scholarly journals BRAFV600E Accelerates Disease Progression and Increases Immune Suppression in a Mouse Model of B-Cell Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1206-1206 ◽  
Author(s):  
Yo-Ting Tsai ◽  
Aparna Lakshmanan ◽  
Amy M. Lehman ◽  
Ellen J. Sass ◽  
Minh Tran ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
B Cell ◽  
Immune Suppression ◽  
Research Funding ◽  
Cell Leukemia ◽  
Wild Type ◽  
B Cell Leukemia

Abstract Background: Mitogen-activated protein kinase (MAPK) pathway components are frequently mutated in cancer, and abnormal MAPK signaling can drive malignancy by promoting tumor survival and proliferation. Furthermore, work in solid tumors shows that the presence of BRAFV600E further enhances tumor-induced immune suppression, but if and how this occurs in a disseminated disease such as leukemia is unknown. MAPK mutations occur in nearly 100% of classic hairy cell leukemia (HCL) and approximately 10% of chronic lymphocytic leukemia (CLL). Furthermore, up to 30% of B cell lymphomas exhibit activated MAPK signaling via expression of a BRAF pseudogene. However, the pathomechanisms of these abnormalities and the clinical utility of MAPK inhibitors in hematologic malignancies are unclear, and few models are available to evaluate this. To study these aspects, we developed in vitro and in vivo models of BRAF-mutated B cell malignancy. Methods: To mechanistically interrogate the role of BRAFV600E in malignant B cells, we transfected the CLL cell line OSUCLL with doxycycline-inducible constructs containing normal BRAF or BRAFV600E and incubated the cells -/+ doxycycline. These cells were also co-cultured with healthy donor T cells pre-incubated with anti-CD3/anti-CD28, and T cell proliferation was measured by flow cytometry. Cytokines and surface proteins were assessed by flow cytometry. To mimic human HCL, we developed a transgenic mouse model of BRAFV600E B cell leukemia. Mice with Cre-activatable BRAFV600E expression were crossed with mice carrying Cre under the control of the CD19 promoter. CD19-Cre x BRAFV600E or CD19-Cre only mice were then crossed with the well-characterized Eµ-TCL1 model of CLL to generate mice with spontaneous B cell leukemia expressing either wild-type BRAF (CD19-Cre x TCL1) or BRAFV600E (BRAFVE x CD19-Cre x TCL1) under the native BRAF promoter. B cell restricted expression of BRAFV600E was confirmed by immunoblots of purified B and T cells using a BRAFV600E specific antibody. Blood and spleen cells were examined by flow cytometry. For adoptive transfer experiments, leukemia cells (2x10^7) from transgenic mice were introduced intravenously into syngeneic healthy adult animals. Results: OSUCLL cells expressing BRAFV600E showed no significant changes in growth vs. wild-type BRAF expressing cells, but more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. Expression of PD-L1 was not different between cells expressing normal and mutated BRAF, indicating this common checkpoint molecule is not the reason for greater immune suppression in this context. TNF levels were higher in BRAFV600E expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E expressing cells on T cell proliferation. BRAFV600E x CD19-Cre x TCL1 mice developed B cell leukemia significantly earlier (median 4.9 vs. 8.1 months; P<0.001) and had significantly shorter lifespan (median 7.3 vs. 12.1 months; P<0.001) than their wild-type BRAF counterparts. In contrast to effects of BRAFV600E described in some solid tumors, BRAFV600E expression in B cells had no impact on the rate of B cell proliferation in vivo and only modestly reduced spontaneous apoptosis. To study the effects of this activating mutation on tumor-mediated immune suppression in vivo, leukemia cells from BRAFV600E x CD19-Cre x TCL1 or CD19-Cre x TCL1 mice were adoptively transferred into syngeneic mice. In 3 separate studies, tumor cells from BRAFV600E mice produced leukemia (>10% CD5+/CD19+ cells in blood) sooner than CD19-Cre x TCL1 tumor cells. In mice matched for disease load, BRAFV600E B cells produced a greater negative impact on T cells as evidenced by lower overall percentage of T cells, increased expression of T cell exhaustion markers PD-1, CD244, and CD160, and higher percentage of CD44+ memory T cells. Current studies are investigating the mechanism of these effects in vivo as well as the potential for pharmacologic reversal. Conclusions: Together, these results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a novel mouse model to develop rational combination strategies to both directly target the tumor cell and overcome tumor-mediated immune evasion. Disclosures Lozanski: Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding.

scholarly journals Azacytidine Sensitizes AML Cells for Effective Elimination By CD123 CAR T-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3904-3904
Author(s):  
Nadia El Khawanky ◽  
Amy Hughes ◽  
Wenbo Yu ◽  
Sanaz Taromi ◽  
Jade Clarson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Clinical Studies ◽  
Research Funding ◽  
De Novo ◽  
Survival Advantage ◽  
Cell Surface Expression ◽  
Third Generation

Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p<.01). Our findings indicate that Aza increases immunogenicity and augments the cell surface expression of CD123 on AML cells, allowing enhanced recognition and elimination of malignant cells by CD123 CAR Tc. This is the first demonstration that HMAs and CAR Tc immunotherapy can be used synergistically to treat AML. Considering HMAs are currently under clinical investigation in AML, our data encourage further clinical evaluation of this dual treatment in r/r AML, including high-risk patients that are chemotherapy or allogeneic transplantation ineligible. Disclosures Hughes: Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Yong:Novartis: Honoraria, Research Funding; Celgene: Research Funding; BMS: Honoraria, Research Funding.

T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 380-380 ◽  
Author(s):  
Stephan A. Grupp ◽  
Shannon L Maude ◽  
Pamela Shaw ◽  
Richard Aplenc ◽  
David M. Barrett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Dose Range ◽  
Single Chain

Abstract BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors. RESULTS 30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. Figure 1 Figure 1. All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONS: CTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. Disclosures Grupp: Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1452-1452
Author(s):  
Tiffany Simms-Waldrip ◽  
Michelle Yoonha Cho ◽  
Kenneth Dorshkind ◽  
Kathleen M Sakamoto
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Research Funding ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Wild Type ◽  
In Vivo Models ◽  
Hematopoietic Stem

Abstract Abstract 1452 The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment. The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 106) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 106) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations. At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 106 cells/mL and 1.3 × 106 cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics. Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

Durable Remissions in Children with Relapsed/Refractory ALL Treated with T Cells Engineered with a CD19-Targeted Chimeric Antigen Receptor (CTL019)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 681-681 ◽  
Author(s):  
Stephan A. Grupp ◽  
Shannon L Maude ◽  
Pamela A Shaw ◽  
Richard Aplenc ◽  
David M. Barrett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Young Adults ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Disease Burden ◽  
Children And Young Adults

BACKGROUND CARs combine a targeting antibody (scFv) domain with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR, which have resulted in up to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 53 pts with relapsed/refractory (r/r) ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with r/r CD19+ ALL. 48/53 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 3.6-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, and not their allo donors. RESULTS We treated 53 children and young adults with CD19+ ALL, median age 11y, (4-24y). To assess disease burden after lymphodepleting chemotherapy, pts had BM aspirations performed 1D prior to 1st CTL019 infusion: 41/53 pts had detectable ALL while 12 were MRD(-). A median of 4.3x106 CTL019 cells/kg (1-17.4x106/kg) were infused over 1-2D (1 pt got cells over 3D). There were no infusional toxicities >gr2, although pts who developed fevers within 24h of infusion did not receive a planned 2nd infusion of CTL019 cells. 50 pts (94%) achieved a CR, including a patient with CD19+ T ALL, 3 did not respond. MRD measured by clinical flow cytometry was <0.01% at D28 in 45 responding pts and positive at 0.024%-1.1% in 5 pts, with 2 patients becoming negative by 3 mo with no further therapy. With median follow up 10.6 mo (1-39 mo), 29 pts have ongoing CR, with only 6 receiving subsequent treatment such as donor lymphocyte infusion or SCT, EFS is 70% at 6 mo (95% CI, 58-85%) and 45% at 12 mo (95% CI, 31-66%), RFS is 72% at 6 mo (95% CI, 59-87%) and 44% at 12 mo (95% CI, 30-65%), and OS is 78% at 12 mo (95% CI, 67-91%). CTL019 was detected by qPCR in the CSF of 46/47 pts and 4 pts with CNS2a ALL experienced a CR in CSF. 20 pts with a CR at 1 mo have subsequently relapsed, with 3 relapses occurring after subsequent therapy (i.e. SCT) and 13 with CD19(-) blasts. 4/5 pts previously refractory to CD19-directed blinatumomab went into CR with CTL019, 3 subsequently relapsed with CD19(-) disease. All but 5 (90%) of pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 28% of patients and was reversed in all cases with the IL6-receptor antagonist tocilizumab, together with short courses of corticosteroids in 9 pts. Although T cells collected from the 35 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells can be detected by flow cytometry and/or QPCR, and results in the pharmacodynamic marker of CTL019 function, B cell aplasia, which continued for 3-39 months after infusion in pts with ongoing responses. B cell aplasia has been treated with IVIg without significant infectious complications. CONCLUSIONS: CTL019 cells can undergo robust in vivo expansion and can persist for 3 years or longer in children and young adults with r/r ALL, allowing for the possibility of long-term disease control without subsequent therapy such as SCT. This approach also has promise as salvage therapy for patients who relapse after allo SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with r/r ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CTL019 resistance. Rapid loss of CTL019 cells (prior to 3 months) is associated with a high risk of CD19+ relapse. CTL019 therapy has received Breakthrough Therapy designation from the FDA in pediatric and adult ALL, and phase 2 multicenter registration trials are well underway. Disclosures Grupp: Novartis: Consultancy, Research Funding. Maude:Novartis: Consultancy, Research Funding. Shaw:Novartis: Research Funding. Aplenc:Sigma Tau: Consultancy. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy; Endo: Other: Husband's employer, has equity interest. Teachey:Novartis: Research Funding. Wood:Novartis Pharmaceuticals Corporation: Employment. Porter:Novartis: Other: IP interest, Research Funding; Genentech: Other: Spouse employment. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding.

scholarly journals In Vivo Expansion and Antitumor Activity of Coinfused CD28- and 4-1BB-Engineered CAR-T Cells in Patients with B Cell Leukemia

2018 ◽  
Vol 26 (4) ◽  
pp. 976-985 ◽  
Author(s):  
Zhi Cheng ◽  
Runhong Wei ◽  
Qiuling Ma ◽  
Lin Shi ◽  
Feng He ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
B Cell ◽  
Cell Leukemia ◽  
Car T Cells ◽  
Car T ◽  
B Cell Leukemia

scholarly journals Strategy to prevent epitope masking in CAR.CD19+ B-cell leukemia blasts

2021 ◽  
Vol 9 (6) ◽  
pp. e001514
Author(s):  
Concetta Quintarelli ◽  
Marika Guercio ◽  
Simona Manni ◽  
Iolanda Boffa ◽  
Matilde Sinibaldi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Leukemia Cells ◽  
Cell Leukemia ◽  
Car T Cell ◽  
Car T ◽  
B Cell Leukemia

Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells.BackgroundMethodsWe evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing.ResultsThe presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells.ConclusionsTaken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.

CD19 chimeric antigen receptor-T cells in B-cell leukemia and lymphoma: current status and perspectives

Leukemia ◽  
2019 ◽  
Vol 33 (12) ◽  
pp. 2767-2778 ◽  
Author(s):  
Mohamad Mohty ◽  
Jordan Gautier ◽  
Florent Malard ◽  
Mahmoud Aljurf ◽  
Ali Bazarbachi ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Current Status ◽  
Cell Leukemia ◽  
B Cell Leukemia

Pentostatin Facilitates Fully MHC-Disparate Murine Mini-Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3539-3539
Author(s):  
Jacopo Mariotti ◽  
Kaitlyn Ryan ◽  
Paul Massey ◽  
Nicole Buxhoeveden ◽  
Jason Foley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Suppression ◽  
Graft Rejection ◽  
Mixed Chimerism ◽  
Post Transplant

Abstract Abstract 3539 Poster Board III-476 Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy. Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures: No relevant conflicts of interest to declare.

Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1633-1633
Author(s):  
Marcelo A. Navarrete ◽  
Benjamin Kisser ◽  
Hendrik J. Veelken
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphomas ◽  
Cd8 Cells ◽  
Fab Fragments ◽  
The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

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