Mol Ther Research Articles

EVEREST-1: A seamless phase 1/2 study of A2B530, a carcinoembryonic antigen (CEA) logic-gated Tmod CAR T-cell therapy, in patients with solid tumors associated with CEA expression also exhibiting human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH).

TPS2698 Background: Chimeric antigen receptor (CAR) T-cell therapies for treating solid tumors are challenging due to a lack of tumor-specific targets that discriminate cancer from normal cells. Previous studies using CEA T-cell receptors and T-cell engagers have resulted in dose-limiting, on-target, off-tumor toxicities (1,2). A2B530 is an autologous logic-gated, CEA-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining a CAR-activating receptor with a blocking receptor to discriminate tumor from normal cells (3). The activator recognizes CEA on the surface of both tumor and normal cells while specificity for tumor cells is provided by a blocker that binds HLA-A*02. In patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). HLA-A*02 LOH can be detected using next-generation sequencing (Tempus AI, Inc.). With this definitive discriminator target, A2B530 can potentially provide a therapeutic window to treat patients with CEA-expressing solid tumors exhibiting HLA LOH. Methods: EVEREST-1 (NCT05736731) is a seamless, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B530 in adult patients. Patients are enrolled through BASECAMP-1 (NCT04981119), a master prescreening study that identifies patients with HLA LOH at any time in the course of their disease. BASECAMP-1–eligible patients undergo leukapheresis and, when clinically appropriate, their banked T cells are manufactured for the EVEREST-1 study. The key inclusion criteria include histologically confirmed recurrent unresectable, locally advanced, or metastatic cancers associated with CEA expression: non-small cell lung, colorectal, or pancreatic cancers. Patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B530 and determine the recommended phase 2 dose (RP2D). The dose-escalation portion is based on the Bayesian optimal interval design. The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B530. Phase 2 will assess overall response rate per RECIST v1.1. As of January 29, 2024, 8 patients have been enrolled on EVEREST-1. A2B530 was successfully manufactured for all patients, and all patients have received A2B530 infusion, with the first patient dosed in May 2023. Dose escalation is ongoing. 1. Parkhurst, et al. Mol Ther. 2022. 2. Tabernero, et al. J Clin Oncol. 2017. 3. Hamburger, et al. Mol Immunol. 2020. 4. Sandberg, et al. Sci Transl Med. 2022. Clinical trial information: NCT05736731 .

EVEREST-2: A seamless phase 1/2 study of A2B694, a mesothelin (MSLN) logic-gated Tmod CAR T-cell therapy, in patients with solid tumors that show MSLN expression and human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH).

TPS2699 Background: Despite the success in hematologic cancers, chimeric antigen receptor (CAR) T-cell therapies are challenging to implement in solid tumors owing to a lack of tumor-specific targets that discriminate cancer from normal cells. MSLN expression normally is limited to the mesothelium of major body cavities but can be upregulated in diverse solid tumor types (TCGA 2022), making it a potential target for cancer therapy. MSLN-targeted cell approaches, including CAR T-cell and T-cell receptor fusion therapies, have shown promising clinical activity; however, on-target, off-tumor toxicity including fatal events have occurred (1-3). A2B694 is an autologous logic-gated, MSLN-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining 2 CARs: an activating and blocking receptor. The activator recognizes MSLN present on the surface of both tumor and normal cells; the blocker binds HLA-A*02 and prevents CAR T-cell activity. Thus, in patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). Through this unique discriminatory mechanism, A2B694 may provide a therapeutic window to treat patients with MSLN-expressing solid tumors exhibiting HLA-A*02 LOH. Methods: EVEREST-2 (NCT06051695) is a first-in-human, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B694 in adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression, including non-small cell lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, mesothelioma, or other solid tumors with MSLN expression. Eligible patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. Enrollment to EVEREST-2 occurs through the prescreening study BASECAMP-1 (NCT04981119), which identifies patients with tumor-associated HLA-A*02 LOH via next-generation sequencing (Tempus AI, Inc.). Eligible patients enroll in the BASECAMP-1 study and undergo leukapheresis. A2B694 is manufactured from cryopreserved T cells when clinically appropriate for patients. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B694 and identify the recommended phase 2 dose (RP2D). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Phase 2 will assess overall response rate per RECIST v1.1. 1. Beatty, et al. Gastroenterology. 2018. 2. Haas, et al. Mol Ther. 2023. 3. Hong, et al. ESMO 2021. Abstract 9590. 4. Hamburger, et al. Mol Immunol. 2020. Clinical trial information: NCT06051695 .

Phase I study of functionally enhanced CD33 CAR T cells in patients with relapsed or refractory acute myeloid leukemia.

6518 Background: Chimeric antigen receptor (CAR) T-cell therapy has shown promising efficacy in B-cell malignancies (Majzner RG, et al. Nat Med. 2019 Sep;25(9):1341-1355). However, its feasibility in acute myeloid leukemia (AML) is not fully established. CD33 CAR T-cell therapy in relapsed or refractory (r/r) AML have reported limited or no significant anti-leukemic effects (Tambaro FP, et al. Leukemia. 2021 Nov;35(11):3282-3286; Wang QS, et al. Mol Ther. 2015 Jan;23(1):184-91). CD123 CAR T-cell therapy has weak proliferation and low CR rates (Wermke M, et al. Blood. 2021 Jun 3;137(22):3145-3148; Cummins, Katherine D. et al. Blood. 2017; 130: 1359), while CLL1 CAR T-cell therapy has induced anti-leukemic responses in a small subset of patients without sustained expansion (Pei K, et al. Cancer Med. 2023 Apr;12(8):9655-9661; Jin X, et al. J Hematol Oncol. 2022 Jul 7;15(1):88). Here, we present early safety and efficacy of functionally enhanced CD33 CAR T cells in AML. Methods: We improved the performance of CD33 CAR T cells by adding a potentiating molecule linked to human CD33 scFv by P2A. The study was registered with ClinicalTrials.gov (NCT04835519). The trial used the “3+3” approach, starting with an initial dose of 5×105 (±20%)/kg. The primary endpoint was safety with efficacy secondary. Results: Four relapsed patients, including three who underwent stem cell transplantation, were enrolled and received initial dose of CAR T cells between April 13, and July 28, 2021. The Data and Safety Monitoring Committee approved the preliminary report. Three patients (75%) experienced grade 1−2 cytokine release syndrome (CRS), while one (25%) had grade 4 CRS. One developed grade 2 CRS after the second infusion. One patient had dose-limiting toxicity. Two patients (50%) had grade 1 neurotoxicity, and two (50%) developed grade 1–2 graft-versus-host disease. All patients experienced grade 2–4 neutropenia, monocytopenia, and thrombocytopenia. One patient developed sepsis. Two patients achieved CR with incomplete hematologic recovery (CRi) and were MRD-negative at day 30, while two had no response. Of these, one patient received a second infusion and achieved MRD+ CRi. Two patients have remained disease free for over two years. One patient remained disease free for one year before CD33+ relapse. The two patients with CRi had peak CAR T cell counts of 192 and 23.5 cells/μl, while the two without CRi had lower peak counts of 2.12 and 10.3 cells/μl. One patient peaked at 1.73 cells/μl after the second infusion. The two patients with CRi also had a higher proportion of CAR T cells among the lymphocytes (46.8% and 53.0%). As expected, peripheral blood CD33+ cells decreased. Conclusions: We report the safety and efficacy of functionally enhanced CD33 CAR T cells. While some patients had CRi, there was also depletion of CD33-positive normal cells. Thus, further research is needed to address the issue of normal cell depletion. Clinical trial information: NCT04835519 .

Improving ethnic and racial diversity in biomarker-driven clinical trials: A proof of concept with the BASECAMP-1 master prescreening study of patients with high-risk solid tumors with human leukocyte antigen-A*02 (HLA-A*02) loss of heterozygosity (LOH).

1604 Background: Diversity in clinical studies is essential to address health disparities, but racial and ethnic minorities are often underrepresented in clinical studies, especially in precision medicine studies using genetic data (1). BASECAMP-1 (NCT04981119) is an ongoing prescreening study to identify patients with unresectable advanced or metastatic solid tumors and tumor-associated HLA-A*02 LOH for the EVEREST-1 and -2 Tmod chimeric antigen receptor T-cell therapy trials. When enrollment began in 2021, eligibility for Part 1 was restricted to patients with germline HLA-A*02:01 based on preclinical data. Additional analysis revealed the blocker in the Tmod construct had activity across HLA-A*02 subtypes (Mock, et al. Mol Ther Oncolytics.2022). Eligibility for Part 1 was then expanded to all HLA-A*02 subtypes, allowing enrollment of more diverse patient populations. HLA-A*02 allele subtypes vary by ethnicity and race; the frequency of HLA-A*02:01 is 96% in non-Hispanic Whites, but ranges from 53% to 73% in other ethnicities and races. The frequency of all other HLA-A*02 allele subtypes (HLA-A*02:XX) is <5% in non-Hispanic Whites but up to 66% in other ethnicities and races (2,3). Methods: Patients eligible for BASECAMP-1 were identified at United States study sites. All patients screened before January 16, 2024, for Part 1 were included for demographic and central lab HLA-A typing. The % increase in eligibility and % of heterozygous HLA-A*02 patients with HLA-A*02:XX were calculated by ethnicity and race. Results: A total of 1124 patients were screened for germline HLA-A*02 typing; 431 patients were HLA-A*02:01 heterozygous, of whom 37 (9%) were Hispanic, 14 (3%) African American (AA), 2 (<1%) American Indian or Alaska Native, 17 (4%) Asian or Pacific Islander (API), and 324 (75%) non-Hispanic White (Table). The eligibility expansion identified 62 patients with HLA-A*02:XX heterozygosity, of whom 6 (10%) were Hispanic, 6 (10%) AA, 19 (31%) API, and 25 (40%) non-Hispanic White. Conclusions: By expanding enrollment to include HLA-A*02:XX, 16% more Hispanic, 43% more AA, and 112% more API patients were identified, improving the diversity in this trial compared with that of initial enrollment. 1. Aldrighetti, et al. JAMA Netw Open 2021. 2. Ellis, et al. Human Immunol. 2000. 3. Gragert, et al. Human Immunol.2013. Clinical trial information: NCT04981119 . [Table: see text]

Abstract 7250: Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors

Abstract GEN2 is a non-replicating retroviral gene transfer vector encoding a sequence-optimized herpes simplex thymidine kinase (HSV-TK), which activates the antiherpetic pro-drug ganciclovir (GCV), resulting in chain termination during DNA replication, triggering cancer cell apoptosis and release of neoantigens (NA). GEN2 also encodes hGM-CSF, which may augment antitumor immunity by enhancing NA presentation to immune effector cells. GEN2 in combination with valganciclovir (an oral formulation of GCV) was evaluated in a dose-escalating Phase 1 clinical trial in 61 adult patients (pts) with an advanced solid tumor (NCT04313868), with serial assessment of PBMCs to detect any GEN2 integration and replication competent retrovirus (RCR). Methods: PBMCs were obtained in Cycle 1 (following 1st/3rd dose), prior to and 2 hours after Cycle 2, and thereafter at 6-month intervals for 15 years, or until death or withdrawal of consent. Digital droplet PCR (ddPCR) for psi and envelope gene sequences in extracted genomic DNA from PBMCs was used to detect vector genome integration and replication competent retrovirus (RCR), respectively. Results: All 82 samples obtained from the intravenous cohorts 1-12 were below LOD (Limit of Detection) for RCR. All results for integration were BLOQ (Below Limit of Quantitation), whereas 4 samples collected at two hours following the end of infusion were above LOD, likely resulting from non-integrated cDNA. GEN2 continues to be studied in clinical trials in adult patients with solid tumors. Per the 2020 FDA Guidance for Industry (Long Term Follow-Up After Administration of Human Gene Therapy Products), serial assessment of vector integration is mandated throughout a pt’s participation in a gene therapy clinical trial and for 15 years following the last dose. These guidance documents were originally conceived when gene therapy was developed for treatment of rare hereditary diseases in pediatric pts; however, adult pts enrolled in early-stage clinical trials for cancer typically do not have a prolonged life expectancy (BMC Cancer 2011 11:426). In the context of proper and warranted usage of pts’ blood for testing, attention should be drawn to the limited expectation for a 2nd malignancy within the lifespan of this pt population, and the absence of detectable RCR in cancer patients receiving retroviral vectors over decades. A recent review noted no evidence of RCR in 1,595 post-treatment PBMCs obtained from 60 clinical trials (Mol Ther 2023 31(3):801). Our results confirm that GEN2 also shows negligible genotoxic risk for gene therapy of cancer at doses tested to date. Citation Format: Alison L. Hannah, Noriyuki Kasahara, Li Liu, Allen Wang, Joe McNulty, Robert G. Johnson. Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7250.

Trans-Amplifying RNA Vaccines Against Infectious Diseases: A Comparison with Non-Replicating and Self-Amplifying RNA.

The recent COVID-19 pandemic as well as other past and recent outbreaks of newly or re-emerging viruses show the urgent need to develop potent new vaccine approaches, that enable a quick response to prevent global spread of infectious diseases. The breakthrough of first messenger RNA (mRNA)-based vaccines 2019 approved only months after identification of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), opens a big new field for vaccine engineering. Currently, two major types of mRNA are being pursued as vaccines for the prevention of infectious diseases. One is non-replicating mRNA, including nucleoside-modified mRNA, used in the current COVID-19 vaccines of Moderna and BioNTech (Sahin et al., Nat Rev Drug Discov 13(10):759-780, 2014; Baden et al., N Engl J Med 384(5):403-416, 2021; Polack et al., N Engl J Med 383(27):2603-2615, 2020), the other is self-amplifying RNA (saRNA) derived from RNA viruses. Recently, trans-amplifying RNA, a split vector system, has been described as a third class of mRNA (Spuul et al., J Virol 85(10):4739-4751, 2011; Blakney et al., Front Mol Biosci 5:71, 2018; Beissert et al., Mol Ther 28(1):119-128, 2020). In this chapter we review the different types of mRNA currently used for vaccine development with focus on trans-amplifying RNA.

Effectiveness and Safety of Orelabrutinib Combined with Rituximab As First-Line Treatment in Marginal Zone Lymphoma

Background: Marginal zone lymphoma (MZL) collectively is the third most common type of B-cell non-Hodgkin's lymphoma (NHL) worldwide, with an increased trend in incidence during the last two decades (Rossi D E et al., N Engl J Med. 2022). The lack of predominant first-line systemic therapy and toxicities associated with current rituximab-based therapies have inspired the exploration of more optimal treatment strategies (Becnel MR et al., Br J Haematol, 2019). Given the crucial role of bruton tyrosine kinase (BTK) in the B-cell receptor signaling pathway, BTK inhibitor has been proven to yield robust outcomes with improved tolerability in the development of NHL (Dhillon S et al., Drugs, 2021; Xu W et al., Blood, 2019; Song Y et al., Blood, 2019). Orelabrutinib is a novel BTK inhibitor with high selectivity and has been approved in China for relapsed/refractory MZL recently (Dhillon S et al., Drugs, 2021). Meanwhile, several clinical studies reported that orelabrutinib in combination with rituximab in NHL could preserve natural killer cell-mediated antibody-dependent cellular cytotoxicity induced by rituximab and enhance the clinical efficacy (Yu H et al., Mol Ther Oncolytics, 2021; Qu C et al., Blood, 2023). However, orelabrutinib plus rituximab as the first-line treatment for MZL remains largely understudied, thus, we conducted a retrospective study to evaluate its effectiveness and safety. Methods: Between July 27, 2021 and March 18, 2023, 10 patients with MZL who received orelabrutinib (150 mg, qd, po) with rituximab (375 mg/m 2, iv, day 1 of each 28-day cycle) as first-line therapy were included in this retrospective study. After 6 cycles of treatment, patients underwent orelabrutinib monotherapy (150 mg, qd, po) as maintenance for a year. Dynamic dose adjustment was performed according to the patient's clinical status. Effectiveness was assessed by positron emission tomography/computed tomography-based 2014 Lugano criteria. The primary endpoint was the overall response rate (ORR); secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. Results: Ten patients (6 males; median age 63.5 years, range 45-76) were included in this study, 4 with extranodal MZL of mucosa-associated lymphoid tissue (40.0%), 2 with nodal MZL (20.0%), 1 with splenic MZL (10.0%), and 3 with unknown subtype (30%). Among a total of 10 patients, 3 (30%) achieved complete response (CR) and 6 (60%) attained partial response (PR) as their best response for an ORR of 90% ( Figure 1). After a median follow-up of 13.0 months (range 7.8-24.7), the median PFS was not reached and a 6-month PFS rate was 100% ( Table 1). OS could not be assessed due to there were no deaths occurred. As of the cut-off date (May 6, 2023), 8 patients received orelabrutinib maintenance treatment and the median duration of maintenance treatment was 9.6 months (range 3.0-17.8). The ORR was 75% (6/8) during maintenance treatment, while 1 patient had stable disease (SD) and 1 developed progressive disease (PD). In addition, abnormal hematology due to disease gradually recovered with the therapy within 4 months. No serious adverse events were observed. The off-target related AEs such as atrial fibrillation, diarrhea, and major hemorrhage were not reported. Conclusion: This retrospective data suggests that orelabrutinib with rituximab has an encouraging anti-tumor activity in MZL, with a favorable safety profile. These results provide a potential first-line treatment strategy in MZL. Further verification in prospective clinical trials of clinical outcomes of orelabrutinib in first-line MZL is warranted in the future.

A Prospective Multicenter Phase II Study of Orelabrutinib-Lenalidomide-Rituximab (OLR) in Patients with Untreated Mantle Cell Lymphoma (MCL) in China (POLARIS Study): Preliminary Analysis on Efficacy, Safety, Mutation Spectrum and Impact of Mutation Profiling on Treatment Responses

Background: Recently, the first-line treatment of MCL has been continuously evolving with the introduction of targeted agents and chemo-free regimens. It is well-known that bruton's tyrosine kinase (BTK) inhibitors were effective with MCL patients (pts). Orelabrutinib (O), a novel highly selective BTK inhibitor, has shown high response rates and good tolerability in R/R MCL (Song et al. Blood. 2020). Besides, O combined with rituximab (R) could preserve NK cell mediated ADCC induced by R and enhance the apoptosis of tumor (Yu et al. Mol Ther Oncolytics. 2021). Thus, we hypothesized that OLR would exhibit synergistic antitumor activity in MCL and improve the depth and durability of responses. Here, we reported the preliminary data on efficacy, safety, and mutation spectrum of OLR regimen in untreated MCL from the POLARIS study (NCT05076097). Methods: Pts ≥18 years with untreated MCL were enrolled in this prospective, multicenter, single-arm phase 2 study. All pts received OLR-induction therapy on a 28-day cycle for 6 cycles (O, 150 mg/d; L, 15 mg on days 1-21, then 20 mg of cycles 2-6 if tolerated or 10 mg if not tolerated; R, 375 mg/m 2 on days 1, 8, 15, 22, then day 1 of cycles 3, 5), followed by OLR-maintenance therapy for up to 18 cycles. Primary endpoint was complete response rate (CRR) after 6 cycles of induction treatment. Secondary endpoints included overall response rate (ORR), time to response (TTR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Exploratory endpoint was to explore the prognostic value of detecting peripheral blood (PB) ctDNA by NGS. Results: As of July 10, 2023,a total of 28 pts (male, 89.3%; median age, 60.0 y [range, 53.0-64.5]) were enrolled. Baseline characteristics included 96.4% Eastern Cooperative Oncology Group performance status of 0-1, 82.1% stage III-IV disease, 57.1% Ki67 index (≥30%), 75.0% low-risk MIPI scores, 42.9% bone marrow involvement, and 60.7% maximum lesion diameter of ≥5 cm. Twenty-one pts (75.0%) who have completed 6 cycles of induction therapy were evaluable for response, 16 (76.2%) had a complete response (CR) and 5 (23.8%) had a partial response (PR), with an ORR of 100% ( Figure 1). In addition, 18 of 21 pts were available for minimal residual disease (MRD) analysis, and PB-MRD and bone marrow MRD (BM-MRD) of the 18 pts were negative. The results of CRR were reported across different subgroups stratified by Ki67 index (<30% vs. ≥30%, 66.7% and 83.3%), MIPI scores (low vs. intermediate, 81.3% and 60.0%), maximum lesion diameter (<5 cm vs. ≥5 cm, 87.5% and 69.2%), and bone marrow involvement (yes vs. no, 83.3% and 73.3%). With a median follow-up of 11.0 months, the median TTR was 3.0 months (range, 2.8-3.4). Median DOR and median PFS were not reached, with the estimated 12-month DOR rate and PFS rate of 90.9% and 92.3%, respectively. Mutationalspectrum (475 panels) was determined in 23 pts. The spectrum of gene mutations is depicted by PB samples and tissue samples at baseline in Figure 2. The most common mutated genes in tissues samples were: ATM (48%), KMT2D (43%), CCND1 (17%), SMARCA4(17%), ARID2 (13%), BCOR (13%), BIRC3 (13%), NFKBIE (13%), NOTCH1 (13%), ACTB (9%), CDK12 (9%), FANCC(9%),FAT1(9%),KLF2(9%),MEF2B(9%),MET(9%),SETD2(9%), TP53 (13%),TERT(9%). The most common mutated genes in PB samples were: ATM (48%), KMT2D (39%), DNMT3A (17%), BIRC3 (13%), CCND1 (13%), TP53 (13%), BCOR (9%), CARD11 (9%), CBLB (9%), CRYBG1 (9%), FAT1(9%), NOTCH1 (9%), GRIN2A (9%), KDM5A (9%), KLHL6(9%), KRAS (9%), MAP3K14 (9%). For all gene mutations, 75.4% (43/57) detected in tissue were also found in PB samples. Further, more additional mutated genes can be detected in plasma. Meanwhile, 4 of 23 pts had a PR, 1 of 4 had multiple gene mutations in PB samples, including BTK and TP53. This suggests that ctDNA detection may potentially predict that these gene mutations are associated with depth of remission. Twenty-six (92.9%) pts experienced adverse events (AEs). The most common AEs (any grades, ≥3 grades) were neutropenia (64.3%, 39.3%), leukopenia (60.7%, 14.3%), thrombocytopenia (35.7%, 7.1%), lymphopenia (35.7%, 3.6%), and COVID-19 infection (28.6%, 7.1%). At the time of data cut-off, 26 pts remained on study. No deaths were observed. Conclusions: The preliminary findings show that the OLR exerted synergistic antitumor activity, with manageable toxicity in untreated MCL. Detection of PB ctDNA by NGS method may help to predict prognosis.

Abstract A14: Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome

Abstract The potential of immunotherapies in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains still under investigation. Clever-1 (also known as Stabilin-1) is a multifunctional scavenger and adhesion receptor expressed by monocytes and immunosuppressive macrophages. Bexmarilimab, a Clever-1 targeting antibody, demonstrates many immunomodulatory effects1 along with promising anti-tumor activity against solid tumors in patients with multiple lines of previous treatment2. In AML, high STAB1 (mRNA) levels associate with poor survival and resistance to therapy3. The aim of this study was to profile Clever-1 expression in AML and MDS and test in preclinical models the growth inhibitory and immunomodulatory potential of bexmarilimab, as a single agent and in combination with azaciditine and venetoclax. AML cell lines (n=11) and frozen mononuclear cells, extracted from AML (n=42) and very high risk MDS (n=4) patient bone marrow (BM) aspirates provided by the Finnish Hematology Registry and Biobank, were used. Samples were treated for 48h with bexmarilimab alone, or in combination with azacytidine and/or venetoclax. Flow cytometry was used to detect different myeloid and TBNK cell populations along with Clever-1, HLA-DR, PD-(L)1 and additional T cell activation markers. Our results confirmed Clever-1 protein expression in AML cell lines and bexmarilimab treatment induced metabolic and growth inhibition of KG1 blasts (Clever-1high). In patients with AML, FAB M4/M5 subtypes exhibited highest Clever-1 levels, along with FAB M2 patients with consequent rapid relapse. Clever-1 expression correlated negatively with monocyte MHC class II molecule, HLA-DR, expression, and BM T-cell frequency, in line with the immunosuppressed state associated with high Clever-1. Ex vivo treatment of the primary AML BM cells with bexmarilimab resulted in a notable, 5-10x fold increase in monocyte HLA-DR in samples with low basal HLA-DR and high Clever-1. The combination of azacitidine with bexmarilimab augmented HLA-DR induction by 20-110% (mean 44%). FAB M1/M2 AML showed increase in activation markers, such as Ki67, CXCR3 and Granzyme B after ex vivo bexmarilimab treatment in CD8+ T-cells. Furthermore, bexmarilimab reduced PD-1 expression in NK- and CD8+CXCR3+ T-cell populations of FAB M0-M2 AML and MDS-EB2. This project is the first comprehensive investigation of Clever-1 expression in AML and MDS patient BM blasts and monocytes. Ex vivo treatment with bexmarilimab, alone or in combination with azacytidine or venetoclax, indicate enhanced antigen presentation capability and immunological activation. These results validate the therapeutic potential of bexmarilimab in myeloid malignancies. The safety, tolerability and preliminary efficacy of bexmarilimab is now further investigated in combination with venetoclax and/or azacytidine in a phase I/II clinical trial BEXMAB (NCT05428969).1Viitala et al., Clin Cancer Res 2019;25:3289-303; 2 Bono et al., Annals of Oncology; 2021. p 32, 5: S1283-S346; 3 Lin et al., Mol Ther Nucleic Acids 2019;18:476-84 Citation Format: Arno Ylitalo, Sofia Aakko, Heikki Kuusanmäki, Mari Björkman, Juho Jalkanen, Marie-Louise Fjällskog, Caroline Heckman, Maija Hollmén, Mika Kontro. Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A14.

Abstract 2698: Improved calcium phosphate delivery system cNaD to aim clinical application

Abstract Super carbonate apatite (sCA) nanoparticle is a superb calcium phosphate delivery system for miRNA and siRNA to tumors (PlosOne 2015). We reported that sCA delivered efficiently siRNA for survivin and PTBP as well as miR-340 (Mol Cancer Ther 2015, 2015) to colon tumors implanted into nude mice. MiR-4689 and miR-29b-1-5p suppressed KRAS mutated colon cancer cells (Mol Ther NA2015, 2018) and miR-302 and miR-369 realized in vivo cancer reprograming (PlosOne 2015). In every case the RNA function was fully realized. Moreover, effect of influenza vaccination was augmented by sCA plus CpG nucleic acid (NA) (Frontiers in Immunol 2018). sCA coupled with miR-29a or miR-29b prevented the DSS (dextran sodium sulfate)-induced inflammatory bowel disease (Mol Ther NA2018). We also applied the sCA-ICG complex to minimally invasive treatment of photodynamic therapy (Mol Cancer Ther 2018). Following these outstanding findings, we tried and succeeded to establish a clinically available novel calcium phosphate delivery system, named cNaD, which controls the nano-particle size and avoids accumulation in normal organs including liver and spleen. It is characterized by its ability to efficiently deliver siRNA and microRNA to cancers and provided prominent therapeutic effect in various mouse models. Safety test has been accomplished in rats and monkeys based on GLP standards. Also, the scale-up manufacturing system >1000 folds has been attained. Our cNaD system will accelerate the development of NA medicine in the world. Citation Format: Hirofumi Yamamoto. Improved calcium phosphate delivery system cNaD to aim clinical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2698.

5613417 CO-TREATMENT OF ERYTHROID CELLS ISOLATED FROM Β039-THALASSEMIA PATIENTS WITH CRISPR-CAS9 GENE EDITING AND FETAL HEMOGLOBIN INDUCTION

Background: Gene editing (GE) can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenetic diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9 gene editing can be proposed for efficient correction of the β039-thalassemia mutation. GE is suitable for personalized approaches in precision medicine for β-thalassemia [1]. Aims: On the other hand, robust evidence demonstrate that increased production of fetal hemoglobin (HbF) can be beneficial for β-thalassemia patients. This can be obtained also using CRISPR-Cas9 based protocols. In this case, the objective of the gene editing strategy is (a) the silencing of transcriptional repressors of the γ-globin genes (such as BCL11A), (b) the disruption of their regulatory binding sites within the γ-globin genes, or (c) the induction of natural hereditary persistence of fetal hemoglobin mutations in the γ-globin gene or the b-like-globin gene cluster. Our study aimed to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols based on the use of repositioned drugs. Results: Gene Editing of the β039-thalassemia mutation was obtained by the CRISPR-Cas9 method; gene correction and transcription of the corrected gene have been analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; production of HbA and HbF was studied by HPLC and Western blotting. For HbF induction, the repurposed drug sirolimus (rapamycin) was used. The data obtained conclusively demonstrate that maximal production of HbA and HbF is obtained in GE-corrected, sirolimus-induced erythroid progenitors from β039-thalassemia patients. In conclusion, GE and HbF in-duction might be used in combination in order to achieve the de novo production of HbA together with increased production of induced HbF. Acknowledgements This study was sustained by the Wellcome Trust (innovator award 208872/Z/17/Z) and by AIFA (AIFA-2016-02364887). The research leading to these results has received funding also from the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Βeta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1). We thanks A.L.T. (Associazione per la lotta alla Talassemia) “Rino Vullo” - Ferrara, and A.V.L.T. (Associazione Veneta per la Lotta alla Talassemia) “Elio Zago” - APS – Rovigo and Association “Tutti per Chiara”, Montagnana, PD, Italy. Reference 1. Cosenza L.C., Gasparello J., Romanini N., Zurlo M., Zuccato C., Gambari R., Finotti A. Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β039-thalassemia patients. Mol Ther Methods Clin Dev 2021;21:507−523. https://doi.org/10.1016/j.omtm.2021.03.025

The Delivery of ABE mRNA to the Adult Murine Liver by Lipid Nanoparticles (LNPs).

A genetic disorder is a disease caused by an abnormal DNA sequence, and almost half of the known pathogenic monogenetic mutations are caused by G-to-A mutation (Landrum et al., Nucleic Acids Res 44:D862-868, 2016). Adenine base editors (ABE), developed from the CRISPR, hold the great promise to mediate the A-to-G transition in genomic DNA while not inducing DNA cleavage (Gaudelli et al., Nature 551:464-471, 2017). Additionally, lipid nanoparticles (LNPs), as a non-viral delivery, are able to deliver the ABE mRNAs and gRNA to the target tissues (Newby and Liu, Mol Ther 29:3107-3124, 2021). This chapter mainly introduces the production and LNP delivery of ABE mRNA and gRNA.

Cardiac and Skeletal Muscle Pathology in the D2/mdx Mouse Model and Caveats Associated with the Quantification of Utrophin.

Duchenne muscular dystrophy (DMD) (the most common form of muscular dystrophy) is caused by a lack of dystrophin protein. Currently, although many therapeutic strategies are under investigation, there is no cure for DMD and unfortunately, patients succumb to respiratory and/or cardiac failure in their second or third decade of life. Preclinical work has focused on the mouse model C57BL/10ScSn-Dmdmdx/J (BL10/mdx), which does not exhibit a robust pathophenotype. More recently, the D2.B10-Dmdmdx/J (D2/mdx) mouse has been utilized, which presents a more severe pathology and therefore more closely mimics the human pathophenotype, particularly in the heart. Here, we outline important considerations when utilizing the D2/mdx model by highlighting the differences between these models in addition to describing histological and immunohistochemical methods utilized in Kennedy et al. (Mol Ther Methods Clin Dev 11:92-105, 2018) for both cardiac and skeletal muscle, which can quantify these differences. These considerations are particularly important when investigating treatment strategies that may be affected by regeneration; such is the case for upregulation of the dystrophin paralogue, utrophin.

Augmenting the Persistence and Efficacy of Engineered Regulatory T Cells to Suppress Anti-Drug Antibody Formation

Background Adoptive cell therapy (ACT) using engineered antigen specific regulatory T cells (Tregs) is a powerful tool for delivering immune tolerance in autoimmune disease, transplantation and in suppressing undesirable anti-drug antibody (ADA) responses against therapeutic proteins. An important example of the latter is hemophilia A (HA), a genetic disorder characterized by deficiency of the blood clotting protein, factor VIII (FVIII). Replacement therapy with recombinant FVIII can be neutralized by ADA development in up to 30% of severe HA patients. We previously showed that ACT with Tregs expressing a chimeric CD3ε based T cell receptor fusion construct (TRuC), encoding extracellular antibody fragments with specificity to human FVIII, suppressed ADA formation to FVIII in a preclinical HA model [Mol Ther. 2021 29(9):2660-2676]. Ensuring adequate in vivo persistence and a suppressive phenotype without risk of plasticity are major concerns that impede translation of ACT with engineered Tregs. Here we evaluate a synthetic single chain antibody fusion IL2 cytokine product (IL2-IC) to selectively expand adoptively transferred FVIII-TRuC Tregs in vivo. We then combine IL2-IC delivery with FVIII-TRuC Tregs expressing the co-inhibitory receptor PD-L1 to test improved suppression to ADA formation. Methods IL2-IC was generated by fusing IL-2 cytokine to the anti-IL-2 monoclonal antibody F5111 via a flexible linker. This complex specifically delivers IL2 to cells that express high levels of IL2 receptor α (IL2Rα) (i.e., Tregs) while avoiding interaction with cells that express low levels (i.e., Tconv, NK and CD8+ T cells). To assess whether IL2-IC could expand TRuC Tregs in vivo, 2x106 FVIII TRuC Tregs were injected intravenously (IV) into HAmice followed 5 hrs later with low dose IL2-IC or IL2 fused to an irrelevant Ab (FITC-IC) as control. 24 hrs later, mice received 2 international units (IU) of FVIII, and spleens were assayed on days 3 and 6. Murine PD-L1 was co-expressed in FVIII TRuC Tregs and in vitro assessed by stimulating TRuC PDL1 Tregs with 2IU FVIII alone or in combination with 2 ug/ml of PD1-Fc to understand the impact of PDL1 signaling on the Treg phenotype. In vivo, 0.5x106 FVIII TRuC PD-L1 Tregs were IV injected in HAmice followed 5 hrs later by mock, IL2-IC or FITC-IC IV injections. 24 hrs later, mice received weekly IV injections of 1IU FVIII. Results IL2-IC treated FVIII TRuC Tregs were preferentially expanded over endogenous Tregs (fold expansion 7.47 vs 4.56 respectively) in spleens on day 3 post ACT. Whereas both IL2-IC and FITC-IC treated FVIII TRuC Tregs had higher division indices over mock treatment (4.047 ± 0.03 and 5.070 ± 0.07 over 2.00 ± 0.00), only IL2-IC treated FVIII TRuC Tregs showed increased frequencies (1.72% ± 0.376% of CD4+ T cells) as compared to FITC-IC (0.28% ± 0.005% of CD4+ T cells) or mock treatment (0.23% ± 0.023% of CD4+ T cells). FITC-IC treatment nonspecifically expanded CD8+ Tconv cells as well as NK cells, whereas IL2-IC selectively expanded Tregs. PD1-Fc triggered PD-L1 signals in FVIII stimulated TRuC Tregs resulted in significant upregulation of Treg associated activation markers FoxP3, CD69, LAP, CTLA4, PD1, ICOS and BCL2 in vitro. In vivo, IL2-IC administration combined with FVIII TRuC PD-L1 ACT led to complete suppression of ADA formation in HA mice. At 4 weeks, mice that only received FVIII infusions developed high functional ADA titers (29.53 ± 19.58 BU/mL), while IL2-IC, TRuC PDL1 Treg and TRuC PDL1 Treg + IL2-IC groups had significantly lower titers of 1.117 ± 0.952 BU/mL, 1.805 ± 0.702 BU/mL and 0.423 ± 0.243 BU/mL, respectively. FITC-IC combined with PDL1 TRuC Tregs resulted in high ADAs of average 109 ± 21.31 BU/mL, confirming the requirement for the IL2Rα targeting antibody to selectively expand Tregs. Conclusion We show that the PD-L1/PD1 axis is an important pathway that can be exploited to improve the suppressive capacity of engineered Tregs. We also demonstrate that exogenous administration of the novel IL2-IC can robustly expand FVIII TRuC Tregs. These 2 strategies can be readily combined to selectively expand and enhance the suppressive capacity of engineered Tregs. The concepts defined here can be extended for improving the persistence and suppressive potential of engineered Tregs for various autoimmune disorders and to prevent transplant rejection. We are currently evaluating long term suppression of ADA responses using this approach.

Abrogation of Immune Effector Cell Neurotoxicity Syndrome (ICANS) By Rimiducid (RIM) in Patients Treated with CD19-Specific Chimeric Antigen Receptor Modified T-Cells (CAR-T) Engineered with an Inducible Caspase 9 (iC9 CAR.19)-Clinical and Pharmacodynamic Correlates

Introduction: CD19-targeted CAR-T cells are highly active in B-cell malignancies. While early use of corticosteroids (CS) and tocilizumab have improved the management of cytokine release syndrome (CRS), CS are the mainstay of ICANS management. Though patients with low-grade ICANS often do not require therapy, severe ICANS that does not respond to CS is life-threatening. Rimiducid (RIM) is a dimerizing drug that induces apoptosis in cells engineered with an inducible caspase-9 switch. We reported the first case of the administration of a potentially ablative dose of RIM (0.4 mg/kg) to a patient with B-lymphoblastic leukemia (B-ALL) with severe, prolonged ICANS (Foster MC et al. Blood 2021 Jun 10;137(23):3306-3309). Given this patient's rapid resolution of steroid-nonresponsive ICANS and reduction in iC9 CAR.19 numbers, we sought to explore the effects of lower, potentially non-ablative doses of RIM in patients with CS-nonresponsive ICANS before the development of life threatening complications, with a goal of clinical resolution of ICANS without ablation of CAR T cells. Here, we report the clinical and pharmacodynamic courses of ICANS for the four patients treated with RIM for ICANS to date, in an ongoing expansion cohort of a phase I/II study of iC9 CAR.19 cells in patients with B-ALL. Methods: Adults with B-ALL in 2nd or greater bone marrow (BM) relapse, relapse >100 days after allogeneic stem cell transplant, disease refractory to ≥2 induction therapies, or with measurable residual disease (MRD) persistence/recurrence were enrolled. T-lymphocytes were collected from the subjects, and CAR-T cell products generated by gene modification with a g-retroviral vector encoding for iC9, ΔNGFR (for selection and tracking purposes) and CAR.CD19 (encoding 4-1BB) genes (Diaconu I et al Mol Ther 2017; 25:580). Subjects underwent lymphodepletion with fludarabine and cyclophosphamide followed by infusion of iC9 CAR.19 cells at one of two dose levels (DL1: 5 x 105 CAR-T cells/kg; DL2: 1 x 106 CAR-T cells/kg). Toxicities were graded by CTCAE v5 or ASBMT consensus grading for CRS and ICANS. CAR-T cell expansion in peripheral blood (PB) was measured by flow cytometry (FC) and droplet digital PCR (ddPCR). Cytokines were measured by commercially available multiplex (Luminex®) cytokine assays. Leukemia response was determined by NCCN criteria. Subjects who experienced grade 3-4 ICANS despite CS for >72 hours received RIM 0.4 mg/kg IV over 2 hours. After protocol amendment, subjects with ≥ grade 2 ICANS for >24 hours despite CS received RIM 0.1 mg/kg IV over 2 hours. FC, ddPCR and cytokine levels were measured prior to RIM, at 4 hours after the end of RIM infusion and daily until ICANS resolution. Results: 13 subjects were treated, 3 on DL1 and 10 on DL2, which was the expansion cohort cell dose. Median age was 31 years (range 21-69). CRS developed in 11, with median duration of 5 days (range 1-8). Maximum CRS grade was 2 in 5 and 1 in 6. ICANS developed in 5 subjects, with maximum grade 4 in 2, and grade 3 in 3. One patient with CS-responsive grade 3 ICANS did not meet criteria for RIM. Two subjects with grade 3-4 ICANS for >72 hours received RIM 0.4 mg/kg, both achieving improvement by at least 1 grade within 12 hours and with resolution within 5 days (FIGURE 1). ddPCR for ΔNGFR showed reduction in transcripts by 4 hours after the end of RIM infusion (by 83.6% and 98.6% respectively, FIGURE 2). In two subjects with grade 3 ICANS for 24 hours treated with RIM 0.1 mg/kg, ICANS improved to grade 1 in 24 hours and grade 0 in 48 hours (FIGURE 1). In these subjects, ddPCR for ΔNGFR showed reduction in transcripts at 4 hours after end of RIM infusion by 88.6% and 88.1% respectively (FIGURE 2). Rates of overall response (CR+CRi+MLFS at 4 weeks) were 3/4 RIM subjects versus 9/9 in other subjects. The subject with grade 3 ICANS who did not receive RIM demonstrated peak ΔNGFR transcripts, IL-6, TNF-α and IFNγ two weeks after iC9 CAR.19 infusion, whereas peak transcripts and cytokine levels occurred at one week, and prior to RIM for RIM-treated subjects. Conclusion: RIM administration to patients with ICANS is associated with abrupt reduction of circulating iC9 CAR.19 cells and ICANS grade. Despite detection of CAR-T cells at 3 weeks post infusion and the observance of remissions after treatment with RIM, further investigation of the effects of RIM on iC9 CAR.19 persistence and antileukemic activity is warranted. Lower, potentially less ablative doses of RIM are being explored. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Key Role of Kupffer Cells in IL-1 Dependent Activation of CD8+ T Cell Responses to AAV Transgene Product in Liver

Introduction: Adeno-associated virus (AAV) based viral vectors have emerged as preferred vectors for in vivo gene therapy and are being evaluated in multiple phase I/II and phase III clinical studies. However, immune responses to both the AAV capsid and its encoded transgene still remain a major hurdle. Previously, we demonstrated in a murine model that cellular responses to AAV encoded transgene in the liver are dependent on IL-1α/β mediated activation of IL-1R1-MyD88 signaling. Using flow cytometry and tissue staining we provided evidence that plasmacytoid dendritic cells (pDCs) and Kupffer cells are central players in initiation of IL-1 signaling. Further, we showed that cross-presenting XCR1+ dendritic cells (DCs) are critical for antigen presentation to transgene-specific CD8+ T cells (Blood 136 Supplement 1: 2-3, 2020). In the present study we tested the impact of Kupffer cells depletion on transgene-specific CD8+ T cell response. Since Kupffer cells have been implicated in TLR4-MyD88 mediated secretion of IL-1α in a murine model of acetaminophen induced liver injury (Cell Mol Immunol 15(11): 973, 2018). Therefore, we also explored if innate sensor TLR4 is required for IL-1 mediated CD8+ T cell response to AAV encoded transgene in liver microenvironment. Further, we tested if intrinsic IL-1R1-MyD88 signaling in DCs is essential for these cellular responses. Methods: To study the impact of Kupffer cell depletion on transgene-specific CD8+ T cells we intraperitoneally injected wild-type (WT) C57BL6 mice with 200μg of depleting α-mouse F4/80 antibody (BioXcell) one day prior and 2X/week following vector administered for up to 4 weeks. Groups of C57BL6 WT (control), C57BL6 WT (treated with α-mouse F4/80 antibody) and TLR4-/- mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8 vector encoding ovalbumin (OVA) as the transgene (Mol Ther 25(4): 880, 2017). To study the role of MyD88 intrinsic signaling in DCs, we generated mice that lacked adaptor protein MyD88 in DCs (DC-MyD88-/-) using cre-lox system (Blood 129(24): 3184, 2017). Mice were again injected with 1x109vg of AAV8-OVA via IV route. Four weeks after vector administration PBMCs from each group were analyzed for OVA-specific CD8+ T cells using MHC class I tetramer. Results: As expected, ~62% of WT mice developed OVA-specific CD8+ T cell response. Interestingly, only 12% of mice (1 out of 8) treated with α-mouse F4/80 antibody showed CD8+ T cells specific to OVA. Whereas similar to WT mice, 50% of TLR4-/- mice had OVA-specific CD8+ T cell. We conclude that Kupffer cells, which show increased surface expression of IL-1R1 within days after AAV vector administration, are essential for the IL-1R-dependent CD8+ T cells response to the hepatic transgene product but do not function through a mechanism that requires TLR4. This mechanism therefore shares some aspects of acetaminophen-induced liver injury (critical role of KCs and IL-1) but is distinct with regard to TLR involvement. IL-1R1 signaling requires the cytosolic MyD88 adapter molecule. We know from prior studies that MHC I antigen presentation of liver-derived AAV encoded transgene product in lymphoid organs is primarily mediated by cross-presenting XCR1+ DCs. Thus, we tested the requirement of intrinsic MyD88 signaling in DCs. Our results suggest that intrinsic MyD88 signaling in DCs is not required in lymphoid organs, as the CD8+ T cell response in DC-MyD88-/- mice was comparable to that in WT control mice (these mice lack MyD88 in conventional DCs in lymphoid organs but retain MyD88 in hepatic DCs and in pDCs). Conclusions: In summary, we established that Kupffer cells play a central role in elicitation of transgene product-specific CD8+ T cell responses in hepatic AAV gene transfer (through a TLR4-independent mechanisms). While direct sensing of IL-1 by DCs in the hepatic environment may very well play a role, it is not required in lymphoid organs. Following initial priming of CD8+ T cell in the liver, co-stimulation by CD4+ T helper cells may replace IL-1 signaling during the subsequent systemic response.

Trial in Progress: A Phase I/II Study to Assess Safety, Tolerability and Preliminary Efficacy of Bexmarilimab in Combination with Standard of Care in Patients with Hematological Malignancies (BEXMAB)

Background: Emerging data on the role of leukemic cells on shaping the tumor immune environment places macrophages as target cells and immune checkpoints in acute myeloid leukemia (AML) (Miari et. al. Front. Cell Dev. Biol.2021; Sallman et. al. Blood 2019). CLEVER-1 (common lymphatic endothelial and vascular endothelial receptor-1) constitutes such macrophage checkpoint. The receptor contributes to tumor immune tolerance by antigen clearing and T cell inhibition. In multiple models, tumor growth is significantly reduced in CLEVER-1 absence (Viitala et. al. Clin Cancer Res 2019). CLEVER-1 expression level is a prognostic factor for poor outcomes in AML (Pölönen et. al. Cancer Research 2019) and its suppression inhibits leukemia cell growth. Analyses suggests lower expression is associated with venetoclax sensitivity (Lin et al. Mol Ther 2019). Bexmarilimab (FP-1305; BEX), an IgG4 monoclonal antibody, binds CLEVER-1 and alters the function of macrophages and inactive T cells. BEX was shown to increase antigen presentation, induce proinflammatory cytokines and infiltration of activated T cells (Viitala et. al. Clin Cancer Res 2019). The therapeutic potential of BEX is supported with data from the MATINS study (NCT03733990) evaluating single agent in advanced solid tumors. Up to 40% of patients in selected indications experience disease control (stable disease/partial response) associated with a 65% chance of 1-year overall survival (OS) compared to 11% of patients without disease control. BEX treatment is well tolerated without any dose limiting toxicity (DLT) reported. CLEVER-1 is highly expression on malignant blasts and monocytes of bone marrow from AML and myelodysplastic syndrome (MDS) patients. Treatment of these BM cells with BEX alone or in combination with azacitidine or venetoclax results in enhanced antigen presentation capacity and increased activation markers on effector T cells with synergistic effects (EHA 2022 P380). Study Design and Methods: The PhI/II study investigates BEX treatment plus standard of care in MDS, chronic myelomonocytic leukemia (CMML) frontline or hypomethylating agent (HMA)-failed patients or newly diagnosed AML or relapsed/refractory (r/r) AML patients (NCT05428969). Enrollment is ongoing. Ph I is a dose escalation using a Bayesian optimal interval (BOIN) design to explore 4 dose levels of BEX in a cohort size of 5 with 1.0 mg/kg as starting dose. BEX plus azacitidine (doublet) is assessed in a mixed cohort of MDS, CMML frontline and hypomethylating agent (HMA)-failure patients or r/r AML patients. Dose determination may then be performed in separate indications. BEX plus azacitidine + venetocolax (triplet) will follow a similar BOIN design for dose determination in patients with newly diagnosed AML unfit for induction chemotherapy. BEX is dosed in 28-day cycles Q1W during cycles 1-3 followed by Q2W dosing, until disease progression or intolerable toxicity. Standard of care therapy is administered as per label. During Ph II, selected indications are explored for efficacy when BEX is administered at recommended phase 2 dose (RP2D) determined in the doublet and triplet. Dose expansion follows a 2-Stage design with indication-specific target efficacy rates, including complete remission +/- incomplete hematologic recovery, overall response rate or minimal residual disease (MRD)-negativity. Primary endpoints are safety and tolerability, RP2D of BEX and preliminary efficacy. Secondary endpoints include extended efficacy for duration of response, event free survival and OS. Exploratory endpoints are focused on pharmacodynamic parameters for immunological and cellular read-outs and the association of predictive biomarker of BEX clinical activity in different patient sub-groups. Key eligibility includes patient ≥ 18 years of age presenting with morphologically confirmed diagnosis of either MDS (intermediate-very high rIPSS risk), CMML-2 with indication for azacitidine treatment; CMML and MDS patients with failure to HMA therapy, confirmed diagnosis of r/r AML following at least 1 prior treatment line or diagnosis of AML in patients unfit for induction therapy with indication for azacitidine + venetoclax. Patients with APL or myeloproliferative CMML, active auto-immune disorder or requiring systemic corticosteroid or other immunosuppressive treatment are excluded. Approximately 181 patients at 10 US and EU sites will be enrolled.

Transient Responses and Significant Toxicities Due to Cytopenias and Rashes with Anti-CD30 CAR T Cells for CD30-Expressing Lymphomas: Results of a Phase I Clinical Trial

Although several effective therapies are available for Hodgkin lymphoma (HL), new treatments are needed, especially for chemotherapy-refractory HL. We developed a novel anti-CD30 CAR, designated 5F11-28Z, with a human antibody-derived binding domain, CD28 hinge, transmembrane, and costimulatory domains, and a CD3-ζ domain (Choi et al., Hum Gene Ther 2021; 32(13-4): 730-43). T cells expressing 5F11-28Z are designated 5F11-28Z-T; 5F11-28Z-T exhibited a full range of T-cell functions in vitro and eradicated tumors from mice. Safety and feasibility of 5F11-28Z-T were evaluated in a phase I clinical trial for patients (Pts) with CD30-expressing lymphomas (NCT03049449). Pts were divided into 2 cohorts with separate dose escalations based on prior allogeneic stem cell transplant status and received 5F11-28Z-T on one of 4 dose levels (Table). The treatment protocol was 300 mg/m2 or 500 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine on days -5 to -3 followed by infusion of 5F11-28Z-T on day 0. Normal organ function and absence of baseline Grade (Gr) 3-4 cytopenias were required for eligibility. Twenty-one Pts received 5F11-28Z-T infusions, 20 with HL and 1 Pt with anaplastic large cell lymphoma (Table). The median prior lines of therapy was 7 (4-15). Forty-three percent of Pts (9 of 21) obtained a response of partial remission or better with one (4.8%) obtaining a complete remission. The median event-free survival was 13 (4-22) weeks. Eleven Pts (52%) had cytokine release syndrome (CRS): 4 (19%) with Gr 1, 6 (29%) with Gr 2, 1 (4.8%) with Gr 3, and no Pts had grade 4-5. Two Pts (9.5%) received tocilizumab; no Pts received corticosteroids for CRS. Neurologic toxicity possibly related to CAR T cells (graded by CTCAE v 5) occurred at Gr 2 in 5 (24%) Pts, with no Gr 3-4 events. There were no treatment related deaths. Nine Pts (43%) had new onset maculopapular or pustular rashes after 5F11-28Z-T infusion (Figure). Rashes occurred a median of 11 (7-55) days after 5F11-28Z-T infusion and lasted at Gr of 2 or higher for a median of 7 (3-39) days. Six Pts had Gr 2 rashes; 3 Pts had Gr 3 rashes. Two Pts with Gr 3 rashes lasting 29 and 39 days received systemic corticosteroids for management of rash. One Pt received phototherapy. Nineteen Pts (90%) had Gr 3-4 neutropenia; 5 (24%) had Gr 3-4 thrombocytopenia; and 6 (29%) had Gr 3-4 anemia. Three Pts (14%) had Gr 3-4 cytopenias lasting more than 30 days. Two Pts had prolonged pancytopenia, lasting 77 and > 82 days; both Pts had infections requiring intensive care while neutropenic. Three Pts received eltrombopag; 2 Pts received corticosteroids for management of prolonged cytopenias. Eleven bone marrow (BM) biopsies were performed for 5 Pts to evaluate cytopenias. All biopsies showed hypocellularity; 10/11 (4 Pts) showed trilineage or myeloid hypoplasia, 4/11 (2 Pts) showed fibrosis, 1 showed stromal gelatinous changes, and none showed HL or dysplasia. Blood 5F11-28-T were detected in all 15 patients assessed so far. A median of 2.2% (range 0.1-29.4%) of peripheral blood mononuclear cells contained the CAR gene. An immunohistochemical stain for CAR+ cells was performed with an antibody against the CAR linker. Seven Pts had technically adequate tumor biopsies within 30 days following 5F11-28Z-T; infiltrating CAR+ cells were not detected in any of these biopsies. For comparison, post-treatment tumor biopsy specimens from Pts with B-cell lymphoma were collected within 30 days after infusion of anti-CD19 CAR T-cells (Brudno et al., Nat Med 2020; 26: 270-80). These specimens were stained for CAR+ cells with the same antibody used to stain for 5F11-28Z-T. Two of 5 Pts (40%) had one or more tumor biopsy samples with detectable CAR+ cells. Four Pts with rashes after 5F11-28Z-T had skin biopsies, which showed a spongiotic dermatitis. CD30 was detected on mononuclear cells in 2 biopsies (2 Pts). Of 3 evaluable skin biopsies, 1 performed at Day 7 had detectable CAR+ cells in the dermis; none from later time points had CAR+ cells. Three Pts with Gr 4 cytopenias had 5 evaluable BM biopsies after 5F11-28Z-T. The earliest biopsy, performed at Day 16, had CAR+ cells. In conclusion, 5F11-28Z-T cells have limited anti-malignancy efficacy. Severe toxicities from rashes and myelosuppression may be due to CD30 expression on dermal mononuclear cells and on hematopoietic stem cells (Hombach et al., Mol Ther 2016; 24(8):1423-34). These toxicities along with limited anti-lymphoma activity prompted us to end this clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First-in-human clinical trial of an oncolytic adenovirus armed with TNFa and IL-2 in patients with advanced melanoma receiving adoptive cell transfer of tumor-infiltrating lymphocytes.

TPS9590 Background: The long-term complete remission experienced by some cancer patients after receiving immunotherapies, such as adoptively transferred tumor-infiltrating lymphocytes (ACT-TIL), represent the ideal outcome to pursue for the development of therapies for oncology. At the same time, those responses are limited to a minority of treated patients, and adverse events resulting from preconditioning chemotherapy and postconditioning IL2 are a rather common scenario. TILT-123 is an oncolytic adenovirus (Ad5/3-E2F-D24-TNFa-IRES-IL2) designed to enable T-cell therapies and checkpoint inhibition against cancer. Ultimately, the aim of this approach is to expand the proportion of patients benefiting from immunotherapies. Extensive preclinical studies with this technology showed that the virus repolarizes the tumor’s immune microenvironment in a way that favors T-cell presence and their activity against tumor cells. When TILT-123 was used together with TILs in preclinical in vivo models, animals had a higher chance to display curative results while showing reduced toxicity as the use of TILT-123 replaces the pre and post conditioning (Havunen R. et al Mol Ther Oncolytics 2016, Santos J.M. Mol Ther 2018). A Phase I clinical trial (NCT04217473) is ongoing to evaluate safety of the approach in advanced melanoma patients. Extensive biological assays of patient aim to characterize viral transduction of tumors through the intravenous and intratumoral routes, and the recruitment of activated lymphocytes to tumors in order to elucidate the immunological impact of the drug. Methods: The primary aim of NCT04217473 is to evaluate safety of TILT-123 in advanced melanoma patients. Refractory or recurrent stage III/IV patients, which cannot be treated with curative intent with available therapies, and are eligible for ACT-TIL therapy, can potentially participate in the study. TILT-123 administration begins while the manufacturing of ACT-TILs takes place and continues after the TIL-therapy is administered to the patient. In contrast with standard ACT-TIL therapy, patients are not conditioned with lymphodepletion or IL-2 in this approach. Patients must present at least one biopsiable/operable tumor for the generation of TILs and another injectable lesion for intratumoral administration of TILT-123. The open label, dose escalation trial has the main endpoint of establishing TILT-123 safety by day 36 (prior to TIL administration), which is based on the incidence of adverse events, severe adverse events, vital signs, ECG, and safety laboratory results. Secondary endpoints include safety and tolerability after TIL therapy has been administered, evaluation of antitumor responses and studies of tumor immune repolarization. Cohorts 1-3 have been completed without DLTs. Enrollment in cohort 4 was initiated in December 2021. Clinical trial information: NCT04217473.

BASECAMP-1: Leveraging human leukocyte antigen (HLA) loss of heterozygosity (LOH) in solid tumors by next-generation sequencing (NGS) to identify patients with relapsed solid tumor for future logic-gated Tmod CAR T-cell therapy.

TPS2676 Background: Solid tumors comprise > 90% of cancers. Metastatic colorectal cancer, non-small cell lung cancer, and pancreatic cancer are among the leading causes of cancer-related mortality (5-year overall survival: 14%, 6%, and 3%, respectively) (ACS. 2021). Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies (Neelapu S. et al. N Engl J Med. 2017). Translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities (Parkhurst M, et al. Mol Ther. 2011; Tanyi J. Cellicon Valley '21). To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust NOT logic gate capable of killing tumor cells, while leaving healthy cells intact (Hamburger A, et al. Mol Immunol. 2020). The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells. The frequency of HLA LOH among advanced GI solid tumor cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%-20.8% between colorectal, pancreatic, and gastroesophageal tumors (Hecht R. et al. ASCO-GI 2022. Abstract #190). As such, HLA LOH offers a definitive tumor versus normal discriminator target for CAR T-cell therapy. Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives of 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) To obtain leukapheresis and feasibility for the future EVEREST Tmod CAR T-cell trial. Methods: BASECAMP-1 (NCT04981119) patient eligibility has 2 parts: 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xT-Onco NGS testing. 2) If the tumor demonstrates HLA-A*02 LOH and the patient is eligible after screening, the patient will undergo leukapheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. Clinical trial information: NCT04981119.