Degree Of Antigen Expression Research Articles

IMMUNOGLOBULIN INDICATORS TO VIRUSES CYTOMEGAL AND GENITAL HERPES IN THE BLOOD SERUM OF WOMEN WITH NON-ATIPICAL ENDOMETRIAL HYPERPROLIFETATIVE PATHOLOGY

The aim of the study was to analyze the level of Ig M- and Ig G-antibody (Ab) for cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV-2) in serum of women with non-atypical endometrial hyperproliferative pathology. Materials and methods: The analysis of immunoglobulin indices to CMV and HSV-2 in serum of women with non-atypical endometrial hyperproliferative pathology. In women with uterine body polyps the presence of CMV in the uterine cavity was found in 54.8% of cases, in women with non-atypical endometrial hyperplasia in 38.3% of cases. The levels of Ig G-Ab and Ig M-Ab to CMV in serum have a clear dependence on the degree of antigen expression in endometrial tissue. HSV-2 antigens were determined in 22.58 ± 5.31% of women with uterine body polyps and in 8.33 ± 3.57% of patients with non-atypical endometrial hyperplasia while increasing serum specific antibodies to HSV-2. Results: The results indicate that there is a clear link between viral infection of hyperproliferatively altered endometrium and the determination of positive immunoglobulin levels in peripheral blood, which may be a reliable marker of chronic persistent viral infection in a woman's body. Conclusions: In women with uterine body polyps, the presence of CMV in the uterine cavity was found in 54.84 ± 6.32% of cases, in women with non-atypical endometrial hyperplasia in 38.33 ± 6.28% of cases. The levels of Ig G-Ab and and Ig M-Ab to CMV in serum have a clear dependence on the degree of antigen expression in endometrial tissue. HSV-2 antigens were determined in 22.58 ± 5.31% of women with uterine body polyps and in 8.33 ± 3.57% of patients with non-atypical endometrial hyperplasia while increasing serum specific antibodies to HSV-2. The results indicate that there is a clear link between viral infection of the target tissue (hyperproliferatively altered endometrium) and the determination of a positive level of peripheral blood immunoglobulin, which may be a reliable marker of chronic persistent viral infection in a woman.

Preclinical Development of a T-Cell ALL CAR Demonstrates That Differences in CAR Membrane Distribution May Impact Efficacy

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression.Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone.Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface.Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells. DisclosuresOrentas:Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.

Flow cytometric study of potential target antigens (CD19, CD20, CD22, CD33) for antibody-based immunotherapy in acute lymphoblastic leukemia: analysis of 552 cases

Monoclonal antibody (MoAb)-based therapies have opened innovative treatment avenues that have impacted on the management of patients with both neoplastic and non-neoplastic hematological diseases. The aim of our study was to evaluate in a large series of cases of acute lymphoblastic leukemia (ALL) the expression of specific antigens, CD19, CD20, CD22, and CD33, for which MoAbs are available for clinical use. For each antigen, evaluation was based on the percentage of positive leukemic cells and the degree of antigen expression by mean fluorescence intensity (MFI) and antibody binding capacity (ABC) that were correlated with age, immunophenotype, and presence/absence of particular molecular markers. We can document that some of the analyzed antigens showed a degree of expression related to the B-cell maturation profile, and that the antigen expression intensity appeared to vary according to the presence of specific genetic markers. These findings suggest that the possible clinical use of a given MoAb in patients with ALL should take into account both the maturation profile of the leukemic cells and the presence of a given molecular transcript. Only clinical studies will conclusively demonstrate whether the differences in antigenic expression truly correlate with the different therapeutic efficacies of the various clinical grade MoAbs.

High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextran and glycophorin A–M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles (< 0.2 μm) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish separation conditions that allowed recovery of cells that differ only in the degree of antibody labelling.

Relationship between internalization and cytotoxicity of ricin A-chain immunotoxins.

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A-chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB-ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti-CD3 IT, anti-CD5 IT and anti-CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti-CD4 IT inhibited poorly and anti-CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine-labelled antibodies. Anti-CD7 showed the fastest internalization rate, followed by anti-CD3 and anti-CD5. Anti-CD4 and anti-CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.

Immunohistochemical localization of melanoma-associated antigen p94 kd200 with the use of a modified avidin-biotin-complex lectin method.

The immunohistochemical localization of melanoma-associated antigen p94 kd200 was investigated in frozen sections of 3 congenital nevi, 4 benign intradermal nevi, 1 regressing nevus, 1 blue nevus, 1 dysplastic nevus, 1 lentigo maligna, 1 superficial spreading melanoma and 2 metastatic melanomas. The original avidin-biotin complex lectin method (Hsu SM, Raine L, Fanger H: Am. J. Clin. Pathol., 75: 734-738, 1981) was modified to detect the antigen. The sections were exposed to the monoclonal antibody to p94 kd200 (Hybritech Inc.), the linking biotin-labelled anti-mouse IgG, the avidin-biotin peroxidase complex and the 3-amino-9-ethylcarbazole solution in an incubator at 37 degrees C and 100% humidity. We found that the percentage of cells expressing p94 kd200 varied between 0 and 100% in congenital nevi, between 80 and 100% in benign intradermal nevi, between 0 and 20% in the regressing, blue and dysplastic nevi, and in the lentigo maligna, 80 to 100% in the superficial spreading melanoma, and between 0 and 40% in the metastatic melanomas. Positive cells were found to be hypomelanotic (did not have heavy melanin content). The intensity of labelling or the degree of antigen expression on benign and malignant hypomelanotic cells was also found to vary. These findings 1) reinforce the concept of quantitative rather than qualitative antigenic differences in benign and malignant cells 2) suggest that kd200 is lost with increasing pigment production 3) offer a potentially significant tool to investigate the antigenic changes during cell differentiation.

Tumor‐specific and forssman antigens of guinea‐pig hepatoma cells: Comparison of tumor cells grown in vivo and in vitro

Cloned tumor-cell lines were derived from two antigenically distinct ascites variants of diethylnitrosamine-induced guinea-pig hepatomas (designated line 1 and line 10). Cell-surface antigens on the ascites and in vitro-grown tumor cells were analyzed with immunofluorescence, C1 fixation and transfer, and antibody-complement-mediated cytotoxity tests. Tumor-specific and Forssman antigens continued to be expressed during 3-6 months in vitro cultivation. Differences between ascites and cultured cells were noted in the degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity. Cells grown in vitro exhibited a greater number of Forssman and tumor-specific antigen sites than in vivo grown cells as determined by the quantitative C1 fixation and transfer test. Immunofluorescent staining indicated that some cloned lines were considerably more homogeneous in terms of antigen expression than were the cultured, non-cloned parent cells or ascites-grown cells. Cloned lines were frequently more sensitive to the cytotoxic action of antibody and complement than were the in vivo grown and cultured non-cloned parent tumor cells. Sensitivity to cytotoxicity did not necessarily correlate, however, with the degree of antigen expression. These results suggest that; (1) the expression of Forssman and tumor-specific antigens does not diminish on cells cultivated in vitro and (2) ascites hepatoma cells in vivo are a heterogeneous population of cells differing in their degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity.