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Immune complexes formed between monoclonal antibodies (mAbs) and toxins can neutralize toxicity in vivo by multiple mechanisms. Toxin sequestration and clearance by mAbs may be improved by enhancing their ability to bind to red blood cells (RBCs) through immune adherence. This can be achieved by converting the mAbs to heteropolymers (HPs), which are antigen-specific mAbs cross-linked to mAbs targeting the complement receptor (CR1), a protein that is expressed on the surface of RBCs in primates and mediates delivery of complement C3b-containing immune complexes to tissue macrophages. Conversion of mAbs to HPs has been shown to enhance clearance of multivalent antigens from the blood circulation, but the interaction of HPs with monovalent toxins has not been examined. Using botulinum neurotoxin (BoNT) as a model system, we studied the effect of conversion of a pair of BoNT-specific mAbs into HPs on toxin neutralization and handling in vivo. Two HPs given in combination had 166-fold greater potency than un-modified mAbs, neutralizing 5,000 LD50 BoNT, when tested in transgenic mice expressing human CR1 on RBC membranes. Improvement required adherence of BoNT to the RBC in vivo and 2 HPs, rather than an HP + mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2 hours, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages in vitro, compared to pairs of mAbs or mAb + HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from a lethal BoNT dose up to 3 hours after toxin exposure. In a pre-exposure prophylaxis model, mice given HP up to 5 days prior to BoNT administration were fully protected from a lethal BoNT dose. These studies elucidate general mechanisms for the neutralization of toxins by HP pairs and demonstrate the potential utility of HPs as BoNT therapeutics.

Affinity-matured human antibodies have demonstrated efficacy as countermeasures for exposure to botulinum neurotoxin (BoNT), which is the cause of the disease botulism category A select bioterror agent. Little is known, however, about the potential role of natural (un-mutated) antibodies in the protective immune response to BoNT. Here we describe the cloning of two human IgM antibodies that bind serotype A BoNT. Both are un-mutated IgM antibodies, consistent with an origin in naive B cells. One of the antibodies is able to fully neutralize a lethal dose of serotype A BoNT in vivo. These results suggest that the natural human antibody repertoire may play a role in protection from exposure to biological toxins.

Background: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

Native human antibodies are defined as those that arise naturally as the result of the functioning of an intact human immune system. The utility of native antibodies for the treatment of human viral diseases has been established through experience with hyperimmune human globulins. Native antibodies, as a class, differ in some respects from those obtained by recombinant library methods (phage or transgenic mouse) and possess distinct properties that may make them ideal therapeutics for human viral diseases. Methods for cloning native human antibodies have been beset by technical problems, yet many antibodies specific for viral antigens have been cloned. In the present review, we discuss native human antibodies and ongoing improvements in cloning methods that should facilitate the creation of novel, potent antiviral therapeutics obtained from the native human antibody repertoire.

Introduction: New cancer-specific antigens are needed to fully realize the potential of antibody-based cancer therapies. Cancer cells express novel antigens that are characteristic of the transformed state and may engender an antibody response in patients. We hypothesize that some of these antibodies detect neo-antigens shared by diverse tumor types. We optimized a hybridoma method for cloning human IgG monoclonal antibodies (mAbs) by screening for binding to heterologous cell lines. Here, we describe four mAbs, isolated from cancer patients, that bind to cancer cells. Methods: We developed immunofluorescence binding assays with the A431, MCF-7, and MDA-MB-231 cell lines, detecting cell-reactive human IgG with the Operetta (Perkin Elmer). The assays were adapted for extracellular binding as well as internalization using specific culture conditions. We tested sera from 59 lung cancer patients with diverse clinical features. 24 of the samples contained IgG reactivity with A431 cells at > 1:100 titer, and 17 had evidence of IgG internalization. We generated hybridomas using peripheral blood B-cells isolated from two of these lung cancer patients, as well as B-cells from a lymph node of a stage I, ER+PR+ breast cancer patient. We characterized the binding of four mAbs using immunohistochemistry (IHC), microscopy, flow cytometry and immunoblotting. One mAb was used to identify its cognate antigen by immunoprecipitation and mass spectrometry. Results: In a panel of 59 lung cancer patient sera, 24 had IgG that bound the A431 cell line, and 17 of these had evidence of internalization. We isolated three human mAbs from two lung cancer patients and one mAb from the breast cancer lymph node. We performed IHC with three of the mAbs in formalin fixed, paraffin embedded normal and tumor tissues and found primarily tumor cell binding to a variety of tumor types. The 5A6 (lung) bound 3 of 4 non-small cell lung, 3 of 4 colon, 2 of 4 ovarian and 1 of 4 small cell lung cancers. 19F10 (lung) bound 1 of 4 colon and 2 of 4 small cell lung. 2G4 (breast) bound 4 of 4 non-small cell lung, 3 of 4 prostate, 3 of 4 ovarian, and 2 of 4 colon cancers. Some normal tissue binding was observed, such as small intestine and distal renal tubule (2G4 and 19F10), pancreatic islet (19F10), and prostate (2G4). The 6A10 mAb (lung) bound to the constitutive heat shock protein, HSC70, which was localized in the plasma membrane of cancer cell lines. Conclusion: We have cloned 4 human mAbs from cancer patients. This supports the hypothesis that patients with active malignancies make antibodies capable of binding cancer-specific antigens. We identified HSC70, a protein that has an altered cell localization in cancer cells, as one such antigen. The three mAbs tested by IHC bound to diverse tumor types that often differed from the tumors of the patients where the B-cells originated. The human anti-cancer antibody repertoire holds promise as a tool to identify new targets for mAb-based cancer diagnostics and therapeutics. Citation Format: Huiwu Zhao, Jiping Zhang, Ramdev Puligedda, Cezary Swider, Paul Simon, Baron Heimbach, Sharad Adekar, Maureen Murphy, Hossein Borghaei, Scott Dessain. Tumor-specific human monoclonal antibodies isolated from cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2014-3625

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

In heparin-induced thrombocytopenia (HIT), patients receiving heparin develop IgG antibodies that bind complexes formed between heparin and platelet factor 4 (PF4). Most patients are asymptomatic, but some develop life- and limb-threatening arterial or venous thrombosis. We previously described a transgenic mouse model of HIT that demonstrated four factors are necessary and sufficient to recapitulate the clinical manifestations of HIT with thrombosis: heparin, human PF4, a heparin/PF4 antibody, and platelet activation via FcγRIIA. We hypothesize that specific quantitative and qualitative characteristics of heparin/PF4 antibodies determine which patients with HIT will develop thrombosis. Because heparin/PF4 antibodies isolated from patients with HIT are generally polyclonal and heterogeneous, it has been impossible to directly test this hypothesis. We recently developed a novel means of cloning human antibodies, in which we fuse primary human B-cells to a murine cell line that ectopically expresses human telomerase (hTERT) and murine interleukin-6 (mIL-6). This method readily generates heterohybridoma cells that stably secrete human antibodies. We have used this method to clone human heparin/PF4 antibodies from patients with HIT and thrombosis. We identified a patient with clinical HIT, exhibiting a heparin-dependent drop in platelet counts, plasma heparin/PF4 IgG antibodies by ELISA, and a deep venous thrombosis. We fused peripheral blood lymphocytes from this patient to our novel fusion partner cell line, and isolated 2 independent hybridomas that express IgM antibodies that strongly bind heparin/PF4 complexes. We assayed the specificity of the cloned antibodies by testing serial dilutions for heparin/PF4 binding activity by ELISA. Each cloned antibody bound heparin/PF4 with an absorbance >2X background at dilutions that ranged from 1:32–1:64, whereas three negative control antibodies gave no detectable binding at dilutions of 1:2 or greater. cDNA sequencing indicated that both antibodies have the same heavy chain sequences, including a novel CDR3 region, suggesting that the antibodies were derived from post-germinal center memory B-cells rather than from naive B-cells. Using recombinant DNA techniques and ectopic expression in CHO cells, we produced an IgG1 version of one of these antibodies (8E5). By ELISA, the 8E5 IgG1 antibody binds human PF4 at optimal ratio of 10 μg/ml to 0.4 U/ml of heparin (see Figure). We are currently exploring the nature of the 8E5 IgG/heparin/PF4 complex and its platelet activating/thrombotic potential. Figure Figure