Therapeutic Antimyeloma Effect of Dendritic-Tumor Cells Hybrids Transduced with Adenovirus Encoding CD40L.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1709-1709
Author(s):  
Eva Alvarez ◽  
Esther Moga ◽  
Jorge Sierra ◽  
Javier Briones
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Tumor Cells ◽  
Antitumor Effect ◽  
Tumor Antigens ◽  
Recombinant Adenovirus ◽  
Lymphoid Tissues ◽  
Term Survival ◽  
Mhc Ii

Abstract Dendritic cells (DCs) are the main antigen presenting cells and play a pivotal role in the stimulation of T-cell immune responses. DCs cultured in the presence of a single tumor antigen can elicit an immune response against tumor cells expressing that antigen. However, simultaneous use of several tumor antigens may be advantageous since polyclonal activation of T cells against different tumor antigens may be a better approach to eradicate tumor cells. In this sense, fusions of dendritic and tumor cells (FCs) show a broad spectrum of tumor antigens, both known and unidentified, to be presented by class I and II MHC. Although prophylactic vaccines were successful in murine models, the results in the therapeutic setting have been unsatisfactory. We hypothesised that enhancing costimulation of FCs would help to break tumor tolerance once the tumor is established. To this purpose, we transduced FCs with a recombinant adenovirus encoding CD40L (AdvCD40L or AdvGFP as control) and we studied the therapeutic antitumoral effect of the administration of FC-CD40L in a murine model of myeloma. DCs obtained from day 7-bone marrow cultures of Balb/c mice were fused with tumor cells, a syngeneic murine myeloma cell line (4TOO). FCs hybrids were generated with PEG and selected after culturing in HAT medium plus GM-CSF for 7 days. FC were quantified by determining the percentage of cells that coexpress specific DC (CD11c) and tumor markers (CD138). Mean fusion efficiency was 30% (20–40%) and FCs expressed moderate levels of CD80, CD83, CD86, CD54, CD40 and MHC II and did not express CD40L. FC-CD40L showed a significant increase of expression of costimulatory molecules (CD80, CD86, CD54, and MHC II) compared to FC-GFP (p=0.011). Moreover, in a syngeneic mixed lymphocyte reaction, FC-CD40L induced a two-fold higher T-cell proliferation than FC-GFP or FC alone. In addition, FC-CD40L had improved migration to lymphoid tissues, preferentially to spleen, in comparison with FC-GFP (2.8% versus 1.6%). The antitumor effect of FC-CD40L was analyzed in vivo. Mice (n=10 per group) were injected i.v. with 2.5×105 tumor cells and treated with irradiated FC, FC-GFP or FC-CD40L (1×106 cells each) on days 2, 6 and 10 after tumor challenge. 40% of mice treated with FC-CD40L had long-term survival (>120 days). In contrast, all of mice treated with FC or FC-GFP died between days 25 and 35 (p=0.012). In parallel, treatment with mixed cells (not fused DC+ tumor cells), mix transduced with AdvGFP, or mix transduced with AdvCD40L did not provide any significant antitumor effect. We conclude that FCs transduced with AdvCD40L better stimulate in vitro and in vivo immune responses than FC alone and may provide a new strategy for treating patients with multiple myeloma or lymphoma.

scholarly journals New differentiation pathway for double-negative regulatory T cells that regulates the magnitude of immune responses

Blood ◽  
2006 ◽  
Vol 109 (9) ◽  
pp. 4071-4079 ◽  
Author(s):  
Dong Zhang ◽  
Wei Yang ◽  
Nicolas Degauque ◽  
Yan Tian ◽  
Allison Mikita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Surface ◽  
Immune Responses ◽  
Lymphoid Tissues ◽  
Double Negative ◽  
Healthy Humans

Abstract Recent studies have demonstrated that in peripheral lymphoid tissues of normal mice and healthy humans, 1% to 5% of αβ T-cell receptor–positive (TCR+) T cells are CD4−CD8− (double-negative [DN]) T cells, capable of down-regulating immune responses. However, the origin and developmental pathway of DN T cells is still not clear. In this study, by monitoring CD4 expression during T-cell proliferation and differentiation, we identified a new differentiation pathway for the conversion of CD4+ T cells to DN regulatory T cells. We showed that the converted DN T cells retained a stable phenotype after restimulation and that furthermore, the disappearance of cell-surface CD4 molecules on converted DN T cells was a result of CD4 gene silencing. The converted DN T cells were resistant to activation-induced cell death (AICD) and expressed a unique set of cell-surface markers and gene profiles. These cells were highly potent in suppressing alloimmune responses both in vitro and in vivo in an antigen-specific manner. Perforin was highly expressed by the converted DN regulatory T cells and played a role in DN T-cell–mediated suppression. Our findings thus identify a new differentiation pathway for DN regulatory T cells and uncover a new intrinsic homeostatic mechanism that regulates the magnitude of immune responses. This pathway provides a novel, cell-based, therapeutic approach for preventing allograft rejection.

scholarly journals A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses

2021 ◽  
Author(s):  
Tahoora Mousavi ◽  
Reza Valadan ◽  
Alireza Rafiei ◽  
Ali Abbasi ◽  
Mohammad Reza Haghshenas
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Fusion Protein ◽  
Protein Vaccine ◽  
Term Survival ◽  
Ifn Γ ◽  
E7 Proteins ◽  
Hiv 1

Abstract Objectives Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. Results In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E.coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD+8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8+ T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. Conclusion Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8+ T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

scholarly journals A Novel Recombinant Protein Vaccine Containing the Different E7 Proteins of the HPV16, 18, 6, 11 E7 Linked to the HIV-1 Tat (47-57) Improve Cytotoxic Immune Responses

2020 ◽  
Author(s):  
Tahoora Mousavi ◽  
Reza Valadan ◽  
Alireza Rafiei ◽  
Ali Abbasi ◽  
Mohammad Reza Haghshenas
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Fusion Protein ◽  
Protein Vaccine ◽  
Term Survival ◽  
Ifn Γ ◽  
E7 Proteins ◽  
Hiv 1

Abstract Objective: Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. Methods: In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E.coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD+8 cytotoxicity assay and tumor challenge experiment. Results: Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8+ T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. Conclusion: Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8+ T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

scholarly journals Characterization of IL-10-producing neutrophils in cattle infected with Ostertagia ostertagi

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lei Li ◽  
Hongbin Si ◽  
Shu-Wei Wu ◽  
Jonatan Orangel Mendez ◽  
Dante Zarlenga ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Ostertagia Ostertagi ◽  
Mhc Ii

AbstractIL-10 is a master regulator of immune responses, but its cellular source and function in cattle during the initial phase of immune priming have not been well established. Despite a massive B cell response in the abomasal draining lymph nodes in Ostertagia ostertagi (OO)-infected cattle, protective immunity is slow to develop, and partial protection requires years of repeated exposure. In addressing this problem, our initial hypothesis was that B cells produce IL-10 that downregulates the host protective immune response. However, our results showed that neutrophils made up the majority of IL-10-producing cells in circulation and in secondary lymphoid tissues, particularly the spleen (80%). Conversely, IL-10-producing B cells were rare. In addition, approximately 10% to 20% of the neutrophils in the blood and spleen expressed MHC II and were IL-10 negative, suggesting that neutrophils could also participate in antigen presentation. In vitro investigation of bovine neutrophils revealed that exposure thereof to OO extract increased IL-10 and MHC II expression in these cells in a dose-dependent manner, consistent with IL-10+/MHC II+ neutrophils detected in cattle shortly after experimental OO infection. Co-culture of untreated neutrophils with anti-CD3 antibody (Ab)-stimulated CD4+ T cells led to enhanced T cell activation; also, IL-10 depletion with neutralizing Ab enhanced the stimulatory function of neutrophils. OO extract depressed neutrophil stimulation of CD4+ T cells in the presence of IL-10-neutralizing Ab, suggesting that OO utilizes both IL-10-dependent and independent mechanisms to manipulate the bovine immune response. Finally, contact and viability were required for T cell-stimulatory neutrophil function. This report, to the best of our knowledge, is the first to demonstrate that neutrophil-derived IL-10 is directly involved in T cell regulation in cattle. Our data suggest that neutrophils and neutrophil-derived IL-10 are co-opted by nematode parasites and other pathogens to attenuate host immune responses and facilitate pathogen survival.

Real-Time In Vivo Biodistribution of Multipotent Adult Progenitor Cells (MAPC): Role of the Immune System in MAPC Resistance in Non-Transplanted and Bone Marrow Transplanted Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 507-507
Author(s):  
Jakub Tolar ◽  
Scott Bell ◽  
Ron McElmurry ◽  
Lily Xia ◽  
R. Scott McIvor ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Nk Cells ◽  
Nk Cell ◽  
Sleeping Beauty ◽  
Whole Body ◽  
Term Survival ◽  
Hematopoietic Stem

Abstract MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion>> Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.

In Vivo Demonstration of Donor Idiotype Specific T Cells That Kill Primary CLL Cells Post Allogeneic Transplant.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 62-62
Author(s):  
Rifca Ledieu ◽  
Gullu Gorgun ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Allogeneic Transplant ◽  
Histocompatibility Antigens ◽  
Post Transplant ◽  
Cell Responses

Abstract Following allogeneic stem cell transplantation there is evidence of a graft versus leukemia effect (GVL). Whether this is mediated by an immune response against histocompatibility antigens also expressed on the tumor cells, i.e. by graft versus host disease (GVHD) or might be contributed to by response against specific tumor associated antigens (TAA) is currently under intense investigation. If the latter is the case, then it may be possible to generate tumor specific immune responses, thereby decreasing tumor relapse and minimizing GVHD. We and others have previously demonstrated that it is possible ex vivo to characterize CD8+ T cell responses against idiotype (Id) determinants and that these T cells can be quantified and further characterized by tetramer and ELISPOT assays. We now sought to determine whether it was possible to demonstrate evidence of in vivo immune responses against Id in patients with chronic lymphocytic leukemia (CLL) after reduced intensity conditioning (RIC) allogeneic transplant from HLA matched donors. To be included in this study, patients had to express HLA-A*0101, HLA-A*0201 or HLA-A*0301 for which tetramer constructs were available, have an IgH sequence containing Id determinants that could bind to the appropriate HLA-Class I and have serial PB and BM samples after RIC transplantation available for analysis. Of 36 patients who had undergone transplant, 21 fulfilled these criteria. No Id specific T cells could be detected in PB or BM samples from any of these patients before transplantation, although it was possible to generate autologous Id specific T cells ex vivo. Id specific cells could be detected by tetramer staining at some point post transplant in 17 of these 21 patients (80%), with a frequency ranging from 0.2 to 2.9% of CD8+ T cells. In all cases these cells were of donor origin, demonstrated either by microchimerism, or in the case of sex mismatched donors, by FISH. The earliest appearance of these specific T cells was from 80–120 (median 100) days post RIC transplant, and of note an increased frequency of Id specific cells was often co-incident with subsequent development of chronic GVHD. In some cases, the Id specific cells remained detectable for up to one year post transplant, but the detection of these cells appeared to require persistence of tumor in vivo, and once patients no longer had PCR detectable disease, Id specific T cells could no longer be detected. Id specific T cells could also be further amplified ex vivo using peptide pulsed antigen presenting cells and cytokines. In all cases we were able to demonstrate that the tetramer sorted T cells could kill the patients’ primary CLL cells in vitro, but we have no direct evidence that this was occurring in vivo. Indeed it is unlikely that these Id specific T cell responses could solely be responsible for the anti-tumour activity, and although we did not examine specific immune responses against minor histocompatibility antigens, these would undoubtedly be present since the majority of these patients developed clinically evident GVHD. However, the finding that we are able to demonstrate in vivo donor specific immune responses against TAAs such as Id, that are capable of killing primary tumor cells, provides the rational for the development of clinical programs aimed at maximizing specific immune responses. We are currently performing pre-clinical studies aimed at generating TAA specific T cells for subsequent infusion to patients as an alternative to non-specific donor lymphocyte infusions.

Three Steps to Breaking Immune Tolerance: A Microparticle Approach

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Amani Makkouk ◽  
Vijaya B Joshi ◽  
Caitlin D Lemke ◽  
Amaraporn Wongrakpanich ◽  
Alicia K Olivier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Tumor Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Optimal Size ◽  
Step Therapy ◽  
Three Components

Abstract Lymphoma immunotherapy can result in durable immune and clinical responses. Nevertheless, the clinical impact of such therapy remains suboptimal and there is still a need to apply our growing understanding of cancer immunity to the design and testing of rational combination approaches to lymphoma immunotherapy. This includes consideration of the three steps necessary to generate an antitumor response: (1) providing tumor antigens to dendritic cells (DCs) in a manner that enhances their ability to process and present antigens to T cells; (2) enhancing T cell activation; and (3) overcoming immunosuppression so that activated anti-lymphoma T cells can proceed unrestrained. In situ immunization generates systemic immune responses through local injection of agents into the tumor, thus providing DCs with the patient’s own tumor antigens. We evaluated a three-step approach to in situ immunization against lymphoma using Doxorubicin, anti-CTLA-4 and anti-OX40: (1) Doxorubicin (Dox) to induce the expression of “eat-me” signals by dying tumor cells, facilitating their phagocytosis by DCs; (2) anti-OX40 antibody to augment OX40-mediated stimulation of T cells; (3) anti-CTLA-4 antibody to block immunosuppression imposed by CTLA-4 on T cells. While Dox is highly effective as a systemic anti-lymphoma agent and has been reported to induce immunogenic cell death, intratumoral injection of soluble Dox is not clinically feasible due to its strong vesicant effect. Poly(lactide-co-glycolide) (PLGA) is an FDA-approved polymer that is used in biodegradable surgical sutures and microparticles (MPs) with sustained-release properties. Thus, PLGA MPs loaded with Dox (Dox MPs) represent a clinically translatable approach for delivering Dox, as its slow release would decrease likelihood of vesication. In addition, PLGA MPs at an optimal size of 1-µm enhance antigen-specific immune responses by activating the NALP3 inflammasome in DCs. While both tumor cells and DCs are exposed to Dox released by MPs in the tumor, we found in vitro that Dox MPs (1-µm) are less cytotoxic to DCs than to A20 B-lymphoma cells and do not require internalization for their cytotoxic activity. Dox MPs significantly enhanced phagocytosis of A20 by DCs as compared to soluble Dox. In vivo, we used the two-tumor mouse model to assess immune responses. This allowed us to monitor the local effect (on the tumor injected with MP) and the systemic effect (on the distant tumor that was not injected with MP) of therapy. Dox MPs injected intratumorally do not induce vesication even at doses as high as 100 µg Dox. Using a low dose of Dox MPs (2 µg Dox) and of antibody to limit systemic toxicity, we found that three-step therapy induced CD4- and CD8- T cell-dependent systemic immune responses that enhanced T cell infiltration into distant tumors. This led to their eradication and significantly improved survival as compared to antibody-only therapy (87% of mice treated with all three components became tumor-free). Moreover, all three components were required for maximum efficacy (Figure 1). These results demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy. The potential value of this approach to immunotherapy is not limited to lymphoma and merits further evaluation in lymphoma and other cancers. Figure 1. Mice challenged with two A20 tumors were treated with PBS (control) or Dox MP into one tumor and antibodies (anti-CTLA-4 and/or anti-OX40) given systemically. Figure 1. Mice challenged with two A20 tumors were treated with PBS (control) or Dox MP into one tumor and antibodies (anti-CTLA-4 and/or anti-OX40) given systemically. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cytotoxic T Lymphocytes to An Unmutated Tumor Rejection Antigen P1A: Normal Development but Restrained Effector Function In Vivo

1999 ◽  
Vol 189 (5) ◽  
pp. 811-820 ◽  
Author(s):  
Supria Sarma ◽  
Yong Guo ◽  
Yannik Guilloux ◽  
Cheng Lee ◽  
Xue-Feng Bai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Tumor Antigens ◽  
Effector Function ◽  
Tumor Rejection ◽  
Lymphoid Tissues ◽  
Clonal Deletion ◽  
Tumor Rejection Antigen

Unmutated tumor antigens are chosen as primary candidates for tumor vaccine because of their expression on multiple lineages of tumors. A critical issue is whether unmutated tumor antigens are expressed in normal cells, and if so, whether such expression imposes special restrictions on cytotoxic T lymphocyte (CTL) responses. In this study, we use a transgenic approach to study the development and effector function of T cells specific for P1A, a prototypical unmutated tumor antigen. We report here that although P1A is expressed at low levels in normal tissues, including lymphoid tissues, the P1A-specific transgenic T cells develop normally and remain highly responsive to the P1A antigen. The fact that transgenic expression of P1A antigen in the thymus induces T cell clonal deletion demonstrates that normal hematopoietic cells can process and present the P1A antigen and that P1A-specific T cells are susceptible to clonal deletion. By inference, P1A-specific T cells must have escaped clonal deletion due to low expression of P1A in the thymus. Interestingly, despite the fact that an overwhelming majority of T cells in the T cell receptor for antigen (TCR)–transgenic mice are specific for P1A, these mice are no more resistant to a P1A-expressing plasmocytoma than nontransgenic littermates. Moreover, when the same TCR-transgenic mice were challenged simultaneously with B7-1+ and B7-1− tumors, only B7-1+ tumors were rejected. Therefore, even though P1A can be a tumor rejection antigen, the effector function of P1A-specific CTL is restrained in vivo. These results have important implications for the strategy of tumor immunotherapy.

scholarly journals DDRE-18. DEVELOPMENT OF EGFRvIII AND EGFR DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii65-ii65
Author(s):  
Michael Ruff ◽  
Reona Sakemura ◽  
Claudia Manriquez-Roman ◽  
Mehrdad Hefazi-Torghabeh ◽  
Kendall Schick ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Chimeric Antigen Receptor ◽  
Tumor Antigens ◽  
Antigen Receptor ◽  
Receptor Expression ◽  
T Cell Therapy ◽  
Multiple Tumor

Abstract BACKGROUND Chimeric antigen receptor T cell (CART) therapy has revolutionized the treatment landscape for hematological malignancies, its efficacy remains limited in solid tumors. EGFRvIII is a truncated version of the wild type EGFR in which deletion of exons 2–7 of the extracellular domain leads the variant (EGFRvIII) that is strongly antigenic and is mostly expressed on tumor cells. EGFR amplification (tEGFR) almost uniformly precedes the presence of EGFRvIII on tumor cells. The heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment underscore the need to develop CART strategies to target multiple tumor antigens simultaneously. METHODS we generated tEGFR/EGFRvIII directed CAR construct by cloning a clinically relevant tEGFR and EGFRvIII specific scFv into a second generation CAR construct (41BB stimulated) in a third generation lentivirus backbone. This was used to transfect 293T cells and the generated lentivirus particles were used to transduce T cells and generate EGFRvIII/tEGFR CART cells. GBM primary patient derived cell lines were used in these experiments. These cells were passaged and maintained in patient derived xenograft models. RESULTS EGFRvIII/tEGFR directed CART cells exhibited potent antitumor activity against EGFRvIII/tEGFR + GBM cell lines: with 100% killing at 1.25:1, 2.5:1, 5:1 and 10:1 E:T ratio on multiple PDX cell lines with EGFRvIII expression and EGFR over-expression (greater than five copies of EGFR gene) at 24 hours of incubation. Conclusion: We demonstrate that targeting EGFRvIII and over-expressed EGFR with CART cells is feasible, efficacious and represents a promising therapeutic strategy to target GMB. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

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