Isolation of human antibodies against the central DNA binding domain of p53 from an individual with colorectal cancer using antibody phage display.

PURPOSE: Serum anti-p53 antibodies are detected in approximately 30% of patients with cancer, and most of these antibodies are directed against the NH2- and COOH-terminal domains of p53. The aim of this study was to identify individuals with serum reactivity against the central and COOH-terminal domains of p53 and to isolate and characterize these recombinant human antibodies. EXPERIMENTAL DESIGN: The serum of individuals with colorectal cancer was screened for the presence of anti-p53 antibodies. Antibody phage display libraries were constructed from four immunoreactive individuals, and the libraries were panned against the central and COOH-terminal domains of p53. RESULTS: An antibody fragment (1159.8) that was specific for the whole molecule as well as the central domain was isolated from one of the libraries. The serum from the original individual inhibited the binding of this Fab fragment to p53, thus indicating that the antibody reflected the specificity of the in vivo immune response. The VL and VH genes from Fab 1159.8 matched germ-line genes from the VH3 and VK2 families, respectively. Competition analysis with monoclonal antibodies showed that Fab binding could be inhibited most effectively with DO11 and, to a lesser extent, Pab240, indicating an epitope within or adjacent to residues 181-190 of p53. CONCLUSIONS: The successful isolation of this human anticentral domain Fab provides additional insight into the nature and specificity of the tumor-specific immune response. This antibody could be used as a reagent for functional studies of p53, or it may be a candidate for use as an anticancer idiotypic vaccine.

Immunoglobulin genes expressed by B-lymphocytes infiltrating cervical carcinomas show evidence of antigen-driven selection.

Lymphocyte infiltration is often present in cervical cancer lesions, possibly reflecting an ongoing (but ineffective) immune response to the tumour. B-lymphocytes are the predominant lymphocyte infiltrate in pre-malignant cervical lesions, where they are thought to comprise the host immune response to active human papillomavirus (HPV) infection. Although B cells are less frequently detected in cervical tumours, a high proportion of terminally differentiated plasma cells expressing tumour-specific immunoglobulin (Ig) remain. The antigen specificity and functional significance of the antibody response to cervical tumours is unknown. As part of a study to characterise the antibodies expressed by the tumour-infiltrating B cells (TIL-B) in cervical tumours using antibody phage display, we examined expressed Ig gene sequences to determine if there was molecular evidence of a selective response to antigenic changes in the transformed epithelial cells. We found that biased variable region gene usage by the B cells and the rate of somatic hypermutation in the rearranged Ig heavy chain variable regions (VH) both indicated antigenic selection of the B cells. We also found evidence of affinity maturation, as indicated by the detection of antibodies of the IgG1, IgG2 and IgA isotypes, and possible clonal selection of the Ig receptors. These data support the notion that B-lymphocytes and plasma cells infiltrating cervical carcinomas are the result of an antigen-induced response to HPV infection or transformation.

Generation of anti-p53 Fab fragments from individuals with colorectal cancer using phage display.

Although many individuals with malignancy develop Abs against p53, little is currently known of the structural features, V gene usage, and degree of somatic mutation of these Abs. Such information is critical to any meaningful understanding of the nature and significance of this humoral immune response to p53. We have constructed phage display libraries from six individuals with colorectal cancer and a demonstrable serum immune response against p53. Following panning with recombinant p53, a total of 43 binding Fab were identified. Four of these Abs bound with high affinity to wild-type denatured p53 (1.19 x 10-8 - 1.57 x 10-8), as determined by BIAcore analysis, and were highly specific for both recombinant and cell line-derived p53, as determined by ELISA and immunoprecipitation. Epitope mapping showed they were reactive with the N terminus of human p53 between residues 27 and 44. Sequence analysis showed that the heavy chains were derived from the VH1 gene family, and the light chains from VL4. The pattern of replacement and silent mutations in the Fab sequence indicated that negative selection had occurred in the framework regions of all the VH genes. We show that lymphocytes from individuals with cancer represent a valuable source of high affinity human Abs against p53. This approach provides an opportunity to examine the genetic structure of these naturally occurring Abs, and to draw inferences regarding the nature of the immune response that produced them. Abs identified in this way have a number of potential therapeutic applications.

The significance of anti-sialyl-Tn antibodies in patients with colorectal and breast cancer.

The sera of 54 individuals with colorectal or breast cancer, and 50 healthy volunteers were assayed for the presence of anti-bovine submaxillary mucin antibodies using an enzyme linked immunoassay. The serum levels of these antibodies were found to be significantly lower in people with breast (p < 0.001) or colorectal cancer (p < 0.001) with respect to healthy individuals. Within the colorectal cancer group the presence of antibodies was significantly lower in those individuals with poorly differentiated tumors compared to other histological grades (p < 0.05), but did not correlate with the presence of local or distant metastases or anatomical location of the tumor (p > 0.05). No correlation was found with respect to the age of the patient and the level of anti-sialyl-Tn antibodies (p > 0.05). Competition analysis with the anti-sialyl-Tn monoclonal antibody 3C2 indicated that the activity against bovine submaxillary mucin was primarily due to specificity for the sialyl-Tn epitope of the glycoprotein. In contrast to findings with other tumor associated antigens, we could find no evidence of an increase in the level of antibodies against this epitope.

Adenosine analogues as antimetabolites against Plasmodium falciparum malaria.

Analogues of purine nucleosides and deoxynucleosides were tested for toxicity against the intraerythrocytic parasite Plasmodium falciparum in vitro culture. Sangivamycin (7-deaza-7-amido-adenosine) (IC37 of 0.3 microM), tubercidin (7-deaza-adenosine) (IC37 of 0.7 microM), 6-methylamino-deoxyadenosine (IC37 of 10 microM), 8-aza-2-amino-deoxy-adenosine (IC37 of 11 microM) and 2-chloro-adenosine (IC37 of 11 microM) were found to be the most toxic towards the parasite. Structure-activity analysis suggested that alteration of the purine ring at the 7 or 8 position significantly increased the toxicity of the compound against P. falciparum. Analysis by HPLC of parasite lysates which had been subjected to the cytotoxic compounds confirmed that alterations in the flux of the purine salvage pathways of the parasite had occurred. Comparison of the toxicity of these compounds against P. falciparum with the toxicity against a similar intraerythrocytic parasite, Babesia bovis, or human melanoma cell lines indicated a differential toxicity, in that many of the compounds toxic towards P. falciparum were relatively non-toxic towards human melanoma cell lines or B. bovis and vice versa. The mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids appears to vary significantly between P. falciparum, B. bovis and mammalian cells.