Modeling Sézary Syndrome for Immunophenotyping and Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy

Background: Sézary syndrome (SS) is a highly-morbid T cell leukemic lymphoma with no widely-effective treatments and few preclinical models. We demonstrated effective T cell lymphoma therapy with allogeneic gene-edited anti-CD7 CARTs (Cooper et al, Leukemia, 2018). However, 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 NOD scid IL2Rgamma^null (NSG) mice expressing SCF, GM-CSF, and IL-3 (NSG-SGM3) with ~2x10⁶ 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 (Staser et al, Cytometry A, 2018). To generate UCART2s, we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9, a TRAC-targeted gRNA, and a CD2-targeted gRNA followed by viral transduction with an anti-CD2 scFv 3^rd generation CAR. For initial UCART2 testing, we injected NSG mice with 5x10⁵ cells from a human Sézary cell line transduced with click beetle red luciferase (HH^CBR-GFP) four days prior to UCART2 treatment. Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART2 infusion.

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 dermo-epidermal junction. Second generation PDXs showed infiltration of the spleens, blood, and bone marrow with CD2+CD7- human cells and developed alopecia, scaling, and subcutaneous and intraperitoneal masses, with immunophenotyping, sequencing, and UCART treatment studies ongoing.

To test UCART2's efficacy in killing SS cells in vivo, we injected NSG mice with HH^CBR-GFP+ cells. UCART2-treated HH^CBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HH^CBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p<0.0001, UCART2 vs. UCART19). Moreover, UCART2-treated HH^CBR-GFP mice survived ~40 days as compared to ~21 days in the UCART19 group. Remarkably, UCART2-treated HH^CBR-GFP mice receiving NT-I7 showed virtually no tumor burden (maximum 10⁶ photon flux/s vs. 10¹⁰ photon flux/s, UCART2+NT-I7 vs. UCART2 only groups) with 100% of UCART2- and NT-I7-treated HH^CBR-GFP mice surviving beyond 49 days (Figure 1).

Discussion: We describe the generation of physiologically-relevant SS preclinical models, comprehensive immunophenotyping of patient SS samples, clonal SS PDX outgrowth, and the highly effective anti-tumor activity of UCART2- plus NT-I7-mediated killing of SS cells in vivo using an NSG xenograft model. Ongoing studies involve treating primary SS and CTCL PDX models with UCART2 and NT-I7. These preclinical data validate the use of allogeneic "off-the-shelf" adoptive immunotherapy for the treatment of Sézary syndrome, while demonstrating the dramatic enhancement of CART efficacy using a dose-adjustable, clinic-ready long-acting interleukin-7 agonist given in an adjuvant setting.

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