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r- R TIII: COUNCIL FOR TOBACCO RESRARCH-U.S.A., INC. January 23, 1985 TO: Dr. S. C. Sommers FROM: D. H. Ford RE: Site visit with Dr. Regina Santolla, Columbia University College of Physicians and Surgeons, January 17, 1985 Grant No. 1483A entitled, "Development of Monoclonal Antibodies to Carcinogen Adducts" Goal: To develop antibodies to DNA and protein carcinogen adducts which can then be used to create an assay which can be used to survey large groups of individuals in the 'work place' wherein they may be exposed to various carcinogens. Further, Dan Santella wishes to determine the relative degree to which each worker forms such adducts, which could then be used as an index ofrisk for developing cancer. Personnel: Dr. Santella, the Pl,has taken over the direction of a program originally conceived by Dr. I. Weinstein, who will continue his association with Dr. Santella and serve in an advisory capacity. There are in addition two technicians (Chen-Dao Lin and S. Allen), both of whom appear quite knowledgeable about the program. Dr. Alan Jeffrey is a bio- chemist who will be involved in determining which proteins (other than hemoglobin), present in various organs, are involved in forming carcinogen adducts, as well as determining the binding site on each protein. Progress: Considerable progress has been achieved in assaying DNA- carcinogen adducts, using white cells obtained from blood samples. Dr. Santella reports that they can detect the presence of as little as one adduct in 108 nucleotides (about 100 adducts/cell) after exposure to benzo(a)pyrene diol epoxide (BPDE). Measurable DNA adducts have also been found in lung and in lung tumor tissue from 4 to 14 lung cancer patients. The method she uses is the enzyme-linked immunosorbent assay (ELISA). (See review article in the December 7, 1984 issue of Science, Vol. 226, p. 1183 by T. H. Maugh...attached). Dr. Santella currently has 4 monoclonal antibodies which recognize the DNA-carcinogen adduct complex and a fifth antibody which recognizes the guanosine base of the DNA complexed with BSA. Problem: While Dr. Santella and others have been successful in creating antibodies to detect binding of BPDE to the DNA of white cells, the amount of blood needed is considerable (50-60m1), which would make the method difficult to apply to large groups. Using hemoglobin, perhaps as little as one drop of blood would provide enough protein to detect the presence of any adduct formed by BPDE or other potential carcinogen. Therefore, the current thrust of the program is to develop an ELISA for a BPDE-protein adduct, initially focusing on hemoglobin as an easily obtained protein. Once a sufficiently sensitive methodology is established for hemoglobin, they will attempt to develop similar assays for other carcinogen-protein adducts with proteins from various tissues, i.e. skin.

SITE VISIT-Grant #1483A January 23, 1985 Progress on the BPDE-protein adduct: Initial studies show that such an adduct is formed (see attached abstract of a paper to be presented at the AACR meeting this Spring). Despite this early success, the assay, as currently performed, is not sufficiently sensitive for application. The specificity seems to be more to the BP chromophore than to the adduct. Therefore, Dr. Santella is planning new approaches with her 2B6 and 2G4 clones, seeking to modify the antibody. She also plans to determine if digestion of the protein to various peptides or denaturation will improve specificity. She is also considering utilizing a fluorescent or radio- active assay, either of which would be more sensitive than the ELISA. Future: Once a satisfactory assay for a protein-carcinogen adduct is developed, Dr. Santella plans to proceed with 1) Animal studies wherein she will compare the relative sensitivity of DNA and hemoglobin-carcinogen adducts. 2) Hemoglobin adducts will also be compared with adducts formed with proteins from other tissues. 3) Determine what level of protein adduct formation indicates a risk to develop cancer. 4) What sort of BP adduct is bound to which protein (so far the only adducts known are for BPDE with DNA and RNA, aside from the preliminary report of protein binding in the abstract). Comment: Dr. Santella is a young, enthusiastic, well-informed investigator with well-defined goals. This seems to be an exciting project with which she is making excellent progress. She may well develop an easy routine assay which could be used for testing large numbers of individuals exposed to various "questionable" environmental hazards to determine the relative risk of each individual to developing cancer. Merits continued support. DHF DHF/s Enc.