Antibody immunoglobulin G (IgG) against human prostatic specific antigen (PSA) as a carrier protein for chemotherapeutic drugs to human prostate tumors: Part 1. A double immunofluorescence analysis.
BACKGROUND
Adenocarcinoma of the prostate (CaP) is the the second highest cause of cancer deaths in U.S. males. Current chemotherapeutic and/or endocrine treatments do not specifically and selectively target tumor cells of prostate cancer and benign prostatic hyperplasia (BPH). We hypothesized that because of the specific binding characteristics of antibody immunoglobulin G (IgG) to human prostatic-specific antigen (PSA), PSA-IgG could function as a carrier protein for conjugated chemotherapeutic drugs and that the immunoconjugate would selectively bind to prostatic epithelial cells and their tumors, but not to epithelial cells of unrelated organs. Our objective was to test the hypothesis using human prostatectomy specimens.


METHODS
WE used several derivatives of 5'-fluorouracil, namely, 5'-fluoro- 2'-deoxyuridine (5'-Fu-2'-d), 5'-fluoro-2'-deoxyuridine-5' monophosphate (5'-Fu-2'-d-5'-mp), 5'-fluoro-2'-deoxyuridine-5'-(p-aminophenyl) monophosphate (5'-Fu-2'-d'-5'-amp), to conjugate with rabbit anti-PSA-IgG together with fluorescent markers (such as rhodamine and fluorescein or fluorescein isothiocyanate: FITC). Prostate specimens were obtained from prostatectomy patients who had not been treated with cytotoxic drugs before surgery. We evaluated formalin-fixed and paraffin-embedded sections as well as cryostat sections of frozen specimens for localization of PSA-IgG alone and PSA-IgG-drug immunoconjugate using immunoperoxidase (IP) and single and/or double immunofluorescence (IF) localization techniques.


RESULTS
Our study showed that the immunoconjugate (PSA-IgG-5'-Fu-2'-d) bound to PSA (molecular size of approximately 34 KDa) on nitrocellulose sheets in Western immunoblots of extracts of BPH and CaP tissues. This binding of immunoconjugate to PSA on immunoblots was similar to that of the unconjugated PSA-IgG. Immunostaining patterns for rabbit anti-PSA-IgG and PSA-IgG-5'-Fu-2'-d immunoconjugate were similar and specific for prostate epithelial cells and their tumors, as revealed by IP techniques. To demonstrate that both the antibody and drug localized in the same group of prostatic epithelial cells, we used an immunoconjugate in which the PSA-IgG was labeled with rhodamine and 5'-Fu-2'-d-5'-amp with FITC. Our study showed that fluorescence for rhodamine and FITC was present in the same group of prostatic epithelial cells. Phase contrast microscopy demonstrated details of prostatic glandular epithelium and connective tissues. Our study showed that fluorescence for rhodamine and FITC and immunostaining by IP techniques were not observed in prostate sections incubated with normal rabbit serum.


CONCLUSIONS
We have shown that conjugation of 5'-Fu derivatives to PSA-IgG did not affect either the selectivity or specificity of the antibody for prostatic epithelial cells. Differential immunofluorescence study has shown that PSA-IgG may function as a carrier protein for chemotherapeutic drugs to prostate epithelial cells and their tumors. Furthermore, FITC-labeled 5'-Fu-2'-d did not specifically localize in prostatic glands, kidney, lungs, bladder, or colon. Because of the specificity and selectivity of the immunoconjugate for prostatic epithelial cells and their tumors, the immunoconjugate could be used in small dosages to treat prostatic tumors and such treatment would greatly reduce many unpleasant side effects in patients. This is the first report to show that PSA-IgG can function as an organ specific carrier protein for chemotherapeutic drugs to human prostate epithelium and its tumors.
Sinha
A A
AA
Research Service, Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.
Sackrison
J L
JL
DeLeon
O F
OF
Wilson
M J
MJ
Gleason
D F
DF
D000906:Antibodies / Q000276:immunology*; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D002352:Carrier Proteins / Q000378:metabolism*; D004337:Drug Carriers; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D000922:Immunotoxins / Q000378:metabolism; D008297:Male; D017430:Prostate-Specific Antigen / Q000276:immunology* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism*; D012235:Rhodamines; D014018:Tissue Distribution
D000906:Antibodies; D000970:Antineoplastic Agents; D002352:Carrier Proteins; D004337:Drug Carriers; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D000922:Immunotoxins; D012235:Rhodamines; D017430:Prostate-Specific Antigen; D016650:Fluorescein-5-isothiocyanate