Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial.

In this prospective, interventional phase 1 study for individuals with advanced sarcoma, we infused autologous HER2-specific chimeric antigen receptor T cells (HER2 CAR T cells) after lymphodepletion with fludarabine (Flu) ± cyclophosphamide (Cy): 1 × 108 T cells per m2 after Flu (cohort A) or Flu/Cy (cohort B) and 1 × 108 CAR+ T cells per m2 after Flu/Cy (cohort C). The primary outcome was assessment of safety of one dose of HER2 CAR T cells after lymphodepletion. Determination of antitumor responses was the secondary outcome. Thirteen individuals were treated in 14 enrollments, and seven received multiple infusions. HER2 CAR T cells expanded after 19 of 21 infusions. Nine of 12 individuals in cohorts A and B developed grade 1-2 cytokine release syndrome. Two individuals in cohort C experienced dose-limiting toxicity with grade 3-4 cytokine release syndrome. Antitumor activity was observed with clinical benefit in 50% of individuals treated. The tumor samples analyzed showed spatial heterogeneity of immune cells and clustering by sarcoma type and by treatment response. Our results affirm HER2 as a CAR T cell target and demonstrate the safety of this therapeutic approach in sarcoma. ClinicalTrials.gov registration: NCT00902044 .

Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma.

Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report.

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy.

BACKGROUND AIMS: EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1. METHODS: To investigate whether BARF1 is a potential T-cell immunotherapy target, we determined the frequency of BARF1-specific T-cell responses in the peripheral blood of EBV-seropositive healthy donor and patients with EBV-positive malignancies, mapped epitopes and evaluated the effector function of ex vivo-generated BARF1-specific T-cell lines. RESULTS: BARF1-specific T cells were present in the peripheral blood of 12/16 (75%) EBV-positive healthy donors and 13/20 (65%) patients with EBV-positive malignancies. Ex vivo expanded BARF1-specific T-cell lines contained CD4- and CD8-positive T-cell subpopulations, and we identified 23 BARF1 peptides, which encoded major histocompatibility complex class I- and/or II-restricted epitopes. Epitope mapping identified one human leukocyte antigen (HLA)-A*02-restricted epitope that was recognized by 50% of HLA-A*02, EBV-seropositive donors and one HLA-B*15(62)-restricted epitope. Exvivo expanded BARF1-specific T cells recognized and killed autologous, EBV-transformed lymphoblastoid cell lines and partially HLA-matched EBV-positive lymphoma cell lines. DISCUSSION: BARF1 should be considered as an immunotherapy target for EBV type II (and III) latency. Targeting BARF1, in addition to EBNA1, LMP1 and LMP2, has the potential to improve the efficacy of current T-cell immunotherapy approaches for these malignancies.

Estrogen receptor expression in chronic hepatitis C and hepatocellular carcinoma pathogenesis.

AIM: To investigate gender-specific liver estrogen receptor (ER) expression in normal subjects and patients with hepatitis C virus (HCV)-related cirrhosis and hepatocellular carcinoma (HCC). METHODS: Liver tissues from normal donors and patients diagnosed with HCV-related cirrhosis and HCV-related HCC were obtained from the NIH Liver Tissue and Cell Distribution System. The expression of ER subtypes, ERα and ERß, were evaluated by Western blotting and real-time RT-PCR. The subcellular distribution of ERα and ERß was further determined in nuclear and cytoplasmic tissue lysates along with the expression of inflammatory [activated NF-κB and IκB-kinase (IKK)] and oncogenic (cyclin D1) markers by Western blotting and immunohistochemistry. The expression of ERα and ERß was correlated with the expression of activated NF-κB, activated IKK and cyclin D1 by Spearman's correlation. RESULTS: Both ER subtypes were expressed in normal livers but male livers showed significantly higher expression of ERα than females (P < 0.05). We observed significantly higher mRNA expression of ERα in HCV-related HCC liver tissues as compared to normals (P < 0.05) and ERß in livers of HCV-related cirrhosis and HCV-related HCC subjects (P < 0.05). At the protein level, there was a significantly higher expression of nuclear ERα in livers of HCV-related HCC patients and nuclear ERß in HCV-related cirrhosis patients as compared to normals (P < 0.05). Furthermore, we observed a significantly higher expression of phosphorylated NF-κB and cyclin D1 in diseased livers (P < 0.05). There was a positive correlation between the expression of nuclear ER subtypes and nuclear cyclin D1 and a negative correlation between cytoplasmic ER subtypes and cytoplasmic phosphorylated IKK in HCV-related HCC livers. These findings suggest that dysregulated expression of ER subtypes following chronic HCV-infection may contribute to the progression of HCV-related cirrhosis to HCV-related HCC. CONCLUSION: Gender differences were observed in ERα expression in normal livers. Alterations in ER subtype expression observed in diseased livers may influence gender-related disparity in HCV-related pathogenesis.

HER2-Specific Chimeric Antigen Receptor-Modified Virus-Specific T Cells for Progressive Glioblastoma: A Phase 1 Dose-Escalation Trial.

IMPORTANCE: Glioblastoma is an incurable tumor, and the therapeutic options for patients are limited. OBJECTIVE: To determine whether the systemic administration of HER2-specific chimeric antigen receptor (CAR)-modified virus-specific T cells (VSTs) is safe and whether these cells have antiglioblastoma activity. DESIGN, SETTING, AND PARTICIPANTS: In this open-label phase 1 dose-escalation study conducted at Baylor College of Medicine, Houston Methodist Hospital, and Texas Children's Hospital, patients with progressive HER2-positive glioblastoma were enrolled between July 25, 2011, and April 21, 2014. The duration of follow-up was 10 weeks to 29 months (median, 8 months). INTERVENTIONS: Monotherapy with autologous VSTs specific for cytomegalovirus, Epstein-Barr virus, or adenovirus and genetically modified to express HER2-CARs with a CD28.ζ-signaling endodomain (HER2-CAR VSTs). MAIN OUTCOMES AND MEASURES: Primary end points were feasibility and safety. The key secondary end points were T-cell persistence and their antiglioblastoma activity. RESULTS: A total of 17 patients (8 females and 9 males; 10 patients ≥18 years [median age, 60 years; range, 30-69 years] and 7 patients <18 years [median age, 14 years; range, 10-17 years]) with progressive HER2-positive glioblastoma received 1 or more infusions of autologous HER2-CAR VSTs (1 × 106/m2 to 1 × 108/m2) without prior lymphodepletion. Infusions were well tolerated, with no dose-limiting toxic effects. HER2-CAR VSTs were detected in the peripheral blood for up to 12 months after the infusion by quantitative real-time polymerase chain reaction. Of 16 evaluable patients (9 adults and 7 children), 1 had a partial response for more than 9 months, 7 had stable disease for 8 weeks to 29 months, and 8 progressed after T-cell infusion. Three patients with stable disease are alive without any evidence of progression during 24 to 29 months of follow-up. For the entire study cohort, median overall survival was 11.1 months (95% CI, 4.1-27.2 months) from the first T-cell infusion and 24.5 months (95% CI, 17.2-34.6 months) from diagnosis. CONCLUSIONS AND RELEVANCE: Infusion of autologous HER2-CAR VSTs is safe and can be associated with clinical benefit for patients with progressive glioblastoma. Further evaluation of HER2-CAR VSTs in a phase 2b study is warranted as a single agent or in combination with other immunomodulatory approaches for glioblastoma.

Targeting EBV's Achilles' heel with antigen-specific T cells.

Evaluation of: Icheva V, Kayser S, Wolff D et al. Adoptive transfer of Epstein-Barr virus (EBV) nuclear antigen 1-specific T cells as treatment for EBV reactivation and lymphoproliferative disorders after allogeneic stem-cell transplantation. J. Clin. Oncol. 31(1), 39-48 (2013). Adoptive transfer of donor-derived EBV-specific T cells is an effective strategy for the prevention and treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD). However, the time-consuming process of EBV-specific T-cell generation using standard protocols has limited their broader use. Ex vivo IFN-γ capture assay is an attractive alternative for the rapid isolation of EBV-specific T cells. In the present study, Icheva et al. employ this method to rapidly isolate clinical-grade T cells that are specific for EBNA1. Adoptive transfer of EBNA1-specific T cells was safe and resulted in clinical benefit in 7 out of 10 patients with EBV-viremia and/or PTLD. Thus, T-cell therapy targeting a single EBV antigen, EBNA1, which is critical for EBV episome maintenance, may be sufficient for EBV-PTLD therapy in hematopoietic stem cells transplantation recipients.

Development of an ultrasensitive polymerase chain reaction-amplified immunoassay based on mycobacterial RD antigens: implications for the serodiagnosis of tuberculosis.

Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex-specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 10(7) times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA.

Status of bacterial colonization, Toll-like receptor expression and nuclear factor-kappa B activation in normal and diseased human livers.

Epidemiological data on bacterial translocation (BT), colonization and inflammation in normal human livers is lacking. In this study we investigated the status of bacterial colonization and inflammation in the normal, cirrhotic primary biliary cirrhosis (PBC), and nonalcoholic steatohepatitis (NASH) human liver tissues. Comparatively normal livers showed increased bacterial colonization than PBC and NASH. We analyzed mRNA levels of Toll-like receptors (TLR) 2 and TLR4, and protein levels of TLR4. Phosphorylated IKKα (pIKKα) protein estimation served as a marker for nuclear factor-kappa B (NF-κB) activation. In spite of the increased bacterial colonization in normal liver tissues, lower levels of TLR2/4 mRNA and TLR4 and pIKKα proteins were found compared to PBC and NASH indicating the maintenance of suppressed inflammation and immune tolerance in normal livers. To our knowledge, this is the first clinical evidence showing suppressed inflammation despite bacterial colonization in normal human livers thus maintaining liver immune homeostasis.

Evaluation of Mycobacterium tuberculosis specific RD antigens for delayed type hypersensitivity responses in guinea pig.

Tuberculin skin test (TST), an age old method is based on measuring delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). However, inspite of simplicity, ease and cost effectiveness, the usefulness of PPD test is limited due to its inability to distinguish among a protective immune response, latent infection and active tuberculosis disease. On the other hand, a skin test based on RD antigens would add advantages of a high specificity of antigens with the logistics of a skin test. However, except few reports, in vivo data of intradermal use of RD antigens for skin testing is limited. Therefore, in the present study, four M. tuberculosis (Mtb) specific antigens (ESAT6, CFP10, CFP21 and MPT64) were evaluated for their diagnostic utility based on DTH response. These antigens alone and their multiple combinations induced strong DTH response in Mtb infected guinea pigs and the response was negligible in BCG vaccinated and sham immunized animals.

Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for tuberculosis.

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity.

Role of sex steroid receptors in pathobiology of hepatocellular carcinoma.

The striking gender disparity observed in the incidence of hepatocellular carcinoma (HCC) suggests an important role of sex hormones in HCC pathogenesis. Though the studies began as early as in 1980s, the precise role of sex hormones and the significance of their receptors in HCC still remain poorly understood and perhaps contribute to current controversies about the potential use of hormonal therapy in HCC. A comprehensive review of the existing literature revealed several shortcomings associated with the studies on estrogen receptor (ER) and androgen receptor (AR) in normal liver and HCC. These shortcomings include the use of less sensitive receptor ligand binding assays and immunohistochemistry studies for ERalpha alone until 1996 when ERbeta isoform was identified. The animal models of HCC utilized for studies were primarily based on chemical-induced hepatocarcinogenesis with less similarity to virus-induced HCC pathogenesis. However, recent in vitro studies in hepatoma cells provide newer insights for hormonal regulation of key cellular processes including interaction of ER and AR with viral proteins. In light of the above facts, there is an urgent need for a detailed investigation of sex hormones and their receptors in normal liver and HCC. In this review, we systematically present the information currently available on androgens, estrogens and their receptors in normal liver and HCC obtained from in vitro, in vivo experimental models and clinical studies. This information will direct future basic and clinical research to bridge the gap in knowledge to explore the therapeutic potential of hormonal therapy in HCC.

Supplementation with RD antigens enhances the protective efficacy of BCG in tuberculous mice.

Different combinations of ESAT-6, CFP-10, CFP-21, MPT-64, encoded by RD1 and RD2 of Mycobacterium tuberculosis were evaluated on the basis of antigenicity in PPD positive TB contacts and immunogenicity in C57BL/6J mice immunized with the combination of all four RD antigens. The peripheral blood mononuclear cells of TB contacts showed maximum recognition in response to the combination of ESAT-6+MPT-64 in terms of predominant lymphoproliferation, IFN-gamma levels and the number of responders. On the contrary, the combination of ESAT-6+CFP-21+MPT-64 was found to be most immunogenic based on both T-cell and antibody responses in immunized mice. Prophylactic potential of the selected combinations was assessed as supplementation vaccines to BCG against intravenous challenge with M. tuberculosis in mice. BCG supplementation with the selected combinations resulted in significantly greater protection as compared to BCG alone against experimental tuberculosis and thus appears to be a promising approach to enhance the protective efficacy of the existing vaccine.

Tuberculosis subunit vaccine design: the conflict of antigenicity and immunogenicity.

The attempts to find an effective antituberculous subunit vaccine are based on the assumption that it must drive a Th1 response. In the absence of effective correlates of protection, a vast array of mycobacterial components are being evaluated worldwide either on the basis of their ability to be recognized by T lymphocytes in in vitro assays during early stage of animal or human infection (antigenicity) or their capacity to induce T cell response following immunization in animal models (immunogenicity). The putative vaccine candidates selected using either of these strategies are then subjected to challenge studies in different animal models to evaluate the protective efficacy. Here we review the outcome of this current scheme of selection of vaccine candidates using an 'antigenicity' or 'immunogenicity' criterion on the actual protective efficacy observed in experimental animal models. The possible implications for the success of some of the leading vaccine candidates in clinical trials will also be discussed.

Peripheral blood and pleural fluid mononuclear cell responses to low-molecular-mass secretory polypeptides of Mycobacterium tuberculosis in human models of immunity to tuberculosis.

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H(37)Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.