DNA Immunization Against Melanoma Antigens Enhances Tumor Immunity in Mouse Models of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 304-304
Author(s):  
Adi Diab ◽  
Miguel-Angel Perales ◽  
Adam Cohen ◽  
Vanessa M. Hubbard ◽  
Jeff Eng ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
T Cell ◽  
Dna Vaccines ◽  
Adoptive Cell Therapy ◽  
Dna Immunization ◽  
Hematopoietic Stem ◽  
Free Survival ◽  
Allogeneic Hsct ◽  
Gm Csf

Abstract Allogeneic HSCT is an important therapy with curative potential for a variety of malignant diseases, including leukemias, lymphomas and some solid tumors. Despite significant progress in reducing treatment-related mortality, malignant relapse remains a major problem. We are developing DNA vaccines that encode gene products closely related to self-antigens, including xenogeneic DNA and mutated DNA, and have initiated clinical trials of DNA vaccines in patients with advanced melanoma or prostate cancer. Using the B16 mouse melanoma model, we have shown that immunization with human TRP-2 DNA (xenogeneic melanoma differentiation antigen - MDA) or Opt-Tyrp1 DNA (a mutated MDA related to TRP-2, which we have optimized for CD8 epitopes), can induce tumor protection, including against established tumors. We hypothesized that immunization of allogeneic HSCT recipients (or their donors) against specific tumor antigens could decrease the risk of relapse without enhancing graft-versus-host disease (GVHD). In an MHC-matched minor antigen-mismatched mouse HSCT model (LP into B6), we found that: (1) by day 28 after transplant, recipients of an allogeneic T-cell depleted (TCD)-HSCT have considerable numbers of splenic T cells, including de novo generated donor T cells, which suggests that vaccination aimed at T cells might be feasible; (2) post-HSCT DNA immunization against a single tumor antigen can provide protection from a tumor challenge that is comparable to that observed with a whole cell vaccine (B16-GM-CSF) and significantly greater than HSCT alone; (3) DNA immunization post-HSCT can induce tumor-specific CD8+ T cells of donor origin (detected by ELISPOT or intracellular cytokine flow cytometry assay); (4) the combination of donor leukocyte infusion (DLI) and post-HSCT DNA immunization further enhances tumor-free survival (Figure); (5) there is no evidence of GVHD in multiple experiments using a clinical GVHD score to monitor recipients; and (6) the effects of post-HSCT DNA immunization on both tumor-free survival and CD8+ T cell responses have been validated for two different DNA vaccine strategies (hTRP-2 + GM-CSF DNA, or Opt-Tyrp1 DNA). These results demonstrate that DNA immunization after allogeneic TCD-HSCT can induce potent anti-tumor effects without the induction of GVHD. This and similar investigations provide a strong rationale for the development of novel therapeutic strategies that combine allogeneic HSCT, post-transplant tumor vaccination and adoptive cell therapy in human clinical trials. Figure Figure

Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 144-144
Author(s):  
Mohammad S Hossain ◽  
David L Jaye ◽  
Brian P Pollack ◽  
Alton B Farr ◽  
John Roback ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Viral Immunity ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Radiation Chimeras ◽  
Donor T Cells

Abstract Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus. To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

Molecular Remission and B Cell Aplasia Induced in a First Cohort of Adults with Relapsed B-ALL Treated with 19–28z CAR-Targeted T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3566-3566
Author(s):  
Marco L Davila ◽  
Isabelle Riviere ◽  
Xiuyan Wang ◽  
Jae H. Park ◽  
Jolanta Stefanski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Induction Chemotherapy ◽  
High Dose ◽  
Molecular Remission ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Initial Cohort

Abstract Abstract 3566 B cell acute lymphoblastic leukemia (B-ALL) in adults has a dismal prognosis. Intensified, combinatorial chemotherapy yields remarkable results in pediatric ALL but less so in adults. As allogeneic hematopoietic stem cell transplantation (HSCT) is curative in only a minority of patients, there remains a dire need for effective therapies in this patient population. Building on our results obtained with genetically targeted T cells in xenogeneic models of ALL and our experience in patients with chronic lymphocytic leukemia (Brentjens, Rivière, Blood, 2011), we have recently initiated a clinical trial (NCT01044069) treating adults with relapsed or refractory B-ALL with autologous T cells expressing the 19–28z chimeric antigen receptor (CAR), a dual CD3zeta/CD28 signaling receptor specific for the B cell antigen CD19. Human peripheral blood T cells retrovirally transduced to express the 19–28z receptor specifically lyse normal and malignant B cells in vitro and eradicate established tumors in vivo in pre-clinical animal models. We report the results from the initial cohort of patients treated on this protocol. The first two patients had relapsed disease that achieved morphologic remission following re-induction chemotherapy. Following adoptive therapy with CAR-modified T cells, one patient achieved a B cell aplasia and molecular remission documented by deep sequencing, which demonstrated the disappearance of IgH rearrangements associated with the malignant clone. The other patient had achieved a molecular remission after re-induction chemotherapy but further developed a complete B cell aplasia following modified T cell infusion. While in molecular remission, both patients successfully underwent allogeneic HSCT and were therefore removed from the study. In contrast to these two patients, the third patient failed to achieve a morphologic remission after re-induction chemotherapy and had >60% blasts in the bone marrow (BM) at the time of T cell infusion. Within 12 hours of completing T cell infusion, the patient developed high-grade fevers, hypotension, and rigors. Serum analyses demonstrated a sharp rise in cytokines (IFNg, TNFa, IL6, IL2, and IL8), reflecting rapid and robust onset of T cell activation. High-dose corticosteroids initiated 5 days after T cell infusion controlled these symptoms. Post-T cell BM aspirate on day 8 demonstrated undetectable blasts. Flow cytometry could not detect blasts or B lineage cells, but readily identified 19–28z CAR+ T cells. Significantly, the patient's B-ALL tumor cells harbored a unique 9p21 cytogenetic deletion that was detected by FISH in 28% of nuclei immediately prior to T cell infusion and was reduced to 5% 8 days after T cell infusion. The next BM aspirate, performed on day 24 after T cell infusion, demonstrated a persistent morphologic remission, a recovering BM, and complete absence of any detectable 9p21 deletions. Furthermore, PCR amplification for IgH rearrangements in the BM 24 days after T cell infusion confirmed a molecular remission and a B cell aplasia. Collectively, the clinical outcomes from this initial cohort of 3 patients demonstrate for the first time the in vivo efficacy of CD19-targeted T cells to induce clinical and molecular remissions as well as B cell aplasia in adults with relapsed or refractory B-ALL. These early results strongly support further investigation of 19–28z targeted T cells to treat leukemias and suggest this is a potential salvage therapy for patients with relapsed/refractory B-ALL who are failing chemotherapy or are ineligible for allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 573-584 ◽  
Author(s):  
Nicoletta Cieri ◽  
Barbara Camisa ◽  
Fabienne Cocchiarella ◽  
Mattia Forcato ◽  
Giacomo Oliveira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Adoptive Cell Therapy ◽  
Cell Subset ◽  
Gene Expression Signature ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Hematopoietic Stem ◽  
T Cell Subset

Abstract Long-living memory stem T cells (TSCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8+ TSCM lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L+CCR7+CD45RA+CD45R0+IL-7Rα+CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, TSCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived TSCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified TSCM are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

Prospective Molecular Identification of Alloreactive CTL Clones in Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4972-4972
Author(s):  
Christine L. O’Keefe ◽  
Ronald Sobecks ◽  
Alexander Rodriguez ◽  
Julie Curtis ◽  
Elizabeth Kuckowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Molecular Analysis ◽  
Cd8 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Donor Cells

Abstract The process of immune recovery after allogeneic HSCT can be characterized by an often profound oligoclonality of the TCR spectrum which may reflect: 1) A decreased diversity within the T cell population or 2) Expansion of individual clones that may be caused by specific antigenic drive exerted by pathogens (e.g., CMV) or alloantigens during the process of GvHD. Novel technologies based on the molecular analysis of the TCR repertoire can be applied to study clonal responses, including multiplex amplification of rearranged TCR VB chains followed by sequencing and quantitation of their contribution to the entire T cell repertoire. We initially studied the T cell repertoire after allogeneic HSCT in sibling (N=20) and matched unrelated (N=9) transplants. VB spectratyping was performed on CD8+ T cells in 22 patients; of the expanded VB families tested, 61.2% (30 of 49) were mono- or oligoclonal by genotyping. The clonal size and structure was determined by sequencing. Immunodominant clones contributed up to 5.4% (avg. 1.4%; range 0.1–5.4%) of all CD8+ T cells, indicating that certain stimuli may drive expansion of immunodominant clones. We originally hypothesized that these expanded clones were allospecific and likely played a role in GvHD; however, we found no correlation between the presence of significant expansions and grade III/IV GvHD. Therefore, in order to identify alloreactive CTL clones and their clonotypic markers, an alternative approach was devised. The proposed technique utilizes an allostimulation step: recipient cells serve as targets to induce activation of allospecific donor cells. Donor alloreactive cells are identified by their expression of activation markers, such as CD25 or CD69. After sorting, allospecific T cells are used as a source of cDNA for identification and quantitation of allospecific clonotypes. In this fashion, we have analyzed patients undergoing allogeneic sibling and matched unrelated donor grafting (N=7). Prior to transplant, allostimulation was performed and alloreactive CD8-derived clonotypes were subjected to molecular analysis. VB families represented within alloresponsive CTL populations that were oligoclonal by genotyping were subcloned and a large number of CDR3 clones were sequenced to identify the immunodominant clonotypes. Sequences have been derived from activated CD8+ donor cells in 6 cases; an average of 4 (range 1–7) VB families per pair have been characterized.. Although the presence of multiple VB families with a diversified CDR3 spectrum suggests the polyclonal nature of alloresponsive clones, immunodominant clones were identified. A total of 13 immunodominant clonotypes have been identified in 5 patients. Five such clones were identified in one donor/recipient pair; in each pair at least one immunodominant clonotype was isolated. Up to 18 clones per VB family were sequenced, and the average expansion contributed 56% to the entire VB family (range 15–100%). Clonotype-specific primers have been designed from two expanded clones and used to detect the allospecific clones in post-transplant blood samples in one patient/donor pair. In sum, molecularly defined marker clonotypes indicative of alloresponsive CTLs in HSCT can be individually and prospectively isolated. Such clonotypes may find application in tissue and blood diagnosis of GvHD.

Hematopoietic Multipotent Progenitor Cells Mobilized by G-CSF Can Inhibit Gvhd

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2969-2969
Author(s):  
Maud D'Aveni ◽  
Julien Rossignol ◽  
Ruddy Montandon ◽  
Marie Bouillie ◽  
Flora Zavala ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Mechanisms Of Action ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Ifn Γ

Abstract Abstract 2969 Backgound. Acute graft-versus-host-disease (aGVHD) is a frequent life threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Despite the infusion of higher doses of T cells with the use of G-CSF-mobilized HSC grafts, the incidence of aGVHD is not increased. The mechanisms by which G-CSF-mobilized HSC can control GVHD are imperfectly elucidated. We previously described the mobilization of murin hematopoïetic progenitor cells (HPCs) by G-CSF and FLT3 ligand capable of inducing tolerance against autoimmune diabetes in the nude mice (Kared, Immunity 2006). We now show that G-CSF can mobilize murin HPCs with immunoregulatory functions in the allogeneic immune response and describe their mechanisms of action. Methods. Mobilization of HPCs is performed by subcutaneous administration of human recombinant G-CSF at 200μg/kg per day, for 4 consecutive days in the C57BL6 (H-2b) mouse. HPCs are collected in the spleen by FACS sorting according to their phenotype: Lin- Sca1high cKithigh FLT3low CD34+ CD106+ CD127−. In vitro, functions and mechanisms of action were analyzed by co-cultures with i) T cells (from C57BL6) activated by anti-CD28 and -CD3 mAbs or activated by BALB/c (H-2d) allogeneic splenic LPS matured dendritic cells, ii) C57BL6 splenic selected CD4+CD25high T regulatory T cells activated by anti-CD28 and -CD3 mAbs iii) activated antitumor specific CD8 T cells (C57BL6 ovalbumin specific TCR transgenic T cells). These different cultures were performed in the presence or absence of inhibitors of selective cytokines or other regulatory molecules. In vivo, we assessed the effect of donor HPCs on GVHD development by injecting C57BL6 derived HPCs (0.5×106/mouse), splenic T cells (1×106/mouse) and T depleted bone marrow cells (5×106/mouse) into lethally irradiated (8 Gy) Balb/c recipients. Results. In vitro, as compared to controls without HPCs, after 3 days of culture, HPCs: 1) promote the proliferation of natural T regs activated by anti-CD3 and anti-CD28 (>80% at 3 days of culture compared to control <50%), 2) inhibit the proliferation of activated T cells (>80% T cells blocked before 4 divisions as compared to control-T cells alone >80% after 4 divisions- p<0, 001) and 3) induce the apoptosis of activated T cells (30% increased, p=0, 01). The proliferation of T regs was cell contact dependant and required the presence of TGF-b. The inhibition of T cell activation required IFN γ produced by activated T-cells and some contact-dependent stimuli. In such pro-inflammatory conditions, HPCs differentiate after 4 days in myeloid derived suppressor cells (MDSC). These cells could then produce NO in response to IFN γ and suppress the proliferation of activated T cell. However, T cell suppression was not dependant on L-arginine depletion. Induction of apoptosis of T cells was Fas/Fas-L dependant. Although in the presence of HPCs the proliferation of CD8+ T TCR transgenic against the dominant ovalbumin epitope SIINFEKL was reduced, the cytotoxic response against the SIINFEKL-pulsed EL4 cell line was enhanced (cytotoxicity >90% with HPCs versus <90% w/o HPCs, p<0, 001). In addition, HPCs express CCR7 and CD62L, which should allow their migration to the sites of allopriming. In vivo, none of the mice that had received allogeneic HSCT with HPCs developed clinical or histological GVHD signs as compared to 50% of the control allografted mice without HPCs. Conclusion. Hematopoietic progenitor cells acquire an immunosuppressive potential after G-CSF mobilization. These cells can be isolated from mobilized peripheral stem cells and suppress GVHD while possibly preserving the GVL effect. Work is underway in humans to identify and amplify this population ex vivo for potential therapeutic application in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Differential Plasma Cytokine Levels in Patients with and without Chronic Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4476-4476
Author(s):  
Caron A. Jacobson ◽  
Joshua Geary ◽  
Haesook T Kim ◽  
Sean McDonough ◽  
Carol G Reynolds ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Interferon Γ ◽  
Immune Homeostasis ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Graft Versus Host ◽  
Gm Csf ◽  
Cytokine Levels

Abstract Abstract 4476 Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HSCT). It is not known why some patients develop cGVHD and others do not, but identifying predictive biomarkers would facilitate the development of pre-emptive therapeutic strategies. Investigation into the pathophysiology of cGVHD has revealed different patterns of immune reconstitution in patients who develop cGVHD following HSCT. This suggests that the development of cGVHD occurs as a result of altered immune homeostasis and the inability to establish B and T cell tolerance. We prospectively examined 88 patients with hematologic malignancies following allogeneic HSCT at the Brigham and Woman's Hospital/Dana-Farber Cancer Institute between the years 2004 and 2008 and measured plasma cytokines known to modulate effector T cell, regulatory T cell, and B cell homeostasis and function. Seventy-six patients (86%) received reduced intensity conditioning; 84 (95%) received filgrastim-mobilized peripheral blood stem cells. Median follow-up for all patients was 5 years (range 2.9 to 7.3 years). Fifty-nine percent developed cGVHD. Plasma samples were collected at 1, 3, 6, and 12 months after HSCT and multiplex Luminex bead assays were used to measure levels of the following cytokines: Interferon-γ, IL-2, IL-7, IL-1β, IL-12, TNFα, IL-4, IL-5, IL-6, IL-10, and GM-CSF. Cytokine levels in patients who developed cGVHD were compared to patients who did not develop cGVHD. Results are shown in figure 1. Significantly higher levels of IL-4, IL-6, IL-12, and IL-1β were observed at 1 and/or 3 months after transplantation in patients who subsequently developed cGVHD. IL-4 and IL-6 are characteristic of T-helper-2 (TH2) cellular and humoral immune responses previously associated with cGVHD and fibrogenesis. IL-12 and IL-1β have heterogeneous functions including stimulating interferon-γ and TNFα production, enhancing cytotoxic and helper T cells, and promotion of autoimmunity. IL-1α also stimulates IL-6 production and activates fibroblasts. Finally, GM-CSF was significantly elevated in patients who do not develop cGVHD at 1 year following HSCT (2.5 v 1.46 pg/mL, p=0.017). As shown in figure 1, we observed a general pattern for many cytokines, including IFNγ, IL-2, IL-4, IL-5, IL-6, and IL-1β, in which these cytokines are initially similar in both groups, or lower in patients without cGVHD, but gradually increase over time in patients who do not develop cGVHD. In contrast, cytokine levels remain stable in patients with cGVHD and are generally lower 1 year after transplantation in these patients. These late differences may reflect immune suppressive therapies as well as persistent abnormalities of immune homeostasis. Taken together, these results support the hypothesis that alterations in immune homeostasis early after allogeneic HSCT are significantly associated with the subsequent development of cGVHD. Manipulation of the cytokine environment early after HSCT can modulate immune homeostasis and may be of potential prophylactic value. Administration of homeostatic cytokines late after HSCT may also have therapeutic benefit. Disclosures: No relevant conflicts of interest to declare.

Striking Clinical, Molecular and Immunological Outcomes in Non-Human Primate Hematopoietic Stem Cell Transplantation Using a Novel OX40L Blockade Agent, KY1005, Combined with Rapamycin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3341-3341
Author(s):  
Victor Tkachev ◽  
Scott N. Furlan ◽  
Ben Watkins ◽  
Betty Zheng ◽  
Daniel Hunt ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Free Survival ◽  
The Impact

Abstract While calcineurin inhibition (CNI)-based strategies remain the mainstay for GVHD prevention, CNI are notoriously antagonistic to immune tolerance induction. Rapamycin (Rapa) has been shown to be more pro-tolerogenic; however, the best agents to combine with Rapa are still undetermined, and it remains a second-line GVHD prevention strategy without clear superiority over CNI. Finding tolerogenic partners for Rapa, therefore, represents a critical unmet need in the field. Of the possible partners for Rapa, the OX40/OX40L pathway represents an important target. OX40 is a costimulatory receptor expressed on activated human T cells, which, upon interaction with OX40L delivers activation signals to conventional T cells (Tconv) promoting their proliferation, survival and clonal expansion. Notably, these same OX40/OX40L signals may either inhibit or promote Treg functions, depending on context, suggesting that blockade of this pathway may simultaneously control Tconv activation while permitting Treg homeostasis. During GVHD in non-human primates (NHP), we found OX40L upregulation on myeloid dendritic cells and OX40 upregulation on activated T cells in recipients treated with multiple immunosuppressive agents, including Rapa (Fig 1). These data provided strong rationale for testing KY1005, a novel human monoclonal antibody that binds to OX40L and blocks its interaction with OX40, as a potential partner with Rapa. We tested the outcomes of prophylactic blockade of this pathway on NHP GVHD, using KY1005 alone and in combination with Rapa. These experiments utilized our previously published NHP GVHD model, in which GVHD is studied after T cell-replete haplo-identical HCT. KY1005 was dosed at 10mg/kg weekly from days -2ˆ+54 and Rapa was continued through Day +100. Prophylaxis with KY1005 alone provided initial evidence for its in vivo activity, with control of CD4>CD8 T cell proliferation and mitigation of the expansion of CD4>CD8 T effector/memory cells. Consistent with the partial control of T cell activation, these recipients demonstrated improved GVHD-free survival versus unprophylaxed controls, but disease ultimately broke through (Median Survival Time (MST) = 19.5 days with KY1005 (n=4) compared to 8 days in unprophylaxed recipients (n= 10, Fig 2)). We next investigated the impact of OX40L blockade + Rapa. We have published that Rapa as a monotherapy minimally controlled both immunologic and clinical disease, with an MST = 14 days (n=6). Combined prophylaxis was striking: recipients given KY1005+Rapa (n=5) maintained robust health throughout the entire experiment (MST >100d), and demonstrated high levels of donor T cell chimerism (86 +/- 3% at Day 100), rapid hematopoietic reconstitution, and had a terminal GVHD Grade of 0, compared to a Grade of III-IV in both KY1005- and Rapa-monotherapy cohorts. Immunologic analysis demonstrated synergistic control of both CD4 and CD8 T cell proliferation, restoring it to the level observed during autologous immune reconstitution, and resulting in a concomitant abrogation of CD4 and CD8 memory/effector expansion while preserving T cells with a na•ve phenotype. In striking contrast to the inhibition of Tconv activation by KY1005+Rapa, recipients of dual therapy demonstrated intact Treg reconstitution post-HCT, which resulted in a favorable Treg:Tconv ratio of 5.4 vs 1.4:100 in KY1005+Rapa treated compared to untreated recipients (p < 0.05). Transcriptomic analysis confirmed the unique immunologic state conferred by KY1005+Rapa on purified T cells, with gene arrays from these recipients demonstrating separation from all other transplant cohorts in Principal Component space (Figure 3A) and Class Neighbor Analysis identifying unique expression modules that tracked with KY1005 + Rapa prophylaxis (Figure 3B red and blue boxes). These results underscore the critical role of OX40/OX40L signaling in the development of GVHD and demonstrate the striking control of GVHD in KY1005+Rapa recipients. They represent the first demonstration of uniform, long-term GVHD-free survival in the primate model of high-risk haplo-identical HCT, and the first therapeutic strategy that simultaneously controls Tconv activation while supporting Treg homeostasis in this model. They suggest that OX40L blockade + Rapa is a novel, evidence-based combinatorial strategy to control GVHD that is an exceptional candidate regimen for clinical translation. Disclosures Tkachev: Kymab Ltd: Patents & Royalties: US Patent 9,382,325, Research Funding. Casson:Kymab Ltd: Employment. Kirby:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Bland-Ward:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Kean:Juno Therapeutics, Inc: Research Funding.

scholarly journals Sequential CD19- and CD22-CART Cell Therapies for Relapsed B-Cell Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2126-2126 ◽  
Author(s):  
Shuangyou Liu ◽  
Biping Deng ◽  
Yuehui Lin ◽  
Zhichao Yin ◽  
Jing Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Free Survival ◽  
Car T Cells ◽  
Car T

Abstract With traditional therapies, the prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is extremely poor. Chimeric antigen receptor (CAR) T cell therapy targeting at CD19 has demonstrated a significant efficacy on refractory/relapsed (r/r) B-ALL, but single-target CART could not maintain a long-term remission. Recently, CD22-CART has also shown an exciting result in r/r B-ALL. Here we sequentially applied CD19- and CD22-specific CART cells to treat relapsed B-ALL post-HSCT and observed the therapeutic effect. From June 30,2017 through May 31,2018, twenty-four B-ALL patients (pts) relapsing after allo-HSCT with both antigens CD19 and CD22 expression on blasts were enrolled, the median age was 24 (2.3-55) years. Seventeen pts had hematologic relapse, 6 with both bone marrow and extramedullary (EM) involvements and 1 with EM disease (EMD) only. Fourteen pts had failed to previous therapies including chemotherapy, donor lymphocyte infusion, interferon and even murinized CD19-CART in other hospitals. Recipient-derived donor T cells were collected for producing CAR-T cells, which were transfected by a lentiviral vector encoding the CAR composed of CD3ζ and 4-1BB. Eighteen pts were initially infused with murinized CD19-CART, then humanized CD22-CART; while 6 pts (5 failed to prior murinized CD19-CART and 1 had bright CD22-expression) were initially infused with humanized CD22-CART, then humanized CD19-CART. The time interval between two infusions was 1.5-6 months based on patients' clinical conditions. The average dose of infused CAR T cells was 1.4×105/kg (0.4-9.2×105/kg) for CD19 and 1.9×105/kg (0.55-6.6×105/kg) for CD22. All patients received fludarabine with or without cyclophosphamide prior to each infusion, some pts accepted additional chemo drugs to reduce the disease burden. Treatment effects were evaluated on day 30 and then monthly after each CART, minimal residual disease (MRD) was detected by flow cytometry (FCM) and quantitative PCR for fusion genes, EMD was examined by PET-CT, CT or MRI. Sixteen patients finished sequential CD19- and CD22-CART therapies. Three cases could not undergo the second round of CART infusion (1 died, 1 gave up and 1 developed extensive chronic graft-versus-host disease (GVHD)). The rest of 5 pts are waiting for the second CART. After first T-cell infusion, 20/24 (83.3%) pts achieved complete remission (CR) or CR with incomplete count recovery (CRi), MRD-negative was 100% in CR or CRi pts, 3 (12.5%) cases with multiple EMD obtained partial remission (PR), and 1 (4.2%) died of severe cytokine release syndrome (CRS) and severe acute hepatic GVHD. Sixteen patients (15 CR and 1 PR) underwent the second CART therapy. Before second infusion, 3/15 pts in CR became MRD+ and others remained MRD-. On day 30 post-infusion, 1 of 3 MRD+ pts turned to MRD-, 1 maintained MRD+ ( BCR/ABL+) and 1 had no response then hematologic relapse later. The PR patient still had not obtained CR and then disease progressed. As of 31 May 2018, at a median follow-up of 6.5 (4-10) months, among 16 pts who received sequential CD-19 and CD-22 CART therapies, 1 had disease progression, 2 presented with hematological relapse and 2 with BCR/ABL+ only, the overall survival (OS) rate was 100% (16/16), disease-free survival (DFS) was 81.3% (13/16) and MRD-free survival was 68.8% (11/16). CRS occurred in 91.7% (22/24) pts in the first round of T-cell infusion, most of them were mild-moderate (grade I-II), merely 2 pts experienced severe CRS (grade III-IV). The second CART only caused grade I or no CRS since the leukemia burden was very low. GVHD induced by CART therapy was a major adverse event in these post-HSCT patients. After the first CART, 7/24 (29.2%) pts experienced GVHD, of them, 4 presented with mild skin GVHD, 2 with severe hepatic GVHD (1 recovered and 1 died), and 1 developed extensive chronic GVHD. No severe GVHD occurred in the second infusion. Our preliminary clinical study showed that for B-ALL patients who relapsed after allo-HSCT, single CD19- or CD22- CART infusion resulted in a high CR rate of 83.3%, sequentially combined CD19- and CD22-CART therapies significantly improved treatment outcome with the rate of OS, DFS and MRD-free survival being 100%, 81.3% and 68.8%, respectively, at a median follow-up of 6.5 months. The effect of CART on multiple EMD was not good and CART induced GVHD needs to be cautious. Disclosures No relevant conflicts of interest to declare.

scholarly journals Generation of Memory Stem T Cells Specific for Tumor Antigens and Resistant to Inhibitory Signals By Genome Editing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2202-2202
Author(s):  
Beatrice Claudia Cianciotti ◽  
Alessia Potenza ◽  
Valentina Vavassori ◽  
Zulma Magnani ◽  
Luigi Naldini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Gene Editing ◽  
Tumor Antigens ◽  
Lentiviral Vector ◽  
Adoptive Cell Therapy ◽  
Inhibitory Receptors ◽  
Hematopoietic Stem ◽  
Β Chain

Abstract Tumor-infiltrating lymphocytes (TILs) may express different inhibitory receptors (IRs) on the cell surface that are exploited by cancer cells to evade the immune attack. Thus, exhausted T cells display an unresponsive functional profile and fail to kill tumor cells. TIM-3 and LAG-3 represent highly expressed inhibitory receptors in exhausted T cells infiltrating solid tumors. We recently showed that a large fraction of bone marrow infiltrating CD8+ cells co-express multiple inhibitory receptors, including TIM-3, in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation. These observations advocate for strategies to generate innovative tumor-specific T cell products resistant to inhibitory signals and able to persist long-term in treated patients. To reach these objectives we developed a strategy to simultaneously redirect T cell specificity by TCR gene transfer and permanently disrupt IRs by CRISPR/Cas9 system in long-living memory stem T cells (TSCM) for adoptive cell therapy. Primary T cells were activated with CD3/CD28-conjugated beads and cultured with low doses of IL7 and IL15, to preserve their TSCM phenotype, and electroporated with Cas9/gRNA ribonucleoproteins (RNPs) targeting a coding sequence of TIM-3,LAG3, 2B4 and of the TCR α and β chain constant region (TRAC and TRBC1/2) genes. Each gene was edited alone or in combination in a multiplex approach. The efficiencies of NHEJ-mediated inactivation of each gene were assessed by cytofluorimetric analysis and confirmed at molecular level by surveyor assay or ddPCR. In order to avoid mispairing between the endogenous TCR and the tumor-specific TCR, we simultaneously inactivated the constant regions of α and β chain of the endogenous TCR genes. Efficiency of TRAC and/or TRBC1/2 gene disruption was 98%, when measured as % of CD3neg cells. TCR knocked-out cells could be efficiently (70-85%) transduced with a lentiviral vector encoding for TCR specific for an HLA-A2 restricted peptide from NY-ESO1. We then focused on IR gene inactivation. Single gene editing produced up to 98%, 93% and 63% genetic knock-out of the TIM-3 , the LAG-3 and the 2B4 genes, respectively. We finally combined one IR inactivation with TCR gene editing in a single protocol. We obtained an average of 68% and 69% of TIM3neg and LAG3neg NY-ESO1-TCR redirected T cells, respectively, and more than 90% of edited cells showed a TSCM phenotype. When tested in functional assays, TCR-IR-edited TSCM cells proved highly effective and specific in killing HLA-A2+ NY-ESO1+ multiple myeloma cells. Of notice, TIM-3neg and LAG-3neg cells proved more effective in producing IFNg, TNFa and CD107a, once challenged with tumor cells. In conclusion, by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to inhibitory signals. Ongoing in vivo evaluation of tumor-specific IR disrupted memory stem T cells will be reported and discussed. Disclosures Bonini: Intellia Therapeutics: Research Funding.

Enhanced T Cell Function in Patients with Hematological Malignanicies Undergoing Mobilization for Autologous Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3288-3288
Author(s):  
Adriana I. Colovai ◽  
Joseph Schwartz ◽  
Ronit Slotky ◽  
Zhouru Liu ◽  
Jianshe Fan ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Function ◽  
Stem Cell Mobilization ◽  
Cell Reactivity ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
T Cell Function ◽  
Cell Mobilization

Abstract Various regimens used for hematopoietic stem cell mobilization have been optimized to mobilize a maximum number of CD34+ hematopoietic stem cells (HSC) into the peripheral blood. However, the effects of mobilization regimens on other bone marrow derived cell populations have not been carefully examined. Since T cells from HSC grafts play an important role in anti-tumor responses, we studied T cell function in 11 adult patients with hematological malignancies undergoing mobilization for autologous stem cell transplantation (SCT). Mobilization regimens consisted of G-CSF alone (3 patients) or in combination with: GM-CSF (1 patient), chemotherapy (2), GM-CSF and chemotherapy (3), or AMD3100 (2). T cell function was determined using ImmuKnow (Cylex) assay, an FDA approved test, which measures immune cell function in whole blood by quantifying the amount of ATP released by PHA-stimulated CD4+ T helper (Th) cells. Prior to mobilization treatment, average Th reactivity was 242±128 ng ATP/ml (normal range: 200–525 ng ATP/ml). With mobilization, Th reactivity increased gradually in all patients, reaching peak reactivity values (>525 ng ATP/ml) on day 4–6 of treatment. Efficient stem cell mobilization (≥10 CD34+ cells/ul of blood) was accomplished in 8 out of 11 patients. These 8 patients exhibited an average Th peak reactivity level of 701±126 ng ATP/ml. Stem cell mobilization failed in the remaining 3 patients (<10 CD34+ cells/ul). Two of these patients had received AMD3100 (a CXCR4 inhibitor) and G-CSF, and showed unusually high ATP levels (>1000 ng/ml). Chi-Square analysis indicated that there was an inverse correlation between CD34 stem cell counts and peak ATP levels (p=0.01). However, there was no direct relationship between the number of CD3+ or CD3+CD4+ T cells and Th reactivity. Immunophenotypic studies performed in 4 patients with CD34 counts ≥10 cells/ul and 2 patients with CD34 counts <10 cells/ul indicated that the agents used for stem cell mobilization triggered the expansion of CD4+CD25+ T cell population. However, the vast majority of these T cells did not co-express FoxP3, the characteristic marker of regulatory T cells, consistent with the enhanced T cell reactivity detected in these patients. It, therefore, results that mobilization agents have a “priming” effect on CD4+ T helper cells, inducing their robust activation. This effect may lead to improved anti-tumor surveillance and might contribute to the higher rate of complete remission and overall survival observed in patients with hematological malignancies, who have received autologous SCT in addition to chemotherapy. Thus, monitoring of T cell reactivity by Immuknow assay might help tailor therapy to maximize the patient’s potential to mount an immune response against the tumor and, at the same time, obtain efficient stem cell mobilization.

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