Target For Immunotherapeutic Approaches Research Articles

High CD49a+ NK cell infiltrate is associated with poor clinical outcomes in Hepatocellular Carcinoma

NK cells infiltrating Hepatocellular Carcinoma (HCC) may express residency markers such as Integrin Subunit Alpha 1 (CD49a) that have been associated with nurturing functions in the decidua, and characterized by the production of angiogenic factors as well as loss of cytotoxicity. CIBERSORT, a computational analysis method for quantifying cell fractions from bulk tissue gene expression profiles, was used to estimate the infiltrating immune cell composition of the tumor microenvironment from gene expression profiles of a large cohort of 225 HCCs in the public GEO database. Decidual-like CD49a+ NK cells, in addition to another 22 immune cell populations, were characterized and thoroughly investigated so that HCC cell heterogeneity in a large cohort of 225 HCCs from the public GEO database could be studied. An inverse correlation of the expression of CD49a+ NK-cells and CD8+ T-cells suggested a negative association with clinical outcomes. This result was confirmed in a further validation cohort of 100 HCC patients from The Cancer Genome Atlas, Liver Hepatocellular Carcinoma (TCGA-LIHC). Cox regression analysis did not identify CD49a+ cells as a variable independently associated with survival. However, a more abundant infiltrate of this subset was present in patients at a more advanced pathological and clinical HCC stage. In conclusion, we found that NK cells, with a decidual-like gene expression profile, are enriched in HCC, and their abundance increases not only in tumor size but also at advanced stages of the disease suggesting that these cells play a role in tumor growth. For this reason, these NK cells may represent a possible new target for immunotherapeutic approaches in HCC.

The Immunological Facets Of Chondrocytes In Osteoarthritis: A Narrative Review.

Osteoarthritis (OA) is a disease in which the pathogenesis affects the joint and its surrounding tissues. Cartilage degeneration is the main hallmark of OA and chondrocytes within the cartilage regulate matrix production and degradation. In OA patients and animal models of OA, the pathology of the disease relates to disequilibrium between anabolic and catabolic states of the cartilage. Moreover, chondrocyte phenotype and function are also immunologically altered. Under inflammatory conditions, chondrocytes increase production levels of inflammatory cytokines and cartilage-degrading enzymes, which further drive cartilage destruction. Chondrocytes also have an innate immune function and respond to DAMPs and cartilage fragments via innate immune receptors. In addition, chondrocytes play a role in adaptive immune responses by acting as antigen presenting cells and presenting cartilaginous antigens to T cells. Indirectly, chondrocytes are stimulated by pathogen-associated molecular patterns (PAMPs) present in the joints, a result of the microbiota in the host. Chondrocytes have both direct and indirect relationships with immune cells and the immune compartment of OA patients. Therefore, chondrocytes serve as a target for immunotherapeutic approaches in OA. In this narrative review, we cover the aforementioned immune-related aspects of chondrocytes in OA.

Artificial intelligence: accelerating translational cell therapy development through computational methods for neoepitope discovery

A surge in the efforts to identify cancer-specific antigens as targets for immunotherapeutic approaches has characterized the past few decades. However, the clinical use of tumor-associated antigens has been mostly limited to cancer/testis antigens, restricting its application to certain cancers and limiting the weak immune response elicited by self-antigens. Neoantigens resulting from somatic mutations in cancer cells represent an alternative immunotherapy target voided of such limitations. Similarly, cancer-specific protein isoforms or mutational hotspot antigens provide the advantage of tumor tissue specificity coupled with low thymic selection and central tolerance. Choosing the best antigenic determinants is critical for successful adoptive cell therapy; the availability of algorithms for clinical data mining and to predict the most immunogenic epitopes is thus a powerful promise for the rapid advancement of cancer medicine. The state of the art of computational neo-antigen research is discussed here.

Evaluation of the PBP2 transglycosylase region of Staphylococcus aureus as a target for immunotherapeutic approaches

Infections caused by Staphylococcus aureus are increasingly prevalent, and treatment has become more difficult due to the emergence of strains that are resistant to multiple drugs, such as methicillin-resistant Staphylococcus aureus (MRSA). Penicillin-binding proteins (PBPs) are essential enzymes in peptidoglycan biosynthesis. Only found in bacteria, they are an excellent target for the development of bacterial control strategies. S. aureus has 4 PBPs, and only PBP2 has transglycosylation activity, making it a good model to evaluate whether the inactivation of the transglycosylase domain (PBP2t) could lead to bacterial death. (His6)-tagged PBP2t was purified from the E. coli cell lysate using Ni-charged resin, and ELISA and immunoblotting assays demonstrated that PBP2t is immunogenic. Flow cytometry analysis was performed to verify the binding of polyclonal antibodies to the bacterial cell surface. In order to verify the ability to provide protection, immunized mice were challenged with a sublethal dose of MRSA, and the bacterial loads in kidneys and spleen were evaluated. A reduction of 2–2.5 logs was seen in organs from immunized mice compared with the negative controls in two independent assays (p < 0.01). Our results demonstrate that the PBP2t is a promising target for the development of novel antimicrobial strategies, but further testing should be performed to validate the protection conferred by immunization with this protein.

Bispecific NKG2D-CD3 and NKG2D-CD16 Fusion Proteins as Novel Treatment Option in Advanced Soft Tissue Sarcomas.

Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.

In silico analysis reveals EP300 as a panCancer inhibitor of anti-tumor immune response via metabolic modulation

The tumor immune microenvironment (TIME) of head and neck squamous cell carcinomas (HNSCC) and other solid malignancies is a key determinant of therapy response and prognosis. Among other factors, it is shaped by the tumor mutational burden and defects in DNA repair enzymes. Based on the TCGA database we aimed to define specific, altered genes associated with different TIME types, which might represent new predictive markers or targets for immuno-therapeutic approaches. The HNSCC cohort of the TCGA database was used to define 3 TIME types (immune-activated, immune-suppressed, immune-absent) according to expression of immune-related genes. Mutation frequencies were correlated to the 3 TIME types. Overall survival was best in the immune-activated group. 9 genes were significantly differentially mutated in the 3 TIME types with strongest differences for TP53 and the histone-acetyltransferase EP300. Mutations in EP300 correlated with an immune-activated TIME. In panCancer analyses anti-tumor immune activity was increased in EP300 mutated esophageal, stomach and prostate cancers. Downregulation of EP300 gene expression was associated with higher anti-tumor immunity in most solid malignancies. Since EP300 is a promoter of glycolysis, which negatively affects anti-tumor immune response, we analyzed the association of EP300 with tumor metabolism. PanCancer tumor metabolism was strongly shifted towards oxidative phosphorylation in EP300 downregulated tumors. In silico analyses of of publicly available in vitro data showed a decrease of glycolysis-associated genes after treatment with the EP300 inhibitor C646. Our study reveals associations of specific gene alterations with different TIME types. In detail, we defined EP300 as a panCancer inhibitor of the TIME most likely via metabolic modulation. In this context EP300 represents a promising predictive biomarker and an immuno-therapeutic target.

NKG2E comprises an immunogenic peptide, derived from an alu-retrotransposon: An attractive novel target for immunotherapeutic approaches

Abstract Background Endogenous retroelements are a main source of targetable tumor-specific antigens that have the potential to augment host adaptive antitumor responses. KLRC3 gene encodes for the NKG2E and NKG2H isoforms of NKG2 family of transmembrane receptors. Current data support that NKG2E represents the main protein isoform. Methods Genome, mobilome and molecular evolutionarily analysis were performed through the corresponding tracks of UCSC Genome Browser Database. NKG2E protein expression data were downloaded from the Human Protein Atlas. NetMHCII 2.3 was used to predict MHC class II binding scores. Results In normal tissues, NKG2E protein is scarcely expressed in a rare subset of immune cells in the intestinal tract and lymphoid tissue. Vice versa, in tumor tissues NKG2E expression is detected in > 75% of tumor cells in cases of liver, breast and ovarian cancer as well as in 25-75% of tumor cells in cases of thyroid, stomach and prostate cancer. NKG2E protein is encoded by the long isoform of KLRC3 gene. The last exon of this mRNA is derived via an Alu-element exonization, which provides the last 14 aa of the coding region. Previous analysis showed that the 14 aa Alu-peptide is hydrophobic and impels the intracellular retention of NKG2E protein thereby impeding its function as a transmembrane receptor. A previous study revealed the presence of highly-specific IgG autoantibodies against the Alu-peptide in a disease known to stimulate Alu RNAs overexpression, but not in healthy individuals. The latter signifies not only a high immunogenicity but also that the host is mostly immunologically ignorant rather than tolerant of the Alu-peptide. Accordingly NetMHCII analysis identifies the Alu-peptide as a HLA-DRB1_0103 and -DRB1_1101 high binder. Conclusion NKG2E protein represents an aberrant molecule that is misexpressed in multiple cancers, including immunologically “cold” tumor types. Characterization of the rare immune cells producing the aberrant NKG2E molecule in normal tissues warrants further research. Subsequent studies could inquire into a TCD4-specific TCR usage that would allow for the inoculation of cancer patients with autologous CD4-T cells reactive against the Alu-peptide. Legal entity responsible for the study Spyros I. Papamichos. Funding Has not received any funding. Disclosure The author has declared no conflicts of interest.

Abstract 2306: CAR-T cell therapies targeting aspartyl β-hydroxylase (ASPH)

Abstract Aspartate β-hydroxylase (ASPH) is a unique cancer target that is expressed exclusively on the surface of cancer cells. It is a type II transmembrane protein predominantly expressed during embryogenesis where it promotes cell migration for organ development, but has very low expression in healthy adult tissue, and is localized to the intracellular compartment of the endoplasmic reticulum. However, re-expression and translocation to the tumor cell surface has been detected in more than 20 different types of cancers, with expression levels inversely correlated with disease prognosis. Promising phase I clinical trial results have been achieved with our ASPH-targeted vaccine, SNS-301, in the treatment of biochemical recurrence of prostate cancer (reported previously: Nordquist, et al. (2018) J Clin Oncol. 36, suppl; abstr e15166; Nordquist, et al. Annals of Oncology, 29, suppl_8, 1 October 2018, mdy279.404). This study demonstrated reductions in disease-specific biomarkers (i.e. PSA) which correlated to the breaking of immune self-tolerance as indicated by stimulation of both cellular and humoral ASPH-specific immune responses. Importantly the vaccine was well tolerated with minimal numbers of adverse events or off-target effects. Thus, ASPH represents a promising target for immunotherapeutic approaches to human cancers. Here we present studies that we have initiated towards the development of ASPH-targeted CAR (chimeric antigen receptor)-T cell therapies. A series of ASPH-targeting chimeric antigen receptor constructs have been produced which combine various ASPH-specific scFv domains and either the 4-1BB or CD28 co-stimulatory domains. The ASPH-specific scFv explored include several scFv that we previously isolated from a fully human non-immune scFv library displayed on the surface of yeast and targeted at different epitopes within the extracellular domain of ASPH as well as several affinity matured variations of these scFv (Yeung, et al. (2007) Human Antibodies 16, 163-76). All constructs were incorporated into a lentiviral vector which was used to transfect human CD4+ and CD8+ T-cells. CAR expression and affinities were determined by flow cytometry using a fluorescently labeled antigen. Cell killing efficacy and cytokine responses of the resulting CAR-T cells were observed in co-culture experiments with a series of ASPH-expressing human cancer cell lines allowing for the ranking of the CAR constructs in terms of overall extent of immune stimulation ex vivo. It is our expectation that the unique features of the ASPH tumor specific antigen will help overcome one of the major hurdles in T-cell therapy: the identification of antigens that permit effective targeting of tumors in the absence of non-tolerable and/or off-target toxicities to essential tissues and organs. Experiments to further characterize ASPH-targeted CAR-T cells are ongoing with the goal of moving these promising therapeutics into clinic. Citation Format: Thomas Thisted, Kanam Malhotra, Michael S. Lebowitz, Hossein Ghanbari. CAR-T cell therapies targeting aspartyl β-hydroxylase (ASPH) [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 2306.

Dysregulation of helper T lymphocytes in esophageal squamous cell carcinoma (ESCC) patients is highly associated with aberrant production of miR-21.

Dysregulation of helper T (Th) cell subsets has been contributed to the initiation and propagation of esophageal squamous cell carcinoma (ESCC). Different microRNAs (miRNAs) have been reported to control the development and functions of tumor-associated immune cells in ESCC. Here, we aimed to assess the IL-10, TGF-β, IFN-γ, and IL-17a-producing CD3+CD8- T cells in association whit miR-21, miR-29b, miR-106a, and miR-155 expression in ESCC patients. A total of 34 ESCC patients including 12 newly diagnosed (ND) and 22 under-treatment (UT) cases and also34 age-matched healthy donors were enrolled. Flow cytometric characterization of stimulated T cells was performed by staining of the cells with fluorescent conjugated specific anti-human CD3 and CD8 cell surface markers as well as IL-17a, IFN-γ, IL-10, and TGF-β intracytoplasmic cytokines. Circulating RNA was extracted from the plasma, and qRT-PCR was used to evaluate the expression of microRNAs. TGF-β plasma levels were also assessed by ELISA. Results showed that the frequency of Th cells was significantly reduced in patients. A significant increase in Treg as well as Th17 cells population in both patient subgroups was observed. ND patients showed elevated level of Th1 cells and IL-10. However the mean expression of IFN-γ was significantly decreased in Th cells. We also detected higher level of miR-21 in the ESCC patients which was significantly correlated with different subsets of Th cells. Our findings revealed that immune response relatedto the Th cells is highly impaired in ESCC patients. Association between miR-21 and Th subsets could be correlated with the impairment of anti-tumor immunity and ESCC pathogenesis, which could be potentially used as an important target for immunotherapeutic approaches.

Nedd4-Binding Protein 1 and TNFAIP3-Interacting Protein 1 Control MHC-1 Display in Neuroblastoma.

: Neuroblastoma is the second most common tumor in children. The cause of neuroblastoma is thought to lie in aberrant development of embryonic neural crest cells and is accompanied by low MHC-1 expression and suppression of the NF-κB transcription factor, thereby gearing cells toward escape from immunosurveillance. Here, we assess regulation of the MHC-1 gene in neuroblastoma to enhance its immunogenic potential for therapeutic T-cell targeting. A genome-wide CRISPR screen identified N4BP1 and TNIP1 as inhibitory factors of NF-κB-mediated MHC-1 expression in neuroblastoma. Patients with advanced stage neuroblastoma who expressed high levels of TNIP1 and N4BP1 exhibited worse overall survival. Depletion of N4BP1 or TNIP1 increased NF-κB and MHC-1 expression and stimulated recognition by antigen-specific CD8+ T cells. We confirmed that TNIP1 inhibited canonical NF-κB member RelA by preventing activation of the RelA/p50 NF-κB dimer. Furthermore, N4BP1 inhibited both canonical and noncanonical NF-κB through binding of deubiquitinating enzyme CEZANNE, resulting in stabilization of TRAF3 and degradation of NF-κB-inducing kinase NIK. These data suggest that N4BP1/CEZANNE or TNIP1 may be candidate targets for immunotherapy in neuroblastoma tumors and should lift NF-κB suppression, thereby triggering increased peptide/MHC1-mediated tumor reactivity to enhance therapeutic T-cell targeting. SIGNIFICANCE: Aberrant regulation of NF-κB and MHC-1 in neuroblastoma tumors provides new targets for immunotherapeutic approaches against neuroblastoma.

CD38: A Target for Immunotherapeutic Approaches in Multiple Myeloma.

Multiple Myeloma (MM) is a hematological cancer characterized by proliferation of malignant plasma cells in the bone marrow (BM). MM represents the second most frequent hematological malignancy, accounting 1% of all cancer and 13% of hematological tumors, with ~9,000 new cases per year. Patients with monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic smoldering MM (SMM) usually evolve to active MM in the presence of increased tumor burden, symptoms and organ damage. Despite the role of high dose chemotherapy in combination with autologous stem cell transplantation and the introduction of new treatments, the prognosis of MM patients is still poor, and novel therapeutic approaches have been tested in the last years, including new immunomodulatory drugs, proteasome inhibitors and monoclonal antibodies (mAbs). CD38 is a glycoprotein with ectoenzymatic functions, which is expressed on plasma cells and other lymphoid and myeloid cell populations. Since its expression is very high and uniform on myeloma cells, CD38 is a good target for novel therapeutic strategies. Among them, immunotherapy represents a promising approach. Here, we summarized recent findings regarding CD38-targeted immunotherapy of MM in pre-clinical models and clinical trials, including (i) mAbs (daratumumab and isatuximab), (ii) radioimmunotherapy, and (iii) adoptive cell therapy, using chimeric antigen receptor (CAR)-transfected T cells specific for CD38. Finally, we discussed the efficacy and possible limitations of these therapeutic approaches for MM patients.

HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors

Capture of retroviral envelope genes is likely to have played a role in the emergence of placental mammals, with evidence for multiple, reiterated, and independent capture events occurring in mammals, and be responsible for the diversity of present day placental structures. Here, we uncover a full-length endogenous retrovirus envelope protein, dubbed HEMO [human endogenous MER34 (medium-reiteration-frequency-family-34) ORF], with unprecedented characteristics, because it is actively shed in the blood circulation in humans via specific cleavage of the precursor envelope protein upstream of the transmembrane domain. At variance with previously identified retroviral envelope genes, its encoding gene is found to be transcribed from a unique CpG-rich promoter not related to a retroviral LTR, with sites of expression including the placenta as well as other tissues and rather unexpectedly, stem cells as well as reprogrammed induced pluripotent stem cells (iPSCs), where the protein can also be detected. We provide evidence that the associated retroviral capture event most probably occurred >100 Mya before the split of Laurasiatheria and Euarchontoglires, with the identified retroviral envelope gene encoding a full-length protein in all simians under purifying selection and with similar shedding capacity. Finally, a comprehensive screen of the expression of the gene discloses high transcript levels in several tumor tissues, such as germ cell, breast, and ovarian tumors, with in the latter case, evidence for a histotype dependence and specific protein expression in clear-cell carcinoma. Altogether, the identified protein could constitute a "stemness marker" of the normal cell and a possible target for immunotherapeutic approaches in tumors.

CD70 as a critical mediator of tumor progression and immunosuppression in gliomas.

18 Background: For malignant gliomas, current treatment modalities are rarely curative, necessitating development of novel therapies. Cancer immunotherapy has represented one of the most promising new treatment strategies for patients with gliomas based on the evidence that patients with brain tumors are able to mount immune responses against the autologous tumors. However, very few tumor specific targets have been discovered in this malignancy. Thus, identifying clinical useful targets for immunotherapeutic approaches is desperately needed. Methods: CD70 expression was tested from primary GBM and low grade gliomas patient tissues. CD70 gene expression and clinical outcomes were culled from TCGA datasets. CD70 inducing CD8 T cells death was performed by flowcytometry. Results: We demonstrate that CD70, a member of the TNF ligand family, was constitutively overexpressed by primary IDH-wild-type LGG and GBMs with mesenchymal gene signatures. Elevated CD70 expression was also found in recurrent tumors and correlated with tumor progression and poor survival outcome in LGGs and GBMs. CD70 was shown to be directly involved in tumor-chemokine production and associated with sustained T regulatory cells in tumor. Importantly, CD70 played a role inducing CD8+T- specific cell death via engagement of the EREG-EGFR axis in glioma. Conclusions: CD70 is a multi-pronged modulator of immunosuppression in gliomas and enhances tumor progression.

Cancer-Testis Antigens: A Novel Group of Tumor Biomarkers in Ovarian Cancers

Context: Ovarian cancer is the most fatal gynecological malignancy with no effective screening strategy for early detection. As most cases are being detected in advance stages, conventional therapies are not beneficial for the majority of patients. Cancer-testis antigens (CTAs) are a group of tumor associated antigens with specific expression pattern in cancers which potentiate them for application as cancer biomarkers and targets for immunotherapy. Evidence Acquisition: We performed a computerized search of the MEDLINE/PUBMED databases with key words: ovarian cancer, cancer-testis antigen, biomarker and immunotherapy. Results: Thirty five CTAs have been shown to be expressed in ovarian cancer. At least 13 of them have been shown to elicit immune responses in different studies. The pattern of expression for some of them may facilitate molecular classification of different histologic classes of ovarian cancer. In addition, some CTAs such as NY-ESO-1 and MAGE have been used as targets for immunotherapeutic approaches with promising results. Conclusions: The expression pattern of CTAs in ovarian cancer and the preliminary results of clinical trials indicate that CTAs can be used as targets for immunotherapy of ovarian cancer patients.

Wilms´Tumor 1 Protein Is Highly Expressed on Malignant Plasma Cells and Provides a Novel Target for Immunotherapeutic Approaches

Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect.Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter.Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years.Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. DisclosuresO'Reilly:Atara Biotherapeutics: Research Funding.

Abstract 2479: Obinutuzumab compared to Rituximab significantly enhances cell death, antibody dependent cytotoxicity (ADCC) and improves overall survival against CD20+ rituximab-sensitive/-resistant Burkitt Lymphoma (BL) and precursor Lymphoblastic L

Abstract Background: CD20, expressed on normal and malignant B-cells, has proven to be a useful target for immunotherapeutic approaches in hematological malignancies. (Dalle, S et al. Mol. Can. Ther., 2011) Patients who relapse with CD20+ BL/ B-ALL have a dismal prognosis, often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. Blood, 2007, Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013,Goldman/Cairo et al. Leukemia, 2013). Obinutuzumab, a novel glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Can Res, 2013), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant/sensitive BL and pre-B-ALL tumor targets in-vitro/ in-vivo in xenografted NSG mice. Methods: Raji (CD20+), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab (Hoffmann La Roche (Switzerland)), and/or RTX for 48 hrs. Cell death was evaluated by staining with Ann.V/7AAD by flow-cytometry. ADCC were performed with K562-IL-15-41BBL expanded NK cells at 20:1E: T ratio. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab (10 and 30 mg/kg), and RTX (30 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH and U698M cells at 5×106 tumor cells/mouse. Mice were treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Results: Obinutuzumab vs. RTX (100 μg/ml), significantly enhanced cell death in Raji, 35.6 ± 3.1 vs. 25.1± 2.0% (p = 0.001), Raji2R, 18.2 ± 0.9 vs. 7.8 ± 2.4% (p = 0.001), Raji4RH, 19.7 ± 2.2 vs. 7.97 ± 3.4% (p = 0.001) and U698-M 47.3 ± 4.9 vs 23.2 ± 0.50%, (p = 0.001). Obinutuzumab vs RTX also elicited a significant increase in ADCC in expanded NK cells, Raji 73.8± 8.1% vs 56.81± 4.6% (p = 0.001), Raji-2R, 38.0 ± 2.0 vs 21.6 ± 1.2% (p = 0.0001), Raji-4RH 40.0±1.6% vs 0.5±1.1%, (p = 0.001), and U-698-M 70.0±6% vs. 45.56± 0.1%, (p = 0.001). Further, overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX; Raji (p = 0.05), Raji4RH (p = 0.024) and U698-M (p = 0.03). Conclusion: Obinutuzumab significantly enhances cell death and NK mediated ADCC in RTX sensitive/ resistant CD20+ BL and pre-B-ALL vs. RTX. Furthermore, obinutuzumanb significantly increased survival and decreased tumor burden in BL and Pre-B-ALL xenografts compared to an equal dose of RTX. Citation Format: Aradhana Awasthi Tiwari, Janet Ayello, Carmella Vandeven, Mona Elmacken, Matthew J. Barth, Christian Klein, Mitchell S. Cairo. Obinutuzumab compared to Rituximab significantly enhances cell death, antibody dependent cytotoxicity (ADCC) and improves overall survival against CD20+ rituximab-sensitive/-resistant Burkitt Lymphoma (BL) and precursor Lymphoblastic L [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2015-2479

Upregulated expression of NKG2D and its ligands give potential therapeutic targets for patients with thymoma.

The activating receptor NKG2D (natural killer group 2, member D) of natural killer (NK) cells promotes tumor immune surveillance by targeting ligands selectively induced on cancer cells, and thus having an important role in antitumor immune response. Because these ligands are not widely expressed on healthy adult tissue, NKG2D ligands may present as useful target for immunotherapeutic approaches in cancer. In this study, to elucidate the role of NKG2D-NKG2D ligand interaction in thymoma tissues and to evaluate the potential role of NKG2D ligands as therapeutic target for thymoma, we examined the expression of NKG2D and its specific ligands: MICA (major histocompatibility complex class I chain-related protein A), MICB (major histocompatibility complex class I chain-related protein B) and ULBP (UL16-binding protein) in 36 thymomas (6 subtype A, 6 subtype AB, 8 subtype B1, 5 subtype B2, 6 subtype B3 and 5 subtype C), 15 thymic atrophy and 8 thymic hyperplasia by immunohistochemistry and reverse transcription-real-time-PCR methods. We demonstrated that both mRNA and protein levels of NKG2D, MICA, MICB and ULBP were upregulated in six types of thymomas compared with those in atrophic thymus or proliferating thymus. Furthermore, the NKG2D ligands were found to be frequently coexpressed on thymoma cells. Furthermore, the expression of MICA, MICB and ULBP in subtype C was higher compared with those in subtype A, AB, B1, B2 and B3. Thus, we concluded that high expressions of NKG2D, MICA, MICB and ULBP1 were shown in patients with thymoma, and this may enhance the recognition function of NK cells to eliminate tumor cells. MICA, MICB and ULBP presented an attractive target for thymoma therapy. The abnormal expression of NKG2D, MICA, MICB and ULBP1 can provide us with evidence of the occurrence of thymoma and could also be used as a target in the treatment of thymoma.

A Comprehensive Expression Analysis of Cancer Testis Antigens in Head and Neck Squamous Cell Carcinoma Revels MAGEA3/6 as a Marker for Recurrence.

Despite significant advances in the treatment of head and neck squamous cell carcinoma (HNSCC), the survival rate has not changed in the last decades. Therefore, the development of novel therapeutic strategies is pursued. Cancer-testis antigens (CTA) are strong immunogenic proteins with a tumor-restricted expression pattern, and are considered ideal targets for tumor-specific immunotherapeutic approaches. In this study, using an in silico approach, we selected, among 139 previously described CTA, candidates to be evaluated in 89 HNSCC and 20 normal mucosa samples. SPANX-CD (71.9%), MAGEB2 (44.9%), MAGEA1 (44.9%), MAGEB6 (32.6%), and CXORF48 (27.0%) were found frequently expressed in HNSCC, and over 85% of the tumors expressed at least one of these five CTAs. The mRNA positivity of CXORF48, MAGEB6, and CRISP2 presented significant associations with recognized clinical features for poor outcome. Furthermore, MAGEA3/6 positivity was associated with significantly better disease-free survival (DFS, P = 0.014), and the expression of this antigen was shown to be an independent prognostic factor for tumor recurrence. In conclusion, one of five selected CTAs is expressed in at least 85% of the HNSCCs, suggesting a possible usage as target for immunotherapeutic approaches, and the mRNA-positivity for MAGEA3/6 is shown to be an independent marker for DFS.

Wilms’ tumor 1 protein is highly expressed on malignant plasma cells and provides a novel target for immunotherapeutic approaches

Abstract Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect. Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter. Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years. Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. Disclosures O'Reilly: Atara Biotherapeutics: Research Funding.

IT-28 * VACCINATION AGAINST EPIDERMAL GROWTH FACTOR RECEPTOR VARIANT III IN GLIOBLASTOMA: THE RINDOPEPIMUT COMPASSIONATE USE EXPERIENCE

The tumor-specific epidermal growth factor receptor variant III mutation (EGFRvIII) is widely expressed in glioblastoma and thus represents an attractive target for immunotherapeutic approaches. The investigational vaccine rindopepimut is an EGFRvIII peptide sequence conjugated to keyhole limpet hemocyanin and is administered intradermally with GM-CSF. Previous single-arm studies of rindopepimut in newly diagnosed, resected, EGFRvIII+ glioblastoma have shown encouraging PFS and OS. This compassionate use program provided rindopepimut to 61 EGFRvIII+ glioblastoma patients who were ineligible for ongoing clinical trials. Data are currently available on 42 patients, 12 (29%) with newly diagnosed glioblastoma (resected or inoperable) and 30 (71%) with recurrent disease. MGMT methylation was seen in 9/17, while all tested patients were negative for IDH1 (12/12) and IDH2 (7/7). Median age was 53 years (15-70) and median time from diagnosis was 14.5 months (2.7-58.7). Rindopepimut, administered in combination with temozolomide (57%), bevacizumab (57%), and/or other (17%), was well tolerated, with frequent mild injection site reactions and one potentially treatment-related, serious event of cerebral edema. Median peak rindopepimut-induced anti-EGFRvIII titer was 1:1,200 ( 50% shrinkage in measurable disease) was observed in six patients receiving rindopepimut with other agents. One inoperable glioblastoma patient experienced a CR during treatment with rindopepimut, erlotinib, temozolomide, and bevacizumab, and has continued rindopepimut for >5 years without significant toxicity or disease recurrence. Biopsy at recurrence showed EGFRvIII was eliminated in a patient who received rindopepimut and temozolomide for ∼9 months. In patients with newly diagnosed and recurrent glioblastoma, respectively, median PFS was 9.1 and 2.5 months, and median OS was 15.7 and 8.7 months from first vaccination. In conclusion, rindopepimut in combination with various anticancer therapies resulted in robust anti-EGFRvIII humoral response with minimal toxicity. PFS and OS appear promising in this heterogeneous, poor prognosis population.