scholarly journals Donor-Derived Adoptive T-Cell Therapy Targeting Multiple Tumor Associated Antigens to Prevent Post-Transplant Relapse in Patients with ALL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 471-471
Author(s):  
Swati Naik ◽  
Spyridoula Vasileiou ◽  
Ifigeneia Tzannou ◽  
Manik Kuvalekar ◽  
Ayumi Watanabe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Management Support ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Post Infusion ◽  
T Cell Lines

Abstract Background: Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk Acute Lymphoblastic Leukemia (HR-ALL), but relapse remains a major cause of treatment failure. Strategies to enhance the graft-versus-leukemia (GVL) effect have been employed to prevent relapse, including modulating immune suppression post-HSCT to hasten immune reconstitution or with the use of donor lymphocyte infusions (DLIs). However, DLIs carry a significant risk of graft-versus-host disease (GVHD) due to the concurrent transfer of alloreactive T cells. To enhance the GVL effect while minimizing GVHD, we developed a protocol for the generation of ex vivo expanded, donor-derived T-cell lines targeting PRAME, WT1 and Survivin - tumor associated antigens that are frequently expressed in both B- and T-cell ALL. These multi-antigen-targeted T cells (multiTAAs) were adoptively transferred to pediatric and adult patients with HR-ALL who had undergone an allogeneic HSCT. Methods: Donor-derived multiTAA-specific T cells were generated by co-culturing PBMCs with autologous DCs loaded with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. Following 2-4 rounds of stimulation these multiTAA-specific T cells were infused to patients with ALL who had undergone an HSCT but remained at a high risk for disease relapse. Results: We have generated 15 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 95.1±1.9%) with a mixture of CD4+ (mean 22.8±6.3%) and CD8+ (mean 52.5±5.3%) cells, which expressed central [CD45RO+/CD62L+: 13.5±2.8%] and effector memory markers [CD45RO+/CD62L-: 56.4±3.8%]. The expanded lines recognized the targeted antigens PRAME (range 0-370 SFC/2x10 5), WT1 (0-363 SFC/2x10 5), and Survivin (0-65 SFC/2x10 5) in an IFNg ELIspot. None of the lines reacted against non-malignant patient-derived cells (3.7±0.8% specific lysis; E: T 20:1) - a study release criterion indicating lack of alloreactivity. We have infused 11 HR-ALL patients (8 pediatric and 3 adult) with donor-derived multiTAA-specific T cells to prevent disease relapse (Table 1). Patients were administered with up to 4 infusions of cells at 3 escalating dose levels, ranging from 0.5 - 2x10 7 cells/m 2. Infusions were well tolerated with no dose-limiting toxicity, GVHD, cytokine release syndrome or other adverse events. Three patients were not evaluable per study criteria as they received >0.5mg/kg of steroids (2 patients received stress doses for septic shock and 1 for elevated liver enzymes presumed to be GVHD that was later ruled out) within 4 weeks of infusion and were replaced. Six of the 8 remaining patients infused remain in CR on long-term follow up at a median of 46.5 months post-infusion (range 9-51 months). In patients who remained in long term CR we detected an expansion of tumor-reactive T cells in their peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading, which correlated temporally (within 4 weeks) with multiTAA infusions. By contrast in the two patients who relapsed we saw no evidence of in vivo T cell amplification within the first 4 weeks after infusion. Conclusion: The preparation and infusion of donor-derived multiTAA-specific T cells to patients with B- and T-ALL post allogeneic HSCT is feasible, safe and as evidenced by in vivo tumor-directed T cell expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Figure 1 Figure 1. Disclosures Vasileiou: Allovir: Consultancy. Tzannou: Gileas: Honoraria; Allovir: Current equity holder in publicly-traded company. Kuvalekar: Allovir: Consultancy. Watanabe: Allovir: Consultancy. Grilley: QB Regulatory Consulting: Other: Ownership, project management support, Research Funding; Marker: Consultancy, Other: Regulatory and project management support; Allovir: Current equity holder in publicly-traded company, Other: Leadership. Hill: Incyte: Membership on an entity's Board of Directors or advisory committees. Omer: Allovir: Research Funding. Gottschalk: Tessa Therapeutics: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Tidal: Consultancy; Novartis: Consultancy; Catamaran Bio: Consultancy. Heslop: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Kuur Therapeutics: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company; Fresh Wind Biotherapies: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Rooney: Allogene: Patents & Royalties; Bellicum: Patents & Royalties; Bluebird: Current equity holder in publicly-traded company; Allovir: Current equity holder in publicly-traded company; Alimera: Consultancy; Memgen: Consultancy; TScan Therapeutics: Consultancy; Takeda: Patents & Royalties; Marker: Current equity holder in publicly-traded company; Tessa: Consultancy, Other: Leadership, Research Funding. Vera: Allovir: Consultancy, Current equity holder in publicly-traded company, Other: Leadership, travel , accomodations, expenses, Patents & Royalties; Marker: Current Employment, Other: Travel, Accomodations, Expenses, Patents & Royalties, Research Funding. Leen: Allovir: Consultancy, Current equity holder in publicly-traded company; Marker: Current equity holder in publicly-traded company.

scholarly journals Adoptive T-Cell Therapy for Acute Lymphoblastic Leukemia Targeting Multiple Tumor Associated Antigens

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2693-2693
Author(s):  
Swati Naik ◽  
Premal Lulla ◽  
Ifigeneia Tzannou ◽  
Robert A. Krance ◽  
George Carrum ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Disease Relapse ◽  
Advisory Committees ◽  
Leukemic Relapse ◽  
Post Infusion ◽  
Equity Ownership ◽  
T Cell Lines

Abstract Background: Leukemic relapse remains the major cause of treatment failure in hematopoietic stem cell transplant (HSCT) recipients. While the infusion of donor lymphocytes to prevent and treat relapse has been clinically implemented this strategy does not provide durable remissions and carries the risk of life-threatening graft-versus-host disease (GVHD). More recently the adoptive transfer of T cells that have been engineered to express CD19-targeted chimeric antigen receptors (CARs), has shown potent anti-leukemic activity in HSCT recipients with recurrent disease. However, disease relapse with the emergence of CD19 negative tumors is an emerging clinical issue post-administration of these mono-targeted T cells. To overcome these limitations, we developed a protocol for the generation of donor-derived T cell lines that simultaneously targeted a range of tumor associated antigens (multiTAAs) that are frequently expressed by B- and T-cell ALL including PRAME, WT1 and Survivin for adoptive transfer to high risk recipients transplanted for ALL. Methods/Results: We were consistently able to generate donor-derived multiTAA-specific T cells by culturing PBMCs in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail, using autologous DCs as APCs and loading them with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens. The use of whole antigen increases the range of patient HLA polymorphisms that can be exploited beyond those matched to single peptides, while targeting multiple antigens simultaneously reduces the risk of tumor immune evasion. To date, we have generated 14 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 94±9%) with a mixture of CD4+ (mean 21±28%) and CD8+ (mean 52±24 %) cells, which expressed central [CD45RO+/CD62L+: 14±9%] and effector memory markers [CD45RO+/CD62L-: 80±11%] associated with long term in vivo persistence. The expanded lines recognized the targeted antigens WT1, PRAME and Survivin by IFNg ELIspot with activity against >1 targeted antigens in all cases. None of the lines reacted against non-malignant patient-derived cells (4±3% specific lysis; E: T 20:1) - a study release criterion. Thus far we have treated 8 high risk ALL patients with donor derived TAA T cells post-transplant to prevent disease relapse (Table 1). Infusions were well tolerated with no dose-limiting toxicity, GVHD, CRS or other adverse events. Two patients were not evaluable per study criteria as they received >0.5mg/kg of steroids within 4 weeks of infusion and were replaced. Five of the 6 remaining patients infused remain in CR a median of 11.2 months post-infusion (range 9-22 months). We detected the expansion of tumor-reactive T cells in patient peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading. The single patient who relapsed showed no evidence of tumor-directed T cell expansion despite receiving 3 additional infusions at 4 week intervals. Conclusion: In summary, infusion of donor multi-TAA-specific T cells to patients with ALL post allogeneic HSCT is feasible, safe and as evidenced by expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Table 1. Table 1. Disclosures Vera: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Cytosen: Membership on an entity's Board of Directors or advisory committees; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Viracyte: Equity Ownership. Leen:Marker: Equity Ownership.

scholarly journals Targeting Lymphomas Using Non-Engineered, Multi-Antigen Specific T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1685-1685 ◽  
Author(s):  
George Carrum ◽  
Premal Lulla ◽  
Ifigeneia Tzannou ◽  
Ayumi Watanabe ◽  
Manik Kuvalekar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Post Infusion ◽  
Equity Ownership ◽  
T Cell Lines ◽  
Large B Cell

Abstract Immunotherapy is emerging as a potent therapy for a range of hematologic malignancies including lymphomas. Indeed adoptive transfer of T cells genetically engineered to express the CD19 chimeric antigen receptor (CAR) has now received FDA approval for the treatment of patients with refractory diffuse large B cell lymphomas (DLBCL). We have developed a non-engineered T cell-based therapy to treat patients with all types of lymphomas: Hodgkin's (HL) and non-Hodgkin's lymphoma (NHL). The approach uses single T cell lines that simultaneously target a range of tumor-associated antigens (TAAs) that are frequently expressed by these tumors, including PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin. We can consistently prepare these lines by culturing PBMCs in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail, and adding autologous DCs as APCs that are loaded with pepmixes (15mer peptides overlapping by 11 amino-acids) spanning all 5 target antigens. The use of whole antigen should remove the HLA restriction imposed by the use of transgenic TCRs specific for single peptides, while targeting multiple antigens simultaneously would reduce the risk of tumor immune evasion. We have generated 42 clinical-grade multiTAA-specific T cell lines, comprising CD3+ T cells (mean 98±1.1%) with a mixture of CD4+ (mean 48±4.3%) and CD8+ (mean 37±4%) T cells, which expressed central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- mean 14±3%; CD45RO+/CD62L+/CCR7- -- 10±2.2%; CD45RO+/CD62L-/CCR7- -- 28.3±3.6%) (n=42). The expanded lines recognized the targeted antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin (range 0-463, 0-496, 0-330, 0-379 and 0-304 spot forming units (SFU)/2x105 input cells, respectively in IFNg ELIspot, n=34). None of the lines reacted against non-malignant autologous recipient cells (3±3.8% specific lysis; E:T 20:1). We have treated 33 patients: 13 with HL, 17 with aggressive NHL (diffuse large B-cell, mantle cell, or T cell lymphomas) and 3 with indolent NHLs (FL and marginal zone lymphoma). Patients received 0.5-2x107 multiTAA-T cells/m2. Of 18 patients who were infused as adjuvant therapy all but 2 remain in remission (range 3-42 months post-infusion). Fifteen patients have received multiTAA-specific T cells to treat active disease, all of whom had failed a median of 4 lines of prior therapy. Of these, 5 had transient disease stabilization followed by disease progression, 4 have ongoing stable disease, 3-18 months post-multiTAA-specific T cells while the remaining 6 (3 with HL and 3 with DLBCL) have all had complete and durable responses ( 4 to 41 months), as assessed by PET imaging. These clinical responses correlated with the detection of tumor-reactive T cells in patient peripheral blood post-infusion directed against both targeted antigens as well as non-targeted TAAs including MAGEA2B and MAGE C1, indicating induction of antigen/epitope spreading. Notably, no patient, including the complete responders, had infusion-related systemic- or neuro-toxicity. Thus, infusion of autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin has been safe and provided durable clinical benefit to patients with lymphomas. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Cell Medica: Research Funding; Tessa Therapeutics: Research Funding; Viracyte: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Cytosen: Membership on an entity's Board of Directors or advisory committees. Rooney:Marker: Equity Ownership. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

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

scholarly journals Gabarap Loss Mediates Immune Escape in High Risk Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 891-891
Author(s):  
Annamaria Gulla ◽  
Eugenio Morelli ◽  
Mehmet K. Samur ◽  
Cirino Botta ◽  
Megan Johnstone ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Clinical Outcome ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Escape ◽  
Publicly Traded ◽  
Advisory Committees

Abstract Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Clinical Efficacy of Anti-CD22 Chimeric Antigen Receptor T Cells for B-Cell Acute Lymphoblastic Leukemia Is Correlated with the Length of the Scfv Linker and Can be Predicted Using Xenograft Models

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 807-807
Author(s):  
Marco Ruella ◽  
Shannon L Maude ◽  
Boris Engels ◽  
David M. Barrett ◽  
Noelle Frey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Xenograft Models

Abstract Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19 or CTL019) have shown impressive clinical activity in B-cell acute lymphoblastic leukemia (B-ALL) and are poised to receive FDA approval. However, some patients relapse after losing CD19 expression. Since CD22 remains highly expressed in relapsed/refractory (r/r) B-ALL even in these patients, anti-CD22 CART (CART22) have been developed. The National Cancer Institute (NCI) reported 4/9 complete remission (CR) in patients receiving CART22, with 100% CR at the highest T cell dose (NCT02315612)(S hah NN, ASH 2016 #650). Patients and Methods. We generated a second-generation CAR22 differing from that used by the NCI only by the use of a longer linker [4x(GGGGS); LL vs. 1x(GGGGS); SL] between the light and heavy chains of the scFv (Fig. 1 A). This construct was tested in two pilot clinical trials in adults (NCT02588456)and children with r/r-ALL (NCT02650414). CART22 cells were generated using lentiviral transduction as in our previous studies. The protocol-specified CART22 dose was 2x106-1x107 cells/kg for pediatric patients <50kg and 1-5x108 for pediatric patients ≥50kg and adult patients,. infused after lymphodepleting chemotherapy. Patient characteristics are described in Table 1. For the adult trial, 5 patients were screened, 4 enrolled (1 patient withdrew consent) and 3 infused (1 manufacturing failure). For the pediatric trial, 9 patients were screened, 8 enrolled (1 screen failure) and 6 infused (two patients were not infused for disease progression). For the preclinical studies, we generated CART22LL and CART22SL and tested them in vivo using xenograft models. NOD-SCID gamma chain deficient (NSG) mice were engrafted with either a luciferase+ standard B-ALL cell line (NALM6) or primary B-ALL cells obtained from a patient relapsing after CART19 (CHP110R). We also used 2-photon imaging to study the in vivo behavior and immune synapse formation and flow cytometry to asses T cell activation. Results. CART22 cells were successfully manufactured for 10/12 patients. In the adult cohort 3/3 patients developed CRS (gr.1-3) and no neurotoxicity was observed; in the pediatric cohort out of 5 evaluable patients (1 discontinued for lineage switch to AML on pre-infusion marrow), 3/5 developed cytokine-release syndrome (CRS) (all grade 2) and 1 patient had encephalopathy (gr.1). CART22 cells expanded in the PB with median peak of 1977 (18-40314) copies/ug DNA at day 11-18. Interestingly, in an adult patient who had previously received CART19 a second CART19 re-expansion was observed following CART22 expansion (Fig 1 B). At day 28, in the adult cohort the patient who was infused in morphologic CR remained in CR, while the other 2 had no response (NR); in the pediatric cohort 2/5 patients were in CR, 1 in partial remission (PR) that then converted to CR with incomplete recovery at 2 months, and 2 NR. No CD22-negative leukemia progression was observed. Since our results with a long linker appeared inferior compared to the previously reported CART22 trial (short linker), we performed a direct comparison of the 2 different CAR22 constructs. In xenograft models, CART22SL significantly outperformed CART22LL (Fi 1 C) with improved overall survival. Moreover, CART22SL showed higher in vivo proliferation at day 17 (Fig 1 D). Mechanistically, intravital 2-photon imaging showed that CART22SL established more protracted T cell:leukemia interactions than did CART22LL, suggesting the establishment of productive synapses (Fig 1 E). Moreover, in vivo at 24 hrs higher T cell activation (CD69, PD-1) was observed in CART22SL from the BM of NALM-6-bearing mice. Conclusions. Here we report the results of two pilot clinical trials evaluating the safety and feasibility of CART22 therapy for r/r B-ALL. Although feasible and with manageable toxicity CART22LL led to modest clinical responses. Preclinical evaluation allowed us to conclude that shortening the linker by 15 amino acids significantly increases the anti-leukemia activity of CART22, possibly by leading to more effective interactions between T cells and their targets. Finally, with the caveats of cross-trial comparison, our data suggest that xenograft models can predict the clinical efficacy of CART products and validate the use of in vivo models for lead candidate selection Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Maude: Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Engels: Novartis: Employment. Frey: Novartis: Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Brogdon: Novartis: Employment. Young: Novartis: Research Funding. Porter: Incyte: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Immunovative Therapies: Other: Member DSMB; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement. June: WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp: Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; University of Pennsylvania: Patents & Royalties; Adaptimmune: Consultancy. Gill: Novartis: Patents & Royalties, Research Funding.

NME-2 Protein Functions as a Tumour Associated Antigen in HLA-A2+ Cells and Is Over Expressed in CML Via a Bcr/Abl Dependent Post Transcriptional Mechanism.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3266-3266
Author(s):  
Sabine Tschiedel ◽  
Melanie Adler ◽  
Karoline Schubert ◽  
Annette Jilo ◽  
Enrica Mueller ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Research Funding ◽  
Kinase Activity ◽  
T Cell Response ◽  
Advisory Committees

Abstract Abstract 3266 Poster Board III-1 Introduction: NmE2 (Nm23-H2, NDP kinase B) is one of a family of proteins that catalyze the transfer of gamma-phosphate between nucleoside-triphosphates and diphosphates. The two major family members, NmE1 and NmE2 are strongly implicated in the control of differentiation, proliferation, migration and apoptosis via interactions which are often independent of their kinase activity, NmE2 being a transcriptional activator of the c-myc gene. We recently identified NmE2 as a tumour associated, HLA-A32+ restricted, antigen in a patient with CML and found the protein (but not the mRNA) to be generally over expressed in CML but not in other haematological malignancies. We also detected a specific T-cell response in peripheral blood cells of a patient 5 years after transplantation. This identifies NmE2 as a potential target for both molecular and immunotherapy of CML. However, the development of immunotherapeutic approaches will depend on the ability of NmE2 to function as a tumour antigen in common HLA backgrounds. The aims of this study were firstly to investigate the antigenicity of NmE2 in the HLA-A2 background (which accounts for more than 50% of the Caucasian population), and secondly to characterise the regulatory relationship between Bcr/Abl and NmE2 using a cell line model of CML. Materials and Methods: 5 nonameric NmE2 peptides with predicted anchor amino acids for HLA-A2 were loaded at concentrations of 10μM separately onto HLA-A2 expressing antigen presenting cells. Elispot Assays were carried out with CD8+ MLLCs (for the identification of antigenic peptides) or CD8+ cells isolated directly from a CML patient at different time points after HCT. Ba/F3 cells stably expressing wild type and mutant forms of Bcr/Abl were treated with imatinib and nilotinib (0 – 10 μM) for 48h. Bcr/Abl activity was assessed by FACS using antibodies specific for the phosphorylated forms of CrkL and Stat5. NmE2 and c-Myc protein were detected by immunocytochemistry and Western blotting with specific antibodies [Santa Cruz, clones L-16 and 9E10 respectively]. Levels of nme2 and c-myc mRNA were determined by quantitative real time PCR. Results: Full length NmE2 protein and 2 of 5 HLA-A2 anchor-containing peptides tested (NmE2132–140 and NmE2112–120) were specifically recognized by the HLA-A2+ CD8+ MLLC, demonstrating the antigenicity of NmE2 in the HLA-A2 background in vitro. Furthermore, while CD8+ T-cells from a transplanted HLA-A2+ CML patient showed little or no specific reactivity in the first 10 months after HCT, a distinct reactivity (up to 0.6 % NmE2 reactive CD8+ T cells) became apparent at later stages, consistent with the development of an immune response against NmE2-expressing cells in vivo. The patient remained negative for bcr/abl transcripts throughout this period. BA/F3 Bcr/Abl cells expressed increased levels of NmE2 protein (but not mRNA) compared to the parent BA/F3 line. Interestingly, treatment with imatinib or nilotinib reduced NmE2 protein expression in BA/F3 Bcr/Abl, but not in cells expressing Bcr/Abl mutants resistant to the respective inhibitors. Treatment of BA/F3 Bcr/Abl cells with the PI3K inhibitor Ly294002 resulted in reduced Bcr/Abl activity and a corresponding reduction in both c-Myc and NmE2 protein levels, without affecting mRNA levels. Conclusion: The over expression of NmE2 is closely linked to Bcr/Abl kinase activity, the predominant level of regulation being post-transcriptional and dependent on PI-3K activity. The NmE2 protein is restricted by HLA-A2 as well as by HLA-A32. The development of an NmE2-specific T-cell response in a CML patient after stem cell transplantation suggests that NmE2 functions as a tumour antigen in HLA-A2+ patients in vivo and may be relevant to the long term immune control of CML. NmE2 is therefore a promising candidate for the development of new immunotherapeutic strategies for the treatment of CML. Disclosures: Lange: BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Niederwieser:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

scholarly journals Phase 1 Study of CD19 Targeted 4-1BBL Costimulatory Agonist to Enhance T Cell (Glofitamab Combination) or NK Cell Effector Function (Obinutuzumab Combination) in Relapsed/Refractory B Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17 ◽  
Author(s):  
Martin Hutchings ◽  
Fritz C. Offner ◽  
Francesc Bosch ◽  
Giuseppe Gritti ◽  
Carmelo Carlo-Stella ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Dose Escalation ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Anti Tumor Activity

Background: Up to 50% of patients suffering from Non-Hodgkin`s lymphoma (NHL) become refractory to or relapse after treatment (M. Crump, Blood 2017). With this, the lack of curative outcomes for patients with both indolent and aggressive NHL subtypes remains an unmet medical need. The CD20 CD3 T cell bispecific antibody glofitamab induces specific T-cell activation and has demonstrated significant single agent activity in r/r NHL patients (NP30179 study, M. Dickinson, EHA 2020, Abstract S241). RO7227166, a CD19 targeted 4-1BBL (CD137) costimulatory agonist has shown synergistic anti-tumor activity when combined with glofitamab in preclinical models (fig 1). RO7227166 is a bispecific antibody-like fusion protein composed of a split trimeric 4-1BB ligand, a tumor antigen-targeting moiety recognizing CD19, and a silent Fc part preventing Fc-mediated toxicity. 4-1BB is an inducible co-stimulatory molecule expressed by activated T-cells or NK cells. Through CD19-binding, the 4-1BB ligand moiety can deliver co-stimulatory signals to activated T- and NK-cell subsets in the tumor. The expected mode of action (MoA) for this molecule is to deliver a costimulatory signal 2 to enhance the effector function of tumor-infiltrating T cells or NK cells upon their activation (signal 1) by a T-cell bispecific antibody (e.g. glofitamab, RO7082859) or a tumor-targeted ADCC antibody (e.g. obinutuzumab). By delivering direct T-cell-target cell engagement followed by costimulatory activation the aim is to offer a highly active off-the-shelf immunotherapy combination. Methods: RO7227166 is being developed in combination with glofitamab and obinutuzumab in a phase I, open-label, dose-escalation study BP41072 (NCT04077723). The study is designed to evaluate the combination maximum tolerated dose (MTD), safety, tolerability, pharmacokinetic (PK), and/or pharmacodynamic (PD) profile of escalating doses of RO7227166, and to evaluate preliminary anti-tumor activity in participants with r/r NHL. The dose escalation stage is divided into Part I (combination with obinutuzumab) and Part II (combination with glofitamab) followed by an expansion stage (Part III). During Part I patients receive 1000mg obinutuzumab intravenously (IV) at a q3w schedule in combination with CD19 4-1BBL IV. During part II glofitamab is given in a q3w schedule with RO7227166 introduced at C2D8 and administered concomitantly from C3D1 onwards. A fixed dose of obinutuzumab (Gpt; pre-treatment) is administered seven days prior to the first administration of RO7227166 and seven days prior to the first administration of glofitamab (M. Bacac, Clin Cancer Res 2018; M. Dickinson, EHA 2020, Abstract S241). Patients will initially be recruited into part I of the study only using single-participant cohorts, where a rule-based dose-escalation is implemented, with dosing initiated at 5 μg (flat dose). As doses of RO7227166 increase, multiple participant cohorts will be recruited and dose-escalation will be guided by the mCRM-EWOC design for overdose control. Commencement of Part II including decision on the RO7227166 starting dose will be guided by safety and PK data from Part I. Patients with r/r NHL meeting standard organ function criteria and with adequate blood counts will be eligible. The maximum duration of the study for each participant will be up to 24 months in Part I (excluding survival follow-up) and up to 18 months in Part II and Part III. Tumor biopsies and peripheral blood biomarker analyses will be used to demonstrate MoA and proof of concept of an off the shelf flexible combination option providing signals 1 and 2. Disclosures Hutchings: Takeda: Honoraria; Takeda: Research Funding; Genmab: Honoraria; Roche: Honoraria; Genmab: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Sankyo: Research Funding; Roche: Consultancy; Genmab: Consultancy; Takeda: Consultancy; Roche: Research Funding; Celgene: Research Funding; Daiichi: Research Funding; Sanofi: Research Funding. Bosch:Hoffmann-La Roche: Research Funding. Gritti:Italfarmaco: Consultancy; F. Hoffmann-La Roche Ltd: Honoraria; Jannsen: Other: Travel Support; Autolus: Consultancy; IQVIA: Consultancy; Kite: Consultancy; Takeda: Honoraria; Amgen: Honoraria. Carlo-Stella:Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca: Honoraria; Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics and Rhizen Pharmaceuticals: Research Funding; Boehringer Ingelheim and Sanofi: Consultancy. Townsend:Roche, Gilead: Consultancy, Honoraria. Morschhauser:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Consultancy; Janssen: Honoraria; Epizyme: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy. Cartron:Celgene: Consultancy, Honoraria; F. Hoffmann-La Roche: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Honoraria; Jansen: Honoraria; Gilead: Honoraria. Ghesquieres:CELGENE: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Roche: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Gilead: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Janssen: Honoraria. de Guibert:Gilead Sciences: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Herter:Roche Glycart AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Korfi:Roche Diagnostics GmbH: Consultancy. Craine:Roche: Current Employment. Mycroft:Roche: Current Employment. Whayman:Roche: Current Employment. Mueller:Roche: Current Employment. Dimier:Roche: Current Employment. Moore:Roche: Current Employment. Belli:Roche Pharma: Current Employment. Kornacker:Hoffmann-La Roche Ltd.: Current Employment, Current equity holder in publicly-traded company. Lechner:Roche Diagnostics GmbH: Current Employment, Current equity holder in publicly-traded company. Dickinson:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck Sharp & Dohme: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 75-75
Author(s):  
Jooeun Bae ◽  
Shuichi Kitayama ◽  
Laurence Daheron ◽  
Zach Herbert ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
T Cell Differentiation ◽  
Immune Checkpoints ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Induced Pluripotent

Abstract T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. Figure 1 Figure 1. Disclosures Munshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Hong Mu-Mosley ◽  
Lauren B Ostermann ◽  
Ran Zhao ◽  
Challice L. Bonifant ◽  
Stephen Gottschalk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Vehicle Control ◽  
Advisory Committees ◽  
Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Peri-Transplant Alemtuzumab Levels Predict Risk of Secondary Graft Failure and Inversely Impact CXCL9 Levels after RIC HCT (A Correlative Biology Study to BMT-CTN 1204 RICHI)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 748-748
Author(s):  
Ashley V Geerlinks ◽  
Brooks Scull ◽  
Christa Krupski ◽  
Ryan Fleischmann ◽  
Michael A. Pulsipher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Interferon Gamma ◽  
Research Funding ◽  
Graft Failure ◽  
Mixed Chimerism ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Donor Chimerism

Abstract Introduction The BMT-CTN 1204 study for Hemophagocytic Syndromes or Selected Primary Immune Deficiencies (NCT01998633) (RICHI) was a single arm study testing safety and efficacy of reduced intensity conditioning (RIC) with alemtuzumab (1mg/kg), fludarabine (150 mg/m2) and melphalan (140 mg/m2). Survival was favorable compared to historical studies, but patients experienced high rates of mixed chimerism (MC) and ultimate secondary graft failure (GF). Mechanisms for GF are not known. Expansion of recipient T cells and interferon-gamma pathway activation have been proposed as drivers for GF. However, high peri-transplant alemtuzumab levels have been associated with higher risk of MC and eventual secondary GF, suggesting an inverse relationship between GF and immune activation in the context of RIC. In order to elucidate mechanisms of GF for patients on the RICHI study, we systematically evaluated cytokine patterns and alemtuzumab levels and their association with durable engraftment. Methods Serial blood samples were collected, processed, and stored for consenting patients at day -14 (window: day -28 to -14), day -7 (+/- 1 day), day -1 (+/- 1), day +1 (+1 to +3), day +14 (+/- 2), day +28 (+/- 2), day +42 (+/- 3), day +70 (+/- 10), and day +100 (+/- 10). Alemtuzumab levels were measured using a flow cytometric assay as previously described. Comprehensive cytokine analysis was performed for over 100 analytes using the MagPix platform. Primary GF was defined as donor chimerism <5% prior to day +42. Secondary GF was defined as donor chimerism <5% after initial engraftment and/or requirement of donor lymphocyte infusion (DLI) or second HCT (with or without conditioning) to manage MC or graft loss. Mixed chimerism (MC) was defined as donor chimerism <95% on at least one occasion. Results Thirty-three patients were included in this study with HLH (n=25), CAEBV (n=3), CGD (n=2), HIGM (n=2), and IPEX (n=1). All patients received bone marrow grafts and 27 (82%) patients had unrelated donors. Twenty-one grafts were 8/8 or 6/6 HLA-matched (64%) and 12 grafts were 7/8 HLA-matched (36%). Among all patients, 1 patient (3%) developed primary GF, 22 (67%) developed mixed chimerism (MC), and 11 patients (33%) developed secondary GF. Survival with sustained engraftment without DLI or second HCT was 40.0%. We first evaluated peripheral blood levels of 100+ cytokines. Analysis revealed significant differences between patients with and without GF as shown in Figure 1A. Notably, on day +14 and +28, patients with secondary GF had significantly lower CXCL9 levels than those without GF. We then estimated the cumulative incidence (CI) of secondary GF among patients with CXCL9 levels above and below the day +14 median level of 2394pg/mL. The CI of secondary GF in patients with a day +14 CXCL9 level ≤2394pg/mL was 73.6% vs 0% in patients with a level >2394pg/mL (p=0.002). The CI of secondary GF in patients with a day +28 CXCL9 level ≤2867pg/mL (day +28 median) was 64.3%, vs 0% in patients with levels >2867pg/mL (p=0.004). We then sought to correlate CXCL9 levels with alemtuzumab exposure, as high alemtuzumab levels would result in more efficient T cell depletion of donor grafts that could lead to lower CXCL9 levels. Indeed, CXCL9 levels inversely correlated with day 0 alemtuzumab levels. Patients with day 0 alemtuzumab levels >0.32µg/mL had lower CXCL9 levels compared to patients with levels ≤0.32µg/mL (Figure 1B). Finally, we examined the impact of alemtuzumab levels on MC and secondary GF. Patients with day 0 alemtuzumab levels ≤0.32µg/mL had a lower CI of MC compared to patients with levels >0.32µg/mL, 14.3% vs 90.9%, respectively (p=0.03). The CI of secondary GF was 0% in patients with day 0 alemtuzumab levels ≤0.32µg/mL compared to 54.3% in patients with levels >0.32µg/mL (p=0.08). Conclusions This study demonstrates a strong relationship between alemtuzumab levels and durable engraftment. Further, interferon gamma activity, as reflected by CXCL9, inversely correlates with peri-transplant alemtuzumab levels in this prospective cohort treated with RIC. Our findings support the paradigm that higher alemtuzumab levels drive efficient T cell depletion of the stem cell product which increases the risk of MC and secondary GF, suggesting that donor T cells promote engraftment via a graft versus hematopoiesis function. Precision alemtuzumab dosing strategies may offer an opportunity to improve outcomes for patients who undergo RIC HCT. Figure 1 Figure 1. Disclosures Pulsipher: Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees; Jasper Therapeutics: Honoraria. Bollard: Neximmune: Current equity holder in publicly-traded company; Catamaran Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Repertoire Immune Medicines: Current equity holder in publicly-traded company; ROCHE: Consultancy, Honoraria; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kean: Regeneron: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Bluebird Bio: Research Funding; Gilead: Research Funding; Vertex: Consultancy; Novartis: Consultancy; EMD Serono: Consultancy. Jordan: Sobi: Consultancy. Allen: Sobi: Consultancy. OffLabel Disclosure: Alemtuzumab, humanized monoclonal antibody against CD52, used as part of allogeneic HCT conditioning

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