A phase I dose escalation trial of MAGE-A3- and HPV16-specific peptide immunomodulatory vaccines in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN).

BACKGROUND: We conducted a phase I dose escalation study to evaluate the safety and immunologic response to peptide immunomodulatory vaccines GL-0810 (HPV16) and GL-0817 (MAGE-A3) in HPV16 and MAGE-A3-positive RM-SCCHN patients, respectively. METHODS: Three dose levels (500, 1,000, and 1,500 µg) of GL-0810 or GL-0817 with adjuvants Montanide (1.2 ml) and GM-CSF (100 µg/m2) were administered subcutaneously q2 weeks for a total of four vaccinations in HPV16 and MAGE-A3-positive RM-SCCHN patients, respectively. RESULTS: Nine and seven patients were enrolled in the HPV16 and MAGE-A3 cohorts, respectively. No dose-limiting toxicities were observed, and toxicity was predominantly local and grade 1 (erythema, pain, and itching at the injection site). In those patients who received all four vaccinations, 80 % (4/5) of the HPV16 cohort and 67 % (4/6) of the MAGE-A3 cohort developed antigen-specific T cell and antibody responses to the vaccine. Significant concordance between T cell and antibody responses was observed for both groups. No clear dose-response correlation was seen. All patients progressed by RECIST at first repeat imaging, except for one patient in the MAGE-A3 500 µg cohort who had stable disease for 10.5 months. The median PFS and OS for the MAGE-A3 cohorts were 79 and 183 days, respectively, and for the HPV16 cohort 80 and 196 days, respectively. CONCLUSIONS: GL-0810 and GL-0817 were well tolerated in patients with RM-SCCHN with T cell and antibody responses observed in the majority of patients who received all four vaccinations.

The IL-23/Th17 axis is involved in the adaptive immune response to Bacillus anthracis in humans.

The neutralization of toxins is considered essential for protection against lethal infection with Bacillus anthracis (BA), a select agent and bioterrorism threat. However, toxin-neutralizing activity alone would not be expected to provide sterile immunity. Therefore, we hypothesized that the development of an adaptive immune response against BA is required for bacterial clearance. We found that human monocyte-derived dendritic cells (hDCs) kill germinated BA bacilli, but not nongerminated BA spores. hDCs produce IL-1ß, IL-6, IL-12, and IL-23, and these cytokines are differentially regulated by germination-proficient versus germination-deficient BA spores. Moreover, the IL-23 response to BA spores is regulated by IL-1R-mediated signaling. hDCs infected with germinating BA spores stimulated autologous CD4(+) T cells to secrete IL-17A and IFN-γ in a contact-dependent and antigen-specific manner. The T-cell response to BA spores was not recapitulated by hDCs infected with germination-deficient BA spores, implying that the germination of spores into replicating bacilli triggers the proinflammatory cytokine response in hDCs. Our results provide primary evidence that hDCs can generate a BA-specific Th17 response, and help elucidate the mechanisms involved. These novel findings suggest that the IL-23/Th17 axis is involved in the immune response to anthrax in humans.

Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma.

BACKGROUND: We performed a pilot study using Trojan vaccines in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). These vaccines are composed of HLA-I and HLA-II restricted melanoma antigen E (MAGE)-A3 or human papillomavirus (HPV)-16 derived peptides, joined by furin-cleavable linkers, and linked to a "penetrin" peptide sequence derived from HIV-TAT. Thirty-one patients with SCCHN were screened for the trial and 5 were enrolled. METHODS: Enrolled patients were treated with 300 µg of Trojan peptide supplemented with Montanide and granulocyte-macrophage colony-stimulating factor (GM-CSF) at 4-week intervals for up to 4 injections. RESULTS: Following vaccination, peripheral blood mononuclear cells (PBMCs) from 4 of 5 patients recognized both the full Trojan constructs and constituent HLA-II peptides, whereas responses to HLA-I restricted peptides were less pronounced. CONCLUSION: This treatment regimen seems to have acceptable toxicity and elicits measurable systemic immune responses against HLA-II restricted epitopes in a subset of patients with advanced SCCHN.

Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity.

BACKGROUND: The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i) if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii) their ability to kill allogeneic and autologous tumor targets by direct cytotoxicity and by antibody-mediated cellular cytotoxicity and (iii) defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. METHODS: Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. RESULTS: Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. CONCLUSION: These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical evidence necessary to support clinical trials utilizing autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy in select solid tumors.

Phase 1 and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias.

MS-275 is a benzamide derivative with potent histone deacetylase (HDAC) inhibitory and antitumor activity in preclinical models. We conducted a phase 1 trial of orally administered MS-275 in 38 adults with advanced acute leukemias. Cohorts of patients were treated with MS-275 initially once weekly x 2, repeated every 4 weeks from 4 to 8 mg/m2, and after 13 patients were treated, once weekly x 4, repeated every 6 weeks from 8 to 10 mg/m2. The maximum-tolerated dose was 8 mg/m2 weekly for 4 weeks every 6 weeks. Dose-limiting toxicities (DLTs) included infections and neurologic toxicity manifesting as unsteady gait and somnolence. Other frequent non-DLTs were fatigue, anorexia, nausea, vomiting, hypoalbuminemia, and hypocalcemia. Treatment with MS-275 induced increase in protein and histone H3/H4 acetylation, p21 expression, and caspase-3 activation in bone marrow mononuclear cells. No responses by classical criteria were seen. Our results show that MS-275 effectively inhibits HDAC in vivo in patients with advanced myeloid leukemias and should be further tested, preferably in patients with less-advanced disease.

Absence of Spiroplasma or other bacterial 16s rRNA genes in brain tissue of hamsters with scrapie.

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained > or =10(10) infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.

Analysis of the relationship between Scl transcription factor complex protein expression patterns and the effects of LiCl on ATRA-induced differentiation in blast cells from patients with acute myeloid leukemia.

Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+ myelomonocytic leukemia cells interferes with their capacity to respond to all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias.