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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.

Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

Worldwide, the incidences of cancer are rising. Various environmental and genetic factors are predisposing individuals to cancer. Majority of these factors result in upregulation of pro-survival pathways, downregulation of tumor suppressors, and chronic inflammation. The ratio of w-6/w-3 polyunsaturated fatty acids (PUFAs) plays very crucial role in the initiation and progression of cancer. Low w-6/w-3 PUFA ratio has been shown beneficial in managing the hallmarks of cancer cell. Enormous data from cancer cell line and in vivo cancer models have given insight into the mechanisms underlying the anticancer effects of w-3 PUFAs. Here, we discussed major possible mechanisms for beneficial effects of w-3 PUFAs as evidenced by the preclinical in vitro cancer cell line models and in vivo models.

Supplementation of all essential nutrients in daily diet is must, and omega-3 is the most crucial nutrient today. Anti-nutrients, pollutants should be avoided. Good nutrition helps to renew our body with healthy cells. Seemingly, opposite acting metabolism in both plants and animal life-forms works for creating reducing atmosphere, by deriving NADH and NADPH, reduced molecules, and by processing the food within plant cell and animal cell. Life is a process of defeating entropy, to keep the oxidative stress in check. Vegetarianism is healthier, naturally richer in antioxidants, the reduced molecules. Unlike plant forms, we are crippled in evolution and we need 40 odd essential nutrients to keep our body in a healthy state. Adequate supply of all the essential nutrients, at all times, is pivotal to keep all our faculties fit and fine till last. Although homeostasis keeps metabolites within normal limits, occasional drift by a process called allostasis may also be necessary by a way of readjustment. Redox seems to be playing a very vital role in every cell’s decision, to either remain in a healthy state of life, disease state or die or become immortal and assume a cancerous state.

Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potentialin vitroandin vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aβ and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aβ and LC amyloid formationin vitroand abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aβin vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aβ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

An essential requirement for eradication of poliomyelitis is the elimination of circulating vaccine derived polioviruses (cVDPV) and polioviruses excreted by chronically infected individuals with immunodeficiencies (iVDPV). As part of a post-eradication risk management strategy, a human monoclonal antibody (mAb) therapeutic could play a role in halting excretion in asymptomatic carriers and could be used, in combination with vaccines and antiviral drugs, to protect polio-exposed individuals. Cross-neutralizing mAbs may be particularly useful, as they would reduce the number of mAbs needed to create a comprehensive PV therapeutic. We cloned a panel of IgG mAbs from OPV-vaccinated, IPV-boosted healthy subjects. Many of the mAbs had potent neutralizing activities against PV wild-type (WT) and Sabin strains, and two of the mAbs, 12F8 and 1E4, were significantly cross-reactive against types 1 and 2 and types 1 and 3, respectively. Mapping the binding epitopes using strains resistant to neutralization (escape mutants) suggested that cross-specific PV binding epitopes may primarily reside within the canyon region, which interacts with the cellular receptor molecule CD155 and the cross-neutralizing chimpanzee/human mAb, A12. Despite their close proximity, the epitopes for the 12F8 and 1E4 mAbs on Sabin 1 were not functionally identical to the A12 epitope. When tested together, 12F8 and 1E4 neutralized a diverse panel of clinically relevant PV strains and did not exhibit interference. Virus mutants resistant to the anti-poliovirus drug V-073 were also neutralized by the mAbs. The 12F8 and 1E4 mAbs may suitable for use as anti-PV therapeutics.

We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of Abeta, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig gamma heavy chains. A gamma(1) heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloid-laden tissue sections. F1 did not bind to native Abeta monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig gamma heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble Abeta oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig gamma heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules.

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

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