Human antiserum against E. coli J5, a mutant strain whose endotoxin (LPS) contains core determinants common to all gram-negative bacteria but lacks serotype-specific oligosaccharide, protects patients from shock and death due to gram-negative bacteremia. The antitoxic effect of passive J5 immunization has been demonstrated not only in treatment of established bacteremia but also when prophylactic antiserum is administered frequently to patients at high risk of fatal endotoxin shock because of trauma or abdominal surgery. Recent construction of a stable human-mouse heteromyeloma cell line has allowed us to make hybridomas producing human anti-J5 LPS monoclonal antibodies (mAb). One of these, an IgM, binds to a wide variety of gram-negative bacteria and their lipopolysaccharides and prevents the dermal Shwartzman reaction and death from experimental E. coli, Klebsiella and Pseudomonas bacteremia. Such mAb's offer not only unlimited production of safe immunotherapeutic agents but also the chance to probe LPS antigenic relatedness and mechanisms of LPS biologic activities with new precision. We plan to use our heteromyelomas to generate a library of human anti LPS core monoclonal antibodies which are diverse with respect to isotype, epitope specificity and binding characteristics. We will attempt fusions with splenocytes and peripheral B cells sensitized in vitro as well as in vivo. Then we will use these antibodies to pursue six Specific Aims: 1) To correlate epitope specificity with the extent of cross-reactivity between J5 LPS and the core determinants (core sugars + lipid A) of endotoxins from clinical gram-negative isolates; 2) To dissect the mechanisms by which LIPS core antibody protects against LPS and infection; 3) To study whether epitope specificity is a significant determinant of LPS biologic activities such as mitogenicity, complement activation and pyrogenicity; 4) To raise rodent anti-idio-type antibodies against LPS mAb's and to see if any of them mimic LPS; 5) To adapt human hybridomas to growth in serum-free media; and 6) To explore the possibility of preserving antibody production permanently by cloning J5 Immunoglobulin genes. We have sufficient preliminary data in most of these areas to encourage us that substantial progress can be made toward our long-term goals of undertanding the pathogenesis of endotoxin shock and providing a safe, stable source of protective anti-LPS antibody to prevent the toxic consequences of gram-negative bacteremia in patients.
