scholarly journals 183 Therapeutic T cells exhibit distinct vulnerability to glucose deprivation in tumors which can be overcome with an engineered glucose transporter

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A194-A194
Author(s):  
Jessica Jana ◽  
Yiyang Wang ◽  
Ashley Menk ◽  
Andrew Frisch ◽  
Greg Delgoffe
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Tumor Cells ◽  
Glucose Transporter ◽  
Effector Function ◽  
Checkpoint Blockade ◽  
Cell Therapies ◽  
Effector Functions

BackgroundWhile checkpoint blockade cancer immunotherapies reawaken dormant antitumor immunity, adoptive cell therapies (ACT) bolster an immune response through infusion of expanded tumor infiltrating lymphocytes (TIL), or healthy T cell redirected to tumors via chimeric antigen receptor (CAR), or T cell receptor (TCR) expression. However, the harsh, nutrient depleted tumor microenvironment (TME) creates metabolic barriers for T cell persistence and effector function. We and others have shown that glucose availability is key for T cell effector functions through multiple non-redundant mechanisms. We thus asked whether therapeutic T cells harbor increased sensitivity for glucose and whether this could be mitigated to increase efficacy.MethodsB16 and Pten deficient, Braf mutant melanomas were used as models in C57BL/6 mice. Tumor cell glucose uptake was inhibited using stable expression of shRNA to Slc2a1, encoding Glut1. Glut1 was retrovirally overexpressed in therapeutic, Pmel-1 (gp100-specific) T cells, and phosphomimetic mutations were engineered (S226D) into Glut1, stabilizing cell surface trafficking. Glucose uptake and glycolysis were measured using fluorescent glucose tracers and Seahorse analysis, respectively.ResultsHere we sought to equip glucose sensitive therapeutic T cells with heightened ability to compete for glucose within the TME. Murine therapeutic T cells, expanded in hyperglycemic conditions in vitro, that infiltrate solid tumors compete poorly for glucose tracers compared to endogenous T cells. Knockdown or deletion of Glut1 in tumor cells sensitizes tumors to T cell therapies, but not checkpoint blockade, highlighting a role for glucose competition specifically in ACT. Overexpression of WT Glut1 in therapeutic T cells yields only modest increased glucose competition due to various modes of Glut1 regulation. We thus engineered a cell surface stabilized Glut1 construct. This construct’s competitive advantage manifests robust increases in glucose uptake and glycolytic capacity, leading to superior effector functions even in extremely hypoglycemic conditions. This enhanced effector function manifests in therapeutic efficacy in highly glycolytic melanomas and with heightened competition for glucose tracers, tumor infiltration, and effector function in vivo.ConclusionsOur study suggests that, due to the hyperglycemic conditions of their ex vivo expansion, therapeutic T cells display a distinct metabolic disadvantage when they enter the nutrient poor TME in comparison to their endogenous counterparts. Overexpression of our surface engineered Glut1 in these therapeutic T cells rescues their ability to compete with highly glycolytic tumor cells for glucose, resulting in robust glycolytic metabolism and curative response to immunotherapy for cancer.

scholarly journals Beyond the Lactate Paradox: How Lactate and Acidity Impact T Cell Therapies against Cancer

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 25
Author(s):  
Violet Y. Tu ◽  
Asma Ayari ◽  
Roddy S. O’Connor
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Biology ◽  
Redox Balance ◽  
Hematologic Malignancies ◽  
Activated T Cells ◽  
Unique Challenge ◽  
Cell Therapies

T cell therapies, including CAR T cells, have proven more effective in hematologic malignancies than solid tumors, where the local metabolic environment is distinctly immunosuppressive. In particular, the acidic and hypoxic features of the tumor microenvironment (TME) present a unique challenge for T cells. Local metabolism is an important consideration for activated T cells as they undergo bursts of migration, proliferation and differentiation in hostile soil. Tumor cells and activated T cells both produce lactic acid at high rates. The role of lactic acid in T cell biology is complex, as lactate is an often-neglected carbon source that can fuel TCA anaplerosis. Circulating lactate is also an important means to regulate redox balance. In hypoxic tumors, lactate is immune-suppressive. Here, we discuss how intrinsic- (T cells) as well as extrinsic (tumor cells and micro-environmental)-derived metabolic factors, including lactate, suppress the ability of antigen-specific T cells to eradicate tumors. Finally, we introduce recent discoveries that target the TME in order to potentiate T cell-based therapies against cancer.

scholarly journals Checkpoint blockade accelerates a novel switch from an NKT-driven TNFα response toward a T cell driven IFN-γ response within the tumor microenvironment

2021 ◽  
Vol 9 (6) ◽  
pp. e002269
Author(s):  
Shota Aoyama ◽  
Ryosuke Nakagawa ◽  
Satoshi Nemoto ◽  
Patricio Perez-Villarroel ◽  
James J Mulé ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
T Cell Response ◽  
Effector Function ◽  
Checkpoint Blockade ◽  
Necrosis Factor Alpha ◽  
Ifn Γ

BackgroundThe temporal response to checkpoint blockade (CB) is incompletely understood. Here, we profiled the tumor infiltrating lymphocyte (TIL) landscape in response to combination checkpoint blockade at two distinct timepoints of solid tumor growth.MethodsC57BL/6 mice bearing subcutaneous MC38 tumors were treated with anti-PD-1 and/or anti-CTLA-4 antibodies. At 11 or 21 days, TIL phenotype and effector function were analyzed in excised tumor digests using high parameter flow cytometry. The contributions of major TIL populations toward overall response were then assessed using ex vivo cytotoxicity and in vivo tumor growth assays.ResultsThe distribution and effector function among 37 distinct TIL populations shifted dramatically between early and late MC38 growth. At 11 days, the immune response was dominated by Tumor necrosis factor alpha (TNFα)-producing NKT, representing over half of all TIL. These were accompanied by modest frequencies of natural killer (NK), CD4+, or CD8+ T cells, producing low levels of IFN-γ. At 21 days, NKT populations were reduced to a combined 20% of TIL, giving way to increased NK, CD4+, and CD8+ T cells, with increased IFN-γ production. Treatment with CB accelerated this switch. At day 11, CB reduced NKT to less than 20% of all TIL, downregulated TNFα across NKT and CD4+ T cell populations, increased CD4+ and CD8+ TIL frequencies, and significantly upregulated IFN-γ production. Degranulation was largely associated with NK and NKT TIL. Blockade of H-2kb and/or CD1d during ex vivo cytotoxicity assays revealed NKT has limited direct cytotoxicity against parent MC38. However, forced CD1d overexpression in MC38 cells significantly diminished tumor growth, suggesting NKT TIL exerts indirect control over MC38 growth.ConclusionsDespite an indirect benefit of early NKT activity, CB accelerates a switch from TNFα, NKT-driven immune response toward an IFN-γ driven CD4+/CD8+ T cell response in MC38 tumors. These results uncover a novel NKT/T cell switch that may be a key feature of CB response in CD1d+ tumors.

A New Platform For Trivalent Bispecific Antibodies Used For T-Cell Redirected Killing Of B-Cell Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3078-3078
Author(s):  
Diane L Rossi ◽  
Edmund A Rossi ◽  
David M Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Stock Option ◽  
B Cell Malignancies ◽  
Hla Dr ◽  
Close Proximity

Abstract Background Various formats of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise both pre-clinically and clinically. The scFv-based constructs, including BiTE and DART, which bind monovalently to CD3 on T cells and to the target antigen on tumor cells, exhibit fast blood clearance and neurological toxicity due to their small size (∼55 kDa). Herein, we describe the generation of novel T-cell redirecting trivalent bsAbs comprising an anti-CD3 scFv covalently conjugated to a stabilized F(ab)2. The design was initially characterized with a prototype construct designated (19)-3s, which specifically targets CD19 on B cells. A panel of trivalent bsAbs was evaluated for their potential use in targeted T-cell immunotherapy of various B-cell malignancies. Potential advantages of this design include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. Methods The DOCK-AND-LOCKTM (DNLTM) method was used to generate a panel of B-cell targeting bsAbs, (19)-3s, (20)-3s, (22)-3s, and (C2)-3s, which target CD19, CD20, CD22, and HLA-DR, respectively. This was achieved by combining a stabilized anti-X F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by LC-MS, SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Each construct can mediate the formation of immunological synapses between T cells and malignant B cells, resulting in T-cell activation. At an E:T ratio of 10:1, using isolated T cells as effector cells, the bsAbs induced potent T-cell-mediated cytotoxicity in various B-cell malignancies, including Burkitt lymphomas (Daudi, Ramos, Namalwa), mantle cell lymphoma (Jeko-1), and acute lymphoblastic leukemia (Nalm-6). A non-tumor binding control, (14)-3s, induced only moderate T-cell killing at >10 nM. The nature of the antigen/epitope, particularly its size and proximity to the cell surface, appears to be more important than antigen density for T-cell retargeting potency (Table 1). It is likely that (20)-3s is consistently more potent than (19)-3s and (C2)-3s, even when the expression of CD19 or HLA-DR is considerably higher than CD20, as seen with Namalwa and Jeko-1, respectively. This is likely because the CD20 epitope comprises a small extracellular loop having close proximity to the cell surface. When compared directly using Daudi, (22)-3s was the least potent. Compared to CD19 and CD20, CD22 is expressed at the lowest density, is a rapidly internalizing antigen, and its epitope is further away from the cell surface; each of these factors may contribute to its reduced potency. Finally, sensitivity to T-cell retargeted killing is cell-line-dependent, as observed using (19)-3s, where Raji (IC50 >3 nM) is largely unresponsive yet Ramos (IC50 = 2 pM) is highly sensitive, even though the former expresses higher CD19 antigen density. Conclusions (19)-3s, (20)-3s, (22)-3s, and (C2)-3s can bind T cells and target B cells simultaneously and induce T-cell-mediated killing in vitro. The modular nature of the DNL method allowed the rapid production of several related conjugates for redirected T-cell killing of various B-cell malignancies, without the need for additional recombinant engineering and protein production. The close proximity of the CD20 extracellular epitope to the cell surface results in the highest potency for (20)-3s, which is an attractive candidate bsAb for use in this platform. We are currently evaluating the in vivo activity of these constructs to determine if this novel bsAb format offers additional advantages. Disclosures: Rossi: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other.

SOX2 as a target for immunotherapy of pediatric gliomas.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e22012-e22012 ◽  
Author(s):  
Juan Vasquez ◽  
Anita Huttner ◽  
Lin Zhang ◽  
Asher Marks ◽  
Amy Chan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Tumor Immunity ◽  
Immune Checkpoint ◽  
Cell Mass ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Low Grade ◽  
Glial Tumors

e22012 Background: New treatments are needed to improve outcomes for pediatric gliomas. Immune checkpoint inhibitors are effective therapies in tumors with a high mutation burden that express multiple neo-antigens. However, for pediatric tumors that carry few mutations, there is a need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors, and the capacity of the immune system in these patients to recognize SOX2, has not been studied. Methods: We examined the expression of SOX2 on paraffin-embedded tissue from pediatric glial tumors (n = 30). The presence of T cell immunity to SOX2 was examined in both blood and tumor-infiltrating T cells using antigen-dependent cytokine and T cell proliferation assays (n = 15). The nature of tumor-infiltrating immune cells in glial tumors (n = 4) was analyzed using single cell mass cytometry. Results: SOX2 is expressed by tumor cells but not surrounding normal tissue in all low grade gliomas (n = 15), high grade gliomas (n = 7), ependymomas (n = 3) and in 60% of oligodendrogliomas (n = 5). T cells against SOX2 can be detected in blood and tumor tissue in 33% of patients. CD4 and CD8 tumor infiltrating T-cells display a higher proportion of PD-1 expression compared to circulating T cells (p < 0.05). Glial CD4 and CD8 T cells are enriched for tissue resident memory phenotype (TRM; CD45RO+, CD69+, CCR7-) and the expression of PD-1 is primarily on these TRM cells (p < 0.05). A subset of CD4 and CD8 TRM cells also co-express multiple inhibitory checkpoints including PD-L1 and TIGIT. Glial tumors also contain NK cells with reduced expression of lytic granzyme (p < 0.05). Conclusions: Our data demonstrate in vivo immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Our data also suggest that the TRM subset of tumor-infiltrating T cells may be key targets for immune checkpoint blockade, and harnessing tumor immunity will likely require the combined targeting of multiple inhibitory checkpoints. Future efforts to target SOX2 with dendritic cell vaccines combined with immune checkpoint blockade could provide effective tumor immunity and improve outcomes in pediatric brain tumors.

scholarly journals Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160293 ◽  
Author(s):  
Lee Kim Swee ◽  
Zhen Wei Tan ◽  
Anna Sanecka ◽  
Nagisa Yoshida ◽  
Harshil Patel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Effector Function ◽  
Effector Functions ◽  
Physiological Conditions ◽  
Cell Clones

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii . We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro . Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds.

scholarly journals T Cell Metabolism and Memory

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-6-SCI-6
Author(s):  
Jeffrey C Rathmell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Glucose Transporter ◽  
Cell Metabolism ◽  
Effector Function ◽  
Glutamine Metabolism ◽  
T Cell Subsets ◽  
Cd4 T Cell ◽  
Th1 Cells ◽  
Inflammatory Models

Abstract Lymphocyte activation leads to rapid proliferation and differentiation and we have shown that CD4 T cell subsets are metabolically distinct. These specific metabolic programs may allow new understanding and approaches to manipulate immunity. Using metabolic network analysis of metabolomics and proteomics we defined several metabolic nodes differentially utilized by CD4 T cell subsets, including glutamine metabolism. By genetically deleting the glucose transporter Glut1 or the glutaminolysis enzyme, Glutaminase (GLS), we have shown that glycolysis and glutaminolysis are both used by activated T cells. All effector T cells require glycolysis, but we found that Th17 cells preferentially require GLS while Th1 cells are actively impaired by this enzyme. Thus, inhibition of GLS both reduces Th17 responses and promotes differentiation of Th1 cells and can lead to signs of T cell exhaustion. We show that GLS-deficiency can protect against Th17-mediated inflammatory models and also can augment effector function of Th1 CAR-T cells against B cell targets. Understanding mechanisms that regulate T cell metabolism may provide new tools to modulate immunity the balance of T cell effector populations to both suppress inflammation or promote effector function. Disclosures Rathmell: Calithera: Research Funding.

TAMI-27. METABOLIC MANIPULATION OF T CELLS TO TREAT BRAIN TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi203-vi204
Author(s):  
Guimei Tian ◽  
Linchun Jin ◽  
Devshri Doshi ◽  
Aida Karachi ◽  
Mariana Dajac ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Brain Tumors ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Disease Recurrence

Abstract INTRODUCTION Glioblastoma are a challenge for neuro-oncologists and current therapies are minimally effective. Standard-of- care treatment is almost inevitably followed by disease recurrence. Adoptive T cell transfer has emerged as a viable therapeutic for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. OBJECTIVES The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T cells. The overall goal of this project is to test new treatments to reverse immune dysfunction in cancer through the regulation of T cell metabolic signaling. We propose that modulating glucose pathway in T cells can potentiate their anti-tumor activity once adoptively transferred. METHODS T cells glucose metabolic pathway was modulated via glucose transporters overexpression. The functionality of metabolically modified T cells was investigated in murine and human models. RESULTS We demonstrated the existence of a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T cell glucose utilization and provide survival/growth advantage and enhanced T cell activation in glucose-restricted conditions. We also established that glucose transporter overexpression improves intratumoral infiltration of adoptively transferred T cells. CONCLUSION This project integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

Development of Novel Non-Immunoglobulin Centyrin-Based Cars (CARTyrins) Targeting Human Bcma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4557-4557 ◽  
Author(s):  
Burton Earle Barnett ◽  
Xinxin Wang ◽  
David L. Hermanson ◽  
Yening Tan ◽  
Eric M. Osertag ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Surface Expression ◽  
Domain Swapping ◽  
Target Cells ◽  
High Quality ◽  
Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapy is a promising type of cancer therapy and substantial progress has been made in developing adoptive T cell approaches for B cell malignancies. B cell maturation antigen (BCMA) is an attractive target for patients with multiple myeloma (MM) due to its high level of expression on tumor cells and restricted expression on normal tissues. Traditionally, the antigen-binding domain of a CAR is a single chain variable fragment (scFv) comprised of heavy chain (HC) and light chain (LC) variable fragments joined by a flexible linker that has been derived from a non-human monoclonal Ab (mAb). However, there are a number of disadvantages to scFv-based CARs including the limited availability of scFv, their potential to elicit antibody responses, and their association with tonic signaling due, in part, to inherent instability and flexibility of the structure and the potential for both HC/LC domain swapping and multimer formation through framework region interactions. Thus, replacement with alternative binding technologies may improve CAR-T efficacy in the clinic. Centyrins are alternative scaffold molecules that bind protein targets with high affinity and specificity, similar to scFv molecules. However, unlike scFv, Centyrins are smaller, derived from human consensus tenascin FN3 domains and are predicted to have decreased immunogenicity. Additionally, a monomeric Centryin in CAR format (i.e. CARTyrin molecule) is less likely to engage in domain swapping or interact with other Centyrins at the cell surface, thereby limiting the potential for the tonic signaling that drives the functional exhaustion of CAR T cells. Centyrins can be isolated against virtually any antigen through ex vivo panning of an extensive Centyrin library, yielding many distinct binders with a range of affinities and target epitopes. Panning with soluble BCMA protein yielded a large pool of BCMA-specific Centyrins, from which 11 distinct monomeric binders and 1 non-monomeric binder were selected for further study in CAR format. In addition, we tested numerous signal peptides, linkers, transmembrane domains and signaling domains to determine optimal configuration. We then created all CARTyrins by fusing each Centyrin with a CD8a leader peptide, spacer and transmembrane domain, as well as an intracellular signaling domain derived from both 4-1BB and CD3ζ. High quality mRNA of each CARTyrin construct was produced in order to rapidly screen CARTyrin cell surface expression and functionality in human pan T cells against BCMA+ targets. We also constructed scFv-based CARs against CD19 and BCMA for comparison. Previously CD3/CD28-stimulated T cells were electroporated (EP) with mRNA encoding each of the 12 anti-BCMA CARTyrins and, the following day, analyzed for surface expression of CARTyrin and their ability to degranulate against BCMA+ tumor cells. All 12 CARTyrins were detected on the cell surface and the 11 monomeric CARTyrins imparted BCMA-specific killing capacity to T cells. Notably, in these assays, CARTyrins were functionally comparable to scFv-based CARs against BCMA or to CD19-specific scFv-based CARs in a parallel assay with CD19+ tumor cells. The 11 functional anti-BCMA CARTyrins were further characterized for functional avidity by determining their activity against a panel of target cells with titrated levels of surface BCMA expression. To create this panel, various amounts of high quality BCMA mRNA were electroporated into BCMA- K562 tumor cells. After 4 hours of co-culture with the panel of BCMA expressing cells, CARTyrin+ T cell activity was measured as a function of CD107a expression. We observed a range of activities by each CARTyrin and show that this assay can be utilized to determine the minimal effective dose of BCMA needed to induce killing by CARTyrin+ cells. Furthermore, we establish that certain BCMA-specific CARTyrins are responsive to target cells with extremely low levels of surface BCMA expression. These results confirm that Centyrins are viable replacements for scFv in the construction of functional CARs and establish their potential utility in generating novel BCMA-specific CAR molecules, as well as other novel targetable tumor antigens. Disclosures Barnett: Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

scholarly journals T Cells Expressing Engager and Costimulatory Molecules for the Immunotherapy of CD19+ Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2433-2433
Author(s):  
Mireya Paulina Velasquez ◽  
Kota Iwahori ◽  
David L Torres ◽  
Sunitha Kakarla ◽  
Caroline Arber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Xenograft Model ◽  
Retroviral Vector ◽  
Effector Function ◽  
Target Cells ◽  
Antitumor Effects

Abstract Background: Immunotherapy with anti-CD19/anti-CD3 bispecific engager molecules has shown promise in clinical studies for CD19+ malignancies. However engager molecules have short half-lives and do not accumulate at tumor sites. In addition, co-delivery of other immunostimulatory molecules to enhance antitumor effects is difficult to achieve. We have recently shown that T cells can be genetically modified to secrete bispecific engager molecules (ENG-T cells). ENG-T cells are activated by tumor cells in an antigen-dependent manner, redirect bystander T cells to tumor cells, and have antitumor activity in preclinical models. We now wanted to explore if additional genetic modifications of ENG-T cells can enhance their effector function in vitro and in vivo. Since bispecific engager molecules do not provide co-stimulation, we focused on the provision of co-stimulatory signals by coexpressing CD80 and CD137L on the cell surface of ENG-T cells. Thus, the aim of the study was to compare the effector function of CD19-specific T-cell engagers (CD19-ENG T cells) and CD19-ENG T cells co-expressing CD80 and 41BBL (CD19-ENG/Costim T cells). Methods: CD19-ENG T cells were generated by transducing T cells with a retroviral vector encoding a CD19-specific T-cell engager and mOrange separated by an IRES (SFG.CD19-ENG-I-mO), and CD19-ENG/Costim T cells were generated by double transducing T cells with SFG.CD19-ENG-I-mO and a 2nd retroviral vector encoding 41BBL and CD80 separated by an IRES. The effector function of ENG T-cells was evaluated in vitro and in a leukemia xenograft model. Results: After single or double transduction 60-80% of T cells were positive for mOrange, and ~80% of CD19-ENG/Costim T cells were positive for CD80 and 30-40% positive for 41BBL. In coculture assays CD19-ENG and CD19-ENG/Costim T cells recognized CD19+ lymphoma (Daudi, Raji) and acute leukemia (BV173) cells as judged by IFN-g secretion in contrast to negative controls. While CD19+ target cells that express CD80 and CD86 (Daudi and Raji) induced robust IL2 production of CD19-ENG and CD19-ENG/Costim T cells, CD19-ENG/Costim T cells produced significantly higher levels of IL2 in comparison to CD19-ENG T cells after stimulation with CD19+/CD80-/CD86- negative target cells (BV173). Cytokine production was antigen dependent since ENG and ENG/Costim T cells specific for an irrelevant antigen (EphA2) did not produce cytokines. Specificity was confirmed in cytotoxicity assays. In transwell assays containing inserts preventing T-cell migration, only ENG T cells redirected bystander T cells in the bottom well to CD19+ tumor cells. To assess in vivo anti-tumor activity of CD19-ENG T cells and CD19-ENG/Costim T cells we used the BV173/NSG mouse xenograft model in which BV173 cells are genetically modified with firefly luciferase (ffLuc-BV173) to allow for serial bioluminescence imaging. While therapy with CD19-ENG T cells on day 7 post ffLuc-BV173 injection resulted in the cure of all mice, when therapy was delayed to day 14, only 1/10 mice was alive on day 80. In contrast therapy of mice on day 14 with CD19-ENG/Costim T cells resulted in long-term survival of 7/10 mice. Control T cells (EphA2-ENG T cells or EphA2-ENG/Costim T cells) had no antitumor effects. Conclusions: We have generated CD19-ENG T cells and CD19-ENG/Costim T cells with the ability to direct bystander T cells to CD19+ malignancies. Both ENG T-cell populations had potent antitumor activity in a preclinical ALL model, and provision of costimulation further enhanced antitumor effects. Genetically modifying T cells to express engager molecules and additional molecules to enhance their effector function may present a promising alternative to current CD19-targeted immunotherapies. Disclosures Velasquez: Celgene, Bluebird bio: Other. Iwahori:Celgene, Bluebird bio: Other. Kakarla:Celgene, Bluebird bio: Other. Song:Celgene, Bluebird bio: Other. Gottschalk:Celgene, Bluebird bio: Other.

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