ALBERT

Description: Key Points The authors developed a novel method for isolating tumor membrane antigens. LCP1 is functionally relevant to leukemia chemokine induced migration.

Description: Introduction Treatment of CLL is rapidly evolving, now including oral targeted agents and novel combinations. However, complete response (CR) rates remain low and continuous therapy is required. Eradication of minimal residual disease (MRD) is an increasingly important endpoint and an independent predictor of improved survival. Lisocabtagene maraleucel (liso-cel; JCAR017) is an investigational CD19-directed 4-1BB CAR T cell product administered in a defined composition of CD8:CD4 CAR T cells. TRANSCEND CLL 004 is an open-label Phase 1/2 trial of liso-cel in patients (pts) with relapsed/refractory (R/R) CLL (NCT03331198). Preliminary safety, pharmacokinetic (PK), and efficacy results from the Phase 1 monotherapy dose-finding portion of this study are reported. Methods Pts with CLL/SLL were eligible if they had received 3 (standard risk) or 2 [high risk: del(17p), TP53 mutation, unmutated IGVH, or complex karyotype] prior lines of therapy, including a Bruton's tyrosine kinase inhibitor (BTKi) unless medically contraindicated. Pts with active untreated CNS disease, ECOG 〉1, or Richter's transformation were excluded. After 3 days of lymphodepletion with fludarabine and cyclophosphamide, pts received liso-cel infusion. Two dose levels (DL) have been tested (DL1=5 × 107 CAR T cells; DL2=1 × 108 CAR T cells). Dose escalation followed a modified toxicity probability-interval-2 (mTIPI-2) algorithm. Dose-limiting toxicities (DLTs) were evaluated for 28 days post liso-cel infusion. Responses were assessed by iwCLL 2008 criteria. MRD was assessed at 10-4 sensitivity by 6-color flow cytometry using peripheral blood and at 10-6 sensitivity by clonoSEQâ (Adaptive) deep sequencing of bone marrow (BM) aspirates. Blood PK of liso-cel was determined using flow cytometry. Serum soluble chemokine and cytokine profiles for 39 analytes were assessed using V-PLEX immunoassays (MSD). Results At the time of data cut, 10 pts received liso-cel: 6 pts treated with DL1 and 4 pts with DL2. The median age was 64.5 years (range 51-76); 7/10 pts had high-risk disease. Pts had received a median of 4 prior therapies (range 3-8), including 9/10 pts who had received prior ibrutinib and 6/10 who previously received venetoclax and ibrutinib. No DLTs were identified. The most common adverse events (AEs) were cytokine release syndrome (CRS) (8/10 pts; all grade [G] 1/2), anemia (7/10 pts), thrombocytopenia (6/10 pts), and leukopenia (5/10 pts). Neurologic events (NE) were reported in 3/10 pts: G1 impaired concentration and aphasia, G3 encephalopathy, and G3 aphasia. The median time to onset of CRS and NE was 4.5 (range 1-9) and 11 (range 11-21) days respectively, and the median duration of CRS and NE was 5.5 (range 3-30) and 6 (range 2-20) days respectively. Six pts received tocilizumab and/or steroids for the management of CRS and/or NE. Serious AEs, all of which were G3/4, were reported in 5/10 pts. At 30 days post-dose, 6 of 8 pts who were evaluable for response had an objective response (75%), including 4 CRs (50%). Six of 7 pts (85.7 %) evaluable for MRD had undetectable disease by flow at the day 30 assessment. Of the 5 pts evaluable for response at 3 months post-dose, 4 had ongoing response and 1 progressed with Richter's transformation. All 4 pts with ongoing response continued to have undetectable MRD by flow at 3 months post-dose. Responses, including CRs and undetectable MRD responses, occurred in pts with high-risk and with standard-risk disease. Available BM analyses from clonoSEQâ data corroborate these findings and will be reported. Median Cmax was 219 CAR T cells/µl (range 0.35-583.46). Median time to peak expansion was 15.5 days (range 13-19) and median AUC was 1528 cells*day/μL (range 590-2847). In the one pt with a best response of progressive disease, minimal CAR T cell expansion was observed. CAR T cells persisted in pts maintaining their response at 3 months post-dose. Serum analysis showed elevated levels of multiple biomarkers, including CRP, IL6, PLGF, IL16, and IL15, in conjunction with CRS and NE. Conclusion Liso-cel toxicities were manageable, including events of CRS and NE, in these heavily pretreated pts with CLL. CRs and undetectable MRD were rapidly achieved in pts with both high-risk and standard-risk CLL who previously received ibrutinib, with the majority also having had received venetoclax. These preliminary data support continued development of single-dose liso-cel treatment in CLL. Disclosures Siddiqi: Juno Therapeutics: Other: Steering committee. Wierda:Genentech: Research Funding; AbbVie, Inc: Research Funding. Dubovsky:Juno Therapeutics: Employment, Equity Ownership. Gillenwater:Juno Therapeutics: Employment, Equity Ownership. Gong:Juno Therapeutics: Employment, Equity Ownership. Mitchell:Juno Therapeutics: Employment, Equity Ownership. Thorpe:Juno Therapeutics: Employment, Equity Ownership. Yang:Juno Therapeutics: Employment, Equity Ownership.

Description: Background: The availability of chimeric antigen receptor (CAR)-modified T cells (CAR T) has profoundly increased therapeutic options for patients (pts) with B cell malignancies, including DLBCL. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB, CAR T cell product administered at a target dose of CD4+ and CD8+ CAR T cells. To understand tumor microenvironmental (TME) factors affecting short-term and durable responses in pts with R/R DLBCL who received liso-cel in the TRANSCEND NHL 001 study, we conducted multiplexed IF analyses of 111 DLBCL biopsies for 83 pts obtained at baseline (n=58) and approximately 11 days (D11) (n=53; 28 paired) after liso-cel infusion (NCT02631044). Methods: We employed three 5-plex IF panels, consisting of antibodies detecting (1) B cell (CD19, CD20) and T cell lineage (CD4, CD8, EGFR) markers, (2) immunosuppressive markers (CD163, FoxP3, CD73, IDO1, PD-L1), and (3) functional markers (CD3, Ki67, GZMB, PD-1, EGFR). Liso-cel expresses a truncated EGFR (EGFRt), and fluorescent anti-EGFR was used to identify CAR T cells within the tumor biopsies. We also performed bulk tumor RNA profiling for an overlapping subset of 50 baseline biopsies and 37 D11 biopsies (11 paired). We investigated the association of differences in marker densities for pts with best overall response (BOR) of complete response (CR), and progressive disease (PD). Baseline and D11 biopsy findings were correlated with early responses at ~1 month (M1) posttreatment (PD n=16; CR n=42) and durable responses at ~9 months (M9) posttreatment (PD n=76; CR n=32; 55 pts evaluated at both M1 and M9). We investigated how baseline and D11 densities, with spatial distinction between tumoral and peritumoral regions, correlated with early and durable responses. All comparisons describe differences in median densities, and have statistical significance reported with uncorrected P values assessed via the (unpaired) Wilcoxon-Mann-Whitney nonparametric test. Results: Signals in baseline biopsies that correlated with early (M1) response differed from those that correlated with durable (M9) CR. A 21% higher baseline presence of PD-1+ T cells was associated with pts who achieved early CR at M1 vs pts who had PD at M1 (P=0.007). Pts with durable CR at M9 had 39% lower baseline levels of CD163+ macrophages (P=0.019) and 270% higher levels of CD73+ cells (P=0.028) than those with PD at M9. On-treatment (D11) tumors of pts with both early and durable CR had 28% higher levels of EGFRt+ (CAR T) CD8+ T cells (P=0.022), and 810% higher EGFRt- (non-CAR T) CD4+ (but notably, not CD8+; P=0.28) T cells (P=0.009). We also investigated changes in marker densities between baseline and on-treatment (D11) biopsies, and found that pts with durable CR at M9 had decreased on-treatment B cell densities (P=0.029), and increased densities of CD8+ GZMB+, Ki67+, and/or PD-1+ CAR (P=0.001) as well as non-CAR T (P=0.017) cells. Pts with durable CR also had a 29% increase in tumor-associated CD163+ macrophages at D11 relative to baseline (P=0.033). While the accessibility of spatial arrangements and multilabeled cells from IF enables a more nuanced picture of the TME, many of the general trends described above are concordant with those observed in bulk tumor RNA sequencing. Lower baseline expression of CD163 (P=0.021) and higher expression of CD73 (P=0.054) were seen in pts with durable CR. Additionally, elevated on-treatment (D11) expression of CD3E, CD4, and liso-cel (P

Description: On the forefront of targeted cancer therapy are second generation tyrosine kinase inhibitors (TKIs) which impact the outcome of chronic, accelerated, and blast phase chronic myelogenous leukemia (CML). Among these TKIs dasatinib is particularly effective for the treatment of imatinib-resistant Philadelphia chromosome-positive CML; although this drug has been associated with the generation of pleural effusions, fevers, and colitis in some patients. Recent data links these side effects to oligoclonal expansions of large granular lymphocyte (LGL) immunophenotype. To further characterize these events we conducted a comprehensive immunophenotye, T-cell receptor variable region, KIR, and HLA analysis of seven CML patients receiving dasatinib, two of which developed adverse reactions in association with lymphocytosis. Flow cytometric analysis confirmed these proliferative events and elucidated a previously undescribed CD3-CD8+CD56+ population. In addition, we identified multiple patients with decreased CD4 to CD8 T-cell ratios and a shift in CD4+ and CD8+ lymphocyte populations towards an effector and terminal memory phenotype (CD62L-CD45RA-and CD62L-CD45RA+ respectively). Further analysis of the T-cell receptor beta chain showed that all seven patients had oligoclonal populations of either CD4 or CD8 T-cells significantly greater than that of healthy donors. Most of these findings were independent of the occurrence of adverse reactions. All in all our data implicates dasatinib in generating oligoclonal lymphocyte expansions of various phenotypes in all CML patients. It is still unclear as to the underlying molecular mechanisms inducing these expansions, but further exploration could help to prevent or alleviate these reactions and could conceivably provide new tools for future directed immunotherapy.

Description: Abstract 2204 Poster Board II-181 Tyrosine kinase inhibitor (TKI) therapy has become the standard treatment of chronic myelogenous leukemia (CML). Although all of these drugs directly inhibit bcr-abl, off target effects are potentially responsible for their side effects. Of these TKIs, dasatinib is effective for the treatment of imatinib-resistant Philadelphia chromosome-positive CML. It has been shown that some dasatinib treated patients' exhibit adverse side effects resulting in colitis, pleural effusions and fevers. In conjunction with these adverse side effects, patients have been shown to present with oligoclonal expansions of large granular lymphocytes (LGL). We have now further characterized this phenomenon in fifty-six patients treated with five different TKIs. Age matched healthy controls as well as untreated newly diagnosed CML patients were used for comparisons. TCR clonality was determined using total cellular DNA subjected to amplification with a series of oligonucleotide primers for regions of the T-cell receptor genes and a flow cytometry based V-beta repertoire panel. Levels of activated nuclear T-bet and NFκB were analyzed by utilizing the respective Active Motif's Trans-AM T-bet and NFκB family kit in conjunction with the nuclear extraction kit according to established protocol. An analysis of membrane-bound IL-15 in LGL cells was determined by flow cytometry with commercially available mouse anti-human IL-15 in conjunction with CD3, CD8, CD16, CD56 and CD57. PDGF BB plasma levels were determined with ELISA techniques using plates generated from commercially available antibodies. Regulatory T cell percentages were investigated also by flow cytometry using mouse anti-human CD4, CD25 and CD127. Patients on dasatinib therapy with and without side effects show an elevated membrane bound IL-15, as well as modest elevated PDGF-BB plasma levels, both of which have been shown to be important in LGL survival. Patients with LGL expansions had constitutively active NFκB and variable T-bet expression in the CD8+ compartment in the setting of alpha/beta TCR clonality. Imatinib treated patients without significant side effects show broader plasma levels of PDGF-BB as well as an elevated membrane bound IL-15 expression with variable T-bet and NFκB expression. All patients treated with nilotinib showed low expression of IL-15, T-bet and NFκB expression as well as low levels of PDGF-BB. TCR clonality with or without LGL expansions is a relatively common phenomenon in CML patients under therapy with TKI's. In patients treated with dasatinib, some adverse side effects typically related with this drug are more frequently associated with this event. However, the ultimate mechanism of these clonal expansions is still unclear. A decreased percentage of regulatory T-cells or enhancement of antigen presentation capability that overcomes tolerance mechanisms has been described by our group and could play an important role. Finally, the inhibition of tyrosine kinases involved in the regulation of memory lymphocytes development is under investigation in our laboratory. Disclosures: Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Description: Abstract 183 CLL is a common leukemia which demonstrates variable reactivity of the B cell receptor (BCR) to antigen ligation, but constitutive activation of this pathway. Bruton's Tyrosine Kinase (BTK) is a member of this pathway which shows transcriptional upregulation and constitutive activity in CLL. Ongoing trials of ibrutinib, an orally bioavailable inhibitor of BTK, have shown outstanding preliminary activity in CLL. However results with other known reversible and more selective irreversible BTK inhibitors have had variable results. As ibrutinib potentially has other targets in addition to BTK, this raises the question of whether BTK is an important target in CLL. To answer this question and determine the role of BTK in the development and expansion of CLL, we have used Eμ-TCL1 (TCL1) transgenic mouse model of CLL, where the TCL1 oncogene is under the control of the VH promoter-IgH-Eμ enhancer and adult transgenic mice spontaneously develop CLL. To determine whether BTK is an important target in CLL as opposed to an alternative target of ibrutinib, we crossed the B6/TCL1 mouse with the XID mouse, which harbors a mutation in the PH domain of BTK, rendering it kinase-inactive. In B-cells derived from these mice we observed continued TCL1 expression and diminished BCR signaling with BCR ligation. XID/TCL1 mice (n=65) had a significantly prolonged overall survival (OS) compared to those with wild-type (WT) BTK (n=79) (Median not reached versus (vs) 13.4 months respectively, p

Description: Abstract 2376 Poster Board II-353 Background: Patients with relapsed or refractory CLL/SLL and patients with mantle cell lymphoma (McL) have a poor prognosis. Overall response rate (ORR) to salvage therapy for refractory patients is approximately 10-30%, and survival benefit with current treatment approaches is limited. Phase II studies of single agent lenalidomide in patients with relapsed or refractory CLL revealed an ORR of 32-58% (7-17% CR), depending on treatment dose, scheduled used and duration of treatment with lenalidomide. In patients with refractory or relapsed McL, lenalidomide treatment resulted in an ORR of 53% (CR 20%, PR 33%), and a 14-month median duration of response (Habermann et al 2009). Recent in vitro studies have shown that lenalidomide enhances the rituximab-induced killing of NHL cell lines and B-CLL cells via ADCC by restoring the defective T-cell and NK-cell mediated ability to form immune synapses to exert tumor cell cytotoxicity. Methods: Patients with relapsed or refractory CLL/SLL or McL received oral lenalidomide via dose escalation as follows, 2.5 mg on days 1-7, 5mg on day 8-14 and 10mg on day 15-21 followed by 7 days of rest in 28-day cycle; for cycle 2 and beyond 20mg was given on day 1-21 on a 28 day cycle. Rituximab was dosed at 375mg/m2 IV weekly for 4 weeks starting on day 15 of cycle 1. Treatment was continued until disease progression or toxicity. All patients were given allopurinol 300mg orally twice per day starting 3 days prior to first dose of lenalidomide. CT scans, and bone marrow biopsies were done every 2 months to assess for response. Primary objectives were overall response rate (CR+PR) and safety and tolerability of the combination regimen. Results: 17 patients were enrolled on study (13 patients with CLL/SLL and 4 patients with McL). Median number of prior chemotherapies was 3 (range 1-5). Median age was 64 years (range 42-80). Among patients with CLL, the most common cytogenetic abnormalities were trisomy 12 (isolated n=3, associated with other abnormalities n=4), del11q (isolated n=1, with others n=3), isolated del13q (n=1), complex cytogenetics with 3 or more abnormalities (n=4 including 1 patient with del 17p). Responses were assessed every 2 months after initiation of therapy. Response rate for 13 evaluable patients (10 with CLL and 3 with McL) relative to months on treatment with lenalidomide are summarized in the table. Although all responses were PR, the rate of PR improved with continued therapy suggesting increased responses with a longer duration of treatment with lenalidomide. Currently, 7 patients are still receiving active treatment on study, all with CLL (3 achieved a PR and 4 have SD). Of the 4 patients with McL enrolled on study, 1 achieved a PR after 2 months of therapy; 1 achieved SD after 2 months of therapy with a sustained SD after 6 months; 1 patient achieved SD after 2 months, but progressed after 6 months on treatment. The regimen was well tolerated. Most common (〉5%) toxicities include neutropenia (35% grade 3, 6% grade 4), fatigue (17% grade 1-2, 6% grade 3), tumor flare (12% grade 2, 12% grade 3), acute renal insufficiency (6% grade 1, 12% grade 3), rituximab related infusion reactions (6% grade 2, 6% grade 3), flu-like symptoms (6% grade 2, 6% grade 3), venous thromboembolic disease (6% grade 2, 6% grade 3), infections (11% including 1 patient with fatal endocarditis), and hypercalcemia (11% grade 4). Correlative studies are ongoing. Conclusions: The combination of lenalidomide with Rituximab is a promising combination regimen in CLL patients with very poor prognosis who have undergone multiple lines of therapy. This treatment combination appears tolerable with observed events consistent with the use of these two agents in other studies. Further investigation is warranted, possibly in the front line setting and in combination with other agents. Disclosures: Lancet: Celgene: Research Funding.

Description: Abstract 3866 Chronic lymphocytic leukemia (CLL) is an incurable accumulation of malignant B-lymphocytes in peripheral circulation, which home to supportive leukemic microenvironments within the bone marrow, lymph nodes, and spleen. Membrane associated antigens are critical to the pathogenesis of CLL since they facilitate leukemic cell signaling, microenvironment homing, proliferation, and survival. Targeting CLL surface molecules and associated signaling patterns is a current focus of CLL therapeutic development with antibodies and vaccine-based approaches. Rituximab, ofatumumab, and alemtuzumab are widely used antibody-based therapeutics that are proven to be effective at targeting CLL membrane associated antigens. However, despite enhanced patient survival, targeting of non-obligate antigens such as CD20 ultimately leads to antibody resistance. In addition, the expression of CD52 across multiple healthy immune cells often produces unacceptable off-target toxicity. Targeting immunosurveilance antigens may provide a key to overcome these obstacles. A number of relevant tumor antigens such as p53, EGFR, WT1, BCR-Abl and CLL-specific ROR1 elicit robust autoimmune responses in cancer patients due to high levels of ectopic expression, high relative immunogenicity and obligate oncogenic signaling that forces expression despite autoimmune recognition. In many cases, autoantibody signatures were realized long after identification of the oncologic target antigens; however, the convergence of these two datasets implies that such signatures could be utilized to identify novel targets. To further explore this possibility, we interrogated anti-tumor humoral reactivity specifically directed against autologous CLL membrane antigens. Immunoreactive leads were identified by mass spectrometry. Potential membrane antigens were further confirmed using immunoblot and ELISA based techniques. Our analysis revealed Lymphocyte Cytosolic Protein 1 (LCP1), a membrane associated lymphocyte-specific target that is constitutively expressed on CLL and ectopically expressed in cancers of various histological subtypes. Subsequent confirmatory assays unveiled high frequency, robust, LCP1-specific IgG autoimmune responses in CLL patients despite a profound absence of reactivity to common viral and vaccine antigens, implying continued antigenic stimulation in an immunosuppressed host. LCP1 plays a critical role in B-cell biology by cross linking f-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for CLL-critical signaling pathways such as PKC. In healthy B-cells phosphorylation of LCP1 at serine 5 is a key step in responding to CXCL12, a powerful lymph node and stromal cell chemokine. To further explore the role of LCP1 in CLL microenvironment recall signaling, we developed stable LCP1 knockdown and Ser-5 phosphomimetic CLL cell lines. Transwell assays confirmed that LCP1 activity contributed to the migration of CLL cells towards CXCL12. Our data reveals that LCP1 is a novel membrane associated target antigen in CLL with evidence of differential immune response as compared to common vaccine antigens. We also demonstrate that LCP1 plays a role in CLL microenvironment signaling. Future studies will focus on targeting specific moieties within LCP1 which are critical for its oncogenic signature. Disclosures: No relevant conflicts of interest to declare.

Description: Background: The advent of oral-targeted drugs has improved treatment outcomes for patients (pts) with CLL. Nonetheless, some pts prove intolerant or resistant to therapy and/or fail to achieve complete response (CR) with uMRD. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB CAR T cell product administered at a target dose of CD4+ and CD8+ CAR T cells. TRANSCEND CLL 004 is an open-label phase 1/2 study of liso-cel in pts with R/R CLL/SLL (NCT03331198). Methods: Eligible pts with CLL/SLL had received at least 3 (standard-risk disease) or at least 2 (high-risk disease: del[17p], TP53 mutation, unmutated IGHV, or complex karyotype) prior lines of therapy, including a Bruton's tyrosine kinase inhibitor unless contraindicated. Pts with active untreated central nervous system disease, Eastern Cooperative Oncology Group performance status 〉1, or Richter's transformation were excluded. After 3 days of lymphodepletion with fludarabine/cyclophosphamide, pts received liso-cel infusion; 2 dose levels (DLs) were tested: DL1=50×106 and DL2=100×106 CAR+ T cells. Dose-limiting toxicities (DLTs) were evaluated for 28 days postinfusion. Responses were assessed by 2018 International Workshop on CLL criteria. MRD was assessed at a sensitivity of ≤10-4 in blood and/or bone marrow (BM) lymphocytes. Liso-cel CAR T cells were monitored by flow cytometry of blood cells from treated pts over time. Serum cytokines and chemokines were assessed via an electrochemiluminescence platform. Results: At data cutoff, 23 pts were evaluable for safety and 22 for efficacy. Median age was 66 (range, 49‒79) years; 83% (19/23) of pts had high-risk disease. Pts had a median of 5 (range, 2‒11) prior therapies. All pts had received prior ibrutinib; 56.5% (13/23) had progressed on ibrutinib and received therapy with venetoclax; 91% (21/23) were refractory to, or had relapsed on, ibrutinib; and 9% (n=2) were intolerant to ibrutinib. Liso-cel was successfully manufactured in 96% of pts, with the established process. Nine pts were treated at DL1 and 14 pts at DL2. Two pts had DLTs (all at DL2: grade 4 hypertension in 1 pt; grade 3 encephalopathy, grade 3 muscle weakness, and grade 4 tumor lysis syndrome in 1 pt). The most common grade 3/4 treatment-emergent adverse events were thrombocytopenia, 70%; anemia, 96%; neutropenia, 56.5%; leukopenia, 43.5%. Two pts had grade 3 cytokine release syndrome (CRS); 5 pts had grade ≥3 neurological events (NEs), including encephalopathy (n=3). Median time to onset of CRS and NE was 4 (range, 1-10) days and 4 (range, 2-21) days, respectively. The median duration of CRS and NE was 5 (range, 2-30) days and 21 (range, 6-169) days, respectively. To manage CRS and/or NE, 61% (n=14) of pts received tocilizumab and 48% (n=11) received corticosteroids. Lymph node tumor burden correlated with NE (P=0.025). Additional disease distribution correlates are being evaluated and will be presented. Cytokine analyses revealed that NE onset was preceded by elevated TNFα and IL16 early after liso-cel infusion (P

Description: Key Points PI3K p110δ/γ inhibitor IPI-145 abrogates prosurvival signals and induces apoptosis in CLL cells. IPI-145 overcomes BTK C481S mutation conferring ibrutinib resistance.