Recruitment of NK Cells during Autologous Apheresis Allows Efficient NK Cell Collection for Immunotherapy

Abstract Background: Natural killer (NK) cell-based immunotherapies are emerging as a new, cutting-edge, promising cancer treatment. NK cells have been used to generate autologous as well as allogenic chimeric antigen receptor NK (CAR-NK) cells, and are currently being tested in multiple clinical trials. The advantage of these cells over CAR-T cells is that they show a superior toxicity profile, less on-target/off-tumor effects, and their toxicity mechanism could be independent of CAR. Additionally, allogenic CAR-NK cells are less immunogenic and cause less graft versus host disease in comparison to CAR-T cells. NK-cell collection during apheresis is the first step in the production of CAR-NK-cells. Efficient collection of NK-cells is paramount to successful manufacture of a CAR-NK cell product. Here, we present our single-center experience on individualized high-flow autologous lymphocyte apheresis in heavily pre-treated patients who qualified for a CAR-T treatment. In this abstract, we demonstrate data on NK-cell collections that derived from our research on T-cell collections. Study design and methods: In this retrospective, descriptive study, we compared collection efficiencies (CE%), absolute efficiencies (AE; absolute yield) and relative efficiencies (RE; AE/total number of absolute circulating pre-apheresis cells) for NK- and T-cell collections. Recruitment of cells was assumed if RE>1. The analysis was performed for 9 heavily pre-treated diffuse large B-cell lymphoma patients who underwent autologous T-cell collections for CAR-T treatment between October 2018 and November 2019. These 9 patients were chosen because the data on pre-apheresis NK- and T-cell concentrations, and harvest NK- and T-cell yield, were available for them. For calculation of CE% a standard formula was used (Figure 1, A). Results: A total of 9 high-flow lymphocyte collections achieved the cell yield of 1x10 9 cells for T- cells and 88% (n=8) achieved the cell yield of 1x10 9 cells for NK-cells. The median pre-apheresis cell concentration for NK-cells was 0.21 x10 9/L (range 0.05 - 0.29) and for T-cells was 0.69 x10 9/L (0.11 - 2.09). The median average blood flow was 77 ml/min (49 - 132), the median processed blood volume was 13.51L (7.35 - 31.17), while the median ratio of processed blood volume to total blood volume was 2.57 (1.46 - 6.67). The median AE for NK-cells with 1.58x10 9 (0.55 - 5.9) was markedly lower but not statistically significant (p=0.14) than that of T-cells with 5.5x10 9 (1.88 - 18.41). The CE of NK-collections was median 89% (29 - 125), while the CE of T-cell collections was median 68% (31 - 93) and they were not statistically different (p=0.605). The same was observed for RE which was 3.04 (0.63 - 4.31) for NK-cells and 1.91 (0.68 - 4.52) for T-cells with no statistically significant difference (Independent-Samples Mann-Whitney U Test, p=0.836). Figure 1 presents the differences in AE (B), CE (C) and RE (D) between NK- and T-cell collections. Conclusions: During high-flow lymphocyte apheresis, NK- as well as T-cells are recruited, as in the majority of collections the cell yield is greater than the absolute circulating pre-apheresis cell count (when RE was > 1). Thus, the origin and immunophenotype of these NK- and T-cells should be subject of further investigation. CE for NK- and T-cells are similar and high enough to perform efficient cell collections of both cell types for immunotherapy. Figure 1 Figure 1. Disclosures Jalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Daskalakis: Amgen: Other: Travel grant; Roche: Other: Travel grant; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Celgene/BMS: Consultancy, Other: Travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; NovoNordisk: Other: Travel grant.

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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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Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Blood ◽

10.1182/blood-2019-127338 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1571-1571

Author(s):

Patrick P. Ng ◽

Mehrdad Mobasher ◽

Kitman S. Yeung ◽

Andrew N. Hotson ◽

Craig M. Hill ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Phase 1 ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression of this gene has been reported in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Genomic analyses have demonstrated the contribution of aberrant TCR signaling in the pathogenesis of T-cell lymphomas (TCL). RLK, a closely related kinase, is co-expressed with ITK in T and NK cells, and is partially functionally redundant with ITK signaling. In NK cells, ITK has been shown to be involved in FcγRIII signaling and antibody-dependent cellular cytotoxicity (ADCC). However, the relative contribution of ITK vs RLK in ADCC is not well understood. Thus, selective inhibition of ITK, but not other signal transduction components such as RLK, may be an effective strategy to treat TCL while preserving normal T and NK cell functions. CPI-818 is an orally bioavailable, covalent inhibitor of ITK with >100-fold selectivity over RLK and BTK. It was well tolerated and exhibited anti-tumor activity in companion dogs with spontaneous TCL (2019 AACR Annual Meeting Abstract #1313). A phase 1/1b trial with CPI-818 in human TCL has been initiated (NCT03952078). Here we present preclinical evidence that CPI-818 inhibits the proliferation of human malignant T cells with relative sparing of normal lymphocytes and report early results from the clinical trial. Methods Eligible patients for the dose-escalation/expansion trial of CPI-818 have relapsed/refractory TCL (PTCL, CTCL and others). Starting dose of CPI-818 is 100 mg BID continuously. The objectives of the study are to evaluate the safety and tolerability of CPI-818 in ascending dose levels; evaluate pharmacokinetics/pharmacodynamics and potential biomarkers. In in vitro studies, T cells from the blood of Sézary syndrome patients were stimulated for 6 days with αCD3/CD28. Sézary cells were identified by antibodies to specific TCR Vβ. For assays of ADCC, αCD20-coated lymphoma B cells were cultured with NK cells from multiple healthy donors for 18 h with inhibitors. In animal studies, mice received control or CPI-818-formulated diet (300 mg/kg/day). C57BL/6 mice were vaccinated with keyhole limpet hemocyanin (KLH) or subcutaneously implanted with the TCL line EL4. MRL/lpr mice began treatment at 9 weeks old. Lymph nodes were calipered weekly. Spleens and lungs were harvested at 22 weeks. Results Mouse models were studied to assess the impact of CPI-818 on normal, autoreactive and malignant T cells in vivo. No changes in total blood cell counts or T, B, NK cell subsets in lymphoid organs were seen in normal mice receiving daily doses of CPI-818 sufficient to continuously inhibit ITK for 28 days. Immune responses to antigen re-challenge were not affected in these mice, as determined by levels of antibody or CD4 T cell response to vaccination with KLH. In mice with established EL4 lymphoma, administration of CPI-818 reduced the growth of tumors at the primary site and in the draining lymph nodes (P values <0.033). CPI-818 also reduced lymphadenopathy and expansion of autoreactive T cells in the spleens of MRL/lpr mice (P values <0.0001), without affecting CD4 or CD8 cells. Sézary cells from 3 of 3 patients tested in vitro were more sensitive to growth inhibition with CPI-818 than autologous normal CD4 or CD8 cells, or T cells from a healthy donor (Figure 1). CPI-818 showed minimal inhibition of NK-mediated ADCC (5%), whereas CP-2193, an ITK/RLK dual inhibitor with an IC50 for ITK comparable to CPI-818, reduced ADCC by 50%. CPI-818 has been administered to two patients at the first dose level cohort (100 mg BID) with no DLTs, and with no changes to B, T, and NK cell counts in blood during the first dosing cycle (21 days). Pharmacokinetic and occupancy studies have revealed 80% and 50% occupancy of ITK at peak and trough drug levels, respectively in peripheral blood T cells. Conclusions CPI-818 is a selective covalent ITK inhibitor that has greater antiproliferative effects on malignant and autoreactive T cells compared to normal T cells. The drug has a minimal impact on NK mediated ADCC compared with a less selective inhibitor that also blocks RLK. Preliminary data from a phase 1/1b study shows CPI-818 at 100 mg BID was tolerable with acceptable bioavailability and ITK occupancy. Further dose escalation is ongoing. Disclosures Ng: Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Mobasher:Corvus Pharmaceuticals: Employment, Equity Ownership. Yeung:Corvus Pharmaceuticals: Employment, Equity Ownership. Hotson:Corvus Pharmaceuticals: Employment, Equity Ownership. Hill:Corvus Pharmaceuticals: Employment, Equity Ownership. Madriaga:Corvus Pharmaceuticals: Employment, Equity Ownership. Dao-Pick:Corvus Pharmaceuticals: Employment, Equity Ownership. Verner:Corvus Pharmaceuticals: Employment, Equity Ownership. Radeski:Corvus Pharmaceuticals: Research Funding. Khodadoust:Corvus Pharmaceuticals: Research Funding. Kim:Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Horizon: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galderma: Research Funding; Elorac: Research Funding; Soligenix: Research Funding; Medivir: Honoraria, Membership on an entity's Board of Directors or advisory committees; miRagen: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Research Funding; Portola Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Corvus: Honoraria, Membership on an entity's Board of Directors or advisory committees; Trillium: Research Funding. Miller:Corvus Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Buggy:Corvus Pharmaceuticals: Employment, Equity Ownership. Janc:Corvus Pharmaceuticals: Employment, Equity Ownership.

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Two-Pronged Cell Therapy for B-Cell Malignancies: Engineering NK Cells to Target CD22 and Redirect Bystander T Cells to CD19

Blood ◽

10.1182/blood.v128.22.4560.4560 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4560-4560 ◽

Cited By ~ 1

Author(s):

Mireya Paulina Velasquez ◽

Arpad Szoor ◽

Challice L. Bonifant ◽

Abishek Vaidya ◽

Lorenzo Brunetti ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Nk Cells ◽

Immune Escape ◽

Nk Cell ◽

Antitumor Effects ◽

B Cell Malignancies ◽

Cell Therapies ◽

Car T

Abstract Background: T-cell therapy with CD19-CAR T cells has been very successful for the treatment of B-cell derived malignancies in humans. However, cytokine release syndrome, neurotoxicity, and development of CD19- escape variants have emerged as potential limitations. Developing CAR NK-cell based therapies might overcome some of these side effects since NK cells do not rapidly expand or persist long-term after adoptive transfer. However, CAR NK-cell therapies are less effective than CAR T-cell therapies in preclinical models. To overcome these limitations we have devised a strategy to genetically modify NK cells with CD22-CARs and CD19/CD3-bispecific T-cell engager (CD19-ENG) molecules. These NK cells should not only kill CD22+ B cells directly, but also redirect bystander T cells to malignant CD19+ B cells, enhancing antitumor effects and preventing immune escape. Methods: NK cells were generated using K562s expressing 41BBL and membrane bound IL15, and genetically modified with a retroviral vector encoding a CD22-CAR with a 41BB.ζ endodomain and/or a retroviral vector encoding CD19-ENG and mOrange separated by an IRES. To mimic immune escape, CD19 or CD22 knockout (ko) Ph+ leukemia cells (BV173) were generated by CRISPR/cas9. The effector function of genetically modified NK cells was evaluated using standard immunological assays. Results: After transduction 70-80% of NK cells expressed CD22-CARs, and ~50% expressed CD22-CARs and CD19-ENGs as judged by FACS analysis. We performed coculture and cytotoxicity assays using non-transduced (NT), CD22-CAR, CD19-ENG, and CD22-CAR/CD19-ENG NK cells as effectors and BV173 (CD19+/CD22+), BV173.koCD19, BV173.koCD22, Daudi (CD19+/CD22+), and KG1a (CD19-,CD22-) as targets. Cocultures were preformed +/- T cells and after 24 hours IFNγ and IL2 was determined by ELISA. In the absence of T cells, CD22-CAR and CD22-CAR/CD19-ENG NK cells only recognized CD22+ targets as judged by IFNγ production. Moreover, CD22-CAR/CD19-ENG and CD19-ENG NK cells efficiently redirected T cells to secrete IFNγ in the presence of CD19+/CD22- targets. No NK-cell population produced IL2, however CD22-CAR/CD19-ENG and CD19-ENG NK cells induced IL2 production of T cells in the presence of CD19+ targets. No significant cytokine production was observed in the absence of antigen (media, KG1a). Specificity of generated NK cells was confirmed in cytotoxicity assays. In vivo studies to confirm our in vitro findings are in progress. Conclusions: We have generated for the first time NK cells that kill B-cell malignancies through a CAR (CD22) and simultaneously redirect bystander T cells to a 2nd B-cell antigen (CD19) to enhance antitumor effects and prevent immune escape. Genetic modification of NK cells to enhance their antitumor activity and redirect bystander T cells may present a promising addition to current cell therapies for B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

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Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-03001-7 ◽

2021 ◽

Author(s):

Shannon L. McArdel ◽

Anne-Sophie Dugast ◽

Maegan E. Hoover ◽

Arjun Bollampalli ◽

Enping Hong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Blood Cell ◽

Nk Cells ◽

Red Blood Cell ◽

Safety Profile ◽

Nk Cell ◽

Cell Activity ◽

In Vivo Studies

AbstractRecombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

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DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

Journal of Experimental Medicine ◽

10.1084/jem.20081752 ◽

2008 ◽

Vol 205 (13) ◽

pp. 2965-2973 ◽

Cited By ~ 195

Author(s):

Susan Gilfillan ◽

Christopher J. Chan ◽

Marina Cella ◽

Nicole M. Haynes ◽

Aaron S. Rapaport ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Nk Cell ◽

Cd8 T Cell ◽

Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

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Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-141540 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Pinar Ataca Atilla ◽

Mary K McKenna ◽

Norihiro Watanabe ◽

Maksim Mamonkin ◽

Malcolm K. Brenner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Control ◽

Publicly Traded ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p<0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P<0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p<0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

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Interferon Alpha Enhances NK Cell Function and the Suppressive Capacity of HIV-Specific CD8+T Cells

Journal of Virology ◽

10.1128/jvi.01541-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 7

Author(s):

Abena K. R. Kwaa ◽

Chloe A. G. Talana ◽

Joel N. Blankson

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Effector Cell ◽

Cell Function ◽

Nk Cell ◽

Suppressive Capacity ◽

Short Course ◽

Shock And Kill ◽

Hiv 1

ABSTRACTCurrent shock-and-kill strategies for the eradication of the HIV-1 reservoir have resulted in blips of viremia but not in a decrease in the size of the latent reservoir in patients on suppressive antiretroviral therapy (ART). This discrepancy could potentially be explained by an inability of the immune system to kill HIV-1-infected cells following the reversal of latency. Furthermore, some studies have suggested that certain latency-reversing agents (LRAs) may inhibit CD8+T cell and natural killer (NK) cell responses. In this study, we tested the hypothesis that alpha interferon (IFN-α) could improve the function of NK cells from chronic progressors (CP) on ART. We show here that IFN-α treatment enhanced cytokine secretion, polyfunctionality, degranulation, and the cytotoxic potential of NK cells from healthy donors (HD) and CP. We also show that this cytokine enhanced the viral suppressive capacity of NK cells from HD and elite controllers or suppressors. Furthermore, IFN-α enhanced global CP CD8+T cell cytokine responses and the suppressive capacity of ES CD8+T cells. Our data suggest that IFN-α treatment may potentially be used as an immunomodulatory agent in HIV-1 cure strategies.IMPORTANCEData suggest that HIV+individuals unable to control infection fail to do so due to impaired cytokine production and/cytotoxic effector cell function. Consequently, the success of cure agendas such as the shock-and-kill strategy will probably depend on enhancing patient effector cell function. In this regard, NK cells are of particular interest since they complement the function of CD8+T cells. Here, we demonstrate the ability of short-course alpha interferon (IFN-α) treatments to effectively enhance such effector functions in chronic progressor NK cells without inhibiting their general CD8+T cell function. These results point to the possibility of exploring such short-course IFN-α treatments for the enhancement of effector cell function in HIV+patients in future cure strategies.

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Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽

10.1182/blood-2019-129640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 776-776

Author(s):

Claire Roddie ◽

Maeve A O'Reilly ◽

Maria A V Marzolini ◽

Leigh Wood ◽

Juliana Dias Alves Pinto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Peak Car ◽

Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

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Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

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Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Blood ◽

10.1182/blood-2019-124031 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 196-196

Author(s):

Bishwas Shrestha ◽

Kelly Walton ◽

Jordan Reff ◽

Elizabeth M. Sagatys ◽

Nhan Tu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Graft Versus Host ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Fund Research

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

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