The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4⁺ and CD8⁺ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no “antigenic competition” in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.     

Switching to BCL-6 Negativity in Relapsed Diffuse Large B Cell Lymphoma Correlated with More Aggressive Disease Course

Diffuse large B-cell lymphoma (DLBCL) is the most frequent, complex and heterogeneous lymphoma of adulthood. Heterogeneity is expressed at clinical, genetic, and molecular levels. It is known that BCL-6 expression is a favorable prognostic factor in DLBCL. However, the underlying mechanisms of BCL-6 expression in DLBCL relapse are not yet elucidated. Here, we present so far undescribed clinical phenomenon of switching BCL-6⁺ protein expression into BCL-6⁻ expression in 19 of 41 relapsed DLBCL patients. The switch in relapsed DLBCL was associated with more aggressive clinical course of the disease. Bone marrow infiltration and high IPI risk were more often present in BCL-6⁻ patients. Significantly increased biochemical parameters, such as LDH, beta-2 macroglobulin, CRP, and ferritin have been found, as well as significantly decreased serum Fe, TIBC, and hemoglobin. A Ki-67 proliferation marker was considerably high in relapsed DLBCL, but without significant differences between BCL-6⁺ and BCL-6⁻ groups of patients. Thus, switching of the positive into negative BCL-6 expression during DLBCL relapse could be used as a prognostic factor and a valuable criterion for treatment decision.     

Induction of immune tolerance in patients with hemophilia A and inhibitors treated with porcine VIIIC by home therapy

Home therapy with porcine factor VIIIC was safe and effective when administered to five hemophilic patients over periods of 8 1/2, 6, 4, 3 1/2, and 2 years. No significant transfusion reactions occurred. Before treatment with porcine factor VIIIC, all five had high-level, high- responding anti-human VIIIC inhibitors initially lacking anti-porcine factor VIIIC activity. Although specific anti-porcine VIIIC inhibitors arose in all patients, these were generally transient, and only one patient became refractory to treatment. We believe that porcine factor VIIIC is the treatment of choice in patients whose inhibitors do not cross-react. All five patients lost their original anti-human VIIIC inhibitors after starting treatment with porcine VIIIC, permitting the reintroduction of human VIIIC in three of them. There has been no recurrence of anti-human VIIIC inhibitor activity during 2 to 3 years of regular treatment with human VIIIC in these patients. This suggests that tolerance to human VIIIC has arisen as a result of treatment with porcine VIIIC. Porcine VIIIC may have a role in the desensitization of some factor VIIIC inhibitor patients.     

Carrier detection in hemophilia A: a cooperative international study. I. The carrier phenotype

Eight laboratories in six countries cooperated to clarify several issues concerning the phenotypes of heterozygous carriers of hemophilia "A." Plasma levels of factor VIII (F.VIII:C, formerly VIII:C) and von Willebrand factor (VWF:Ag, formerly VIIIR:Ag) of carriers and normal women were determined by various "in-house" methods; a single lyophilized plasma standard was used for all assays. Analysis of the collated data from 336 carriers (296 obligatory carriers and 40 sporadic carriers) and 137 normal women showed that there was no difference in the F.VIII:C levels of "paternal" carriers (women who had obtained the abnormal gene from their fathers) and "maternal" carriers. Neither was there a difference in the VWF:Ag levels of normal women and either type of carrier. Age was found to have a significant effect on both F.VIII:C and VWF:Ag, values being higher at very young and very old ages, the minima occurring in the 25- to 30-year range. ABO blood type had a striking effect. Women of types A, B, and AB (designated non- O in the study), both normals and carriers, had significantly higher levels of both factors than did women of type O. Analysis by laboratories showed that differences in mean levels of both factors between laboratories were highly significant. It was concluded that age, ABO blood type, and laboratory variation should be taken into account in carrier detection.     

Lymphokine-induced phagocytosis in angiocentric immunoproliferative lesions (AIL) and malignant lymphoma arising in AIL

A factor that augmented the phagocytosis of IgG-coated ox red blood cells by the human monocyte/macrophage line U937 was identified in cell culture supernatants from two of two patients with angiocentric peripheral T cell lymphomas, three of three patients with angiocentric immunoproliferative lesions that were not frankly malignant, and one of two patients with T lymphoblastic malignancies. The factor was not present in supernatants derived from 14 nonangiocentric peripheral T cell lymphomas of other histologic types nor in ten cases of B cell lymphoma and two cases of Hodgkin's disease. A similar factor was present in the supernatants of concanavalin A (Con A)-stimulated normal peripheral blood mononuclear cells and in the supernatants of IL-2- dependent T cell lines derived from normal peripheral blood. The factor had an apparent mol wt of greater than 50,000 daltons, was heat labile (100 degrees C for two minutes), and stable at pH 2.0. Its stimulation of phagocytosis was independent of any increase in number of Fc receptors. Thus, this factor is probably not gamma-interferon. This factor may play a pathogenetic role in the hemophagocytic syndromes associated with certain T cell malignancies and immunodeficient states.     

Traumatic brain injury-induced cerebral microbleeds in the elderly

Traumatic brain injury (TBI) was shown to lead to the development of cerebral microbleeds (CMBs), which are associated with long term cognitive decline and gait disturbances in patients. The elderly is one of the most vulnerable parts of the population to suffer TBI. Importantly, ageing is known to exacerbate microvascular fragility and to promote the formation of CMBs. In this overview, the effect of ageing is discussed on the development and characteristics of TBI-related CMBs, with special emphasis on CMBs associated with mild TBI. Four cases of TBI-related CMBs are described to illustrate the concept that ageing exacerbates the deleterious microvascular effects of TBI and that similar brain trauma may induce more CMBs in old patients than in young ones. Recommendations are made for future prospective studies to establish the mechanistic effects of ageing on the formation of CMBs after TBI, and to determine long-term consequences of CMBs on clinically relevant outcome measures including cognitive performance, gait and balance function.

     

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell- depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B- cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.