Parikh, Sameer A.

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding; Morphosys: Research Funding.

Background The CLL international prognostic index (CLL-IPI) is a validated staging system for newly diagnosed CLL patients that combines age, clinical stage, serum Beta-2 microglobulin, TP53, and IGHV mutation status into a single score and stratifies CLL patients into four prognostic risk groups with a c-statistic=0.72. Next-generation sequencing identified ~60 putative driver genes recurrently mutated in CLL, and genome-wide association studies (GWAS) identified 41 single nucleotide polymorphisms (SNPs) associated with CLL risk. We previously showed that a polygenic risk score (PRS) of the weighted average of the number of risk alleles among these 41 SNPs is associated with risk of both CLL and MBL. We and others have also shown that the tumor mutational load (TML), i.e., the total number of somatically mutated driver genes, is associated with time to first treatment (TTT). Herein, we examine whether TML and PRS add prognostic value beyond CLL-IPI for disease progression in a cohort of CLL and high-count MBLs. Methods We used the Mayo Clinic CLL resource to identify newly diagnosed (<2 years) previously untreated CLL and high-count MBL patients based on the 2008 International Workshop CLL (IWCLL) criteria . We extracted DNA from CD5+/CD19+ clonal B-cells and sequenced the coding regions of 59 recurrently mutated CLL driver genes using a custom SureSelect panel and Illumina HiSeq 4000. The median coverage depth per sample was 2737X. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies >0.01%, and by those identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering high/moderate impact mutations, we calculated the TML. Peripheral blood DNA was genotyped on Illumina Omni Express array; after extensive quality control metrics, we calculated the germline PRS as the weighted average of the number of risk alleles among the CLL susceptibility SNPs. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT. We analyzed the low/intermediate risk groups and the high/very high risk groups in stratified analyses and tested for interactions between CLL-IPI and TML or PRS. Survival curves are displayed using the Kaplan-Meier method using P-values from the log-rank test; the PRS is displayed by tertile categories. Results Of 166 patients (120 CLL, 46 MBL), 67% were male and the median age at diagnosis was 62 years (range: 28-85). The median follow-up was 5.3 years, and 73 were treated for progressive disease as defined by the 2018 IWCLL criteria. Low risk CLL-IPI comprised 51%, intermediate risk 27%, high risk 18%, and very-high risk 4%. The most commonly somatically mutated genes were TP53 (8%), CHD2 (8%), BIRC3 (8%), SF3B1 (7%), and NOTCH1 (7%). We found that 42%, 32%, and 26% of patients had 0, 1, or 2+ genes with somatic mutations, respectively. The median PRS was 8.3 (range: 6.1-11.3) which is consistent with our prior PRS study on CLL/MBL and higher than 7.53 found in 7983 controls from our prior work. When we modeled TML, PRS, sex, CLL/MBL status and CLL-IPI together with the joint effects of CLL-IPI with TML and PRS, both interactions were significant in the model (P=0.008 for TML*CLL-IPI and P=0.014 for PRS*CLL-IPI), with c-statistic=0.81 (CI:0.77-0.85). When we stratified by CLL-IPI, TML was associated with shorter TTT in the low/intermediate risk (N=129, continuous HR=2.36 per TML category, CI: 1.61-3.47, P=1.3x10-5, Figure 1.A), but there was no evidence of an association in the high/very-high risk (N=37, continuous HR=1.12 per TML category, CI: 0.70-1. 78, P=0.64, Figure 1.B), after adjusting for PRS, sex, and CLL/MBL status. However, the PRS was associated with a shorter TTT in the high/very-high risk (continuous HR=1.77 per SD, CI: 1.00-3.12, P=0.048, Figure 1.D), with no evidence of an association in the low/intermediate risk (continuous HR=0.95 per SD, CI: 0.95-1.29, P=0.74, Figure 1.C), after adjusting for TML, sex, and CLL/MBL status. Conclusions In our study, TML was prognostic among the low/intermediate CLL-IPI risk groups while the PRS was prognostic among the high/very-high CLL-IPI risk groups. Sequencing CLL driver genes and genotyping relevant germline SNPs at time of diagnosis may provide incremental value over the CLL-IPI in predicting TTT among patients with CLL/MBL. Disclosures Parikh: MorphoSys: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Acerta Pharma: Research Funding. Ding:Merck: Research Funding; DTRM Biopharma: Research Funding. Cerhan:Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB.

Treatment with CD19-directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes and was FDA-approved in acute lymphoblastic leukemia and non-Hodgkin B-cell lymphoma. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and in part may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. In this study, we aimed to investigate the interaction between circulating EVs isolated from CLL patient plasma (designated as CLL-derived EVs) and CART19 cells. We enumerated and immunophenotyped circulating EVs from platelet free plasma in untreated patients with CLL. We determined their interaction with CART19 cells using second generation, 41BB co-stimulated, lentiviral transduced CART19 cells generated in the laboratory from normal donors (FMC63-41BBζ CART cells). Our findings indicate that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation against the CD19+ mantle cell lymphoma cell line Jeko-1 (Figure 1A). Next, we characterized CLL-derived EVs using nanoscale flow cytometric analysis of surface proteins and compared to healthy controls. Although the total EV particle count was not different between CLL and healthy controls (Figure 1B), there were significantly higher PD-L1+ EVs in patients with CLL (Figure 1C). Based on these results, we sought to assess the physical interaction between CLL-derived EVs and CART cells from normal individuals. When CLL-derived EVs were co-cultured with CART19 and CLL B cells and imaged with super-resolution microscopy, EVs were localized at the T cell-tumor junction (Figure 1D). Furthermore, CLL-derived EVs are captured by T cells as indicated by a significant reduction in the absolute count of EVs when co-cultured with resting T cells (Figure 1E). Having demonstrated that 1) there is an excess of PD-L1+ EVs in patients with CLL (Figure 1C) and 2) CLL-derived EVs physically interact with CART cells (Figures 1D-E), we sought to establish their functional impact on CART19 cells. Here, CART19 cells were stimulated with irradiated CD19+ JeKo-1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL-derived EVs. There was a significant upregulation of inhibitory receptors such as PD-1 and CTLA-4 on the T cells (Figure 1F). This is associated with a reduction in CART effector cytokines (i.e., TNFβ) at higher concentrations of EVs (Figure 1G), suggesting a state of exhaustion in activated CART19 cells in the presence of CLL-derived EVs. This was further supported by transcriptome interrogation of CART19 cells. Here, CART19 cells were stimulated via 24-hour co-culture with the irradiated CD19+ cell line JeKo-1, in the presence of CLL-derived EVs at ratios of 10:1 and 1:1 EV:CART19 and then isolated by magnetic sorting. RNA sequencing of these activated CART19 cells indicated a significant upregulation of AP-1 (FOS-JUN) and YY1 (Figures 1H), known critical pathways in inducing T cell exhaustion. Finally, to confirm the impact of CLL-derived EVs on CART19 functions in vivo, we used our xenograft model for relapsed mantle cell lymphoma. Here, immunocompromised NOD-SCID-ɣ-/- mice were engrafted with the CD19+ luciferase+ cell line JeKo-1 (1x106 cells I.V. via tail vein injection). Engraftment was confirmed through bioluminescent imaging and mice were randomized to treatment with 1) untreated, 2) CART19 cells, or 3) CART19 cells co-cultured ex vivo with CLL-derived EVs for six hours prior to injection. A single low dose of CAR19 (2.5x105) was injected, to induce relapse. Treatment with CART19 cells that were co-cultured ex vivo with CLL-derived EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Figure 1I). Our results indicate that CLL-derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, through a direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way through which circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. Kenderian:Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

Despite the success of chimeric antigen receptor T (CART) cell therapy, it is limited by 1) lower rates of durable responses related to inadequate CART cell expansion and trafficking to tumor sites and 2) development of life-threatening complication such as cytokine release syndrome (CRS). Development of a strategy to efficiently and robustly image and track CART cells in the clinic would allow the in vivo characterization of T cell expansion and trafficking to tumor sites as well as the development of strategies to potentially overcome these limitations. The sodium iodide symporter (NIS) is a characterized and sensitive reporter system that has been used for cell imaging in the clinic. We hypothesized that the incorporation of NIS into CART cells would be a sensitive and efficient way to assess CART cell expansion, trafficking, and toxicity. To test our hypothesis, we used two CART cell constructs that are characterized in preclinical models and studied extensively in the clinic: CART19 (41BB costimulated) and BCMA-CART cells (41BB costimulated). First, we generated NIS+CART19 and NIS+BCMA-CART cells through dual transduction of lentiviral vectors (Fig A) and revealed the exclusive 125I uptake by these NIS+CARTs and its inhibition by the NIS inhibitor KClO4in vitro (Fig B). We then analyzed T cell functions of NIS+CART cells. Here, NIS+CART19 or CART19 cells were cultured with the CD19+ acute lymphoblastic leukemia (ALL) cell line NALM6. There was no difference in CART cell cytotoxicity (Fig C), proliferation, or cytokine production (not shown) between NIS+CART19 and CART19. This indicates that the incorporation of NIS into CART cells does not impair their antitumor activity. Next, we evaluated the sensitivity of NIS+CART19 cell detection by TFB-PET in vivo; imaging was performed using an Inveon TFB-PET/CT scanner. Mice received 250 μCi 18F-TFB 45 minutes prior to image acquisition. NIS+CART cells were detectable with TFB-PET when cells were subcutaneously injected at a dose of 1.25x106 cells (Fig D). Having demonstrated that the incorporation of NIS in CART cells provides a sensitive way of their detection by TFB-PET and does not interfere with their effector functions, we tested its efficiency to assess CART cell trafficking in vivo, using multiple myeloma (MM) xenografts. Here, immunocompromised NOD-SCID-ɣ-/- (NSG) mice were engrafted with the BCMA+OPM2 MM cell line (1x106 IV). After engraftment, the tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive 1) BCMA-CART or 2) NIS+BCMA-CART cells (5x106 IV). Mice were then serially imaged for 1) bioluminescence as a measure of disease burden, and 2) with TFB-PET to assess CART cell expansion and trafficking. As expected, BLI demonstrated that MM predominantly engrafts in bones (Fig E). TFB-PET confirmed trafficking of the NIS+BCMA-CART cells to the bones, corresponding to the areas involved by MM based on BLI (Fig E, right). Both BCMA-CART and NIS+BCMA-CART cells exhibited similarly potent antitumor activity in this model (not shown). Finally, we aimed to explore whether TFB-PET can detect CART massive expansion in vivo and predict the development of CRS. Here, we used an established CRS model in our laboratory. NSG mice were engrafted with patient derived relapsed ALL blasts (5x106 IV). Engraftment was confirmed by peripheral blood sampling. When the leukemic burden is >10 CD19+ cell/µl, mice were treated with either high dose NIS+CART19 cells (5x106 IV) or PBS control. One week after NIS+CART19 cell treatment, mice developed muscle weakness, hunched bodies, and weight loss (Fig F), which correlate with an extreme elevation of cytokines (Sterner et al. Blood 2018). TFB-PET revealed a significant uptake in the bone marrow, spleen, liver, and lungs (Fig G) of the diseased mice but not control mice. Mice were then euthanized, and tissues were harvested. Flow cytometric analysis confirmed an extensive infiltration of CART cells in the liver and spleen. This demonstrates the ability of TFB-PET to detect NIS+CART cell expansion in vivo, correlating with the development of CRS. In summary, our results robustly show that the incorporation of NIS into CART cells provides a sensitive, clinically applicable platform to image CART cells using TFB-PET and to assess their expansion, trafficking to tumor sites, and the development of CRS. These studies illuminate a novel way to noninvasively assess CART cell functions in vivo. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Suksanpaisan:Imanis: Employment. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board. Peng:Imanis: Equity Ownership. Russell:Imanis: Equity Ownership. Kenderian:Kite/Gilead: Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding.

Abstract Background: Hemophagocytic lymphohistiocytosis (HLH) is a severe clinical syndrome characterized by the activation of mononuclear phagocytic system. Patients (pts) typically present with fever, cytopenias, splenomegaly, hyperferritinemia and hemophagocytosis. There are two categories of HLH: primary (familial) and secondary (acquired). Primary HLH (FHL) is caused by genetic defects in proteins involved in cytotoxic function in T and NK cells, and occurs most commonly in children; though hypomorphic mutations in the same genes may cause adult-onset FHL. In contrast, secondary HLH typically occurs in adults, and is due to underlying infectious, autoimmune, and malignant disorders. The limited knowledge about secondary adult HLH is derived from case reports or case series involving few pts. The current study is the first collaborative effort to describe the etiology, clinical characteristics and outcomes in a large cohort of adult HLH pts. Methods: All adult (≥18 years) pts with a diagnosis of HLH according to the HLH-04 criteria seen at Barnes Jewish Hospital, St. Louis, MO (from October 2003 through June 2014) and at Mayo Clinic, Rochester, MN (from January 1996 through May 2014) were identified. Five of the following eight criteria are considered diagnostic of HLH according to the HLH-04 criteria: 1) fever ≥38.50C; 2) splenomegaly; 3) cytopenia of two or three lines: ANC <1 × 109/L, hemoglobin <9 g/dL, platelet count <100 × 109/L; 4) serum triglycerides ≥265 mg/dL or serum fibrinogen ≤150 mg/dL; 5) serum ferritin ≥500 mcg/L; 6) soluble IL-2 receptor ≥2400 U/mL; 7) low/absent NK cell activity; and 8) biopsy-proven hemophagocytosis. Pts who were treated as HLH but did not fulfill the diagnostic criteria were excluded. The most likely etiology of HLH (malignancy, infection, autoimmune, and idiopathic) was assigned for each patient based on standard diagnostic criteria. Clinical characteristics were compared using Chi-square, student’s t-test and Mann-Whitney test, as appropriate. Overall survival (OS), defined as time from HLH diagnosis to death from any cause, was assessed using Kaplan-Meier survival analysis and log-rank tests. The Institutional Review Boards of both institutions approved this study. Results: One hundred and sixteen adult pts with HLH were identified. Their median age at diagnosis was 50 years (range, 18-82); 69 (58%) pts were male. The clinical and laboratory findings of pts according to HLH-04 criteria are summarized in Table 1. At diagnosis, the median serum ferritin was 18510 mcg/L (range, 561-684,000), median serum fibrinogen was 169 mg/dL (range, 50-766), and median serum triglycerides was 307 mg/dL (range, 68-1475). Ninety-one (78%) pts had transaminitis; 97/106 (92%) pts had hypoalbuminemia; 86/114 (75%) pts had elevated PT and/or aPTT; and 51 (44%) pts had renal insufficiency. The causes of HLH were as follows: 43 (37%) malignancies, 42 (36%) infections, 15 (13%) attributed to autoimmune disorders and primary immunodeficiencies, and 16 (14%) were idiopathic. After a median follow-up of 1.5 months, 70 (60.3%) pts have died. The median OS of the entire cohort was 2.8 months (range, 0.1-73.1 months), with a 5-year overall survival of 20%. Pts with malignancy-associated HLH had a markedly worse survival (median=1.2 months) compared to pts with non-malignancy-associated HLH (median=13.8 months, p < 0.0001, Figure 1). In a multivariable analysis, malignancy-associated HLH (hazard ratio [HR] = 4.4; 95% CI: 1.7-11.3; p = 0.002) and male gender (HR = 2.8, 95% CI: 1.2-6.5; p = 0.02) were independently associated with shorter survival. Conclusions: The triad of fever, cytopenias and elevated ferritin (present in >80% of pts in this series) should alert physicians to a possible diagnosis of adult HLH. Survival after a diagnosis of HLH is dismal, especially among those with malignancy-associated HLH. The development of a national registry for adults with HLH will be beneficial to further our understanding of this condition and develop effective treatment strategies. Table 1: Clinical and laboratory findings according to the HLH-04 criteria Characteristic No. (%) of patients Fever 112/116 (97) Splenomegaly 75/116 (65) Bicytopenia or pancytopenia 95/116 (82) Hypertriglyceridemia 71/107 (66) Hypofibrinogenemia 50/107 (47) Hemophagocytosis 95/111 (86) Hyperferritinemia 115/115 (100) Elevated soluble IL-2 33/40 (82) Absent or decreased NK cell function 12/22 (54) Disclosures No relevant conflicts of interest to declare.

Background: The 2018 iwCLL guidelines recommend close observation of early stage CLL pts who do not meet indications for therapy ("watch and wait" strategy). Prior attempts at early therapeutic intervention in unselected early-stage CLL pts using alkylating agents such as chlorambucil and chemoimmunotherapy failed to show a significant benefit; and these therapies were associated with substantial toxicities. The introduction of Bruton tyrosine kinase (BTK) inhibitors has revolutionized the treatment landscape of CLL. The German CLL Study group recently presented results of the CLL12 study which demonstrated a significant improvement in event-free survival (EFS) among untreated early stage high risk CLL pts who received ibrutinib compared to placebo (median EFS not reached vs. 47.8 months, respectively; Langerbeins, EHA, 2019). Acalabrutinib is a more selective second-generation BTK inhibitor that has fewer adverse events in pts with CLL, and has shown equally impressive clinical activity for CLL pts. We are conducting an investigator initiated randomized phase 2 study to compare acalabrutinib with or without obinutuzumab (a glycoengineered anti-CD20 monoclonal antibody approved in the treatment of CLL) among high risk early stage CLL pts. Study Design: All pts with newly diagnosed (<2 years from registration) previously untreated early stage CLL who do not meet the 2018 iwCLL guidelines for initiation of therapy are eligible for enrollment in this study. Pts undergo risk stratification according to the CLL-International prognostic index (CLL-IPI) which consists of the following 5 variables: age >65 years (1 point), Rai stage I-IV (1 point), del17p and/or TP53 mutation (4 points), unmutated immunoglobulin heavy chain (IGHV) genes (2 points), and serum β2-microglobulin >3.5 g/dL (2 points). Pts with high (4-6) and very high (7-10) risk CLL-IPI are randomly (1:1) assigned to acalabrutinib 100 mg orally twice daily (Arm A) or acalabrutinib with obinutuzumab at a standard approved schedule (Arm B). Treatment with acalabrutinib is continued for a minimum of 2 years unless the patient has unacceptable side effects or withdraws consent. Pts with low (0-1) and intermediate (2-3) risk CLL-IPI score are assigned to an observation arm with follow-up once every 6 months for 2 years, and then according to routine clinical practice (Arm C). Endpoints: The primary endpoint of the intervention arms (Arms A and B) is the achievement of minimal residual disease (MRD)-negative complete remission in the bone marrow after 2 years of therapy. MRD is assessed using an 8-color flow cytometry with a detection limit of 0.01%. The primary endpoint of the observation arm (Arm C) is time to first therapy. Secondary endpoints for all arms are: progression-free survival, overall survival, and safety. Statistical design: A randomized trial comparing the experimental arm (acalabrutinib plus obinutuzumab) against the control arm (acalabrutinib alone) will be conducted as described by Rubinstein. A sample size of 36 pts per arm provides 80% power to detect an improvement in MRD-negative complete response rate from 10% to 30%, using a one-sided test at a significance level of 0.10 (EAST 6.4). We anticipate accruing an additional 8 high/very high risk pts (4 in each arm) to account for ineligibility, cancellation, or major treatment violation. The total enrollment for Arms A and B will be 80 pts. We will enroll 40 pts to Arm C; for a total enrollment of 120 pts. Key correlatives: Comprehensive profiling to assess the innate and adaptive immune system in paired peripheral blood and bone marrow will be conducted in all pts registered to Arms A and B at baseline, 12 and 24 months after enrollment. In addition, bone marrow hematopoietic function will be assessed in all pts at these time points. We will employ a CLL focused custom 61-gene panel to estimate tumor mutational burden as well as mutational profiles for each patient at baseline, 12, and 24 month time points, as well at the time of disease progression. Preliminary results: The trial is registered at clinicaltrials.gov NCT03516617. The trial opened to accrual at all three Mayo Clinic sites (Rochester, MN; Jacksonville, FL; and Scottsdale, AZ) in September 2018. Twenty-eight pts have been registered; the baseline characteristics are shown in Table 1. The trial is expected to complete enrollment in September 2020; and the primary analysis will be available in September 2022. Disclosures Parikh: Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Ding:DTRM Biopharma: Research Funding; Merck: Research Funding. Ailawadhi:Pharmacyclics: Research Funding; Cellectar: Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Chanan-Khan:Mayo Clinic: Employment; Ascentage: Research Funding; Millennium: Research Funding; Jansen: Research Funding; AbbVie: Research Funding; Xencor: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding. Kay:Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. OffLabel Disclosure: Drug: Acalabrutinib Purpose: treatment of CLL

Introduction: Patients (pts) with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) who failed treatment with the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib have a poor outcome and are difficult to treat. This ongoing, two-cohort, Phase II trial evaluates the safety and preliminary efficacy of tafasitamab (MOR208), an Fc-enhanced anti-CD19 monoclonal antibody in combination with idelalisib (IDE) (Cohort A) or venetoclax (VEN) (Cohort B) in R/R CLL pts previously treated with a BTKi. Preliminary results were published at EHA 2018 for Cohort A and at ASH 2018 for Cohort B. Here, we report the results of the primary analysis for both cohorts. Methods and Patients: Pts who either progressed or were intolerant to BTKi were enrolled at 12 sites in six countries in Europe and the US from Nov 2016 to Apr 2018. The primary endpoint is the incidence and severity of adverse events (AEs); secondary endpoints include overall response rate (ORR) as per investigator assessment according to International Workshop on CLL (IWCLL) 2008 guidelines. Complete response (CR) was confirmed by computed tomography assessment and by bone marrow (BM) biopsy. The exploratory endpoint minimal residual disease (MRD) was assessed centrally by quantitative allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) in peripheral blood (PB) and BM. Each treatment cycle (C) lasts 28 days (D). Dose and administration: tafasitamab intravenous infusion, 12 mg/kg weekly in C1-C3, every other week in C4-C6 and monthly from C7D1; IDE orally, 150 mg twice daily; VEN orally, weekly ramp up starting on C1D8 at 20 mg to full daily dose of 400 mg. Patients: mean time since first CLL diagnosis was 135 months (mos) for pts in Cohort A and 105 mos in Cohort B. Median number of prior therapy lines was five (2-9) and three (1-5), respectively. All pts had received ibrutinib; one pt had subsequent acalabrutinib treatment as last prior therapy line. Mutations of BTK and PLCγ2 were assessed in nine pts in Cohort A and 13 pts in Cohort B. BTK/PLCγ2 mutations were centrally detected in 4/3 pts in Cohort A and in 2/3 pts in Cohort B, respectively. Complex karyotype was observed in six (54.5%) pts in Cohort A and 12 (92.3%) pts in Cohort B. Results with a data cut-off date of 9 Nov 2018 are presented. Results: Cohort A: Median time on study was 9.9 mos (95% confidence interval [CI]: 5.7-not reached). Eleven pts were enrolled and received tafasitamab and IDE. Two pts discontinued treatment due to AEs (aspartate-aminotransferase increased; acute pancreatitis), two due to progressive disease (PD) and one pt by physician's decision. One pt died due to PD and one pt due to cardiac failure. At the cut-off date, treatment was ongoing in four pts. Table 1 summarizes treatment-emergent adverse events (TEAEs) with neutropenia Grade ≥3 being most common (5 [46%]). Fourteen treatment-emergent serious AEs (SAEs) were reported in eight (72.7%) pts. ORR was 90.9% (CR=9.1%, partial response [PR]=81.8%), disease control was achieved in all 11 pts. One of eight pts (12.5%) assessed for MRD status reached MRD-negativity in PB at C14. Cohort B: Median time on study was 12 mos (95% CI: 2.8-not reached). Eleven of 13 enrolled pts received tafasitamab and VEN while two pts received tafasitamab only. Three pts discontinued treatment due to AEs (infusion-related reactions [two pts], diarrhea [one pt], one due to PD and one withdrew consent. At the cut-off date, treatment was ongoing in eight patients. Table 2 summarizes TEAEs, neutropenia Grade ≥3 was most commonly observed (six [46%] pts). Fourteen SAEs were reported in nine (69.2%) pts. The ORR in all 13 pts was 76.9% (CR=23.1%, PR=53.8%, not evaluable=23.1%). Six of seven pts assessed for MRD in PB (46.2% of 13 [100%] pts) reached negative status in PB by C7 at the latest. One of three pts assessed for MRD in BM (7.7% of 13 [100%] pts) reached MRD negative status in BM at C15. Conclusions: This trial demonstrates that in heavily pretreated pts with R/R CLL who failed prior BTKi, tafasitamab in combination with IDE or VEN is a potential therapeutic option. The safety profiles of the combinations are influenced by the combination partner, but both combinations are manageable. The response rates and MRD-negativity rates indicate that combinations of targeted agents with anti-CD19 tafasitamab have valuable antitumor activity and warrant further investigation of tafasitamab-based combinations in CLL. Disclosures Staber: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Jurczak:Celtrion: Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morphosys: Research Funding; Celgene Corporation: Research Funding; Incyte: Research Funding; Servier: Research Funding; Roche: Research Funding; Novo Nordisk: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Gilead: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; TG Therapeutics: Research Funding. Brugger:AstraZeneca: Equity Ownership; MorphoSys: Employment. Chanan-Khan:Pharmacyclics: Research Funding; Merck: Research Funding; Jansen: Research Funding; Mayo Clinic: Employment; Ascentage: Research Funding; Millennium: Research Funding; Xencor: Research Funding; AbbVie: Research Funding. Greil:Mundipharma: Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Sanofi Aventis: Honoraria; Sandoz: Honoraria; AstraZeneca: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Celgene: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Eisai: Honoraria; Janssen-Cilag: Honoraria; Cephalon: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Boehringer Ingelheim: Honoraria; Amgen: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Ratiopharm: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; MSD: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Pfizer: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Roche: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; GSK: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Dirnberger-Hertweck:MorphoSys: Employment. Kelemen:MorphoSys: Employment. Middeke:Janssen: Consultancy, Speakers Bureau; MSD: Consultancy; AbbVie: Consultancy, Speakers Bureau; Gilead: Consultancy; Roche: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau. Montillo:Roche: Consultancy, Honoraria, Research Funding; Acerta: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Versatem: Membership on an entity's Board of Directors or advisory committees. Munir:Acerta: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: TBC; AbbVie: Honoraria; Alexion: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy. Parikh:Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; MorphoSys: Research Funding; Acerta Pharma: Research Funding; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Research Funding. Stilgenbauer:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Weirather:MorphoSys: Employment. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Wendtner:AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffman-La Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

INTRODUCTION Mutations in BTK and PLCG2 have been reported to occur in ~80% of CLL patients (pts) who have progression of disease on ibrutinib therapy (Woyach, JCO 2017; Ahn, Blood 2017). These mutations are described as appearing months before actual relapse and thus considered as a potential predictive biomarker for future relapse (Quinquenel, Blood 2019). However, the outcomes of these pts after disease progression are not well described. In this study, we seek to investigate time to next therapy and overall survival (OS) following progression among CLL pts on ibrutinib therapy with and without these resistance mutations. METHODS Between 10/2012 and 6/2019, we identified 34 pts in the Mayo Clinic clinical CLL resource who progressed while receiving ibrutinib therapy and also had testing for BTK and PLCG2 mutation performed as part of routine clinical practice at either NeoGenomics Laboratories or The Ohio State University. OS was calculated from time of ibrutinib progression to last known alive or death date; OS was plotted using Kaplan Meier methods and was compared using the log-rank test between various groups (e.g., mutation positive vs negative; CLL progression vs Richter's). Cumulative incidence of time to next treatment in those who had a treatment after progression was adjusted for the competing risk of death. RESULTS Of 34 pts who progressed while receiving ibrutinib, 26 pts experienced CLL progression and 8 pts had Richter's transformation; baseline characteristics in Table 1A. The presence of a BTK or PLCG2 mutation was found in 20/34 (59%) pts (specific mutations in Table 1B). BTK mutation alone was present in 9 pts, 7 pts had PLCG2 mutation alone, and 4 pts had both mutations. Median time between a positive test and start of next therapy was 4 months (range 1-19 months) and did not vary between BTK vs PLCG2 mutations. Among the 26 pts with CLL progression, 18 (69%) pts had a mutation present: BTK alone (n=8), PLCG2 alone (n=6), both (n=4). Therapy following progression on ibrutinib in these pts was as follows: venetoclax (n=16; 11 pts who continued ibrutinib in combination), idelalisib (n=4), investigational treatments (n=2), continued ibrutinib alone (n=2), dose-adjusted EPOCH-R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab; n=1), and unknown (n=1). Twelve of the 26 pts with CLL progression on ibrutinib, including 8 pts with a prior resistance mutation detected, had subsequent progression of disease on the aforementioned next line therapy. Treatment of these patients consisted of the following: restarted ibrutinib in addition to current treatment of venetoclax (n=5), venetoclax (n=2), pembrolizumab (n=2; 1 pt with continued ibrutinib), obinutuzumab with continued ibrutinib (n=1), gemcitabine and vinorelbine with continued ibrutinib (n=1), and no further treatment (n=1). Among the 8 pts with Richter's transformation as the initial progression event on ibrutinib after mutation testing, 1 pt had a BTK mutation and 1 pt had a PLCG2 mutation. Treatments following progression on ibrutinib included multi-agent chemoimmunotherapy (n=3; 2 pts received rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone [R-CHOP] with continued ibrutinib and 1 pt received doxorubicin, bleomycin, vinblastine, dacarbazine [ABVD] alone), pembrolizumab (n=3; 1 pt in combination with continued ibrutinib), venetoclax in combination with continued ibrutinib (n=1), and venetoclax and obinutuzumab (n=1). The median time to next treatment (second line of treatment beyond ibrutinib) for the 31 pts who started another therapy following progression on ibrutinib was 16.7 months (95% CI 9.6-NE; Figure 1A) and was not significantly different for pts with or without a resistance mutation (p=0.57). Median OS for all 26 pts with CLL progression was 28.7 month and there was no difference according to presence or absence of a resistance mutation (median 28.7 months vs 18.2 months, p=0.53; Figure 1B). The 8 pts with Richter's transformation had a median OS of 7.1 months (95% CI 2.0-NE). CONCLUSION Approximately 60% of pts tested in this progression cohort had a BTK or PLCG2 mutation at time of or preceding progression on ibrutinib therapy. OS and time to next therapy did not differ statistically between pts with mutated vs non-mutated clones; however, caution should be applied with the conclusions given the limited sample size. Disclosures Ding: DTRM Biopharma: Research Funding; Merck: Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:Ascentage Pharma: Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding.