A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1∶106 with E. coli O157:H7 and affinity constants ranged from 1.57×1010 to 2.79×1010 L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food

Abstract P2-09-04: Anti-Trastuzumab Antibodies Are Associated with Longer Progression Free Survival in Patients Treated with Trastuzumab

Abstract Recombinant methodologies have allowed the production of monoclonal antibodies (MAbs) with higher percentages of human content to decrease antibody immunogenicity and reduce or eliminate the anti-antibody response. Trastuzumab (Tz) is a humanized MAb approved for treatment of HER2-overexpressing breast cancer. We detected anti-Tz antibodies in the serum of patients treated with Tz. We initially studied the effects of these antibodies on the response to Tz in a transgenic mouse model of breast cancer. Twenty-two wild-type FVB mice were transplanted with mammary tumors arising in FVBMMTV/HER2 transgenic mice. Mice with established tumors were treated with Tz (n=12) or PBS (n=10) for 4 weeks. Five of 12 mice responded to treatment while 7/12 mice exhibited progressive disease (PD). All 5 responders had elevated anti-Tz antibodies, whereas in mice that had tumor progression anti-Tz levels were low or undetectable (p=0.002). These findings were confirmed in a second mouse cohort (n=16), in which anti-Tz antibodies were detected in responder mice as early as one week after starting treatment. MAbs produced by hybridomas stemming from spleen cells derived from responder mice bound Tz. These findings argue against the current understanding that an anti-xenoglobulin response to Tz would produce blocking antibodies. Therefore, we addressed if detectable anti-Tz antibodies would predict benefit from antibody therapy in patients with HER2+ breast cancer. Samples from 21 patients with stage IV HER2 positive breast cancer experiencing clinical response or disease stabilization upon treatment with Tz that had been enrolled in an anti-HER2 vaccine phase I trial (J Clin Oncol 2009;4685) were retrospectively analyzed in a blinded fashion. With a median follow-up of 36 months, 14 patients had had disease progression while on Tz plus the vaccine. The presence of anti-Tz F(ab')2 antibodies was quantified among the enrolled patients. A univariate Cox regression analysis of the progression free survival (PFS) was performed. Analysis revealed that higher anti-Tz F(ab')2 antibodies were significantly associated with lower risk of disease progression (p=0.023). Fine specificity studies confirm the presence of a human polyclonal response to Tz, directed to epitopes present in both its Fc and Fab regions. In conclusion, in mice, response to Tz was significantly associated with the presence of anti-Tz antibodies in serum, while in patients with stage IV breast cancer treated with Tz, the presence of anti-Tz F(ab')2 antibodies was associated with longer PFS. The fine specificity of anti-Tz antibodies reveals epitopes that could be potentially relevant for vaccination strategies. Information about cognate epitopes on Tz may be also useful in the design of new therapeutic antibodies. These data also support prospective evaluation of patients undergoing therapy with other chimeric or humanized antibodies to address if detectable anti-therapeutic antibodies would predict benefit from long antibody therapy regimens, especially in the adjuvant setting, in which clinical benefit can only be measured by survival rates. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-04.</jats:p