Suitable Targets For Immunotherapy Research Articles

Mass cytometry reveals immune atlas of urothelial carcinoma

Immunotherapy has emerged as a robust clinical strategy for cancer treatment. PD1/PD-L1 inhibitors have been used as second-line therapy for urothelial carcinoma due to the high tumor mutational burden. Despite the efficacy of the treatment is significant, the response rate is still poor. The tumor immune microenvironment plays a key role in the regulation of immunotherapeutic efficacy. However, a comprehensive understanding of the intricate microenvironment in clinical samples remains unclear. To obtain detailed systematic tumor immune profile, we performed an in-depth immunoassay on 12 human urothelial carcinoma tissue samples and 14 paratumor tissue samples using mass cytometry. Among the large number of cells assayed, we identified 71 T-cell phenotypes, 30 tumor-associated macrophage phenotypes. T cell marker expression profiles showed that almost all T cells in the tumor tissue were in a state of exhaustion. CD38 expression on tumor-associated macrophages (TAMs) was significantly higher than PDL1, and CD38+ TAMs were closely associated with immunosuppression. CD38 may be a more suitable target for immunotherapy in urothelial carcinoma compared to PD1/PDL1. This single-cell analysis of clinical samples expands our insights into the immune microenvironment of urothelial carcinoma and reveals potential biomarkers and targets for immunotherapy development.

A Tetravalent Biparatopic Antibody Causes Strong HER2 Internalization and Inhibits Cellular Proliferation.

The overexpression of tyrosine kinase HER2 in numerous cancers, connected with fierce signaling and uncontrolled proliferation, makes it a suitable target for immunotherapy. The acquisition of resistance to currently used compounds and the multiplicity of signaling pathways involved prompted research into the discovery of novel binders as well as treatment options with multiple targeting and multispecific agents. Here we constructed an anti-HER2 tetravalent and biparatopic symmetrical IgG-like molecule by combining the Fab of pertuzumab with a HER2-specific Fcab (Fc fragment with antigen binding), which recognizes an epitope overlapping with trastuzumab. In the strongly HER2-positive cell line SK-BR-3, the molecule induced a rapid and efficient reduction in surface HER2 levels. A potent anti-proliferative effect, specific for the HER2-positive cell line, was observed in vitro, following the induction of apoptosis, and this could not be achieved with treatment with the mixture of pertuzumab and the parental Fcab. The inhibitory cytotoxic effect of our antibody as a single agent makes it a promising contribution to the armory of anti-cancer molecules directed against HER2-addicted cells.

Abstract 247: Identification of an ovarian cancer selective splice variant of mesothelin utilizing the Kiromic proprietary search engine CancerDiff

Abstract The advancement of cures for cancer needs the development of novel, more efficacious, and more specific immunotherapeutic approaches through the discovery of novel target candidates displaying differential expression between healthy and malignant tissues. CancerDiff is a proprietary software module for the identification of potential new immunotherapeutic cancer targets that originate from differentially expressed, alternatively spliced transcripts. When utilized to analyze Ovarian Cancer (OV) datasets, CancerDiff identified a selectively upregulated mesothelin (MSLN) splice variant translated into a protein isoform (IsoMSLN) bearing a distinct unique peptide absent in the canonical protein sequence. To validate this prediction and to confirm the upregulation of IsoMSLN in OV, datasets from publicly available proteomic repositories were searched for its unique signature peptide. In agreement with CancerDiff prediction, IsoMSLN peptide was detected in 71% of OV samples and 61% of adjacent normal tissues. Molecular modeling tools predicted this peptide to be part of the extracellular portion of the protein in an antibody accessible region. These results indicate IsoMSLN unique peptide as a suitable target for immunotherapy for OV cancer. Citation Format: Lucia Piccotti, Leonardo Mirandola, Maurizio Chiriva-Internati. Identification of an ovarian cancer selective splice variant of mesothelin utilizing the Kiromic proprietary search engine CancerDiff [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 247.

In pancreatic cancer, chemotherapy increases antitumor responses to tumor-associated antigens and potentiates DNA vaccination

BackgroundPancreatic ductal adenocarcinoma (PDA) is an almost incurable tumor that is mostly resistant to chemotherapy (CT). Adaptive immune responses to tumor-associated antigens (TAA) have been reported, but immunotherapy (IT) clinical...

A Potential Role for NKG2C+ γδ T Cells as a Cellular Therapy in Combating Post-Lung Transplant Cytomegalovirus Infection

Cytomegalovirus (CMV) is the most clinically significant pathogen following lung transplantation (LTx). While currently used anti-viral treatments are efficacious, their use is associated with toxicity and antiviral resistance. Given early promising results, immunotherapy is an attractive option to complement current antiviral therapies. We assessed the phenotype of circulating T cells following LTx with the aim of identifying novel cell subsets that may be candidate population as anti-viral immunotherapy. Peripheral blood mononuclear cells (PBMC) were isolated from lung transplant patients from a single centre at pre-transplant, 0.5, 1.5, 3, 6, 9, 12- and 18-months post-LTx. Patients that were at risk of developing CMV disease were included in the study and were stratified based on the risk - moderate risk (Recipient CMV positive, n=15) or high risk (Recipient CMV negative/Donor CMV positive, n =11). CMV reactivation was classified as CMV PCR positive (>150 copies/ml) in blood and/or BAL within the first 12-months post-LTx. The phenotype of T cells in PBMC was assessed by multi-colour flow cytometry with a particular focus on γδT cells. We observed that recipient's γδT cell subset increased markedly following cessation of anti-viral prophylaxis and in CMV seropositive recipients, this subset is prominent pre-LTx, being highest in those who did not experience CMV reactivation. These cells are enriched in Vδ1 TCR-expressing population. Furthermore, a strong bias towards NKG2C+ cells was observed. NKG2C is a well-defined activation marker on NK cells and has been implicated in CMV immunity. In vitro assays showed that NKG2C+Vδ1+ γδT cells from healthy individuals exhibit functional capacity in line with anti-viral immunity. Our results indicate that NKG2C+Vδ1+ cells may be a suitable target for immunotherapy. The increase of blood NKG2C+Vδ1+ γδT cells in the presence of CMV indicates their contribution in this infection. As these cells could act in HLA-independent manner, we propose they may be an ideal target for cellular therapy against CMV in the transplant setting.

Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer

ObjectivesCancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. Materials and methodsTo comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. ResultsAltogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. ConclusionWe identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.

Affinity proteomic profiling of plasma for proteins associated to pancreatic cancer reveal candidates for patient survival

Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies.To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases.Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients.We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.

104 Melanoma associated antigen A3 (MAGE-A3) influences proliferation of metastatic squamous cell carcinoma (SCC) in vitro

104 Melanoma associated antigen A3 (MAGE-A3) influences proliferation of metastatic squamous cell carcinoma (SCC) in vitro A Santana, N Roudiani, J Therrien, JH Lee, D Felsen and J Carucci 1 Dept. of Derm., NYU Langone Med. Ctr., New York, NY, 2 Stiefel Laboratories, Research Triangle Park, NC and 3 Inst. for Ped. Urol., Weill Cornell Medicine, New York, NY In order to identify novel targets for cancer immune therapy, our lab has profiled the expression of cancer testis antigens (CTA) in primary human SCC. CTAs are immunogenic and may be suitable targets for immunotherapy, or as biomarkers for aggressive biological behavior. We recently showed that MAGE-A3 is highly expressed in aggressive SCC with poor prognosis. We hypothesized that MAGE-A3 may be a novel target for immunotherapy and/or a biomarker for metastatic SCCs. In this study we determined MAGE-A3 expression in and the effect of antibody treatment on growth of human (A431) and murine (Pam 212 and LY2, BALB/c) SCC cell lines. Pam 212 cells develop locally invasive SCC in syngeneic hosts and LY2 were isolated from a rare Pam 212 metastasis (Yuspa et al., JID, 1983; Dong et al., J Cell Biochem 1997). A431, Pam 212, and LY2 cell lines express MAGE-A3 at the transcript and protein level as measured by qRT-PCR and immunoblot, respectively. Moreover, A431 SCC growth and proliferation was significantly inhibited after MAGE-A3 antibody treatment measured by MTT (P 0.001) and crystal violet (P 0.001). We found that cell growth and proliferation of locally invasive Pam 212 cells was unaffected by anti-MAGE-A3 treatment. However, metastatic tumor derived LY2 cell growth and proliferation was significantly inhibited after MAGE-A3 antibody treatment measured by MTT (P 0.01) and crystal violet (P 0.01). MAGE-A3 expression could potentially serve both as a cancer biomarker and vaccine candidate due to its expression by SCC cells in vitro. Interestingly, MAGE-A3 antibody treatment inhibited the growth of A431 and LY2, SCCs that metastasize in vivo. These results suggest that metastatic SCC may rely on signaling events downstream of MAGE-A3 to support proliferation. Ongoing studies focus on elucidation of pathways involved in MAGE driven SCC proliferation in vivo using immune competent BALB/c mice. 105 Keratinocyte p38a loss results in increased tumor initiation, decreased malignant progression, and altered tumor type specification during two-stage chemical carcinogenesis in murine skin A Kiss, AC Koppel, C Cataisson, J Anders, S Yuspa, P Bible, M Kellett, MI Morasso and T Efimova 1 Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, 2 Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD and 3 Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD The p38a signaling pathway not only regulates the inflammation and stress responses, but also controls, in a context-specific manner, the homeostatic balance of cell proliferation, differentiation and survival, deregulation of which is known to contribute to tumor development. Studies in mouse models revealed both tumor-suppressing and tumor-promoting roles of p38a, depending on tissue and tumor type, and tumor stage. However, the in vivo functional contributions of p38a to the skin carcinogenesis remain incompletely understood. We explored the in vivo role of p38a in two-stage chemical skin carcinogenesis using mice with conditional epidermal keratinocyte-specific genetic ablation of p38a (p38a-cKO). Mutant mice exhibited a significantly increased tumor multiplicity, and, in addition to characteristic benign and malignant squamous tumors, developed benign well-differentiated sebaceous adenomas containing a signature activating Hras mutation. Only 4% of tumors in p38a-cKO mice underwent malignant conversion to squamous cell carcinomas (SCCs), compared with 38% of tumors in wild-type (WT) mice (p<0.0001). p38a deficiency resulted in a significantly reduced size and a more differentiated morphology of p38a-cKO SCCs, relative to WT SCCs. RNA sequencing revealed p38a-dependent transcriptional changes in genes involved in cell differentiation, development, proliferation, cell death, and inflammation, both in control and in v-ras-transformed keratinocytes. Altogether, our data reveal novel roles for p38a signaling in control of tumor initiation, malignant progression, and tumor type specification.

T Cell Receptor Gene Therapy Targeting the Intracellular Transcription Factor Bob1 for the Treatment of Multiple Myeloma and Other B Cell Malignancies

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR.Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated.Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells.In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

T Cell Receptors Specific for the Intracellular Transcription Factor Bob1 Allow Efficient Targeting of Human B Cell Leukemia and Multiple Myeloma

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. However, loss of CD20 and CD19 expression or absence of these molecules on other malignancies such as multiple myeloma restricts their application.Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to T cell receptors (TCRs) and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a total of 3 x 109 peripheral blood mononuclear cells from 6 different HLA-A2/B7-negative healthy donors, we isolated and clonally expanded more than 1000 CD8+ T cells binding to peptide-MHC-tetramers composed of the Bob1-derived peptides bound to HLA-A2 or HLA-B7. The T cell clones were tested for stringent peptide-specificity by stimulation with Bob1-negative K562 cells expressing either HLA-A2 or B7 unloaded or pulsed with Bob1-derived peptides. This resulted in the selection of 15 T cell clones highly specific for Bob1. To identify the T cell clones of highest avidity, T cell clones were compared for peptide-sensitivity by testing the recognition of stimulator cells loaded with titrated amounts of Bob1-derived peptides and of Bob1-expressing HLA-A2/B7-positive EBV-transformed B cells. T cell clone 4G11 was selected because of high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7 and T cell clone 3C10 specifically recognized peptide Bob1245 bound to HLA-A2. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. To investigate whether harmful toxicities could be caused by these T cell clones, we tested their reactivity against a wide panel of Bob1-negative stimulator cells demonstrating absence of recognition of HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. These data illustrate a safe reactivity profile with little chance of off-target toxicity. To test their clinical applicability, clone 4G11 and 3C10 were tested for recognition of various primary B cell malignancies. Clone 4G11 efficiently recognized HLA-B7-positive primary ALL, CLL and mantle cell lymphoma while clone 3C10 recognized HLA-A2-positive primary B cell malignancies albeit to a lesser degree. Furthermore, reproducible strong recognition of purified primary HLA-B7-positive multiple myeloma could be demonstrated for clone 4G11. Therefore, T cell clone 4G11’s TCR may be used for immunotherapy by administering TCR-transduced T cells to multiple myeloma patients. To test whether introduction of 4G11’s TCR confers Bob1-reactivity onto recipient cells, the TCR was cloned into a retroviral vector. Highly specific reactivity against HLA-B7-positive Bob1-expressing target cells could be installed to TCR-transduced recipient T cells.In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies and multiple myeloma. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. TCR gene transfer approaches using Bob1-specific TCRs can bring novel treatment modalities for patients with B cell malignancies or multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

A CSPG4-specific immunotoxin kills rhabdomyosarcoma cells and binds to primary tumor tissues

The treatment of rhabdomyosarcoma (RMS) remains challenging, with metastatic and alveolar RMS offering a particularly poor prognosis. Therefore, the identification and evaluation of novel antigens, which are suitable targets for immunotherapy, is one attractive possibility to improve the treatment of this disease. Here we show that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on RMS cell lines and RMS patient material. We evaluated the immunotoxin (IT) αMCSP-ETA′, which specifically recognizes CSPG4 on the RMS cell lines RD, FL-OH1, TE-671 and Rh30. It is internalized rapidly, induces apoptosis and thus kills RMS cells selectively. We also demonstrate the specific binding of this IT to RMS primary tumor material from three different patients.

Immunotherapy for prostate cancer: lessons from responses to tumor-associated antigens.

Prostate cancer (PCa) is the most common cancer in men and the second most common cause of cancer-related death in men. In recent years, novel therapeutic options for PCa have been developed and studied extensively in clinical trials. Sipuleucel-T is the first cell-based immunotherapeutic vaccine for treatment of cancer. This vaccine consists of autologous mononuclear cells stimulated and loaded with an immunostimulatory fusion protein containing the prostate tumor antigen prostate acid posphatase. The choice of antigen might be key for the efficiency of cell-based immunotherapy. Depending on the treatment strategy, target antigens should be immunogenic, abundantly expressed by tumor cells, and preferably functionally important for the tumor to prevent loss of antigen expression. Autoimmune responses have been reported against several antigens expressed in the prostate, indicating that PCa is a suitable target for immunotherapy. In this review, we will discuss PCa antigens that exhibit immunogenic features and/or have been targeted in immunotherapeutic settings with promising results, and we highlight the hurdles and opportunities for cancer immunotherapy.

Clinical and Immunological Results Of a Phase I/IIa Study Of Allogeneic Dendritic Cell (DC) Vaccination, An “off The shelf” Treatment To Prevent Or Delay Relapse In Elderly Patients With Acute Myeloid Leukemia

Vaccines against tumor associated antigens represent an appealing strategy for preventing tumor recurrence. A novel immunotherapy platform is represented by a dendritic cell vaccine that originates from a human myeloid leukemia cell line, DCOne, which endogenously expresses a range of leukemia associated antigens, including PRAME and WT-1, and can be differentiated into mature dendritic cells (DCs). Mature DC derived from DCOne are being developed to replace patient-derived DC vaccines. The first indication in which vaccination with mature DCs derived from DCOne has been tested clinically is Acute Myeloid Leukemia (AML) because of the high unmet medical need and substantial evidence that AML is a suitable target for immunotherapy. A Phase I/IIa study enrolled 12 AML patients (age range 58-71) who were either in CR1/CR2 (n=5) or had smoldering disease (n=7). Patients had received all available standard care, were at high risk of relapse and ineligible for all available post-remission therapies, including allogeneic stem cell transplantation. Patients were selected for the study because they had an estimated life expectancy of 3-6 months.A standard 3+3 design was used, starting with 4 bi-weekly intradermal DCOne DC vaccinations of 10E6 (n=3), 25E6 (n=3) or 50E6 (n=6) cells. Patients were monitored for clinical and immunological responses for 126 days and surviving patients underwent long-term follow-up after study completion. Primary endpoints were safety and feasibility; secondary endpoints were clinical and immunological responses. Treatment was well tolerated in all patients, with related adverse events mainly limited to injection site reactions. During the 5 months duration of the study 3 patients died: 2 from infections and 1 from leukemia. Clear evidence for induction of multi-functional immune responses was obtained, including increased post vaccination delayed type hypersensitivity reactions, increases in CD4+ and CD8+ T cell proliferative responses and/or sero-conversion to DCOne DCs and/or AML blasts in 6 out of 9 patients. Three of 7 patients who were evaluable by IFNgELISpots showed vaccination-induced reactive T cell responses to WT-1 and/or PRAME, antigens which are present in DCOne. The patients who survived more than 6 months post-vaccination showed strongly prolonged survival. Four patients are still alive 28, 22, 12 and 10 months after study entry and 1 patient survived for 23 months. Patients in CR1 or CR2 at study entry were all in CR at the end of the study and these patients were more likely to exhibit prolonged survival. Patients not in CR at study entry, with one exception, all had persistence of disease at the end of the study and died due to disease progression.Together, these results suggest that patients who have a capable immune system can induce a multi-functional and lasting immune response to the vaccine. As expected this most likely translates into long-term clinical benefit if patients are in CR at the time of vaccination. This is in keeping with the expected kinetics of cancer immunotherapy. We conclude that vaccination with DCOne derived DCs is safe and feasible in elderly AML patients, and generates both cellular and humoral immune responses. The hypothesis that DCOne-derived DCs induce immune responses against the patients' leukemia cells, translating into clinical benefit in patients with a capable immune system who are in CR at the time of vaccination, will now be investigated in a multi-center randomized Phase II trial in AML patients in first remission. Disclosures:van Wetering:DCPrime: Employment. Kaur Singh:DCPrime: Employment. Hall:DCPrime: Consultancy. Kruisbeek:DCPrime: Employment.

Survivin in Adrenocortical Tumors - Pathophysiological Implications and Therapeutic Potential

Treatment options for adrenocortical carcinoma (ACC) are very limited. In other solid tumors, small vaccination trials targeting the anti-apoptotic molecule survivin suggested immunological and clinical benefit in selected patients. Therefore, we investigated whether survivin might be a suitable target for immunotherapy in ACC. Survivin mRNA and protein expression was assessed in adrenal tissue specimens [by real-time-PCR in 29 ACC, 24 adrenocortical adenomas (ACA) and 12 normal adrenal glands; by immunohistochemistry in 167 ACCs, 15 ACA, and 5 normal adrenal glands]. Expression was correlated with clinical outcome using Kaplan-Meier and Cox regression analyses. The anti-apoptotic role of survivin was investigated in the SW13 ACC cell line using survivin siRNA. The presence of spontaneous survivin specific T-cells in peripheral blood was assessed by FACS dextramere staining in 29 ACC patients in comparison to healthy controls. Survivin mRNA in ACC was significantly overexpressed when compared with ACA or normal adrenal glands. Immunohistochemistry confirmed survivin protein expression in 97% of the ACCs. In 83% of samples, staining was moderate or high and clinical outcome in this subgroup showed a trend towards poorer prognosis [hazard ratio for death 2.28 (95% CI 0.99-5.28); p=0.053]. Survivin knockdown in SW-13 cell significantly increased the rate of apoptosis. Finally, spontaneous survivin-reactive T cells were detectable in 3 of 29 ACC patients. In conclusion, our data suggest that survivin could play an important role in the anti-apoptotic mechanisms in ACC and provide first hints that targeting survivin might be an interesting new therapeutic approach in this rare disease.

Abstract LB-155: A new syngeneic MYCN-overexpressing neuroblastoma mouse model and MYCN-DNA vaccine

Abstract High-level expression of MycN caused by amplification of the gene characterizes the malignant phenotype of neuroblastoma. Recent studies suggest that MYCN is a suitable target for immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN is not available to examine immunotherapeutic strategies in vivo. Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a MYCN-DNA vaccine. For this purpose, the murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector. Stable transfection was verified by real-time PCR and Western-Blot, revealing high expression levels of MYCN RNA and protein, respectively. Furthermore, a MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN vaccine effectively lysed C1300-MYCN +tet cells in cytotoxicity assays in contrast to wild type C1300 cells and lymphocytes from control mice. Lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells than lymphocytes from control mice. Interestingly, vaccine induced cytotoxic T lymphocytes also kill parental C1300 tumor cells which show low MYCN expression. We expect that this effect will be enhanced when MYCN-over expressing C1300 cells are employed in a similar cytotoxicity assay which will be reported at the meeting. In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an useful syngeneic mouse model for in vivo evaluation of MYCN directed immunotherapeutic strategies. 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 LB-155. doi:10.1158/1538-7445.AM2011-LB-155

Current Immunotherapeutic Approaches in Pancreatic Cancer

Pancreatic cancer is a highly aggressive and notoriously difficult to treat. As the vast majority of patients are diagnosed at advanced stage of the disease, only a small population is curative by surgical resection. Although gemcitabine-based chemotherapy is typically offered as standard of care, most patients do not survive longer than 6 months. Thus, new therapeutic approaches are needed. Pancreatic cancer cells that develop gemcitabine resistance would still be suitable targets for immunotherapy. Therefore, one promising treatment approach may be immunotherapy that is designed to target pancreatic-cancer-associated antigens. In this paper, we detail recent work in immunotherapy and the advances in concept of combination therapy of immunotherapy and chemotherapy. We offer our perspective on how to increase the clinical efficacy of immunotherapies for pancreatic cancer.

Abstract 4758: A new syngeneic MYCN-overexpressing neuroblastoma mouse model and MYCN-DNA vaccine

Abstract Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. High-level expression of MycN caused by amplification of the gene plays an important role in maintaining the malignant phenotype of NB and is the most significant marker for poor prognosis overall. Recent studies suggested that MYCN is a suitable target for immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN and is not available to examine immunotherapeutic strategies in vivo. Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a new MYCN-DNA vaccine. The murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector system T-RExTM (Invitrogen). Stable transfection was verified by real-time PCR and Western-Blot, revealing significant high expression levels of MYCN RNA and protein, respectively. Characterization of the murine MYCN expressing cells in vitro and in vivo will be reported. Furthermore, a new MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN minigen vaccine effectively lysed wild type C1300 cells in cytotoxicity assays in contrast to lymphocytes from control mice. Additionally, lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells, than lymphocytes from control mice, indicating the induction of cytotoxic T lymphocytes by the vaccine that actively kill even parental C1300 tumor cells with relatively low MYCN expression. We suspect that this effect will be enhanced when the newly established high MYCN-expressing C1300 cells are employed in a similar cytotoxicity assay. Results to support this hypothesis will be reported at the meeting. In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an important new syngeneic mouse model for in vivo evaluation of new immunotherapeutic strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4758.

Abstract 2323: Inhibition of in vitro growth of human mesothelioma cells by CSPG4-specific monoclonal antibodies

Abstract The membrane bound chondroitin sulphate protidoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), plays a role in tumor cell proliferation, growth and differentiation, as well as in angiogenesis and extracellular matrix remodeling. Contrary to the early reports which had described CSPG4 expression restricted to melanoma cells, this molecule is expressed by various types of malignant tumors. Because of its high expression on tumor cells, including cancer stem cells, and its restricted distribution in normal tissues, CSPG4 represents an attractive target for immunotherapy. The lack of effective conventional therapy and the paucity of suitable targets for immunotherapy in mesothelioma have prompted us to test whether CSPG4 is expressed by mesothelioma cells and mediates the anti-tumor activity of CSPG4-specific monoclonal antibodies (mAb). Immunohistochemical staining with mAb showed that CSPG4 was expressed on mesothelioma cells in 24 out of 40 MM biopsies with minimal expression on surrounding normal cells. Furthermore FACS analysis and western blots detected CSPG4 in all the 9 human MM cell lines tested and that the expression was induced on MM cells by their interactions with the extracellular matrix (ECM). To test the sensitivity of MM cells to the anti-tumor activity of CSPG4-specific mAb, 3 MM cell lines were incubated with mAb TP41.2 (final concentration 10ug/ml) for up to 72hrs. Cell proliferation was determined by MTT assay. Cells incubated with normal mouse IgG antibodies were used as controls. Addition of mAb TP41.2 to cells already attached to plates had no detectable effect on their proliferation. On the other hand incubation of MM cells with mAb TP41.2 before seeding in plates markedly inhibited their growth. The effect was time-dependent. These results were observed in all 3 cell lines and each cell lines was tested 3 times. The data suggest that CSPG4 promotes MM cell growth by mediating their interactions with ECM. This conclusion is corroborated by the growth of CSPG4-positive MM cells in serum-free medium in plates coated with collagen I, collagen IV or fibronectin. Furthermore CSPG4-specific mAb decreased the expression of the angiogenic growth factors VEGF and HEY1, as determined by RT-PCR, in MM cells. These results suggest that it may be worthwhile to test the hypothesis that CSPG4-specific mAb-based immunotherapy may represent a viable approach for the treatment of MM. 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 2323.

Sperm associated antigen 9 expression and humoral response in chronic myeloid leukemia

Early diagnosis and cure for patients with chronic myeloid leukemia (CML) exhibit significant clinical challenges because of the disease progression from chronic phase (CP) into a rapidly fatal blast crisis. Our earlier studies suggested an association of sperm associated antigen 9 (SPAG9) with various human malignancies. The present investigation revealed that SPAG9 mRNA and protein are expressed in CML patients (88%), in K562 and KCL-22 cells. Further, SPAG9 protein expression was also detected on cell surface suggesting that this molecule may be a suitable target for immunotherapy. Interestingly, 90% CML-CP patients showed humoral response against SPAG9, suggesting its important role in early diagnostic of CML-CP. Further investigation is warranted in establishing the potential of SPAG9 as a biomarker and immunotherapeutic target for early treatment of CML-CP patients.

The forkhead box M1 transcription factor as a candidate of target for anti‐cancer immunotherapy

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty‐three human FOXM1‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their ability to induce HLA‐A2‐restricted T cells in HLA‐A2 transgenic mice. FOXM1362‐370 (YLVPIQFPV), FOXM1373‐382 (SLVLQPSVKV), and FOXM1640‐649 (GLMDLSTTPL) peptides primed HLA‐A2‐restricted cytotoxic T lymphocytes (CTLs) in the HLA‐A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA‐A2‐positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA‐A2‐positive FOXM1‐transfectants by peptide‐induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.