468 Enhancers and repressors of immunotherapy: translational perspectives on gene-mediated cytotoxic immunotherapy in glioblastoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A498-A498
Author(s):  
Sean Lawler ◽  
Marilin Koch ◽  
Mikolay Zdioruk ◽  
Estuardo Aguilar-Cordova ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
Cell Killing ◽  
Future Application ◽  
Cytotoxicity Assays

AcknowledgementsThis was supported by NCI P01CA069246 (Chiocca)ConclusionsOur data suggest that dexamethasone may decrease the efficacy of immunotherapy for glioma through impaired T cell function: this emphasizes the need in identifying alternatives to dexamethasone to prevent attenuated responses in immunotherapies. The combination of GMCI with ATRi however points to additional therapeutic benefit through enhanced cytotoxic efficacy, improved immunogenicity in vitro and increased long-term survival in vivo, making it a promising future approach for the treatment of glioblastoma.ResultsCytotoxicity assays showed that dexamethasone has a slight impact on GMCI in vitro. In T-cell-functional assays, we observed a significantly impaired tumor cell killing. Immune cell response assays revealed a reduced immune cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells. In vivo, while treatment with GMCI alone resulted in longer median symptom-free survival (39.5d) versus no treatment (23d), the combination of GMCI and dexamethasone resulted in the significant reduction of this effect (29d vs 39.5d ; p = 0.0184).The combination of ATRi with GMCI proved to be synergistic in cytotoxicity assays. Flow cytometry revealed a significant increase in DSB-associated H2AX foci as well as an improved immune profile by downregulation of GMCI-induced PD-L1 expression. In vivo, the combination with ATRi led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant compared to the untreated control (p=0.0022).MethodsWe investigated the effects of ATR-inhibition and dexamethasone on GMCI in vitro using cytotoxicity, flow cytometry and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone and ATRi in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. Tumor immune infiltrates were analyzed with flow cytometry.BackgroundGene-mediated cytotoxic immunotherapy (GMCI) is a local tumor immunotherapy that uses aglatimagene besadenovec (a non-replicating serotype 5 adenovirus, expressing HSV1 thymidine kinase) with the prodrug ganciclovir to induce DNA double strand breaks (DSB), leading to immunogenic tumor cell death and intratumoral immune cell invasion. Here we investigate potential repressors and enhancers of GMCI’s effectiveness. GMCI is currently in clinical trials in combination with immune checkpoint blockade in glioblastoma. Thus we set out to identify potential areas to improve this approach for future application. Dexamethasone is used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. However, the influence of dexamethasone on the efficacy of GMCI has not been explored. In contrast, DNA damage response inhibitors like the ATR inhibitor (ATRi) AZD6738 might not only amend the cytotoxic but also the immunogenic profile of GMCI, rendering it an attractive combination partner.

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.

In Vivo Tracking of T Cells in Humans Unveils Decade-Long Survival and Activity of Genetically Modified T Memory Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 547-547
Author(s):  
Serena Scala ◽  
Luca Biasco ◽  
Luca Basso Ricci ◽  
Francesca Dionisio ◽  
Cristina Baricordi ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Future Application ◽  
Long Survival ◽  
Healthy Donors ◽  
Integration Sites

Abstract A deeper understanding of T lymphocytes survival and differentiation potential in humans is paramount for the development of effective gene/cell therapies based on T-cell engineering. We here performed a comprehensive study of T-cells dynamics and plasticity in humans by a unique combination of phenotypic/functional studies and high-throughput integration sites (IS) analyses. We analyzed samples from hematopoietic stem cells (HSC) (n=10) or mature lymphocytes (PBL) gene therapy (GT) (n=4) treated ADA (adenosine deaminase) deficient-SCID patients. For comparative analyses, we also collected data from pediatric (n=19) and adult (n=52) healthy donors (HD), and from bone marrow transplanted patients (BMT) with primary immunodeficiencies (n=10, 4 with ADA-SCID). We observed that vector-positive CD62L+/CD45RA+ putative T naïve cells were detectable 12 years after last infusion of gene-corrected lymphocytes in peripheral blood of PBL-GT patients that lack the support of transduced lymphocytes precursors. We then unveiled that the vast majority of these CD62L+/CD45RA+ cells (80.3%) in PBL-GT patients could be actually classified phenotypically (CD95, IL2Rβ and IL7Rα surface expression) and functionally (IFNγ production and aCD3/aCD28 in vitro differentiation) as active long-lasting T memory stem cells (Tscm). The peculiar Tscm frequency found in PBL-GT patients was most likely due to a combinatorial in vitro and in vivo effect. Indeed, by a series of in vitro assays, we showed that Tscm relative enrichment in CD45RA+CD62L+ compartment have occurred during the in vitro manipulation of T cells before infusion. Additionally, we found higher-then-normal Tscm contribution among CD45RA+/CD62L+ cells even in ADA-SCID patients receiving HSC-GT and BMT, suggesting a role of disease background on in vivo Tscm persistence. Analyzing our cohorts of healthy donors and treated individuals we were able to further correlate Tscm contribution in vivo with age, conditioning regimen, disease background, cell source, and long-term T-cell reconstitution. One unique aspect of our study consisted in the opportunity to track Tscm clonal dynamics in vivo in humans since each gene-corrected cell infused in our GT patients is univocally and permanently tagged by a retroviral integration site.To perform in vivo molecular tracing of individual T-cell clones we sorted T naïve, Tscm, central memory and effector memory subtypes. We then collected from these subpopulations, by LAM-PCR+Illumina-Miseq sequencing, 2.584.137 integration sites (IS) sequences mapped to 1.746 unique chromosomal positions, corresponding to 910 integrations from 5 HSC-GT patients in vivo, 79 integrations from 2 PBL-GT samples of transduced cell products prior to infusion and 754 integrations from 4 PBL-GT patients in vivo. Firstly, to establish a relationship between precursors and terminally differentiated T cells we searched for the presence of identical insertion sites detected in multiple T-cell subtypes, applying stringent analytical filters for cross-contaminations. Strikingly, the level of shared integrations in each subtype was directly correlated to its stage of differentiation with Tscm, isolated from PBL-GT patients, showing the highest proportion of integration sites shared with the other T-cell subsets. Importantly, the results of the same analysis performed on HSC-GT patients were outstandingly coherent with the progressive developmental model of memory T-cell differentiation. We then assessed the survival of individual Tscm clones by performing a longitudinal IS analysis of different T-cell subtypes isolated from 3 PBL-GT patients over a 2 to 5 years timeframe up to 12 years after last infusion. We were able to formally prove the persistence of individual Tscm by re-capturing identical IS tagging specific Tscm clones in two independent timepoints in a 5- years window. Importantly, the same IS were also detected in multiple T-cell subtypes, representing the best indirect evidence that these clones were endowed with long-term precursor activity. We also documented, by IS sequencing reads, the long-term polyclonal composition of each subtype and we did not observe enrichment for IS flanking proto-oncogenes. Overall, this study validates, for the first time in humans, the safe and functional decade-long survival of engineered Tscm, paving the way for their future application in clinical settings. Disclosures No relevant conflicts of interest to declare.

scholarly journals 888 An integrative approach to optimize a synthetic EphA2/CD137 agonist: balancing potency, physiochemical properties, and pharmacokinetics to achieve robust anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A931-A931
Author(s):  
Punit Upadhyaya ◽  
Gemma Mudd ◽  
Kristen Hurov ◽  
Johanna Lahdenranta ◽  
Elizabeth Repash ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
List Type ◽  
Continuous Exposure ◽  
Target Engagement ◽  
In Vivo Models ◽  
Anti Tumor Activity

BackgroundCD137 (4-1BB) is a resurging target in immunotherapy after the first generation of monoclonal antibodies were limited by hepatotoxicity1 or lack of efficacy.2 A new generation of CD137 agonists are now in clinical development but they exclusively utilize large molecules derived from recombinant technology and are associated with long circulating terminal half-lives.3–6 Unlike checkpoint inhibition where complete saturation of the receptors drives the reversal of immunosuppression, intermittent target engagement that reflects the physiological context of T cell co-stimulation may be more appropriate for a CD137 agonist.7 Bicyclic peptides or Bicycles are a class of small (MW~2kDa), highly constrained peptides characterized by formation of two loops cyclized around a symmetric scaffold. To develop a differentiated tumor antigen dependent CD137 agonist for treating EphA2 expressing solid tumors, we integrated structure activity relationship (SAR) data from biochemical binding studies and in-vitro and in-vivo models to understand the relationship between exposure, target engagement and preclinical efficacy.MethodsOver 150 different EphA2/CD137 tumor-targeted immune cell agonists (Bicycle TICAs) were synthesized by linking Bicycle® binders to EphA2 to those binding CD137.8 The molecules were assessed in vitro using a CD137 reporter assay and by measuring cytokine production from primary human PBMC in tumor cell co-cultures. The pharmacokinetics were evaluated in rodents using Phoenix WinNonlin. The in vivo activity was determined in syngeneic mouse tumor models by measuring tumor growth kinetics and using tumor immune cell and transcriptional profiling by IHC and NanoString.ResultsEvaluation of the Bicycle TICAs in co-culture assays with EphA2-expressing tumor cell lines and Jurkat reporter cells overexpressing CD137 or human PBMCs demonstrated that constructs bearing two CD137 binding Bicycles to one EphA2 binding Bicycle (1:2 format) were more potent than the 1:1 format.8 Several Bicycle TICAs with amino acid substitutions to the EphA2 binding Bicycle maintained sub-nanomolar potency in-vitro and exhibited a plasma terminal half-life (t1/2) in rodents ranging from 0.4 and 4.0 h. Modifications that conferred aqueous solubility of greater than 10 mg/mL were considered suitable for further development. Treatment of MC38 tumors in immunocompetent mice with this series of molecules demonstrated that low MW Bicycle TICAs with sub-nanomolar potency and a t½ of ~1 h in mouse maintained target coverages necessary to produce robust modulation of the tumor immune microenvironment and tumor regression.ConclusionsA differentiated EphA2-dependent CD137 agonist was developed that exploits intermittent rather than continuous exposure for robust anti-tumor activity.ReferencesSegal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res 2017;23(8):1929–1936.Segal NH, Aiwu RH, Toshihiko D, et al. Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res 2018;24(8):1816–1823.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Hinner MJ, Aiba RSB, Jaquin TJ, et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res 2019;25(19):5878–5889.Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019;11(496):eaav5989.Eskiocak U, Guzman W, Wolf B, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020;5(5):e133647.Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonist antibody development in cancer. Nat Rev Drug Discov 2018;17:509–27.Upadhyaya P, Lahdenranta J, Hurov K, et al. Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist. J Immunother Cancer 2021;9:e001762.

102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A113-A113
Author(s):  
Mireia Bachiller García ◽  
Lorena Pérez-Amill ◽  
Anthony Battram ◽  
Alvaro Urbano-Ispizua ◽  
Beatriz Martín-Antonio
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Cord Blood ◽  
Immune Cells ◽  
In Vivo Models ◽  
Cell Clusters ◽  
Cytotoxicity Assays ◽  
Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals 228 Antagonistic pH-selective VISTA antibody SNS-101 potentiates anti-PD-1/PD-L1-induced anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A243-A243
Author(s):  
Thomas Thisted ◽  
Arnab Mukherjee ◽  
Kanam Malhotra ◽  
Zuzana Biesova ◽  
Yuliya Kleschenko ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Activation ◽  
Drug Disposition ◽  
Checkpoint Inhibitors ◽  
Tumor Rejection ◽  
Therapeutic Window ◽  
Drug Candidate

BackgroundImmunotherapies, especially immune checkpoint inhibitors, have become a cornerstone of cancer treatment. Remarkable clinical responses have been observed blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis across a spectrum of indications. However, innate and/or acquired resistance to anti-PD-1 blockade remains a major challenge. V-domain Ig suppressor of T-cell activation (VISTA) is a B7-family member, which promotes T-cell and myeloid quiescence and represents a promising target, particularly in combination with anti-PD-1/PD-L1 treatment. Recently, the interaction of VISTA with its receptor PSGL-1 was demonstrated to be significantly enhanced by the acidic tumor microenvironment (TME). As VISTA is highly expressed on myeloid cells, including those in the blood, antibodies binding VISTA at physiological pH 7.4 could result in rapid elimination from circulation through targeted-mediated drug disposition, making efficacious drug occupancy levels difficult to reach and potentially narrowing the therapeutic window. An antibody engineered to selectively bind and block VISTA at low pH in the TME may therefore be an ideal drug candidate.MethodsIn this study, fully human anti-VISTA antibodies were generated through pH-selective enrichment strategies of a yeast-based display library comprising highly diverse synthetic immune repertoires. The ‘parental’ antibodies have been extensively characterized using in vitro flow-cytometry, surface-plasmon resonance (SPR) and PSGL-1/VISTA inhibition assays in primary human CD4 and CD8 T-cells at pH 6.0 and pH 7.4. Eight parental antibodies were identified and tested for combinatorial efficacy with anti-PD-1 in vivo in human VISTA knock-in mice inoculated with syngeneic MC-38 tumors. These antibodies underwent further optimization for enhanced binding affinity at pH 6.0 and decreased binding at pH 7.4. ‘Progeny’ antibody ranking was based on the same in vitro and in vivo characterization as parental antibodies.ResultsEighty four parental antibodies were initially discovered. Flow-cytometry and SPR analysis revealed candidates displaying pH-dependent binding to endogenously expressed native VISTA on cells, and a PSGL-1/VISTA inhibition assay at pH 6.0 was run to identify and rank potent interface blockers. Eight candidate antibodies were tested in an in vivo intervention study in combination with anti-murine PD-1 demonstrating varied combinatorial efficacy with a subset leading to superior tumor rejection. Characterization of optimized progeny antibodies led to identification of anti-VISTA antibody SNS-101.ConclusionsEnrichment of highly diverse antibody libraries led to the identification of a pH-selective inhibitory anti-VISTA antibody SNS-101, which exerts excellent combinability with anti-PD-1 leading to superior anti-tumor activity in a mouse model.

scholarly journals IMMU-46. EXAMINATION OF THE EFFECTS OF DEXAMETHASONE ON THE EFFICACY OF IMMUNOTHERAPY IN GLIOMA USING GENE-MEDIATED CYTOTOXIC IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi129-vi129
Author(s):  
Marilin Koch ◽  
Mykola Zdioruk ◽  
M Oskar Nowicki ◽  
Estuardo Aguilar ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Symptomatic Treatment ◽  
Tumor Cell Death ◽  
The Impact

Abstract RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 866-866
Author(s):  
Carolina Berger ◽  
Michael C. Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Annexin V ◽  
T Cell Memory ◽  
T Cell Clones ◽  
Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for &gt;30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

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