MONOCLONAL-ANTIBODY FAB' FRAGMENT CROSS-LINKING USING EQUILIBRIUM TRANSFER ALKYLATION REAGENTS - A STRATEGY FOR SITE-SPECIFIC CONJUGATION OF DIAGNOSTIC AND THERAPEUTIC AGENTS WITH F(AB')(2) FRAGMENTS

An investigation was conducted to evaluate the feasibility of site-sel ective addition of diagnostic and therapeutic agents to monoclonal ant ibody F(ab')2 fragments through cross-linking of antibody Fab' fragmen ts. In the investigation, trifunctional equilibrium transfer alkylatio n cross-link (ETAC) reagents, 4-[2,2-bis[(p-tolylsulfonyl)methyl]acety l]benzoic acid, yl]-benzoyl]-4-(tri-n-butylstannyl)phenethylamine, 3a, and (p-tolylsulfonyl)methyl]acetyl]-benzoyl]-4-[I-125, I-131]iodophen ethylamine, 3b, were synthesized. The ETAC derivatives were reacted wi th Fab' fragments of an antirenal cell carcinoma antibody (A6H) produc ed from reduction of F(ab')2 using 1,4-dithiothreitol. Cross-linking o f Fab' was obtained to yield a radioiodinated modified F(ab')2, [mF(ab ')2], fragment. The cross-linking reaction produced mixed addition pro ducts, requiring the desired mF(ab')2 to be separated from radioiodina ted Fab' by size exclusion HPLC. Tumor cell binding immunoreactivities varied (60-90%) for five isolated mF(ab')2 preparations but were cons istent with other radiolabeled antibody preparations tested on the sam e day. In vitro stability testing indicated that the mF(ab')2 was reas onably stable toward loss of the ETAC cross-linking reagent, except un der strongly basic conditions. Under reducing sodium dodecylsulfate-po lyacrylamide gel electrophoresis (SDS-PAGE) analyses, protein bands be lieved to be cross-linked heavy chain dimers were observed. Biodistrib ution of purified radioiodinated A6H mF(ab')2 was conducted in athymic mice bearing a renal cell carcinoma xenograft (TK-82). A nonmodified control A6H F(ab')2, radioiodinated as a p-[I-125, I-131]-iodobenzoyl conjugate, was coinjected for comparison. The radioiodinated mF(ab')2 had a similar distribution to the radioiodinated control at 3.5, 19, a nd 43 h postinjection. In another study, the distribution of radioiodi nated A6H Fab' was evaluated at 4 and 24 h to establish clearance and pharmacokinetics for comparison with the data obtained from the mF(ab' )2. The biodistribution data indicated that A6H mF(ab')2 was quite dif ferent from that of A6H Fab'. The results from this preliminary study suggest that it may be possible to attach (large polymeric) diagnostic or therapeutic agents to monoclonal antibody F(ab')2 fragments throug h the use of ETAC reagents.