RECOMBINANT THYROID PEROXIDASE-SPECIFIC AUTOANTIBODIES .2. ROLE OF INDIVIDUAL HEAVY AND LIGHT-CHAINS IN DETERMINING EPITOPE RECOGNITION

Most thyroid peroxidase (TPO) autoantibodies in man recognize closely associated epitopes in two domains (A and B) on TPO. These epitopes we re defined by recombinant monoclonal human autoantibodies expressed as antigen-binding fragments [F(ab)]. Only five heavy (H) and light (L) chain gene combinations encoded 34 F(ab), all of which have high affin ity (K-d, similar to 10(-10) M) for TPO. We, therefore, investigated t he roles of H and L chain genes in TPO domain recognition in two ways. First, we created hybrid F(ab) by forced recombination of H and L cha in genes from 4 F(ab) recognizing the A or B domains. These hybrid F(a b) proteins, expressed in bacteria, bound extremely poorly (or not at all) to TPO, even at concentrations more than 100-fold higher than tho se required for detection of TPO binding by the original F(ab). Nucleo tide sequencing of the cDNA as well as gel electrophoresis of the expr essed proteins confirmed that poor hybrid F(ab) binding to TPO was not the result of cloning artifacts. Therefore, contrary to prevailing vi ews on combinatorial libraries, we found no tolerance for H and L chai n cross-combinations in high affinity TPO binding. These observations strengthen the likelihood that the H and L chain combinations from com binatorial libraries reflect those of TPO autoantibodies in vivo. In a second approach to examine the roles of H and L chains in TPO binding , we focused on three original F(ab) with similar L chains (encoded by KL012-like germline genes) and similar H chains (encoded by V1-3B-lik e germline genes), but different diversity (D) regions. All F(ab) boun d predominantly to TPO domain A, as observed previously for a F(ab) wi th a KL012 L chain and a different H chain. Conversely, a F(ab) with a V1-3B-like H chain but a different L chain (A') bound to TPO domain B . These data indicate that the L chain plays a major role in defining TPO epitope recognition.