Manufacture and In Vivo Support of Gene-Edited, Regulatory-like, T-Cells (edTreg) Using a Synthetic IL2 Receptor

Regulatory T-cells (Treg) depend on signals from IL2 and their endogenous T-cell receptors to survive, proliferate, and maintain suppressive activity. We have developed a strategy for engineering effector CD4 T-cells into edited, regulatory-like T-cells (edTregs) for treatment of autoimmune disease. edTregs contain a rapamycin-activated synthetic IL2 receptor (designated a chemical-induced signaling complex or CISC) for use in selective cell manufacturing and in vivo trophic support. Our strategy uses gene editing to integrate an MND/ CISC expression cassette into the native FOXP3 locus, resulting in the CISC system cis-linked to expression of the native FOXP3 gene. The resulting high-level and stable expression of FOXP3 converts peripheral T-cells to edTregs with immunosuppressive activity. Cis-linked expression of the CISC system allows for selective cell expansion and in vivo support using rapamycin. Using an optimized protocol, we obtained efficient HDR rates across cells from multiple healthy donors. Edited cells were enriched to >90% purity and expanded 20-50-fold over a 10 day period of culture in rapamycin. Importantly, we demonstrated sustained in vivo suppressive activity of edTregs in a xeno-GvHD mouse model, and successful trophic support of these edTregs by rapamycin (via the CISC) in the absence of effector CD4 T-cells. Along with preliminary data showing successful editing of CD4-positive T-cells from auto-immune disease patients, our data provide pre-clinical proof-of-concept data supporting clinical use of CISC-edTreg in conjunction with rapamycin support for therapy of autoimmune disease. Disclosures Uenishi: Casebia Therapeutics: Employment. Mallari:Casebia Therapeutics: Employment. Gamboa:Casebia Therapeutics: Employment. Boucher:Casebia Therapeutics LLc: Employment. Chin:Casebia Therapeutics LLc: Employment. Aeran:Casebia Therapeutics: Employment. Wodziak:Casebia Therapeutics: Employment. Gebremeskel:Casebia Therapeutics: Employment. Vo:Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Patel:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Stankovich:Casebia Therapeutics LLc: Employment. Torgerson:Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership. Cost:Casebia Therapeutics: Employment.

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A combination of cyclophosphamide, interleukin-2 allow CD4+ T cells converted to Tregs to control scurfy syndrome

Blood ◽

10.1182/blood.2020009187 ◽

2021 ◽

Author(s):

Marianne Delville ◽

Florence Bellier ◽

Juliette Leon ◽

Roman Klifa ◽

Sabrina Lizot ◽

...

Keyword(s):

T Cells ◽

Autoimmune Disease ◽

Adoptive Transfer ◽

Cd4 T Cells ◽

Lentiviral Vector ◽

Interleukin 2 ◽

Foxp3 Expression ◽

In Vivo Model ◽

Loss Of Function

Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome is caused by mutations in FOXP3, which lead to the loss of function of regulatory T cells (Treg) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide conditioning and interleukin-2 treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and as a reporter a truncated form of the 5 low-affinity nerve growth factor receptor (DLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Treg. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Treg. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Treg capable of controlling severe autoimmune disease.

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Enabling Gene-Edited, Regulatory-like, T Cells (edTreg) for Treatment of IPEX and Other Autoimmune Disorders

Blood ◽

10.1182/blood-2019-131946 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2071-2071 ◽

Cited By ~ 1

Author(s):

Yuchi Honaker ◽

Karen Sommer ◽

Noelle Dahl ◽

Yufei Xiang ◽

Christina Lopez ◽

...

Keyword(s):

T Cells ◽

Safety Data ◽

Autoimmune Disorder ◽

Foxp3 Expression ◽

Effector T Cells ◽

Therapy Strategy ◽

Equity Ownership ◽

High Level ◽

And Function

IPEX (immunedysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a severe congenital autoimmune disorder in males resulting from hemizygous inheritance of a mutant FOXP3 allele. FOXP3 encodes a transcription factor that governs the development, maintenance, and function of regulatory T cells (Treg). We have developed a cell therapy strategy for treatment of IPEX using a gene-editing approach in which CRISPR/Cas9 RNPs are co-delivered with an AAV6 donor template designed to integrate into the FOXP3 locus an expression cassette containing the MND promoter driving expression of a functional FOXP3 cDNA and a surface LNGFR tag linked by a 2A ribosomal skip peptide. This approach enforces heterologous FOXP3 expression in IPEX CD4 effector T cells (Teff), while simultaneously eliminating expression of the endogenous FOXP3 allele. The resultant high level and stable expression of functional FOXP3 converts Teff to Treg-like cells with immunosuppressive activity. Using an optimized protocol, we obtained efficient HDR rates across multiple healthy donors. Edited cells were consistently enriched to >95% purity by a magnetic LNGFR antibody selection and expanded 50-fold in a week. Expression of FOXP3 cDNA in edited cells was sufficient to enforce Treg-like phenotypes including the up-regulation of Treg-associated markers (CD25, CTLA-4, and ICOS), and down-regulation of CD127 and inflammatory cytokines (IL2, IFNgamma, TNFalpha). Importantly, we demonstrate sustained in vivo suppressive activity of these edited Treg-like cells (edTreg) in a xeno-GvHD mouse model. edTreg (as well as expanded natural Treg) limited effector T cell expansion and tissue infiltration and significantly protected mice from xeno-GvHD induced by co-transferred autologous effector T cells. Along with preliminary data showing successful editing in CD4 T cells from IPEX patients, our data provide key pre-clinical proof-of-concept and safety data supporting use of edTreg in a clinical trial for IPEX and, potentially, for use in other autoimmune diseases. Disclosures Torgerson: Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership; Generation Bio: Equity Ownership.

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MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Blood ◽

10.1182/blood.v126.23.4134.4134 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4134-4134

Author(s):

Mirco di Marco ◽

Serena Veschi ◽

Rosa Visone ◽

Giuseppe Leone ◽

Paola Lanuti ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Healthy Donors ◽

Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

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Single Cell RNA Sequencing Identifies Transcriptional Programs That Enhance Anti-Tumor Function of Transgenic CD4+ T Cells Redirected with TCR and CD8αβ

Blood ◽

10.1182/blood-2019-124714 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 250-250

Author(s):

Jan A Rath ◽

Gagan Bajwa ◽

Benoit Carreres ◽

Isabelle Gruber ◽

Elisabeth Hoyer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Leukemia Cells ◽

Advisory Committees ◽

Equity Ownership

Introduction:Transgenic co-expression of a major histocompatibility complex class I restricted tumor associated antigen specific TCR and CD8αβ (TCR8) has been previously proposed as a strategy to redirect CD4+ T cells to tumors. However, it is unknown whether forced TCR8 expression induces more fundamental transcriptional consequences in both CD4+ and CD8+ T cells, and whether T cell lineage origin affects this outcome. Here we deeply interrogate the effects of transgenic TCR and TCR8 in human CD4+ and CD8+ T cells upon leukemia challenge by single cell RNA sequencing (scRNAseq) and investigate T cell function in vitro and in vivo. We identify profound changes of gene expression that have significant functional consequences. Methods:A previously characterized HLA-A*02:01 restricted survivin-specific TCR was used (Arber et al, JCI, 2015 Jan;125(1):157-68) and a new polycistronic vector with this TCR and CD8αβ was generated. CD4+ and CD8+ T cells were isolated and scRNAseq (25'474 cells in total) was performed on (1) freshly isolated cells, (2) retrovirally transduced (TCR or TCR8) expanded cells, and (3) TCR+CD8+, TCR8+CD8+ and TCR8+CD4+ T cells co-cultured with BV173 leukemia cells (HLA-A*02:01+survivin+). scRNAseq results were cross-validated in independent experiments with FACS analysis of selected markers, in vitro stress-killing assays, analysis of cytokine production, and assessment of anti-tumor function in vivo in xenograft mice. Results:CD4+ T cells only killed BV173 leukemia cells when redirected with TCR8 but not with TCR alone (p=0.0004, n=7), while killing by TCR+CD8+ and TCR8+CD8+ T was comparable (p=NS). To explore some of the possible underlying mechanisms, we used dimensionality reduction and unsupervised clustering of the scRNASeq data and identified 19 distinct cell clusters. CD4+ and CD8+ lineage origin clearly separated the samples, but separation by transgene type only became apparent upon co-culture. Analyzing differentially expressed genes, we found that co-cultured samples contained clusters with high expression of cytotoxic markers but with significant differences between CD4+ and CD8+ lineages (e.g. transcription of GZMB in CD4+ T cells and GNLY, NKG7, GZMK in CD8+ T cells). Next, we analyzed which genes were upregulated from the expanded to co-cultured states. Co-cultured TCR8+CD4+ T cells had more upregulated genes with a broader diversity compared to TCR+CD8+ or TCR8+CD8+ T cells. Among these upregulated pathways were cytotoxicity, co-stimulation, oxidative phosphorylation, NFkB regulation, cell growth and transcription factors. TCR8+CD4+ T cells also retained a less differentiated phenotype (e.g. high IL7R, SELL, CCR7, CXCR4) with preservation of replicative potential. Furthermore, co-cultured TCR8+CD4+ T cells expressed more co-stimulatory and less activation/ exhaustion markers. In addition, co-cultured TCR8+CD4+ T cells heavily relied on oxidative phosphorylation and had higher mitochondrial activity compared to co-cultured TCR+ or TCR8+ CD8+ T cells. In stress co-cultures with multiple rounds of tumor challenge, TCR8+CD4+ T cells outperformed TCR+CD8+ T cells (number of killings TCR8+CD4+ vs TCR+CD8+: 3.3±0.5 vs 1.3±1.1, p=0.01, n=7), but were comparable to TCR8+CD8+ T cells (TCR8+CD4+ vs TCR8+CD8+: 3.3±0.5 vs 2±1.4,p=NS, n=7). TCR8+CD4+ T cells expanded significantly better than TCR+CD8+ T cells (p=0.002) and TCR8+CD8+ T cells (p=0.015) and produced TH1 type cytokines. In the xenograft mouse model, we observed significant BV173 leukemia control in mice treated with TCR+CD8+ T cells compared to controls (NT), and further enhancement in mice treated with TCR8+CD8+ T cells (NT vs TCR: p=0.0002, NT vs TCR8: p<0.0001, TCR vs TCR8: p=0.01, n=5). TCR8+CD4+ T cells also significantly delayed leukemia progression compared to TCR+CD4+ or NT T cells (p=0.001, n=5). Conclusion:Transgenic TCR8 expression has previously been proposed as a strategy to enhance TCR-pMHC recognition. Here we identify profound transcriptional changes involving multiple pathways that are important for sustained anti-tumor function upon adoptive T cell transfer in vivo, such as cytotoxicity, co-stimulation, cell cycle and metabolism. Our results point towards previously unrecognized mechanisms by which TCR8 transgenes mediate their beneficial effect in both CD4+ and CD8+ T cells. Disclosures Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees. Arber:Cell Medica: Patents & Royalties.

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The involvement of T cell receptor peptide-specific regulatory CD4+ T cells in recovery from antigen-induced autoimmune disease.

Journal of Experimental Medicine ◽

10.1084/jem.178.3.909 ◽

1993 ◽

Vol 178 (3) ◽

pp. 909-916 ◽

Cited By ~ 149

Author(s):

V Kumar ◽

E E Sercarz

Keyword(s):

T Cells ◽

T Cell ◽

Autoimmune Disease ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Gene Segment ◽

Cell Receptor ◽

Histocompatibility Complex ◽

Peripheral T Cells

Experimental allergic encephalomyelitis (EAE) is a prototype for CD4+ T cell-mediated autoimmune diseases. Immunization with myelin basic protein (MBP) in B10.PL mice results in EAE, and a majority of animals recover permanently from the disease. Most MBP-reactive encephalitogenic T cells recognize an immunodominant NH2-terminal peptide, Ac1-9, and predominantly use the T cell receptor (TCR) V beta 8.2 gene segment. Here we report that in mice recovering from MBP-induced EAE, peripheral T cells proliferate in response to a single immunodominant TCR peptide from the V beta 8.2 chain (amino acids 76-101), indicating natural priming during the course of the disease. Cloned T cells, specific for this TCR peptide, specifically downregulate proliferative responses to Ac1-9 in vivo and also protect mice from MBP-induced EAE. These regulatory T cells express CD4 molecules and recognize a dominant peptide from the TCR variable framework region of V beta 8.2, in the context of the major histocompatibility complex class II molecule, I-Au, and predominantly use the TCR V beta 14 gene segment. This is the first demonstration of the physiological induction of TCR peptide-specific CD4+ T cells that result from MBP immunization and that are revealed only during the recovery from disease. The downregulation of disease-causing T cells by TCR peptide-specific T cells offers a mechanism for antigen-specific, network-induced recovery from autoimmune disease.

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