scholarly journals Optimizing Ex-Vivo Expanded NK Cell- Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Combined with NKTR-255 in Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), and Burkitt Lymphoma (BL)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-24
Author(s):  
Yaya Chu ◽  
Susiyan Jiang ◽  
Jian Jiang ◽  
Meijuan Tian ◽  
Dean Anthony Lee ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Type I ◽  
Type Ii ◽  
Advisory Committees ◽  
Anti Cd20

Background: The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and expand natural killer (NK) cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p<0.001), PGA-1 (E:T=3:1, p<0.001), and DOHH2 (E:T=3:1, p<0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p<0.05; perforin: p<0.001), PGA-1(granzyme: p<0.05; perforin: p<0.05), DOHH2 (granzyme: p<0.05; perforin: p<0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups (Fig.1B). NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells in vitro with enhanced IFN-g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

scholarly journals FT596: Translation of First-of-Kind Multi-Antigen Targeted Off-the-Shelf CAR-NK Cell with Engineered Persistence for the Treatment of B Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 301-301 ◽  
Author(s):  
Jode P Goodridge ◽  
Sajid Mahmood ◽  
Huang Zhu ◽  
Svetlana Gaidarova ◽  
Robert Blum ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Advisory Committees

Induced pluripotent stem cell (iPSC)-derived effector cells offer distinct advantages for immune therapy over existing patient- or donor- derived platforms, both in terms of scalable manufacturing from a renewable starting cellular material and precision genetic engineering that is performed at the single-cell level. iPSC derived natural killer (iNK) cells offer the further advantage of innate reactivity to stress ligands and MHC downregulation and the potential to recruit downstream adaptive responses. These unique features form the basis of our multi-antigen targeted chimeric antigen receptor (CAR) CAR-iNK cell product candidate, termed FT596, which is further combined with additional functionality to enhance effector function. FT596 is consistently manufactured from a master iPSC line engineered to uniformly express an NK cell-calibrated CD19-targeting CAR (CD19-CAR), an enhanced functioning high-affinity, non-cleavable CD16 (hnCD16) and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) for cytokine-autonomous persistence. The design of the CD19-CAR involved exploiting the intrinsic polyfunctionality of NK cells, which function by engaging multiple signaling pathways activated through combinations of distinct germline encoded receptors. Using this approach, the transmembrane region of activating receptor NKG2D, combined with the intracellular signaling domains of SLAM co-receptor 2B4 and CD3ζ, proved the most effective in triggering antigen specific functional responses in NK cells. Chimerization of an anti-CD19 scFv onto this NKG2D-2B4-CD3ζ signaling platform produced specific in vitro recognition of CD19+ B cell lymphoma cells in short-term and long-term NK cytotoxicity assays (>80% and <40% clearance of tumor cells at 60H, p<0.001 respectively). The functionality of the CD19-CAR was further enhanced in combination with autonomous IL-15 signaling. Introduction of the IL-15RF enabled expansion of iNK cells without addition of soluble cytokine and greatly improved longevity and functional persistence of iNK cells both in vitro and in animal models. Moreover, iNK cells modified with IL-15RF showed enhanced functional maturation, including upregulated expression of effector molecules such as granzyme B. iNK cells with both CD19-CAR and IL-15RF resulted in enhanced CAR functionality in vitro, and mouse models for B cell malignancy demonstrated that treatment with iNK cells engineered with CD19-CAR and IL-15RF were curative against B cell lymphoma (p<0.002), when compared with iNK cells alone or iNK cells modified with CD19-CAR alone. In combination with hnCD16, co-expression of CD19-CAR and IL15-RF culminates in iNK cells capable of dual-specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. In long term killing assays, FT596 alone demonstrated equivalent levels of CD19 targeted anti-tumor activity as primary CD19-targeted CAR (CAR19) T cells when tested against CD19+ CD20+ B lymphoblast target cells and demonstrated enhanced levels of activity when used in combination with anti-CD20 (rituximab). When targeting CD19- CD20+ B lymphoblast target cells and used in combination with rituximab, only FT596 was able to effectively eliminate the CD19 antigen escaped target cell (64% vs 30% clearance of tumor cells at 36H vs rituximab alone). In vivo FT596 showed equivalent levels of tumor cell clearance as primary CAR19 T cells against the CD19+ acute lymphoblastic leukemia cell line NALM6 and CD19+CD20+ Burkitts lymphoma cell line RAJI, and enhanced clearance of RAJI tumor cells in combination with rituximab (p=0.0002). Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab versus RAJI tumor cells. Together, these studies demonstrate FT596 provides a multi-antigen targeting, potent and persistent engineered immune cell that is derived from a master iPSC line which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform and supports the rational for a first-of-kind Phase I Study as a monotherapy and in combination with CD20-targeted mAbs including rituximab in subjects with relapsed/refractory B-cell lymphoma and leukemia. Figure Disclosures Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

scholarly journals CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 477-477
Author(s):  
Shih-Shih Chen ◽  
Constantine S. Tam ◽  
Alan G. Ramsay ◽  
Priyadarshini Ravichandran ◽  
Natalia C. Couto-Francisco ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Subset ◽  
Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Extended Survival in CLL Patients Who Discontinued Idelalisib Because of Colitis Correlates with Higher Th17 Levels in Blood and Colon Tissue

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1848-1848
Author(s):  
Rukhsana Aslam ◽  
Jacqueline C Barrientos ◽  
Gerardo Ferrer ◽  
Pui Yan Chiu ◽  
Kanti R Rai ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Th17 Cells ◽  
Research Funding ◽  
Treg Cells ◽  
Drug Discontinuation ◽  
Advisory Committees ◽  
Extended Survival

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. Despite significant clinical and molecular advances, CLL remains an incurable disease. In CLL there is ongoing communication between leukemic B cells and non-leukemic cells in the tissue microenvironment. Idelalisib, a PI3Kδ inhibitor, is efficacious in CLL although side effects like inflammatory reactions in different tissues (e.g., colitis, transaminitis and pneumonitis) and progression of disease can lead to discontinuation of the drug. We previously presented an intriguing finding that a cohort of patients who discontinued idelalisib therapy due to colitis had extended survival compared to other patients who discontinued drug but did not have colitis. Of note, the colon tissues from patients with ulcerative colitis (UC) often contain elevated levels of Th17 cells and decreased levels of Treg (Foxp3+) cells. Moreover, Th17 cells from humans and mice have been shown to promote autoimmune B-cell maturation. In this regard, we have previously published that circulating Th17 levels are elevated in CLL patients and Th17 numbers correlate with extended survival. For these reasons, we have now quantified the numbers of Th17 and Treg cells in the blood and colon tissues of a cohort of CLL patients with drug-associated colitis and extended survival and have investigated the effects of in vitro exposure to idelalisib on Th17 generation. Circulating Th17A levels were quantified in a cohort of patients (n=11) before starting idelalisib-treatment and at the time the drug was discontinued due to colitis by flow cytometric analysis of intracellular IL-17A in CD4+ T cells. In addition, colon tissues from those CLL patients who developed colitis during idelalisib treatment taken at the time of drug discontinuation and patients with UC were examined to quantify the infiltration of T (CD3+) cells, Th17 (IL17A+) cells and Treg (Foxp3+) cells by immunohistochemistry (IHC). As negative control tissues (CT) for these studies we used colectomy samples from patients with inactive diverticulosis. FACS analysis of peripheral blood mononuclear cells from the CLL patients treated with idelalisib and having extended survival showed significantly higher circulating Th17A levels (P < 0.01) at the time of drug discontinuation compared to pre-treatment levels. In addition, IHC analyses on tissues from a subset of these same patients indicated that the ratio of % IL-17A+ to CD3+ cells was significantly higher in CLL than in CT (P = 0.0002). It was also significantly higher in CLL than UC (P = 0.001), even though the average number of CD3+ cells in UC was higher than in CLL (P = 0.0001). We also determined the ratio of % FoxP3+ to CD3+ cells in the colon tissues of 12 CT, 16 UC and 6 CLL patients from the cohort studied above. This revealed the average percentage of FoxP3+ cells within the total number of infiltrating T cells was significantly higher in CLL than CT (P = <0.0001), but not significantly different from UC patients (P = 0.90). When analyzing solely the CLL samples, there was a reciprocal relationship between Th17A+ cells and FoxP3+ cells, with the former being higher and the latter lower. To examine the effect of idelalisib on Th17-cell generation, naïve CD4+ CLL T cells from 6 patients (3 M-CLL + 3 U-CLL) were activated in vitro by anti-CD3/28 ligation plus IL-2 in the presence or absence of idelalisib. After 7 days, T helper subset profiling was performed by intracellular cytokine staining. Significantly higher percentages of Th17A cells and significantly lower percentages of Th1 cells (IFNg+) (P= 0.006) were found in cultures containing idelalisib. The percentages of Tregs were unchanged in the same cultures. These findings suggest that the higher numbers of Th17s in blood and tissue of patients after idelalisib treatment may reflect the action of idelalisib on Th17 generation. In conclusion, CLL patients who discontinued idelalisib due to colitis and yet had extended survival have increased circulating and tissue-resident Th17 cells and decreased levels of Treg cells. These findings suggest that an immune imbalance between Th17 and Treg cell numbers and function promotes colitis and at the same time favors improved clinical outcome. This is consistent with the known action of Th17 cells enhancing autoimmune B-cell responses. Disclosures Barrientos: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

scholarly journals A High Dose of Naïve CD8+ T-Cells Increases the Incidence of Graft-Versus-Host Disease (GvHD) after Donor Lymphocyte Infusions (DLIs) from HLA Identical Sibling Donors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3292-3292
Author(s):  
Guillermo Ortí ◽  
Carlos Palacio ◽  
Irene García-Cadenas ◽  
Isabel Sánchez-Ortega ◽  
María-José Jimenez ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Mixed Chimerism ◽  
High Dose ◽  
Cellular Composition ◽  
Advisory Committees

Introduction DLIs represent a major therapeutic approach for relapse and mixed chimerism (MC) after allogeneic hematopoietic cell transplant (AlloHCT). DLI studies have identified several variables with impact on response and GvHD. Despite some studies having explored the role of T-cells and other cell subsets, such as mononuclear cells (MNCs), comprehensive data regarding the cellular composition of DLI and its role in GvHD remains incomplete, as the development of GvHD post DLI is often unpredictable. Herein we analyzed the cellular composition of DLI from fully human leukocyte antigen (HLA) identical sibling (HLA Id Sib) donors and its impact on the development of GvHD in patients who underwent AlloHCT for hematological malignancy, and its impact on the development of GvHD. Methods Inclusion criteria were as follows: 1) Patients ≥ 18 years-old, 2) AlloHCT, 3) HLA Id Sib donor; 4) treatment with DLI; and 5) signed informed consent of patient and donor. Exclusion criteria were: 1) unrelated or mismatched related donors, 2) HCT2 prior to DLI, or 3) GvHD at DLI. For the purpose of avoiding bias, only the cell composition of the first DLI (DLI1) was analyzed. The following cell subsets of the DLI were studied: CD3+, CD4+, CD8+, CD16+CD56+CD3+ (NKT-cell), CD3+CD45RA+CCR7+ (TN), CD3+CD45RA+CCR7+CD31+ (TRTE), CD3+CD45RA+CD95+CD27+ (TSCM), CD3+CD45RA-CCR7+ (TCM), CD3+CD45RA-CCR7- (TEM), CD3+CD45RA+CCR7- (TTE), CD3+CD4+CD25brightCD127dim (TREG), CD3+CD4+CD25brightCD45RA+CD127dim (naïve TREG). The TN, TCM, TEM and TEM compartment was analyzed for both CD4+ and CD8+. We also analyzed the MNCs, CD19+ (B-cell), CD27+CD19+ (mature B-cell), CD16+CD56+CD27- (natural killer (NK+) cell) and CD16+CD56+CD27+ (CD27+NK+cell). Results Fifty-six DLIs were infused in 36 patients; the median number of DLI was 1 per patient (range, 1-3). Diagnoses were as follows: 13 AML/MDS, 6 HL, 5 MPN, 4 NHL, 4 CLL, 3 MM and 1 B-ALL. For the study, a landmark analysis was performed from the DLI date. The median follow up from DLI was 282 days (range, 9-5,560 days). Overall response rate in relapsed patients was 29% (9 of 31 patients; 6 CR and 3 PR, most responses being observed after DLI1. Further, five patients had DLI for MC and full donor chimerism was achieved in all patients. Thirteen patients (36%) developed GvHD post DLI. Two patients had GvHD before DLI, but there was no case of GvHD at DLI. The median time interval form DLI to GvHD was 76 days (range, 7-261). As per clinical presentation, 10 patients (27%) had acute GvHD, whereas eight patients (22%) had chronic GvHD. The 6-month and 1-year cumulative incidence (CI) of GvHD was 33% and 46%, respectively. When the risk of GvHD was analyzed according to DLI cell subsets, we observed that a DLI1 containing >3x106 CD8+TN correlated with an increased incidence of GvHD (Figure 1a). Also, a DLI1 with >0.8x108 MNCs/Kg (Figure 1b), >2.6x106 mature B-cell/Kg, or >0.35x106 CD27+NK+cells/Kg were linked to the development of GvHD (Table 1). Noteworthy, CD3+, TN (both CD4+ and CD8+ combined) or CD4+TN had no impact on the development of GvHD; and a high proportion of TREG was not protective for the development of GvHD (Table 2). Finally, there was no statistically significant association between any clinical variable and GvHD. Conclusion In conclusion, in this cohort of AlloHCT patients from HLA Id Sib donors, a DLI1 containing a high proportion of CD8+TN, but not CD4+TN, increased the probability of developing GvHD. Further, a DLI1 containing a high dose of MNCs, CD27+NK+cells and mature B-cell also associated with GvHD. These data provide novel insight for the understanding of GvHD post DLI. A DLI1 containing a lower dose of CD8+TN could reduce the risk of GvHD, but this asset warrants further validation in larger cohorts, and within a controlled randomized trial setting. Disclosures Bosch: F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

scholarly journals Rapid and Robust CD4+ and CD8+ T-, NK-, B- and Monocyte Cell Reconstitution after Nicotinamide-Expanded Cord Blood Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2123-2123
Author(s):  
Jaap Jan Boelens ◽  
Coco de Koning ◽  
Mitchell E. Horwitz ◽  
Guillermo Sanz ◽  
Madan Jagasia ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cord Blood ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Monitoring ◽  
Cell Recovery ◽  
Advisory Committees

Abstract Introduction Nicotinamide-expanded cord blood (NiCord) is a promising alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-matched donor is unavailable. A phase 1/2 trial with standalone NiCord HCT showed rapid neutrophil engraftment (median 11 days) and platelet engraftment (median 34 days). However, successful CD4+ immune reconstitution (IR) has shown to be crucial for infectious and relapse control associated with favorable survival (Admiraal JACI 2017). We performed unique in-depth immune monitoring to evaluate and compare the recovery of immune subsets after NiCord and conventional HCT. Methods In the phase1/2 multicenter trial, patients (n=36) with hematologic malignancies received NiCord-HCT after myeloablative (MA) conditioning without antithymocyte globulin (ATG). Immune monitoring was performed (harmonized sampling, handling and analyses in a central lab) in a random subgroup. The primary endpoint was probability of achieving CD4+ IR (>50*106/L within 100 days). Secondary endpoints were subset recovery over time of B-cells, CD4+ and CD8+ T-cells, natural killer (NK), monocytes, and dendritic cells (DC), during 7-365 days after HCT. In addition, TREC analyses are pending and will be available at the meeting. Data were compared with IR in cohorts of adolescent and young adult (AYA) patients at the UMC Utrecht receiving either unmanipulated cord blood transplantation (unCBT) or T-repleted unrelated bone marrow transplantation (BMT) for hematological malignancy after MA conditioning without ATG. Linear-mixed effects modelling in LOESS-regression curves and two-sided log-rank test for univariate comparisons in cumulative incidence plots were used. Results 24 NiCord recipients (median 41.5; 13.4-61.7 yrs) had blood samples available for in-depth early immune monitoring. NiCord cell dose consisted of median 6.4*106 CD34+/kg, and 2.3*106 CD3+T-cells/kg of the co-infused negative fraction (following CD133+-selection). 91% of patients achieved successful early CD4+ IR after NiCord (Fig 1). When comparing the NiCord with 27 unCBT (median age 15.4; range 12.2-22.1 yrs) and 20 BMT (median age 14.3; range 12.1-19.7 yrs), no difference in probability of early CD4+ IR was noted (p=0.76: Fig 1). Overall T-cell reconstitution was similar; CD3+ (p=0.99), CD4+ (p=0.71), CD8+ (p=0.08), although effector and central memory CD4+ and CD8+ T-cells, Tregs, gamma-delta T-cells, Th2, and Th17 recovered somewhat faster after NiCord. Recovery of conventional- (p=0.41) and plasmacytoid DCs (p=0.52) was similar as well. Overall reconstitution of NK-cells (p<0.001); especially naïve NK-cells, monocytes (p<0.001); mostly classical, and B-cells (p=0.026) was faster after HCT with NiCord, compared to unCBT and BMT cohorts (Fig 2). In B-cell recovery, strikingly faster early recovery of follicular B-cells (p=0.04), memory B-cells (p=0.003), and plasma cells (p=0.003) was observed in NiCord recipients. Conclusions In-depth immune monitoring reveals rapid and robust immune reconstitution in NiCord recipients, with high early CD4+ IR probability, and comparable recovery of T-cell-, NK-cell-, monocyte-, and DC-subsets to AYA controls receiving unCBT and BMT. Interestingly, B-cell recovery consisted of markedly higher follicular B-cell, memory B-cell, and plasma cell levels in NiCord recipients. Next to higher B-cell recovery, monocyte and NK-cells also recovered faster after NiCord transplantation, despite the younger age of the AYA cohort (expected to reconstituted faster). This may be explained by the higher stem cell dose and higher proliferative capacity of the NiCord- expanded product. Optimal comparison of IR in NiCord vs. unCBT in a randomized phase 3 trial is underway. Disclosures Horwitz: Gamida Cell: Research Funding. Jagasia:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding. Cilloni:Janssen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees. Nierkens:Gamida: Research Funding.

Expression of the Tigit/CD226/CD155 Receptors/Ligand System in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5454-5454 ◽  
Author(s):  
Francesca Arruga ◽  
Giulia Guerra ◽  
Denis Baev ◽  
Catherine Hoofd ◽  
Marta Coscia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees

Introduction: T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a surface receptor mainly expressed by CD8+, regulatory T lymphocytes and natural killer (NK) cells, but not by normal B cells. It performs as an inhibitory immune checkpoint, activated through binding of CD155. TIGIT competes with CD226 for CD155 binding, resulting in opposite outcomes: while CD226 enhances cytotoxicity of T lymphocytes and NK cells, TIGIT exerts immunosuppressive effects. Whether TIGIT engagement triggers an alternative signaling cascade, or whether it simply prevents CD226 activation, remains an open point. Tumor-infiltrating T lymphocytes generally express high levels of the molecule, together with the other checkpoint inhibitor PD-1. On this basis, antagonist antibodies targeting TIGIT are under evaluation to restore immunity and treat cancer patients, alone or in various combinations. Chronic lymphocytic leukemia (CLL), the most common adult leukemia, is characterized by a highly heterogeneous clinical outcome. Several molecular markers can help in stratifying patients, including the presence or absence of somatic mutations in B cell receptor, cytogenetic aberrations and single gene mutations. Interestingly, CLL cells express several T cell specific antigens, including CD5. A previous report indicates that, in CLL, TIGIT is expressed by circulating CD4+T cells, increasing during disease progression, while nothing is known about its expression on CLL cells. Aim:This work was undertaken with the aim of studying expression of the TIGIT/CD226/CD155 axis in CLL. Methods:We assembled a cohort of 101 primary CLL samples (40% females, mean age of 61). All patients were either untreated or had not received treatment in the 6 months prior to analysis. PBMC samples were tested for expression of TIGIT, CD155 and CD226 in both T and B subsets. A multiparametric flow cytometry strategy was designed, combining anti-TIGIT, anti-CD155 and anti-CD226 antibodies with a panel of B- (anti-CD19, anti-CD5, anti-CD38, anti-CD49d and anti-CD73) and T-mono/NK specific (anti-CD3, anti-CD8, anti-CD4, anti-CD14 and anti-CD56) markers. The number of TIGIT molecules on leukemic cells was estimated by interpolating values of mean fluorescence intensity (MFI) of each sample with that of PE-Quantibrite beads. Results:CLL cells heterogeneously express surface TIGIT, ranging from 0.2 to 81% (mean value 20%, median 10%, SEM ±2.145). The estimated number of molecules per cell was in the range of 32.5-3571 (mean 1140, median 841.1, SEM ±83.6). Expression of TIGIT was independent of gender or age at diagnosis and there was no correlation between TIGIT levels and lymphocyte counts in peripheral blood. In contrast, in this cohort of untreated patients, we observed a significantly lower TIGIT expression in samples with advanced disease (RAI III-IV) compared to early stages (RAI 0-I). Accordingly, low TIGIT associated with unmutated (UM) IGHVgenes and with an unfavorable FISH profile (trisomy 12, deletion 17 and deletion 11 vs. deletion 13 or normal karyotype). Lower, although not significant, TIGIT levels were observed in NOTCH1-mutated CLL samples (n=11) compared to counterpart (n=89). Looking at the T cell population, we observed overall higher TIGIT levels in the CD8+vs CD4+subset (mean %TIGIT+cells in CD8+56.7±1.8 vs 27.2±1.3 in CD4+). In line with reported observations, we found a modest but significant increase of TIGIT+T cells in advanced stage CLLs, at variance with what observed on the leukemic B cell side. Accordingly, we observed higher percentages of TIGIT+/CD4+cells in CLL samples carrying UM IGHVgenes. CD226 and CD155 were more homogeneously expressed in all subsets without significant differences, both in CLL and T cell components. Conclusions: This work shows that CLL cells express the immunomodulatory molecule TIGIT, particularly in the early stages of the disease in untreated patients. While further studies are needed to characterize its functional implications as well as treatment effect on TIGIT expression, it is tempting to speculate that TIGIT expression by CLL cells may serve to trigger an immunosuppressive behavior in these cells, which is no longer needed when the disease becomes advanced. This observation represents a starting point for future studies investigating the role of TIGIT in CLL and hints to a possible use of anti-TIGIT antibodies to target different cellular components of the disease. Disclosures Hoofd: iTeos Therapeutics: Employment. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Research Funding. Gaidano:Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Furman:Acerta Pharma: Consultancy; Beigene: Consultancy; Incyte: Consultancy; Janssen: Consultancy; Oncotracker: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Deaglio:VelosBio Inc.: Research Funding; Verastem Inc: Research Funding; iTeos Therapeutics: Research Funding.

Phase I/Ib Study of the Novel Immunotoxin MT-3724 in Relapsed/Refractory Non-Hodgkin's B-Cell Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5112-5112
Author(s):  
Paul A Hamlin ◽  
Catherine S. Diefenbach ◽  
David J. Valacer ◽  
Jack Higgins ◽  
Michelle A. Fanale
Keyword(s):  
B Cells ◽  
Phase I ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Dose Cohort ◽  
Mixed Response

Abstract Background CD20 is selectively expressed on the surface of early pre-B-cells, remains throughout B-cell development, and is then lost from plasma cells. Because CD20 is present on the majority of B-cell lymphomas, anti-CD20 monoclonal antibody (MAb) therapy is widely employed in the treatment of NHL. However a majority of NHL patients eventually become refractory to CD20 MAb(s). Resistance mechanisms may include increased MAb catabolism, initial or post treatment selection of low CD20 expressing tumor cells, trogocytosis of surface CD20, failure of MAb effector mechanisms and/or impaired patient immune cell function. MT-3724 is a recombinant fusion protein consisting of a CD20 binding variable fragment (scFv) fused to the enzymatically active Shiga-like toxin-I A1 subunit (SLT-I A1). SLT-I A1 is an N-glycosidase that catalytically inactivates 60S ribosomal subunits causing inhibition of protein synthesis. Upon its scFv binding to cell surface CD20 in vitro, SLT-I A1 forces MT-3724 internalization which then routes in a predictable fashion to the cytosol and irreversibly inactivates the cell ribosomes triggering cell death. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro and non-human primate (NHP) B-cells in vivo. MT-3724 was tested for safety in healthy NHPs: 6 intravenous (IV) doses of MT-3724 were given over 12 days at doses of 50, 150, and 450 mcg/kg. There were no deaths or effects on serum chemistries in the NHP studies. The major observed toxicity (inappetence) resolved within 48 hours of last dose. There was a significant, dose-dependent NHP B-cell depletion by Day 3 at all doses. Given the preclinical activity and mechanism of action, a Phase I/Ib study of MT-3724 was initiated in NHL. Methods MT-3724 is being tested for safety and tolerability in a first-in-human, open label, ascending dose study (3 + 3 design) in sequential cohorts of 5, 10, 20 and 50 mcg/kg/dose. Eligible subjects who previously responded to a CD20 MAb containing therapy followed by relapse/recurrence of NHL receive 6 doses by 2 hour IV infusions over the first 12 days of a 28 day cycle (first cycle). With continued safety, tolerability and lack of tumor progression, subjects may receive up to 4 additional 6-dose cycles (21 days) with tumor assessments after cycles 2, 4 and 5. Dose escalation is based on < 33% dose limiting toxicities (DLTs) observed during the first 28 day cycle. Results Three NHL subjects (2 transformed DLBCL, 1 FL) have completed at least one cycle in the 5 mcg/kg/dose cohort with no protocol DLTs or infusion related reactions and are evaluable for safety. Non-DLTs included grade (Gr) 2-3 transient hyperglycemic episodes related to pre-infusion corticosteroid therapy (n=1); transient Gr 4 neutropenia, possibly related to MT-3724 during cycle 1, week 4 (n=1); Gr 4 hypercalcemia and acute kidney injury with Gr 3 hypophosphatemia during cycle 1, week 4 due to leukemic disease progression (n=1). Subject 1 completed 5 cycles of therapy, with a partial response achieved post cycle 2 sustained through cycle 5; Subject 3 had a mixed response (both subjects had transformed DLBCL). Three subjects have now initiated treatment in the 10 mcg/kg/dose cohort with updated data to be presented at the meeting. Conclusions MT-3724 at 5 mcg/kg/dose has been safely administered for up to 5 cycles in this first-in-human study in relapsed/refractory NHL subjects. Treatment with the 10 mcg/kg cohort has commenced with continuing dose ascension planned. There is early evidence of clinical activity. Disclosures Diefenbach: Gilead: Equity Ownership, Research Funding, Speakers Bureau; Jannsen Oncology: Consultancy; Idera: Consultancy; Immunogen: Consultancy; Incyte: Research Funding; Genentech: Research Funding; Celgene: Consultancy; Molecular Templates: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding. Valacer:Molecular Templates: Employment. Higgins:Molecular Templates: Employment. Fanale:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Research Funding; Infinity: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Research Funding; Genentech: Research Funding; Medimmune: Research Funding; Novartis: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Molecular Templates: Research Funding; ADC Therapeutics: Research Funding; Onyx: Research Funding; Gilead: Research Funding.

scholarly journals ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1204-1204
Author(s):  
Bin Cai ◽  
Aaron N Nguyen ◽  
Songmao Zheng ◽  
Jianfeng Shi ◽  
Guizhong Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cytokine Release ◽  
Publicly Traded ◽  
Advisory Committees ◽  
The Past

Abstract Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented. ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells. In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro. ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

scholarly journals 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 ◽  
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.

scholarly journals Clinical Predictors of T Cell Fitness for CAR T Cell Manufacturing and Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1886-1886 ◽  
Author(s):  
Ehren Dancy ◽  
Alfred L. Garfall ◽  
Adam D. Cohen ◽  
Joseph A Fraietta ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Expansion ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

Abstract Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p<0.0001, mid figure). Magnitude of in vitro T cell expansion during manufacturing (measured as population doublings by day 9, or PDL9), which correlated with response to CART19 in CLL, was higher in post-ind vs rel/ref cohort (median PDL9 5.3 vs 4.5, p=0.0008, right figure). Pooling data from both cohorts, PDL9 correlated with both early-mem T cell percentage (Spearman's rho 0.38, multiplicity adjusted p=0.01) and CD4/CD8 ratio (Spearman's rho 0.42, multiplicity adjusted p=0.005). Within the post-ind cohort, there was no significant association between early-mem T cell percentage and time since MM diagnosis, duration of therapy, exposure to specific therapies (including cyclophosphamide, bortezomib, or lenalidomide), or bone marrow plasma cell content at time of apheresis. However, in the post-ind cohort, there was a trend of toward lower percentage early-mem phenotype (29% vs 49%, p=0.07) and lower CD4/CD8 ratio (median 1.4 vs 2.7, p=0.04) among patients who required >2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.

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