A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

Abstract Background: IL-21 expanded NK cells have high expression of CD16 and have demonstrated antibody-dependent cell-mediated cytotoxicity (ADCC) activity in combination with monoclonal antitumor antibodies (mAb). Mogamulizumab (moga) is a mAb targeting CCR4 that is defucosylated to enhance its binding to CD16, thereby enhancing ADCC of NK cells against targets expressing CCR4. We designed a pilot phase I clinical trial studying this combination in patients with relapsed/refractory (r/r) CTCL and ATLL. The study is soon opening to accrual at the OSU James Cancer Center (NCT04848064). Study is conduced under IND 26888. Preclinical data: Allogeneic NK cells obtained from buffy coat (Red Cross Blood Bank), were expanded for 14 days on CSTX002 feeder cells, cryopreserved, and then thawed and recovered for 48 hours prior to testing. Malignant T-cells were incubated with moga (at 10ng/µl) for 30 minutes prior to co-culture with NK cells and cytotoxicity was determined by the calcein release assay (Somanchi et al, J Vis Exp 2011). Malignant T-cells were obtained from peripheral blood from 3 patients with multiply relapsed CTCL and all have circulating Sezary cells and from CCRF-CEM cell line (T-ALL cell line that expresses CCR4). No significant cytotoxicity was observed with moga alone and significant synergy in cytotoxicity was observed between and moga and NK cells in all 3 patient samples and also CCRF-CEM cell line (figure 1: A and B). Two-fold increase in ADCC was observed with addition of moga to NK cells (p=0.0272; figure 1C) Design: Patients will receive lymphodepleting chemotherapy (Fludarabine/Cyclophosphamide) on days -5 to -3 prior to cell infusion, moga weekly for 4 doses starting on day -7 (prior to the first NK cell infusion) and then every 2 weeks until toxicity or progression. Patients will receive third-party ideal-donor mbIL-21 expanded NK cells once every 2 weeks for 6 total doses (Figure 1D). Donors meeting ideal-donor characteristics from National Marrow Donor Program were identified in collaboration with Be The Match Biotherapies. PBMNC were collected by apheresis, CD3-depleted, expanded for 14 days as previously described, and cryopreserved in ready-to-infuse aliquots. NK cells will be thawed and infused in 2 dosing cohorts; 3x10 7 and 1x10 8 cells/kg in a standard dose-escalation design. Primary endpoint is the maximum tolerated dose of NK cells given in combination with standard-dose moga. Dose-limiting toxicity (DLT) is defined as any steroid refractory graft vs host disease (GVHD), severe NK cell-related toxicities, or other unusual events occurring from D-7 until D+84 post last dose of NK cells. Dose-escalation will proceed in the standard 3+3 fashion. Secondary endpoints include overall response rate (ORR) per ISCL/USCLC/EORTC consensus panel, for CTCL, and international consensus panel, for ATLL and progression-free and overall survival. Correlative endpoints include quality of life impact as captured by Skindex-16 score, serum cytokine levels in blood, persistence of NK cells by chimerism studies, correlation between CCR4 staining of tumor cells in skin and trafficking of NK cells to skin by immunohistochemistry or immunofluorescence in serial skin biopsies and ORR (Figure 1D). Abbreviated eligibility: Eligible patients will be 18 years, or older, with biopsy-proven, measurable, stage IB-IVB relapsed or refractory CTCL or ATLL, progressing on at least one standard chemotherapy. Other eligibility criteria include: ECOG performance status of ≤ 1, no systemic anti-neoplastic therapy within a week or 3 half-lives, adequate laboratory parameters including: absolute neutrophil count ≥1000/mm³, platelet count ≥50,000/mm³, total bilirubin ≤ 2 x upper limit of normal (ULN), AST/ALT ≤ 3 x ULN or ≤ 5 x ULN in patients with documented hepatic involvement by lymphoma, and calculated creatinine clearance ≥ 50 ml/min., disease free of prior malignancies for ≥ 2 years with exception of treated basal cell, squamous cell carcinoma of the skin, or carcinoma in situ of the cervix or breast and life expectancy ≥ 90 days. Patients who were pre-treated with moga, pregnant, HIV positive, with active hepatitis B and C, active CNS involvement, active grade II-IV acute or extensive chronic GVHD or other serious medical comorbid conditions are excluded. Figure 1 Figure 1. Disclosures William: Kyowa Kirin: Consultancy; Incyte: Research Funding; Merck: Research Funding; Dova Pharmaceuticals: Research Funding; Guidepoint Global: Consultancy. de Lima: Miltenyi Biotec: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Devine: Be the Match: Current Employment; Johnsonand Johnson: Consultancy, Research Funding; Orca Bio: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Magenta Therapeutics: Current Employment, Research Funding; Tmunity: Current Employment, Research Funding; Vor Bio: Research Funding; Kiadis: Consultancy, Research Funding. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Lee: Courier Therapeutics: Current holder of individual stocks in a privately-held company; Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. OffLabel Disclosure: Will discuss combination of mogamulizumab and NK cells in a context of a clinical trial

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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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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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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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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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Identification of Novel Immune Cell Populations in Lenalidomide Refractory Relapsed Multiple Myeloma Patients Treated with Pomalidomide and Low Dose Dexamethasone

Blood ◽

10.1182/blood-2019-130154 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3186-3186

Author(s):

Anna Kalff ◽

Tiffany Khong ◽

Malarmathy Ramachandran ◽

Sam Norton ◽

Andrew Mitchell ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Statistical Significance ◽

Advisory Committees ◽

Travel Costs ◽

Trial Investigator ◽

Hla Dr ◽

Advisory Role

The ALLG MM14 trial evaluated the impact of low dose dexamethasone (LoDEX) withdrawal in lenalidomide (LEN) refractory and relapsed (RR) multiple myeloma (MM) patients achieving initial disease control with pomalidomide (POM) and LoDEX re-induction. As previously reported, patients continuing with POM LoDEX had superior progression free survival (PFS) compared to maintenance with POM alone, however, this early PFS benefit was lost and by 18m was reversed to favour POM only. In patients who received post-progression therapy, more durable responses (second PFS: 12.7m vs 4.6m, p=0.034) and superior survival (OS: 19.4m vs 12.5m, p=0.092) were seen in those previously treated with POM alone. Here we present findings from the preliminary correlative immune studies of this trial. Aims To undertake mass cytometry (CyTOF) based immune profiling in patients with advanced MM receiving treatment with POM LoDEX. Methods MM14 was a multicentre, open-label, randomised phase 2 study of LEN refractory RRMM patients who had received ≥ 2 prior lines of therapy. Patients were treated with POM 4mg d1-21 (28d cycle) and LoDEX (40mg weekly). After 4 cycles (induction), patients with stable disease or better (≥SD) were randomised to receive maintenance with ongoing POM-LoDEX or POM alone. Therapy continued until toxicity/progression. PBMCs were collected at baseline and sequentially while on treatment. Cells were barcoded using the Cell-ID 20-Plex Pd barcoding kit (Fluidigm) followed by staining with sub-set/function defining antibodies (targeting myeloid, B, T and NK cells: CD16, CD24, CD11c, CD45RO, CD314, CD38, CD336, HLA-DR, CD14, CD56, CD158a, CD27, CD28, CD159a, CD8, CD19, CD45RA, CD11b, CD4, IgD, CD335, FOXP3, CD25, CD66b, CD3, CD337, CD20, CD158b, CD127 CD57, CD197, CD194, CD304 and CD279). Samples were acquired on the Helios instrument. Data were clustered in the VORTEX package. Significant differences in cluster frequency were assessed by Mann-Whitney test for statistical significance. Cluster phenotypes were determined and validated via multiple visualisation approaches. CD3-CD19-CD56+ NK cells were pre-gated from patient datasets. We then performed Boolean gating using seven NK cell activation/inhibitory markers - CD158a, CD158b, CD159a, CD314, CD335, CD336 and CD337. Boolean populations that comprised 3% or greater of the total NK cell population (median) were then compared. A Mann-Whitney test was used to determine statistical significance. Results 154 patients from 11 Australian sites were enrolled. The median number of prior treatment lines was 4.5, 82.5% were double refractory. 78 patients who achieved ≥SD were randomised to maintenance: POM n = 40, Pom LoDEX n = 38. CD336+CD20+ cells ("NK-B-cells") were identified in the pre-induction samples of all patients and were significantly more frequent in responders (median 2% of total cells) than in non-responders (0.8% of total cells, p<0.0001). These cells also variably expressed CD19, IgD, HLA-DR, CD158b, CD38 and CD45RA. Preliminary validation of this observation has also been successfully undertaken in an independent cohort of MM patients utilising multi-parameter flow cytometry. In the patients who achieved ≥SD, 5 out of the 8 large clusters (each at least 3% [median] of total nucleated cells evaluated) that were significantly enriched (p<0.0001) following POM LoDEX induction were neutrophil populations. These populations all expressed CD66b but with variable expression of CD24, CD16, CD11c, CD11b and CD45RO. Inhibited NK cells (CD3-CD19-CD56+) based on CD159a, CD314 and CD158a expression were enriched pre-induction and significantly decreased following POM LoDEX (p<0.0001), while activated NK cells expressing CD337 and CD336 and no inhibitory receptors were significantly increased following POM LoDEX (p<0.0001). Conclusion Utilising CyToF, we have identified a novel "NK B cell" population in RRMM patients, with a higher baseline frequency of these cells being associated with a greater likelihood of response to POM LoDEX. Importantly, we have also confirmed the presence of these cells in an independent MM cohort. Moreover, subsequent to POM LoDEX exposure we have demonstrated the enrichment of heterogeneous neutrophil populations as well as an increase in activated NK cells and commensurate decrease in inhibited NK cells. These novel observations may provide new insights into the mechanisms of action of pomalidomide in MM. Disclosures Kalff: Amgen: Honoraria; Celgene: Honoraria; pfizer: Honoraria. Khong:Novartis Oncology: Research Funding. Reynolds:Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership; Novartis Australia: Honoraria. Quach:GSK: 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; Sanofi: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Ho:Novartis: Other: Trial Investigator meeting travel costs; La Jolla: Other: Trial Investigator meeting travel costs; Celgene: Other: Trial Investigator meeting travel costs; Janssen: Other: Trial Investigator meeting travel costs. Mollee:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Spencer:Takeda: Other: Consulting/advisory role, Research Funding; Janssen Oncology: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Amgen: Other: Consulting/advisory role, Research Funding; AbbVie: Other: Consulting/advisory role, Research Funding; Servier: Other: Consulting/advisory role; Secura Bio: Other: Consulting/advisory role; Haemalogix: Other: Consulting/advisory role; Celgene: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Sanofi: Other: Consulting/advisory role; Specialised Therapeutics Australia: Consultancy, Honoraria.

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Lenalidomide Enhances Multiple Myeloma Cytotoxicity Induced by a Novel Fc Domain-Engineered Anti-HM1.24 Monoclonal Antibody with Augmented NK Cell Degranulation

Blood ◽

10.1182/blood.v116.21.4064.4064 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4064-4064

Author(s):

Sun-Young Kong ◽

Sabikun Nahar ◽

Xian-Feng Li ◽

Weihua Song ◽

Yiguo Hu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Surface ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Flow Cytometric Analysis ◽

Cell Lysis ◽

Myeloma Cell ◽

Advisory Committees

Abstract Abstract 4064 HM1.24, an immunological target highly expressed on majority of multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). Recently, XmAb5592, a novel Fc-domain engineered humanized anti-HM1.24 mAb with specific Fc-domain modification, was shown to induce >10-fold antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis against MM cells, when compared with the humanized normal anti-HM1.24 IgG1 (XmAb5627) from which it is derived (ASH Abstract#609, 2009). Here we investigated whether XmAb5592, when combined with other anti-MM drugs, further enhanced ADCC against MM cell lines and primary patient MM cells using Calcein-AM release ADCC assays and flow cytometric analysis for cell membrane CD107a to specifically quantitate NK cell (CD56+CD3-) activation. Addition of lenalidomide (10 μ M) in standard ADCC assays increased XmAb5592 (0.1 μ g/ml)-induced cell lysis against MM1S, MM1R, and RPMI8226 cells in the presence of peripheral blood mononuclear cells (PBMCs) from normal donors (n=2). Specifically, XmAb5592 (0.01, and 0.1 μ g/ml) combined with lenalidomide (2.5, 5, 10 μ M), in the presence or absence of IL-2 (100 units/ml), synergistically induced NK-mediated RPMI8226 MM cell lysis, as evidenced by combination index (CI) < 1 (0.08-0.89). Pre-incubation of PBMCs with additional IL-2 (100 units/ml) enhanced even stronger XmAb5592-induced cytotoxicity against MM cells than pretreatment with lenalidomide alone. Lenalidomide also enhanced PBMC effectors to kill primary patient MM cells. Cell surface CD107a, as a functional marker for NK cell activation dependent on granzyme B secretion, was further determined following target myeloma cell incubation, with or without NK cells and in the presence of mAbs. NK cells were activated by as low as 0.001 μ g/ml of XmAb5592 only in the presence of MM1S tumor cells. In contrast, neither XmAb6166, an Fc-domain knockout of XmAb5592 without NK binding, nor XmAb4614, a similar Fc-engineered mAb targeting respiratory syncytial virus (RSV) antigen, induced any cell surface CD107a on NK cells incubated with MM cells. Specific XmAb5592-induced CD107a-dependent NK cell degranulation further correlated with IFNγ secretion (r=0.7, p=0.03). XmAb5592 induced > 10-fold more potent NK degranulation than XmAb5627, which significantly correlated with MM cell lysis by ADCC, even in the presence of bone marrow stromal cells (BMSCs). Moreover, no significant HM1.24 was expressed on NK cells stimulated with or without IFN-α, suggesting minimal NK toxicity. XmAb5592 more potently (>10-fold) than XmAb5627 induces homotypic aggregation and adhesion of NK cells, which was further enhanced by lenalidomide. Finally, minimal HM1.24 expression was confirmed on different PBMC subsets including CD15+ (PMN), CD19+ (B), CD14+ (MC), and CD3+ (T) cells. These results indicate that lenalidomide further potentiates XmAb5592-induced myeloma cell killing via NK-mediated ADCC, providing a rationale to combine both novel drugs to improve patient outcome in MM. Disclosures: Muchhal: Xencor Inc: Employment. Desjarlais:Xencor Inc: Employment. Richardson:Gentium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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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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