Intracellular Tumor Antigens Research Articles

PF207 TARGETING WILMS TUMOR 1 WITH A T CELL BISPECIFIC ANTIBODY (WT1‐TCB): EX VIVO AND IN VIVO POTENCY BY BIVALENT RECOGNITION OF PEPTIDE‐MHC COMPLEXES FROM AN INTRACELLULAR TUMOR ANTIGEN

Background:Antibody‐based immunotherapy represents a promising strategy to target chemo‐resistant leukemic cells. However, classical antibody‐based approaches are restricted to cell surface antigens, even though targeting of intracellular antigens would allow further access to a large number of tumor‐specific target structures.Aims:In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody based on T cell receptor (TCR)‐like antibodies for immunotherapy of acute myeloid leukemia (AML). The TCB targets the intracellular tumor antigen Wilms Tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A∗02:01 (HLA‐A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR‐specificity.Methods:WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real‐time PCR, western blot and immunohistochemical staining. WT1‐TCB‐mediated cytotoxicity was analyzed by co‐cultivation of WT1‐expressing HLA‐A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCB‐mediated cytotoxicity against primary AML cells was evaluated in our pre‐established feeder layer‐based ex vivo long‐term culture system. For in vivo testing, NSG mice (NOD.Cg‐Prkdcscid‐Il2rgtm1Wjl/SzJ) were humanized with human HLA‐A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1‐expressing HLA‐A2+ SKM‐1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1‐TCB.Results:In accordance with previous reports, we observed WT1 expression in 79% (n = 38) of cancer cell lines and in 92% (n = 65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n = 26), whereas WT1 was expressed again at time of relapse (n = 21). WT1‐TCBs elicited antibody‐mediated T cell cytotoxicity against peptide‐pulsed T2 cells and AML cell lines in a WT1 and HLA‐restricted manner. Equally, TCR downstream signaling was observed in a WT1‐restrictive manner by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67 ± 6% after 13–14 days; ± SEM; n = 18). Correspondingly, up‐regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3 ± 1.5, PD‐1: 5.1 ± 0.7, TIM‐3: 4.7 ± 0.6; ± SEM; n = 22). WT1‐TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38 ± 13% after 13–14 days; ± SEM; n = 5). In comparison with WT1RMF‐specific T cells, only bivalent binding by WT1‐TCB induced efficient lysis of primary AML cells. Furthermore, WT1‐TCB‐treated humanized mice bearing SKM‐1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control.Summary/Conclusion:TCR‐like TCBs targeting of intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide‐MHC complex on the cell surface validating WT1‐TCB as a promising therapeutic agent for the treatment of AML.

Percutaneous ablative cryoimmunotherapy for micrometastaic abscopal effect: No complications

Recent studies have shown efficacy of nivolumab, a monoclonal antibody specific for an immunoregulatory protein termed programmed death 1 (PD-1), against metastatic renal cell carcinoma. PD-1 is a transmembrane protein expressed on T cells that suppresses activation upon binding to its ligands, which may be secreted paraneoplastically by various cancers. Inhibition of PD-1 signaling via nivolumab may sensitize tumor-specific T lymphocytes in the immunosuppressive tumor microenvironment. Systemic elimination of micrometastases requires robust activation and proliferation of tumor antigen stimulated T cells. Cryoablation lyses tumor cell membranes but leaves intracellular tumor antigens intact for recognition by effectors systemically, while eliminating permissive T cell subtypes locally. This single case report describes CT-guided percutaneous cryoablation of a metastatic renal cell carcinoma with local administration of nivolumab to simultaneously debulk the primary tumor, sensitize effector T cells against tumor antigens, and augment the systemic immune response elicited against established metastases. One month follow up PET scan revealed decreased uptake in the two smaller metastatic bone lesions with the smallest lesion completely eliminated. The largest metastatic bone lesion was slightly decreased in size and exhibited slightly increased uptake. The patient's presenting complaint of hip pain was abrogated, allowing her to resume independent ambulation. CT-guided percutaneous cryoablation is minimally invasive and preserves tumor antigens, which are subsequently presented to tumor-specific T cells; their differentiation into cytotoxic T cells may be guided and their proliferation may be augmented by local administration of immunostimulatory pharmacotherapy at the time of the procedure.

Therapeutic Antibodies against Intracellular Tumor Antigens.

Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8–10 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I) molecules. These tumor-associated peptide–MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm) or T-cell receptor (TCR)-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.

Innate and adaptive immunity in human epilepsies.

SummaryInflammatory mechanisms have been increasingly implicated in the origin of seizures and epilepsy. These mechanisms are involved in the genesis of encephalitides in which seizures are a common complaint. Experimental and clinical evidence suggests different inflammatory responses in the brains of patients with epilepsy depending on the etiology. In general, activation of both innate and adaptive immunity plays a role in refractory forms of epilepsy. Epilepsies in which seizures develop after infiltration of cells of the adaptive immune system in the central nervous system (CNS) include a broad range of epileptic disorders with different (known or unknown) etiologies. Infiltration of lymphocytes is observed in autoimmune epilepsies, especially the classical paraneoplastic encephalitides with antibodies against intracellular tumor antigens. The presence of lymphocytes in the CNS also has been found in focal cerebral dysplasia type 2 and in cortical tubers. Various autoantibodies have been shown to be associated with temporal lobe epilepsy (TLE) and hippocampal sclerosis of unknown etiology, which may be due to the presence of viral DNA. During the last decade, an increasing number of antineuronal autoantibodies directed against membranous epitopes have been discovered and are associated with various neurologic syndromes, including limbic encephalitis. A major challenge in epilepsy is to define biomarkers, which would allow the recognition of patient populations who might benefit from immune‐modulatory therapies. Some peripheral inflammatory markers appear to be differentially expressed in patients with medically controlled and medically refractory and, as such, could be used for diagnostic, prognostic, or therapeutic purposes. Establishing an autoimmune basis in patients with drug‐resistant epilepsy allows for efficacious and targeted immunotherapy. Although current immunotherapies can give great benefit to the correctly identified patient, there are limitations to their efficacy and they may have considerable side effects. Thus the identification of new immunomodulatory compounds remains of utmost importance.

Abstract IA21: Immune mobilizing TCR therapies against cancer (ImmTAC): A novel class of antigen-specific tumor immunotherapy

Abstract Cancer immunosuppression prevents the effective activation of antigen-specific cytotoxic T cell responses against tumor cells. The activation of tumor-specific responses has the potential to halt the progression of cancer and, in some circumstances, eradicate it. Designing the therapeutic interventions that can achieve this intended mechanism of action (T cell eradication of tumors) aims to harness the known specificity of the T cell: target cell interaction to simultaneously a) lead to death of tumor target cells expressing the antigen and b) to limit surrounding tissue damage and thus manage potential toxicity. Previous design approaches of bispecific therapeutic molecules to redirect T cell responses against cancer cells have utilized antibody binding domains to recognize tumor cell-surface proteins. These first generation bi-specifics have proven efficacy in hematologic malignancies, and novel approaches such as TCR targeting have the goal of enhancing activity in solid tumor settings. The ImmTACs (immune-mobilizing monoclonal TCRs against cancer) are a new class of bispecific therapeutic molecule, utilizing the physiologic antigen recognition system of the T cell, the monoclonal T cell receptor (TCR), as the targeting effector fused to an anti-CD3 domain. The soluble TCRs have been engineered to possess extremely high affinity for their tumor antigen peptide-HLA complex. ImmTACs thus overcome the problem of low affinity tumor-specific T cells imposed by thymic selection, and provide a means to target intracellular tumor antigens not accessible to antibody binding domains. The ImmTACs drive recruitment of polyclonal T cells to the tumor, resulting in potent redirected T cell response and tumor cell destruction in an antigen-specific manner. The first ImmTAC tested in the clinic is IMCgp100, an engineered TCR recognizing the gp100 melanoma antigen in the context of HLA-A2 with promising, preliminary single agent activity in both cutaneous and uveal melanoma. Citation Format: Christina M. Coughlin. Immune mobilizing TCR therapies against cancer (ImmTAC): A novel class of antigen-specific tumor immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA21.

Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell-based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

ImmTACs: bi-specific TCR-based reagents for targeted cancer immunotherapy (VAC7P.1037)

Abstract ImmTACs (immune mobilizing monoclonal T cell receptors against cancer) are a new class of soluble bi-specific reagent that exploit the natural antigen recognition pathway mediated by the T cell receptor (TCR). As TCRs recognise HLA-presented peptides, ImmTACs can access the larger pool of intracellular tumor antigens unavailable to antibody based therapies. ImmTACs target tumors via a soluble monocloncal TCR, engineered to recognise a given tumor associated peptide-HLA complex with exceptionally high sensitivity and specificity. Once bound to their target, ImmTACs redirect host polyclonal T cells via an anti-CD3 antibody fragment. In this way ImmTACs facilitate targeted T cell recognition of tumors leading to potent T cell activation and tumor cell killing, with the potential to break T cell tolerance. ImmTACs are specific for humans, thus efficacy, specificity and off target effects cannot be assessed in animal models. Using antigen positive tumor cells and HLA appropriate primary human cell lines representing a range of tissues, the in vitro pre-clinical package can be predictive of in vivo clinical observations. A first ImmTAC, IMCgp100, has entered phase I/IIa clinical trials for patients with advanced melanoma. The drug is well tolerated and many patients show tumor shrinkage after the first dose of ImmTAC with tumor shrinkage improving with each dose. Emerging data demonstrate durable partial responses in three patients and a complete response in a fourth patient.

Functional autoantibodies against SSX-2 and NY-ESO-1 in multiple myeloma patients after allogeneic stem cell transplantation.

Multiple myeloma (MM) is the malignancy with the most frequent expression of the highly immunogenic cancer-testis antigens (CTA), and we have performed the first analysis of longitudinal expression, immunological properties, and fine specificity of CTA-specific antibody responses in MM. Frequency and characteristics of antibody responses against cancer-testis antigens MAGE-A3, NY-ESO-1, PRAME, and SSX-2 were analyzed using peripheral blood (N = 1094) and bone marrow (N = 200) plasma samples from 194 MM patients. We found that antibody responses against CTA were surprisingly rare, only 2.6 and 3.1 % of patients evidenced NY-ESO-1- and SSX-2-specific antibodies, respectively. NY-ESO-1-specific responses were observed during disease progression, while anti-SSX-2 antibodies appeared after allogeneic stem cell transplantation and persisted during clinical remission. We found that NY-ESO-1- and SSX-2-specific antibodies were both capable of activating complement and increasing CTA uptake by antigen-presenting cells. SSX-2-specific antibodies were restricted to IgG3, NY-ESO-1 responses to IgG1 and IgG3. Remarkably, NY-ESO-1-positive sera recognized various non-contiguous regions, while SSX-2-specific responses were directed against a single 6mer epitope, SSX-2(85-90). We conclude that primary autoantibodies against intracellular MM-specific tumor antigens SSX-2 and NY-ESO-1 are rare but functional. While their contribution to disease control still remains unclear, our data demonstrate their theoretic ability to affect cellular anti-tumor immunity by formation and uptake of mono- and polyvalent immune complexes.

Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8+ T cell responses

DNA vaccination with plasmid has conventionally involved vectors designed for transient expression of antigens in injected tissues. Next generation plasmids are being developed for site-directed integration of transgenes into safe sites in host genomes and may provide an innovative approach for stable and sustained expression of antigens for vaccination. The goal of this study was to evaluate in vivo antigen expression and the generation of cell mediated immunity in mice injected with a non-integrating plasmid compared to a plasmid with integrating potential. Hyperactive piggyBac transposase-based integrating vectors (pmhyGENIE-3) contained a transgene encoding either eGFP (pmhyGENIE-3-eGFP) or luciferase (pmhyGENIE-3-GL3), and were compared to transposase-deficient plasmids with the same transgene and DNA backbone. Both non-integrating and integrating plasmids were equivalent at day 1 for protein expression at the site of injection. While protein expression from the non-integrating plasmid was lost by day 14, the pmhyGENIE-3 was found to exhibit sustained protein expression up to 28 days post-injection. Vaccination with pmhyGENIE-3-eGFP resulted in a robust CD8+ T cell response that was three-fold higher than that of non-integrating plasmid vaccinations. Additionally we observed in splenocyte restimulation experiments that only the vaccination with pmhyGENIE-3-eGFP was characterized by IFNγ producing CD8+ T cells. Overall, these findings suggest that plasmids designed to direct integration of transgenes into the host genome are a promising approach for designing DNA vaccines. Robust cell mediated CD8+ T cell responses generated using integrating plasmids may provide effective, sustained protection against intracellular pathogens or tumor antigens.

Cytometry for cells and particles in disease

CELL therapy is a major hope for future tailored therapy in many human diseases and cytometry is a central player in its development. The technologies and assays developed for quantitative analysis of individual cells and cell systems is of major importance in the development of cell therapy, clearcut identification of cell based diseases like cancer, as well as monitoring success of the therapy. It is crucial for personalized medicine to unequivocally identify disease types for individualized therapy. Until today, state of the art for judging histological specimens in the pathology laboratory is done by the experienced pathologists. Unfortunately, standardization of this expertise is hardly possible, sometimes rendering contradictory results. In addition, institutions that lack such experts need to send their material to other laboratories which becomes difficult for remote places, if possible at all. Manifold approaches have been made to automate and standardize histopathology and make it quantitative (1). Now, Orlov and colleagues from Baltimore and Bethesda, Maryland, Philadelphia, Pennsylvania and Southfield, Michigan, USA (this issue, page 364) developed an image based method for tissue microarrays in order to automate detection of the progression of melanomas. The authors analyzed automatically acquired microscopic images of hematoxylin/eosin stained tissue microarrays containing melanoma sections at different stages of the disease progression. Using their algorithm they could ‘‘teach’’ the software to learn differences between various tumor stages and healthy tissue. This algorithm was then able to identify unknown specimens in a test set with high sensitivity and specificity. It turned out that color is an important discriminator. Whereas the above approach was based on morphological criteria, single cell analysis in tissues may have additional benefits for classification. However, single cell identification by segmentation algorithms is still challenging and prone to failure (2). Mashburn and colleagues from Nashville, Tennessee, USA (this issue, page 409) developed a new interactive algorithm, termed SeedWater Segmenter to identify and track individual cells in cell cultures. Their test system was a primary strain of Drosophila melanogaster expressing cadherinGFP fusion protein so that cell boundaries were visible in confocal fluorescence images. The authors report that their approach is based on a simple and useful paradigm, the interactive manual segmentation correction using direct seed manipulation. It fills a niche as a convenient tool for extremely accurate tracking of cells in living tissues. Cell based therapy of tumors and metastasis by preconditioned lymphocytes that become primed for the specific tumor cells of the excised tumor of a patient is an emerging technology for personalized therapy of minimal residual disease (3). This so-called adoptive cell therapy requires thorough quality control which is the detailed characterization of the cell products that are to be administered to the patient. Among others, purity of the lymphocytes and freedom from live tumor cells needs to be proven. By a sophisticated panel of live/dead discrimination as well as an antibody cocktail staining for extraand intracellular tumor antigens, Richards and colleagues from Milwaukee, Wisconsin, USA (this issue, page 374) present an important flow-cytometry assay to characterize and quantitate contaminating tumor cells from primary cultures of excised tumors. The authors conclude that flow cytometry could be an alternative method to immunohistochemistry due to its higher sensitivity and statistical accuracy. Important additional information that would be useful in targeted therapy is to characterize the lymphocytes intended for adoptive transfer with respect to their more detailed phenotyping and biological function. This functional characteri-

Intracellular Tumor-Associated Antigens Represent Effective Targets for Passive Immunotherapy

Monoclonal antibody (mAb) therapy against tumor antigens expressed on the tumor surface is associated with clinical benefit. However, many tumor antigens are intracellular molecules that generally would not be considered suitable targets for mAb therapy. In this study, we provide evidence challenging this view through an investigation of the efficacy of mAb directed against NY-ESO-1, a widely expressed immunogen in human tumors that is expressed intracellularly rather than on the surface of cells. On their own, NY-ESO-1 mAb could neither augment antigen-specific CD8(+) T-cell induction nor cause tumor eradication. To facilitate mAb access to intracellular target molecules, we combined anti-NY-ESO-1 mAb with anticancer drugs to accentuate the release of intracellular NY-ESO-1 from dying tumor cells. Strikingly, combination therapy induced a strong antitumor effect that was accompanied by the development of NY-ESO-1-specific effector/memory CD8(+) T cells that were not elicited by single treatments alone. The combinatorial effect was also associated with upregulation of maturation markers on dendritic cells, consistent with the organization of an effective antitumor T-cell response. Administration of Fc-depleted F(ab) mAb or combination treatment in Fcγ receptor-deficient host mice abolished the therapeutic effect. Together, our findings show that intracellular tumor antigens can be captured by mAbs and engaged in an efficient induction of CD8(+) T-cell responses, greatly expanding the possible use of mAb for passive cancer immunotherapy.

Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.

Abstract 4741: Therapeutic efficacy of a human-derived antibody against cancer-testis antigen NY-ESO-1

Abstract Rationale: It is accepted that the immune system can control cancer and a favorable clinical course correlates with high infiltration by CD8+ T cells in different cancer entities. Thus, boosting cancer-specific immunity is an interesting therapeutic modality. Cancer-testis antigens (CTA) are promising target antigens due to their tumor-restricted expression pattern and high immunogenicity. NY-ESO-1 is one of the best characterized CTA, and spontaneous anti-NY-ESO-1 humoral and cellular responses were described in patients with various cancers. We propose that antibodies (Abs) against intracellular CTA have therapeutic potential because they may facilitate the uptake and presentation of antigens by dendritic cells (DC), which supports cancer-specific T cell responses. This effect may be more pronounced when Abs are used in combination with therapies that result in death, and thus antigen release, of cancer cells. We report here the characterization of the first human anti-NY-ESO-1 monoclonal Ab. Methods: Human-derived monoclonal Ab 12D7 was obtained by molecular cloning from memory B cells of a melanoma patient, ZH-311. To characterize the binding of 12D7 we performed ELISA, Biacore analysis and tissue staining. In addition, we used 12D7 to immunoprecipitate native NY-ESO-1 from human melanoma cell line SK-MEL-37. As a proof of concept, we compared the activation of NY-ESO-1-specific CD8+ T cell clones by DC fed with NY-ESO-1 vs. NY-ESO-1/12D7 immune complexes (IC) in vitro. To determine the therapeutic effect in vivo, we treated BALB/c mice bearing syngeneic CT26/NY-ESO-1 colorectal tumors with 5-FU +/- 12D7. To investigate Ab accumulation in established tumors 12D7 was labeled with FITC and injected into mice 2 days after 5-FU treatment. Results: Human monoclonal 12D7 specifically bound to NY-ESO-1 with high affinity (pM range) and precipitated endogenous NY-ESO-1 from SK-MEL-37. The uptake and cross-presentation of NY-ESO-1 by DC was greatly enhanced when NY-ESO-1 was complexed with 12D7 before feeding. Importantly, uptake of NY-ESO-1/12D7 IC, but not NY-ESO-1, resulted in maturation of DCs as determined by upregulation of co-stimulatory molecules. Finally, 12D7 significantly improved the therapeutic efficacy of 5-FU treatment in established CT26/NY-ESO-1 tumors. Ab accumulation in CT26/NY-ESO-1 tumors was enhanced when mice were treated with 5-FU as compared with untreated mice. Conclusion: We propose that the release of intracellular tumor antigens by chemotherapy and the accumulation of Ab to such antigens results in the local formation of IC. These IC are efficiently cross-presented to tumor-specific CD8+ T cells by DCs and may induce in situ maturation of the latter, both of which support tumor-specific effector function and thus contributes to tumor control. Our results suggest that targeting intracellular antigens, more specifically CTA, with Abs is a useful strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2011-4741

Abstract 2441: An innovative treatment modality of antibody therapy against intracellular tumor antigens

Abstract Therapy with mAb directed against cell surface antigens of cancer is now a well-established treatment modality for human cancer. However, many tumor antigens of great interest have an intracellular location, and therefore not considered suitable targets for antibody therapy. On the other hand, it has been reported that mAbs targeted against specific tumor antigens induce cellular immune response including CD8+ T cells. This raises the possibility that the anti-tumor effects of mAb therapy may partially depend on enhanced cross-priming mediated by APCs following the uptake of antigen released from tumors complexed with Ab (immune-complex). Here, we investigated whether mAb therapy could be applicable to intracellular antigens using a transplantable colon carcinoma CT-26 expressing NY-ESO-1 (a well-studied human cancer/testis antigen), an intracellular antigen lacking cell surface expression. Immune responses to NY-ESO-1 in the murine model closely parallel NY-ESO-1 humoral and cellular immune responses in humans. To accentuate the natural release of intracellular NY-ESO-1 from dying tumor cells to facilitate immune-complex formation, we employed the effect the anti-cancer drug on the response to NY-ESO-1 mAb. The combination of NY-ESO-1 mAb and 5-fluorouracil exhibited a strong and long-lasting anti-tumor effect that could not be achieved by treatment with either 5-fluorouracil or mAb alone. The anti-tumor effect induced by combination therapy was no longer observed when Fc-depleted Fab antibodies were used, indicating a Fc-dependent action on APCs. Furthermore, augmented anti-tumor effects observed by the combination therapy was associated with strong NY-ESO-1-specific CD8+ T cells in draining lymph nodes and tumor sites, and upregulation of maturation markers on dendritic cells. The opsoniziation of released intracellular tumor antigens with injected mAb facilitates uptake by APC and presentation to CD8+ T cells, leading to accelerated and augmented active immunization. The data indicate that mAb to intracellular molecules of cancer represents a promising approach to cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2441.

Antitumoral Immune Response by Recruitment and Expansion of Dendritic Cells in Tumors Infected with Telomerase-Dependent Oncolytic Viruses

Virotherapy can potentially be used to induce tumor-specific immune responses and to overcome tumor-mediated tolerance mechanisms because apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor-specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide-specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T-cell responses against intracellular tumor antigens. Triggering of DC and T-cell infiltration to the oncolytic tumors by macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Although immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1alpha/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1alpha/FLT3L virotherapy, we observed systemic antitumoral effects by MIP-1alpha/FLT3L virotherapy on uninfected lung metastasis in immunocompetent mice but not in nude mice. Furthermore, MIP-1alpha/FLT3L virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long-term survival of 50% of treated animals. In summary, improvement of cross-presentation of tumor antigens by triggering of DC and T-cell infiltration during virotherapy enhances antitumoral immune response that facilitates an effective viroimmunotherapy of primary tumors and established metastases.

Antitumoral immune response by recruitment and expansion of dendritic cells in tumors infected with telomerase-dependent oncolytic viruses

Virotherapy can potentially be used to induce tumor specific immune responses and to overcome tumor-mediated tolerance mechanisms, since apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T cell responses against intracellular tumor antigens. Triggering of DC and T cell infiltration to the oncolytic tumors by Macrophage Inflammatory Protein 1a (MIP-1a, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Though immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1a/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1a/FLT3L-virotherapy, we observed systemic antitumoral effects by MIP-1a/FLT3L-virotherapy on uninfected lung metastasis in immunocompetent mice, but not in nude mice. Furthermore, MIP-1a/FLT3L-virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long term survival of 50% of treated animals.

T cell memory and protective immunity by vaccination: is more better?

Protection against intracellular pathogens or tumor antigens requires T-cell mediated responses. Recently, it has become apparent that protection against disease correlates with T cells of the central memory type in many instances. Here, we analyze current data to distill a set of rules for the induction and maintenance of central memory T-cell responses. Recent studies show that T-cell help and the lack of overt inflammation at the time of priming are prerequisite for the induction, maintenance and expansion of memory T cells. Central to our hypothesis is that, in addition to these factors, successful vaccination in the immunologically inexperienced individual should be based on low antigen dose, to decelerate replicative senescence in responding cells and favor lineage differentiation of central memory T cells. In the immunologically experienced individual, it will be necessary, in addition, to abate the antigen load in plasma before vaccination. These guiding principles might help to raise improved protective T-cell responses by vaccination in humans.

Vaccine Therapy in NHL: Future Promises and Current Limitations

Recent advances in genomics are revealing new molecular targets for vaccination strategies in cancer, and unraveling the immune pathways that must be activated. Initially, work focused on idiotypic antigen expressed by the clonal immunoglobulin (Ig) of B-cell tumors. This involved assembling the encoding VH and VL genes as single-chain Fv (scFv), which has been fused to Fragment C (FrC) of tetanus toxin to enhance recognition and response. The fusion gene induces strong anti-idiotypic protection against lymphoma in experimental models, and is now in clinical trials with encouraging results emerging. This principle of fusing a pathogen-derived sequence is widely applicable. A coat protein from a potato virus substituted for the FrC sequence has also been found to be capable of promoting anti-tumor immunity. The current design is effective in inducing anti-idiotypic (Id) antibody and CD4+ T-cell attack. For intracellular tumor antigens expressed as MHC Class I-associated peptides, a design consisting of a gene encoding a single domain of FrC linked to a candidate peptide sequence has been developed. This induces fast cytotoxic T-cell attack on peptide-expressing tumor cells. Injection of DNA appears safe and clinical trials will test the efficacy of this approach, and probably lead to improvements in these flexible vaccines.