Moreno-Cortes, Eider

Abstract Background: Chimeric Antigen Receptor (CAR) T cell therapy is arguably one of the most significant breakthroughs in cancer treatment. There are currently five FDA-approved products that are commercially available. However, despite their success, these CAR T-cell therapies cannot induce long-term durable responses in approximately 50% of leukemia or lymphoma-treated patients. Similarly, the results of CAR T-cells in solid tumors have been somewhat disappointing. Therefore, there is an urgent need to design and develop novel CAR T cells with improved efficacy in hematologic malignancies and solid tumors. ROR1 is a carcinoembryonic antigen expressed in different cancers and is associated with tumor stemness, proliferation, metastatic transformation, and treatment resistance. In this project, we optimize an anti-ROR1 CAR using a humanized single-chain variable fragment (scFv) with second (2G) or third-generation (3G) costimulatory domains. Methods: Several optimization steps in silico were performed using a selected scFv binding domain that targets ROR1. Those included codon optimizations, positional arrangement of heavy-light chains, evaluation of the ideal length of linkers based on tridimensional modeling of the docking between the antibody-like paratope with the target antigen (Figure 1A). After this initial scFv optimization process, we constructed a lentiviral vector that encodes CARs using the selected scFv linked to a transmembrane domain CD28 and different signaling endodomains for 2G and 3G variants (CD28, 41BB, ICOS, OX40), each linked to the T cell receptor CD3z domain. The cytotoxic activity of these constructs was evaluated using an in vitro rechallenge luciferase assay in ROR1 expressing JeKo-1 cells and ROR1(negative) controls. Results: The 2G 41BB-z construct with V H-V L scFv orientation and a long linker (V H-L-V L) showed optimal cytotoxicity with a CAR expression level in T cells of 36% (Range 28-49% for other constructs, Figure 1B-C). The V H-L-V L 41BB-z construct was evaluated comparatively using a rechallenge cytotoxic assay with 3G constructs that expressed CD28, ICOS, or OX40 signaling domains using JeKo-1 and ROR1(negative) target cells as controls. All the tested constructs showed specific ROR1 medicated cytotoxicity. CD28-41BB-z and ICOS-41BB-z showed the lowest cytotoxicity levels during the Day 1 of the repetitive rechallenge. However, the cytotoxicity levels of those constructs gradually increased during the 7 days of rechallenge and were closed to the levels induced by the 2G- 41BB-z construct (>80% of cytotoxicity). There were no significant differences in CAR T cells subsets generated by the different constructs during the 7 days of rechallenge with a predominance of effector memory phenotype (CCR7-, CD45RA-) and no difference in PD1 expression. Conclusions: Our results demonstrate that optimization of the CAR constructs enhances T-cell effector function and cytotoxicity against ROR1+ target cells. In previous studies, 3G CARs have shown longer persistence of the transduced T cells in peripheral blood, sustained and regulated cellular activation, improved solid tumor infiltration, and positive modulation of the tumor microenvironment. Our preclinical in vitro optimization demonstrates strategies to generate 3G constructs with a progressive and modulated cytotoxic profile that may confer benefits when tested in vivo in terms of enhanced persistence and lower adverse events profile. Additional experiments in vivo will be presented during the meeting to corroborate our findings. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective/background: Despite the success of chimeric antigen receptor (CAR) T-cell therapy in patients with aggressive non-Hodgkin lymphoma (aNHL), some patients still fail treatment, and their prognosis is dismal. Methods: We performed a retrospective study of aNHL patients treated with axicabtagene ciloleucel (axi-cel) at two Mayo Clinic centers between 2018 and 2020. We evaluated predictive factors, toxicities, and responses to salvage regimens after CAR T-cell therapy. Results: Thirty-four patients received axi-cel with a median length of hospitalization of 14 days. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome of any grade occurred in 91% and 41% of patients, respectively. Furthermore, 71% of patients responded to therapy, with 53% achieving a complete response (CR). The CRS grade and absolute lymphocyte count at leukapheresis (ALC Leuk ) correlated with CR and overall survival (OS), respectively. After a median follow-up of 6.8 months (interquartile range [IQR] 4.6–14.9), 15 patients (44%) showed progressive disease (PD). Most patients (60%) progressed during the first 3 months and had persistent CD19 tumor expression. Elevated C-reactive protein at baseline increased the risk of PD, whereas elevated ferritin increased PD and mortality risk. Twelve patients received salvage therapy, but only three responded. Median OS of relapsed/refractory patients to axi-cel was 3 months (IQR 1.3–5.1). Conclusion: The grade of CRS and ALC Leuk correlated with better outcomes to axi-cel therapy. In addition, elevated inflammatory markers at baseline were associated with PD and shorter survival. Relapses after treatment frequently occur within months after axi-cel infusion; they confer a poor prognosis and create an urgent need for novel and effective treatment options in this patient population.

Abstract Ibrutinib-based therapies are costly and require continuous administration. We hypothesized combining BTK inhibition with anti-CD20 monoclonal antibodies would yield deep remissions allowing discontinuation. We enrolled 32 therapy-naïve CLL patients to receive ibrutinib plus obinutuzumab, followed by single-agent ibrutinib. Patients could discontinue ibrutinib after 36 months with sustained complete response (CR). We evaluated treatment safety, efficacy, and outcomes after ibrutinib discontinuation. The overall response rate was 100%, 28% achieved a CR, and 12.5% achieved bone marrow undetectable minimal residual disease. At a three-year median follow-up, 91% remain in remission with 100% overall survival. Five patients in sustained CR stopped ibrutinib and have not progressed. Eight non-CR patients discontinued for other reasons, with only two progressing. The treatment was safe, with a lower IRR rate. All patients responded to treatment with longer time-to-progression after discontinuation of ibrutinib. Our data support the evaluation of ibrutinib discontinuation strategies in more extensive clinical trials.

Single oral targeted therapies have emerged as a standard of care in chronic lymphocytic leukemia (CLL). However, accessibility, side effects, and financial burden associated with long term administration limit their clinical use. Mainly, it is unclear in what clinical situation discontinuation of oral therapy can be recommended. The combination of type II anti-CD20 antibody obinutuzumab-Gazyva® with ibrutinib (GI) has shown a significant progression-free survival benefit in patients (pts) with CLL, including those with high-risk genomic aberrations. We conducted a phase 1b/2, single-arm, open-label trial to evaluate the safety and efficacy of GI as first-line treatment in 32 CLL pts. We report the outcome in pts that discontinued ibrutinib (either after 3 years of sustained complete response (CR) as stipulated in the clinical protocol, or due to other reasons). CLL pts enrolled in this protocol were ≥65 years old, or unfit/unwilling to receive chemotherapy. Pts received GI for six cycles, followed by daily single-agent ibrutinib. The protocol was designed to ensure that pts with a sustained CR after 36 months were allowed to discontinue ibrutinib. The median age was 66 years (IQR 59-73), and 6% of the evaluated pts had 17p deletion. All pts were able to complete the six planned cycles of obinutuzumab. The combination regimen was well-tolerated, and the most common adverse events (>5% CTCAE grade 3-4) were neutropenia, thrombocytopenia, and hyperglycemia. The rate and severity of infusion-related reactions (IRR) were much lower than expected (Grade≥ 3, 3%), and pts without IRR had lower serum levels of cytokines/chemokines CCL3 (P=0.0460), IFN-γ (P=0.0457), and TNF-α (P=0.0032) after infusion. The overall response rate was 100%, with nine pts (28%) achieving a CR, and four pts (12.5%) with undetectable minimal residual disease (uMRD) in the bone marrow, defined as <10-4 CLL cells on multicolor flow cytometry. At a median follow-up of 35.5 months (IQR 24.5-42.7) after starting treatment, 91% of the enrolled pts remain in remission with a 100% overall survival. Sixteen pts have completed a long-term follow-up of 36 months. Six pts showed CR, with three of them achieving uMRD in the bone marrow. Ten of these pts were in PR, and only one had disease progression and started treatment for symptomatic stage I disease with obinutuzumab plus venetoclax. In total, thirteen pts (41%) have stopped ibrutinib, with a median time on treatment prior to discontinuation of 35 months. Five (16%) of these pts had CRs and discontinued after 36 months. Eight additional pts (25%) had PRs and discontinued ibrutinib without being eligible: three pts discontinued prior to 36 months due to toxicities, and five pts discontinued after 36 months (3 due to side effects, and 2 due to financially driven decision). One patient eligible to discontinue ibrutinib, decided to remain on treatment despite sustained CR. After a median follow up time following ibrutinib discontinuation of 8 months (IQR 3.5-17), only two out of 13 pts have progressed (10 and 17 months after Ibrutinib discontinuation). None of the pts that stopped ibrutinib after achieving a CR have shown signs of disease progression. Of note, the pharmaceutical sponsor provided ibrutinib for the first 36 months, after which pts or their insurer became financially responsible. This particular scenario could bias the discontinuation pattern compared to a real world experience. It also provided us with a perspective about diverse factors affecting the treatment choices of pts. In summary, the obinutuzumab plus ibrutinib combination therapy was well-tolerated, with a much lower IRR rate. Efficacy compares favorably with historical controls with all pts responding to therapy, no deaths associated with treatment or disease progression, and a longer than expected time-to-progression after discontinuation of ibrutinib. The rate of ibrutinib discontinuation was higher than reported in the literature, most likely influenced by the protocol design and financial decisions driven by the switch from sponsor-provided ibrutinib to insurance or self-paid medication. Our observations regarding safety, efficacy and lack of disease progression after ibrutinib discontinuation are encouraging, and warrant confirmation in long-term prospective studies. Clinicaltrials.gov Identifier NCT02315768. Funding: Pharmacyclics LLC. Disclosures Choi: AbbVie: Consultancy, Speakers Bureau. Amaya-Chanaga:AbbVie: Ended employment in the past 24 months, Other: Research performed while employed as an investigator of this study at UCSD.. Kipps:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Castro:Kite Pharma: Research Funding; Pharmacyclics: Research Funding; Fate Therapeutics: Research Funding.

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has changed the treatment landscape for patients with Non-Hodgkin Lymphoma (NHL). Despite the excellent responses in relapsed or refractory (R/R) aggressive NHL (aNHL), the outcome of patients (pts) that fail CAR T-cell therapy remains poor, and there is not a clear path for management of their disease. Methods: We conducted a retrospective analysis of aNHL pts treated with axicabtagene ciloleucel (axi-cel) at the Mayo Clinic campuses in Arizona and Florida between June 2018 and August 2020. We evaluated the predisposing factors, management, toxicities, and response after CAR T-cell therapy failure. Statistical calculations using parametric tests were performed, and survival curves were estimated using the Kaplan-Meier method and compared statistically using the log-rank test and Pearson's correlation. Results: Thirty-four pts with aNHL received axi-cel. The median age was 53 years [IQR 42-63], and 62% were male. All pts received inpatient axi-cel infusions and the median length of hospital stay was 14 days (IQR: 11-17). Cytokine Release Syndrome (CRS) was observed in 91% of pts (3% grade ≥3), while Immune Effector Cell Associated Neurotoxicity Syndrome (ICANS) was observed in 41% (24% grade ≥3). At day 30 response assessment, 16 pts (47%) had complete responses (CR), 9 (26%) had a partial response (PR), 4 (12%) had stable disease (SD), 4 (12%) showed progression with primary refractory disease (PD) and 1 (3%) died before assessment due to grade 5 ICANS (Table 1). After a median follow-up of 178 days, we observed PD in 12 (35%) pts. The median time-to-progression was 72 days (IQR 58-93) and most of the pts (83%) progressed during the first 3 months. None of the patients with more than 5 months of sustained response developed progression of disease. The likelihood of progression during the first 6 months after axi-cel infusion was 19%, 57%, 50% for pts that initially achieved a CR, PR, SD, respectively. Expression of CD19 was observed in 66% (4/6) of pts with available biopsies after axi-cel suggesting a failure mechanism other than antigen escape. The mortality rate of the R/R aNHL group was 58% with a median survival time of 83 days (IQR: 50-109). There was no association between age, stage, number of previous therapies, time from previous therapy to axi-cel infusion, time from apheresis to infusion, use of tocilizumab, or steroids with progression of disease. Of note, no correlation between CRS or ICANS with progression of disease was found (2-way ANOVA test F (1, 4) = 3.802, p=0.1230). Maintaining a response to axi-cel treatment (CR, PR, or SD) for ≥ 3 months was a strong predictor of durable response with an HR of 0.05 (p= <0.0001). Eleven R/R pts received subsequent therapies with a median time to retreatment of 76 days. Those treatments included: Radiotherapy (n=7), pembrolizumab (n=3), polatuzumab-rituximab with (n=3) and without (n=1) bendamustine, obinutuzumab with (n=1) or without (n=1) lenalidomide, Hyper-CVAD (n=2), R-GemOx (n=1), rituximab with lenalidomide (n=1) and intrathecal methotrexate (n=1). Only 2 (17%) patients have responded to salvage therapy achieving PR (one patient treated with radiotherapy and the other with rituximab-lenalidomide after two other salvage therapies). Conclusion: Our experience demonstrates the majority of aNHL patients respond to axi-cel. If patients maintain their response for more than 3 months, the likelihood of progression is very low - 15%. Similar to what has been previously reported in the literature, our series showed that 35% of patients progressed after axi-cel, and subsequently have a poor prognosis with median survival after a relapse of only 83 days (IQR: 50-109). Therapy options following axi-cel were limited due to severe cytopenias, only 2 of 11 patients have responded to salvage therapy, suggesting that conventional treatments are probably not effective/safe in this high-risk group of patients. Interestingly, the majority of R/R pts with available biopsies showed persistent CD19 expression suggesting that CAR T-cell exhaustion, poor in vivo expansion, and inhibitory signals of the tumor microenvironment may contribute to resistance. Additional strategies for monitoring of axi-cel persistence and its immunophenotypic profile could be helpful for prognosis and management of CAR T-cell pts receiving axi-cel. Disclosures Kharfan-Dabaja: Daiichi Sankyo: Consultancy; Pharmacyclics: Consultancy. Castro:Fate Therapeutics: Research Funding; Kite Pharma: Research Funding; Pharmacyclics: Research Funding.

Background: Chimeric Antigen Receptor (CAR) T cell therapy is a promising cancer immunotherapy that is growing exponentially. The doubling time of medical knowledge in 2010 was 3.5 years, and the projection for 2020 is just 73 days. In the last five years, the number of PubMed publications on cancer applications of CAR T cells has tripled. Therefore, to remain updated in the field represents a challenge for patients, care providers and researchers. In this review we provide a focused summary of the currently ongoing clinical trials, with a comprehensive overview of advances in CAR T cell therapy, beyond CD19, emphasizing on antigenic targets, development phases, and leading sponsor pharmaceutical companies. Methods: We retrieved the available data from the national registry of clinical trials (clinicaltrials.gov) using the following keywords: "CAR T cell", "CAR T cell and cancer", "chimeric antigen receptor", "CAR T AND tumor antigen", 'CAR T cell antigens", "Tumor antigens targeted by CAR T cells", "engineered T cells", "modified T cell", "CAR T cells in Cancer", "CAR T cell therapy", "CAR T cell therapy AND Cancer" until December 31, 2018 and manually excluded the trials unrelated to CAR T-cell therapies on cancer, by reviewing the detailed information provided on the website as well as preliminary data published. Results: The analysis included 271 clinical trials posted on the clinicaltrials.gov website from the United States by the cut-off date. For efficacy analysis, we retrieved information from 52 trials, by NCT number on a PubMed search. The majority of CAR T clinical research is focused on hematological cancer (57%), followed by CNS 8%, GI 6%, Skin 5%, Genitourinary 4%, Breast 4%, Gynecologic 4%, Respiratory 3%, Sarcoma 2%, Mesothelioma 2% and others 5%. The most used target in CAR T cell therapy and the leaders in phase 3 trials are CD19 (42%) and BCMA (12%), followed by CD20, NY-ESO-1, Mesothelin, HER2, GD2, MAGE-A3 and CD30. An essential step in CAR T cell therapy development is the selection of the right antigen/target. Here, we provide an overview of the clinically relevant targets that are actively being using by clinical trials in the United States. For example, CD19 appears to be a leading target regarding CAR T cell therapy on cancer with 116 trials (42% of total CAR T cells trials) on going just in the United States with a significant increment in the previous years. Similarly, with BCMA is one of the targets with more phase 3 trials (Figure 1) with promising results on patients with Multiple Myeloma with and the objective response of 85%, CR 45%, and PFS of 11.8 months. Second-generation CARs with either CD28 or 4-1BB as costimulatory signaling domain are preferred, with 4-1BB being the most commonly chosen. Conclusions: Our findings show growing trends in the development of CAR T cell-based therapies, combination and possible retargeting therapies in the future for solid tumor and hematologic malignances; taking into account the amount of important information and the complexity of the database, we have developed this analysis to understand how to generate in the future a friendly platform for researchers and patients to have an detailed overview of the clinical trials in cellular therapies Disclosures No relevant conflicts of interest to declare.

Abstract Obinutuzumab-Gazyva (G) is an anti-CD20 monoclonal antibody that has shown better outcomes in patients (pts) with chronic lymphocytic leukemia and low-grade lymphoma (Goede 2014, Marcus 2017). However, one limitation for its use when compared to Rituximab is the presence of significantly more frequent and more severe infusion related reactions (IRR). Strategies to mitigate this significant adverse event are needed particularly to allow a safer administration of this antibody to elderly pts or those with existing comorbidities. We have observed a reduction of G-induced IRR in previously untreated CLL pts that are enrolled on a phase Ib/II clinical trial (NCT02315768) that combines G with the Bruton's tyrosine kinase inhibitor, ibrutinib (Ibr). Only 5 out of 23 pts treated have developed IRR (Grade 1-2, 17% and Grade 3, 4%). This rate of IRR is much lower as compared with rates previously reported (all grades: 65%, grade 3-4: 20% - Goede 2014), (all grades: 92%, grade 3-4: 26.3% - Freeman,2015). Moreover, there were no pts that require permanent discontinuation of G due to IRR. To understand the biology of this beneficial effect of Ibr, we performed serial cytokine measurements on plasma samples from 23 pts enrolled in this study at different time points during the first week of combined treatment (Cycle 1 prior to the first infusion of G and post G infusion on Day 1, Day 2 and Day 8) - Figure 1. We developed a multiplex assay (Luminex) to measure 7 different cytokines previously reported to be involved in IRR (IFN-g, IL-10, IL6, IL8, CCL3/MIP1-a, CCL4/MIP1-band TNF-a). Standards were set up in duplicate yielding curves from 3.2 pg/ml to 10.000 pg/ml. Assays were performed according to manufacturer's instructions, with undiluted samples and overnight agitated incubation at 4 °C. We identified the maximum peak of cytokine levels post G infusion and compared those values with the baseline cytokine profile obtained prior to the first G infusion on Cycle 1 Day 1. The majority of pts (22 out of 23) showed cytokines maximum peaks in the middle of G-infusion during Cycle 1 Day 1 and this correlated with the onset of IRR associated symptoms in those pts that reacted to G. With the exception of IL-6, we observed statistically higher post vs. pre G infusion levels of TNF-a(p=0.0012), IFN-g(p=<0.0001), CCL3 (p=0.0458), CCL4 (p=<0.0001), IL-8 (p=<0.0001), and IL-10 (p=<0.0001) even in pts that did not develop IRR. However, the post infusion peak levels of TNF-a(p=0.0043), IFN-g(p=0.0457) and CCL3 (p=0.0460) were significantly higher in pts with IRR compared to those that had no IRR. Baseline levels prior to G infusion of TNF-a(p=0.0495) and IFN-g(p=0.0301) were higher in IRR pts, suggesting a possible predictive role in the development of IRR. Figure 1. Our study shows that concurrent administration of Ibr (Initiated on Cycle 1 Day 1 with pre-medications) and G shows a beneficial effect decreasing the rates of IRR (Amaya-Chanaga, 2016). All pts showed a significant increase of cytokine levels post G infusion with IL-6 levels being the exception. When we compared post G infusion cytokine levels, we observed that IRR-pts had a significant increase in TNF-a, IFN-g, and CCL3 suggesting a role of these cytokines in the clinical manifestations associated with IRR. In addition, higher levels of TNF-a and IFN-g, at baseline prior to G infusion appear to be predictive of the development of IRR. Even though our sample size is small, our observations provide additional insights into the biology of G associated IRR and how to decrease effectively those adverse events using Ibr while preserving the immune function needed for the activity of this monoclonal antibody. In addition, Ibr induced modulation of signaling through the B cell receptor and patterns of cytokine release might be efficacious in preventing other monoclonal antibody IRR as well as those reactions observed in pts that receive adoptive cellular therapy (i.e. CART cell treatment). Disclosures Choi: AbbVie, Inc: Consultancy, Speakers Bureau; Gilead: Speakers Bureau; Genentech: Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Rigel: Consultancy. Amaya-Chanaga:AbbVie: Equity Ownership, Other: Research performed while employed as an investigator of this study at UCSD. Review and approval of abstract performed while employed at Pharmacyclics, LLC, an AbbVie Company.; Pharmacyclics, an AbbVie Company: Employment, Other: Research performed while employed as an investigator of this study at UCSD. Review and approval of abstract performed while employed at Pharmacyclics, LLC, an AbbVie Company.. Kipps:Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees. Castro:F. Hoffmann-La Roche: Consultancy; Genentech, Inc: Consultancy; Pharmacyclics, LLC, an AbbVie Company:: Consultancy.