Defined Tumor Antigens Research Articles

Next-Generation Therapies for Multiple Myeloma.

Recent therapeutic advances have significantly improved the outcome for patients with multiple myeloma (MM). The backbone of successful standard therapy is the combination of Ikaros degraders, glucocorticoids, and proteasome inhibitors that interfere with the integrity of myeloma-specific superenhancers by directly or indirectly targeting enhancer-bound transcription factors and coactivators that control expression of MM dependency genes. T cell engagers and chimeric antigen receptor T cells redirect patients' own T cells onto defined tumor antigens to kill MM cells. They have induced complete remissions even in end-stage patients. Unfortunately, responses to both conventional therapy and immunotherapy are not durable, and tumor heterogeneity, antigen loss, and lack of T cell fitness lead to therapy resistance and relapse. Novel approaches are under development to target myeloma-specific vulnerabilities, as is the design of multimodality immunological approaches, including and beyond T cells, that simultaneously recognize multiple epitopes to prevent antigen escape and tumor relapse.

Efficacious Dendritic Cell Based Immunotherapy for Advanced Solid Tumors: Three Case Studies

Background and Aims: Dendritic cell (DC)-based immunotherapy is promising as a viable tool in cancer treatment. Dendritic cell (DC)-based vaccination can provoke antitumor T cell responses in vivo. These case studies examined feasibility and outcome of DC-based tumor vaccination for patients with advanced cancers. This approach has been used mostly in patients in the presence of defined tumor antigens. Experimental Design: Accessible tumor tissue was disrupted into single cell suspensions. Autologous DCs were prepared from adherent peripheral blood mononuclear cells and cultured in granulocyte macrophage colony-stimulating factor, interleukin and autologous plasma. Tumor cells and DCs were co-cultured in the presence of polyethylene glycol to generate the fusions. Fusion cells were quantified by determining the percentage of cells that co-express tumor and DC markers. Patients were vaccinated with three doses of DC (10 X 106 ) were administered after every 2 weeks’ intervals and assessed weekly for toxicity, and tumor response was assessed at 3 months after completion of vaccination. Results: Vaccination was well tolerated. No physical signs of autoimmunity were detected. There was no evidence of significant toxicity from vaccine or adjuvant. There was increased expression of T helper type 1 cytokines. Vaccination resulted in a significant reduction in the level of prostate-specific antigen (PSA) in the prostate cancer patient. Disease was stable upto 6 months in cases of breast and cervical cancer patients. Conclusion: DC-based vaccination can stimulate an antitumoral T cell response in patients with various cancers. This data indicates that vaccination with autologous tumor-pulsed DCs generated from peripheral blood is safe and can induce tumorspecific cellular cytotoxicity. Clinical responses are achievable, even in patients with advanced disease.

Nano-immunotherapy: Overcoming tumour immune evasion.

Immunotherapy is emerging as a groundbreaking cancer treatment, offering the unprecedented opportunity to effectively treat and in several cases, even cure previously untreatable malignancies. Anti-tumour immunotherapies designed to amplify T cell responses against defined tumour antigens have long been considered effective approaches for cancer treatment. Despite a clear rationale behind such immunotherapies, extensive past efforts were unsuccessful in mediating clinically relevant anti-tumour activity in humans. This is mainly because tumours adopt specific mechanisms to circumvent the host´s immunity. Emerging data suggest that the full potential of cancer immunotherapy will be only achieved by combining immunotherapies designed to generate or amplify anti-tumour T cell responses with strategies able to impair key tumour immune-evasion mechanisms. However, many approaches aimed to re-shape the tumour immune microenvironment (TIME) are commonly associated with severe systemic toxicity, require frequent administration, and only show modest efficacy in clinical settings. The use of nanodelivery systems is revealing as a valid means to overcome these limitations by improving the targeting efficiency, minimising systemic exposure of immunomodulatory agents, and enabling the development of novel combinatorial immunotherapies. In this review, we examine the emerging field of therapeutic modulation of TIME by the use of nanoparticle-based immunomodulators and potential future directions for TIME-targeting nanotherapies.

Modulation of tumor microenvironment using a TLR-7/8 agonist-loaded nanoparticle system that exerts low-temperature hyperthermia and immunotherapy for in situ cancer vaccination

Most cancer vaccines under development are associated with defined tumor antigens rather than with all antigens of whole tumor cells, limiting the anti-tumor immune responses that they elicit. This work proposes an immunomodulator (R848)-loaded nanoparticle system (R848@NPs) that can absorb near-infrared light (+NIR) to cause low-temperature hyperthermia that interacts synergistically with its loaded R848 to relieve the tumor-mediated immunosuppressive microenvironment, generating robust anti-tumor memory immunity. In vitro results reveal that the R848@NPs could be effectively internalized by dendritic cells, causing their maturation and the subsequent regulation of their anti-tumor immune responses. Post-treatment observations in mice in which tumors were heat-treated at high temperatures reveal that tumor growth was significantly inhibited initially but not in the longer term, while low-temperature hyperthermia or immunotherapy alone simply delayed tumor growth. In contrast, a combined therapy that involved low-temperature hyperthermia and immunotherapy using R848@NPs/+NIR induced a long-lasting immunologic memory and consequently inhibited tumor growth and prevented cancer recurrence and metastasis. These results suggest that the method that is proposed herein is promising for generating cancer vaccines in situ, by using the tumor itself as the antigen source and the introduced R848@NPs/+NIR to generate a long-term anti-tumor immunity, for personalized immunotherapy.

Abstract 1457: Sustained persistence of IL-2 signaling enhances the antitumor effect of peptide vaccines

Abstract Peptide vaccines can be a successful and cost-effective way of generating T-cell responses against defined tumor antigens, especially when combined with immune adjuvants such as poly-IC. However, strong immune adjuvants can induce a collateral increase in numbers of irrelevant, nonspecific T cells, which limits the effectiveness of the peptide vaccines. Here, we report that providing prolonged IL2 signaling in the form of either IL2/ anti-IL2 complexes or pegylated IL2 overcomes the competitive suppressive effect of irrelevant T cells, allowing the preferential expansion of antigen-specific T cells, and increasing the therapeutic effectiveness of the vaccines against established tumors. This vaccination strategy using peptides and sustained IL2 could be taken into the clinic for the treatment of cancer. Citation Format: Takumi Kumai, Hussein Sultan, Yasuaki Harabuchi, Esteban Celis. Sustained persistence of IL-2 signaling enhances the antitumor effect of peptide vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1457.

Abstract 586: CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival

Abstract The 4T1.2 syngeneic murine breast cancer model is an aggressive model of stage IV triple-negative breast cancer. Despite its significant strength, the 4T1.2 model is poorly immunogenic with few defined tumor antigens. The firefly luciferase gene, luc2, has been transfected into various tumor cell lines as a reporter for bioluminescence imaging. However, studies have shown that the luciferase (Luc) may serve as a tumor antigen and induce CD8+ cytotoxic T cell responses. The goal of the current study was to determine if luciferase expression in 4T1.2 tumor cells would enhance the immunogenicity of the tumor, and explore the role of immune effector cells in the parental 4T1.2 and luciferase-transfected 4T1.2luc tumor model. In vitro proliferation of the 4T1.2 and 4T1.2luc cells was assessed using the MTS cell proliferation assay. For in vivo experiments, female BALB/c mice were randomized into 2 groups and orthotopically injected with 5x104 4T1.2 or 4T1.2luc cells, respectively. Tumor growth was assessed until day 35 post tumor implantation. Each group was further randomized into 5 subgroups and received i.p. injections of PBS, rat IgG2b mAb, anti-CD4 mAb, anti-CD8 mAb, or anti-asialo GM1. Mice were sacrificed between day 28-35 post tumor implantation, and lung metastases were analyzed for each group. To further evaluate the immunogenicity of luciferase in the 4T1.2luc model, a separate cohort of mice were orthotopically injected with 5x104 4T1.2luc cells and sacrificed at day 35 post tumor implantation. Splenocytes were harvested and cultured in the presence of 1 or 10 µg/ml peptides representing MHC I-restricted Luc epitopes for 5 days, and cytotoxicity of the effector cells was assessed by the chromium-release assay. In vitro proliferation rates were not significantly different between 4T1.2 and 4T1.2luc tumor cells. However, 4T1.2luc tumor-bearing mice showed a significant reduction in primary tumor growth compared to 4T1.2 tumor-bearing mice. 4T1.2 primary tumor growth was not altered by the depletion of any immune cell type, while 4T1.2luc primary tumor growth was significantly increased by the depletion of CD8+ T cells, and reduced by the depletion of NK cells. Depletion of CD4+ T cells, CD8+ T cells or NK cells in 4T1.2luc tumor-bearing mice resulted in a significant reduction in median survival. Splenocytes from 4T1.2luc tumor bearing mice demonstrated Luc-specific cytotoxicity upon ex vivo restimulation with Luc peptides, and a stronger response was observed when cells were restimulated with 1 µg/ml compared to 10 µg/ml Luc peptides. These findings suggest that luciferase may serve as a tumor antigen in the 4T1.2luc tumor model, and host immune components (CD4+ and CD8+ T cells) may play a role in controlling 4T1.2luc primary tumor growth and survival. The stronger immunogenicity of 4T1.2luc conferred by luciferase will enable future immunotherapeutic research that ultimately may benefit breast cancer patients. Citation Format: Yitong Xu, Shizhao Duan, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 586.

Sustained Persistence of IL2 Signaling Enhances the Antitumor Effect of Peptide Vaccines through T-cell Expansion and Preventing PD-1 Inhibition.

Peptide vaccines can be a successful and cost-effective way of generating T-cell responses against defined tumor antigens, especially when combined with immune adjuvants such as poly-IC. However, strong immune adjuvants can induce a collateral increase in numbers of irrelevant, nonspecific T cells, which limits the effectiveness of the peptide vaccines. Here, we report that providing prolonged IL2 signaling in the form of either IL2/anti-IL2 complexes or pegylated IL2 overcomes the competitive suppressive effect of irrelevant T cells, allowing the preferential expansion of antigen-specific T cells. In addition to increasing the number of tumor-reactive T cells, sustained IL2 enhanced the ability of T cells to resist PD-1-induced negative signals, increasing the therapeutic effectiveness of the vaccines against established tumors. This vaccination strategy using peptides and sustained IL2 could be taken into the clinic for the treatment of cancer. Cancer Immunol Res; 6(5); 617-27. ©2018 AACR.

Inferring the Impact of Regulatory Mechanisms that Underpin CD8+ T Cell Control of B16 Tumor Growth In vivo Using Mechanistic Models and Simulation.

A major barrier for broadening the efficacy of immunotherapies for cancer is identifying key mechanisms that limit the efficacy of tumor infiltrating lymphocytes. Yet, identifying these mechanisms using human samples and mouse models for cancer remains a challenge. While interactions between cancer and the immune system are dynamic and non-linear, identifying the relative roles that biological components play in regulating anti-tumor immunity commonly relies on human intuition alone, which can be limited by cognitive biases. To assist natural intuition, modeling and simulation play an emerging role in identifying therapeutic mechanisms. To illustrate the approach, we developed a multi-scale mechanistic model to describe the control of tumor growth by a primary response of CD8+ T cells against defined tumor antigens using the B16 C57Bl/6 mouse model for malignant melanoma. The mechanistic model was calibrated to data obtained following adenovirus-based immunization and validated to data obtained following adoptive transfer of transgenic CD8+ T cells. More importantly, we use simulation to test whether the postulated network topology, that is the modeled biological components and their associated interactions, is sufficient to capture the observed anti-tumor immune response. Given the available data, the simulation results also provided a statistical basis for quantifying the relative importance of different mechanisms that underpin CD8+ T cell control of B16F10 growth. By identifying conditions where the postulated network topology is incomplete, we illustrate how this approach can be used as part of an iterative design-build-test cycle to expand the predictive power of the model.

Abstract A53: Combining oncolytic virotherapy with immunotherapy for ovarian cancer treatment.

Abstract While spectacular responses have been observed in some patients following cancer immunotherapy, the majority of responses are short-lived with ultimate tumor relapse. Therefore, it is crucial to identify strategies that will effectively synergize with immunotherapies to improve clinical outcome. Oncolytic viruses (OV) are replication-competent viruses that can infect and kill tumor cells, while also acting as potent immune adjuvants. Additionally, as OV can be engineered to express tumor antigens, we are investigating the use of OV as vaccine vectors with the capacity to enhance immune responses to defined tumor antigen targets. Based on these unique attributes of OV, we hypothesized that OV could be effectively combined with multiple immunotherapeutic strategies currently in clinical testing as a means of enhancing anti-tumor immunity and improving tumor growth control/treatment outcome. Targeting a model tumor antigen in a pre-clinical ovarian cancer model, we observed that delivery of oncolytic Maraba virus (MRB) following vaccination enhanced tumor-specific CD8+ T cell expansion and trafficking to the tumor microenvironment, reduced intratumoral Tregs and MDSC frequencies, and resulted in improved survival compared to vaccination alone. Additionally, when MRB was combined with adoptive T cell transfer (ACT) of tumor-specific CD8+ T cells, this combination resulted in improved persistence of the transferred T cells and enhanced tumor control compared to treatment with MRB or ACT alone. Lastly, a dramatic improvement in treatment efficacy was observed when MRB was combined with checkpoint blockade (anti-PD-L1) compared to either monotherapy. These data suggest that OV can effectively synergize with multiple cancer immunotherapies currently being explored. Our current studies are focused on combining OV with either vaccines or ACT strategies that target clinically relevant ovarian cancer antigens (WT1, Sp17, and NY-ESO-1) using syngeneic and humanized mouse models. Additionally, we are exploring the transcriptional profile of tumors treated with immunotherapy + OV to identify gene signatures that correlate with improved therapeutic efficacy. Citation Format: AJ Robert McGray, Cheryl Eppolito, Kyle Stephenson, Jonathan Pol, Brian Lichty, Kunle Odunsi. Combining oncolytic virotherapy with immunotherapy for ovarian cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A53.

T Cell Redirecting Therapies for Cancer Treatment.

T cell activity has been stimulated to enhance anti-tumor activity for several decades. Tumor infiltrating lymphocytes have been expanded and reinfused to treat melanoma and other solid tumor cancers, and adaptive immune modulators have been used to increase endogenous T cell activity against cancer. Both of these strategies rely on the pre-existence of adequate recognition of cancer cells by the T cells which are being activated. Redirected T cell therapies represent a paradigm shift in treatment. They do not rely on endogenous T cell recognition but instead focus the T cells onto a defined tumor antigen. This review summarizes both molecular and cellular T cell redirecting therapies, compares their success in the clinic and highlights both their limitations and some potential future solutions.

Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI) and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL) for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs), transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR) gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

Abstract 2802: Fine-tuning T cell receptors for adoptive T cell therapy

Abstract Adoptive T cell therapy (ACT) with gene-modified T cells is emerging as a highly promising strategy for the treatment of many types of cancer. Therapies associated with the induction of anti-tumor immunity, such as the administration of vaccines or the ex-vivo expansion of patient-derived TILs, have historically had limited success. This lack of efficacy is almost certainly brought about as a consequence of thymic selection, leading to a paucity of T cells, and T cell receptors, in the circulating repertoire, with a sufficient degree of sensitivity towards tumor-associated antigens. An alternative approach, which has generated promising results, relies on the adoptive transfer of patient-derived T cells that have been first modified to express a chimeric antigen receptor (CAR). Nevertheless, a lack of tumor-specific antibody targets limits the wider applicability of CAR T cells. To overcome these issues we have developed a strategy that involves the transfer of T cells expressing engineered TCRs with optimised affinity for a defined tumor antigen. Since TCRs recognize peptide antigens presented on the cell surface in complex with human leukocyte antigen (HLA), they give access to the large pool of intracellular proteins. Furthermore, the use of TCRs means that the natural interaction between a T cell and the corresponding antigen presenting cell is preserved. Importantly, for any given antigen, engineered TCRs require individual optimization, to ensure the appropriate balance between potency and specificity. Recent and emerging clinical data using engineered TCRs for ACT are beginning to corroborate promising pre-clinical findings, with evidence of both potent and durable responses in vivo. However, experiences have also highlighted the dangers of this approach. The main risks associated with TCR adoptive therapy are target presentation on healthy tissue and TCR cross-reactivity to a look-alike peptide. Understanding these risks and ensuring comprehensive and robust safety testing mechanisms are in place is pivotal to mitigate these risks. Citation Format: Bent Jakobsen. Fine-tuning T cell receptors for adoptive T cell therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2802. doi:10.1158/1538-7445.AM2014-2802

Actual status of therapeutic vaccination in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related death worldwide. Although treatment methods such as surgery, radiotherapy and/or chemotherapy have improved, prognosis remains unsatisfactory, and developing new therapeutic strategies is still an urgent matter. Immunotherapy is a novel therapeutic approach wherein activated immune cells can specifically kill tumour cells. Several lung cancer vaccines have demonstrated prolonged survival time in phase II and III trials, and several clinical trials are under investigation. However, many clinical trials involving cancer vaccination with defined tumour antigens have shown this method to work only in a small number of patients. Cancer immunotherapy is not completely effective in eradicating tumour cells because they evade host immune control.

Immune evasion in acute myeloid leukemia: current concepts and future directions.

Immune responses generated against malignant cells have the potential to inhibit tumor growth, or even eliminate transformed cells before a tumor forms. However, immune tolerance mechanisms that normally protect healthy tissues from autoimmune damage pose a formidable barrier to the development of effective anti-tumor immunity. Because malignant cells are derived from self-tissues, the majority of defined tumor antigens are either shared or aberrantly expressed self-proteins. Eliciting productive T cell responses against such proteins is challenging, as most high-affinity, self-reactive T cells are purged during thymic selection. Some T cells capable of tumor antigen recognition escape thymic deletion, but are functionally inhibited by peripheral tolerance mechanisms which limit their ability to attack a developing malignancy. Alternatively, some tumors express antigens derived from mutated self-proteins, viral proteins or self proteins expressed only during embryonic development. These antigens are recognized by the immune system as foreign and could be recognized by a relatively large number of peripheral T cells. Even in this scenario, tumors evade otherwise effective T cell responses by employing potent immunosuppressive mechanisms within their local environment. In the setting for solid malignancies, such as melanoma, a growing number of putative immune evasion mechanisms have been characterized. However, acute myeloid leukemia (AML) is a systemic disease, and the pathways it exploits to subvert the host immune response may be quite different than those of a solid tumor. Much remains unknown regarding the immune escape mechanisms promoted by AML, and whether efforts to thwart tolerance may influence the progression of this disease. Here, we review current concepts of immune evasion in AML, and speculate how potentially effective immunotherapeutic strategies might be developed to reverse immune tolerance in leukemia patients in the future.

Integrating a ‘danger’ signal into molecular chaperoning to improve vaccination against cancer

Molecular chaperones are essential for maintaining cellular functions by assisting protein folding and translocation, or by preventing protein misfolding and aggregation [1]. Studies over the last two decades support the concept of intracellular chaperone molecules as carriers of the antigenic repertoire of cancer cells which is attributed to their intrinsic property to interact with polypeptide chains [2]. Autologous tumor-derived, chaperone-peptide complex preparations, therefore, provide an innovative immunotherapeutic approach to treatment of cancers. However, only limited improvement in the clinical outcome has been achieved using this approach [3]. Recombinant chaperone complex vaccines, which are built on the superior client or antigen-holding capability of large stress proteins (110 kDa heat shock protein, Hsp110 and 170 kDa glucose-regulated protein, Grp170), present an alternative vaccine strategy that effectively targets defined tumor antigens. Engineering a modified immunochaperone that is able to concurrently promote antigen cross-presentation and innate immune responses may lead to development of a new generation of chaperoning-based vaccine therapy with improved clinical feasibility and vaccine efficacy.

A case of spontaneous systemic immunity to melanoma associated with cure after amputation for extensive regional recurrence

Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation. A 71-year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELISPOT assay, and antibody responses to a panel of tumor antigens were assayed by ELISA. The patient's tumor had minimal lymphocytic infiltrate (immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T-cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery. The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, and then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.

Enhancing the Efficacy of T Cell Receptor (TCR) Gene Therapy by Co-Transfer of TCR and Additional CD3 Molecules Into CD4+ T Cells

AbstractAbstract 2044 Introduction:The specificity of T cells can be redirected using retroviral T cell receptor (TCR) gene transfer. This has the potential to generate tumour specific T cells that can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8 T cells. However the transfer of antigen specific CD8 T cells in the absence of antigen specific CD4 T cells leads to impaired anti-tumour responses and impaired memory development in vivo. Class I restricted TCR can be used to transduce CD4 T cells for use in adoptive transfer. The majority of class I restricted TCRs are CD8 dependent and thus require co-transduction of CD8 to be fully functional in CD4 T cells. CD4 T cells transduced with the class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Dbclass I molecules) produce IL-2 and proliferate in vitro in response to class II negative tumour cells expressing NP peptide but these cells were not able to generate an IFN-γ response. In vivo, F5-TCR CD4 T cells could provide help for F5-TCR CD8 T cell mediated tumour eradication. These F5-TCR CD4 T cells persisted in vivo for up to 90 days post tumour regression and were able to re-expand following tumour challenge. In order to improve the function of class I restricted TCR expressing CD4 T cells, we co-transduced a vector containing all 4 chains of the CD3 complex. High surface expression of TCR has been shown to correlate with increased responsiveness to specific antigen. When additional TCR is introduced into a T cell, the introduced T cell must compete with the endogenous TCR for binding to CD3. The amount of CD3 within the cell will thus be rate limiting for the level of surface expression of the introduced TCR. Method:The retroviral vectors pMP71-F5α-2A-F5β (F5-TCR) and pMP71-CD3-ζ-2A-ε-2A-δ-2A-γ-IRES-GFP (CD3) were used for retroviral transduction. CD4 splenocytes obtained from C57BL/6 mice were activated with CD3/CD28 magnetic beads for 24 hours prior to transduction with either F5-TCR alone or F5-TCR and CD3. 5 days post transduction, transduced T cells were stimulated with C57BL/6 splenocytes loaded with NP (relevant) peptide or WT1 (irrelevant) peptide and cytokine production was measured by ELISA and intracellular cytokine staining and proliferation by [3H] thymidine incorporation. For in vivo tumour challenge, C57BL/6 recipient mice were irradiated with 5.5Gy and injected subcutaneously with 1 × 106 EL4-NP-luciferase cells (a lymphoma cell line stably transfected with NP peptide and luciferase) on day 0. On day 1, mice received 1 × 106 F5-TCR CD3 CD4 T cells or 1 × 106 F5-TCR CD4 T cells or 1 × 106 Mock Transduced T cells. Tumour area was measured by calipers and by bioluminescence imaging. For T cell trafficking experiments, the experimental set up was as above but transgenic CD4 luciferase T cells were used for adoptive transfer and EL4-NP luciferase negative cells were used for tumour challenge. Results:CD4 T cells transduced with F5-TCR and CD3 had a 5-fold higher expression of F5-TCR compared to cells transduced with F5-TCR alone. In vitro, F5-TCR CD3 CD4 T cells showed increased proliferation and increased production of IL-2 and IFN-γ in response to specific antigen compared to F5-TCR CD4 T cells. F5-TCR CD3 CD4 T cells responded to at a 2-fold lower concentration of specific peptide than F5-TCR CD4 T cells. Following adoptive transfer in murine models, F5-TCR CD3 CD4 T cells eradicated NP expressing EL4 tumours but transfer of equivalent doses of F5-TCR CD4 T cells did not lead to tumour regression. Using bioluminescence imaging, F5-TCR CD3 CD4 T cells trafficked to tumour site faster and accumulated in greater numbers than F5-TCR CD4 T cells. Following tumour challenge, there were higher numbers of F5-TCR CD3 CD4 T cells persisting in bone marrow, lymph node and peripheral blood than in mice that received F5-TCR CD4 T cells. Conclusion:Increased surface expression of class I restricted TCR in CD4 T cells leads to increased sensitivity to peptide in vitro and higher levels of proliferation and cytokine production in response to specific peptide. This translates in vivo to enhanced persistence of F5-TCR CD3 CD4 T cells and more efficient trafficking to tumour site and superior tumour protection. Therefore, the co-transduction of additional CD3 can improve the function of class I restricted TCR in CD4 T cells. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

Relapse of melanoma after successful adoptive T‐cell therapy: escape through inflammation‐induced phenotypic melanoma cell plasticity

Advanced stage melanomas are refractory to most available therapies including recently approved targeted therapies. This is also true for most biological (IFN-a or IL-2) treatments and therapy involving adoptive transfer of melanoma-reactive T cells (de Souza et al., 2012). A number of mechanisms have been proposed to explain the lack of T-cell functional activity in vivo. Some of these mechanisms include emergence of antigen-loss tumor variants, induction of immune inhibitory molecules such as CTLA4 or PD1 and induction of T-reg (regulatory) cells or myeloid-derived suppressor cells (MDSC) or presence of immune-suppressive cytokines such as TGF-b or IL-10 (Grivennikov et al., 2010). A number of environmental factors can further contribute to genetic instability and induce epigenetic changes of progressing heterogeneous tumors. Presence of inflammatory factors at the tumor site can cause selection of preexisting tumor cell variants resulting in preferential growth of antigen-negative tumor variants or tumor cells that have lost MHC-class I molecules (Khong and Restifo, 2002). Immune editing or alterations of tumor antigens due to immune pressure can also be one of the reasons for lack of T-cell-mediated tumor cell death in vivo (Singh and Paterson, 2007). In clinical trials, adoptive cell therapies (ACT) with melanoma-reactive tumor-infiltrating lymphocytes (TILs) or polyclonal T cells engineered to express chimeric antigen receptor (CAR) with re-directed specificity to melanoma have shown great promise with dramatic regression of bulky tumors (Robbins et al., 2011). However, in most patients, initial regression of tumor cells was followed by appearance of new lesions with therapy resistant melanoma cells (Robbins et al., 2011). Until now, the exact nature of ACT-related tumor resistance and the cause of emergence of antigen-negative melanoma cells were largely unknown. In the current edition of Nature, Landsberg et al. describe a novel mechanism of T-cell-induced loss of melanocytic antigens as a possible cause of tumor cell resistance to ACT. To demonstrate therapy resistance, the authors have used a previously published genetically engineered hepatocyte growth factor (Hgf)-Cdk4 mouse melanoma model and anti-gp100-specific CTLs obtained from a TcR transgenic pmel-1 (gp100) mouse model. TNF-a, a proinflammatory cytokine predominantly secreted by activated T cells caused down-modulation of gp100 antigen expression on the mouse melanoma cells resulting in loss of recognition by anti-gp100-specific CTLs used in ACT. TNF-a also down-modulated pigmentrelated gene expression and tyrosinaserelated protein (TRP)-2 on melanoma cells. Interestingly, the pro-inflammatory environment of TNF-a did not downmodulate the expression of non-melanocytic antigens. On the contrary, there was an increased expression of nerve growth factor receptor (NGFR) on melanoma cells. Down-modulation of gp100 expression was reversible when the activity of TNF-a was neutralized or the resistant tumor cells were re-transplanted into a normal mouse lacking an inflammatory environment. The mechanism of non-genetic reversible antigen down-modulation is novel and has not been described before. This is in contrast to other genetic heterogeneityand immunoselection-related mechanisms described earlier. Understanding inflammation-induced plasticity and the non-genetic heterogeneity of melanoma cells are critical for the development of new therapeutic strategies for melanoma. The authors then corroborated their mouse studies using human melanoma cell lines where they report similar down-modulation of gp100 and MART1/Melan-A expression in the presence of TNF-a. Similar to mouse studies, human melanoma cell lines also showed up-regulation of NGFR expression in the presence of TNF-a. This was confirmed in melanoma patients’ tumor tissue sections with abundant infiltrating immune cells further supporting the clinical relevance of the above findings. The results of Landsberg et al. have important clinical implications if the phenotypic plasticity of melanoma cells in an inflammatory microenvironment is validated in a larger cohort of patients with melanoma after ACT. Down-modulation of gp100 expression and concurrent up-regulation of NGFR expression of melanoma cells in an inflammatory environment raises the possibility of using ACT with a cocktail of CAR-engineered T cells with dual or multiple specificities to target more than one melanoma antigen to ensure complete regression of tumor lesions. For this, a personalized approach of analyzing melanoma antigen expression on tumor cells before and after ACT therapy will be essential for the selection of CARengineered T cells with defined tumor antigen specificity. The proposed approach may enhance the long term disease-free survival of patients with metastatic melanoma.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

Human dendritic cells adenovirally-engineered to express three defined tumor antigens promote broad adaptive and innate immunity

Dendritic cell (DC) immunotherapy has shown a promising ability to promote anti-tumor immunity in vitro and in vivo. Many trials have tested single epitopes and single antigens to activate single T cell specificities, and often CD8+ T cells only. We previously found that determinant spreading and breadth of antitumor immunity correlates with improved clinical response. Therefore, to promote activation and expansion of polyclonal, multiple antigen-specific CD8+ T cells, as well as provide cognate help from antigen-specific CD4+ T cells, we have created an adenovirus encoding three full length melanoma tumor antigens (tyrosinase, MART-1 and MAGE-A6, “AdVTMM”). We previously showed that adenovirus (AdV)-mediated antigen engineering of human DC is superior to peptide pulsing for T cell activation, and has positive biological effects on the DC, allowing for efficient activation of not only antigen-specific CD8+ and CD4+ T cells, but also NK cells. Here we describe the cloning and testing of “AdVTMM2,” an E1/E3-deleted AdV encoding the three melanoma antigens. This novel three-antigen virus expresses mRNA and protein for all antigens, and AdVTMM-transduced DC activate both CD8+ and CD4+ T cells which recognize melanoma tumor cells more efficiently than single antigen AdV. Addition of physiological levels of interferon-α (IFNα) further amplifies melanoma antigen-specific T cell activation. NK cells are also activated, and show cytotoxic activity. Vaccination with multi-antigen engineered DC may provide for superior adaptive and innate immunity and ultimately, improved antitumor responses.