Combination of Three Unique Anti-Tumor Modalities Engineered into iPSC-Derived T Cells Demonstrate a Synergistic Effect in Overcoming Tumor Heterogeneity and Cancer Escape

Abstract Chimeric antigen receptor (CAR) is known to trigger an effective immune response through surface antigen recognition enhanced by T-cell activation signal one (ex. CD3) and signal two (ex. CD28); however, targeting neoantigens and intracellular antigens remains a challenge. On the other hand, the T-cell receptor (TCR) can target neo/intracellular antigens presented by MHC molecules, but often the response is not as potent. The CD16 Fc receptor, which is naturally expressed on NK cells, mediates antibody-dependent cellular cytotoxicity (ADCC), but its application in T cells is yet not fully appreciated. Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16 (tri-modal), to determine whether challenges associated with the treatment of heterogeneous tumors may be overcome. To evaluate the benefit of the three distinct targeting modalities, we tested the functional activity of individual and various combinations of i) anti-CD19 CAR, anti-MICA/B CAR, and anti-BCMA CAR, ii) high-affinity, non-cleavable CD16 (hnCD16), and iii) MR1 and NYESO1 TCR modalities in iT cells. All tested combinations successfully expressed the designated edits and differentiated into iT cells (T-lymphocytes > 95%). Initially, we tested CD19 CAR and MR1 TCR in a 9-day serial killing assay of Nalm6 leukemia cells (CD19 high, MR1 +), where we observed CD19 CAR-iT cells induce prompt CAR-mediated tumor growth inhibition (TGI), saw similar effective killing by MR1 TCR-iT cells but with a 24-hr delay, and observed the most effective response of tumor cell elimination when both where combined in the same iT cell population (relative tumor counts; Day 1, no stim: 2.6, CAR: 0.28, TCR: 1.02, CAR+TCR: 0.02; Day 2, no stim: 5.36, CAR: 0.25, TCR: 0.05, CAR+TCR: 0.02). All conditions (CAR, TCR, and CAR+TCR) reached and maintained complete TGI by Day 9 of the assay (relative tumor count, Day 9, no stim: 50.97, all other stim conditions: <0.01). In line with killing kinetics, the time for the activation marker CD25 upregulation differed between CAR and TCR (peak time and percentage of CD25 +, CAR: Day 1, 49.9%; TCR: Day 5, 74.6%). Co-triggering of CAR and TCR in combination revealed quickest, highest, and sustained CD25 upregulation levels (CD25 +, CAR+TCR Day 1: 62.5%, Days 3 to 9: >90%), indicating a synergistic effect and compatibility between CAR and TCR. Assessing anti-MICA/B CAR and hnCD16, we confirmed the hnCD16-mediated response in iT cells in the presence of anti-MICA/B CAR when crosslinking hnCD16 via biotinylated anti-CD16 antibody with streptavidin (phosphorylated CD3zeta peaked at 10 min upon ADCC triggering), indicating the compatibility between a CAR-iT cell and the hnCD16 motif. Lastly, combining iT cells expressing anti-BCMA CAR + MR1 TCR + hnCD16 with daratumumab (anti-CD38 mAb) in a 9-day serial killing assay demonstrated the best TGI among the groups with a near-elimination of transgenic Nalm6 cells (area under curve, no stim: 30.29, TCR: 1.564, CAR: 0.7087, hnCD16+mAb: 1.452, trimodal+mAb: 0.5824). To assess the function of the tri-modal iT cells in vivo, we used a disseminated xenograft model of B-cell leukemia where a heterogenous mixture of transgenic Nalm6 leukemia cells was used to mimic tumor heterogeneity. Assessment of the bone marrow revealed the unique capacity of each target modality to eliminate its target designated Nalm6 leukemia group, with tri-modal iT cells effectively clearing all populations (Figure 1). In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape. Figure 1 Figure 1. Disclosures Yang: Fate Therapeutics, Inc.: Current Employment. Lin: Fate Therapeutics, Inc.: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Pribadi: Fate Therapeutics, Inc.: Current Employment. Chu: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Mehta: Fate Therapeutics, Inc.: Current Employment. Avramis: Fate Therapeutics, Inc.: Current Employment. Whitlock: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Witty: Fate Therapeutics, Inc.: Current Employment. Peralta: Fate Therapeutics, Inc.: Current Employment. Hosking: Fate Therapeutics: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

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Patient's T Cell Functionality Determines Blinatumomab-Mediated Killing of B-ALL Leukemia Cells

Blood ◽

10.1182/blood-2018-99-114503 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4079-4079

Author(s):

Hong Yin ◽

Xian Jia ◽

Xiaojie Guo ◽

Yong Zhou ◽

Manman Deng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Patient Selection ◽

Philadelphia Chromosome ◽

Leukemia Cells ◽

Mass Cytometry ◽

Specific Lysis ◽

Killing Assay

Abstract Introduction B cell acute lymphoblastic leukemia (B-ALL) is a severe disease caused by malignant transformation and uncontrolled proliferation of immature B lymphocytes. Blinatumomab, a Bi-specific T cell engager (BiTE®) combining the VH and VL domains of two antibodies against human CD19 and CD3, has been approved by U.S. Food and Drug Administration (FDA) for the treatment of Philadelphia chromosome negative (Ph-) relapsed or refractory B-ALL in 2014, and recently Philadelphia chromosome-positive (Ph+) B-ALL. Blinatumomab brings killer T and target B cells into close proximity, activating patients' autologous T cells to kill both normal and malignant B cells via mechanisms such as cytolytic immune synapse formation and inflammatory cytokine production. Clinical trials have shown that there are still patients refractory to blinatumomab. It is thus of great importance to understand the resistance mechanisms and search for biomarkers for patient selection. Methods and Results In this study, we used Mass cytometry-based immunophenotyping and Luminex based multiple protein quantitation, to search for biomarkers correlated with the killing capacity of blinatumomab. First, we collected PBMCs from 12 B-ALL patients for an in vitro killing assay, focusing on the quantity and quality profiling of T cells. PBMCs were used as the effector cells and NALM-6 as the target cells. The final effector to target (E/T) ratio is 10:1. Blinatumomab were added to the co-cultured cells (final concentration 10 ng/mL) for 18hrs. A 5% specific lysis of NALM-6 was used as cut-off threshold. Samples with specific killing above 5% were classified as "positive" for blinatumomab response, and below 5% as "negative". Among the patients analyzed, 4 out of 12 patients (33.3%) showed apparent and specific lysis of NALM6 leukemia cells, while the remaining samples showed no obvious effect. This divergent killing effect of blinatumomab with the presence of PBMCs from different donors may reflect the differentiated treatment response in B-ALL patients. Concurrently we immunophenotyped each patient's PBMCs by Mass cytometry with a panel of antibodies for surface lineage markers and intracellular effector molecules and cytokines /chemokines. Unsupervised clustering showed that a panel of T-cell associated biomarkers highly significantly correlated with B-ALL cell response to blinatumomab (p=0.001, R=0.839). This panel of biomarkers included T cell markers of CD45, CD3 and CD4; cytolytic proteins of Perforin and GranzymeB; cytokines of IL-2, INFγ and TNFα; and chemokine CCL4 (also known as MIP-1b). In a Luminex analysis examining multiple proteins accumulating in the culture supernatant from the in vitro killing assay, the top biomarkers identified by Mass cytometry also showed significant response to blinatumomab at different time points (12, 24 and 48 hrs). They included TNF-α, CCL4, IL-2, INFγ and Granzyme B. Furthermore, released CCL4 at 24 and 48 hrs and INFγ at 48 hrs significantly correlated with blinatumomab mediated cytotoxicity. Conclusion In summary, the immunophenotyping and multiplexed protein measurement by Mass cytometry and Luminex assay identified key biomarkers that may correlate with blinatumomab-mediated killing of B-ALL leukemia cells. The biomarker panel reflected the importance of primed T cell activation, associated key cytokine/chemokine as well as the consequent cytolytic protein release in the overall response to blinatumomab. Thus, in vitro biomarker profiling focusing on T cell status with these key molecules may facilitate patient selection and predict the response to the immunotherapy. Disclosures Fu: Amgen: Research Funding.

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T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells

PLoS Pathogens ◽

10.1371/journal.ppat.1006030 ◽

2016 ◽

Vol 12 (11) ◽

pp. e1006030 ◽

Cited By ~ 12

Author(s):

Aileen G. Rowan ◽

Aviva Witkover ◽

Anat Melamed ◽

Yuetsu Tanaka ◽

Lucy B. M. Cook ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Cell Receptor ◽

Leukemia Cells ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

T Cell Receptor Vβ

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CAR T cells targeting CD99 as an approach to eradicate T-cell Acute Lymphoblastic Leukemia without normal blood cells toxicity

10.21203/rs.3.rs-753466/v1 ◽

2021 ◽

Author(s):

Jiangzhou Shi ◽

Zijian Zhang ◽

Hong Cen ◽

Han Wu ◽

Shangkun Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Bone Marrow Cells ◽

Clinical Success ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T ◽

Pilot Clinical Study

Abstract CAR T cell therapy has shown dramatic clinical success in relapsed or refractory (r/r) B-ALL and other haematological malignancies. However, the loss of specific antigens, cell fratricide, T cell aplasia, and normal T cell separation are challenges in treating T cell leukemia/lymphoma with CAR T therapy. CD99 is a promising antigen to target T-ALL and AML as it is expressed on the majority of T-ALL and AML. Here, we isolated a low-affinity CD99 (12E7) antibody, which specifically recognizes leukemia cells over normal bone marrow cells. T cells transduced with an anti-CD99-specific CAR that contained the 12E7 scFv expanded with minor fratricide, maintained their cytotoxic function and mediated powerful antitumour effects. Subsequently, we conducted a pilot clinical study to evaluate the safety and feasibility of therapy with anti-CD99 CAR T cells in 4 patients with r/r T-LBL (n=1), AML (n=2) or myeloid sarcoma (MS) (n=1). The clinical overall response rate (ORR) was 50% (2/4 patients), and 1 patients (25%) achieved complete remission (CR) for 2 month. Mild cytokine release syndrome (CRS) occurred in 2 patients and the CRS no more than grade 2. Together, our results demonstrate that anti-CD99 CAR T cells specifically recognize and efficiently eliminate CD99+ leukemia cells.

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Regulation of T cell expansion by antigen presentation dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812800116 ◽

2019 ◽

Vol 116 (13) ◽

pp. 5914-5919 ◽

Cited By ~ 15

Author(s):

Andreas Mayer ◽

Yaojun Zhang ◽

Alan S. Perelson ◽

Ned S. Wingreen

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Essential Feature ◽

Adaptive Immune Response ◽

Adaptive Immune System ◽

Effective Response ◽

Effector Cells ◽

Adaptive Immune ◽

T Cell Expansion

An essential feature of the adaptive immune system is the proliferation of antigen-specific lymphocytes during an immune reaction to form a large pool of effector cells. This proliferation must be regulated to ensure an effective response to infection while avoiding immunopathology. Recent experiments in mice have demonstrated that the expansion of a specific clone of T cells in response to cognate antigen obeys a striking inverse power law with respect to the initial number of T cells. Here, we show that such a relationship arises naturally from a model in which T cell expansion is limited by decaying levels of presented antigen. The same model also accounts for the observed dependence of T cell expansion on affinity for antigen and on the kinetics of antigen administration. Extending the model to address expansion of multiple T cell clones competing for antigen, we find that higher-affinity clones can suppress the proliferation of lower-affinity clones, thereby promoting the specificity of the response. Using the model to derive optimal vaccination protocols, we find that exponentially increasing antigen doses can achieve a nearly optimized response. We thus conclude that the dynamics of presented antigen is a key regulator of both the size and specificity of the adaptive immune response.

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The Determination of Immunomodulation and Its Impact on Survival of Rectal Cancer Patients Depends on the Area Comprising a Tissue Microarray

Cancers ◽

10.3390/cancers12030563 ◽

2020 ◽

Vol 12 (3) ◽

pp. 563 ◽

Cited By ~ 1

Author(s):

Elisabeth S. Gruber ◽

Georg Oberhuber ◽

Dietmar Pils ◽

Theresa Stork ◽

Katharina Sinn ◽

...

Keyword(s):

Rectal Cancer ◽

T Cells ◽

Survival Analysis ◽

T Cell ◽

Cell Density ◽

Tumor Heterogeneity ◽

Tissue Sections ◽

Cell Quantification ◽

Cell Densities ◽

Tumor Area

Background: T cell density in colorectal cancer (CRC) has proven to be of high prognostic importance. Here, we evaluated the influence of a hyperfractionated preoperative short-term radiation protocol (25 Gy) on immune cell density in tumor samples of rectal cancer (RC) patients and on patient survival. In addition, we assessed spatial tumor heterogeneity by comparison of analogue T cell quantification on full tissue sections with digital T cell quantification on a virtually established tissue microarray (TMA). Methods: A total of 75 RC patients (60 irradiated, 15 treatment-naïve) were defined for retrospective analysis. RC samples were processed for immunohistochemistry (CD3, CD8, PD-1, PD-L1). Analogue (score 0–3) as well as digital quantification (TMA: 2 cores vs. 6 cores, mean T cell count) of marker expression in 2 areas (central tumor, CT; invasive margin, IM) was performed. Survival was estimated on the basis of analogue as well as digital marker densities calculated from 2 cores (Immunoscore: CD3/CD8 ratio) and 6 cores per tumor area. Results: Irradiated RC samples showed a significant decrease in CD3 and CD8 positive T cells, independent of quantification mode. T cell densities of 6 virtual cores approximated to T cell densities of full tissue sections, independent of individual core density or location. Survival analysis based on full tissue section quantification demonstrated that CD3 and CD8 positive T cells as well as PD-1 positive tumor infiltrating leucocytes (TILs) in the CT and the IM had a significant impact on disease-free survival (DFS) as well as overall survival (OS). In addition, CD3 and CD8 positive T cells as well as PD-1 positive TILs in the IM proved as independent prognostic factors for DFS and OS; in the CT, PD-1 positive TILs predicted DFS and CD3 and CD8 positive T cells as well as PD-1 positive TILs predicted OS. Survival analysis based on virtual TMA showed no impact on DFS or OS. Conclusion: Spatial tumor heterogeneity might result in inadequate quantification of immune marker expression; however, if using a TMA, 6 cores per tumor area and patient sample represent comparable amounts of T cell densities to those quantified on full tissue sections. Consistently, the tissue area used for immune marker quantification represents a crucial factor for the evaluation of prognostic and predictive biomarker potential.

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Host-Reactive Donor CD8+ T Memory Stem Cells but Not Mature T Memory Cells Primed Against Leukemic Cells Mediate Potent Graft-Versus-Leukemia Effect.

Blood ◽

10.1182/blood.v108.11.190.190 ◽

2006 ◽

Vol 108 (11) ◽

pp. 190-190

Author(s):

Elizabeth O. Hexner ◽

Dale Frank ◽

Stephen G. Emerson ◽

Yi Zhang

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Memory T Cells ◽

Leukemic Cells ◽

Leukemia Cells ◽

Effector Memory ◽

Memory Cells ◽

Alloreactive T Cell

Abstract The potent ability of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and donor leukocyte infusion (DLI) to cure leukemia remains the most striking example of the ability of the immune system to recognize and destroy tumors. Unfortunately, both allo-HSCT and DLI are often complicated by graft-versus-host disease (GVHD). In addition, durable responses to conventional DLI for acute leukemias have been disappointing. A better understanding of the mechanisms of alloreactive T cell-mediated anti-leukemia activity will be important for separating the GVL effect from GVHD. Adoptive transfer of selected subsets of T cells specific for miHA- or leukemia associated antigens might offer the chance to maximize GVL while minimizing GVHD. Using mouse models of human GVHD directed against miHAs, we recently demonstrated that antigen-experienced CD44loCD62LhiCD8+ T cells contain T memory stem cells that have greater ability than naïve T cells and mature memory T cells to proliferate and generate alloreactive effector cells and all memory T cell subsets (Nature Medicine, 2005, 11:1299). Using the same mouse model, we have now found that although B6/SJL mice receiving donor CD44hiCD8+ T cells (mature memory cells) primed against B6 mouse-derived myeloid leukemia C1498 cells do not develop clinical GVHD, most will die from C1498 leukemia by day 45 following injection of C1498 cells. Adoptive transfer of CD44loCD8+ T cells primed against C1498 leukemic cells caused clinical GVHD, but the majority of recipients (75%) survived long term free of C1498 leukemia. Surprisingly, the GVL effect of donor CD44loCD8+ T cells primed against C1498 leukemia cells was significantly inhibited when C1498 leukemia cell-primed CD44hiCD8+ T cells and CD44loCD8+ T cells were co-injected into B6/SJL mice, with only 25% of the mice surviving without leukemia. These results suggest that while the GVL effect is clearly mediated by antigen experienced CD44loCD8+ T cells, CD44hiCD8+ T memory cells primed against tumor cells are not only functionally defective in eliminating leukemia cells but are also potent inhibitors of alloreactive T cell-mediated GVL activity. We found that host-reactive effector memory CD8+ T cells produced 10-fold higher IL-10 than unstimulated naïve T cells and T memory stem cells, while CD8+ T memory stem cells expressed upregulated IL-10 receptors. These findings suggest that the inhibitory effect of mature memory T cells on alloreactive T cell-mediated GVL effect may be associated with increased production of IL-10 by mature memory cells and/or enhanced susceptibility of T memory stem cells to IL-10 secreted by mature memory cells. In addition, host dendritic cell activation of donor CD8+ naïve T cells progressively induced the generation of memory stem cells (CD44loCD62LhiSca-1hi), central memory cells (CD44hiCD62Lhi) and effector memory cells (CD44hiCD62Llo). CD8+ T memory stem cells displayed a TCR V-beta repertoire similar to that of unstimulated naive T cells. In contrast, both central memory and effector memory T cells showed a skewed TCR V-beta repertoire. Thus, selective elimination of suppressive CD44hiCD8+ T cells may represent an approach to augmenting GVL activity while preserving a diverse TCR V-beta repertoire.

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Development of a Cell Based Vaccine for the Immunotherapy of Acute and Chronic Leukemia by the Use of Autologous Dendritic Cells Loaded with Monoclonal Antibody Treated or Irradiated Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.4889.4889 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4889-4889

Author(s):

Caroline J. Duncan ◽

Peter R.E. Johnson ◽

Patrick H. Roddie

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Monoclonal Antibodies ◽

T Cell ◽

Myeloid Leukemia ◽

T Cell Subsets ◽

Leukemia Cells ◽

Cytotoxic T Cell ◽

Dc Vaccine ◽

Ctl Responses

Abstract Dendritic cell (DC) vaccines in leukemia show promise as a novel treatment modality however to date clinical evidence of efficacy has been limited. This is likely to be as a consequence of a combination of factors, which include insufficient immunogenicity of the DC vaccine and vaccination taking place in an environment adverse for generation of effective immune responses i.e. in patients with active disease. Our study aims to generate more efficient cytotoxic T cell (CTL) responses by improving DC uptake and presentation of leukemia cells in the remission state and will be applicable to both acute and chronic leukemias. Monoclonal antibodies (MoAbs) have been used to treat malignant cells prior to co-culture with DCs to enhance cross-presentation and generation of specific CTLs. We investigated whether this approach could improve DC induction of CTL responses in comparison to DCs loaded with UVB irradiated apoptotic leukemia cells. In this in vitro study we generated dendritic cells from adherent mononuclear cells (differentiation with GM-CSF and IL-4) of patients in remission following chemotherapy for acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). The immature DCs were loaded with autologous leukemia cells from the patients’ presentation samples. The presentation leukemia cells were treated with either UVB irradiation or appropriate monoclonal antibodies (the anti-CD33 MoAb Mylotarg in AML and CML; the anti CD20 MoAb Rituximab or the anti-CD 52 MoAb Alemtuzumab in CLL). Apoptosis was assessed by Annexin/Propidium iodide labelling. Treatment of the leukemia cells by different MoAbs induced varying degrees of apoptosis. DC uptake of antibody treated or apoptotic leukemia cells was assessed by dual colour staining. Leukemia cells were stained with PKH and DCs labelled with FITC-CD80 or CD86. DC uptake was more efficient with MoAb treated cells irrespective of the degree of apoptosis induced by the MoAb. DCs were matured with TNFa for two days then co-cultured with autologous T cells for one week. T cell subsets and Regulatory T cells were assessed on the presentation and remission samples.The T cells were harvested and their cytoxicity assessed in an Interferon Gamma (IFNg) ELISPOT assay where the unmodified blasts were used as stimulators. Initial results show enhanced anti-leukemia activity in the MoAb treated group as compared to the irradiated group. A similar set up using allogeneic DCs and T cells confirmed the augmentation of CTL responses with MoAb treatment of leukemia cells.The use of MoAb in this setting shows promise for improvement in the success and applicability of DC vaccine strategies in leukemia.

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Donor Immunization with WT1 Peptide Augments Anti-Leukemic Activity After MHC-Matched Bone Marrow Transplantation

Blood ◽

10.1182/blood.v118.21.1896.1896 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1896-1896

Author(s):

Holbrook E Kohrt ◽

Antonia MS Mueller ◽

Jeanette B Baker ◽

Matthew J Goldstein ◽

Evan Newell ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Tumor Antigen ◽

Cd4 T Cell ◽

Leukemia Cells ◽

Peptide Vaccination ◽

Tumor Bearing ◽

Ifn Γ ◽

Anti Tumor Activity

Abstract Abstract 1896 The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part due to immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilm's Tumor 1 (WT1) peptides, induces a T cell population that is tumor antigen specific. We determined whether BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent anti-tumor activity when combined with allotransplantation. WT1 peptide vaccinations of healthy syngeneic or allogeneic donor mice with a 9-mer WT1 peptide (amino acids 126–134, the WT1 9-mer which has the highest binding affinity for H-2Db) and Incomplete Freund's Adjuvant induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that compared to vaccination with IFA alone, four weekly WT1 vaccinations induced an increased percentage of WT1-tetramer+CD8 T-cells (0.15% vs. 1%) in the peripheral blood 28 days following the first vaccination (Figure A *p<.001). CD8 T-cells producing IFN-γ+ after co-culture with tumor cells were similarly increased (0.11% vs. 13.6%) at this timepoint (Figure B *p<.001). They were CD44hi suggesting a memory phenotype, specifically reactive to WT1-expressing tumor (FBL3 and not H11), and increased in a vaccination dose-dependent fashion (Figure A and B). Four weekly WT1 vaccinations prevented tumor growth in donors following intravenous leukemia challenge. In contrast, in tumor-bearing mice, WT1 vaccinations failed to induce WT1-tetramer+ or IFN-γ+ CD8 T-cells and were ineffective as a therapeutic vaccine based on intensity of bioluminescence from luciferase-labeled FBL3 leukemia and mortality. BMT from WT1 vaccinated MHC-matched donors including LP/J and C3H.SW, but not C57BL/6 syngeneic donors, into C57BL/6 recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of luciferase-labeled FBL3 leukemia and survival of 70–90% of mice. Interestingly, the transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells, likely as a result of alloreactive and tumor-antigen reactive T cells contained with the donor total CD8+ T cells. Total and tetramer+CD8+ T cells required CD4+ T cell help for maximal anti-tumor activity, which was equivalent in efficacy from immunized or unimmunized CD4+ T cell donors. Total CD4+ T cells, alone, from immunized donors provided no anti-tumor activity. The infused donor LP/J or C3H.SW CD8+ T cells collected from cured C57BL/6 recipients, were highly reactive against WT1-expressing FBL3 leukemia cells (14% IFN-γ+) compared to non-WT1-expressing H11 leukemia cells (5% IFN-γ+). The circulating, WT1-tetramer+CD8+ T cell population expanded in cured recipients, peaking at 3.5% on day 50 and contracting through day 100 post-BMT to 0.56%. These findings show that peptide vaccination of donor mice with a tumor antigen dramatically enhances GvT activity and is synergistic with allogeneic BMT. This novel and broadly applicable approach, using leukemia-associated antigen immunization to enhance GvT by creating an “educated” donor T cell graft for allogeneic transplantation of patients with acute myeloid leukemia and myelodysplastic syndrome, is currently being translated to a Phase 1 clinical trial at our institution. Disclosures: No relevant conflicts of interest to declare.

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Clinical Trial Evaluating DC/AML Fusion Cell Vaccination Alone and in Conjunction with PD-1 Blockade in AML Patients Who Achieve a Chemotherapy-Induced Remission

Blood ◽

10.1182/blood.v118.21.948.948 ◽

2011 ◽

Vol 118 (21) ◽

pp. 948-948 ◽

Cited By ~ 2

Author(s):

Jacalyn Rosenblatt ◽

Richard M. Stone ◽

Irit Avivi ◽

Lynne Uhl ◽

Donna Neuberg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Induction Chemotherapy ◽

Peripheral Blood ◽

Allogeneic Transplantation ◽

Leukemia Cells ◽

Vaccine Site ◽

Fusion Cell ◽

The Mean

Abstract Abstract 948 Patients with acute myeloid leukemia (AML) achieve remission following chemotherapy; however, curative outcomes remain elusive due to relapse with chemotherapy-resistant disease. Allogeneic transplantation remains a potentially curative therapy for AML patients, but is associated with significant morbidity and mortality due to the lack of specificity of the alloreactive response. A promising area of investigation is the development of cancer vaccines that educate host immunity to more selectively target leukemia cells, including the stem cell compartment. Our group has developed a cancer vaccine model in which dendritic cells (DCs) are fused to autologous tumor cells, resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to vaccination. We are conducting a clinical trial in which AML patients who are in a first or second complete remission following chemotherapy receive three monthly doses of DC/AML fusion cells alone (Cohort 1) or in conjunction with anti-PD1 antibody, CT-011 (cohort 2). To date, 16 patients (9 males, 7 females; mean age 55 years) have been enrolled to the first cohort. All patients underwent successful tumor collection from either a bone marrow aspirate (N=12), collection of 20 cc of peripheral blood (N=3), or leukapheresis product (N=1) at the time of presentation with newly diagnosed AML (N=15) or first relapsed AML (N=1). The mean yield was 1.45×108 cells, and the mean viability was 90%. Tumor cells were subjected to immunohistochemical analysis to identify antigens unique to the leukemia fusion partner. Those patients achieving complete remission following 1–2 cycles of induction chemotherapy underwent leukapheresis for dendritic cell generation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5–7 days, and then exposed to TNFα for 48–72 hours to generate mature DCs. Mean viability of the DC preparation was 92%. DCs strongly expressed the co-stimulatory molecule CD86 (mean 75% expression). One patient died during remission induction chemotherapy and 3 patients were removed from study after induction chemotherapy to undergo allogeneic transplantation. Vaccine was successfully generated in 9 patients at a dose of 5×106 fusions cells, mean fusion efficiency of 30%, and viability of 87%. As a measure of their activity as antigen presenting cells, the capacity of the fusion cell preparation to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.7), the DC and fusion cell preparation were potent stimulators (mean SI 20.8 and 13.1, respectively). Vaccination with the DC/leukemia fusion vaccine was initiated within 12 weeks from count recovery following their final cycle of chemotherapy. 4 patients have completed vaccinations and are 2, 4, 5 and 6 months following the final vaccine. One patient was taken off study for disease progression one week after receiving his first vaccine. 4 patients experienced grade 1 vaccine site reactions. Biopsy of a vaccine site reaction demonstrated a dense T cell infiltrate. Additional vaccine related adverse events have included grade 1 ankle pain and edema. The remaining patients are undergoing chemotherapy, and when complete, will initiate vaccination. Peripheral blood samples are being collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Immune response targeting leukemia cells, leukemic stem cells, and leukemia associated antigens will be assessed. Levels of circulating regulatory T cells and T cell expression of PD1 will be measured. Time to disease progression will also be determined. Disclosures: Avigan: Curetech: Research Funding.

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Recovery Of Gamma/Delta+ T Cells After Transplantation With Alpha-Beta+/CD19+ Lymphocyte Depleted Hematopoietic Stem Cells From HLA-Haploidentical Donors

Blood ◽

10.1182/blood.v122.21.3245.3245 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3245-3245

Author(s):

Irma Airoldi ◽

Ignazia Prigione ◽

Alice Bertaina ◽

Claudia Cocco ◽

Daria Pagliara ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Zoledronic Acid ◽

Γδ T Cells ◽

Leukemia Cells ◽

Γδ T Cell ◽

Hematopoietic Stem ◽

Functional Studies ◽

Αβ T Cells ◽

Over Time

Abstract HLA-haploidentical hematopoietic stem cell transplantation (HSCT) using CD34+ selected cells is a widely used procedure, which, however, is complicated by delayed immune reconstitution. We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature natural killer (NK) cells and γδ+ T cells in the graft. These cells can exert a graft-versus-leukemia (GvL) effect and reduce the risk of infection. In particular, unconventional γδ T cells play a critical role in both innate and adaptive immunity and exert HLA-unrestricted cytotoxicity against both solid and hematological tumors, thus potentially acting as beneficial effector cells in transplanted patients. Moreover, such grafts may limit the risk of graft-versus-host disease and prevent EBV-related lymphoproliferative disease. We performed phenotypic and functional studies on γδ T cells collected from 20 pediatric patients (pts, 13 males, 7 females, median age 10 years, range 6 months to 16 years) that received this type of allograft. Eighteen pts had acute leukemia and 2 non-malignant disorders. Ex vivo assays of peripheral blood γδ T cell phenotype and function were performed weekly until Hospital discharge and monthly until 6 months after HSCT. Phenotype of γδ T cells was analysed by flow cytometry. Analyses were performed on mononuclear cells labelled with mAb panels (CD3, CD45, pan-γδ, anti-Vδ1, -Vδ2, -Vγ9, CD45RO, CD45RA, CD27, CD16, CD56) allowing the identification of the main γδ+ T cell subsets, including Vδ1+ and Vδ2+ cells, naïve, central memory (CM), effector memory (EM) and terminally differentiated (TD) γδ T cells. Functional studies were performed using γδ T cells shortly after collection from pts, as well as after in vitro expansion with zoledronic acid and IL-2 for 10 days. Cytotoxic activity of γδ T cells was tested against primary leukemia cells, through CD107a degranulation assay and/or standard 51Cr-release assay. In the first 4 weeks after HSCT, T cells were consistently of the γδ subset (>90% of CD45+CD3+ cells); by contrast, αβ+ T cells gradually increased over time. In approximately half of the pts, the percentage of αβ T cells exceeded that of γδ T cells already starting from 30 days after HSCT. γδ T cells consisted of Vδ2+Vγ9+ and Vδ1+Vγ9+/- cells, and marginally of the Vδ1-Vδ2-Vγ9- population. Detailed phenotypic characterization of Vδ1+ and Vδ2+ γδ T cells revealed that, at day +20 after HSCT, 44% of Vδ1+ cells were CM (identified as CD45RO+CD27+ cells), 26% naïve (CD45RO-CD27+), 21.4% TD (CD45RO-CD27-) and 6.1% EM (CD45RO+CD27-). Similarly, 55.4% of Vδ2+ γδ T lymphocytes were CM, 9.8% naïve, 11.4% TD and 23.1% EM. The proportion of the different Vδ2+ γδ T cell subset did not change significantly over time, especially when comparing that present at day +20 after HSCT (time point, TP1) with that measured 30 days after the attainment of a 1:1 ratio of αβ-to- γδ T cells (TP2) (Figure 1, left panel). By contrast, by comparing TP1 and TP2, we found that Vδ1+ CM γδ T cells decreased and EM cells increased over time, while naïve or TD Vδ1+ γδ T cells did not change (Figure 1, right panel). In transplanted pts experiencing cytomegalovirus (CMV) reactivation, γδ T cells mostly consisted of Vδ1+ cells (mean 59.8% of γδ T cells), among which 49% were TD, 22.7% EM, 18.9% CM and 10.1% naïve. Noteworthy, in transplanted pts who did not have CMV reactivation, the main γδ T cells showed a Vδ2+ phenotype. Functional studies revealed that pt-derived γδ T cells consistently expanded in vitro after exposure to zoledronic acid and IL-2, the resulting Vγ9Vδ2 population expressing mainly an EM phenotype. These Vγ9Vδ2 cells exerted cytotoxic activities against primary allogeneic leukemia cells, especially when leukemia cells were pre-treated with zoledronic acid (Figure 2). More importantly, both Vδ1+ and Vδ2+ γδ T cells obtained from transplanted pts showed cytotoxic activity against primary leukemia cells, as assessed by CD107a degranulation assay. In conclusion, we provide the first phenotypic and functional characterization of γδ T cells, analyzed over time in children transplanted with grafts depleted of αβ+ T cells and of B lymphocytes. Our results support the concept that γδ T cells are important effector cells, which can be expanded and activated after exposure to bisphosphonates and IL-2 with the aim of improving their killing capacity against leukemia cells. Disclosures: No relevant conflicts of interest to declare.

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