scholarly journals Lenalidomide Exhibits Activity in Mantle Cell Lymphoma through Increased NK Cell Mediated Cytotoxicity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 821-821 ◽  
Author(s):  
Patrick Hagner ◽  
Hsiling Chiu ◽  
Maria Ortiz-Estevez ◽  
Tsvetan Biyukov ◽  
Carrie Brachman ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Employment Equity ◽  
Mantle Cell ◽  
Apoptosis Assay ◽  
Equity Ownership

Abstract Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170). The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models. Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay. Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm. Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit. The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours. Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells. Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014). Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro-apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B. Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL. Disclosures Hagner: Celgene: Employment, Equity Ownership. Chiu:Celgene: Employment, Equity Ownership. Ortiz-Estevez:Celgene: Employment, Equity Ownership. Biyukov:Celgene: Employment, Equity Ownership. Brachman:Celgene: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Milpied:Celgene: Honoraria, Research Funding. Musto:Sandoz: Consultancy; Celgene: Honoraria; Roche: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Novartis: Honoraria; Janssen: Honoraria; Mundipharma: Honoraria. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Carmichael:Celgene: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Gandhi:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

scholarly journals CC-122 Exhibits Greater Preclinical Activity in Mantle Cell Lymphoma Than Lenalidomide through a Combination of Direct Cell-Autonomous and Increased Antibody Dependent Cell-Mediated Cytotoxicity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4188-4188
Author(s):  
Patrick R. Hagner ◽  
Hsiling Chiu ◽  
Michelle F. Waldman ◽  
Anke Klippel ◽  
Michael Pourdehnad ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Lymphoma ◽  
Annexin V ◽  
Employment Equity ◽  
Mantle Cell ◽  
Apoptosis Assay ◽  
Dependent Cell ◽  
Autonomous Activity ◽  
Equity Ownership

Abstract Introduction: CC-122 and lenalidomide (len) bind the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN) resulting in degradation of the transcription factor Aiolos, leading to direct anti-lymphoma activity and T and NK cell activation. CC-122 degrades Aiolos at a faster rate and to a greater extent compared to len. Len is currently indicated for the treatment of relapsed/refractory (R/R) mantle cell lymphoma (MCL) in the United States and European Union. CC-122 is in development for DLBCL, FL and CLL in combination with the anti-CD20 monoclonal antibodies, rituximab (Rtx) and obinutuzumab (GA101). We compared the ability of len and CC-122 to effect cell autonomous activity and enhance antibody dependent cell-mediated cytotoxicity (ADCC) in pre-clinical models of MCL. Methods: Proliferation was measured by 3H labeling. Apoptosis was measured by Annexin V/ToPro-3 flow cytometry. ADCC was measured by a 4 hour co-incubation of Rtx or GA101 labeled cells with stimulated PBMC treated with DMSO, len or CC-122 for 3 days followed by apoptosis assay. Inducible shRNA targeting luciferase or Aiolos were activated with 10 ng/ml doxycycline for 7 days followed by apoptosis assay. Results: Lenalidomide treatment (10μΜ) for 5 days resulted in a 31% and 49% decrease in proliferation of two of the six MCL cell lines investigated, whereas CC-122 treatment (1.25μΜ) decreased proliferation in four MCL cell lines by 37-81%. There was no increase in Annexin V and ToPro-3 staining in MCL cells treated with len (0.1-10μΜ) for 7 days. By contrast, CC-122 treatment (0.1-10μΜ) reduced viability in four of six cell lines examined by 32-95%. Examination of the biochemical activity of each drug in Mino and Rec-1 cells demonstrated CC-122 (0.1-10μΜ) induced rapid Aiolos degradation at 6 hours (33-88% and 38-85%, respectively) compared to 10μΜ len (27% and 25%, respectively). In Jeko-1 cells, two distinct doxycycline inducible shRNA targeting Aiolos led to 56-97% decreased Aiolos protein expression. Furthermore, shRNA targeting Aiolos led to a 2- to 3-fold increase in apoptosis relative to shLuciferase. In ADCC assays of Z138 and Granta-519 coated with Rtx (1μg/ml) or GA101 (1μg/ml), CC-122 (10-100nM) was more potent than len (0.1-1μΜ). CC-122 treatment of PBMC increased Rtx labeled Z138 and Granta-519 apoptosis (39-59% and 36-48%, respectively) compared to len (24-35% and 33-40%, respectively), versus vehicle controls (13% and 13%, respectively). Additionally, CC-122 treatment increased GA101 mediated ADCC of Z138 and Granta-519 (60-76% and 59-67%, respectively) compared to len (49-61% and 55-63%, respectively), versus vehicle controls (35% and 41%, respectively). Conclusions: CC-122 treatment of MCL cells resulted in considerable cell autonomous activity in in vitro proliferation and viability assays compared to len. Specific targeting of Aiolos, a substrate which is rapidly degraded by CC-122, through inducible shRNA results in increased levels of apoptosis compared to shLuciferase controls. Additionally, the combination of CC-122 with either Rtx or GA101 in in vitro co-culture ADCC assays resulted in greater apoptosis than len combined with either antibody. The combination of both enhanced cell-autonomous activity and α-CD20 mediated ADCC provide a rational combination strategy for CC-122 development in R/R MCL. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Chiu:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership.

scholarly journals 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 ◽  
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 &gt;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 &lt;0.033). CPI-818 also reduced lymphadenopathy and expansion of autoreactive T cells in the spleens of MRL/lpr mice (P values &lt;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.

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals Recombinant Human IL-12 Exerts Differential Effects on Circulating CD56bright and CD56dim NK Cells in Healthy Human Subjects

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 756-756
Author(s):  
Simmy Thomas ◽  
Chris E Lawrence ◽  
Vernon Mar ◽  
Hue Kha ◽  
Lena A Basile
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Human Subjects ◽  
Cell Populations ◽  
Employment Equity ◽  
Healthy Human ◽  
Healthy Human Subjects ◽  
Equity Ownership

Abstract Interleukin-12 (IL-12) has potent immunoregulatory and hematopoietic properties, and exerts significant biological effects on natural killer (NK) cells, inducing IFNγ production and enhancing cytotoxicity. Two distinct NK cell populations correlate with their immunoregulatory functions. Mature CD56dimCD16bright NK cells represent 90% of the NK cells resident in the blood and can exert cytotoxic effects on transformed cells. Cytokine producing immature CD56brightCD16+/- NK cells exist in the blood (10% of total circulating NK cells) but are most prominently located in secondary lymphoid tissues. In the continued clinical development of recombinant human IL-12 (HemaMax™, rHuIL-12), to be used in combination with radiotherapy or chemotherapy for the treatment of cancer patients, we have performed a clinical safety study in healthy human subjects. A single subcutaneous (sc) dose of rHuIL-12 (12μg) was administered to 17 healthy human subjects. Placebo was administered to 5 healthy subjects. Peripheral blood samples were collected before rHuIL-12 administration, and up to Day 14 post administration. Immunophenotyping of blood cell populations was conducted by FACS. rHuIL-12 caused a transient decrease in peripheral blood CD56dimCD16bright NK cells, with a nadir (60% reduction from baseline) reached on Day 2 following rHuIL-12 administration. CD56dimCD16bright NK cell levels returned almost to baseline levels on Day 5. Placebo was without effect. Conversely rHuIL-12 caused an elevation in peripheral blood CD56brightCD16+/- NK cells, particularly between Days 2 and 3 after rHuIL-12 administration, which was sustained until a peak was reached on Day 5 (265% above baseline). Levels returned to baseline by Day 11, while placebo was without effect. rHuIL-12 did not impact the less functional CD56-CD16bright NK cell subset. CD56dimCD16bright NK cells expressing the IL-12 receptor β2 subunit (IL-12Rβ2+) showed a substantial, and transient, decrease in levels on Day 2. The plasma concentration of IFNγ was elevated to a peak over 35 fold above baseline level at 10hr. after rHuIL-12 administration. Human NK cells were negatively selected from highly enriched leukapheresis-derived blood and stimulated in vitro with 10 pM rHuIL-12. After 16hr. incubation these predominantly CD56dimCD16brightNK cells showed enhanced release of IFNγ and the increased killing of K562 cells, a human erythroleukemic cell line, when compared with vehicle controls. qPCR analysis of the human NK cell lysates showed rHuIL-12-induced elevation of CD56 (302%) and IL-12Rβ2 (587%) mRNA, when compared with vehicle controls. rHuIL-12 did not influence CD16 mRNA expression, but did increase the level of CD62L (L selectin, 206%) mRNA. The rapid 60% fall in circulating mature CD56dimCD16bright NK cells after rHuIL-12 administration to healthy human subjects suggests their immediate exit from peripheral blood into the tissue compartments. This could be mediated by the observed increase in NK cell CD62L mRNA expression seen in vitro. The sustained increase in immature CD56brightCD16+/- NK cell levels between Day 3 and 6 suggests their IL-12-induced development from CD34+ hematopoietic progenitor cells. In summary rHuIL-12 administration to healthy human subjects demonstrates differential effects on the two key NK cell populations in peripheral blood, increasing CD56brightCD16+/- NK cell numbers, potentially stimulating IFNγ release from and enhancing the cytotoxicity of the CD56dimCD16bright NK cells, and preparing this population for migration into tissues. rHuIL-12 thus shows excellent potential as an immunotherapeutic and hematopoietic agent for the treatment of cancer patients, by impacting the maturation, activation, immunoregulation, and cytolytic properties of NK cells. Disclosures Thomas: Neumedicines: Employment, Equity Ownership. Lawrence:Neumedicines: Employment, Equity Ownership. Mar:Neumedicines: Employment, Equity Ownership. Kha:Neumedicines: Employment, Equity Ownership. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Enhanced In Vivo Persistence and Proliferation of NK Cells Expanded in Culture with the Small Molecule Nicotinamide: Development of a Clinical-Applicable Method for NK Expansion

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 657-657 ◽  
Author(s):  
Tony Peled ◽  
Guy Brachya ◽  
Nurit Persi ◽  
Chana Lador ◽  
Esti Olesinski ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Culture Method ◽  
Employment Equity ◽  
Advisory Committees ◽  
Nsg Mice ◽  
Equity Ownership

Abstract Adoptive transfer of cytolitic Natural Killer (NK) cells is a promising immunotherapeutic modality for hematologic and other malignancies. However, limited NK cell in vivo persistence and proliferation have been challenging clinical success of this therapeutic modality. Here we present a reliable, scalable and GMP-compliant culture method for the expansion of highly functional donor NK cells for clinical use. Nicotinamide (NAM), a form of vitamin B-3, serves as a precursor of nicotinamide adenine dinucleotide (NAD) and is a potent inhibitor of enzymes that require NAD including ADP ribosyltransferases and cyclic ADP ribose/NADase. As such, NAM is implicated in the regulation of cell adhesion, polarity, migration, proliferation, and differentiation. We have previously reported that NAM augments tumor cytotoxicity and cytokine (TNFα and IFN-γ) secretion of NK cells expanded in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NK cells expanded with NAM underwent typical changes observed with cytokine only-induced NK cell activation with no significant differences in the expression of activating and inhibitory receptors. CD200R and PD-1 receptors were expressed at low levels in resting NK cells, but their expression was up-regulated following activation in typical cytokine expansion cultures. Interestingly, the increase in CD200R and PD-1 was reduced by NAM, suggesting these NK cells to be less susceptible to cancer immunoevasion mechanisms (Fig 1). In vivo retention and proliferation is a pre-requisite for the success of NK therapy. We have reported that NK expanded with NAM displayed substantially better retention in the bone marrow, spleen and peripheral blood of irradiated NSG mice. Using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay, we demonstrated increased in vivo proliferation of NAM-cultured NK cells compared with cells cultured without NAM. These results were recently confirmed using a BrdU incorporation assay in irradiated NSG mice (Fig.2). These findings were mechanistically supported by a substantial increase in CD62L (L-selectin) expression in cultures treated with NAM. CD62L is pivotal for NK cell trafficking and homeostatic proliferation and its expression is down regulated in IL-2 or IL-15 stimulated cultures (Fig. 3). These data provided the foundation for the development of a feeder cell-free scalable culture method for clinical therapy using apheresis units obtained from healthy volunteers. CD3+ cells were depleted using a CliniMACS T cell depletion set. Following depletion, the CD3- fraction was analyzed for phenotypic markers and cultured in closed-system flasks (G-Rex100 MCS, Wilson Wolf) supplemented with 20ng/ml IL-15 or 50ng/ml IL-2 GMP, 10% human serum, minimum essential medium-α and NAM USP for two weeks. While at seeding, NK cells comprised 5-20% of total culture seeded cells, at harvest, NK cells comprised more than 97% of the culture. Although overall contamination of the NK cultures was low with either IL-15 or IL-2, a lower fraction of CD3+ and CD19+ cells was observed with IL-15 vs IL-2 (0.2±0.1% vs. 0.4±0.2% and 1.3±0.4% vs. 2.4±0.6%, respectively). Consequently, we decided to use IL-15 for clinical manufacturing. Optimization of NAM concentration studies showed similar expansion with 2.5 and 5 mM and a decrease in expansion with 7.5 mM NAM. Since NAM at 5 mM had a stronger impact on CD62L expression and on the release of IFNγ and TNFα than NAM at 2.5 mM, we selected 5mM NAM for clinical manufacturing. Overall median NK expansion after two weeks in closed G-Rex flasks supplemented with IL-15 and 5mM NAM was 50-fold (range 37-87). An additional and significant increase in expansion was obtained after doubling the culture medium one week post seeding. While there was a marked advantage for single culture feeding, more feedings had less impact on NK expansion and had a negative effect on the in vivo retention potential. Our optimized expansion protocol therefore involved one feeding during the two weeks expansion duration resulting in 162±30.7-fold expansion of NK cells relative to their input number in culture. Based on these data, we have initiated a clinical trial at University of Minnesota, to test the safety and efficacy of escalating doses (2 x 107/kg - 2 x 108/kg) of our novel NAM NK cell product in patients with refractory non-Hodgkins lymphoma and multiple myeloma (NCT03019666). Disclosures Peled: Gamida Cell: Employment, Equity Ownership. Brachya: Gamida Cell: Employment. Persi: Gamida Cell: Employment. Lador: gamida Cell: Employment, Equity Ownership. Olesinski: gamida cell: Employment. Landau: gamida cell: Employment, Equity Ownership. Galamidi: gamida cell: Employment. Peled: Biokine: Consultancy; Biosight: Consultancy. Miller: Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Bachanova: Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: 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.

scholarly journals The Combination of the BCL-2 Antagonist Venetoclax with the CD70-Targeting Antibody Cusatuzumab Synergistically Eliminates Primary Human Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3918-3918 ◽  
Author(s):  
Carsten Riether ◽  
Tanja Chiorazzo ◽  
Amy J. Johnson ◽  
Christina Diane Drenberg ◽  
Khaja Waheeduddin Syed ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Standard Of Care ◽  
Leukemia Stem Cells ◽  
Clinical Activity ◽  
Employment Equity ◽  
Equity Ownership

Introduction Acute myeloid leukemia (AML) is a very heterogeneous hematological malignancy characterized by the accumulation of myeloid blasts. Treatment options for unfit AML patients greater than 65 year of age are still limited and outcomes are dismal. The current standard of care for older AML patients are hypomethylating agents (HMA) or low dose Ara-C. However, even though inducing hematological remissions in up to 30 percent of the patients, responses are not durable and survival of these patients is only marginally prolonged. The poor durability is due to insufficient action on leukemia stem cells (LSC) which drive and maintain the disease and are resistance to therapy (Craddock et al, 2013 and DiNardo et al, 2019). We described how aberrant CD70/CD27 signaling drives stemness of AML LSCs and identified CD70 as potential new target for the treatment of AML patients (Riether et al, 2017). In a recent Phase 1 clinical trial, treatment of older and unfit AML patients with the ADCC-enhanced humanized monoclonal anti CD70 antibody (mAb) cusatuzumab in combination with HMA demonstrated promising clinical activity and a favorable tolerability profile. The BCL-2 antagonist, venetoclax, targets and eliminates LSCs by suppression of oxidative phosphorylation and demonstrated very promising activity in older AML patients in clinical phase I and II studies in combination with standard of care (Pollyea et al, 2018). However, even with novel agents such as venetoclax, there are still patients that become refractory or relapse. We hypothesized that combining venetoclax and cusatuzumab with distinct but complementary mechanisms of action could successfully eliminate LSCs. Experimental design To test this hypothesis, we performed a drug-combination study according to the Chou-Talalay method (Chou 2010) in CD70-expressing AML cell lines such as MOLM-13, MV4-11, and NOMO-1 cells in vitro. In addition, we tested the effect of the cusatuzumab/venetoclax and the cusatuzumab/venetoclax/HMA combination on colony formation and re-plating capacity of primary CD34+CD38- LSCs from newly diagnosed AML patients. Results We first treated MOLM-13, MV4-11, and NOMO-1 AML cells with vehicle, cusatuzumab alone or in combination with venetoclax or decitabine in a constant ratio in the presence of CFSE-labeled NK cells (ratio 1:1). AML cell numbers were assessed 72 hours later. Cusatuzumab in combination with venetoclax or decitabine and NK cells synergistically eliminated CD70-expressing AML cells in a broad dose range (Figure 1). To assess the effect of the cusatuzumab/venetoclax combination on primary human AML LSCs, we treated CD34+ CD38- LSCs with cusatuzumab or venetoclax monotherapy or in combination in the presence of NK cells and assessed colony formation. Cusatuzumab/venetoclax co-treatment was more efficacious than each monotherapy alone and strongly reduced LSCs and leukemia progenitors (Figure 2). To analyze the effect of the cusatuzumab/venetoclax treatment on LSC function in a more stringent way, we performed serial re-plating experiments in vitro. The impaired colony formation after combination treatment observed after the first plating was maintained during subsequent the re-plating, even though cusatuzumab and venetoclax were not present in the re-plating, indicating an effective reduction of LSCs. Mechanistically, we could show that treatment with venetoclax results in up-regulation of CD70 on LSCs, suggesting that venetoclax renders LSCs more susceptible to cytolytic killing with cusatuzumab. In older AML patients, cusatuzumab and venetoclax each have demonstrated promising clinical activity in combination with HMA. We determined whether addition of HMA to the cusatuzumab/venetoclax co-treatment could more effectively eliminate LSCs. The triplet combination cusatuzumab/venetoclax/decitabine did not further reduce growth of AML cell lines in vitro (Figure 1) nor colony and re-plating capacity of human LSCs compared to the cusatuzumab/venetoclax co-treatment. Conclusions Overall, these results indicate that a combination of cusatuzumab with venetoclax eliminates LSCs synergistically and more efficiently than as a monotherapy. Tolerability and efficacy will be tested in mouse models using both primary AML and cell line xenografts. The results suggest that targeting LSCs by combining venetoclax and cusatuzumab treatments is a promising novel treatment strategy in AML. Disclosures Johnson: Janssen R&D: Employment. Drenberg:Janssen R&D: Employment. Syed:Janssen R&D: Employment. Moshir:Argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Immune Microenvironment Analysis of Bone Marrow By Mass Cytometry and RNA Sequencing in Multiple Myeloma Patients Treated with Daratumumab and Durvalumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3296-3296 ◽  
Author(s):  
Frances Seymour ◽  
Mary H Young ◽  
Mark Tometsko ◽  
Jamie Cavenagh ◽  
Ethan G. Thompson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Immune Microenvironment ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

scholarly journals Targeting Intracellular WT1 in AML Utilizing a T Cell Bispecific Antibody Construct: Augmenting Efficacy through Combination with Lenalidomide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4450-4450
Author(s):  
Christian Klein ◽  
Christian Augsberger ◽  
Wei Xu ◽  
Christina Heitmüller ◽  
Lydia Hanisch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cancer Cell ◽  
Research Funding ◽  
Cell Cytotoxicity ◽  
Employment Equity ◽  
Specific Lysis ◽  
Equity Ownership ◽  
T Cell Cytotoxicity

Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Xu:Roche: Employment, Equity Ownership, Patents & Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents & Royalties. Sam:Roche: Employment, Equity Ownership, Patents & Royalties. Pulko:Roche: Employment, Equity Ownership, Patents & Royalties. Schönle:Roche: Employment, Equity Ownership, Patents & Royalties. Challier:Roche: Employment, Equity Ownership, Patents & Royalties. Carpy:Roche: Employment, Equity Ownership, Patents & Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.

Autologous T Cells From AML Patients Can Be Effectively Recruited for In-Vitro Lysis of Blasts by a Novel CD33/CD3-Bispecific BiTE Antibody

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 14-14 ◽  
Author(s):  
Michael Aigner ◽  
Julian Feulner ◽  
Roman Kischel ◽  
Peter Kufer ◽  
Patrick A Baeuerle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Target Cells ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership ◽  
Bite Antibody

Abstract Abstract 14 Bispecific T cell-engaging (BiTE®) antibodies combine in one polypeptide chain two single chain antibodies, one specific for CD3 on T cells and one for a tumor-associated antigen. The CD19/CD3-bispecific BiTE antibody blinatumomab has shown in phase 1 and 2 clinical trials very high response rates in patients with non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33, an antigen broadly expressed by myeloid cells including acute myelogenous leukemia (AML) blasts, in redirecting autologous T cells for in vitro lysis of blasts from AML patients. In a first step, the cytolytic potential of the CD33-specific BiTE (CD33 BiTE) was investigated in co-cultures of enriched resting CD8+ T cells from healthy donors and CD33+ leukemic cell lines KG-1 and U-937 as target cells. CD33 BiTE concentrations as low as 0.1 ng/ml (1.8 pM) mediated effective lysis of leukemic cell lines at effector to target (E:T) ratios of 1:1, whereas no lysis was observed with a solely CD3-binding control BiTE antibody. Peripheral CD8+ T cells that were pre-activated in cell culture or CD8+ T cell clones were even more potent in target cell lysis than previously resting T cells. Data obtained with a 51Cr release assay were comparable to those from a flow cytometry-based assay. Next, primary samples from AML patients were co-cultured with mononuclear cells (MNC) from healthy donors at an E:T ratio of 1:1. After 48 hrs of incubation in the presence of 1 ng/ml CD33 BiTE, a decrease in CD33+ AML blasts as well as of CD33+ monocytes was observed when compared to samples with control BiTE or vehicle. The CD33 BiTE induced upregulation of activation markers CD25 and CD69 on the majority of T cells. We furthermore investigated whether T cells from AML patients were capable of mediating lysis of CD33+ leukemia cells by CD33 BiTE. Resting or in vitro pre-stimulated CD8+ T cells were prepared from peripheral blood of newly diagnosed AML patients and tested for lysis of U937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls. Finally, we developed a FACS-based assay that allowed studying autologous blast lysis and T cell behaviour using cryo-preserved patient samples. Upregulation of T cell activation markers in cultures of MNC samples from AML patients was evident following addition of 1 ng/ml CD33 BiTE. Fifty five and 85% of CD4+ cells, and 57 and 65% of CD8+ cells expressed CD25 after 24 h and 48 h, respectively, but not with the control BiTE antibody (all <6%). Despite robust T cell activation, only a limited lysis of myeloid blasts was observed, presumably, due to the short incubation periods and low E:T ratios in the range of 1:5-1:21. We therefore investigated whether blast lysis is more effective after prolonged incubation. In the presence of CD33 BiTEs for 6 days, T cell numbers in AML patient samples dramatically expanded; CD8+ cell counts were up 8-fold, and CD4+ cell counts up 11-fold. This was not observed under control conditions. Up to 85% of AML blasts were now lysed. Currently, a larger collection of primary AML patient samples is being analyzed in order to determine an ex-vivo response rate for CD33 BiTE treatment and the impact of the patient samples’ E:T ratio and CD33 expression level on blasts on redirected lysis. Taken together, the novel CD33 BiTE effectively engages and activates autologous T cells for the elimination of AML blasts in vitro and may thereby constitute a novel therapeutic option for the treatment of patients with CD33-expressing myeloid leukemia. Disclosures: Aigner: Micromet Inc.: Research Funding. Kischel:Micromet Inc.: Employment, Equity Ownership. Kufer:Micromet Inc.: Employment, Equity Ownership. Baeuerle:Micromet Inc.: Employment, Equity Ownership. Mackensen:Micromet. Inc.: Research Funding. Krause:Micromet Inc.: Research Funding.

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