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

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

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

Blood ◽

10.1182/blood-2019-131138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 133-133

Author(s):

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Karrune Woan ◽

Frank Cichocki ◽

Greg Bonello ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Pluripotent Stem Cell ◽

Nk Cell ◽

Target Cells ◽

Stem Cell Line ◽

Advisory Committees ◽

Cell Immunotherapy

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

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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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Arming of iPSC-Derived NK Cells Expressing a Novel CD64 Fusion Receptor with Therapeutic Antibodies Represents a Novel Off-the-Shelf, Antigen-Targeting Strategy for Cancer

Blood ◽

10.1182/blood-2021-148642 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 406-406

Author(s):

Kate Dixon ◽

Kristin Snyder ◽

Melissa Khaw ◽

Robert Hullsiek ◽

Zachary Davis ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Live Cell ◽

Cell Analysis ◽

Advisory Committees ◽

High Affinity ◽

Targeting Strategy ◽

Live Cell Analysis

Abstract Natural killer (NK) cells are innate lymphocytes that target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and that recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells exclusively mediate ADCC through the IgG Fc receptor, CD16A, and studies have demonstrated that increasing the binding affinity between CD16A and therapeutic monoclonal antibodies (mAbs), mediated by the high-affinity 158V polymorphism, can augment clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we sought to arm iPSC-derived NK (iNK) cells expressing a high-affinity recombinant FcγR with various mAbs as unique tumor-targeting strategy for various malignancies. As a member of the FcγR family, CD64 (FcγRI) possesses the highest affinity and can uniquely facilitate antibody preabsorption but it is normally expressed by myeloid cells. To leverage CD64 in NK cells, we developed a novel FcγR recombinant fusion comprising the extracellular region of CD64 with the transmembrane and intracellular regions of other NK cell activating receptors, including CD16A (CD64/16A) (figure 1A). The recombinant CD64/16A engineered into a clonal master induced pluripotent stem cell (iPSC) line for mass production of off-the-shelf iPSC-derived CD64/16A NK (iNK-CD64/16A) cells, can be armed with mAbs, including various combinations thereof to enable multi-antigen targeting and to address tumor heterogeneity (figures 1B and 2). To determine optimal binding and FcR saturation of iNK-CD64/16 cells, rituximab (anti-CD20 therapeutic mAb) was added in a two-hour preabsorbtion assay (figure 3A). Using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against Raji cells, a Burkitt Lymphoma cell line, when the iNKs were preabsorbed and armed with rituximab (figure 3B). Considering the high-affinity state of CD64, we examined the effects of free IgG on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis, ADCC was evaluated in the presence of purified human IgG. Despite the high levels of excess IgG, iNK-CD64/16A cells mediated efficient ADCC when rituximab was either added to the assay (figure 4A) or preabsorbed to the cells (figure 4B), demonstrating that saturating levels of free IgG did not prevent ADCC in either setting. To determine the ability of preabsorbed and armed iNK-CD64/16 cells to retain rituximab and perform serial killing, we performed a sequential killing assay using an IncuCyte® Live Cell Analysis where preabsorbed iNK-CD64/16A cells were thawed and co-cultured with or without Raji cells for 48 hours, followed by a second round of co-culture. As shown in figure 5, iNK-CD64/16A cells armed with rituximab retain ADCC capacity and perform serial killing for an extended time. To establish that iNK-CD64/16A cells can be armed with assorted therapeutic mAbs to target other tumor-associated antigens, we next determined the ability of iNK-CD64/16A cells preabsorbed and armed with anti-HER2 mAb, trastuzumab, to target the adenocarcinoma ovarian cancer cell line SKOV-3. Indeed iNK-CD64/16A cells armed with preabsorbed trastuzumab were able to effectively kill SKOV-3 cells via in vitro ADCC by IncuCyte® Live Cell Analysis (figure 6). We next investigated in vivo ADCC using NSG mice implanted with 3x10 5 SKOV-3 cells expressing firefly luciferase intraperitoneally (IP). 10 million iNK-CD64/16A with or without preabsorbed trastuzumab were injected IP (figure 7A), and a significant reduction in tumor volume in animals treated with iNK-CD64/16A cells armed with trastuzumab compared to unarmed iNK-CD64/16A cells (figure 7B). Collectively, our data show that iNK-CD64/16A cells can be armed with various therapeutic mAbs through a unique preabsorption strategy to mediate a potent and durable ADCC activity. The versatility of mAb-armed iNK-CD64/16A cells is being further investigated in various preclinical models to further elucidate the potential of this approach to overcome antigen escape and address tumor heterogeneity. Figure 1 Figure 1. Disclosures Lee: Fate Therapeutics, Inc: Current Employment. Chu: Fate Therapeutics: Current Employment. Rogers: Fate Therapeutics: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Hosking: Fate Therapeutics: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Miller: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees. Valamehr: Fate Therapeutics, Inc.: Current Employment. Walcheck: Fate Therapeutics: Research Funding.

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The JAK Inhibitor Ruxolitinib Substantially Affects NK Cells in MPN Patients

Blood ◽

10.1182/blood.v124.21.3169.3169 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3169-3169

Author(s):

Kathrin Schönberg ◽

Janna Rudolph ◽

Maria Vonnahme ◽

Isabelle Cornez ◽

Sowmya Parampalli Yajnanarayana ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Receptor Expression ◽

Target Cells ◽

Jak Inhibitor ◽

Advisory Committees ◽

Nk Cell Differentiation

Abstract Introduction: Ruxolitinib (INCB018424) is the first JAK inhibitor approved for treatment of myelofibrosis (MF). Ruxolitinib-induced reduction of splenomegaly and symptoms control is linked to a substantial suppression of MF-associated circulating pro-inflammatory and pro-angiogenic cytokines. However, an increased rate of infections in ruxolitinib-exposed patients with MF was recently described. Natural killer (NK) cells are innate immune effector cells eliminating malignant or virus-infected cells. Thus, the aim of this project was to define in more detail the impact of JAK inhibition on NK cell biology both in vitro and in vivo. Methods: 28 patients with myeloproliferative neoplasms (MPN) with or without ruxolitinib therapy and 12 healthy donors were analyzed for NK cell frequency, NK receptor expression and function. Phenotypic and functional NK cell markers (e.g. CD11b, CD27, KIR, NKG2A, NKG2D, NKp46, CD16, granzyme B, and perforin) were analyzed by FACS. NK cell function was evaluated by classical killing assays upon stimulation with MHC class I-deficient target cells K562. Finally, a set of additional in vitro experiments (e.g. analysis of lytic synapse formation by FACS and confocal microscopy) were performed to define in more detail the characteristics and potential mechanisms of ruxolitinib-induced NK cell dysfunction. Results: In addition to our recent finding that ruxolitinib induces NK cell dysfunction in vitro (e.g. reduced killing, degranulation and IFN-γ production), we here demonstrate that NK cell proliferation and cytokine-induced receptor expression as well as cytokine signalling are drastically impaired by ruxolitinib. Interestingly, reduced killing is at least in part due to a reduced capacity to form a mature lytic synapse with target cells. The significance of the in vitrofindings is underscored by a dramatically reduced proportion and absolute number of NK cells in ruxolitinib-treated MPN patients when compared to treatment-naïve patients or to healthy controls (mean percentage of NK cell frequency: ruxolitinib-naïve MPN patients 12.63% ±1.81; healthy donors 13.51% ±1.44; ruxolitinib-treated patients 5.47% ±1.27). A systematic analysis of NK cell receptor expression revealed that the reduction of NK cells in ruxolitinib-exposed individuals is most likely due to an impaired NK cell differentiation and maturation process, as reflected by a significantly increased ratio of immature to mature NK cells. Finally, the endogenous functional NK cell defect in MPN is further aggravated by intake of the JAK inhibitor ruxolitinib. Conclusion: We here provide compelling in vitro and in vivo evidence that inhibition of the JAK/STAT-pathway by ruxolitinib exerts substantial effects on the NK cell compartment in MPN patients due to the inhibition of NK cell differentiation and NK cell key functions. Our data may help to better understand the increased rate of severe infections and complement recent reports on ruxolitinib-induced immune dysfunction. Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel, Accomodation, Expenses Other. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wolf:Novartis: Consultancy, Honoraria, Research Funding, Travel and Accommodation Other.

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The Phenotype of Natural Killer (NK) Cells in Multiple Myeloma (MM) Patients: Impact on Ex Vivo Responsiveness to the Anti-SLAMF7 Antibody, Elotuzumab

Blood ◽

10.1182/blood.v126.23.3024.3024 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3024-3024

Author(s):

Tatiana Pazina ◽

Alexander MacFarlane ◽

Ashley Mentik-James ◽

Clinton Yam ◽

Rebecca Kotcher ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Receptor Expression ◽

Cell Phenotype ◽

Target Cells ◽

Advisory Committees ◽

Disease Evolution ◽

Expression Levels

Abstract Background: Monoclonal antibodies (mAbs) are an emerging therapeutic class for MM patients (pts). Elotuzumab, a mAb in late-phase clinical development, targets the SLAMF7 receptor expressed highly on MM cells. While its primary mechanism of action is through CD16-mediated ADCC, elotuzumab can also directly activate SLAMF7-expressing NK cells. Gaining a greater understanding of phenotypic and functional changes in NK cells over the course of the disease, and how these changes impact capacity for ADCC, may help identify profiles that can better select pts likely to benefit from elotuzumab or other mAb therapies. Methods: We prospectively performed a comprehensive flow cytometry-based analysis of lymphocyte subsets, focusing on expression of NK cell activating and inhibitory receptors, activation and maturation markers, and degranulation in 30 MM pts (12 newly-diagnosed (ND), 18 relapsed/refractory (RR)) and 19 aged-matched healthy donors (HD). Over 140 immune parameters were analyzed, with differences in expression between HD and pt subsets compared by Wilcoxon rank-sum test. We analyzed correlations between expression of certain markers with each other, and with elotuzumab-induced NK cell degranulation against MM cell targets (MM1R) in a 2-hour co-culture assay. We also compared NK cell parameters in blood and bone marrow (BM) from pts with matched samples available. Results: Within the blood, there was no difference in relative NK cell frequency between the groups, and little difference phenotypically between HD and ND pt NK cells, except for decreased DNAM1 expression in ND. In contrast, in comparison to HD, CD56dim NK cells in RR pts were less mature with a higher CD56bright to CD56dim NK cell ratio and reduced expression of the terminal differentiation/maturation markers, CD57 and KLRG1. RR pts also showed increased expression of the activation marker CD69 on all NK cells, and their CD56dim NK cells had increased levels of the natural cytotoxicity receptors, NKp30 and NKp46 and decreased expression of activating receptors DNAM1 and NKG2D. SLAMF7 expression was also increased in RR pts, but only on the CD56bright subset. Consistent changes in NK cell expression of checkpoint/co-stimulatory molecules (eg. PD-1, Tim3, LAG3, CD137) were not seen. Despite these phenotypic changes, no significant differences between groups were noted for elotuzumab-induced ADCC against MM1R targets, as measured by CD107a degranulation by CD56dim NK cells, with significant variability noted within groups. Interestingly, the expression levels of SLAMF7 on CD56dim NK cells directly correlated with CD16 levels, particularly within RR pts (Fig.), suggesting cooperative interactions between these receptors that may be beneficial in MM patients treated with elotuzumab. In addition, degranulation toward elotuzumab-treated MM1R targets was significantly associated with surface expression levels of both SLAMF7 and CD16 on the CD56dim NK cells. The status of NK cells was also compared between matching blood and BM samples from ND (n=7) and RR (n=8) pts. NK cell phenotype and degranulation in blood and BM were similar in ND pts, but in RR pts, expression of CD69 and SLAMF7 were higher on BM-derived NK cells, and CD56dim NK cells from BM demonstrated greater degranulation toward elotuzumab-treated MM1R targets. DNAM1 expression was reduced, but NKG2D, NKp30, and NKp46 were upregulated on various NK cell populations in BM from RR pts compared to peripheral blood. Conclusions: Taken together, our data indicate that NK cells in RR MM pts had increased activation, reduced maturation status, and distinct changes in activating receptor expression levels that are often further enhanced in the BM microenvironment. Furthermore, CD56dim NK cells in many RR pts had parallel increased expression levels of CD16 and SLAMF7, which correlated with enhanced degranulation toward elotuzumab-treated MM target cells. The fact that these changes are seen primarily in RR pts rather than untreated ND pts implies a significant impact of disease evolution and prior therapy on the NK cell compartment, and supports further exploration of these parameters as potential biomarkers of activity of elotuzumab and other therapeutic mAbs in myeloma. Figure 1. Figure 1. Disclosures Campbell: Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cohen:Bristol-Meyers Squibb: 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.

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The IRE1-XBP1s Pathway Impairment Underpins NK Cell Dysfunction in Hodgkin Lymphoma, That Is Partly Restored By PD-1 Blockade

Blood ◽

10.1182/blood-2019-126372 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2795-2795

Author(s):

Karolina Bednarska ◽

Jay Gunawardana ◽

Frank Vari ◽

Qingyan Cui ◽

Gayathri Thillaiyampalam ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Cell Function ◽

Nk Cell ◽

Advisory Committees ◽

Pathway Activation ◽

Pathway Inhibition ◽

The Impact

Background The frequent loss of Major Histocompatibility Complex I molecule (MHC-I) on Hodgkin/Reed-Sternberg (HRS)-cells renders them susceptible to Natural Killer (NK) cell-mediated lysis in Hodgkin Lymphoma (HL). Optimal NK cell function involves migration from peripheral blood to sites of disease, formation of an immune synapse (NKIS) between NK cells and HRS cells, and release of effector molecules. We recently showed that PD-1+ NK cells are expanded in the circulation of patients with HL (Vari, F Blood 2018), and that PD-1 blockade enhances their anti-HRS capabilities. However, mechanisms behind the functional impairment of NK cells in HL patients and the impact PD-1 blockade has on NK cell function remain to be established. Although the IRE1-XBP1s pathway, part of the unfolded protein response (UPR) system, has established and fundamental roles in macrophage, B, T and dendritic cells homeostatic function, its involvement in NK cells remains unknown. We hypothesized that IRE1-XBP1s dysfunction contributes to NK cell impairment and tested the impact of PD-1 blockade on individual components of NK cell function, including migration, NKIS formation, and cytokine release. Methods Ex-vivo functional assays were performed on blood from 20 participants. Confocal microscopy, time-lapse imaging, trans-well migration, and functional in-vitro immune assays were utilized on a range of NK and HRS cell lines, with and without IRE1-XBP1s small molecule inhibitors (4µ8c and 6-bromo) and/or PD-1 blockade (pembrolizumab). Results Stimulation of both NK cell lines and primary NK cells, with HRS lines resulted in marked and rapid IRE1-XBP1s pathway activation. This occurred independently of the canonical UPR and was associated with increased NK cell effector function. However, IRE1-XBP1s pathway inhibition resulted in aberrant NK cell morphology, reduced motility and migration, deficient NKIS formation and impaired interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) release. Next, we tested the IRE1-XBP1s pathway in the pre-therapy blood of patients with HL and compared this with healthy age/gender matched controls. Strikingly, following co-culture with an HRS-line the pathway was not activated, but this abnormality was restricted to the CD56brightCD16-ve subset that we have previously shown to be expanded and enriched in PD-1 (as well as downregulation of the lymphoid migratory chemokine CCR7) in patients with HL. In subsequent experiments using in-vitro expanded populations of primary NK cells from HL patients, IRE1-XBP1s pathway inhibition impaired the migration, NKIS formation, CD107a degranulation, and secretion of IFNγ and TNFα. Effects were partially but not completely restored by addition of PD-1 blockade (Fig 1). Conclusion Here, we outline a hitherto unrecognized mechanism involving the IRE1-XBP1s pathway that is pivotal to NK cell function, including the relatively poorly understood processes of migration, NKIS formation, and cytokine secretion. Notably, IRE1-XBP1s pathway activation is dysfunctional within the PD-1 enriched CD56brightCD16- NK cell subset. Although PD-1 blockade appears to have a multi-faceted beneficial role on NK cell migrational/NKIS and cytokine release capabilities, it is still only capable of partial restoration of NK cell effector function. Further understanding of the pathways operative in NK cells may result in improved immunotherapeutic strategies to enhance this arm of the immune response in patients with HL. Disclosures Gandhi: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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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)

Blood ◽

10.1182/blood-2021-150543 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1388-1388

Author(s):

Basem M. William ◽

John C. Reneau ◽

Amanda Campbell ◽

Anna Vilgelm ◽

Michelle Watts ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Standard Dose ◽

Pilot Phase ◽

Consensus Panel ◽

Advisory Committees ◽

Malignant T Cells

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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FT576: Multi-Specific Off-the-Shelf CAR-NK Cell Therapy Engineered for Enhanced Persistence, Avoidance of Self-Fratricide and Optimized Mab Combination Therapy to Prevent Antigenic Escape and Elicit a Deep and Durable Response in Multiple Myeloma

Blood ◽

10.1182/blood-2020-142750 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-5

Author(s):

Jode P Goodridge ◽

Ryan Bjordahl ◽

Sajid Mahmood ◽

John Reiser ◽

Svetlana Gaidarova ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Publicly Traded ◽

Advisory Committees ◽

Effector Cells ◽

Exogenous Cytokine

Multiple redundancy within the spectrum of an immune response is required to prevent antigen escape or adaptation of the targeted population to host defenses. As adoptive cell therapies continue to evolve, multi-modal engineering of effector cells offers the prospect of tackling increasingly complex disease settings such as multiple myeloma (MM), where targeting of a single tumor associated antigen is frequently confounded by antigen shedding and escape variation resulting in the inability to develop a curative therapy. There are multiple advantages in expanding treatment options beyond autologous primary T and NK cells, including the use of induced pluripotent stem cells (iPSC) to derive effector cells that can be uniformly manufactured at scale from renewable starting cellular material and where precision genetic engineering can be achieved at the clonal level which can be applied sequentially in order to build multiple specificities and functional modalities. To create a platform targeted toward MM, a multiplexed edited base iPSC-derived NK (iNK) cell configuration consisting of a CD38 KO iPSC modified to overexpress a recombinant IL-15 signaling complex (IL15RF) for autonomous persistence and a functionally enhanced high-affinity, non-cleavable CD16 (hnCD16) was developed. Introduction of IL15RF enabled expansion of iNK cells without additional exogenous cytokine support during the manufacturing process and greatly improved functional persistence of iNK cells both in vitro and in various xenograft mouse models (Figure 1). To target MM in a broad and comprehensive manner, we tested our novel BCMA-CAR in combination with different myeloma targeted antibodies. In combination with hnCD16, co-expression of BCMA-CAR and IL15RF culminates in an iNK cell therapeutic, termed FT576, capable of multiantigen-specificity through combinatorial use of CAR and hnCD16 with monoclonal antibodies to tackle antigen escape. Chimerization of an anti-BCMA scFv shown to elicit higher affinity onto the CAR platform produced specific in vitro recognition of BCMA+ myeloma cells in short-term and long-term NK cell cytotoxicity assays. Specificity of the BCMA-CAR was demonstrated using NALM6 overexpressing BCMA using a short range 4H caspase assay (NALM6_BCMA EC50 14.4, NALM6wt EC50 39.1, p*<0.0001). Utilizing a long range clearance assay, serial restimulation by repeated rounds of exposure to fresh MM1S MM target cells was tested, showing remarkable persistence and antigen-mediated expansion of CAR function in isolation or combined with antibody through 3 rounds of stimulation in the absence of exogenous cytokine support (Figure 2). Continuous long-range clearance assays demonstrated levels of BCMA targeting activity of FT576 alone was equivalent to primary BCMA-targeted CAR-T cells against a panel of BCMA+ target cells. Utilizing hnCD16, BCMA-CAR was tested in combination with anti-CD38 (daratumumab), anti-SLAMF7 (elotuzumab), or anti-CD19, showing synergistic increase in tumor targeting through various tumor associated antigens (TAAs). Polyfunctionality of FT576 stimulated either through CAR or ADCC was similarly measured by both Isoplexis and single cell RNA sequencing. Specificity for plasma cells was confirmed using primary bone marrow samples from either healthy donors or patients. In animal models, as a monotherapy, FT576 achieved sustained tumor control against disseminated MM1s with persistence profile suggestive of antigen mediated expansion (Figure 3). In combination with daratumumab, FT576 was able to achieve complete clearance of MM1S. Combination with other monoclonal antibodies displayed a similar response demonstrating the unique ability of FT576 to be directed to target multiple TAAs. Together, these studies demonstrate the versatility of FT576 as a highly effective multi-antigen targeting and cost-effective off-the-shelf BCMA-CAR iNK cell product and supports the rational for a first-of-kind Phase I Study as a monotherapy or in combination with therapeutic mAbs targeted to MM-associated surface antigens, driving a path towards a curative therapeutic in MM. Disclosures Goodridge: Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Reiser:FATE THERAPEUTICS: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Blum:Fate Therapeutics: Current Employment. Cichocki:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Chu:Fate Therapeutics, Inc: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Meza:Fate Therapeutics, Inc: Current Employment. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties. Miller:Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

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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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TNFR2 As a Target to Improve CD19-Directed CART Cell Fitness and Antitumor Activity in Large B Cell Lymphoma

Blood ◽

10.1182/blood-2021-148591 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 901-901

Author(s):

Claudia Manriquez Roman ◽

Michelle J. Cox ◽

Reona Sakemura ◽

Kun Yun ◽

Mohamad M. Adada ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

B Cell Lymphoma ◽

Death Receptors ◽

Target Cells ◽

Advisory Committees ◽

Anti Tumor Activity

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

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