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Duoc-01, a Cord Blood Derived Cell Therapy Product, Ameliorates Experimental Autoimmune Encephalomyelitis
Abstracts / Biol Blood Marrow Transplant 25 (2019) S100 S289
225
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CARTs in B cell malignancies. These findings encourage further study of TP0903 as an enhancer of T cell immunotherapies.
Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies Reona Sakemura MD, PhD1,2, Nan Yang MD1,2,3, Michelle J. Cox1,2, Sutapa Sinha PhD2, Mehrdad Hefazi MD1,2, Michael J. Hansen4, Kendall J. Schick1,2,5, Justin C Boysen2, Renee C Tschumper4, Lars Mouritsen6, Jason M Foulks6, Steven L Warner6, Sameer Parikh MBBS2, Wei Ding MD2, Neil E. Kay MD2, Saad S. Kenderian MD1. 1 T-Cell Engineering Program, Mayo Clinic, Rochester, MN; 2 Division of Hematology, Mayo Clinic, Rochester, MN; 3 Department of Hematology, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi'an, China; 4 Department of Immunology, Mayo Clinic, Rochester, MN; 5 Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN; 6 Tolero Pharmaceuticals, Inc., Lehi, UT Despite the remarkable results of CD19 directed chimeric antigen receptor T cell (CART19) therapy, the durable responses in B cell lymphoma and chronic lymphocytic leukemia (CLL) is limited. This may be due to the loss of CART persistence, poor trafficking and inhibition by the tumor microenvironment. Strategies to enhance CART function are thus needed. Recent data have shown that abnormal expression of receptor tyrosine kinase (RTK) AXL portends poor prognosis. Inhibition of AXL with TP0903, a high affinity AXL inhibitor, has been found to induce robust apoptosis of CLL B cells. We therefore examined the role of AXL inhibition with TP0903 on T cell and CART19 function. First, we investigated the effect of AXL inhibition on na€ıve T cells. Cells stimulated with PMA/Ionomycin and cultured with TP0903 showed significant reduction of Th2 cytokines and inhibitory receptors (Fig 1a). Effector and regulatory T cells (Treg) treated with TP0903 showed a preferential reduction of Tregs (Fig 1b). Next, we investigated the influence of TP0903 on 41BB costimulated CART19. TP0903 led to polarization of CARTs into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP0903 treatment also significantly downregulated inhibitory receptors on activated CARTs, including cytokine release syndrome (CRS) related cytokines (Fig 1c). The combination of CART19 and TP0903 yielded a synergistic cytotoxicity against JeKo in vitro (Fig 1d). We compared the transcriptome of activated CARTs treated with TP0903, and noted >100 genes that were differentially expressed compared to non-treated cells. Among these, cell junction, cell migration, and immune synapse related genes were significantly increased in activated CARTs treated with TP0903. To investigate the effect of AXL inhibition of CARTs with TP0903 in vivo, we established MCL xenografts via the injection of JeKo into NSG mice. The mice were then treated with vehicle alone, TP0903 alone, CART19 alone, or TP0903 + CART19. Three weeks after the treatment, mice were rechallenged with JeKo. Mice treated with CART19 and TP0903 rejected the JeKo challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART persistence (Fig 1e). Finally, we validated our findings in a phase I clinical trial of TP0903 (NCT02729298). T cells isolated from 3 cancer patients showed significant reduction of Tregs. This will be further investigated in a planned phase I clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634). In summary, we showed for the first time that AXL inhibitior polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes
Figure 1. (a) Inhibitory receptors expression in na€ıve T cells after TCR activation with CD3 stimulation in the increasing dose of TP-0903. Na€ıve T cells were incubated with anti CD3/CD28 beads at a 1:3 ratio in the presence of TP0903. At day 3, T cells were stained with PD-1, TIM-3, Lag-3, and ICOS. (b) TP0903 specifically reduces Treg. Effector T cells (Teff) and regulatory T cells (Treg) were stimulated with anti CD3/CD28 beads and 400 U/ml of IL-2. At day 5, total T cells were evaulated via CountBright quantification. (c) CART19 cytokine assay in the presence TP-0903. UTDs or CART19 were incubated with JeKo in the presence of TP-0903 at a 1:1 ratio. Supernatant was harvested and analyzed by ELISA after 3 days. TP-0903 selectively reduced Th2 and cytokine release syndrome (CRS) related cytokines. (d) CART19 cytotoxicity in the presence TP-0903. CART19 or UTDs were cocultured at different effector-totarget ratios (E:T) with luciferase-positive JeKo in the presence of increasing doses of TP-0903. Cell killing was assessed by luminescence at 48 hours (** P<0.001, *P<0.05, one-way ANOVA analysis). (e) TP-0903 enhanced CART cell persistence in vivo. NSG mice were injected with 1.0 £ 106 luciferase-positive Jeko on day -7. At day 0, mice were randomized according to tumor burden to receive vehicle, TP-0903 20mg/kg/day. 1.0£106 CART19, or TP-0903 20mg/kg/day +0.5£106 CART19. At day 21, mice were re-challenged with Jeko.
226 Duoc-01, a Cord Blood Derived Cell Therapy Product, Ameliorates Experimental Autoimmune Encephalomyelitis Arjun Saha PhD1, Li Xu MSc2, Aruni Gunaratne BS2, Andrew E. Balber PhD3, Jesse D Troy PhD4, Christopher Overall5, Anthony Filiano PhD2, Joanne Kurtzberg MD6. 1 Marcus Center for Cellular Therapy, Duke University Medical Center, Durham, NC; 2 Duke, Durham, NC; 3 The Carolinas Cord Blood Bank and Robertson Cell and Translational Therapy Program, Duke University Medical Center, Durham, NC; 4 Pediatric Blood and Marrow Transplant Division, Duke University Medical Center, Durham, NC; 5 UVA, Charlottesville, VA; 6 The Marcus Center for Cellular Cures, Duke University Medical Center, Durham, NC DUOC-01 is a cord-blood derived, macrophage-based cell therapy product, developed by our group to treat demyelinating conditions of the central nervous system. Recently, we demonstrated that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the corpus callosum of immune-incompetent mice treated with cuprizone. To explore the mechanism and investigate whether DUOC-01 will
S166
Abstracts / Biol Blood Marrow Transplant 25 (2019) S100 S289
be effective in other experimental models of demyelination, we tested DUOC-01 in lysophosphatidylcholine (LPC) mediated demyelinated murine organotypic cerebellar brain slices and a mouse model of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. In the cerebellar brain slices, we found that DUOC-01 enhanced remyelination, reduced gliosis, and promoted the proliferation of oligodendrocyte progenitor cells in brain slices demyelinated by LPC compared to the untreated control samples. When we injected DUOC-01 cells into the cerebrospinal fluid of mice immunized for EAE, we found decreased severity of clinical disease compared to the vehicle injected control. Currently we are analyzing single cell sequencing data to determine various populations present in DUOC-01 cultures and to understand the functional pathways responsible for promoting remyelination and reducing neuroinflammation. Overall, our data suggest that DUOC-01 could be beneficial in treating diverse neurological conditions with demyelination.
227 Harnessing MSC Osteoprogenitor Subpopulations to Augment Hematopoietic Transplantations Zhiyong Poon PhD1, Xiubo Fan PhD2, William Ying Khee Hwang FRCP, FAMS, MMed, MRCP, MBBS1. 1 Dept. of Hematology, Singapore General Hospital, Singapore, Singapore; 2 Dept. of Clinical Research, Singapore General Hospital, Singapore, Singapore Adjuvant MSC infusions could improve transplantation outcomes via suppression of graft-vs-host reactions and enhancement of HSPC engraftment. However, bone marrow (BM) MSCs are a heterogeneous cell population, particularly when culture expanded to obtain therapeutically relevant cell numbers. A systematic study of expanded subpopulations is hindered by the lack of unique surface markers. In lieu of immunophenotype, we hypothesized and verified that biophysical properties of MSCs can be a useful strategy for isolating unique subpopulations. Here, we will describe a clinically relevant biophysical strategy for deriving different MSC subpopulations and discuss their potential utility in the context of hematopoietic transplantation. Cell diameter, membrane stiffness and nuclear membrane fluctuations are collectively predictive of at least two MSC subpopulations (multipotent and osteoprogenitors). Both MSC subpopulations are distinct in their transcriptome (different signatures of VEGF, IL8, ANG1, etc) and differentiation potential (osteogenic and adipogenic) but no surface marker reliably distinguishes them apart. In functional assays, osteoprogenitor vs multipotent MSCs show stronger capacities for CD34+CD38¡HSPC expansion (Day 10: 42.7 § 6.21 vs 29.1 § 8.27, n = 5, respectively), but no significant differences (n = 3 biological repeats) in their ability to suppress CD8+T-cell proliferation in mixed lymphocyte reactions. When used as a rescue therapy for lethal irradiation, osteoprogenitors improved survival rates by 30-40% compared to multipotent MSCs. Part of the recovery process was prompted by MSC secretome mediated stimulation of BM tissue repair, including increased cell proliferation, reduced inflammation and the restoration of niche environments. These paracrine effects may be beneficial in fostering favorable post-myeloablative BM environments for donor HSPCs to home, engraft and survive. In murine transplantation models, we observed a higher rate of early engraftment following adjuvant therapy with osteoprogenitors. Week 4 CD45 chimerism in peripheral blood was 2- to 3- fold higher (»11% vs »4%) in the osteoprogenitor treated group compared to the other MSC groups. These results demonstrate heterogeneity in expanded MSCs populations that would lead to inconsistencies in their overall
properties. Newer biophysical technologies have the potential for isolating useful subpopulations defined by key quality attributes for specific forms of MSC-therapy; these capabilities may advance MSC-based therapy for regenerative medicine. Here, we demonstrate the augmentation of HSPC transplants in murine models using a biophysically isolated osteogenic MSC subpopulation (osteoprogenitors) which led to the fastest engraftment rates; this may be due to their superior abilities for promoting BM tissue regeneration as well as hematopoietic expansion.
228 zary Syndrome for Immunophenotyping and Modeling Se Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy Karl William Staser MD, PhD1,2, Matthew Cooper PhD2, Jaebok Choi PhD3, Anand Chukka3, Kidist Ashami BS3, Jessica Niswonger BS2, Jaehan Park PhD4, Byung Ha Lee PhD4, Shunqiang Li PhD3, Amanda Cashen MD3, Amy Musiek MD1, John F. DiPersio MD, PhD2,5. 1 Division of Dermatology, Washington University in St. Louis, St. Louis, MO; 2 Department of Medicine, Division of Oncology, Washington University, Saint Louis, MO; 3 Division of Oncology, Washington University in St. Louis, St. Louis, MO; 4 NeoImmuneTech, Rockville, MD; 5 Section of Bone Marrow Transplantation and Leukemia, Washington University in St. Louis, St. Louis, MO zary syndrome (SS) is a highly-morbid T cell Background: Se leukemic lymphoma with no widely-effective treatments and few preclinical models. SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo. Methods: To generate SS PDX models, we injected NSG-SGM3 mice with »2 £ 106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers. To generate UCART2s, we CRISPR/Cas9 multiplexed edited CD2 and TRAC from CART2. For initial testing, we injected NSG mice zary cell line (HH) on day -4 with 5 £ 105 cells from a human Se from UCART2 treatment, followed by NT-I7 (10mg/kg SC) on days +1, +15 and +29. Results: SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p<0.01, SS vs. normal PBMCs), and loss of effector memory CD4 cells (8% vs 34%, p<0.001, SS vs. normal PBMCs). SS cells were CD3+CD4+CD2+CD5+CD8- with variable CD7 loss and PD1 gain. Four of six unique human SS samples injected in NSG-SGM3 mice engrafted within »6 weeks with no signs of xenogeneic GVHD. Following engraftment, SS cells showed near ubiquitous PD1 expression (>90% vs »20%, p<0.001, SS vs. normal PBMCs), CD7 loss, and increased CD30 and CD26 expression. Immunohistochemistry further revealed atypical CD3+CD4+CD8-CD7- lymphocytes lining the dermoepidermal junction. UCART2-treated HHCBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HHCBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p<0.0001, UCART2 vs. UCART19). Moreover, UCART2-treated HHCBR-GFP mice survived »40 days as compared to »21 days in
225
S165
CARTs in B cell malignancies. These findings encourage further study of TP0903 as an enhancer of T cell immunotherapies.
Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies Reona Sakemura MD, PhD1,2, Nan Yang MD1,2,3, Michelle J. Cox1,2, Sutapa Sinha PhD2, Mehrdad Hefazi MD1,2, Michael J. Hansen4, Kendall J. Schick1,2,5, Justin C Boysen2, Renee C Tschumper4, Lars Mouritsen6, Jason M Foulks6, Steven L Warner6, Sameer Parikh MBBS2, Wei Ding MD2, Neil E. Kay MD2, Saad S. Kenderian MD1. 1 T-Cell Engineering Program, Mayo Clinic, Rochester, MN; 2 Division of Hematology, Mayo Clinic, Rochester, MN; 3 Department of Hematology, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi'an, China; 4 Department of Immunology, Mayo Clinic, Rochester, MN; 5 Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN; 6 Tolero Pharmaceuticals, Inc., Lehi, UT Despite the remarkable results of CD19 directed chimeric antigen receptor T cell (CART19) therapy, the durable responses in B cell lymphoma and chronic lymphocytic leukemia (CLL) is limited. This may be due to the loss of CART persistence, poor trafficking and inhibition by the tumor microenvironment. Strategies to enhance CART function are thus needed. Recent data have shown that abnormal expression of receptor tyrosine kinase (RTK) AXL portends poor prognosis. Inhibition of AXL with TP0903, a high affinity AXL inhibitor, has been found to induce robust apoptosis of CLL B cells. We therefore examined the role of AXL inhibition with TP0903 on T cell and CART19 function. First, we investigated the effect of AXL inhibition on na€ıve T cells. Cells stimulated with PMA/Ionomycin and cultured with TP0903 showed significant reduction of Th2 cytokines and inhibitory receptors (Fig 1a). Effector and regulatory T cells (Treg) treated with TP0903 showed a preferential reduction of Tregs (Fig 1b). Next, we investigated the influence of TP0903 on 41BB costimulated CART19. TP0903 led to polarization of CARTs into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP0903 treatment also significantly downregulated inhibitory receptors on activated CARTs, including cytokine release syndrome (CRS) related cytokines (Fig 1c). The combination of CART19 and TP0903 yielded a synergistic cytotoxicity against JeKo in vitro (Fig 1d). We compared the transcriptome of activated CARTs treated with TP0903, and noted >100 genes that were differentially expressed compared to non-treated cells. Among these, cell junction, cell migration, and immune synapse related genes were significantly increased in activated CARTs treated with TP0903. To investigate the effect of AXL inhibition of CARTs with TP0903 in vivo, we established MCL xenografts via the injection of JeKo into NSG mice. The mice were then treated with vehicle alone, TP0903 alone, CART19 alone, or TP0903 + CART19. Three weeks after the treatment, mice were rechallenged with JeKo. Mice treated with CART19 and TP0903 rejected the JeKo challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART persistence (Fig 1e). Finally, we validated our findings in a phase I clinical trial of TP0903 (NCT02729298). T cells isolated from 3 cancer patients showed significant reduction of Tregs. This will be further investigated in a planned phase I clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634). In summary, we showed for the first time that AXL inhibitior polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes
Figure 1. (a) Inhibitory receptors expression in na€ıve T cells after TCR activation with CD3 stimulation in the increasing dose of TP-0903. Na€ıve T cells were incubated with anti CD3/CD28 beads at a 1:3 ratio in the presence of TP0903. At day 3, T cells were stained with PD-1, TIM-3, Lag-3, and ICOS. (b) TP0903 specifically reduces Treg. Effector T cells (Teff) and regulatory T cells (Treg) were stimulated with anti CD3/CD28 beads and 400 U/ml of IL-2. At day 5, total T cells were evaulated via CountBright quantification. (c) CART19 cytokine assay in the presence TP-0903. UTDs or CART19 were incubated with JeKo in the presence of TP-0903 at a 1:1 ratio. Supernatant was harvested and analyzed by ELISA after 3 days. TP-0903 selectively reduced Th2 and cytokine release syndrome (CRS) related cytokines. (d) CART19 cytotoxicity in the presence TP-0903. CART19 or UTDs were cocultured at different effector-totarget ratios (E:T) with luciferase-positive JeKo in the presence of increasing doses of TP-0903. Cell killing was assessed by luminescence at 48 hours (** P<0.001, *P<0.05, one-way ANOVA analysis). (e) TP-0903 enhanced CART cell persistence in vivo. NSG mice were injected with 1.0 £ 106 luciferase-positive Jeko on day -7. At day 0, mice were randomized according to tumor burden to receive vehicle, TP-0903 20mg/kg/day. 1.0£106 CART19, or TP-0903 20mg/kg/day +0.5£106 CART19. At day 21, mice were re-challenged with Jeko.
226 Duoc-01, a Cord Blood Derived Cell Therapy Product, Ameliorates Experimental Autoimmune Encephalomyelitis Arjun Saha PhD1, Li Xu MSc2, Aruni Gunaratne BS2, Andrew E. Balber PhD3, Jesse D Troy PhD4, Christopher Overall5, Anthony Filiano PhD2, Joanne Kurtzberg MD6. 1 Marcus Center for Cellular Therapy, Duke University Medical Center, Durham, NC; 2 Duke, Durham, NC; 3 The Carolinas Cord Blood Bank and Robertson Cell and Translational Therapy Program, Duke University Medical Center, Durham, NC; 4 Pediatric Blood and Marrow Transplant Division, Duke University Medical Center, Durham, NC; 5 UVA, Charlottesville, VA; 6 The Marcus Center for Cellular Cures, Duke University Medical Center, Durham, NC DUOC-01 is a cord-blood derived, macrophage-based cell therapy product, developed by our group to treat demyelinating conditions of the central nervous system. Recently, we demonstrated that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the corpus callosum of immune-incompetent mice treated with cuprizone. To explore the mechanism and investigate whether DUOC-01 will
S166
Abstracts / Biol Blood Marrow Transplant 25 (2019) S100 S289
be effective in other experimental models of demyelination, we tested DUOC-01 in lysophosphatidylcholine (LPC) mediated demyelinated murine organotypic cerebellar brain slices and a mouse model of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. In the cerebellar brain slices, we found that DUOC-01 enhanced remyelination, reduced gliosis, and promoted the proliferation of oligodendrocyte progenitor cells in brain slices demyelinated by LPC compared to the untreated control samples. When we injected DUOC-01 cells into the cerebrospinal fluid of mice immunized for EAE, we found decreased severity of clinical disease compared to the vehicle injected control. Currently we are analyzing single cell sequencing data to determine various populations present in DUOC-01 cultures and to understand the functional pathways responsible for promoting remyelination and reducing neuroinflammation. Overall, our data suggest that DUOC-01 could be beneficial in treating diverse neurological conditions with demyelination.
227 Harnessing MSC Osteoprogenitor Subpopulations to Augment Hematopoietic Transplantations Zhiyong Poon PhD1, Xiubo Fan PhD2, William Ying Khee Hwang FRCP, FAMS, MMed, MRCP, MBBS1. 1 Dept. of Hematology, Singapore General Hospital, Singapore, Singapore; 2 Dept. of Clinical Research, Singapore General Hospital, Singapore, Singapore Adjuvant MSC infusions could improve transplantation outcomes via suppression of graft-vs-host reactions and enhancement of HSPC engraftment. However, bone marrow (BM) MSCs are a heterogeneous cell population, particularly when culture expanded to obtain therapeutically relevant cell numbers. A systematic study of expanded subpopulations is hindered by the lack of unique surface markers. In lieu of immunophenotype, we hypothesized and verified that biophysical properties of MSCs can be a useful strategy for isolating unique subpopulations. Here, we will describe a clinically relevant biophysical strategy for deriving different MSC subpopulations and discuss their potential utility in the context of hematopoietic transplantation. Cell diameter, membrane stiffness and nuclear membrane fluctuations are collectively predictive of at least two MSC subpopulations (multipotent and osteoprogenitors). Both MSC subpopulations are distinct in their transcriptome (different signatures of VEGF, IL8, ANG1, etc) and differentiation potential (osteogenic and adipogenic) but no surface marker reliably distinguishes them apart. In functional assays, osteoprogenitor vs multipotent MSCs show stronger capacities for CD34+CD38¡HSPC expansion (Day 10: 42.7 § 6.21 vs 29.1 § 8.27, n = 5, respectively), but no significant differences (n = 3 biological repeats) in their ability to suppress CD8+T-cell proliferation in mixed lymphocyte reactions. When used as a rescue therapy for lethal irradiation, osteoprogenitors improved survival rates by 30-40% compared to multipotent MSCs. Part of the recovery process was prompted by MSC secretome mediated stimulation of BM tissue repair, including increased cell proliferation, reduced inflammation and the restoration of niche environments. These paracrine effects may be beneficial in fostering favorable post-myeloablative BM environments for donor HSPCs to home, engraft and survive. In murine transplantation models, we observed a higher rate of early engraftment following adjuvant therapy with osteoprogenitors. Week 4 CD45 chimerism in peripheral blood was 2- to 3- fold higher (»11% vs »4%) in the osteoprogenitor treated group compared to the other MSC groups. These results demonstrate heterogeneity in expanded MSCs populations that would lead to inconsistencies in their overall
properties. Newer biophysical technologies have the potential for isolating useful subpopulations defined by key quality attributes for specific forms of MSC-therapy; these capabilities may advance MSC-based therapy for regenerative medicine. Here, we demonstrate the augmentation of HSPC transplants in murine models using a biophysically isolated osteogenic MSC subpopulation (osteoprogenitors) which led to the fastest engraftment rates; this may be due to their superior abilities for promoting BM tissue regeneration as well as hematopoietic expansion.
228 zary Syndrome for Immunophenotyping and Modeling Se Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy Karl William Staser MD, PhD1,2, Matthew Cooper PhD2, Jaebok Choi PhD3, Anand Chukka3, Kidist Ashami BS3, Jessica Niswonger BS2, Jaehan Park PhD4, Byung Ha Lee PhD4, Shunqiang Li PhD3, Amanda Cashen MD3, Amy Musiek MD1, John F. DiPersio MD, PhD2,5. 1 Division of Dermatology, Washington University in St. Louis, St. Louis, MO; 2 Department of Medicine, Division of Oncology, Washington University, Saint Louis, MO; 3 Division of Oncology, Washington University in St. Louis, St. Louis, MO; 4 NeoImmuneTech, Rockville, MD; 5 Section of Bone Marrow Transplantation and Leukemia, Washington University in St. Louis, St. Louis, MO zary syndrome (SS) is a highly-morbid T cell Background: Se leukemic lymphoma with no widely-effective treatments and few preclinical models. SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo. Methods: To generate SS PDX models, we injected NSG-SGM3 mice with »2 £ 106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers. To generate UCART2s, we CRISPR/Cas9 multiplexed edited CD2 and TRAC from CART2. For initial testing, we injected NSG mice zary cell line (HH) on day -4 with 5 £ 105 cells from a human Se from UCART2 treatment, followed by NT-I7 (10mg/kg SC) on days +1, +15 and +29. Results: SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p<0.01, SS vs. normal PBMCs), and loss of effector memory CD4 cells (8% vs 34%, p<0.001, SS vs. normal PBMCs). SS cells were CD3+CD4+CD2+CD5+CD8- with variable CD7 loss and PD1 gain. Four of six unique human SS samples injected in NSG-SGM3 mice engrafted within »6 weeks with no signs of xenogeneic GVHD. Following engraftment, SS cells showed near ubiquitous PD1 expression (>90% vs »20%, p<0.001, SS vs. normal PBMCs), CD7 loss, and increased CD30 and CD26 expression. Immunohistochemistry further revealed atypical CD3+CD4+CD8-CD7- lymphocytes lining the dermoepidermal junction. UCART2-treated HHCBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HHCBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p<0.0001, UCART2 vs. UCART19). Moreover, UCART2-treated HHCBR-GFP mice survived »40 days as compared to »21 days in