Novel Super-High Affinity Sheep Monoclonal Antibodies Against CEA Bind Colon and Lung Adenocarcinoma

Abstract

We have developed technology to create monoclonal antibodies (MAbs) using lymphocytes from immunized sheep. The affinities of these sheep monoclonal antibodies (SMA) can be several orders of magnitude higher than mouse MAbs. This paper reports the development and validation of a modified enzyme-linked immunoadsorbent assay (ELISA) method to select high-affinity antibodies of the desired specificity at the first screen. Using this method, we have isolated high affinity SMA to carcinoembryonic antigen (CEA), a marker of colon cancer. Comparisons of our novel SMA with mouse MAbs using the new ELISA and BIAcore technology (BIAcore AB, Stevenage, Herts, UK) have confirmed that we have made super-high affinity antibodies to CEA. One of these has a t(1/2) for dissociation of 8 days, which could provide a longer therapeutic window than is available with murine monoclonals currently being used in the clinic. This antibody has a specific tissue staining profile; it thus appears to be an excellent candidate for use in the clinic.

Review: Veterinary Sources of Nonrodent Monoclonal Antibodies: Interspecific and Intraspecific Hybridomas

“…They may have dram atically higher affinities and respond to different epitopes. (20)(21)(22)(23) These aspects have been exploited for veterinary applications, as will be discussed later, but they also provide a valuable source of binding sites for engineered human therapeutic MAbs.…”

“…(62,63) These studies have been extensions of ongoing work on polyclonal antibodies or investigations into particular applications requiring nonmurine antibody-secreting lines. Pig (54) and sheep MAbs (23) have both been suggested as candidates for human immunotherapy. A number of laboratories, primarily Japanese, have also successfully produced chicken MAbs following the isolation of a B lymphoblastoid mutant deficient in thymidine kinase and suitable for use as a fusion partner.…”

“…(87) Work with bacteria and bacterial antigens has generally been confined to infectious diseases of cattle and pigs, including E. coli, (53,78,88) Brucella abortus, (47) and Streptococcus agalacticae, the causitive agent of mastitis, (89 ) Streptococcus pyogenes has also been used as a model antigen in rabbits. (74,90) Antibodies to cellular and tissue antigens have been generated to preparations varying in purity from tissue hom ogenates (37) ; through whole blood (33,49,91) and purified cell populations (28,4 8,90,92) ; to isolated, (23,28,54 ) or recombinant, (41) cellular antigens.…”

“…The advantages apparent with the use of primary heteromyelom a fusion partners have been further investigated by the use of secondary, tertiary, or higher orders of derivatization. This approach has been used with sheep (23,100,118) and cattle. (73,88,118,1 20) Greenlee et al (122) have also looked at the effectiveness of increasing the degree of derivatization of the fusion partner in porcine Mab production, based on an initial mouse 3 bovine fusion partner.…”

“…(31) Kemp et al (48) used an autom ated lymphocytotoxi city test for complement-fixing MAbs. Lumanglas et al (55) and Osborne et al (23) used the Biacore system (Biacore AB, Sweden) to investigate binding rates and affinities of pig and sheep MAbs, respectively. One other point to be remembered in designing a screening assay is the effect of any supplementary serum on the system.…”