ALBERT

Description: Introduction: The autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel (Axi-cel) improved long-term survival of patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the pivotal ZUMA-1 trial indicates a 2-year PFS of ~40% (Locke, Lancet Oncology 2018). Early identification of patients with increased relapse risk may allow for early intervention and improved outcomes. In a pilot study of 6 ZUMA-1 patients, minimal residual disease (MRD) evaluation via a next-generation sequencing MRD assay (Adaptive Biotechnologies, Seattle, WA) to assess for circulating tumor (ct)DNA, mirrored clinical outcome as assessed by PET-CT (Hossain et. al. Leukemia & Lymphoma 2019). Based on these promising results, a multi-institutional prospective study utilizing cell-free MRD assessments to predict outcomes in r/r DLBCL patients after Axi-cel therapy was initiated. Methods: To identify tumor clonotype(s), tumor DNA extracted from archival paraffin-embedded tissue underwent PCR amplification of IgH-VDJ, IgH-DJ and IgKappa/Lambda regions using universal consensus primers. CtDNA levels were measured pre-LD, 0, 7, 14, 21, 28, 56, 90, 180, 270, and 365 days following Axi-cel infusion. PET-CT scans were obtained at baseline, Day 28, Month 3, 6, and 12 with response assessed per Lugano criteria. Deauville 1-3 was considered PET-negative. The protocol prespecified that patients with less than Day 28 follow-up be excluded from analysis. Any detectable ctDNA was considered MRD positive. Results: Here we report on the pre-planned analysis of the first 50 study patients with at least a Day 28 MRD assessment and 3 months of follow up. An additional 4 patients with at least 3 months of follow-up but who did not have a Day 28 MRD assessment were also included. Baseline characteristics and clinical outcomes of patients were similar to ZUMA-1 and a real-world analysis of 295 patient who received Axi-cel (Nastoupil et al ASH 2018). The median age was 61 years old (range 19-76) (53.7% male, 46.3% female) and 59% of patients received 3 or more prior lines of therapy (range 1-6). After a median follow-up of 7.5 months, the best overall response rate was 87% (47 of 54) and complete response rate was 57% (31 of 54). The median OS was not reached and median PFS was 4.6 months (panel A). At Day 28, 56% (28 of 50) of patients were MRD negative (MRD-neg) and 44% (22 of 50) were MRD positive (MRD-pos). As compared to MRD-pos, MRD-neg correlated with improved median PFS (not reached vs. 2.96 months, p

Description: Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

Description: Background: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have significant activity in patients with relapsed/refractory DLBCL (rrDLBCL). While the majority of rrDLBCL patients receiving axicabtagene ciloleucel (Axi-cel)achieve complete responses, a significant subset of patients experience disease progression (Locke FL, et al. Lancet Oncol. 2019). Circulating tumor DNA (ctDNA) analysis has demonstrated utility for predicting therapeutic benefit in DLBCL, as well as for detecting emergent resistance mechanisms to targeted therapies. Here we apply cell-free DNA (cfDNA) analysis to patients receiving Axi-cel, to characterize molecular responses, resistance mechanisms, and to track CAR19 cells. Methods: We performed Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) on DNA from germline and plasma samples collected prior to CAR T-cell infusion, multiple time-points post infusion, and, where available, at the time of relapse from 30 patients receiving Axi-cel for rrDLBCL at Stanford University. We designed a novel hybrid-capture panel and analysis pipeline designed to detect both tumor variants, as well as Axi-cel specific recombinant retroviral sequences to quantify CAR19 levels in cfDNA. Tumor variants were identified prior to and following Axi-cel therapy to assess for emergent variants, and Axi-cel specific sequences were quantified. Results: The median follow-up for the 30 patients after Axi-cel infusion was 10 months, with 47% (14/30) of patients experiencing disease progression after Axi-cel therapy. We identified an average of 164.3 SNVs per case (range:1-685) before Axi-cel therapy; the most common coding variants identified at baseline were in MLL2 (29.2%), BCL2 (22.5%), and TP53 (19.3%). When treated as a continuous variable, pretreatment ctDNA levels were prognostic of PFS (HR 2.16, 95% CI 1.11-4.21, P=0.02). Using a previously established ctDNA threshold to stratify disease burden (2.5 log10(hGE/mL); Kurtz et al. JCO 2018), we observed significantly superior PFS in patients with low pretreatment ctDNA levels treated with Axi-cel (Fig. 1A). In the majority of Axi-cel treated patients (62.9%), ctDNA was detectable at day 28, and PFS was significantly longer in patients with undetectable ctDNA at this time-point (Fig. 1B). Multiple putative resistance mechanisms were identified at relapse after Axi-cel, including emergent variants in CD19, HVEM, and TP53, as well as copy number gains in PD-L1 (Fig. 1C). For example, in one patient, a CD19 stop-gain mutation, which was not detected prior to treatment or at the time of the first interim PET scan, emerged at the time of relapse (Fig. 1D). Finally, we found cfDNA evidence for Axi-cel DNA in 74% of patients 28 days after therapy, including in patients without evidence of circulating CAR T-cells in PBMCs. Axi-cel levels in cfDNA as measured by CAPP-Seq were significantly correlated with CAR19 flow cytometry (Pearson r=0.55, P=.015; Fig. 1E). Conclusions: Baseline and interim ctDNA measurements have prognostic significance in DLBCL patients being treated with CAR19 T-cells, and potential emergent resistance mutations, including in CD19, can be identified in patients via cfDNA analysis. Quantification of CAR19 T-cells using cfDNA is significantly correlated with flow cytometric quantification, indicating that these cells can be quantified via cfDNA. Taken together, these data indicate that cfDNA analysis is a powerful tool for predicting response to CAR19 therapy, identifying genomic determinants of resistance and quantifying CAR19 cells, which may in turn inform the next therapeutic steps. Figure 1: A) Kaplan Meier analysis of PFS, with patients stratified based on pre-Axi-cel therapy ctDNA level, above and below a previously established threshold (2.5 log10[haploid Genome Equivalents/mL]). B) A Kaplan Meier plot depicting PFS stratification for patients with detectable versus undetectable ctDNA at day 28 after Axi-cel infusion. C) Oncoprint depicting selected emergent and baseline tumor variants in progressors and non-progressors after Axi-cel therapy. D) Change in mean ctDNA variant allele frequency (VAF) and emergence of a CD19 stop-gain mutation (CD19 pTrpX) at the time of relapse in a patient who initially achieved a CR at day 28 after CAR19 infusion. E) Relationship between CAR19 T-cell quantification by cfDNA and flow cytometry. (ND: Not detected) Disclosures Kurtz: Roche: Consultancy. Chabon:Lexent Bio Inc: Consultancy. Khodadoust:Corvus Pharmaceuticals: Research Funding. Majzner:Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Diehn:Roche: Consultancy; Quanticell: Consultancy; Novartis: Consultancy; AstraZeneca: Consultancy; BioNTech: Consultancy. Miklos:Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees. Alizadeh:Genentech: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

Description: BACKGROUND: Non-Hodgkin B cell lymphomas are often infiltrated by immune effector cells including T, NK and dendritic cells. But despite their presence within the tumor they fail to control tumor growth. Some T cells can even play a supportive role to maintain the tumor and prevent the function of anti-tumor immune cells. Regulatory T cells (TRegs) function normally to dampen immune responses and prevent auto-immunity by direct cell contact or by secreting suppressive cytokines i.e. TGF-b and Interleukin-10. Follicular Lymphoma tumor cells can induce the conversion of CD4 T cells into TRegs (1-3), thereby evading anti-tumor immune responses. Follicular helper T cells (Tfh), another class of regulatory T cells, are found within normal follicles of lymphoid organs. Migrating into the germinal center, they promote the survival and differentiation of follicular B cells. In lymphoma, Tfh cells can send a survival signal to tumor B cells through CD40L/CD40 interactions (4). STUDY: In an ongoing multi-center phase I clinical trial (NCT02266147), patients with previously untreated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single, tumor site, followed by intratumoral injection into the same site of a CpG-ODN (SD-101, Dynavax Technologies), an immune-modulatory molecule that targets Toll-like Receptor 9 (TLR-9). Doses ranged from 1mg to 8mg per injection in successive cohorts. A fine needle aspirate (FNA) of the treated site is performed before, one week after the first injection, and 1 week after the 5th and final weekly injection of CpG-ODN. FNA samples are sent to a central site by overnight delivery in media with 5% fetal calf serum. They are processed within 24 hours and stained for flow cytometry with panels of antibodies to delineate T, B, NK, dendritic, myeloid cells, and their subsets. Antibodies against activation antigens, as well as T cell exhaustion, inhibition and function are also used to characterize these cells. To date, 12 patients have been entered into the dose escalation phase of this study: 10 follicular lymphoma, 1 chronic lymphocytic lymphoma and 1 small lymphocytic lymphoma. Nine of 12 patients had FNAs that yielded enough viable cells for evaluation pre and 1 week post treatment. RESULTS: Therapy has been well tolerated. Local injection site reactions and fever, the most common side effects, resolved by 48 hours. There have been no related SAEs. The treated site regressed in all patients per CT scan at 3 months post treatment. The early evaluation of untreated lesions documented 1 PR. The remaining patients have stable disease with all but one having systemic tumor regressions ranging from 14% to 28%. Seven out of 9 patients evaluable by flow cytometry had an increase in total T cells at the treated site 1 week following the first dose of CpG ranging from 18 to 〉 300% above baseline in both the CD4 and CD8 compartments. More interestingly, there was a reduction of TRegs as a percentage of total T cells in 7 of 9 patients (average reduction: 21.3 ± 10.6%). A single patient with an increase in untreated tumor burden of 15% showed no reduction in TRegs. There was also a reduction of Tfh cells in 8 of the 9 evaluable patients (average reduction: 87.2 ±7.2%). One patient with SLL had no Tfh cells pretreatment. Figure 1A and B shows a representative example of TReg and Tfh cell depletion within the same FL patient. CONCLUSION: Immune modulating therapies can be delivered directly into a tumor to minimize systemic toxicity and induce changes in the tumor microenvironment while potentially inducing a global anti-tumor immune response. In this case, the combination of intratumoral CpG and low-dose radiation reduced the proportion of TRegs and Tfh cells, thereby modulating their immune inhibitory effects and tumor growth promoting effects, respectively. This clinical trial is ongoing and open to accrual. Monitoring the cell populations in the tumor site by repetitive sampling and analysis by flow cytometry reveals the pharmocodynamic effects of anti-cancer therapies, especially those intended to trigger anti-tumor immune responses. This knowledge can be used to tailor therapies and to influence the choice and scheduling of combinations of agents designed to target specific cell populations. 1. Yang Z-Z, et al. Blood. 2007;110:537-44. 2. Ai WZ, et al. Int J Cancer. 2009 Jan 1;124(1):239-44. 3. Mittal S, et al. Blood. 2008;111:5359-70. 4. AmŽ-Thomas P, et al. Blood. 2015 Apr 9;125(15):2381-5 Figure 1. Figure 1. Disclosures Bartlett: Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding. Gordon:Northwestern University: Employment; Dr Leo I. Gordon: Patents & Royalties: Patent for gold nanoparticles pending. Coffman:Dynavax Technologies: Employment. Janssen:Dynavax Technologies: Employment. Levy:Bullet Biotechnology, Inc.: Consultancy.

Description: Introduction MCL has a poor prognosis. In eligible patients, intensifying frontline, CHOP-like regimens (e.g., cytarabine) as well as high-dose chemotherapy and autologous stem cell transplant (HDT/ASCT) consolidation in first remission have improved progression free survival (PFS) but less so, overall (OS). Preclinical animal models show benefits of adding tumor-specific T-cells to ASCT. CpG (PF-3512676) is a toll-like receptor 9 (TLR9) agonist and an effective vaccine adjuvant that induces costimulatory molecule expression on both antigen-presenting and MCL cells. This phase I/II clinical trial (NCT00490529) adds autologous T-cell transfer, harvested from patients after vaccination with CpG-activated autologous MCL cells - a maneuver termed immunotransplant. This is a planned interim analysis for safety and efficacy triggered by the first 20 patients reaching 1 year post ASCT. Methods Prior to therapy, subjects' tumor cells are collected by biopsy or apheresis and patient-specific vaccine is created by incubating fresh tumor cells with CpG (3 mcg/mL PF-3512676, 72-hr culture), then irradiated and cryopreserved. Patients receive induction chemoimmunotherapy of physician's choice. Patients achieving at least partial response (PR) then receive 3 subcutaneous autologous tumor vaccinations (1 x 108 cells/dose) mixed with CpG (18 mg) every 4-7 days. Primed T-cells (≥ 1 x 1010 CD3 cells) were collected by apheresis 2-4 weeks following vaccine 3 and a rituximab (375 mg/m2) B-cell purge. After standard HDT/ASCT (conditioning = BCNU, cyclophosphamide, etoposide), primed T-cells and a 4th vaccination are given on day+1. A 5th CpG-MCL vaccination followed 3 months post ASCT. The primary endpoint of this study is freedom from minimal residual disease (MRD) at 1 year post ASCT, measured by presence of patient-specific, malignant B-cell VDJ sequence in peripheral blood (ClonoSeq™, analyzed at a sensitivity of ≥ 1 clone/10,000 leukocytes) -an endpoint previously shown to be highly prognostic. Secondary endpoints include PFS, OS, and immune response to vaccine. 59, transplant-eligible, MCL patients are targeted for accrual in this 2-stage design. Results In this interim analysis of 24 patients accrued, 20 have surpassed 1 year post ASCT. All patients had Stage IV disease. Median values included (range): follow-up 43.5 months (11.5-60.1), age 60 years (47-70), and MIPI score 5.9 (5.1-7.8). Vaccine was made from biopsy alone (n=12), apheresis alone (n=9), or both (n=1). Frontline therapy included R-CHOP (n=7), R-hyperCVAD (n=14), alternating R-CHOP/R-DHAP (n=2), and R-EPOCH (n=1). 19 patients achieved complete response while another 3 had PR. All responding patients were vaccinated, able to yield sufficient T-cells for adoptive transfer, and proceeded through standard HDT/ASCT. At 1-year post ASCT, freedom from MRD was 90.5% (n=21). 2 patients did not reach 1-year post ASCT. One died from an ASCT complication (metapneumovirus) while the other relapsed 6 months following ASCT (included in MRD analysis). The most common toxicity due to CpG-MCL vaccine was erythematous rash at injection site (90.9%, n=20, each grade 1). No serious adverse events were seen related to vaccines or adoptive T-cell transfer. All patients had successful hematopoietic recovery, but two were delayed and received backup stem cell infusions with eventual recovery. Though median PFS and OS have not been reached, 3-year PFS and OS at interim analysis are 54.5% and 63.6%, respectively (intention to treat). In this data set, higher expression of co-stimulatory molecules (such as CD40, CD80, and CD86) on a subject's MCL in response to CpG was associated with freedom from MRD (p =0.02). Post-ASCT, higher peripheral T-cell granzyme and perforin response to ex vivo re-challenge with autologous MCL cells was also associated with freedom from MRD (p =0.01). Conclusions The addition of CpG-activated, whole MCL vaccination and autologous, adoptive T-cell transfer to standard therapy appears both feasible and safe. At interim analysis, the 1-year freedom from MRD surpasses rates previously reported in other studies and warrants further investigation. To date, 46 patients have either completed and/or are continuing to undergo study treatment and the study remains open to accrual for patients newly diagnosed with MCL. Disclosures Miklos: Pharmacyclics: Research Funding. Rezvani:Pharmacyclics: Research Funding. Faham:Adaptive Biotechnologies: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

Description: Introduction: Prior studies have shown preliminary clinical efficacy in combining CpG-ODN with radiation therapy (XRT) to patients with indolent B-cell lymphoma. We report interim Phase 1/2 data of combination XRT and SD-101, a synthetic class C CpG-ODN TLR9 agonist, selected for the strong induction of type I interferon. Methods: This dose-escalation Phase 1/2 trial enrolled patients with untreated indolent B-cell lymphoma having at least 2 measurable lesions (one palpable). The primary endpoints were safety and alpha-interferon-gene induction. Secondary endpoints included efficacy assessment using Cheson (1999) criteria and quantification of changes in tumor-infiltrating lymphocytes. A single lesion was treated with XRT (2 Gy daily X 2 days) followed by a single intratumoral dose of SD-101 (same lesion). 4 additional doses of intratumoral SD-101 were given weekly over next 4 weeks. The treated lesion and distal lesions were monitored during the study. Pharmacodynamic assessment included flow cytometry analysis of immune infiltrates in an FNA sample of the treated tumor and RT-PCR RNA assay of whole blood to assess induction of alpha-interferon genes. Efficacy assessment included imaging (CT at 3, 6, and every 6 months thereafter). Results: As of 01 Aug 2016, 13 patients were treated with escalating doses of SD-101 at 1, 2, 4 or 8 mg/dose. There were no dose limiting toxicities. The majority of related adverse events (AEs) were Grade 1 (mild). The most common were flu-like symptoms (chills, headache, malaise, myalgia), typically resolving within ≤ 48 hours without requiring intervention (e.g., acetaminophen). An induction of alpha-interferon genes occurred at all dose levels with a similar level of induction. For the dose expansion phase, 15 patients were enrolled at 1 mg (6) and 8 mg dose group (9). Similar to dose escalation phase, the most common related events were again flu-like symptoms, typically resolving within ≤ 48 hours mitigated with acetaminophen. There was one dose delay for Grade 3 neutropenia in one patient (1 mg) that resolved following drug interruption. Twenty-nine Grade 3 transient flu-like symptoms were reported for four patients (44%) who received the 8 mg dose (e.g., chills headache, malaise, myalgia, and fatigue). Only one of the four patients experienced all 5 Grade 3 flu-like symptoms after intratumoral injection. There were no grade 4 or serious events reported, and no patient discontinued due to an adverse event. A reduction in tumor sizes was observed in the study over time (see Figure 1). At Day 90 the median changes in the product of diameters from baseline were -46.1% and -10.2% for treated tumor and distal tumors, respectively. At Day 540, the median changes were -68.6% and -24.1%, respectively. In patients with follicular lymphoma (largest subgroup), preliminary data suggest a higher percentage of CD8+ T cells in the treated lesion (FNA at day 8) correlated with an increased abscopal response. Conclusions: Intratumoral SD-101 following radiation therapy has been well tolerated and preliminary efficacy, promising. Abscopal anti-tumor activity was observed with preliminary data suggesting that a higher percentage of CD8+ T cells in the treated lesion correlated with an increased abscopal response seen in follicular lymphoma. Enrollment is ongoing with an option for cycle 2 retreatment. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Reagan:Seattle Genetics: Research Funding. Friedberg:Bayer: Other: Data Safety Monitoring Committee. Bartlett:Gilead: Consultancy. Leung:Dynavax: Employment. Peterkin:Dynavax: Consultancy. Xing:Dynavax: Employment. Coffman:Dynavax: Employment. Janssen:Dynavax: Employment. Candia:Dynavax: Employment.

Description: Background: In situ vaccination for the treatment of cancer aims to trigger an immune response locally that propagates systemically. We have shown that local treatment with intratumoral CpG (a TLR9 agonist) and low-dose radiation can elicit antitumor immune responses and global tumor shrinkage in patients with low-grade lymphoma (Brody, J Clin Oncol, 2010; Frank, Cancer Discov, 2018). Ibrutinib is a small molecule that compromises B cell survival by inhibiting Bruton's tyrosine kinase (BTK) but also modulates T cell phenotypes by inhibiting interleukin-2-inducible T-cell kinase (ITK). Specifically, there is evidence for ibrutinib-induced skewing of CD4 T-cells toward a Th1 phenotype, which would be expected to promote anti-tumor immunity (Dubovsky, Blood, 2013). In a mouse model of lymphoma, systemic ibrutinib plus intratumoral CpG was curative of systemic disease in all treated mice, an effect that was fully T-cell dependent (Sagiv-Barfi, Blood, 2015). Study Design: To test the hypothesis that ibrutinib will enhance the efficacy of in situ vaccination via its effects on T-cells, we initiated a phase I/II clinical trial combining oral ibrutinib (560mg), intratumoral CpG (SD-101, 3mg) and local low-dose radiation in patients with recurrent low-grade lymphoma (NCT02927964). Patients with follicular lymphoma, marginal zone lymphoma, or indolent mantle cell lymphoma relapsed or refractory to at least one line of prior therapy and with at least one superficial disease site accessible for injection and one independent measurable disease site are eligible. Patients with significant autoimmune disease, recent stroke or intracranial hemorrhage, and those requiring anticoagulation with vitamin K antagonists or chronic treatment with strong CYP3A inhibitors are excluded. The primary outcome of the study is determination of safety of the combination treatment. Secondary outcomes include overall response rate, progression-free survival, and correlative studies of tumor and blood immune populations. Fourteen patients have been enrolled thus far, with a goal enrollment of 21-30 patients. Treatment consists of two consecutive days of low-dose (2 Gy) radiation to the chosen treatment site, followed by 5 weekly injections of CpG into that same site. Daily oral ibrutinib is begun one day after the second CpG injection. CT scans are performed at 3, 6, 12, 18, and 24 months. Common Terminology Criteria for Adverse Events (CTCAE) are used to track treatment-emergent safety events, and Lugano criteria (Cheson, J Clin Oncol, 2014) are used to assess response to therapy. Correlative Studies: Fine needle aspirates (FNAs) are obtained from CpG-injected and non-injected sites pre-treatment and at weeks 2 and 6. Peripheral blood samples are obtained pre-treatment and weeks 2, 4, 6, and at all response assessment timepoints. When available, tumor cells from an excisional biopsy pre-treatment are viably preserved. FNAs and peripheral blood samples from pre-treatment and at weeks 2 and 6 are being analyzed by both flow cytometry and single cell RNA sequencing. For patients for whom viably preserved tumor cells are available, these are co-cultured with autologous peripheral blood T cells in an in vitro immune response assays to test for tumor-specific T-cell activation. Summary: This innovative study combines in situ vaccination and ibrutinib-induced T-cell modulation and incorporates serial sampling of multiple tumors and peripheral blood using minimally invasive procedures, analyzed by methods that allow for deep profiling of treatment-induced changes. This study is ongoing and currently accruing at the Stanford Cancer Center. Disclosures Khodadoust: Corvus Pharmaceuticals: Research Funding. Levy:Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc: Membership on an entity's Board of Directors or advisory committees; XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Five Prime: Membership on an entity's Board of Directors or advisory committees; Corvus: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees.

Description: BACKGROUND: Low-grade B cell lymphoma is often characterized by an infiltration of immune effector cells including T, NK and dendritic cells. But, despite the presence of effector cells within the tumor, these cells fail to control tumor growth. In a preclinical mouse model, we showed that Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), but also ITK (IL-2-inducible T cell kinase), synergized with intratumoral CpG to facilitate complete regression of tumors at the treated site as well as a distal, non-treated site, curing all the mice. This was accompanied by a potent anti-tumor memory T cell response by both CD4 and CD8 T cells that rejected the tumor on re-challenge. (Sagiv-Barfi I, et al. Blood. 2015 March:125(13):2079-2086) STUDY: In an ongoing clinical trial (NCT02927964), patients with previously treated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 3 mg CpG-ODN (SD-101, Dynavax Technologies) into the same site. 1 day after the second injection, patients begin taking a daily 560 mg dose of Ibrutinib. A fine needle aspirate (FNA) of the injected site and a non-injected site outside the radiation field is performed prior to radiotherapy, one week after the first injection, and at week 6, 1 week after the final injection of CpG. FNA samples are stained for flow cytometry with panels of antibodies to delineate all major cell populations and their subsets. Cellular activation as well as T cell exhaustion, inhibition and function are also characterized. When feasible, a biopsy is performed prior to treatment providing tumor cells to be used in an immune response assay to evaluate induced anti-tumor responses by circulating peripheral blood T cells obtained throughout the study. RESULTS: To date, 12 patients have been entered onto the study. Of these patients, 4 had excisional biopsies and subsequent immune response assays performed. All 4 patients exhibited CD8 anti-tumor responses as determined by an increase in the activation marker CD137 as well as the functional marker granzyme B above pretreatment CD8 T cells. At week 12, 7 weeks after the last CpG injection, the average increase above pretreatment baseline for CD137 and granzyme B was 6.1% and 9.3%, respectively. In 3 of these patients, we observed an increase in CD8 T cells expressing CD137 and granzyme B from the FNA of the non-injected site. The 4th patient did not have adequate number of cells for staining. Two patients exhibited CD4 immune responses as characterized by an up-regulation of CD137 and CD278. FNA of 7 patients produced enough cells to analyze the tumor microenvironment from both the injected and the non-injected sites over all 3 time points; 1 patient was evaluable through week 2 for both sites. In 7 out of these 8 patients, CD3 T cells increased at the injected and non-injected sites by week 6. The proportion of CD4 and CD8 T cells did not stay constant, however, as reflected by the changes in CD4:CD8 ratios. This suggests that the increase in T cells was not purely the result of a loss of tumor B cells in the samples. Notably, we observed a significant increase in the effector CD4 T cells in all patients at the injected site by week 2 (14.1% ± 6.5%, p = 0.005) and 5 of 8 patients in the non-injected site by week 6 (12.4% ± 10.4%). Moreover, the proportion of T follicular helper cells significantly decreased in all patients at the treated site by week 2 (17.7% ± 9.7% of T cells, p = 0.0012) and in 5 of 8 patients at the non-treated site by week 6 (8.1% ± 5.5% of T cells). Tregs were more variable, decreasing in 5 of 8 patients in the treated and 3 of 8 patients in the non-treated site by week 6. CONCLUSION: CpG is known to activate antigen-presenting cells such as dendritic cells and macrophages. Ibrutinib is a small molecule that has been shown to have direct anti-tumor effects in B cell lymphoma and may skew an immune response towards that of a TH1 type. Here we show that together, they can effect changes in the tumor microenvironment in both treated and in untreated sites of disease. This clinical trial is ongoing and open to accrual. Disclosures Khodadoust: Innate Pharma: Research Funding.

Description: Autologous CD19 directed CAR T-cell therapy has response rates of 〉70% in adult acute lymphoblastic leukemia (ALL) and 〉40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 & CD22 with intracellular signaling domains incorporating 4-1BB and CD3ζ (CD19/CD22.BB.z) to overcome CD19 immune escape. Here, we present our Phase I experience with this bi-specific CAR in adults. This is a single institution phase I dose escalation study enrolling patients Age ≥ 18 years with relapsed/refractory B-ALL or DLBCL after standard therapies. Primary aim is to determine feasibility of manufacturing the bi-specific CAR and safety at three dose levels (1 x 106 CAR T cells/kg, 3 x 106 CAR T cells/kg, 1 x 107 CAR T cells/kg). Clinical response was evaluated as a secondary endpoint utilizing standard response criteria for ALL and DLBCL. All patients underwent lymphodepletion with cyclophosphamide (500mg/m2 daily x3 doses) and fludarabine (30mg/m2 daily x 3 doses) followed by CAR infusion two days later. Patients were assessed at pre-defined time-points (Day 28, Month 3, 6, 9, 12 then every 6-12 months) with correlative assessments including immunophenotyping, single cell RNAseq, CAR qtPCR, serum and single cell cytokine analysis. Seven adult patients (5 DLBCL, 2 ALL), aged 35 - 75 years have been enrolled and 6 treated, all at dose Level 1 [Table 1]. The first 3 patients received freshly harvested cells and the remaining received cryopreserved cells (1 patient treated twice received initial fresh then cryopreserved product). None received systemic bridging therapy before CAR T infusion. Six patients developed reversible cytokine release syndrome (CRS,4 with Grade 1, 2 with Grade 2), onset between Day 1 to 13, and 2 patients received tocilizumab & systemic steroids. Three patients developed neurotoxicity (1 with grade 2, Day 8-11 and the others grade 1) with grade 2 neurotoxicity managed with steroids. Four patients required growth factor support beyond Day 28 and all treated patients show persistent B-cell aplasia. Two patients achieved CR: an ALL patient with disease in bone marrow/blood/CNS was MRD negative at day 28 & 60; a 75yo DLBCL patient achieved PR at day 28 and CR at month 3. Three others have ongoing PR and one died of progressive disease after initial PR at Day 28. A patient with PD at Day 28 subsequently treated with radiation and 2-months of revlimid/rituximab, now has no detectable disease 6 months post CAR-T. One patient with initial 6-month PR received a second infusion due to PD, did not develop CRS or CRES with 2nd infusion and has SD at Day 28 Notably, the patient experienced a lack of CAR-T expansion with the second infusion, raising the possibility of an immunogenic response to the CAR-T cell infusion. Flow analysis of all patients' peripheral blood showed CAR expansion peaked at median Day 13 (range Day 10-20) and CARs remained detectable [Figure 1]. Multi-parametric CyTOF phenotyping of the CAR19-22 products showed significant numbers of transduced CAR-T memory stem cells (phenotype: CD3+CD8+CD45RA+CD127+CD27+CCR7+). Single cell cytokine secretion analysis (Isoplexis,Rossi et al Blood 2018) revealed high polyfunctional strength index (PSI) in both CD4+ and CD8+ cell subsets in each patient's pre-infusion CAR product that reflected phenotypic expansion in patients. Additional correlative studies, including cytokine analysis, qtPCR based CAR quantification and CyTOF phenotypic analysis of the CAR-T cells will be presented. This first adult phase I trial of bi-specific CAR targeting CD19 & CD22 shows low toxicity with promising efficacy including achievement of CR in adult DLBCL and ALL patients. We have escalated dose to 3x 106 CAR T cells/kg and an expansion study of 60 patients will follow. CAR-T cells expanded within the first 20 days and continue to be detectable through 6 months. Disclosures Muffly: Shire Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding. Miklos:Janssen: Consultancy, Research Funding; Genentech: Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Description: Background: Axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 chimeric antigen receptor (CAR-T), showed significant clinical responses in patients with relapsed-refractory large-B cell lymphomas in the Zuma-1 trial (Neelapu et al, NEJM 2017). Zuma-1 analysis showed blood CAR-T cell expansion was associated with clinical response and toxicity. Herein, we report on 25 patients treated with commercial axi-cel and describe CAR-T expansion by immunophenotyping and its correlation with clinical outcomes. Methods: Twenty-five patients with aggressive lymphoma consecutively apheresed at Stanford University prior to June 30, 2018 were studied on an IRB approved biorepository-clinical outcome protocol. Cytokine release syndrome (CRS) was graded by Lee criteria (Blood 2014) and neurotoxicity according to Neelapu et. al (Nat. Rev. Clin. Onc. 2017). CAR-T cell immunophenotyping was assessed by peripheral blood flow cytometry on days 7, 14, 21 and 28 and then monthly. CAR-T cells were identified by gating on singlet+, live+, CD45+, CD14-, CD3+, anti-CD19-specific CAR mAb (clone 136.20.1; Jena et. al Plos 2013) and characterized as either CD4+ or CD8+. Results: Of 25 apheresed patients, 3 patients died prior to axi-cel infusion due to progressive lymphoma. Of 22 infused patients, 14 (64%) would have been eligible for the Zuma-1 trial. Reasons for ineligibility included symptomatic DVT (n=2), renal insufficiency (n=1), transaminitis (n=1), thrombocytopenia (n=1), MDS (n=1), pleural effusion (n=1) and 1 was ineligible by multiple criteria. Median time from initial clinic visit to infusion was 47 days (range 34-117); median time from apheresis to infusion was 22 days (range 19-38). Nine patients received bridging therapy prior to lymphodepletion chemotherapy (chemo = 4, radiation = 2, high dose dexamethasone = 3). Axi-cel infusion occurred in hospital and patients were followed expectantly for a minimum of 7 days or until adverse events resolved to 35% of CD3+ T-cells expressed CAR19. As of submission, 34 patients were apheresed and updated blood and FNA results will be presented. Conclusion: Our analysis of 22 infused axi-cel patients showed an ORR of 86% and CR of 45%, despite 36% Zuma-1 ineligibilities and steroid use in 82%. Blood CAR-T expansion was associated with both CRS and neurotoxicity but not clinical response. Detection of high concentration of CAR-T cells in affected lymph nodes 2 days post infusion suggests quantification of CAR-T cells at disease sites could be predictive of clinical responses. J.Y.S and B.S are co-first authors Disclosures Latchford: Kite a Gilead Company: Speakers Bureau. Muffly:Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Research Funding; Novartis: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding.