scholarly journals Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice

2021 ◽  
Author(s):  
Pavlina Chuntova ◽  
Yafei Hou ◽  
Ryosuke Naka ◽  
Yitzhar Goretsky ◽  
Takahide Nejo ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Strain ◽  
Growth Factor Receptor ◽  
Suppressor Cells ◽  
Cytotoxic Activities ◽  
Combination Regimen ◽  
Preclinical Studies ◽  
Term Survival

ABSTRACTBackgroundRigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART)-cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII).MethodsWe first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T-cells and evaluated the function of the CART-cells both in vitro and in vivo. In order to inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208).ResultsBoth RV- and Tg-CART-cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART-cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII− SB28.ConclusionOur new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.Importance of studyThe majority of preclinical studies evaluating CART therapy for GBM have utilized xenografts implanted into immunocompromised mice. Because the successful development of these strategies will depend on the understanding of critical interactions between therapeutic cells and the endogenous immune environment, it is essential to develop a novel immunocompetent system which allows us to study these interactions in a robust and reproducible manner. To this end, we created a Tg mouse strain in which all T-cells express a murinized EGFRvIII-CAR. T-cells derived from these mice demonstrated consistent CAR expression and EGFRvIII-specific cytotoxicity while traditional transduction with a CAR vector showed batch-to-batch variability. The syngeneic system also gave us the opportunity to evaluate a combination regimen with blockade of myeloid-derived suppressor cells. The Tg-CART mice represent a novel system for robust, and reproducible preclinical investigations.

scholarly journals Mechanisms of idiotype suppression. I. In vitro generation of idiotype-specific suppressor T cells by anti-idiotype antibodies and specific antigen.

1979 ◽  
Vol 149 (6) ◽  
pp. 1371-1378 ◽  
Author(s):  
B S Kim
Keyword(s):  
T Cells ◽  
Specific Antigen ◽  
Suppressor Cells ◽  
Culture Period ◽  
Spleen Cells ◽  
Suppressor T Cells ◽  
Myeloma Protein ◽  
Specific Suppressor T Cells

Normal BALB/c spleen cells are unresponsive in vitro to the phosphorylcholine (PC) determinant in the presence of anti-idiotype antibodies specific for the TEPC-15 myeloma protein (T15) which carries an idiotypic determinant indistinguishable from that of most anti-PC antibodies in BALB/c mice. The possibility that idiotype-specific suppressor cells may be generated during the culture period was examined by coculturing the cells with untreated syngeneic spleen cells. Cells that had been preincubated with anti-T15 idiotype (anti-T15id) antibodies and a PC-containing antigen, R36a for 3 d, were capable of specifically suppressing the anti-PC response of fresh normal spleen cells, indicating that idiotype-specific suppressor cells were generated during the culture period. The presence of specific antigen also appeared to be necessary because anti-T15id antibodies and a control antigen, DNP-Lys-Ficoll, were not capable of generating such suppressor cells. Suppressor cells were induced only in the population of spleen cells nonadherent to nylon wool and the suppressive activity was abrogated by treatment with anti-Thy 1.2 serum and complement. These results indicate that anti-idiotype antibodies and specific antigen can generate idiotype-specific suppressor T cells in vitro. These in vitro results may reflect in vivo mechanisms of idiotype suppression.

scholarly journals The critical immunosuppressive effect of MDSC-derived exosomes in the tumor microenvironment

2020 ◽  
Author(s):  
Mohammad H. Rashid ◽  
Thaiz F. Borin ◽  
Roxan Ara ◽  
Raziye Piranlioglu ◽  
Bhagelu R. Achyut ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
Cell Types ◽  
Biological Macromolecules

AbstractMyeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME), and our perception regarding the role of MDSCs in tumor promotion is attaining extra layer of intricacy in every study. In conjunction with MDSC’s immunosuppressive and anti-tumor immunity, they candidly facilitate tumor growth, differentiation, and metastasis in several ways that yet to be explored. Alike any other cell types, MDSCs also release a tremendous amount of exosomes or nanovesicles of endosomal origin and partake in intercellular communications by dispatching biological macromolecules. There has not been any experimental study done to characterize the role of MDSCs derived exosomes (MDSC exo) in the modulation of TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant amount of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those are in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cells in vivo by treating the mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that immunosuppressive and tumor-promoting functions of MDSC are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

scholarly journals GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO

1974 ◽  
Vol 140 (3) ◽  
pp. 648-659 ◽  
Author(s):  
Judith A. Kapp ◽  
Carl W. Pierce ◽  
Stuart Schlossman ◽  
Baruj Benacerraf
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune Responses ◽  
Cell Function ◽  
Suppressor Cells ◽  
Specific Response ◽  
Spleen Cells ◽  
X Irradiation

In recent studies we have found that GAT not only fails to elicit a GAT-specific response in nonresponder mice but also specifically decreases the ability of nonresponder mice to develop a GAT-specific PFC response to a subsequent challenge with GAT bound to the immunogenic carrier, MBSA. Studies presented in this paper demonstrate that B cells from nonresponder, DBA/1 mice rendered unresponsive by GAT in vivo can respond in vitro to GAT-MBSA if exogenous, carrier-primed T cells are added to the cultures. The unresponsiveness was shown to be the result of impaired carrier-specific helper T-cell function in the spleen cells of GAT-primed mice. Spleen cells from GAT-primed mice specifically suppressed the GAT-specific PFC response of spleen cells from normal DBA/1 mice incubated with GAT-MBSA. This suppression was prevented by pretreatment of GAT-primed spleen cells with anti-θ serum plus C or X irradiation. Identification of the suppressor cells as T cells was confirmed by the demonstration that suppressor cells were confined to the fraction of the column-purified lymphocytes which contained θ-positive cells and a few non-Ig-bearing cells. The significance of these data to our understanding of Ir-gene regulation of the immune response is discussed.

scholarly journals Elimination of syngeneic sarcomas in rats by a subset of T lymphocytes.

1980 ◽  
Vol 152 (4) ◽  
pp. 823-841 ◽  
Author(s):  
E Fernandez-Cruz ◽  
B A Woda ◽  
J D Feldman
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Subset ◽  
Suppressor Cells ◽  
Effector Cells ◽  
Long Duration ◽  
Ia Antigens

Established subcutaneous Moloney sarcomas (MST-1) of large size and long duration were eliminated from syngeneic rats by intravenous infusion of varying numbers of specific syngeneic effector T lymphocytes. Spleen cells from BN rats in which tumor had regressed were cultured in an in vitro mixed lymphocyte tumor cell culture (MLTC) to augment cytotoxicity of effector cells. In the MLTC a T cell subset was expanded in response to MST-1 antigens and transformed into blast elements. With these changes, there was an increase in the W3/25 antigen on the T cell surface, a decrease of W3/13 antigen, and an increase in the number of T cells with Ia antigens. The subset associated with elimination of established tumors was a blast T cell W3/25+, W3/13+, as detected by monoclonal antibodies to rat T antigens. The W3/25+ subset was poorly cytotoxic in vitro for MST-1 and apparently functioned in vivo as an amplifier or helper cell in the tumor-bearing host. The W3/25- population was a melange of cells that included (W3/13+, W3/25-) T cells, null cells, Ig+ cells, and macrophages, and was associated with enhancement of tumor in vivo, suggesting the presence of suppressor cells.

Myeloid-derived suppressor cells as a potential treatment target for pancreas adenocarcinoma.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 194-194
Author(s):  
M. R. Porembka ◽  
J. B. Mitchem ◽  
P. S. Goedegebuure ◽  
D. Linehan
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Animal Studies ◽  
Suppressor Cells ◽  
Disease Stage ◽  
Tumor Growth Rate ◽  
Myeloid Derived Suppressor Cells ◽  
Pancreas Adenocarcinoma

194 Background: Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA). Here, we quantify MDSC prevalence in patients with PA and assess the efficacy of MDSC depletion in a murine model of PA. Methods: Peripheral blood and tumor samples were collected from patients with PA, analyzed for MDSC (CD15+11b+) by flow cytometry (FC) and compared to cancer-free controls (CFC). The suppressive capacity of MDSC and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid (ZA), a potent aminobisphosphonate previously shown to target MDSC. Endpoints included tumor size, survival, and MDSC prevalence. Tumor cell infiltrate was analyzed by FC for MDSC (Gr1+CD11b+) and effector T cells; tumor cytokine levels were measured by Luminex assay. Results: Patients with PA demonstrated increased circulating MDSC compared to CFC, which correlated with disease stage (metastatic PA: 68%±3.6% of CD45+ cells, resectable PA: 57%±3.5%, CFC: 37%±3.6%; p<0.0001). Normal pancreas tissue showed no MDSC infiltrate while PA avidly recruited CD11b+15+ cells to the primary tumor. Murine tumors similarly recruited MDSC that actively suppressed CD8+ T cells in vitro measured by CFSE dilution and accelerated tumor growth in vivo by adoptive transfer with Pan02 cells (p<0.001). Treatment with ZA impaired MDSC accumulation in the tumor (Placebo: 78%, ZA: 51%, p<0.05) resulting in delayed tumor growth rate (p<0.0001) and prolonged median survival (Placebo: 59 days, ZA: 73 days, p<0.05). MDSC blockade increased recruitment of T cells to the tumor (CD4: 4.4%±1.1% vs 12.2%±2.0%, p<0.05; CD8: 3.9%±1.3% vs 10.6%±2.2%, p<0.05) and a more robust type 1 response with increased levels of IFN-g (p<0.05) and decreased levels of IL-10 (p<0.05). Conclusions: MDSC are an important mediator of tumor-induced immunosuppression in PA. Treatment with ZA effectively blocks MDSC accumulation improving anti-tumor response in animal studies. Efforts to block MDSC may represent a novel treatment strategy for PA. [Table: see text]

IL15, but Not IL2, Supports Long-Term Survival and Function of Human and Macaque Antigen-Specific CD8+ T Cell Clones.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3237-3237
Author(s):  
Carolina S. Berger ◽  
Michael Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cell ◽  
Term Survival ◽  
Long Term Survival ◽  
Cd8 T Cell Memory ◽  
And Function

Abstract The adoptive transfer of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) clones that have been isolated and expanded in vitro is a promising treatment modality for both human malignancies and infections. However, establishing immunity of sufficient magnitude and persistence for sustained efficacy is a limitation of this approach. Recent studies have identified a critical role for cytokine signaling including that mediated by IL15 in the establishment and maintenance of CD8+ T cell memory, suggesting that protocols for generating and transferring antigen-specific T cells might be improved. Interleukin-2 (IL2) is the T cell growth factor that has been widely used in vitro and in vivo for promoting T cell proliferation and persistence, but prolonged exposure of T cells to IL2 can enhance susceptibility to cell death and limit CD8+ memory T cell survival. IL15 is a novel cytokine that shares some activities with IL2 such as the induction of T cell proliferation, but exerts contrasting effects on the homeostasis of CD8+ T cell memory in experimental models. Here, we study the utility of IL15 to enhance the long-term survival and function of human and macaque antigen-specific CD8+ CTL clones in vitro. Human and macaque CD8+ CTL clones reactive against CMV were isolated by limiting dilution, expanded over 14 days in the presence of IL2 or IL15 (1–10 ng/ml), and then rested for &gt;4 weeks in media alone and with IL2 or IL15 at 0.01–10 ng/ml. Surviving T cells were enumerated at intervals, monitored for cell surface phenotype, and assayed for cytotoxicity by chromium release assay. CTL expanded in IL2 or IL15 proliferated equivalently over 14 days with a median of 1100 and 1400 fold increase in number, displayed surface markers consistent with an effector memory phenotype (CD45RA−CD62L−CCR7−CD28−), and showed comparable cytotoxicity (n=4). However, exposure after 14 days to IL15 at doses as little as 0.05-0.1 ng/ml greatly enhanced the survival of the CD8+ CTL as determined by Annexin V staining. By contrast, cells cultured without cytokines or with IL2 declined &gt;80% in number over 3 or 11 days, respectively. Of note, IL15 at higher doses (&gt;0.5 ng/ml), but not IL2, efficiently promoted sustained cell growth illustrated by labeling cells with CFSE. Cells cultured with IL15 displayed 1.5-fold increased expression of antiapoptotic molecules such as Bcl-xL and Bcl-2 over those plated in IL2 (n=4), indicating IL15 mediated its effects at least in part by preventing apoptosis. Of note, the cytotoxicity of CTL rested in IL2 was markedly reduced (&gt;60%, n=3), while the presence of IL15 permitted for sustained CTL function and expansion after restimulation. The responses of human and macaque CTL clones to IL15 were equivalent suggesting in vivo studies of T cell transfer in macaques may be predictive of results in humans. We have constructed retroviral vectors encoding intracytoplasmic truncated macaque CD34 or CD19 genes that could serve as nonimmunogenic selectable marker to track macaque T cells after transfer. Macaque T cells were efficiently transduced to express CD34t and CD19t (&gt;50%), and enriched to high purity by immunomagnetic selection. Studies to examine the safety and utility of IL15 on the survival of adoptively transferred CTL in macaques are in progress. Collectively, our data support that novel cytokines such as IL15 may prove useful to augment the long-term survival and effector function of ex vivo expanded antigen-specific CD8+ CTL clones after transfer.

scholarly journals EXTH-17. LOCAL DELIVERY OF CYTOKINES AND SYNTHETIC IMMUNOMODULATORS INCREASES T CELL INFILTRATION AND SIGNIFICANTLY IMPROVES SURVIVAL IN A POORLY IMMUNOGENIC MODEL OF GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii90-ii90
Author(s):  
Alexander Haddad ◽  
Jordan Spatz ◽  
Sara Collins ◽  
Matheus Pereira ◽  
Sabraj Gill ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Cd8 T Cells ◽  
Cell Killing ◽  
Cell Infiltration ◽  
Tumor Infiltration ◽  
Term Survival

Abstract BACKGROUND Severe local and systemic immune suppression in glioblastoma (GBM) contributes to the failure of single-agent immunotherapies in clinical trials. In this study, we evaluated the efficacy of locally delivered combination immunotherapy in a poorly immunogenic murine GBM model. METHODS Immunomodulators used in these studies included: IL-15 and IL-7 (T cell activation), LIGHT (T cell tumor infiltration), FLT3L (dendritic cell maturation/proliferation), a surface T cell engager (T cell killing of tumor cells), and a bispecific PD-L1/T cell engager (T cell killing targeted to PD-L1-expressing cells). We first assessed T cell-mediated cytotoxicity in vitro against SB28, a poorly immunogenic murine GBM cell line, after expressing these immunomodulators in combination. Next, tumor growth inhibition in vivo was evaluated in syngeneic C57BL/6 mice, initially by establishment of intracranial tumors with pre-transduced SB28 cells, and subsequently by delivering these immunomodulators to pre-established naïve SB28 tumors using neural stem cells (NSCs) and retroviral replicating vectors (RRV). RESULTS SB28 cells transduced with immunomodulators activated dose-dependent T cell-mediated cytotoxicity in vitro. Mice with pre-transduced intracranial SB28 gliomas showed significantly longer survival (minimum survival: 60 days, long-term survival in 57% of mice) vs. control mice (median survival: 20 days) (p&lt; 0.001), and significantly increased tumor infiltration of CD4+ and CD8+ T cells. NSC- and RRV-mediated immunomodulator delivery to pre-established SB28 gliomas also resulted in significantly increased survival of treated mice vs. controls (median survival: 31 days vs. 22 days, p&lt; 0.001). Immunomodulator-treated tumors again showed significantly increased infiltration of CD4+ and CD8+ T cells, along with decreased CD11b+ cell infiltration. CONCLUSIONS A novel combination therapy for GBM immunotherapy activated T cell killing of SB28 GBM cells in vitro and achieved a significant survival benefit in vivo, associated with anti-tumor alterations to the GBM tumor microenvironment. Further studies to optimize the efficiency of combinatorial immunomodulator delivery are currently underway.

scholarly journals Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Zhifen Yang ◽  
Lingyu Li ◽  
Ahu Turkoz ◽  
Pohan Chen ◽  
Rona Harari-Steinfeld ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor Receptor ◽  
Checkpoint Inhibition ◽  
Single Chain ◽  
Tev Protease ◽  
Car T Cells ◽  
Car T

Abstract Background Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. Methods To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. Results Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. Conclusions As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

The immunological impact of the RAF inhibitor BMS908662: Preclinical and early clinical experience in combination with CTLA-4 blockade.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2521-2521 ◽  
Author(s):  
Margaret Callahan ◽  
Gregg Masters ◽  
Jessica Katz ◽  
Valerie Russell ◽  
Ruth Ann Roman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mapk Pathway ◽  
Preclinical Studies ◽  
Anti Tumor Activity

2521 Background: Two new approaches to treat advanced melanoma have transformed the standard of care: the CTLA-4 blocking antibody, ipilimumab, and the targeted inhibitor of mutated BRAF, vemurafenib. These agents are mechanistically unique and combination therapy is a promising next step. We evaluated the combination of BMS908662 (662), a pan RAF inhibitor, with CTLA-4 blockade in preclinical studies and report first-in-human clinical experience with this combination. Methods: 1) We tested the impact of 662 on T cells in vitro, using cultured human T cells, and in vivo, using OT-1 transgenic mice. T cell activation and MAPK pathway signaling were assessed in parallel. 2) Preclinical studies measuring the anti-tumor activity of combination therapy were performed in CT-26 and SA1N tumor models. 3) Three pts with BRAF mutant stage IV melanoma were treated at MSKCC on CA206005, an IRB-approved protocol, receiving ipilimumab (3 mg/kg) and 662 (25 mg bid) (NCT01245556). Two pts consented to an IRB-approved protocol permitting immune monitoring. Results: 1) In vitro studies demonstrate that 662 can potentiate T cell activation after stimulation. This corresponds with increased MAPK pathway signaling, consistent with paradoxical activation of the MAPK pathway in wild type cells, a class effect of RAF inhibitors. In vivo, enhanced expansion of OT-1 cells after ovalbumin challenge was seen in mice treated with 662. T cell expansion was greatest in mice treated with a combination of CTLA-4 blockade and 662 (p<0.05). 2) Both preclinical models demonstrate superior anti-tumor activity with combination therapy compared to monotherapy (p<0.05). 3) All pts treated on protocol CA206005 tolerated combination therapy. New keratoacanthomas and SCCs, likely related to 662, were identified. One pt has an ongoing response at 10 mos (-85%), one had stable disease for 24 wks (-19%) and a third had disease progression. Enhanced MAPK signaling in PBMCs after treatment with 662 was detected ex vivo. Conclusions: RAF inhibitors may potentiate T cell activation in vitro and in vivo, offering one explanation for the enhanced anti-tumor activity seen in combination with CTLA-4 blockade in pre-clinical models.

Effect of sunitinib pretreatment on yield from tumor-infiltrating lymphocyte (TIL) cultures in patients with renal cell carcinoma.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 419-419 ◽  
Author(s):  
Axel Bex ◽  
Aurelie Guislain ◽  
Hester van Boven ◽  
Christian U. Blank
Keyword(s):  
T Cells ◽  
Interleukin 2 ◽  
Suppressor Cells ◽  
Response Rates ◽  
Rapid Expansion ◽  
Phenotypic Analysis ◽  
Primary Tumors ◽  
Treatment Naïve

419 Background: Targeted therapies achieve response rates up to 50% in metastatic RCC, but durable complete remissions are very rare. Therefore additional therapeutic options are warranted. Adoptive transfer (ACT) of tumor infiltrating lymphocytes (TIL) has revealed response rates of up to 72% in melanoma patients and may lead to consolidation of responses upon sunitinib in RCC. Recent data suggest that sunitinib influences lymphocyte infiltration and regulatory T cells (Treg) into tumor, dendritic cells (DC), and myeloid derived suppressor cells (MDSC) in animal models, but impaired proliferation and function of human T cells and was unable to improve human DC activity in vitro. Methods: We compared TIL grown from primary tumors pretreated with 2 courses sunitinib prior to cytoreductive nephrectomy to TIL generated from treatment-naïve RCC. Patients took part in a phase II trial of presurgical sunitinib registered under EudraCT 2006-006491-38 and gave written informed consent. From 6 pretreated primary tumors and 6 untreated controls sterile tumor material was obtained and digested with collagenase, hyaluronidase and DNase to obtain tumor single cell suspension. This TIL/tumor suspension was cultured for up to 25 days in 6000IU/ml interleukin 2 (IL-2) and rapidly expanded by anti-CD3+IL-2 stimulation. Results: TIL harvested after coculture with tumor cells yielded 36+/−SD fold expansion from non-pretreated tumors versus a 128+/−SD fold expansion from tumors from patients that had been pretreated with sunitinib (p=0.015). Phenotypic analysis of culture product indicated a higher content of natural killer (NK) cells from treatment naïve TIL cultures, while sunitinib pretreatment skewed towards NK T cells. Interferon-gamma-production was not different on a per cell level.Rapid expansion after culture using anti-CD3/IL-2 induced 100-200 fold expansion towards a content of more than 75% T cells with no significant differences between both groups. Conclusions: Sunitinib in vivo pretreatment improves TIL grow during initial culture and does not impair function or rapid expansion, allowing higher final yield of TIL for ACT studies in RCC.

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