the same calculated dose per cell compared to fusosomes produced by the original process (>3-fold higher mean CAR+ percentage after 2-hour exposure to 2 IU/PBMC, n=8). Furthermore, fusosomes produced by the improved process generated higher frequencies of CAR+ CD8 T cells and resulted in increased ability to control tumor growth at lower doses in vivo in a Nalm6 model. Importantly, the ECD approach translated well to a mock clinical setting, resulting in successful generation of CAR T cells (12.6% of CD8 T cells after a 2.5-hour incubation). Conclusion: CD8-targeted fusosome encoding a CD19CAR transgene represents a potential novel therapeutic approach for cancer. This study demonstrates that improvements in the manufacturing process can significantly increase the potency of the fusosome in T cells, and we intend to move forward to clinical trials using this improved process. Citation Format: Jesse Green, Brian Granger, Ouwen Liang, Jesus Moreno, Andrew Tucker, Aesha Vakil, Reuben Burch, Nicholas Mozdzierz, Matthew Scott-Skandera, Stephanie Riofrio, Mike DeCosta, Amey Gaikwad, Sanket Phadke, Kaely Gallagher, Adam Charlton, Sarah Miller, Hosein Kouros-Mehr, Misha Shamashkin, Neal Van Hoeven, Kutlu Elpek, Terry Fry. Increased potency of CD8-targeted fusosomes enhances CAR gene delivery to resting primary T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB311.