scholarly journals T Cells Engineered with T Cell Antigen Coupler (TAC) Receptors for Haematological Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3267-3267
Author(s):  
Christopher Helsen ◽  
Vivian Lau ◽  
Joanne Hammill ◽  
Kenneth Mwawasi ◽  
Danielle Hayes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cytokine Production ◽  
Haematological Malignancies ◽  
Cell Antigen ◽  
Engineered T Cells ◽  
Post Infusion

Abstract Background: We recently described the T cell antigen coupler (TAC) technology (Helsen et. al. Nature Communications) which is a chimeric receptor that targets antigens in an MHC-independent fashion and activates T cells by co-opting the natural TCR receptor. In vitro and in vivo assessments of TAC T cells in solid tumor models have revealed that TACs mediate biological effects that are distinct from conventional chimeric antigen receptors (CARs) and offer safety advantages, including greater target selectivity and reduced off-target toxicity. Here, we present in vitro and in vivo data showing that TAC-engineered T cells directed against CD19 and BCMA demonstrate robust anti-tumor efficacy in haematological malignancies with no detectable side effects. Materials and Methods: T cells from health donors were engineered with TAC receptors directed against CD19 or BCMA using lentivirus vectors. Flow cytometry was employed to measure surface expression of TAC receptors, cytokine production and proliferation of TAC T cells following stimulation with relevant target cells. Antigen-specific toxicity was measured using a luciferase-based killing assay. Anti-tumor activity was measured against acute lymphoblast leukemia for CD19 and multiple myeloma for BCMA xenografts in immunodeficient NRG mice. Results: Engineering T cells with TAC receptors targeted against either CD19 or BCMA revealed antigen-specific activation of cytokine production, cytotoxic function and proliferation. TAC T cells, but not CAR engineered T cells, show significant selectivity towards the context of antigen presentation. This is reflected by the differential proliferative response to a diverse framework of antigen surface arrangement, potentially indicating that TAC T cells are less susceptible to off target activation and the resulting toxicities. Treatment of established NALM-6 xenografts (acute lymphoblastic leukemia) and KMS-11 xenografts (multiple myeloma) with CD19 TAC T cells and BCMA TAC T cells, respectively, resulted in clearance of tumors within a few weeks of T cell infusion. Mice that cleared tumors following TAC T cell treatment were resistant to subsequent challenge with fresh tumor cells demonstrating persistence of TAC T cells. Treatment with control TAC T cells that carry no binding domain had no impact on tumor growth. Monitoring of TAC T cells post-infusion revealed robust expansion that peaked in the peripheral blood 1-2 weeks post-infusion. A clinical manufacturing protocol has been developed for the CD19 TAC T cells in anticipation of human trials. Conclusion: Our pre-clinical evaluation suggests that TAC therapy has the potential to becoming a safer and more effective alternative to conventional CAR therapy. A first in human Phase I/II trial with CD19 TAC T cells is expected to start in the first half of 2019. Disclosures Helsen: Triumvira Immunologics: Employment, Patents & Royalties. Hammill:Triumvira Immunologics: Other: Holding shares, Patents & Royalties. Mwawasi:Triumvira Immunologics: Other: Holding shares, Patents & Royalties. Hayes:Triumvira Immunologics: Employment. Afsahi:Triumvira Immunologics: Patents & Royalties. Denisova:Triumvira Immunologics: Patents & Royalties. Bramson:Triumvira Immunologics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals T Cells Engineered with a Novel Chimeric Receptor Demonstrate Durable In Vivo Efficacy Against Disseminated Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 962-962 ◽  
Author(s):  
Ksenia Bezverbnaya ◽  
Vivian Lau ◽  
Craig Aarts ◽  
Galina Denisova ◽  
Arya Afsahi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Inhibitory Effect ◽  
In Vivo Efficacy ◽  
Engineered T Cells

Abstract Despite recent therapeutic developments, multiple myeloma remains an incurable plasma cell malignancy. Poor prognosis for myeloma patients relapsing post-transplant calls for the need for novel treatment options. Immunotherapy with engineered T cells has proven highly efficacious against B-cell cancers, and early-phase clinical trials suggest that multiple myeloma is susceptible to this form of therapy. We designed a new chimeric T cell receptor, T cell antigen coupler (TAC), which relies upon activation through endogenous T cell receptor complex, thus allowing engineered T cells to auto-regulate their activity (Helsen et al, Nat. Comm., 2018). Using published single-chain antibody fragments (scFvs) C11D5.3 and J22.9-xi, we generated B cell maturation antigen (BCMA)-specific TAC receptors for targeting multiple myeloma. Primary human T cells were transduced with lentiviral vectors carrying different BCMA TAC constructs and assessed for in vitro functionality via cytokine production, cytotoxicity, and proliferation assays. In vivo efficacy and T cell tracking were performed in an established orthotopic xenograft mouse model based on a BCMA-positive KMS-11 cell line. C11D5.3 and J22.9-xi TAC T cells demonstrated comparable in vitro performance with both types of cultures efficiently killing BCMA-expressing targets, producing IFN-γ, TNF-α, and IL-2 cytokines, and undergoing multiple rounds of proliferation. In vivo, TAC T cells carrying either scFv were capable of curing mice bearing disseminated myeloma; however, the TAC T cells carrying J22.9-xi scFv were more potent on a per-cell basis (Figure 1A, top panel). Mice in remission 3 months post-treatment with a single dose of 106 TAC-positive T cells showed evidence of sustained anti-tumor protection upon rechallenge with a fresh dose of 106 KMS-11 tumor cells (Figure 1B). Mice treated with low-dose J22.9-xi T cells were more resistant to rechallenge than mice treated with a comparable dose of C11D5.3 TAC T cells. Tracking of the TAC T cells in vivo revealed that the J22.9-xi TAC T cells expanded to a much larger extent than the C11D5.3 TAC T cells (Figure 1A, bottom panel), indicating that there were likely more J22.9-xi TAC T cells present at the time of tumor rechallenge. To understand whether biological aspects of BCMA may influence the proliferative response of the TAC T cells, we explored the influence of APRIL, the soluble ligand for BCMA, on TAC T cell proliferation in vitro. Strikingly, despite comparable proliferation of both TAC T cell populations following stimulation with KMS-11 tumor cells in the absence of APRIL in vitro, the presence of APRIL had a strong inhibitory effect on proliferation of C11D5.3 TAC T cells and only a modest inhibitory effect on J22.9-xi TAC T cells. Our preclinical findings support further development of TAC T cells for the treatment of multiple myeloma and underscore the importance of T cell expansion in determining the therapeutic activity of engineered T cells. This work further reveals a novel observation that the natural ligand of BCMA can impair the therapeutic impact of T cells engineered with chimeric receptors directed against BCMA and provide a basis for advancing BCMA-specific TAC T cells into the clinic. Disclosures Denisova: Triumvira Immunologics: Patents & Royalties. Afsahi:Triumvira Immunologics: Patents & Royalties. Helsen:Triumvira Immunologics: Employment, Patents & Royalties. Bramson:Triumvira Immunologics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

CD3 limits the efficacy of TCR gene therapy in vivo

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3528-3537 ◽  
Author(s):  
Maryam Ahmadi ◽  
Judith W. King ◽  
Shao-An Xue ◽  
Cécile Voisine ◽  
Angelika Holler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Receptor ◽  
Surface Expression ◽  
T Cell Function ◽  
Engineered T Cells ◽  
Rate Limiting

Abstract The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

scholarly journals P025 Identification and validation of predictors for tofacitinib through supervised and unbiased approaches in Inflammatory Bowel Disease

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S141-S141
Author(s):  
B Liu ◽  
M Spalinger ◽  
L G Perez ◽  
A Machicote ◽  
N Gagliani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Signaling Pathway ◽  
Cd4 T Cells ◽  
Cytokine Production ◽  
Stat Signaling ◽  
Deficient Mice

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by an overwhelming gut inflammation, where CD4+ effector T cells are main mediators of the inflammatory response. Tofacitinib, a small molecular drug recently used in IBD patients, blocks the JAK/STAT signaling pathway necessary for CD4+ effector T-cell activation. However, clinical data show that a percentage of patients do not respond to the treatment. Our main goal is to identify biomarkers predicting the response of patients to tofacitinib. Methods Tofacitinib efficacy was studied in vivo in wild type (WT) and T-cell-specific PTPN2 deficient mice (CD4-Cre;Ptpn2 floxed) in which the JAK/STAT signaling pathway is over activated. WT and PTPN2 deficient mice were gavaged with tofacitinib (50mg/kg, twice daily) or vehicle. Acute DSS-colitis was induced. Colitis development was evaluated by weight loss, colonoscopy and histology. CD4+ T cells were isolated from the colon and analyzed by flow cytometry. To study the effect of tofacitinib on T-cell differentiation, we isolated naïve T cells from mouse spleen and polarized them in vitro to different T-cell subsets with or without tofacitinib. CD4+ T cells differentiation and cytokine production were analyzed by flow cytometry. To evaluate the influence of tofacitinib on human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs) from healthy donors and IBD patients were stimulated in presence of tofacitinib, and analyzed by flow cytometry. Results While no protective effect was found after tofacitinib treatment in WT mice, PTPN2 deficient mice were protected from colitis based on less weight loss, lower endoscopic and histological scores. The expression of pro-inflammatory cytokines such as IL-17 and IFN-γ by colonic CD4+ T cells was also decreased by tofacitinib. Consistent with the in vivo observations, in vitro experiments revealed a strong impact of tofacitinib on CD4+ T-cells cytokine production. In PBMCs from IBD patients, IFN-γ and TNF-α expression was strongly impacted. In contrast, in healthy donors, IL-10 was the most impacted cytokine. Finally, tofacitinib decreased the in vitro differentiation of Th1, Th2, Th17, Th22, Treg and Tr1. Conclusion In the T-cell-specific PTPN2 deficient mice, tofacitinib exerts a protective effect after DSS-induced colitis. In line with the in vivo findings, in vitro experiments show that tofacitinib has a strong impact on pro-inflammatory cytokine production, especially in the IBD patients. Taken together, these data suggest that tofacitinib might be suitable primarily for IBD patients where the JAK/STAT signaling pathway is over activated.

scholarly journals Combinatorial targeting of multiple myeloma by complementing T cell engaging antibody fragments

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Maria Geis ◽  
Boris Nowotny ◽  
Marc-Dominic Bohn ◽  
Dina Kouhestani ◽  
Hermann Einsele ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Specific Immunotherapy ◽  
Antibody Fragments ◽  
Cytokine Release ◽  
Major Drawback ◽  
Single Antigen

AbstractBispecific T cell engaging antibodies (BiTEs) address tumor associated antigens that are over-expressed on cancer but that can also be found on healthy tissues, causing substantial on-target/off-tumor toxicities. To overcome this hurdle, we recently introduced hemibodies, a pair of complementary antibody fragments that redirect T cells against cancer-defining antigen combinations. Here we show that hemibodies addressing CD38 and SLAMF7 recruit T cells for the exquisite elimination of dual antigen positive multiple myeloma cells while leaving single antigen positive bystanders unharmed. Moreover, CD38 and SLAMF7 targeting BiTEs, but not hemibodies induce massive cytokine release and T cell fratricide reactions, a major drawback of T cell recruiting strategies. Together, we provide evidence in vitro and in vivo that hemibodies can be developed for the effective and highly specific immunotherapy of multiple myeloma.

Small Molecule Inhibitor of ITK Disrupts TCR Signaling and Demonstrates Anti-Tumor Efficacy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3932-3932
Author(s):  
Mary Faris ◽  
Uriel M Malyankar ◽  
Qingping Zeng ◽  
Gary A Flynn ◽  
Gerold Feuer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Production ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
T Cell Malignancies ◽  
Dose Dependent ◽  
Leukemias And Lymphomas

Abstract Abstract 3932 ITK (Interluekin-2 Inducible Tyrosine Kinase) is a member of the TEC family of intracellular protein tyrosine kinases. ITK is highly expressed in T cells and NK cells, with expression detected in mast cells. ITK plays a key role in several aspects of T cell biology, including T cell development, differentiation, migration, proliferation and activation. The function of ITK in immunity and allergy is well documented. T cells from ITK knock out mice show several developmental and functional defects, including defective signal transduction, altered CD4+ to CD8+ T cells ratios, reduced Th2 lineage differentiation, diminished IL4 and IL2 production and reduced T cell proliferation. Importantly ITK deficient mice fail to mount an immune response to infection and show reduced allergic asthma reactions. In contrast to its well described role in immune function, ITK's function in cancer biology is still emerging. Recent studies had reported enhanced ITK expression and activation of the ITK pathway in several types of leukemias and lymphomas. In addition, the dependence of T cell malignancies on an ITK-regulated pathway, namely the IL2/IL2R (CD25) pathway, has also been observed. Taken together, this information indicates that ITK is a therapeutic target, with applicability in leukemias and lymphomas. MannKind scientists have developed a series of selective small molecule ITK inhibitors, including the orally available tool compound described within, and evaluated their activity in enzyme, cell-based and in vivo studies. In cellular assays, the compounds showed significant inhibition of the T cell-receptor mediated activation of the ITK pathways and related downstream cytokine production. In addition to inhibiting the phosphorylation of ITK and its downstream mediator, PLCg, our tool compounds inhibited the production of IL2 and expression of CD25 in a dose dependent manner. Importantly, our compound regulated the in vitro growth of tumor T cells but not that of unrelated control cells. In vivo studies revealed that the tool compounds inhibited the growth and progression of patient derived ATL tumors in a xenograft pre-clinical model, and prolonged the survival of treated mice in a dose dependent manner, in addition to regulating cytokine production in vivo. In summary, our team has identified ITK selective compounds with demonstrated on-target and anti-tumor activity in vitro and preclinical T cell tumor models, and validated this pathway relative to T cell malignancies. This effort provides a platform for further compound optimization and evaluation for hematologic malignancies. Disclosures: Faris: MannKind Corp: Employment. Malyankar:MannKind Corp: Employment. Zeng:MannKind Corp: Employment. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Vuga:MannKind Corp.: Employment. Rosario:MannKind Corp: Employment. Bot:MannKind Corp: Employment.

A Novel Bcma-Specific, Centyrin-Based CAR-T Product for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2127-2127 ◽  
Author(s):  
David L. Hermanson ◽  
Burton Earle Barnett ◽  
Srinivas Rengarajan ◽  
Rebecca Codde ◽  
Xinxin Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
M Protein ◽  
Manufacturing Processes ◽  
Protein Levels ◽  
Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapies have been remarkably effective in treating acute lymphoblastic leukemia. However, current strategies generally suffer from difficult, inefficient and costly manufacturing processes, significant patient side effects and poor durability of response in some patients. Here, we report for the first time a CAR-T cell therapeutic comprising a non-immunoglobulin alternative scaffold Centyrin molecule (a "CARTyrin") manufactured with a novel non-viral piggyBacTM (PB) transposon-based system. Our lead candidate, P-BCMA-101, encodes a CARTyrin that targets the B cell maturation antigen (BCMA) for the treatment of multiple myeloma (MM) and has several unique aspects that improve upon earlier CAR-T products. First, P-BCMA-101 is manufactured using only in vitro transcribed mRNA and plasmid DNA without the need for lentivirus or g-retrovirus, resulting in time and cost savings. Importantly, PB is also safer than viral systems due to a less mutagenic insertional profile and is non-oncogenic. Furthermore, PB can efficiently deliver transgenes as large as several hundred kilobases, and, once inserted, transgenes demonstrate more stable, prolonged and higher expression when compared to those delivered by virus. Second, a mutein of the dihydrofolate reductase (DHFR) gene is included in the P-BCMA-101 transgene that can be used in combination with the non-genotoxic drug methotrexate (MTX) to provide a simple and effective method of CARTyrin+ cell enrichment and reduces variability in patient product material. Third, P-BCMA-101 incorporates a safety switch for optional depletion in vivo in case of adverse events. Lastly, the CARTyrin is comprised of a BCMA-specific Centyrin, which are based on a human tenascin fibronectin type III (FN3) consensus sequence. Centyrins have similar binding affinities to the antibody-derived single chain variable fragments (scFv), but are smaller, more thermostable and predicted to be less immunogenic. Importantly, no signs of tonic signaling leading to T cell exhaustion have been observed with CARTyrins unlike scFv-based CAR molecules, which can interact with each other on the surface causing non-specific CAR signaling. The manufacture process of P-BCMA-101 from primary human T cells is straightforward, employs no cytokines, and easily produces enough CARTyrin+ cells to treat patients. Within 18 days of electroporation of purified T cells, we demonstrate > 95% of the cell product is positive for CARTyrin expression and ready to be administered. Notably, our manufacturing process results in > 60% of CARTyrin+ T cells exhibiting a stem-cell memory phenotype (i.e. CD45RA+ CD62L+). P-BCMA-101 cells exhibit specific and robust in vitro activity against BCMA+ tumor targets, ranging from high to very low levels of BCMA, as measured by target-cell killing and CARTyrin-T cell proliferation. Importantly, proliferating P-BCMA-101 cells were highly sensitive in vitro to activation of the safety switch. Finally, we have evaluated the anti-tumor efficacy of P-BCMA-101 in a model of human MM. NSG™ mice were injected IV with 1.5x106 luciferase+ MM.1S cells, an aggressive human MM-derived cell line. After the tumor cells were allowed to grow for 21 days, animals received a single IV administration of 5x106 P-BCMA-101 cells. All untreated control animals demonstrated a marked increase in serum M-protein levels, rapid growth of tumor cells demonstrated by bioluminescent imaging (BLI), and death within four weeks. In stark contrast, 100% of animals that received P-BCMA-101 rapidly eliminated tumors within 7 days as measured by BLI and serum M-protein levels and improved survival out to at least 60 days post-treatment. P-BCMA-101 is the first-in-class of Centyrin-based CAR therapeutics. The CARTyrin, combined with our advanced manufacturing processes, represents a significant improvement over first generation, immunoglobulin-based and virally-transduced CAR-T products. P-BCMA-101 exhibited an advantageous stem-cell memory phenotype and demonstrated specific and potent anti-tumor efficacy against BCMA+ myeloma cells both in vitro and in vivo. Based on these results, we plan to initiate a phase I clinical trial of P-BCMA-101 for the treatment of patients with relapsed and/or refractory MM. Disclosures Hermanson: Poseida Therapeutics: Employment. Barnett:Poseida Therapeutics: Employment. Rengarajan:Poseida Therapeutics: Employment. Codde:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

Artificial Thymic Organoids Permit Allelic Exclusion and Efficient Generation of Naïve TCR-Engineered T-Cells from Human Hematopoietic Stem Cells In Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4553-4553
Author(s):  
Christopher S Seet ◽  
Chongbin He ◽  
Michael Bethune ◽  
Suwen Li ◽  
Brent Chick ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Intellectual Property ◽  
Positive Selection ◽  
Allelic Exclusion ◽  
Hematopoietic Stem ◽  
Engineered T Cells ◽  
Selection Of

Abstract Engineered adoptive immunotherapies have shown unprecedented activity in the treatment of cancer and chronic viral infections. Current approaches rely on individualized ex vivo genetic modification of autologous T cells due to the risk of graft-versus-host disease from allogeneic T cells. These processes furthermore require activation and prolonged expansion of T cells, which may reduce in vivo efficacy and persistence. Direct in vitro differentiation of engineered T cells from hematopoietic stem and progenitor cells (HSPCs) may overcome these problems by permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. Existing methods of in vitro human T cell differentiation are subject to wide experimental variability and do not adequately support the positive selection of immature T cell precursors to mature T cells, and thus have not been suitable for clinical-scale production of engineered T cells. We report here the preclinical development of an artificial thymic organoid (ATO) system using off-the-shelf, serum-free components and a standardized stromal cell line that supports highly efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from cord blood (CB), bone marrow, and mobilized peripheral blood CD34+ HSPCs, and purified CD34+CD38- hematopoietic stem cells. ATOs closely recapitulated thymic T cell commitment and differentiation, resulting in greater than 80% CD7+CD5+ T-lineage cells and 50% CD4+CD8+ double positive (DP) T cell precursors by 4 weeks. By 6 weeks, 30-40% of ATO cells were CD3+TCRαβ+ T cells, of which 20-30% were mature CD8 single positive (SP) T cells. CD4SP cells were generated at a lower frequency and later in culture (2-14% of CD3+TCRαβ+ cells). ATO-derived T cells exhibited a naïve CD45RA+CD27+CCR7+CD62L+ phenotype, a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. Transduction of CB CD34+ HSPCs with an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell output per ATO (>400-fold, relative to input HSPCs) and enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. Positive selection of TCR-engineered naïve T cells could be further enhanced by expression of cognate HLA-A*02:01 in ATO stromal cells. ATO-derived TCR-engineered T cells exhibited a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. ATO-derived engineered T cells underwent antigen-specific cytotoxic priming, polyfunctional cytokine release, and proliferation in response to artificial APCs; and exhibited antigen-specific killing of NY-ESO-1+ tumor cells in vitro and in vivo. ATOs thus present a highly efficient off-the-shelf platform for the generation of clinically relevant numbers of naïve and potentially non-alloreactive engineered T cells for adoptive immunotherapy. Clinical translation of the ATO system will be aided by its simplicity, scalability, use of serum-free components, and compatibility with irradiated stromal cells. In addition, genetic manipulation of stem or stromal cell components can be easily incorporated into the system to further enhance downstream T cell engraftment or function. Disclosures Seet: Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Montel-Hagen:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Crooks:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property, Research Funding.

96 Decitabine gene modulation sensitizes human non–small cell lung cancer (NSCLC) to NY-ESO-1 TCR immunotherapy (letetresgene autoleucel; GSK3377794) in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A107-A107
Author(s):  
Dmitry Pankov ◽  
Ioanna Eleftheriadou ◽  
Anna Domogala ◽  
Sara Brett ◽  
Lea Patasic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Human Leukocyte Antigen ◽  
Human Leukocyte ◽  
List Type ◽  
Leukocyte Antigen ◽  
Engineered T Cells

BackgroundNY-ESO-1–specific T cells (letetresgene autoleucel [lete-cel] GSK3377794) are autologous CD4+ and CD8+ T cells transduced to express a high-affinity T-cell receptor (TCR) capable of recognizing NY-ESO-1 and LAGE-1a antigens in complex with human leukocyte antigen (HLA)-A*02. NY-ESO-1 (CTAG1B) and LAGE-1a (CTAG2) are tumor-associated antigens (TAA) that share the SLLMWITQC peptide bound to human leukocyte antigen HLA-A*02 and are expressed in various cancers. Emerging evidence suggests that TCR-engineered T cells targeting NY-ESO-1 hold promise for patients with solid tumors.1 Approximately 75% of synovial sarcomas can over-express NY-ESO-1 vs 12% of NSCLC,2 however, NSCLC expression of NY-ESO-1/LAGE1-a may have therapeutic potential.3 A separate study using engineered T cells targeting NY-ESO-1 has shown a partial response in a patient with advanced lung adenocarcinoma.4 Decitabine (DAC) is a hypomethylating agent and potent inducer of TAA, including NY-ESO-1.5 We have reported in vitro use of DAC to selectively modulate TAA expression in TAA low-expressing tumor cell lines in order to enhance lete-cel therapy.3 The aim of this study was to assess enhancement of combination therapy with lete-cel and DAC in an in vivo NSCLC model.MethodsNOD scid gamma (NSG) mice were injected subcutaneously with the human NSCLC tumor cell line NCI-H1703. Upon engraftment, tumor-bearing mice were treated with a 5-day course of DAC or vehicle control followed by 2 days of rest. Lete-cel was infused on Day 8. RNA was isolated from tumor formalin-fixed paraffin-embedded blocks, and levels of NY-ESO-1 and LAGE-1a transcript were measured by RT-qPCR. Expression pattern of the NY-ESO-1 protein was assessed via immunohistochemistry. Efficacy was defined by changes in tumor volume and systemic IFN-γ secretion.ResultsConsistent with our previous in vitro studies, DAC treatment in vivo resulted in induction of NY-ESO-1 and LAGE-1a in NSCLC tumors. Lete-cel in combination with DAC significantly enhanced antitumor efficacy in vivo compared with lete-cel alone. This was associated with increased interferon-γ secretion. Mice that received DAC treatment only did not show statistically significant tumor reduction compared with untreated mice.Ethics ApprovalAll animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an Institutional Review Board/Ethics Committee approved protocol.ConclusionsGSK is currently enrolling a Phase Ib/IIa, multi-arm, open-label pilot study (NCT03709706) of lete-cel as a monotherapy or in combination with pembrolizumab in HLA-A*02–positive patients with NSCLC whose tumors express NY-ESO-1/LAGE-1a. This work may support rationale for the use of DAC in combination with lete-cel to improve adoptive T-cell therapy by increasing levels of target antigens and antitumor effect in NSCLC.AcknowledgementsFunding: GSKReferencesD’Angelo SP, Melchiori L, Merchant MS, et al. Cancer Discov 2018;8:944–957.Kerkar SP, Wang Z-F, Lasota J, et al. J Immunother 2016;39:181–187.Eleftheriadou I, Brett S, Domogala A, et al. Ann Oncol 2019:30(Suppl 5):v475–v532.Xia Y, Tian X, Wang J, et al. Oncol Lett 2018;16:6998–7007.Schrump DS, Fischette MR, Nguyen DM, et al. Clin Cancer Res 2006;12:5777–5785.

ISB 1342: A first-in-class CD38 T cell engager for the treatment of relapsed refractory multiple myeloma.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 8044-8044
Author(s):  
Marie-Agnès Doucey ◽  
Blandine Pouleau ◽  
Carole Estoppey ◽  
Cian Stutz ◽  
Amelie Croset ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Killing ◽  
Fragment Antigen Binding ◽  
Tumor Cell Killing

8044 Background: ISB 1342 is a bispecific antibody heterodimer based on the Ichnos proprietary Bispecific Engagement by Antibodies based on T cell receptor (BEAT) platform. ISB 1342 is a first-in-class CD38 T cell engager under investigation in subjects with relapsed multiple myeloma refractory to proteasome inhibitors (PIs), immunomodulators (IMiDs) and daratumumab (study ISB 1342-101). Methods: ISB 1342 was engineered with a single chain variable fragment (scFv) arm that specifically recognizes a cluster of differentiation (CD)3-epsilon (CD3ε) and a fragment antigen binding (Fab) arm which specifically recognizes CD38 and does not compete with daratumumab. By co-engaging CD3ε on T cells and CD38 on tumor cells, ISB 1342 redirects T cells to kill CD38-expressing tumor cells. This mechanism of action is differentiated from existing monospecific CD38 targeting therapies and was designed to overcome resistance to daratumumab in multiple myeloma. Results: In vitro, ISB 1342 killed a large range of CD38-expressing tumor cell lines (EC50:12 to 90 pM) with 8 to 239-fold superior efficacy than daratumumab. ISB 1342 was also able to efficiently kill CD38 low-intermediate-expressing tumor cells that were poorly killed by daratumumab. ISB 1342 retained the potency to kill CD38 low-intermediate-expressing tumor cells when used in sequential or concomitant combination with daratumumab. In addition, the presence of soluble CD38 or glucocorticoid did not impact ISB 1342 killing potency. ISB 1342 was constructed with a double LALA mutation that dampens the binding to Fcγ receptors and C1q. Consistently, ISB 1342 showed only residual Fc-mediated effector functions and its mechanism of tumor cell killing critically relies on the engagement and the activation of T lymphocytes. ISB 1342 showed a favorable on target specificity profile in vitro and was unable to activate T cells in the absence of CD38 positive target cells. Further, ISB 1342-induced tumor cell killing was not associated with a detectable T cell fratricide in vitro. Finally, the potency of ISB 1342 was assessed in vivo in a therapeutic model of a subcutaneously established Daudi tumor co-xenografted with human PBMCs. In marked contrast to daratumumab, which induced only a partial tumor control, ISB 1342 induced complete tumor eradication when injected intravenously weekly at 0.5 mg/kg. As anticipated, the ISB 1342 control molecule (ISB 1342_13DU) made of an irrelevant CD38 binder failed to control tumor growth. The release of the Granzyme A and B, TNF-alpha and CXCL-10 in the tumor micro-environment one week post-treatment was strongly and significantly increased by ISB 1342 but not by daratumumab and ISB 1342_13DU; this represents a correlate of anti-tumor immunity associated with ISB 1342 efficacy in vivo. Conclusions: Hence the higher potency of ISB 1342 relative to daratumumab supports the ongoing clinical development in multiple myeloma patients.

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