Significant In Vivo Sensitivity to Aurora Kinase Inhibition in TCF3-Hlf rearranged Acute Lymphoblastic Leukemia

Abstract Acute Lymphoblastic Leukemia (ALL) harboring the t(17;19)(q22;p13) is a rare subtype of leukemia with a dismal prognosis. This recurring translocation produces an aberrant TCF3-HLF fusion with distinct gene expression profiles and drug sensitivity. Recent studies have shown that this subtype of ALL might be targeted using therapies inhibiting BCL-2 and the pre-B cell receptor through inhibition of SRC family kinases. However, preliminary validation of these studies have revealed significant heterogeneity of response to BCL-2 and SRC inhibitors. As such, we sought to identify other possible targets that could overcome this heterogeneity and improve response to therapy. Methods: One local as well as four other samples from the Children's Oncology Group's ALL Biorepository with TCF3-HLF ALL were expanded in immunodeficient NSG mice. All samples were verified by RT-PCR and Sanger sequencing for the fusion transcript. Samples were then interrogated with our functional drug screen that is comprised of compounds with activity against two-thirds of the tyrosine kinome as well as other non-tyrosine kinase pathways, including RAF/MEK/MAPKs, PI3K/AKT/mTOR, AMPK, ATM, Aurora kinases, CAMKs, CDKs, GSK3a/b, IKK, PKA, PKC, PLK1, and RAF as well as BCL2 family, BRD4, IDH1/2, Hedgehog, HSP90, NOTCH/g-secretase, proteasome, survivin, STAT3, and WNT/b-catenin. The samples were sequenced using the Agilent SureSelect Strand-Specific RNA Library Preparation Kit on the Bravo robot (Agilent). All five patient samples successfully engrafted into NSG mice and were tested for in vivo sensitivity as assessed for disease burden or survival. Results: Three patient samples were identified to carry Type I translocations fusing exon 13 of TCF3 with variable intronic insertions followed by exon 4 of HLF. All three type I translocations produced different fusions due to different lengths within the variable region. One sample predicted a truncation product of TCF3 ending in exon 13 with an early stop codon within the variable region. Two patient samples carried the identical type II translocation fusing Exon 12 of TCF3 with exon 4 of HLF. RNA-seq results of the five samples identified other individual translocations, but none involved other specific disease related lesions. Results from our drug screen showed significant heterogeneity in response to the majority of drugs assayed including the ABL/SRC inhibitor dasatinib and the BCL-2 inhibitor venetoclax. Further, in vivo studies exposing cohorts of animals to vehicle (n=5), dasatinib (40mg/kg/day; n=5), venetoclax (25-100mg/kg/day; n=5) or combination of dasatinib and venetoclax (n=5) identified only two samples with treatment benefit. Interestingly, review of the results of the drug screen suggested hypersensitivity to aurora kinase inhibitors. Each sample was tested in vivo in cohorts of vehicle (n=5) and alisertib (30mg/kg/day; n=5). All five ALL samples showed significant response (p<0.01 for all five samples compared to their respective vehicle controls by Chi Square analysis). All animals tolerated treatment and no animal showed significant hematologic toxicity from treatment with drugs. Conclusion: Our results suggest that TCF3-HLF ALL is a heterogeneous subset of ALL with both different gene expression patterns from TCF3-HLF to other fusions as well as functional drug response. In vivo validation in the murine model with these five samples suggests significant heterogeneity to current pursued targets such as BCL-2 and SRC compared to previously published reports. Most intriguing, all samples tested with alisertib identified significant in vivo response suggesting unique preclinical support to pursue further clinical testing within this rare and lethal subtype of ALL. Disclosures Leonard: Amgen: Research Funding. Mullighan:Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy. Tyner:Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding. Druker:GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Patient True Talk: Consultancy; Fred Hutchinson Cancer Research Center: Research Funding; ARIAD: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Celgene: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties.

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Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽

10.1182/blood-2018-99-118367 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1442-1442

Author(s):

Xiangmeng Wang ◽

Po Yee Mak ◽

Wencai Ma ◽

Xiaoping Su ◽

Hong Mu ◽

...

Keyword(s):

Progenitor Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Advisory Committees ◽

Equity Ownership ◽

Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

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Bone and Joint Health Markers in Persons with Hemophilia A (PwHA) Treated with Emicizumab in HAVEN 3

Blood ◽

10.1182/blood-2019-123689 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 626-626 ◽

Cited By ~ 1

Author(s):

Anna Kiialainen ◽

Markus Niggli ◽

Christine L. Kempton ◽

Giancarlo Castaman ◽

Tiffany Y. Chang ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

Study Entry ◽

Type I ◽

Healthy Individuals ◽

Advisory Committees ◽

Joint Health ◽

Equity Ownership

Introduction Emicizumab is a recombinant, humanized, bispecific monoclonal antibody that restores the function of missing activated factor VIII (FVIII) by bridging activated FIX and FX in persons with hemophilia A (PwHA). Prophylaxis with emicizumab once weekly or every two weeks resulted in significant reductions in bleeds, including joint bleeds, and a favorable safety profile in PwHA without FVIII inhibitors in the HAVEN 3 study (NCT02847637; Mahlangu et al. 2018). Recurrent joint bleeds in PwHA can lead to hemophilic arthropathy, and hemophilia A has been associated with decreased bone mineral density (Kempton et al. 2014). To explore the potential effect of emicizumab prophylaxis on bone and joint health beyond bleed prevention, we measured joint health scores and bone and joint biomarkers in HAVEN 3. Methods Hemophilia joint health scores (HJHS; v2.1) were evaluated at baseline and Week 49 of emicizumab prophylaxis in 107 PwHA in HAVEN 3. Biomarkers of bone formation (osteocalcin [OC], N-terminal propeptide of type I procollagen [P1NP]), bone resorption (C-terminal telopeptide of type I collagen [CTX-I]), osteoblasts (osteoprotegerin), osteoclastogenesis (soluble receptor activator of nuclear factor- kappaB Ligand [sRANKL]), cartilage turnover (cartilage oligomeric matrix protein [COMP]), and inflammation (interleukin 1 beta, interleukin 6, and tumor necrosis factor) were measured in 117 PwHA (Table 1) receiving emicizumab at baseline and after 3, 6, 12, and 18 months of treatment. In all, 94 of 117 PwHA with samples for biomarker analysis were part of the HJHS evaluation. Results PwHA previously on FVIII prophylaxis and those with no target joints at study entry had lower (indicating healthier) HJHS scores at baseline. Mean improvements from baseline of −2.25 (95% confidence interval [CI]: −4.12, −0.39) in total HJHS and −2.23 (95% CI: −4.07, −0.38) in HJHS joint-specific domain (excluding gait) were observed after 49 weeks of emicizumab prophylaxis in PwHA with one or more target joints at study entry (n=71). Improvement was consistent across HJHS for different locations (knee, ankle, elbow). No significant differences in the measured biomarkers between PwHA previously on FVIII prophylaxis or on on-demand treatment, or in those with or without target joints, were seen at baseline. Mean baseline values of most bone and joint biomarkers were within normal ranges, or similar to published levels in healthy individuals, although large variability was observed between individuals. None of the measured biomarkers changed significantly during emicizumab prophylaxis. Higher OC, P1NP, and CTX-I levels were observed in adolescent vs adult PwHA at all time points, which is consistent with reported increases of these biomarkers during skeletal growth. Data suggest a potential association of COMP levels with HJHS scores at baseline (Pearson correlation coefficient 0.46, p=0.0001). Data on two additional cartilage biomarkers, CTX-II (C-terminal telopeptide of type II collagen) and CS-846 (a chondroitin sulfate epitope) are being generated. Conclusions Reduction in joint bleeds was previously reported in HAVEN 3, including over 99% target joint resolution with long-term follow up (Callaghan et al. 2019). This analysis provides further evidence of the positive effect of emicizumab on joint health, showing significant and clinically relevant improvements in HJHS (defined as a ≥2-point reduction in HJHS joints domain [Kuijlaars et al. 2017]) after as few as 49 weeks of emicizumab prophylaxis. The biomarkers measured in blood as surrogates of bone and joint health did not show significant changes over the first 18 months of emicizumab prophylaxis. This may reflect heterogeneity between individuals, and effects on the measured biomarkers by factors other than joint health. However, improvement in bone and joint biomarkers would have been unexpected as the observed means at baseline were already similar to levels reported in healthy individuals. Although data from animal models have suggested that FVIII may play a role in bone health beyond protection against bleeds, in this study we observed no indication of worsening in any of the measured bone and joint health markers that might have resulted from reduced exposure to FVIII in PwHA who switched to emicizumab prophylaxis. Additional data are needed to better understand the long-term effect of emicizumab prophylaxis on bone and joint health. Disclosures Kiialainen: F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Niggli:F. Hoffmann-La Roche Ltd: Employment. Kempton:Novo Nordisk: Research Funding; Octapharma: Honoraria; Pfizer: Honoraria; Genentech, Inc.: Honoraria. Castaman:Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Uniqure: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kedrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Werfen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda (SHIRE): Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sobi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Chang:Genentech/Roche: Equity Ownership; Genentech, Inc.: Employment. Paz-Priel:Genentech, Inc.: Employment. Adamkewicz:F. Hoffmann-La Roche Ltd: Equity Ownership; Genentech, Inc.: Employment. Levy:F. Hoffman La Roche: Equity Ownership; Genentech, Inc: Employment.

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T-Cell Suppression By Src/ABL Kinase Inhibitors When Combined with Blinatumomab in Ph+ Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-119301 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2694-2694 ◽

Cited By ~ 1

Author(s):

Jessica Leonard ◽

Yoko Kosaka ◽

Pavani Malla ◽

Brandon Hayes-Lattin ◽

Adam J. Lamble ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Kinase Inhibitors ◽

Cell Function ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

Abl Kinase ◽

Equity Ownership ◽

Abl Kinase Inhibitors

Abstract Introduction: Targeted ABL kinase inhibitors (TKIs) have shown great activity in Ph+ Acute Lymphoblastic Leukemia (Ph+ ALL), however relapsed disease remains an unmet need. The bispecific antibody blinatumomab was recently approved as a single agent for use in patients with Ph+ ALL and there is much interest in combining this with targeted therapies. Second generation ABL kinase inhibitors inhibit both Src and LYN in addition to ABL. This is of particular interest in Ph+ ALL as LYN is important for leukemogenesis. T cell receptor (TCR) signaling is also dependent upon Src family kinase activity, and Src inhibitors may impact the efficacy of immunotherapies reliant on native T cell function. We sought to investigate the in vitro effects of ABL specific vs dual Src/ABL kinases on blinatumomab efficacy in both healthy donor as well as primary patient samples. Methods: We isolated peripheral blood mononuclear cells (PBMC) via Ficoll-Hypaque gradient from five healthy donors as well as from two patients with de novo and one patient with relapsed Ph+ ALL who harbored a T315I mutation. PBMC were labeled with CellTrace Violet and cultured for 5 days with no stimulation, blinatumomab, or blinatumomab in combination with imatinib, dasatinib, ponatinib or nilotinib at varying concentrations. Immunophenotyping was performed using multi-parameter flow cytometry for the following cell surface markers: CD45, CD3, CD4, CD8, CD56, and CD19. Blinatumomab efficacy was assessed by comparing the numbers of CD19+ / CD3- cells in untreated samples to those that had been treated with blinatumomab in the presence or absence of TKIs. Cell division of T cells was measured by CellTrace Violet dilution. Cytokine production was assessed via LEGENDplex Human Th Cytokine Panel. Levels of total Src, phospho-Src, total LCK and phospho-LCK were assessed via immunoblot. Results: After 5 days of exposure, blinatumomab led to T-cell proliferation in both healthy donor and patient PBMCs. Proliferation was observed in both CD8+ and CD4+ T cell subsets, although the effect was more pronounced in CD8+ cells. T cell proliferation, however, was completely suppressed by either dasatinib or ponatinib at nanomolar concentrations. This effect was far less pronounced with the ABL kinase inhibitors imatinib and nilotinib. Treatment of PBMCs with blinatumomab led to increased production of the cytokines IFN-g, IL-17-a and IL-22 in patient samples and healthy donors, while levels of IL-6 were increased in the patient samples only and levels of IL-10 in healthy subjects only. Cytokine production was absent in samples treated with blinatumomab and either dasatinib or ponatinib, while levels of IFN-g, IL-17a and IL-22 were minimally affected when blinatumomab was combined with imatinib. Immunoblots confirmed that dasatinib and ponatinib but not imatinib nor nilotinib inhibited phosphorylation of total Src as well as of LCK, likely explaining the inhibitory effects of these agents. In patient samples, blinatumomab alone and the TKIs alone greatly reduced the number of CD19+ cells. However, when dasatinib and blinatumomab were combined in the sample with a T315I mutation, there was little reduction in the percentage of CD19+ cells and no amplification of CD3+ cells, suggesting that dasatinib was able to inhibit the cytotoxic effects of blinatumomab with no effect to the leukemic cells. Discussion: Our results suggest that the combination of dual Src/ABL inhibitors with blinatumomab may abrogate the effects of blinatumomab by directly inhibiting T cell function. This is likely via inhibition of LCK, a known member of the TCR signaling pathway. Although small case series have reported responses in patients treated with blinatumomab and TKIs, it is possible that the majority of the response is from the TKI rather than blinatumomab. Only a randomized trial of a TKI +/- blinatumomab would be able to discern whether there is benefit of adding a dual Src/ABL TKI to bispecific antibody therapy. While our data are limited by sample numbers and by the fact that responses in living subjects may differ according to many other complex interactions in the in vivo immune microenvironment, the potential immunomodulatory effects of targeted therapies should be taken into consideration before they are combined with immunotherapies. Disclosures Leonard: Amgen: Research Funding. Druker:McGraw Hill: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Monojul: Consultancy; Millipore: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Oregon Health & Science University: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Meyers Squibb: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Henry Stewart Talks: Patents & Royalties; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Constellation: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Array: Research Funding; Takeda: Research Funding; AstraZeneca: Research Funding. Lind:Celgene: Research Funding; Janssen Pharmaceutical R&D: Research Funding; Amgen: Research Funding; Fluidigm: Honoraria; Monojul: Research Funding.

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Clinical Efficacy of Anti-CD22 Chimeric Antigen Receptor T Cells for B-Cell Acute Lymphoblastic Leukemia Is Correlated with the Length of the Scfv Linker and Can be Predicted Using Xenograft Models

Blood ◽

10.1182/blood.v130.suppl_1.807.807 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 807-807

Author(s):

Marco Ruella ◽

Shannon L Maude ◽

Boris Engels ◽

David M. Barrett ◽

Noelle Frey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

Xenograft Models

Abstract Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19 or CTL019) have shown impressive clinical activity in B-cell acute lymphoblastic leukemia (B-ALL) and are poised to receive FDA approval. However, some patients relapse after losing CD19 expression. Since CD22 remains highly expressed in relapsed/refractory (r/r) B-ALL even in these patients, anti-CD22 CART (CART22) have been developed. The National Cancer Institute (NCI) reported 4/9 complete remission (CR) in patients receiving CART22, with 100% CR at the highest T cell dose (NCT02315612)(S hah NN, ASH 2016 #650). Patients and Methods. We generated a second-generation CAR22 differing from that used by the NCI only by the use of a longer linker [4x(GGGGS); LL vs. 1x(GGGGS); SL] between the light and heavy chains of the scFv (Fig. 1 A). This construct was tested in two pilot clinical trials in adults (NCT02588456)and children with r/r-ALL (NCT02650414). CART22 cells were generated using lentiviral transduction as in our previous studies. The protocol-specified CART22 dose was 2x106-1x107 cells/kg for pediatric patients <50kg and 1-5x108 for pediatric patients ≥50kg and adult patients,. infused after lymphodepleting chemotherapy. Patient characteristics are described in Table 1. For the adult trial, 5 patients were screened, 4 enrolled (1 patient withdrew consent) and 3 infused (1 manufacturing failure). For the pediatric trial, 9 patients were screened, 8 enrolled (1 screen failure) and 6 infused (two patients were not infused for disease progression). For the preclinical studies, we generated CART22LL and CART22SL and tested them in vivo using xenograft models. NOD-SCID gamma chain deficient (NSG) mice were engrafted with either a luciferase+ standard B-ALL cell line (NALM6) or primary B-ALL cells obtained from a patient relapsing after CART19 (CHP110R). We also used 2-photon imaging to study the in vivo behavior and immune synapse formation and flow cytometry to asses T cell activation. Results. CART22 cells were successfully manufactured for 10/12 patients. In the adult cohort 3/3 patients developed CRS (gr.1-3) and no neurotoxicity was observed; in the pediatric cohort out of 5 evaluable patients (1 discontinued for lineage switch to AML on pre-infusion marrow), 3/5 developed cytokine-release syndrome (CRS) (all grade 2) and 1 patient had encephalopathy (gr.1). CART22 cells expanded in the PB with median peak of 1977 (18-40314) copies/ug DNA at day 11-18. Interestingly, in an adult patient who had previously received CART19 a second CART19 re-expansion was observed following CART22 expansion (Fig 1 B). At day 28, in the adult cohort the patient who was infused in morphologic CR remained in CR, while the other 2 had no response (NR); in the pediatric cohort 2/5 patients were in CR, 1 in partial remission (PR) that then converted to CR with incomplete recovery at 2 months, and 2 NR. No CD22-negative leukemia progression was observed. Since our results with a long linker appeared inferior compared to the previously reported CART22 trial (short linker), we performed a direct comparison of the 2 different CAR22 constructs. In xenograft models, CART22SL significantly outperformed CART22LL (Fi 1 C) with improved overall survival. Moreover, CART22SL showed higher in vivo proliferation at day 17 (Fig 1 D). Mechanistically, intravital 2-photon imaging showed that CART22SL established more protracted T cell:leukemia interactions than did CART22LL, suggesting the establishment of productive synapses (Fig 1 E). Moreover, in vivo at 24 hrs higher T cell activation (CD69, PD-1) was observed in CART22SL from the BM of NALM-6-bearing mice. Conclusions. Here we report the results of two pilot clinical trials evaluating the safety and feasibility of CART22 therapy for r/r B-ALL. Although feasible and with manageable toxicity CART22LL led to modest clinical responses. Preclinical evaluation allowed us to conclude that shortening the linker by 15 amino acids significantly increases the anti-leukemia activity of CART22, possibly by leading to more effective interactions between T cells and their targets. Finally, with the caveats of cross-trial comparison, our data suggest that xenograft models can predict the clinical efficacy of CART products and validate the use of in vivo models for lead candidate selection Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Maude: Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Engels: Novartis: Employment. Frey: Novartis: Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Brogdon: Novartis: Employment. Young: Novartis: Research Funding. Porter: Incyte: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Immunovative Therapies: Other: Member DSMB; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement. June: WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp: Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; University of Pennsylvania: Patents & Royalties; Adaptimmune: Consultancy. Gill: Novartis: Patents & Royalties, Research Funding.

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CDK9 As a New Therapeutic Vulnerability for Ibrutinib Resistance Mantle Cell Lymphoma (MCL)

Blood ◽

10.1182/blood-2020-141386 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-35

Author(s):

Michelle Wang ◽

Xiaohong Zhao ◽

Huijuan Jiang ◽

Joy Catherine Yan ◽

Eduardo Sotomayor ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Driver Mutations ◽

Cancer Center ◽

Drug Screen ◽

Advisory Committees

MCL is an aggressive B-cell lymphoma with poor prognosis due to emergence of drug resistant populations and lymphoma progression. Ibrutinib is a bruton's tyrosine kinase (BTK) inhibitor that was shown to have high response rates in MCL patients. However, as the use of this drug continues to grow in MCL and other B-cell lymphomas, emergence of resistance and fatal progression are of increasing clinical concern. Currently the mechanisms driving IR are poorly understood and no recurrent driver mutations have been identified in MCL. We have modeled acquired resistance to ibrutinib and implemented chemical proteomics and a cell-based drug screen approaches in IR MCL lines and primary samples, we have shown that MCL cells become resistant to ibrutinib through a kinome-adaptive reprogramming mechanism that lead to constitutive activation of the PI3K/AKT/mTOR pathway with increased levels of Myc and sustained transcription activation. The acquired IR MCL cells have increased rates of growth and augmented adhesion to stromal cells. Collectively, our published and preliminary studies indicate that IR MCL relies on global transcriptome remodeling and subsequent kinome reprogramming, leading to molecularly and clinically aggressive phenotype and resistance to ibrutinib therapy. Rather than there being a single mechanism of acquired IR, kinase networks are rewired in a plethora of ways in MCL cells as they become resistant to ibrutinib treatment. Adaptive remodeling of the kinome creates therapeutic challenges, where even combination targeted kinase inhibitor treatments are unlikely to be successful. Here, using genomic, chemical proteomic and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally and importantly, we provided that a novel and facile ex vivo image-based functional drug screening platform could predict clinical therapeutic responses of IR MCL and identified vulnerabilities that can be targeted to disable the evolution of IR. Disclosures Shain: Amgen: Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding; Karyopharm: Research Funding, Speakers Bureau. Shah:Kite/Gilead, Jazz, Incyte: Research Funding; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria; NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Moffitt Cancer Center: Current Employment.

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Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-131288 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3950-3950

Author(s):

Christopher A. Eide ◽

Stephen E Kurtz ◽

Andy Kaempf ◽

Nicola Long ◽

Jessica Leonard ◽

...

Keyword(s):

Clinical Trial ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Mononuclear Cells ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Advisory Committees ◽

Equity Ownership

Background: In patients with acute lymphoblastic leukemia (ALL), patient outcomes vary considerably by patient age group, specific genetic subtypes, and treatment regimen. Large-scale sequencing efforts have uncovered a spectrum of mutations and gene fusions in ALL, suggesting that combinations of agents will be required to treat these diseases effectively. Previous preclinical studies have shown efficacy of the BCL2 inhibitor venetoclax alone or in combination in ALL cells (Chonghaile et al., Can Disc 2014; Leonard et al, STM 2018), and the multi-kinase inhibitor ibrutinib (approved for patients with chonic lymphoblastic leukemia (CLL)) has also shown potent activity in subsets of ALL (Kim et al., Blood 2017). However, the combination of ibrutinib and venetoclax has not been evaluated to date in patients with ALL. Methods: We used a functional ex vivo screening assay to evaluate the potential efficacy of the combination of ibrutinib and venetoclax (IBR+VEN) across a large cohort (n=808) of patient specimens representing a broad range of hematologic malignancies. Primary mononuclear cells isolated from leukemia patients were plated in the presence of graded concentrations of venetoclax, ibrutinib, or the combination of both FDA-approved drugs. IC50 and AUC values were derived from probit-based regression for each response curve. A panel of clinical labs, treatment information, and genetic features for tested ALL patient specimens was collated from chart review. Single and combination drug treatment sensitivity were compared within groups by Friedman test, across groups by Mann-Whitney test, and with continuous variables by Spearman rank correlation. Results: Consistent with clinical data and previous literature, IBR+VEN was highly effective in CLL specimens ex vivo (median IC50=0.015 µM). Intriguingly, among specimens from 100 unique ALL patients, we also observed that IBR+VEN demonstrated significantly enhanced efficacy by AUC and IC50 compared to either single agent (p<0.001; median IC50=0.018 µM). In contrast, evaluation of this combination on primary mononuclear cells from two healthy donors showed little to no sensitivity. Breakdown of combination sensitivity (as measured by AUC) by a variety of clinical and genetic features revealed no associations with gender or specimen type. Among continuous variables tested, age was modestly correlated with combination AUC (Spearman r = 0.26) and increased blasts in the bone marrow were associated with increased sensitivity to the combination (Spearman r = -0.41; p = 0.0068). More broadly, specimens from patients with B-cell precursor disease (B-ALL) were more sensitive to IBR+VEN than those with T-cell precursor leukemia (T-ALL) (p = 0.0063). Within the B-ALL patient samples, those harboring the BCR-ABL1 fusion were significantly less sensitive to IBR+VEN than other subtypes of B-ALL (p = 0.0031). Within the T-ALL subset, there was a trend toward reduced sensitivity in patients with evidence of mutations in NOTCH1, though statistical significance was not reached. Evaluation of the combination using an expanded 7x7 concentration matrix in human ALL cell lines revealed varying degrees of sensitivity. For example, IBR+VEN showed enhanced efficacy in RCH-ACV B-ALL cells and showed synergy for the majority of drug-pair concentrations by the highest single agent (HSA) method (ibrutinib, venetoclax, and combination IC50: 0.60, 3.4, and 0.28 uM, respectively). Conclusion: Our findings suggest that the IBR+VEN combination, currently approved for patients with CLL, also demonstrates impressive efficacy against primary leukemia cells from ALL patients, warranting further investigation as a treatment strategy in the clinic to continue to improve outcomes for patients. Disclosures Leonard: Amgen: Research Funding. Druker:Cepheid: Consultancy, Honoraria; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Gilead Sciences: Other: former member of Scientific Advisory Board; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees. Tyner:Petra: Research Funding; Agios: Research Funding; Array: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Array: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Syros: Research Funding; Takeda: Research Funding; Petra: Research Funding; Agios: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Gilead: Research Funding; Incyte: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Janssen: Research Funding; Genentech: Research Funding.

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Enhanced In Vivo Persistence and Proliferation of NK Cells Expanded in Culture with the Small Molecule Nicotinamide: Development of a Clinical-Applicable Method for NK Expansion

Blood ◽

10.1182/blood.v130.suppl_1.657.657 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 657-657 ◽

Cited By ~ 1

Author(s):

Tony Peled ◽

Guy Brachya ◽

Nurit Persi ◽

Chana Lador ◽

Esti Olesinski ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Culture Method ◽

Employment Equity ◽

Advisory Committees ◽

Nsg Mice ◽

Equity Ownership

Abstract Adoptive transfer of cytolitic Natural Killer (NK) cells is a promising immunotherapeutic modality for hematologic and other malignancies. However, limited NK cell in vivo persistence and proliferation have been challenging clinical success of this therapeutic modality. Here we present a reliable, scalable and GMP-compliant culture method for the expansion of highly functional donor NK cells for clinical use. Nicotinamide (NAM), a form of vitamin B-3, serves as a precursor of nicotinamide adenine dinucleotide (NAD) and is a potent inhibitor of enzymes that require NAD including ADP ribosyltransferases and cyclic ADP ribose/NADase. As such, NAM is implicated in the regulation of cell adhesion, polarity, migration, proliferation, and differentiation. We have previously reported that NAM augments tumor cytotoxicity and cytokine (TNFα and IFN-γ) secretion of NK cells expanded in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NK cells expanded with NAM underwent typical changes observed with cytokine only-induced NK cell activation with no significant differences in the expression of activating and inhibitory receptors. CD200R and PD-1 receptors were expressed at low levels in resting NK cells, but their expression was up-regulated following activation in typical cytokine expansion cultures. Interestingly, the increase in CD200R and PD-1 was reduced by NAM, suggesting these NK cells to be less susceptible to cancer immunoevasion mechanisms (Fig 1). In vivo retention and proliferation is a pre-requisite for the success of NK therapy. We have reported that NK expanded with NAM displayed substantially better retention in the bone marrow, spleen and peripheral blood of irradiated NSG mice. Using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay, we demonstrated increased in vivo proliferation of NAM-cultured NK cells compared with cells cultured without NAM. These results were recently confirmed using a BrdU incorporation assay in irradiated NSG mice (Fig.2). These findings were mechanistically supported by a substantial increase in CD62L (L-selectin) expression in cultures treated with NAM. CD62L is pivotal for NK cell trafficking and homeostatic proliferation and its expression is down regulated in IL-2 or IL-15 stimulated cultures (Fig. 3). These data provided the foundation for the development of a feeder cell-free scalable culture method for clinical therapy using apheresis units obtained from healthy volunteers. CD3+ cells were depleted using a CliniMACS T cell depletion set. Following depletion, the CD3- fraction was analyzed for phenotypic markers and cultured in closed-system flasks (G-Rex100 MCS, Wilson Wolf) supplemented with 20ng/ml IL-15 or 50ng/ml IL-2 GMP, 10% human serum, minimum essential medium-α and NAM USP for two weeks. While at seeding, NK cells comprised 5-20% of total culture seeded cells, at harvest, NK cells comprised more than 97% of the culture. Although overall contamination of the NK cultures was low with either IL-15 or IL-2, a lower fraction of CD3+ and CD19+ cells was observed with IL-15 vs IL-2 (0.2±0.1% vs. 0.4±0.2% and 1.3±0.4% vs. 2.4±0.6%, respectively). Consequently, we decided to use IL-15 for clinical manufacturing. Optimization of NAM concentration studies showed similar expansion with 2.5 and 5 mM and a decrease in expansion with 7.5 mM NAM. Since NAM at 5 mM had a stronger impact on CD62L expression and on the release of IFNγ and TNFα than NAM at 2.5 mM, we selected 5mM NAM for clinical manufacturing. Overall median NK expansion after two weeks in closed G-Rex flasks supplemented with IL-15 and 5mM NAM was 50-fold (range 37-87). An additional and significant increase in expansion was obtained after doubling the culture medium one week post seeding. While there was a marked advantage for single culture feeding, more feedings had less impact on NK expansion and had a negative effect on the in vivo retention potential. Our optimized expansion protocol therefore involved one feeding during the two weeks expansion duration resulting in 162±30.7-fold expansion of NK cells relative to their input number in culture. Based on these data, we have initiated a clinical trial at University of Minnesota, to test the safety and efficacy of escalating doses (2 x 107/kg - 2 x 108/kg) of our novel NAM NK cell product in patients with refractory non-Hodgkins lymphoma and multiple myeloma (NCT03019666). Disclosures Peled: Gamida Cell: Employment, Equity Ownership. Brachya: Gamida Cell: Employment. Persi: Gamida Cell: Employment. Lador: gamida Cell: Employment, Equity Ownership. Olesinski: gamida cell: Employment. Landau: gamida cell: Employment, Equity Ownership. Galamidi: gamida cell: Employment. Peled: Biokine: Consultancy; Biosight: Consultancy. Miller: Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Bachanova: Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: 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.

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T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Blood ◽

10.1182/blood-2019-131491 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2673-2673

Author(s):

Jessica Liegel ◽

Dina Stroopinsky ◽

Haider Ghiasuddin ◽

Adam Morin ◽

Marzia Capelletti ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Cellular Therapy ◽

Ex Vivo ◽

Advisory Board ◽

Adoptive Cellular Therapy ◽

Advisory Committees ◽

Equity Ownership

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

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Therapeutic Targeting of Mertk and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-151726 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1184-1184

Author(s):

Ryan J Summers ◽

Juhi Jain ◽

Eleana Vasileiadi ◽

Brittany Smith ◽

Madison Stout ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Prolonged Survival ◽

Cell Phenotype ◽

Advisory Committees ◽

Equity Ownership

Abstract Introduction T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL and is associated with inferior outcomes relative to B-cell ALL. Early T-precursor ALL (ETP-ALL) is a subset of T-ALL characterized by an immature T cell phenotype, resistance to therapy, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T cell phenotype, suggesting a role in ETP-ALL. Inhibition of MERTK using shRNA delayed leukemia progression and prolonged survival in a T-ALL xenograft model, implicating MERTK as a therapeutic target. MRX-2843 is an orally available dual MERTK/FLT3 inhibitor currently in phase I clinical trials. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is specifically expressed in immature T cell precursors, is preferentially expressed in ETP-ALL compared to other T-ALLs, is essential for ETP-ALL cell survival, and is regulated downstream of MERTK in acute leukemia cells. Thus, combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods Loucy and PEER ETP-ALL cell lines were cultured with vehicle or MRX-2843. Phosphorylated and total MERTK were assessed by immunoblot. Relative cell numbers were measured using Presto Blue reagent. Cells were stained with PoPro-1-iodide and propidium iodide and apoptotic and dead cells were quantitated by flow cytometry. T-ALL patient samples were cultured with UNC2025, a close analogue of MRX-2843, and relative cell numbers were assessed using MTS reagent. Orthotopic xenografts were established in NSG or NSGS mice using luciferase-expressing Jurkat cells (T-ALL), luciferase-expressing Loucy cells (ETP-ALL) or an ETP-ALL patient sample and leukemia burden was monitored by bioluminescence imaging or flow cytometry. MRX-2843 (65 mg/kg or 75 mg/kg) or saline vehicle were administered orally once daily. Differences in disease burden were assessed with the Mann-Whitney-U test or one-way ANOVA. Survival was determined by Kaplan-Meier analysis. Loucy and PEER cells were plated and screened in quadruplicate against >150 pairwise combinations of MRX-2843 and the BCL-2 inhibitor venetoclax in a high-throughput format. Synergy was calculated using the response additivity model. Results Treatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK, inhibited expansion of ETP-ALL cells, and induced cell death in vitro. Fifty-four percent (21/39) of primary T-ALL patient samples were sensitive to UNC2025 with an IC 50≤550 nM, including 2/5 (40%) pediatric samples and 10/19 (53%) adolescent/young adult samples. Treatment with MRX-2843 significantly reduced leukemia burden in cell line-derived T-ALL and ETP-ALL xenograft models and prolonged survival by 50% and 13% in the T-ALL (n=10, p<0.0001) and ETP-ALL (n=10, p=0.0136) models, respectively. Similarly, in a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 reduced peripheral blood disease burden by 83% and spleen weight by 64% compared to vehicle-treated mice (n=8, p<0.001) and prolonged survival by 41% (n=8, p=0.0016). MRX-2843 mediated anti-leukemia activity in combination with venetoclax and a dose ratio of 1:20 MRX-2843:venetoclax provided optimal synergy in Loucy and PEER ETP-ALL cells in vitro (Figure 1). Conclusions MRX-2843 has therapeutic activity in ETP-ALL cell culture and xenograft models and over half of T-ALL patient samples were sensitive to MERTK/FLT3 inhibition. MRX-2843 also mediated synergistic anti-leukemia activity against ETP-ALL cells in combination with venetoclax, with an optimal molar ratio of 1:20. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL, suggest that MRX-2843 may be particularly active alone and in combination with venetoclax in the ETP-ALL subset, and provide rationale for clinical testing of MRX-2843, with the ultimate goal to progress to trials evaluating MRX-2843 in combination with other agents. Toward this end, MRX-2843 monotherapy will be tested in patients with relapsed leukemia in an upcoming clinical trial (NCT04872478). Figure 1 Figure 1. Disclosures Wang: Meryx: Other: Equity ownership; University of North Carolina: Patents & Royalties. Frye: University of North Carolina: Patents & Royalties; Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Earp: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Tyner: Petra: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Constellation: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Agios: Research Funding. DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

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Antibody-Interferon-Alpha Fusion Protein Therapy for the Treatment of B-Cell Non-Hodgkin Lymphoma: Enhanced ADCC, Inhibition of Proliferation, and In Vivo Eradication of CD20+ Human Lymphomas

Blood ◽

10.1182/blood.v126.23.2762.2762 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2762-2762 ◽

Cited By ~ 1

Author(s):

John M. Timmerman ◽

Kristopher K Steward ◽

Reiko E Yamada ◽

Patricia A Young ◽

Dena M. Minning ◽

...

Keyword(s):

Fusion Protein ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Term Survival ◽

Equity Ownership

Abstract Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab. Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice. Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and < 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p < 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression. Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. Disclosures Timmerman: Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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