scholarly journals Effect of Ara-C on T-Cell Function and Flotetuzumab Activity in Pediatric Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1387-1387
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Anne Kisielewski ◽  
Ezio Bonvini ◽  
John Muth ◽  
Jan K Davidson-Moncada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Cell Function ◽  
Concurrent Administration ◽  
Employment Equity ◽  
Group 3 ◽  
Equity Ownership ◽  
Group 2 ◽  
Pediatric Aml

Acute myeloid leukemia (AML) in children still has a poor prognosis despite the use of maximally intensive chemotherapy associated with severe short-term and long-term side effects. Therefore, development of targeted therapeutics is necessary to improve outcomes in pediatric AML. CD123 (IL3RA) is overexpressed in most of pediatric AML patients (Bras et al., Cytometry B Clin Cytom, 963:134, 2019) and has been pursued as a target for immunotherapy. The efficacy of a dual affinity retargeting agent (CD123xCD3; MGD006 or flotetuzumab), was evaluated in two patient-derived xenograft models of pediatric AML. In addition, concurrent administration of cytarabine with MGD006 was performed to determine the effect of cytarabine on T-cell function and flotetuzumab efficacy. NSG-SGM3 mice were transplanted with 2.5 x 106 cells AML PDX cells. After 18 days post transplant, when human cells were detectable in mouse blood, mice were randomly assigned to one of 8 treatment groups - 1) untreated, 2) T-cells, 3) T-cells with MGD006 (0.5 mg/Kg, Q5d), 4) T-cells with Ara-C (50 mg/Kg, Q5d), 5) T-cells with concurrent administration of Ara-C and MGD006, 6) MDG006 and 7) Ara-C. Mice belonging to groups 2-5 were intravenously injected with 2.0 x 106 human pan T-cells (StemCell Technologies, Cat No. 70024.1), prior to i.p. administration of MGD006 and/or Ara-C. Mice were monitored daily and peripheral blood was collected periodically to evaluate leukemia progression (CD45+CD3-) and T-cell expansion (CD3+CD45+) by flow cytometry. Mice were euthanized when they showed systemic signs of leukemia based on weight and body condition score. The growth of human cell percentage in mouse blood over time was plotted and Kaplan-Meier survival plots were generated. On the day after treatment was terminated, AML cell percentage was greatly reduced, in mice treated with T-cells + MGD006 (Fig. 1, group 3) or T-cells + MGD006 + Ara-C (group 5), compared to the other groups. In addition, exposure to MGD006 (groups 3 and 5) enhanced expansion of adoptively transferred T-cells compared to AML PDX mice receiving T-cells alone (group 2). The ability of MDG006 to enhance the expansion of T-cells in vivo was not attenuated by treatment with Ara-C. Similar results were obtained in a second PDX model (Fig. 2). Taken together, MGD006 enhanced T-cell engraftment with or without Ara-C accompanied by marked reduction in the burden of AML blats in the peripheral blood. As expected, MGD006 in the absence of the effector T-cells (group 6) had minimal effect on reducing leukemic burden or survival (Fig. 3A, B). Mice injected with T-cells alone (group 2) showed 40-day improvement in survival, likely due to the allogeneic effect of T-cells. Regardless, the addition of MGD006 with T-cells (group 3) amplified the effect as mice did not reach experimental endpoints upon study termination at 210 days (Fig. 3B, brown line). Ara-C treatment (group 7) delayed leukemia progression and prolonged median survival by 22.5 days compared to untreated mice (Fig. 4A, B). Consistent with the T-cell expansion induced by Ara-C (Fig. 1), mice treated with T-cells + Ara-C (group 4) survived longer (median survival 180 days) than those treated with Ara-C (group 7) or T-cells alone (group 2) (median survival 116 and 135 days respectively). Mice administered with MGD006 concurrently with Ara-C following T-cell injection (group 5) also did not reach experimental endpoints upon study termination (Fig. 3B, purple line). These mice had 0.1% residual AML cells when the study was terminated (Fig. 3A, solid purple line), which was significantly lower than mice receiving T-cells + MGD006 (group 3, 2% AML cells, P=0.0047). These data demonstrate the activity of MGD006 in the presence of T-cells in prolonging survival in pediatric AML PDX models. Inclusion of Ara-C to this regimen was more efficient in reducing AML burden. Disclosures Bonvini: MacroGenics, Inc.: Employment, Equity Ownership. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership.

scholarly journals Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1881-1881
Author(s):  
Yuxin Feng ◽  
Austin Goodyke ◽  
Marlee Muilenberg ◽  
Kelli Cole ◽  
Kathleen Cannady ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
T Cell Proliferation ◽  
Group 3 ◽  
Group 2 ◽  
Dc Maturation ◽  
Pro Inflammatory Cytokines ◽  
Group 1

Abstract Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model. Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4. Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice. Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. Disclosures Al-Homsi: Millennium Pharmaceuticals: Research Funding.

scholarly journals Helper and suppressor T-cell function in HIV-infected hemophilia patients

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 298-302 ◽  
Author(s):  
R Weimer ◽  
T Schweighoffer ◽  
K Schimpf ◽  
G Opelz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Subset ◽  
Pokeweed Mitogen ◽  
Suppressor Activity ◽  
T Cell Subset ◽  
Group 3 ◽  
Group 2 ◽  
Hiv Negative

Abstract T-lymphocyte helper and suppressor functions were assessed in 61 hemophilia patients. Twenty one patients were HIV-negative (Group 1), 27 were HIV-positive without having AIDS-related complex (ARC)/AIDS (Group 2), and 13 had ARC/AIDS (Group 3). T, CD4-positive, or CD8- positive T lymphocytes were cocultured with B lymphocytes and pokeweed mitogen for 6 days and immunoglobulin producing cells were assessed in a reverse hemolytic plaque assay. In HIV-infected patients, T cells as well as the CD4-positive T cell subset exhibited reduced helper (P less than .01, Group 2; P less than .0005, Group 3) and elevated suppressor activity (P less than .02, Group 2; P less than .005, Group 3), whereas no significant difference was found between HIV-negative patients and controls. The number of CD4-positive cells was not correlated with CD4 cell function. CD4-positive cells showed no helper activity (less than 10% of control T cells) in 8/11 (73%), but an excessive suppressor activity (greater than 80% suppression of plaque formation) in 6/11 (55%) Group 3 patients. Our results show that defective helper and elevated suppressor functions of T cells in HIV-infected patients are caused not only by a change in the CD4/CD8 cell counts but also by functional abnormalities of the CD4-positive T-cell subset. These abnormal helper and suppressor functions may play a role in the development of the immunodeficiency state of AIDS patients.

scholarly journals Helper and suppressor T-cell function in HIV-infected hemophilia patients

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 298-302
Author(s):  
R Weimer ◽  
T Schweighoffer ◽  
K Schimpf ◽  
G Opelz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Subset ◽  
Pokeweed Mitogen ◽  
Suppressor Activity ◽  
T Cell Subset ◽  
Group 3 ◽  
Group 2 ◽  
Hiv Negative

T-lymphocyte helper and suppressor functions were assessed in 61 hemophilia patients. Twenty one patients were HIV-negative (Group 1), 27 were HIV-positive without having AIDS-related complex (ARC)/AIDS (Group 2), and 13 had ARC/AIDS (Group 3). T, CD4-positive, or CD8- positive T lymphocytes were cocultured with B lymphocytes and pokeweed mitogen for 6 days and immunoglobulin producing cells were assessed in a reverse hemolytic plaque assay. In HIV-infected patients, T cells as well as the CD4-positive T cell subset exhibited reduced helper (P less than .01, Group 2; P less than .0005, Group 3) and elevated suppressor activity (P less than .02, Group 2; P less than .005, Group 3), whereas no significant difference was found between HIV-negative patients and controls. The number of CD4-positive cells was not correlated with CD4 cell function. CD4-positive cells showed no helper activity (less than 10% of control T cells) in 8/11 (73%), but an excessive suppressor activity (greater than 80% suppression of plaque formation) in 6/11 (55%) Group 3 patients. Our results show that defective helper and elevated suppressor functions of T cells in HIV-infected patients are caused not only by a change in the CD4/CD8 cell counts but also by functional abnormalities of the CD4-positive T-cell subset. These abnormal helper and suppressor functions may play a role in the development of the immunodeficiency state of AIDS patients.

scholarly journals Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3921-3921 ◽  
Author(s):  
Cesar Sommer ◽  
Hsin-Yuan Cheng ◽  
Yik Andy Yeung ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

scholarly journals Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5603-5603 ◽  
Author(s):  
Cherie Tracy Ng ◽  
Jeanette Ampudia ◽  
Robert J. Soiffer ◽  
Jerome Ritz ◽  
Stephen Connelly
Keyword(s):  
Weight Loss ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Chronic Gvhd ◽  
Vehicle Control ◽  
Control Group ◽  
Employment Equity ◽  
Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

Mesothelin Targeting Bites for Pediatric AML: In Vivo Efficacy and Specificity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3925-3925
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Colin Correnti ◽  
Anne Kisielewski ◽  
Allison Kaeding ◽  
Soheil Meshinchi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Single Chain ◽  
Pediatric Leukemia ◽  
Effector Cells ◽  
Cell Counts ◽  
Human T Cells ◽  
Pediatric Aml

Acute myeloid leukemia (AML) remains the type of pediatric leukemia with poorest outcome. Despite maximally intensive therapy, approximately 20% of patients experience recurrent disease. Novel targeted therapies are needed to improve survival. We recently identified that mesothelin, a well-validated target in some cancers, is also highly expressed in a subset of pediatric AML samples (Tarlock et al., Blood, 128:2873, 2016). Considering that it is not expressed in normal tissues in children (Fan et al., Blood, 130:3792, 2017), MSLN is a viable target for immunotherapies such as Bispecific T-cell Engaging antibodies (BiTEs) that combine antibody single chain variable (scFv) regions targeting a cancer antigen and the T-cell co-receptor CD3. We designed and tested the efficacy and specificity of BiTEs targeting MSLN in disseminated xenograft models of pediatric AML. Using scFv sequences derived from Amatuximab, which recognizes the N-terminal domain of the GPI-linked ectodomain of MSLN, targeting region 1 of MSLN, and from Blinatumomab/AMG-330 targeting CD3, we engineered and expressed two kinds of BiTE molecules - a canonical BiTE and an IgG BiTE, a larger molecule with improved serum half life in vivo. To evaluate the specificity and efficacy of canonical BiTEs, MV4;11-MSLN cell line was generated by lentiviral transduction of parental MV4;11 cells which do not constitutively express MSLN (Fig. 1A, B). These two cell lines were injected i.v. into NSG-SGM3 mice. Once engraftment was confirmed, purified human T cells (3 x 106) were injected to act as effector cells. Mice were then treated with the canonical αMSLN-αCD3 BiTE at a dose of 3 mg/kg/day daily for 6 days. A cohort of mice that were untreated or received BiTE or T-cell infusion only served as controls. Mice from both treated and untreated groups had to be euthanized when they presented with distended abdomens due to myeloid sarcomas and no significant differences in survival were observed. Post euthanasia, bone marrows were flushed and evaluated for the percentage of AML cells (human CD45+CD33+) and T cells (human CD45+CD3+). We observed that the αMSLN-αCD3 BiTE was effective in promoting T-cell activation (based on high T-cell counts compared to mice injected with T-cells alone) and greatly reducing leukemic burden in mice injected with MV4;11 cells engineered to express MSLN (Fig. 1C, D). Similar results were obtained using BiTEs targeting a different MSLN epitope. No T-cell expansion and anti-leukemic effect was observed in mice engrafted with parental MV4;11 cells. Although, there were no significant differences between the median survival of untreated and treated miceThese data highlight the specificity and efficacy of the aMSLN-CD3 BiTEs. Among a panel of 8 AML patient-derived xenograft (PDX) lines generated in the laboratory, NTPL-146 bearing MLL-ENL fusion was found to have endogenous MSLN expression (Fig. 1E). We evaluated the efficacy of αMSLN-αCD3 canonical BiTE (3 mg/Kg Qdx6) against NTPL-146 PDX line in NSG-B2m mice by transfusing human CD3+ T-cells to act as effector cells. A Kaplan-Meier survival plot based on the time when each mouse reached experimental end-point (reduced body weight greater than 20%, impaired mobility, hind limb paralysis) showed that the survival benefit for mice receiving BiTE in the presence of human T-cells (4/6 mice survived at the end of experiment) greatly exceeded the efficacy of T-cells alone (22-day improvement in median survival with no surviving mice), or BiTE treatment alone (no improvement in survival) compared to untreated mice (Fig. 1F, P<0.001). These data validate the efficacy of MSLN targeting BiTEs in a PDX model with endogenous MSLN expression. The efficacy of canonical vs IgG BiTEs was evaluated in MV4;11-MSLN xenografted mice. Mice were dosed Qd5x3 for canonical BiTE and Q7dx3 for IgG BiTE as shown (Fig. 1G). IgG BiTE treatment along with T-cell infusion significantly prolonged survival in mice transplanted with MV4;11-MSLN (Fig. 1H), suggesting that IgG BiTE was far more efficacious than canonical BiTEs (P<0.01). Taken together, these data indicate that MSLN-targeting BiTEs could be used as novel immunotherapy for pediatric AML with MSLN expression. Figure 1 Disclosures Kaeding: Celgene: Employment.

Hydroxyurea Is Most Suitable for Cytoreduction of AML Prior to CD33/CD3 Bispecific BiTE® Antibody (AMG 330) Therapy: Uncompromised T-Cell Proliferation Ex-Vivo and CD33 Upregulation on AML Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 986-986 ◽  
Author(s):  
Christina Krupka ◽  
Franziska Brauneck ◽  
Felix S Lichtenegger ◽  
Peter Kufer ◽  
Roman Kischel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cell Function ◽  
Expression Level ◽  
T Cell Proliferation ◽  
Hl60 Cells ◽  
T Cell Function ◽  
Equity Ownership

Abstract Bispecific T-cell engager (BiTE®) antibodies represent a promising tool for anti-leukemic immunotherapy. The CD19/CD3-bispecific antibody blinatumomab was shown to be active in refractory and relapse patients with B-precursor acute lymphoblastic leukemia (Topp et al, ASCO 2014). Transient, blinatumomab-mediated cytokine release syndrome has been linked to target cell numbers as this phenomenon is predominantly observed within the first treatment cycle. In our previous work, we demonstrated that the bispecific CD33/CD3 BiTE® antibody AMG 330 is able to induce activation and proliferation of residual autologous T-cells and effectively mediates lysis of primary acute myeloid leukemia (AML) cells (Krupka et al, Blood 2014; 123(3):356-65). We hypothesize that in AML patients with high initial leukocyte counts (WBC > 30.000/μl) a cytoreductive phase prior to AMG 330 therapy might be beneficial to reduce the incidence and severity of cytokine mediated toxicity. Ideally, the cytoreductive drug does not impair T-cell function or reduce target antigen expression level. In the current study, we evaluated the effect of cytarabine (20 µM), decitabine (5 µM), azacitidine (1 µM and 5 µM) and hydroxyurea (10 µM and 100 µM) on T-cell proliferation and function in close analogy to potential treatment algorithms for AML. Healthy donor (HD) T-cells were pre-incubated with the cytoreductive drugs for 72 hours. T-cells were CFSE-labeled and co-cultured with either HL60 or MV4-11 cells (effector cell:target (E:T) ratio 1:1) in the presence or absence of AMG 330 (5 ng/ml). After 3 days of co-culture, lysis of HL60 cells and T-cell proliferation was assessed by flow cytometry. Pretreatment of T-cells with cytarabine completely abrogated T-cell function (lysis of HL60 cells: untreated (UT): 96.9% vs 20 µM: 4.2%) and significantly impaired T-cell proliferation (UT: 31.2% vs 20 µM: 4.6%). These findings correlated to data using primary AML samples collected 3 and 6 days after discontinuation of cytarabine treatment. After a 3-day chemotherapy-free interval, we observed no relevant T-cell proliferation and lysis of AML cells upon the addition of AMG 330 to the ex-vivo long-term culture system (lysis of AML cells on day 12: 30%; fold change T-cell expansion 0.9). After a 6-day treatment-free interval, high T-cell proliferation and cytotoxicity against primary AML cells were observed (lysis of AML cells on day 12: 61%; fold change T-cell expansion: 3.1). In contrast to cytarabine, decitabine treatment only marginally impaired T-cell function. Similarly, pre-incubation with azacitidine did not convey a negative effect on T-cell function (lysis of HL60 cells: UT: 100% vs 1 µM: 94.9% vs 5µM: 86.8%; proliferation: UT: 90.9% vs 1 µM: 80% vs 5 µM: 66.8%). Pretreatment with hydroxyurea had the least impact on T-cell performance. It did not impair T-cell function (lysis of HL60 cells: UT: 100% vs 10 µM: 100% vs 100 µM: 100%) and proliferation compared to untreated controls (UT: 92.9% vs 100 µM 90.8% vs 10 µM 92.9%). As we have previously shown that the level of CD33 expression correlates to kinetics of AMG 330-mediated lysis (Krupka et.al, EHA 2014), we analyzed the effect of the cytoreductive agents on CD33 expression level in AML cell lines and primary AML cells. Five AML cell lines (HL60, MV4-11, PL21, OCI-AML3, KG1a) and a primary AML patient sample were cultured in the presence or absence of decitabine (5 µM and 50 µM), azacitidine (1 µM and 5 µM) or hydroxyurea (10 µM and 100 µM) for 72 hours. The change of CD33 expression level was evaluated by flow cytometry (median fluorescence intensity, MFI). No significant changes in CD33 expression level were observed after culture of AML cell lines and primary AML cells with decitabine or azacitidine. In contrast, hydroxyurea upregulated surface expression of CD33 on 2/5 cell lines (HL60 and PL21) in a dose dependent manner (HL 60 MFI Ratio: UT 134.9 vs 10 µM 171.3 vs 100 µM 210; PL21 MFI Ratio: UT 166.9 vs 10 µM 177.9 vs 100 µM 191.8). In summary, we could show that pretreatment with hydroxyurea did not impair T-cell function and proliferation. In addition, we observed an upregulation of CD33 expression on AML cell lines. As the BiTE® technology relies on T-cell function and target antigen expression level, sequential and combinatorial immuno-chemotherapeutic approaches need to address both issues. Our data support the use of hydroxyurea in AML patients that require cytoreduction prior to AMG 330 treatment. Disclosures Krupka: AMGEN Inc.: Research Funding. Kufer:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Zugmaier:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Sinclair:AMGEN Inc.: Employment, Equity Ownership. Newhall:AMGEN Inc.: Employment, Equity Ownership. Frankel:AMGEN Inc.: Employment, Equity Ownership. Baeuerle:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership. Subklewe:AMGEN Inc.: Research Funding.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3094-3094 ◽  
Author(s):  
Alena A. Chekmasova ◽  
Holly M. Horton ◽  
Tracy E. Garrett ◽  
John W. Evans ◽  
Johanna Griecci ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Drug Product ◽  
Single Chain ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T Cell Effector Function in Mice: Implications for Combination with Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2525-2525
Author(s):  
Paul M Tyler ◽  
Mariah M Servos ◽  
Boris Klebanov ◽  
Trinayan Kashyap ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
T Cell Development ◽  
Immune Homeostasis ◽  
Dosing Regimen ◽  
Employment Equity ◽  
Equity Ownership

Abstract Selinexor (KPT-330) is a first in class nuclear transport inhibitor of exportin-1(XPO1) currently in advanced clinical trials to treat patients with solid and hematological malignancies. To determine how selinexor might impact anti-tumor immunity, we analyzed immune homeostasis in mice treated with high selinexor doses (15 mg/kg, three times a week: M, W, F) and found disruptions in T cell development, a progressive loss of CD8 T cells and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by high doses of selinexor, suggesting that normal immune homeostasis could recover. We found that high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week (7.5 mg/kg, M, W, F) was not able to restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that XPO1 function is required for T cell development and function. We then determined the minimum concentration of selinexor required to block T cell activation, and showed that T cell inhibitory effects of selinexor occur at levels above 100nM, corresponding to the first 24 hours post-oral dosing of 10 mg/kg. In a model of implantable melanoma, we used selinexor treatment at the clinically relevant dosing regimen of 10 mg/kg with a 5-day drug holiday (M, W selinexor treatment). After two weeks of treatment, tumors were harvested and tumor infiltrating leukocyte (TIL) populations were analyzed. This treatment led to intratumoral IFNg+, granzyme B+ cytotoxic CD8 T cells that were comparable to vehicle treated mice. Overall, selinexor treatment leads to transient inhibition of T cell activation but the clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T cell functioning and development of anti-tumor immunity. These results provide additional support to the recommended selinexor phase 2 dosing regimen, as was determined recently (Razak et al. 2016). Disclosures Klebanov: Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership. Dougan:Karyopharm Therapeutics: Consultancy. Dougan:Karyopharm Therapeutics: Consultancy.

Identification of Small Molecule Modulators to Enhance the Therapeutic Properties of Chimeric Antigen Receptor T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4712-4712
Author(s):  
Jonathan Rosen ◽  
Betsy Rezner ◽  
David Robbins ◽  
Ian Hardy ◽  
Eigen Peralta ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Small Molecules ◽  
Small Molecule ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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