Sequential Immune Cell Modulation in Nordic Follicular Lymphoma Patients in the SAKK 35/10 Randomized Trial with Rituximab and Lenalidomide

Abstract Background Follicular lymphoma (FL) is the second most common lymphoma in adults. Although responsive to therapies it is still considered incurable. The introduction of the CD20 antibody rituximab is well known to have improved outcome. Rituximab acts through complement-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To enhance the efficacy of rituximab, different combination regimens have been used, mostly with chemotherapy but also with cytokines. Lenalidomide, an immunomodulatory agent commonly used in the treatment of multiple myeloma, has been shown to induce durable responses with manageable toxicity in indolent lymphomas and mantle cell lymphoma, especially in combination with rituximab. It acts on both the malignant cells and their microenvironment. The drug modulates signaling pathways, enhances the capacity of T cells and increases ADCC by natural killer (NK) cells, as well as suppresses angiogenesis. When combined with rituximab, the clinical effects seem to be synergistic (Fowler 2014). We aimed to investigate the dynamics of immune cell subsets in peripheral blood in patients given rituximab with or without lenalidomide. Patients and Methods FL patients included in a multicenter randomized phase II trial performed by the Swiss Group for Clinical Cancer Research (SAKK) in collaboration with the Nordic Lymphoma Group (NLG) were randomized 1:1 to treatment either with rituximab alone or rituximab and lenalidomide. Inclusion criteria were histologically confirmed CD20+ FL grade 1, 2 or 3A in disease stage Ann Arbor III-IV (or II not suitable for radiotherapy). Patients had to be in need of systemic therapy because of clinical symptoms, cytopenia, bulky disease or significant disease progression. In both treatment arms rituximab was administered as 4 single infusions of 375 mg/m2 weeks 1, 2, 3 and 4; in patients who showed at least a minor response 4 additional infusions were administered at weeks 12, 13, 14 and 15. In the combination arm, lenalidomide, 15 mg p.o. daily, was started 14 days before the first infusion and given continuously until 14 days after the last. Peripheral blood cells were sequentially sampled: at baseline, after 2 weeks' use of lenalidomide, 24 hours after first rituximab infusion and at follow-up at weeks 10 and 23. Analyses of CD3+, CD4+, CD8+ and CD56+CD3- (NK) cells were performed with flow cytometry. Results Immune cell activity was assessed on blood samples of 28 Norwegian and Swedish patients until July 2015. In all patients, irrespective of treatment arm, NK cell numbers markedly decreased at 24 hours after the first rituximab infusion compared to baseline counts (P=0.046), but returned to baseline levels by week 10 in most. However, patients in the combination arm exhibited a heterogeneous response with a diverse NK cell depletion/proliferation pattern, some showing a transient rise already after 14 days of lenalidomide use (Figure 1). CD3 levels were not affected at 24 hours after rituximab but increased over time in 15 of 18 patients (without differences between treatment arms). The increase at week 23 was statistically significant (P=0.004) with a median of 1.4 x 109 /L CD3+ cells compared to a baseline median of 0.88 x 109/L. In all patients, independent of treatment arm, the CD4/CD8 ratio increased compared to baseline already 24 hours after rituximab (P=0.011) and persisted throughout the study (week 10, P=0,005; week 23, P=0.019). The increased ratio was due to a large rise in CD4 counts (week 10, P=0.014; week 23, P=0.003), and a less pronounced rise in CD8 counts (week 10, P=0.094; week 23, P=0.007; Figure 2). Conclusion We found changes in the composition of immune cell subsets in peripheral blood in rituximab treated FL patients, with a larger interindividual variation when combined with lenalidomide. Ongoing analyses will reveal whether these patterns of immune cell response correlate with clinical outcome and long-term treatment effects. Figure 1. NK cell absolute counts (x 109/L) in (a) patients treated with rituximab and in (b) patients treated with rituximab plus lenalidomide. 1=baseline, 2=after 14 days of lenalidomide (b only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 1. NK cell absolute counts (x 109/L) in (a) patients treated with rituximab and in (b) patients treated with rituximab plus lenalidomide. 1=baseline, 2=after 14 days of lenalidomide (b only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 2. CD4/CD8 ratios in all 28 patients. The y scale is logarithmic. 1=baseline, 2=after 14 days of lenalidomide (14 patients only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 2. CD4/CD8 ratios in all 28 patients. The y scale is logarithmic. 1=baseline, 2=after 14 days of lenalidomide (14 patients only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 3. Figure 3. Disclosures Off Label Use: Lenalidomide was used together with rituximab in a randomized clinical trial.. Kimby:Gilead: Membership on an entity's Board of Directors or advisory committees; Jansen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

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Inhibition of Immune Cell Subsets Is Differentially Affected By Dasatinib Dosage in Patients with Chronic Phase CML

Blood ◽

10.1182/blood-2020-139501 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 51-53

Author(s):

Patrick Harrington ◽

Richard Dillon ◽

Deepti H. Radia ◽

Donal P. McLornan ◽

Philippe Rousselot ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Function ◽

Immune Cell ◽

Healthy Controls ◽

Advisory Committees ◽

Immune Cell Subsets

Dasatinib is a multi-kinase inhibitor with inhibitory activity against Src kinases in addition to the BCR-ABL1 oncoprotein. The Src kinase Lck plays a pivotal role in signalling from the T cell receptor (TCR) and Src kinases also play a central role in signalling from NK cell activating receptors, with key downstream signalling molecules including ZAP70 and LAT. The STAT5 pathway is essential for NK cell function via IL-15, and T cell function through IL-2 signalling. Immune effector cells are thought to play a role in chronic myeloid leukaemia (CML) disease response, with correlation between the frequency of effector cells, including NK cells and CD8+ T cells, and improved outcome (Hughes A., Blood, 2017; Mustjoki, Blood, 2009). Standard licensed dose of dasatinib is 100mg OD but a reduced dose of 50mg OD can also be used (Naqvi, Cancer, 2019). Methods: 18 patients with chronic phase CML on TKI therapy (Dasatinib N=14, Imatinib N=2, Nilotinib N=2) and 7 healthy controls were included. Of the patients on dasatinib, 10 were taking a dose of 100mg OD and 4 were taking 50mg OD. A two-phase ex vivo functional analysis of immune cell subsets, including CD4+ and CD8+ T cells, NK cells and T regulatory cells (Tregs) was performed. We analysed expression of the cytokines, TNFa, IFNg and IL-2, after treatment of cells with OKT3. We also analysed signalling within cells after treatment with the phosphatase inhibitor H2O2. The relative fluorescence intensity (RFI) was calculated as MFI H2O2 sample/MFI unstimulated sample. Results: Patients on dasatinib had lower frequency of total Tregs and effector Tregs than controls and patients with CML taking other TKIs. However, there was no difference in the frequency of total Tregs between patients on 50mg and 100mg doses of dasatinib. Dasatinib significantly inhibited signalling from the TCR and of the STAT5 pathway when compared with patients on other TKIs and healthy controls, in CD4+ and CD8+ T cells, Tregs and NK cells. Patients on 50mg dasatinib had significantly higher RFI than patients on 100mg in CD4+ cells for pZAP70, and in CD4+ and CD8+ cells for pLAT. Similarly, patients on 50mg dasatinib had a higher RFI for pSTAT5 than those on 100mg in CD4+ and CD8+ T cells and also NK cells. Of note, the difference in the RFI of pSTAT5 between Tregs, and that of both CD8+ T cells and NK cells, was significantly higher in patients on 50mg dasatinib compared with 100mg, suggesting a relative sparing of effector immune cell inhibition at the lower dose. Expression of TNFa, IFNg and IL-2 were significantly reduced in patients taking dasatinib compared with healthy controls and patients on other TKIs. Patients on a 50mg dose of dasatinib had significantly higher proportional increase in IL-2 expression after OKT3 activation in CD4+ and CD8+ cells compared with patients on 100mg. Five patients on dasatinib 100mg OD with increased CD8+ T cells were confirmed to have clonal CD8+ lymphocytosis by performing TCR gene rearrangement analysis. These patients had a lower proportion of Tregs, compared to patients on dasatinib without CD8+ lymphocytosis. Importantly, a significantly lower RFI for pZAP70 and pLAT, within isolated Tregs, was also seen in this group, when compared with patients on dasatinib without CD8+ lymphocytosis. Discussion: Dasatinib inhibits key signalling pathways in T cells and NK cells and suppresses pro-inflammatory cytokine expression in T cells, compared to healthy controls and patients with CML taking other TKIs. However, patients taking a 50mg dose appear to have significantly less inhibition of effector cell function. In contrast, dasatinib may enhance immune surveillance mechanisms due to inhibition of Treg suppressive function, by reducing T effector IL-2 production, as well as STAT5 signalling within Tregs, required for FOXP3 transcription. Patients on dasatinib with clonal CD8+ lymphocytosis, as well as a lower frequency of Tregs, have an additional functional deficit within Tregs, with reduced signalling from the TCR. The presence of clonal CD8+ lymphocytosis is linked to improved outcomes, and typically occurs at the approved 100mg dosage. The dose of dasatinib strongly affects the activity of different immune cell subsets with the lower dosage allowing improved effector cell function. However greater inhibition of Tregs is seen in patients with clonal lymphocytosis, typically at the higher dosage, demonstrating the complexity of the immunomodulatory effect of dasatinib. Disclosures Harrington: Bristol Myers Squibb: Research Funding; Incyte: Honoraria, Speakers Bureau. Dillon:Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria, Research Funding; Astellas: Honoraria; Pfizer: Honoraria, Research Funding; Menarini: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Research Funding. Radia:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Education events; Blueprint Medicines Corporation: Membership on an entity's Board of Directors or advisory committees. McLornan:CELGENE: Honoraria, Speakers Bureau; NOVARTIS: Honoraria, Speakers Bureau; JAZZ PHARMA: Honoraria, Speakers Bureau. Rousselot:Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Rezvani:GemoAb: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing agreement; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Formula Pharma: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: Educational grant; Affimed: Other: Educational grant; Virogen: Membership on an entity's Board of Directors or advisory committees. Kordasti:Novartis: Research Funding; Celgene: Research Funding; Alexion: Honoraria. Harrison:Novartis: Honoraria, Research Funding, Speakers Bureau; Incyte Corporation: Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Promedior: Honoraria; Roche: Honoraria; Celgene: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Sierra Oncology: Honoraria; Janssen: Speakers Bureau. de Lavallade:Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.

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PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

Blood ◽

10.1182/blood-2019-127261 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 621-621

Author(s):

Zachary Davis ◽

Martin Felices ◽

Todd R Lenvik ◽

Sujan Badal ◽

Peter Hinderlie ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Fold Increase ◽

Advisory Committees ◽

Fund Research

Checkpoint blockade has become a promising immunotherapy for the treatment of a variety of malignancies. In particular, the receptor programmed death-1 (PD-1) has become a focus of intense study due to its expression on and negative regulation of T-cell function. The ligand for PD-1, PD-L1, is upregulated on many tumors and, as a result, can suppress antigen-specific T-cells thereby limiting their anti-tumor response. Pharmacological PD-1/PD-L1 axis disruption can occur with either Pembrolizumab and Nivolumab (PD-1 antagonists) and Avelumab and Atezolizumab (PD-L1 antagonists). These antibodies (mAbs) are being used to treat melanoma, non-small cell lung cancer, kidney, bladder and head and neck cancer with varying degrees of success. Like T-cells, natural killer cells (NK) also have potent antitumor cytolytic properties. The expression and functional effects of PD-1 on NK cells remain unclear due to difficulties in receptor detection and efficacy of receptor blockade by available commercial reagents. While some studies have been unable to detect PD-1 on resting NK cells, others have identified PD-1 expression only on specific NK populations under certain conditions (e.g. Cytokine stimulation or virus infection). Here, we identify PD-1 expression on peripheral blood NK cells. Using commercial reagents (Figure 1A) and a FITC-labeled clinical mAb (Pembrolizumab, Pembro), we detect low yet consistent PD-1 expression on all circulating, resting NK cells. Since FITC-Pembro mean fluorescent intensity was low and a high proportion of FITC labeled NK cells overlapped with the isotype control (Figure 1B), we designed a short-chain variable fragment (scFv) of the mAb to determine whether the smaller scFv molecule has better binding and functional activity than the intact mAb. The Pembro scFv bound to resting NK cells with a distinct fluorescent peak compared to the native Prembro from which the scFv was derived (Figure 1B). Compared to intact Prembro, use of the Pembro scFv as a PD-1 antagonist resulted in a 2-fold increase of NK cell cytolytic activity and a 3-4 fold increase in cytokine production against the PD-L1 expressing CML target, K562 (Figure 1C-D) and the AML target, THP-1 (Figure 1E-F). While PD-1 blockade enhanced NK cell degranulation and target cell killing, a greater functional enhancement was seen for interferon-γ production. PD-1 signaling inhibits PI3K induced pAkt and NK function. PD-1/PD-1 ligand blockade by the Pembro scFv resulted in increased NK cell pAKT in the presence of PD-L1 and NK activating NKG2D-ligand-expressing THP-1 cells. In addition to natural cytotoxicity, NK-mediated ADCC was also enhanced with PD-1 blockade. CD33 mAb immunoconjugates have been used to treat AML. Combined anti-CD33 mAb and PD-1 blockade against THP-1 cells resulted in a small but significant increase in NK cell degranulation and a 4-fold increase in cytokine production compared to anti-CD33 mAb without PD-1 blockade (Figure 1G-H). Since stimulation with IL-15, a cytokine that effectively lowers the NK activation threshold, abrogated the benefits of Pembro scFv in diminishing PD-1 inhibitory effects on NK cells, PD-1 control of NK function appears limited to be mostly relevant to resting NK cells. To understand the physiologic expression of PD-1 in vivo, we studied samples taken from AML patients receiving matched sibling donor transplantation at the University of Minnesota. Increased PD-1 on reconstituting NK cells in BMT recipients up to day 100 post-transplant was shown by both flow-cytometric (Figure 2A) and mass-cytometric (CyTOF) analyses (Figure 2B). Blockade of PD-1 on these cells significantly enhanced both NK degranulation (Figure 2C) and cytokine production (Figure 2D) against K562 targets. A similar increase in NK function was observed with PD-1 blockade in AML patients receiving umbilical cord transplants (not shown). These data indicate that PD-1 is present on human NK cells and PD-1 ligation negatively regulates NK function against PD-L1 expressing tumor targets. The observation that functional PD-1 is expressed on NK cells under resting conditions strongly suggests that the use of a PD-1 antagonist, in combination with NK cell therapy, should be clinically effective for treatment of cancer. Disclosures Felices: GT Biopharma.: Other: consulting funds, Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Vallera:GT Biopharma, Inc.: Consultancy, Research Funding. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda. PD-1 blockade on NK cells for tumor immunotherapy

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Efficacy Outcomes of Treatments for Double Relapsed/Refractory Follicular Lymphoma (R/R FL): A Systematic Literature Review

Blood ◽

10.1182/blood-2020-136228 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Brad S. Kahl ◽

Anik R. Patel ◽

Omer Zaidi ◽

Sonya J. Snedecor ◽

Anna G. Purdum

Keyword(s):

Clinical Trials ◽

Follicular Lymphoma ◽

Board Of Directors ◽

Clinical Data ◽

Research Funding ◽

Treatment Options ◽

Unmet Need ◽

Progression Free Survival ◽

Disease Stage ◽

Advisory Committees

ABSTRACT Introduction: Patients with indolent non-Hodgkin lymphomas (iNHL), including follicular lymphoma (FL), have high response to first-line treatment. However, retreatment is often required when relapses occur, and those with multiple relapses represent a patient population with an unmet need for effective treatment. Clinical data for several treatment options exist for the general relapsed and refractory (R/R) population; however, there are relatively fewer data specific to FL patients with ≥2 lines of prior treatment. This work systematically identified the available efficacy data in the double R/R FL population. Methods: The MEDLINE and EMBASE databases were searched through February 10, 2020. Studies were limited to interventional clinical trials of R/R FL patients (or mixed histologies with a predominance of FL) and articles published in English. Studies also must have reported one or more efficacy measures, such as overall response rate (ORR), complete response (CR), duration of response (DoR), time to next treatment (TTNT), progression-free survival (PFS), and overall survival (OS). Potential interventions of interest were lenalidomide ± rituximab (R), duvelisib, ibrutinib, venetoclax, polatuzumab vedotin + R, obinutuzumab, copanlisib, umbralisib, idelalisib, and tazemetostat. Results: Of 35 publications examining treatment outcomes in R/R FL patients, only 14 (representing 5 unique clinical trials) were specific to the ≥ 2-line population. These trials were: CHRONOS Part B (copanlisib), DAWN (ibrutinib), DELTA (idelalisib), DYNAMO (duvelisib), and Morschhauser et al. 2019 (tazemetostat) and included a total of 605 participants. All studies used similar inclusion criteria, and patients included were similar in age (median 62-65), disease stage (III/IV), and ECOG score (0-2). Patients in the CHRONOS study had a median number of prior treatments of 2, whereas those in the DELTA study had 5. ORR ranged from 21% (ibrutinib) to 59% (copanlisib) (Table). The DoR ranged from 8.3 months in tazemetostat patients with EZH2 gene mutation to 19.4 months for ibrutinib. PFS ranged from 5.7 months in tazemetostat patients with wild-type EZH2 to 11.2 months for copanlisib. Median TTNT was only reported in the DAWN study (16 months). Conclusions: Very few clinical data exist reporting efficacy outcomes specific to the double R/R FL population. The limited data indicate that current treatments do not produce durable responses for most double R/R FL patients, demonstrating an unmet need. Further research is needed to fully understand the efficacy and safety of other potential interventions for this population. Disclosures Kahl: Genentech:Consultancy;Pharmacyclics LLC:Consultancy;AstraZeneca Pharmaceuticals LP:Consultancy, Membership on an entity's Board of Directors or advisory committees;ADC Therapeutics:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Celgene Corporation:Consultancy;AbbVie:Consultancy;Roche Laboratories Inc:Consultancy;BeiGene:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Janssen:Consultancy, Membership on an entity's Board of Directors or advisory committees;Acerta:Consultancy, Research Funding.Patel:Kite, a Gilead Company:Current Employment.Zaidi:BMS:Consultancy.Snedecor:Pharmerit - an OPEN Health Company:Other: Employment at consultancy paid by Kite Pharma to conduct this work.Purdum:Kite, a Gilead Company:Current Employment.

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The Influence Of KIR Haplotype In ITP Incidence, Treatment Response and Bleeding Symptoms

Blood ◽

10.1182/blood.v122.21.2316.2316 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2316-2316

Author(s):

Bethan Psaila ◽

Nayla Boulad ◽

Emily Leven ◽

Naznin Haq ◽

Christina Soo Lee ◽

...

Keyword(s):

Platelet Count ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Kir Genes ◽

Number Of Patients

Abstract The pathogenesis of immune thrombocytopenia (ITP) is multifactorial, with both cellular and humoural immune dysfunction. The role of NK cells has not been well defined in ITP but in other diseases NK cells have a role in rejecting “foreign” eg transplanted organ or tumor, and also acting against self as occurs in autoimmunity. NK cell activity is orchestrated by the balance of activating vs. inhibitory signalling, in particular via the killer cell immunoglobulin-like receptor (KIR) family of receptors. Significant variation exists in KIR allelic subtype and copy number for the KIR between individuals, and associations have been made with certain haplotypes and a number of autoimmune disorders including rheumatoid arthritis, scleroderma and diabetes. Previous reports have demonstrated a reduction in natural killer (NK) cell number and function in ITP and expression of inhibitory KIR genes is increased in patients in remission vs. active ITP. Methods To explore whether a particular KIR haplotype might predispose to ITP, and also affect response to ITP treatment, we performed KIR genotyping using the Invitrogen SSP kit on 92 patients attending a haematology centre in New York and compared the results to data from 213 controls taken from the USA Eastern Database. Genomic DNA was typed for the inhibitory KIR genes KIR2DL1, KIR2DL2, KIR2DL5A (alleles 001 and 002), KIR2DL5B (alleles 002-004, 06, and 007), KIR3DL1, KIR3DL3; the activating KIR genes KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1; the framework genes KIR2DL3, KIR2DL4, KIR3DL2, KIR3DP1; and the pseudogene KIR2DP1. The patients with ITP had been or were receiving treatment with IVIG (n=64), corticosteroids (72) and rituximab (37). Bleeding symptoms were recorded. Response to treatment was defined as complete - platelet count increase to > 100 x 109/mL; partial - platelet count increase to > 50 x 109/mL; or no response. For the purpose of analysis, PRs and CRs were combined. A comprehensive database allowed a logistic regression, assessing both responses to treatments, platelet counts, neutrophil counts, CRP, lymphocyte subsets and bleeding symptoms. Results The expression of two inhibitory KIR genes, 2DL1 and 3DL1, was significantly lower in the patients with ITP as compared to controls (87% 2DL1 and 87% 3DL1 compared to 99% in controls - P < 0.02). Response to rituximab was strongly related to KIR haplotype expression. 2DL1 expression was higher among nonresponders to Rituximab (100% of non responders compared to 82% of responders), whereas 2DL3 expression was significantly lower (79% compared to 90%) (P < 0.05, Figure 1B). Separately, patients with the 2DS3 allele, an activatory KIR, were 5.5 times more likely to have experienced significant bleeding. Conclusions Although these findings are preliminary and require further investigation, these data suggest that increased cytotoxic autoimmunity due to reduced KIR inhibition may be associated with the development of ITP and possibly contribute importantly to the pathogenesis. Anti-CD20 targeting therapy directed at B cells was strongly influenced by 2 different KIRs (1 upregulated and one down-regulated) emphasizing the potential role of NK cells in elimination of tissue-based (nodal) B cells. Finally a more pronounced clinical phenotype with a markedly higher incidence of severe bleeding associated with an increased activatory KIR expression demonstrates the role of NK cells in bleeding presumably via their effects on either endothelial cells or platelet function. These exciting findings will be pursued for confirmation in a larger number of patients. Disclosures: Bussel: Amgen: Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; GlaxoSmithKline: Family owns stock, Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

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Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Blood ◽

10.1182/blood-2021-148654 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 407-407

Author(s):

Frank Cichocki ◽

Jode P Goodridge ◽

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Sajid Mahmood ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Abstract Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

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Gene Set Enrichment Analysis Suggests That Increased Rituximab-Mediated NK Cell Cytotoxicity after Vitamin D Substitution Is Driven By Upregulation of Interferon Alpha (IFN-a) Isoforms

Blood ◽

10.1182/blood-2018-99-111817 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3696-3696

Author(s):

Konstantinos Christofyllakis ◽

Frank Neumann ◽

Stephan Stilgenbauer ◽

Dominic Kaddu-Mulindwa ◽

Evi Regitz ◽

...

Keyword(s):

Vitamin D ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Advisory Committees ◽

Vitamin D Levels ◽

Nk Cell Cytotoxicity

Abstract Introduction: We recently showed that vitamin D deficiency leads to decreased overall survival of DLBCL-patients treated with rituximab-chemotherapy (Bittenbring et al, JCO, 2014). We hypothesized that rituximab-mediated NK cell-cytotoxicity is more effective at higher vitamin D levels. This was confirmed by vitamin D substitution of healthy volunteers, which increased their rituximab-mediated cytotoxicity in vitro against the Daudi lymphoma cell line. To unveil the molecular mechanisms behind this finding, resting NK cells before and after vitamin D supplementation were isolated from those volunteers and a whole transcriptome analysis was performed. Methods: We collected PBMCs from eight healthy volunteers with vitamin D deficiency before and after vitamin D substitution to > 30 ng/ml 25-OH vitamin D3. NK cells were isolated from PBMCs by magnetic depletion of all non-NK cells. Purity of the CD16+ cells was confirmed by flow cytometry. After isolating total RNA, we performed a microarray analysis using an Affymetrix Gene-Chip 2.0 ™. The signals were normalized using the LMA algorithm. For pathway analysis, gene set enrichment analysis (GSEA) was used. A two-step approach was chosen. Firstly, we separated 7.705 genes due to their involvement in the NK cell-mediated immune response according to the Gene Ontology database, irrespective of their differential expression. This dataset was used separately for specific analysis of the NK cell-cytotoxicity pathway to increase sensitivity. Secondly, the complete data set of 48.145 genes was used in an exploratory analysis in an attempt to screen for other dysregulated pathways involved in the immune response and vitamin D homeostasis. We used gene sets provided from the Molecular Signature Database. A significance level of < 0.05 for p and False Discovery Rate (FDR) was chosen. Real-time quantitative PCR was performed to confirm the results. Results: The NK cell-associated cytotoxicity pathway was found to be significantly upregulated after restoration of normal vitamin D levels in the specific analysis. The most significantly overexpressed genes in the gene set were five IFN-α subtypes (IFN-α2, IFN-α4, IFN-α6, IFN-α7, and IFN-α10) as well as IFN-κ. The exploratory analysis showed an upregulation of the response to type I interferon pathway and regulation of type I interferon mediated signaling pathway. The most upregulated genes in those pathways were again the IFN-α subtypes mentioned above. Other pathways involved in the immune response were found to be downregulated after vitamin D substitution, like interferon gamma response; cytokine production and chemotaxis. The common denominator of these pathways was the downregulation of three toll-like receptor genes (TLR-8, TLR-7, TLR-2). Conclusion: The increased expression of specific IFN-α subtypes could explain the increased rituximab-mediated NK cell-cytotoxicity after vitamin D substitution in deficient individuals. To the best of our knowledge, this is the first study to suggest a role for vitamin D in IFN-α regulation. TLRs are known to stimulate cytokine production in NK cells including IFN-α. It can be assumed, that the observed upregulation of IFN-α genes after vitamin D substitution leads to a negative feedback on positive regulators of cytokine production like TLR, causing their downregulation once vitamin D levels are restored. This implies a comprehensive role of vitamin D in IFN-α biosynthesis in human NK cells. Disclosures Stilgenbauer: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Results of a Phase 1 Trial of Gda-201, Nicotinamide-Expanded Allogeneic Natural Killer (NK) Cells in Patients with Refractory Non-Hodgkin Lymphoma (NHL) and Multiple Myeloma

Blood ◽

10.1182/blood-2020-142419 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-6 ◽

Cited By ~ 1

Author(s):

Veronika Bachanova ◽

Joseph Maakaron ◽

David H. McKenna ◽

Qing Cao ◽

Todd E. DeFor ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Phase 1 ◽

Advisory Committees ◽

Non Hodgkin Lymphoma ◽

Cell Manufacturing ◽

Cell Current

Background: The innate capacity of natural killer (NK) cells to kill tumor targets has been translated into cancer immunotherapy. GDA-201 is a novel allogeneic NK cell product derived from NK cells from healthy donors, expanded ex-vivo with nicotinamide (NAM) and IL-15. We previously reported improved killing function, in vivo proliferation, organ trafficking, and augmented resistance against exhaustion in pre-clinical models. We conducted a phase 1 study of GDA-201 in combination with monoclonal antibodies to enhance NK cell targeting through antibody-dependent cellular cytotoxicity (ADCC). We now report safety data in patients (pts) with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), and report efficacy outcomes in pts with NHL. Methods: Following donor apheresis, CD3-depleted mononuclear cells were cultured for 14-16 days with NAM (5mM) and IL-15 (20ng/ml), resulting in a 40-fold increase in NK cells and increased expression of CD62L from 2.9% to 21%. GDA-201 contained ~98% NK cells, and CD3 content was maintained at <0.5% (<5x105/kg/dose). Pts with R/R B-cell NHL or MM received lymphodepleting (LD) therapy with cyclophosphamide (400mg/m2 IV x 3d) and fludarabine (30 mg/m2 /d IV x 3d), followed by GDA-201 (days 0 and 2) and low-dose IL-2 (6 million units sc x 3 doses). Pts with NHL or MM received rituximab (375 mg/m2) or elotuzumab (10 mg/kg), respectively, x 3 weekly infusions. Results: 30 pts were enrolled:15 with NHL and 15 with MM, in 3 cohorts of escalating GDA-201 dose; 15 pts received the maximum target dose (median dose 12.4 [range 2.0-26.0] x 107 cells/kg). There were no dose limiting toxicities. The most common grade 3/4 adverse events were thrombocytopenia (n=9), hypertension (n=5), neutropenia (n=4), febrile neutropenia (n=4), and anemia (n=3). There were no neurotoxic events, confirmed cytokine release syndrome, graft versus host disease, or marrow aplasia. One patient died of E-coli sepsis. In pts with NHL, histologies included diffuse large B cell lymphoma (DLBCL) (de novo n=5, transformed n=3), follicular lymphoma (FL) (n=6), and mantle cell lymphoma (n=1). Median age was 64 (range 48-83 years). Pts had a median of 3 lines of prior therapy (range 1-8); most were multiply relapsed or refractory (n=2), and 87% had advanced stage. Median follow-up was 10.8 months (range 4.3-27.5 months). Ten pts had complete response (CR): 6/6 pts with FL and 4/8 with DLBCL; 1 pt had partial response (PR), and overall response rate in pts with NHL was 73.3%. Median duration of response was 8.7 months (range 4.3-25 months). Flow cytometry confirmed the persistence of GDA-201 in peripheral blood for 7-10 days (range 2-92% donor NK cells on day 7), as well as enhanced in vivo proliferation (median Ki 67 99%). Flow cytometry of biopsied tissues at day 4 demonstrated trafficking to bone marrow and lymph nodes. Four pts underwent re-treatment with GDA-201 without LD chemotherapy; GDA-201 cells were detectable in blood after the re-treatment and likely contributed to deepening of response in 2 patients. Post-GDA-201 therapy included allogeneic (n=2) and autologous (n=1) hematopoietic stem cell transplantation. One-year estimates of progression-free survival and overall survival were 66% (95% CI 36-84%) and 82% (95% CI 42-95%), respectively. Conclusions: Cellular therapy using GDA-201 with monoclonal antibodies to enhance ADCC was well-tolerated, and demonstrated significant clinical activity in heavily pretreated pts with advanced NHL. Data support the future testing of multiple infusions to potentially enhance anti-tumor effect. The omission of lymphodepleting chemotherapy is feasible and contributes to safety of this approach. Phase II studies in aggressive and indolent NHL cohorts are planned. Disclosures Bachanova: Incyte: Research Funding; FATE: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Karyopharma: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. McKenna:Gamida: Other: Cell Manufacturing; Fate Therapeutics: Other: Cell Manufacturing; Intima: Other: Cell Manufacturing; Magenta: Other: Cell Manufacturing. Janakiram:Takeda, Fate, Nektar: Research Funding. Simantov:Gamida Cell: Current Employment. Lodie:Gamida Cell: Current Employment. Miller:Vycellix: Consultancy; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, 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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Results of a Phase II Study of Obinutuzumab in Combination with Lenalidomide in Previously Untreated, High Tumor Burden Follicular Lymphoma (FL)

Blood ◽

10.1182/blood-2019-129422 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 125-125 ◽

Cited By ~ 5

Author(s):

Loretta J. Nastoupil ◽

Jason R. Westin ◽

Fredrick B. Hagemeister ◽

Hun Ju Lee ◽

Luis Fayad ◽

...

Keyword(s):

Follicular Lymphoma ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Tumor Burden ◽

Advisory Board ◽

Advisory Committees ◽

High Tumor Burden ◽

Equity Ownership ◽

Grade 3

Introduction: FL, the most common indolent non-Hodgkin lymphoma, is characterized by a defective immune microenvironment that suppresses normal T-cell and natural-killer (NK)-cell activity. The clinical course is often depicted by high initial response rates coupled with a prolonged natural history and repeated relapses with most patients (pts) succumbing to their disease. Effective, well tolerated therapies are desirable. Obinutuzumab (O) is a humanized, type II anti-CD20 monoclonal antibody glycoengineered for enhanced antibody-dependent cellular cytotoxicity (ADCC). Lenalidomide (len) is an immunomodulatory agent that binds the cereblon E3 ubiquitin ligase complex resulting in recruitment, ubiquitination, and degradation of transcription factors Aiolos and Ikaros resulting in T-cell and NK-cell activation. Therefore, combining O with len is anticipated to be synergistic in augmenting the innate and adaptive immune response in FL. The combination has been shown to be well tolerated and effective in relapsed FL (Fowler ICML 2017). Therefore, we sought to explore the efficacy and safety of O-len in previously untreated, high tumor burden FL. Methods: We conducted as single-center, phase 2 study in previously untreated, stage II, III, or IV, high tumor burden (defined by GELF) FL (grade 1, 2 or 3A). Pts received 1000mg of O on days 1, 8, and 15 of cycle 1, day 1 of cycles 2-6, and day 1 of even numbered cycles, cycle 8-30. Cycle length was 28 days. Len was administered as 20mg on days 1-21 of cycles 1-6. Pts in a complete response (CR) after 6 cycles received reduced dose len (10mg on days 1-21) for cycles 7-18. Among pts in a partial response (PR) after 6 cycles, len was continued at 20mg for the next 3-6 cycles or until CR, whichever occurred first, len was then dose reduced to 10mg on days 1-21 for the remainder of 18 cycles. The primary endpoint was progression-free survival (PFS) at 2 years (according to Lugano 2014 criteria). Secondary endpoints included: safety, CR, PR, overall response (ORR), and overall survival (OS). Results: 90 pts with high tumor burden FL were enrolled. Median age was 58 years (range 33-84), 52% (N=47) were male, 67 (74%) had an ECOG performance status of 0, 9 (10%) had stage II, 23 (26%) stage III, and 58 (64%) had stage IV disease. The majority had grade 1/2 FL (80%). Twenty-one percent had low risk FLIPI scores, 37% intermediate risk, and 42% were high risk. With a median follow-up of 22 months (range 1-30 months), the 2-year PFS estimate is 96% (95% CI 92-100%) with only 2 pts experiencing progression to date. The ORR is 98% (85 CR, 1 PR), 92% achieved a CR at the first response assessment (cycle 4, day 1). Correlative studies are underway including serial circulating tumor DNA measurements. No deaths have been observed to date. Eleven pts (12%) discontinued therapy as a result of an adverse event (AE), upper respiratory infection was the most common reason (N=5). Other reasons included bradycardia with sick sinus syndrome, urinary tract infection, constipation, abdominal pain, fatigue, foot neuroma (N=1 for each instance). The most common grade 3 or higher AEs include neutropenia (16%, grade 3 N=5, grade 4 N= 9), rash (10%), lung infection (4%), neutropenic fever (1%). Conclusions: O-Len was associated with very high CR rates and 2-year PFS estimates in untreated, high tumor burden FL. The toxicity profile was manageable. Further study of this effective, immune therapy approach in untreated FL is warranted. Figure Disclosures Nastoupil: Bayer: Honoraria; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Honoraria; TG Therapeutics: Honoraria, Research Funding; Spectrum: Honoraria. Westin:Genentech: Other: Advisory Board, Research Funding; Unum: Research Funding; Novartis: Other: Advisory Board, Research Funding; Janssen: Other: Advisory Board, Research Funding; Juno: Other: Advisory Board; 47 Inc: Research Funding; MorphoSys: Other: Advisory Board; Kite: Other: Advisory Board, Research Funding; Curis: Other: Advisory Board, Research Funding; Celgene: Other: Advisory Board, Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wang:Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta Pharma: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; MoreHealth: Consultancy, Equity Ownership; Kite Pharma: Consultancy, Research Funding; Guidepoint Global: Consultancy; BioInvent: Consultancy, Research Funding; VelosBio: Research Funding; Loxo Oncology: Research Funding; Celgene: Honoraria, Research Funding; Juno Therapeutics: Research Funding; Aviara: Research Funding; Dava Oncology: Honoraria. Neelapu:Acerta: Research Funding; Celgene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Allogene: Consultancy; Cell Medica: Consultancy; Unum Therapeutics: Consultancy, Research Funding; Pfizer: Consultancy; Poseida: Research Funding; Karus: Research Funding; Novartis: Consultancy; Incyte: Consultancy; BMS: Research Funding; Cellectis: Research Funding; Precision Biosciences: Consultancy; Merck: Consultancy, Research Funding. Fowler:ABBVIE: 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, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: 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; Novartis Pharmaceuticals Corporation: Consultancy. OffLabel Disclosure: Lenalidomide in untreated follicular lymphoma

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