Enhancing the Immune Surveillance in Multiple Myeloma Via CDK4/6 Inhibition

Deregulation of cyclin D genes is a uniform event in multiple myeloma (MM) and represent a striking addiction as observed in pan-cancer genome-wide CRISPR screening data. However, early stage Cyclin D and other cell cycle kinases inhibitors have shown a lack of single agent activity suggesting that targeting of cell cycle regulation is insufficient to produce a durable response in MM. Recent evidence recognizes the Cyclin D and CDK4 activities within the immune tumor microenvironment, supporting a previously unrecognized immunomodulatory functions of CDK4/6. This is particularly important in MM, a highly heterogeneous disease that resides in a complex ecosystem comprising of immune, endothelial, and stromal cells. We here evaluated the tumor intrinsic and extrinsic effects of CDK4/6 inhibition in MM with the goal to define rationally designed combination strategies to effectively impact MM growth. We have evaluated MM cell sensitivity to CDK4/6 inhibitors (both Palbociclib and Abemaciclib) in a panel of 32 MM cell lines and primary MM patients' samples. As expected, both inhibitors were mostly cytostatic with G0/G1 cell cycle arrest and significant impact on the pRB-E2F axis both in vitro as well as in vivo. In luminescence subcutaneous SCID models engrafted with H929 or MM1S MM cells expressing an E2F-driven luciferase reporter, treatment with low dose Palbociclib or Abemaciclib caused regression of bulky tumors, evidenced by a ~40% reduction in tumor volume at the 14-day end-point and a decreased E2F1 reporter activity. We next studied genome-wide transcriptional response to treatment using RNA-seq analysis in two MM cell lines using multiple doses and duration (24 and 72 hours) to evaluate the effect of short and long exposure to the drug. Gene set enrichment analysis (GSEA) of RNA-seq data confirmed significant downregulation of proliferation and E2F target genes at 24 hours post treatment. Interestingly, after longer exposure to both drugs we observed modulation of signatures for Ag presentation (including upregulation of HLA-A,B, and C; B2MG), innate immune response (ICAM-1 and 2; IL-8 and several CCLs) and interferon inducible genes IFN response (IRF1, IRF9, STAT1, STAT2, STAT4, OSA1, OSA2, MX1). To confirm genomic data and evaluate if CDK4/6i promotes the induction of the senescence-associated secretory phenotype (SASP) program in MM cells, we performed cytokine profile to assess the secretion of 174 soluble factors in the MM cell supernatant upon CDK4/6i. We confirmed a significant increase of chemokines involved in NK cell recruitment (CCL2, CCL4, CCL5), as well as cytokines that promote NK cell proliferation and activation. Moreover, we found that intercellular adhesion molecule-1 (ICAM-1) and the NKG2D ligands ULBP2 and MICA, required for activation of NK cell cytotoxicity and tumor cell targeting, were induced after CDK4/6i in MM cells. Although not secretory per se, these NK cell ligands are part of the transcriptional module linked to the SASP. Overall, these data suggest that, in addition to a more stable cell cycle arrest, CDK4/6i may promote MM cell immune surveillance through induction of the SASP program. To further confirm the immune effects, we tested freshly isolated NK cells from MM patients and healthy donors using in vitro MM-NK cell coculture assay and observed enhanced degranulation and cytokine production (intracellular IFN-γ and TNFα) by NK cells in response to MM cells. We finally investigated the potential of combining CDK4/6i with daratumumab in a standard 4-h ADCC assays using NK cells from MM patients as effector and MM cell lines as target. Daratumumab-mediated ADCC against MM cells was significantly augmented in the combination compared to single agent. In conclusion, we here report a novel anti-MM activity of CDK4/6i which is beyond the previously reported growth arrest. The observed ability to directly modulate the immune system along with decrease in the proliferative potential of MM cells may provide opportunities to develop unique combination approaches in MM. Disclosures Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: 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; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Fulciniti:NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Legend: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company.

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Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽

10.1182/blood-2020-141459 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-7

Author(s):

Mao Yu Peng ◽

Yasmin Abaza ◽

Martina Mcdermott ◽

Monica Mead ◽

Dennis J. Slamon ◽

...

Keyword(s):

Cell Cycle ◽

Synergistic Effect ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Fatty Acid Synthase ◽

Single Agent ◽

Therapeutic Strategies ◽

Private Company ◽

Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates >85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

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Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Blood ◽

10.1182/blood-2020-142592 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Yao Yao ◽

Woojun D Park ◽

Eugenio Morelli ◽

Mehmet Kemal Samur ◽

Nicholas P Kwiatkowski ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Mononuclear Cells ◽

Luciferase Reporter ◽

G1 Arrest ◽

Normal Donor ◽

Private Company ◽

Advisory Committees

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: 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; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

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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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CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽

10.1182/blood-2018-99-118926 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3199-3199 ◽

Cited By ~ 2

Author(s):

Subhashis Sarkar ◽

Sachin Chauhan ◽

Arwen Stikvoort ◽

Alessandro Natoni ◽

John Daly ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Advisory Committees ◽

Cd38 Expression ◽

Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor

Blood ◽

10.1182/blood-2019-126613 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 138-138

Author(s):

John Daly ◽

Subhashis Sarkar ◽

Alessandro Natoni ◽

Robert Henderson ◽

Dawn Swan ◽

...

Keyword(s):

Multiple Myeloma ◽

Sialic Acid ◽

Nk Cells ◽

Board Of Directors ◽

Cell Lines ◽

Immune Evasion ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees

Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (>85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p<0.01 at 0.5:1, p<0.001 at 1:1, p<0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p<0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p<0.05 at 0.5:1, p<0.01 at 1:1) or 3Fax-Neu5Ac (p<0.01 at 0.5:1, p<0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.

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A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Blood ◽

10.1182/blood.v114.22.3830.3830 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3830-3830

Author(s):

Gullu Gorgun ◽

Elisabetta Calabrese ◽

Teru Hideshima ◽

Jeffrey Ecsedy ◽

Giada Bianchi ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Cell Lines ◽

Mitotic Spindle ◽

Research Funding ◽

Thymidine Incorporation ◽

Aurora A Kinase ◽

Advisory Committees

Abstract Abstract 3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

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Targeting CD38high Acute Myeloid Leukaemia with "Affinity Optimized" Chimeric Antigen Receptor and Membrane Bound TRAIL Expressing Natural Killer Cells

Blood ◽

10.1182/blood-2019-128605 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5536-5536 ◽

Cited By ~ 1

Author(s):

Emma Nolan ◽

Arwen Stikvoort ◽

Mark Gurney ◽

Nutsa Burduli ◽

Lucy Kirkham-McCarthy ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Cytotoxicity Assays ◽

Cd38 Expression ◽

Membrane Bound

Introduction: Chimeric Antigen Receptor (CAR) based cellular-immunotherapies have demonstrated significant clinical efficacy in haematological malignancies. However, the progress of cellular-immunotherapy for the treatment of Acute Myeloid Leukaemia (AML) has failed to gain momentum due to the lack of targetable tumour specific antigens. CD38 is a transmembrane glycoprotein expressed in lymphoid and myeloid cells with high expression in plasma B-cells, and is a well validated target for anti-CD38 therapy in Myeloma. A recent study has furthermore shown that a proportion of AML patients express CD38 on their leukemic blasts. TNF-related apoptosis-inducing ligand (TRAIL) receptor DR4 is another targetable antigen which has been shown to be expressed in 70% of AML patients. In this study, we investigate the therapeutic efficacy of "affinity-optimized" variant(s) of CD38 CAR and membrane bound TRAIL on NK-cell based platforms which can target AML blasts with high expression of CD38 (CD38high AML). The CAR variant is a CAR which binds with lower affinity to CD38 expressed on healthy immune cells such as CD38positive NK cells, while targeting CD38high AML. The membrane bound TRAIL variant (TRAIL4c9) is a mutant which binds with higher affinity to TRAIL-DR4 on AML cells, whilst avoiding binding to decoy receptors. We hypothesize that genetically modifying NK cells to express "affinity optimized" CD38 CARand/or TRAIL4c9 can effectively eliminate CD38high AML cells. Methods: AML cell lines THP-1, U937, and KG1a were immunophenotyped for CD38 and TRAIL-DR4 expression. Retrovirally transduced CD38 CAR-KHYG1 NK cells were used as immune effector cells and were co-cultured with AML cell lines in cytotoxicity assays. CD38low AML cell line KG1a was pre-treated with 10nM all-trans-retinoic acid (ATRA) to upregulate CD38 expression and were subsequently co-cultured with CD38 CAR-KHYG1 in cytotoxicity assays. CD38 CAR-KHYG1 was also co-cultured with n=4 patient derived AML cells in cytotoxicity assays. Using Maxcyte GT electroporation system primary donor derived IL-2 activated NK cells were either mock electroporated, or electroporated with TRAIL4c9 m-RNA orCD38 CAR m-RNA and subsequently co-cultured with THP-1 or ATRA pre-treated KG1a in a cytotoxicity assay. Expression of pro-apoptotic, anti-apoptotic and ligands for checkpoint inhibitory receptors was analysed by immunoblotting or flowcytometry. Results: Based on immunophenotyping, we classified AML cell lines as CD38high (THP-1), CD38moderate (U937) and CD38low (KG1a). CD38 CAR-KHYG1 was significantly more cytotoxic than MOCK KHYG1 against CD38high THP-1, at E:T ratios of 2.5:1, 5:1 and 10:1. CD38 CAR-KHYG1 were also more cytotoxic than MOCK KHYG1 against CD38moderate U937 at multiple E:T ratios; albeit the increase in cytotoxicity was at a much lower level in comparison to THP-1 (Fig 1a). Pre-treatment of CD38low KG1a cells with 10nM ATRA upregulated the cell surface expression of CD38, which were subsequently eliminated by CD38 CAR KHYG1 at E:T ratios of 2.5:1, 5:1 and 10:1. KG1a was intrinsically resistant to NK cells as compared to THP-1 and U937 (Fig 1b). This could partly be explained by the high intracellular expression of Bcl-xL, and higher cell surface expression of Nectin-1 and Sialic acid which are the ligands for checkpoint inhibitory receptors CD96 and Siglec-7/9 respectively on NK cell (Fig 1c). CD38 CAR-KHYG1 mounted a potent cytotoxic response against primary CD45intermediate AML blasts (n=4 patients) at multiple E:T ratios, and the extent of CAR induced cytotoxicity correlated with the cell surface CD38 expression on the primary AML blasts (R2=0.87) (Fig 1d,e). TRAIL4c9 or CD38 CAR m-RNA electroporated primary donor-derived NK cells were also potent in eliminating THP-1 and ATRA pre-treated KG1a at multiple E:T ratios (Fig 1f). This demonstrates the potential of therapeutically treating AML patients, with high CD38 expression, with a combination of NK cells expressing "affinity-optimized" CD38 CAR and membrane bound TRAIL variant. Conclusion: The study demonstrates the therapeutic potential of an "affinity-optimized" CD38 CAR NK cell-based therapy, which can potentially be combined with membrane bound TRAIL expressing NK cells to target CD38high AML. In patients with CD38low expressing AML blasts, patients could be pre-treated with ATRA followed by the combination therapy of CD38 CAR and TRAIL expressing NK cells. Disclosures Stikvoort: Onkimmune Ltd., Ireland: Research Funding. Kirkham-McCarthy:Onkimmune Ltd., Ireland: Research Funding. Van De Donk:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Mutis:Celgene: Research Funding; Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; BMS: Research Funding; Novartis: Research Funding; Aduro: Research Funding; Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy; BMS: Research Funding.

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Engineered iPSC-Derived NK Cells Expressing Recombinant CD64 for Enhanced ADCC

Blood ◽

10.1182/blood-2020-139760 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Kate Dixon ◽

Robert Hullsiek ◽

Kristin Snyder ◽

Zachary Davis ◽

Melissa Khaw ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Advisory Committees ◽

High Affinity ◽

Adcc Assay

Natural killer (NK) cells are innate cytotoxic lymphocytes. They target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and also recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). While ADCC by NK cells is a key mechanism of several clinically successful therapeutic monoclonal antibodies (mAbs), most patients exhibit or acquire resistance to mAb therapies. ADCC by human NK cells is exclusively mediated by the IgG Fc receptor, CD16A (FcγRIIIA). Studies have demonstrated that increasing the binding affinity between CD16A and therapeutic mAbs can augment their clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we are interested in enhancing the ADCC potency of NK cell-based therapies for various malignancies. CD64 is the only high affinity FcγR family member and binds to the same IgG isotypes as CD16A (IgG1 and IgG3) but with > 30-fold higher affinity. CD64 (FcγRI) is normally expressed by certain myeloid cells but not by NK cells. We generated a recombinant version of this receptor consisting of the extracellular region of CD64 and the transmembrane and intracellular regions of human CD16A, referred to as CD64/16A (figure 1A). An important feature of CD64/16A is that due to its high affinity state, soluble monomeric anti-tumor mAbs can be pre-adsorbed to engineered NK cells expressing the recombinant FcγR, and these pre-absorbed mAbs can be switched or mixed for universal tumor antigen targeting (figure 1B). The engineered NK cells used in our study were derived from genetically edited and clonally derived induced pluripotent stem cells (iPSCs) through a series of stepwise differentiation stages (figure 2). Engineered iPSC-derived NK (iNK) cells can be produced in a uniform and clinically scalable manner (figure 2). In Figure 3, using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against SKOV3 cells, an ovarian adenocarcinoma cell line, in the presence of the anti-HER2 therapeutic mAb trastuzumab (Herceptin) or anti-EGFR1 therapeutic mAb cetuximab (Erbitux), when either added to the assay or pre-adsorbed to the iNK cells (figure 3). Considering the high affinity state of CD64, we examined the effects of free IgG in human serum on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis System, ADCC was evaluated in the presence or absence of 5% human AB serum, in which free IgG was approximately 50-fold higher than the IgG saturation level of the CD64/16A receptors on iNK cells (data not shown). Despite the high levels of excess free IgG, iNK-CD64/16A cells mediated efficient ADCC when Herceptin was either added to the assay or pre-adsorbed to the cells (figure 4). ADCC assays were also performed with Raji cells, a Burkitt lymphoma cell line, as target cells and the therapeutic mAb rituximab (Rituxan). iNK-CD64/16A cells were added with or without pre-adsorbed Rituxan and the assay was performed in 10% AB serum. Again, iNK-CD64/16A cells mediated effective target cell killing in the presence of serum IgG (figure 5), demonstrating that saturating levels of free IgG did not prevent ADCC. To determine if we can further optimize the function of recombinant CD64, we engineered CD64 with the transmembrane regions of CD16A or NKG2D and signaling/co-signaling domain from CD28, 2B4 (CD244), 4-1BB (CD137), and CD3ζ (figure 6). CD64/16A signals by non-covalent association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapters CD3ζ and FcRγ found in the cell membrane, whereas the other recombinant CD64 constructs use ITAM and non-ITAM regions to mediate their signaling. The various recombinant CD64 constructs were initially expressed in NK92 cells (lacks expression of endogenous FcγRs) (figure 7). Using the Delfia® ADCC assay system, we examined the function of each recombinant CD64 construct and found all combinations are able to effectively induce ADCC (figure 8). We are in the process of generating iNK cells with these constructs and testing their ability to kill hematologic and solid tumors in vitro and in vivo. Our goal is to utilize this docking approach to pre-absorb mAbs to iNK cells for adoptive cell therapy. The mAbs would thus provide tumor-targeting elements that could be exchanged as a means of preventing tumor cell escape by selectively and easily altering NK cell specificity for tumor antigens. Figure Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Chu:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Dinella:Fate Therapeutics: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding.

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FT538: Preclinical Development of an Off-the-Shelf Adoptive NK Cell Immunotherapy with Targeted Disruption of CD38 to Prevent Anti-CD38 Antibody-Mediated Fratricide and Enhance ADCC in Multiple Myeloma When Combined with Daratumumab

Blood ◽

10.1182/blood-2019-131138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 133-133

Author(s):

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Karrune Woan ◽

Frank Cichocki ◽

Greg Bonello ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Pluripotent Stem Cell ◽

Nk Cell ◽

Target Cells ◽

Stem Cell Line ◽

Advisory Committees ◽

Cell Immunotherapy

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, though there remains meaningful opportunity to improve mAb efficacy by optimizing the interaction with natural killer (NK) cells to enhance antibody-dependent cellular cytotoxicity (ADCC). NK cells are an ideal effector cell for combined use with tumor-targeting mAbs, as NK cells effect both innate tumoricidal capacity and ADCC. CD38-targeting mAbs, such as daratumumab, are effective in treating multiple myeloma (MM) and achieve their efficacy through multiple mechanisms, including ADCC. However, because activated NK cells express high levels of CD38, daratumumab induces NK cell depletion through fratricide, potentially reducing treatment effectiveness. Adoptive NK cell immunotherapy therefore has the potential to augment daratumumab's ADCC activity if fratricide can be reduced or prevented. FT538 is an off-the-shelf adoptive NK cell immunotherapy product candidate designed for enhanced cellular persistence and ADCC while avoiding anti-CD38 mAb induced fratricide. It is derived from induced pluripotent stem cells (iPSC) engineered to lack CD38 expression, which we have previously shown to eliminate daratumumab-induced fratricide among iPSC-derived NK cells, resulting in enhanced long-term daratumumab-mediated ADCC. FT538 is engineered to express an IL-15 receptor alpha fusion protein (IL-15RF; IL-15 tethered to IL-15 receptor α) to enhance persistence and a high-affinity non-cleavable CD16 (hnCD16, FcRγIII) to increase ADCC. To support the clinical translation of FT538, and to enable the repeatable and scalable cell production to support off-the-shelf availability of a uniform NK cell product, a clinical-grade master pluripotent stem cell line was developed. The FT538 master pluripotent stem cell line was created by reprogramming donor fibroblasts into iPSCs using our non-integrating cellular reprogramming platform, and cells were further genetically edited by targeting IL-15RF and hnCD16 to the CD38 locus. Clonal iPSC lines were generated and screened for precise knock-in and knock-out edits at the CD38 locus and a lack of off-target genome integration (15% total success rate for CD38-/-IL-15RF+CD16+). Selected engineered iPSC clones were confirmed to be free of reprogramming transgenes and to maintain genomic stability. Engineered iPSC clones were additionally tested for their NK cell differentiation potential and function, and a single clone was selected to serve as the renewable starting material for cGMP manufacturing and clinical development. Upon differentiation and expansion FT538 demonstrated a mature NK cell phenotype with expression of NK cell receptors including NKp30, NKp46, NKG2D, KIR, NKG2A, and DNAM-1. The functional impact of CD38 knockout on FT538 NK cells was confirmed in an in vitro fratricide assay, where peripheral blood (PB)-NK cells exhibited fratricide at a frequency of 33% after 3 hr culture with increasing daratumumab concentrations. In contrast, FT538 cells were entirely resistant (<1% specific cytotoxicity) to daratumumab-induced fratricide. In vitro cytotoxic re-stimulation assays showed that repeat exposure of PB-NK cells to daratumumab plus MM target cells resulted in a loss of cytotoxic capacity (from 74% to 58% upon re-stimulation), and a similar effect was seen for non-engineered iPSC-derived NK cells. In contrast, FT538 NK cells maintained robust ADCC in during primary and secondary exposure to MM target cells and daratumumab. FT538 with daratumumab resulted in 86% cytotoxicity against MM target cells upon first exposure and 92% cytotoxicity upon re-stimulation, with a 20-fold increase in viable NK cells at the conclusion of the assay compared to non-engineered iPSC-derived NK cells. Additionally, the combined survival benefit of IL-15RF expression and fratricide resistance mediated by the CD38 knockout as well as the enhanced hnCD16-mediated ADCC allowed for greater cytotoxicity of FT538 against MM tumor spheroids. Together, these preclinical data support the clinical translation of FT538, an off-the-shelf adoptive NK cell immunotherapy product engineered for uniform hnCD16 and IL-15RF expression with CD38 elimination for enhanced ADCC in combination with daratumumab and other anti-CD38 mAbs for the treatment of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Fong:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:CytoSen: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

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Anti-CD123 Targeted Therapy with Talacotuzumab in Advanced MDS and AML after Failing Hypomethylating Agents - Final Results of the Samba Trial

Blood ◽

10.1182/blood-2018-99-113112 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4045-4045 ◽

Cited By ~ 9

Author(s):

Anne Sophie Kubasch ◽

Freya Schulze ◽

Katharina S. Götze ◽

Jan Krönke ◽

Katja Sockel ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Treatment Initiation ◽

Response Rate ◽

Nk Cell ◽

Single Agent ◽

Hypomethylating Agents ◽

Advisory Committees ◽

Disease Stabilization

Abstract Introduction Recently, progress has been made in the treatment of patients with higher risk myelodysplastic syndromes (HR MDS) and acute myeloid leukemia (AML). Nevertheless, patients failing hypomethylating agents (HMA) have a dismal prognosis and very limited treatment options. Targeting CD123 on leukemic stem cells (LSC) is one promising approach in MDS and AML. Talacotuzumab (TAL, JNJ-56022473) is an IgG1 monoclonal antibody targeting CD123 preferentially via antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer cells (NKs). Aim The SAMBA trial, a phase II study of the German and French MDS study groups within the EMSCO network assessed the overall hematological response rate after 3 months of single agent TAL treatment in AML or HR MDS patients failing hypomethylating agents (HMAs). Methods TAL was given IV at a dose of 9 mg/kg once every two weeks for a total of 6 infusions, responders received up to 20 additional infusions. After the first 3 months, overall hematological response rate (either CR, PR, marrow-CR, HI, SD) was evaluated by bone marrow biopsy. The study was accompanied by an immune monitoring via flow cytometric analysis to investigate the distribution of T- and NK cells in peripheral blood (PB) and bone marrow (BM) at the time of screening and during therapy in comparison with healthy, age-matched controls. Results 24 patients (19 AML and 5 HR MDS) with a median age of 77 (range 71-90) years, who either failed to achieve complete- (CR), partial response (PR), hematological improvement (HI) or relapsed after HMA therapy were included in the study. After TAL administration, 14 patients could be assessed for response after 4 infusions and 10 patients after 6 infusions. The overall response rate (ORR) was 20.8% including 1 complete remission (CRi), 1 patient with hematologic improvement (HI-E) and additionally 3 patients with disease stabilization. The median duration of response in these patients was 3 months (range 3-14 months). Two patients are still on treatment, one patient despite losing objective response (14 months) and one patient with disease stabilization (13 months). The median overall survival for the entire cohort of patients was 3.2 months (range 0.4-11.2 months). In 10 patients (41.6%), therapy with TAL resulted in grade 3/4 infusion related side effects (pneumonia, n=1; infusion-related reaction, n=8; septic shock, n=1). Before treatment initiation, patients had lower levels of CD56dim NK-cells in PB (82% vs. 89% of NK-cells; p=0.069) expressing significantly more inhibiting NK-cell receptors like KIR2DL2 (8.8% vs. 3.2% of NK-cells; p<0.001) and less activating NK-cells receptors like NKG2D (95% vs. 99% of NK-cells; p<0.01) compared to healthy controls. Moreover, expression of PD-1 on lymphocytes and monocytes as well as their matching ligands PD-L1 and PD-L2 on blasts and monocytes in PB was significantly higher in patients compared to healthy controls (p<0.01), another evidence for an exhausted T-cell immune status in our patients prior to treatment initiation. We could not detect any difference in NK-cell levels in responding patients compared to non-responders. Interestingly, pre-treatment expression (MFI and percentage) of CD123 on immature myeloid derived suppressor cells (iMDSC) was higher in responders than in non-responders (p<0.01). Anti-CD123 targeted therapy with TAL resulted in a decreased CD123+ MFI (4239 vs. 2910; p<0.01) on iMDSCs as well as lower levels of iMDSCs in PB and BM (p<0.05).Responding patients displayed a 10-fold reduction of CD123 MFI after 3 months of treatment (2565 vs. 236; p=0.06), indicating that the CD123 molecule on immature MDSCs is targeted effectively by TAL. Conclusion Single agent TAL has limited efficacy in patients with advanced myeloid malignancies failing HMA. Expression of CD123 on immature MDSCs might serve as a biomarker of response for future anti-CD123 targeted approaches. Disclosures Götze: Celgene: Honoraria, Research Funding; JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria; Takeda: Honoraria, Other: Travel aid ASH 2017. Krönke:Celgene: Honoraria. Middeke:Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fenaux:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding. Schlenk:Pfizer: Research Funding, Speakers Bureau. Ades:JAZZ: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; silent pharma: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Platzbecker:Celgene: Research Funding.

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