PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages.

The immune suppressive microenvironment is a major culprit for difficult-to-treat solid cancers. Particularly, inhibitory tumor-associated macrophages (TAM) define the resistant nature of the tumor milieu. To define tumor-enabling mechanisms of TAMs, we analyzed molecular clinical datasets correlating cell surface receptors with the TAM infiltrate. Though P-selectin glycoprotein ligand-1 (PSGL-1) is found on other immune cells and functions as an adhesion molecule, PSGL-1 is highly expressed on TAMs across multiple tumor types. siRNA-mediated knockdown and antibody-mediated inhibition revealed a role for PSGL-1 in maintaining an immune suppressed macrophage state. PSGL-1 knockdown or inhibition enhanced proinflammatory mediator release across assays and donors in vitro. In several syngeneic mouse models, PSGL-1 blockade alone and in combination with PD-1 blockade reduced tumor growth. Using a humanized tumor model, we observed the proinflammatory TAM switch following treatment with an anti-PSGL-1 antibody. In ex vivo patient-derived tumor cultures, a PSGL-1 blocking antibody increased expression of macrophage-derived proinflammatory cytokines, as well as IFNγ, indicative of T-cell activation. Our data demonstrate that PSGL-1 blockade reprograms TAMs, offering a new therapeutic avenue to patients not responding to T-cell immunotherapies, as well as patients with tumors devoid of T cells. SIGNIFICANCE: This work is a significant and actionable advance, as it offers a novel approach to treating patients with cancer who do not respond to T-cell checkpoint inhibitors, as well as to patients with tumors lacking T-cell infiltration. We expect that this mechanism will be applicable in multiple indications characterized by infiltration of TAMs.

Age-associated insolubility of parkin in human midbrain is linked to redox balance and sequestration of reactive dopamine metabolites.

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.

Effector function of anti-pyroglutamate-3 Aß antibodies affects cognitive benefit, glial activation and amyloid clearance in Alzheimer's-like mice.

BACKGROUND: Pyroglutamate-3 Aß (pGlu-3 Aß) is an N-terminally truncated and post-translationally modified Aß species found in Alzheimer's disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. We address the question of how the effector function of an anti-pGlu-3 Aß antibody influences the efficacy of immunotherapy in mouse models with AD-like pathology. METHODS: We compared two different immunoglobulin (Ig) isotypes of the same murine anti-pGlu-3 Aß mAb (07/1 IgG1 and 07/2a IgG2a) and a general N-terminal Aß mAb (3A1 IgG1) for their ability to clear Aß and protect cognition in a therapeutic passive immunotherapy study in aged, plaque-rich APPSWE/PS1ΔE9 transgenic (Tg) mice. We also compared the ability of these antibodies and a CDC-mutant form of 07/2a (07/2a-k), engineered to avoid complement activation, to clear Aß in an ex vivo phagocytosis assay and following treatment in APPSLxhQC double Tg mice, and to activate microglia using longitudinal microPET imaging with TSPO-specific 18F-GE180 tracer following a single bolus antibody injection in young and old Tg mice. RESULTS: We demonstrated significant cognitive improvement, better plaque clearance, and more plaque-associated microglia in the absence of microhemorrhage in aged APPSWE/PS1ΔE9 Tg mice treated with 07/2a, but not 07/1 or 3A1, compared to PBS in our first in vivo study. All mAbs cleared plaques in an ex vivo assay, although 07/2a promoted the highest phagocytic activity. Compared with 07/2a, 07/2a-k showed slightly reduced affinity to Fcγ receptors CD32 and CD64, although the two antibodies had similar binding affinities to pGlu-3 Aß. Treatment of APPSLxhQC mice with 07/2a and 07/2a-k mAbs in our second in vivo study showed significant plaque-lowering with both mAbs. Longitudinal 18F-GE180 microPET imaging revealed different temporal patterns of microglial activation for 3A1, 07/1, and 07/2a mAbs and no difference between 07/2a-k and PBS-treated Tg mice. CONCLUSION: Our results suggest that attenuation of behavioral deficits and clearance of amyloid is associated with strong effector function of the anti-pGlu-3 Aß mAb in a therapeutic treatment paradigm. We present evidence that antibody engineering to reduce CDC-mediated complement binding facilitates phagocytosis of plaques without inducing neuroinflammation in vivo. Hence, the results provide implications for tailoring effector function of humanized antibodies for clinical development.

An in vitro paradigm to assess potential anti-Aß antibodies for Alzheimer's disease.

Although the amyloid ß-protein (Aß) is believed to play an initiating role in Alzheimer's disease (AD), the molecular characteristics of the key pathogenic Aß forms are not well understood. As a result, it has proved difficult to identify optimal agents that target disease-relevant forms of Aß. Here, we combined the use of Aß-rich aqueous extracts of brain samples from AD patients as a source of human Aß and live-cell imaging of iPSC-derived human neurons to develop a bioassay capable of quantifying the relative protective effects of multiple anti-Aß antibodies. We report the characterization of 1C22, an aggregate-preferring murine anti-Aß antibody, which better protects against forms of Aß oligomers that are toxic to neurites than do the murine precursors of the clinical immunotherapeutics, bapineuzumab and solanezumab. These results suggest further examination of 1C22 is warranted, and that this bioassay maybe useful as a primary screen to identify yet more potent anti-Aß therapeutics.

An anti-pyroglutamate-3 Aß vaccine reduces plaques and improves cognition in APPswe/PS1ΔE9 mice.

Pyroglutamate-3 amyloid-beta (pGlu-3 Aß) is an N-terminally truncated Aß isoform likely playing a decisive role in Alzheimer's disease pathogenesis. Here, we describe a prophylactic passive immunization study in APPswe/PS1ΔE9 mice using a novel pGlu-3 Aß immunoglobulin G1 (IgG1) monoclonal antibody, 07/1 (150 and 500 µg, intraperitoneal, weekly) and compare its efficacy with a general Aß IgG1 monoclonal antibody, 3A1 (200 µg, intraperitoneal, weekly) as a positive control. After 28 weeks of treatment, plaque burden was reduced and cognitive performance of 07/1-immunized Tg mice, especially at the higher dose, was normalized to wild-type levels in 2 hippocampal-dependent tests and partially spared compared with phosphate-buffered saline-treated Tg mice. Mice that received 3A1 had reduced plaque burden but showed no cognitive benefit. In contrast with 3A1, treatment with 07/1 did not increase the concentration of Aß in plasma, suggesting different modes of Aß plaque clearance. In conclusion, early selective targeting of pGlu-3 Aß by immunotherapy may be effective in lowering cerebral Aß plaque burden and preventing cognitive decline in the clinical setting. Targeting this pathologically modified form of Aß thereby is unlikely to interfere with potential physiologic function(s) of Aß that have been proposed.

IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aß and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aß- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aß and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

Anti-Aß antibodies incapable of reducing cerebral Aß oligomers fail to attenuate spatial reference memory deficits in J20 mice.

Compelling genetic evidence links the amyloid precursor protein (APP) to Alzheimer's disease (AD). A leading hypothesis proposes that a small amphipathic fragment of APP, the amyloid ß-protein (Aß), self-associates to form soluble assemblies loosely referred to as "oligomers" and that these are primary mediators of synaptic dysfunction. As such, Aß, and specifically Aß oligomers, are targets for disease modifying therapies. Currently, the most advanced experimental treatment for AD relies on the use of anti-Aß antibodies. In this study, we tested the ability of the monomer-preferring antibody, m266 and a novel aggregate-preferring antibody, 1C22, to attenuate spatial reference memory impairments in J20 mice. Chronic treatment with m266 resulted in a ~70-fold increase in Aß detected in the bloodstream, and a ~50% increase in water-soluble brain Aß--and in both cases Aß was bound to m266. In contrast, 1C22 increased the levels of free Aß in the bloodstream, and bound to amyloid deposits in J20 brain. However, neither 1C22 nor m266 attenuated the cognitive deficits evident in 12month old J20 mice. Moreover, both antibodies failed to alter the levels of soluble Aß oligomers in J20 brain. These results suggest that Aß oligomers may mediate the behavioral deficits seen in J20 mice and highlight the need for the development of aggregate-preferring antibodies that can reach the brain in sufficient levels to neutralize bioactive Aß oligomers. Aside from the lack of positive effect of m266 and 1C22 on cognition, a substantial number of deaths occurred in m266- and 1C22-immunized J20 mice. These fatalities were specific to anti-Aß antibodies and to the J20 mouse line since treatment of wild type or PDAPP mice with these antibodies did not cause any deaths. These and other recent results indicate that J20 mice are particularly susceptible to targeting of the APP/Aß/tau axis. Notwithstanding the specificity of fatalities for J20 mice, it is worrying that the murine precursor (m266) of a lead experimental therapeutic, Solanezumab, did not engage with putatively pathogenic Aß oligomers.

A human monoclonal IgG that binds aß assemblies and diverse amyloids exhibits anti-amyloid activities in vitro and in vivo.

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 potential in vitro and in 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 Aß 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 Aß and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aß in 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 Aß 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.

Specific amyloid ß clearance by a catalytic antibody construct.

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid ß (Aß)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aß C terminus noncovalently and hydrolyzed Aß rapidly, with no reactivity to the Aß precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aß dipeptide unit. The catabody dissolved preformed Aß aggregates and inhibited Aß aggregation more potently than an Aß-binding IgG. Intravenous catabody treatment reduced brain Aß deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aß hydrolysis appears to be an innate immune function that could be applied for therapeutic Aß removal.

Tau immunization: a cautionary tale?

The amyloid ß (Aß)-protein and microtubule-associated protein, tau, are the major components of the amyloid plaques and neurofibrillary tangles that typify Alzheimer's disease (AD) pathology. As such both Aß and tau have long been proposed as therapeutic targets. Immunotherapy, particularly targeting Aß, is currently the most advanced clinical strategy for treating AD. However, several Aß-directed clinical trials have failed, and there is concern that targeting this protein may not be useful. In contrast, there is a growing optimism that tau immunotherapy may prove more efficacious. Here, for the first time, we studied the effects of chronic administration of an anti-tau monoclonal antibody (5E2) in amyloid precursor protein transgenic mice. For our animal model, we chose the J20 mouse line because prior studies had shown that the cognitive deficits in these mice require expression of tau. Despite the fact that 5E2 was present and active in the brains of immunized mice and that this antibody appeared to engage with extracellular tau, 5E2-treatment did not recover age-dependent spatial reference memory deficits. These results indicate that the memory impairment evident in J20 mice is unlikely to be mediated by a form of extracellular tau recognized by 5E2. In addition to the lack of positive effect of anti-tau immunotherapy, we also documented a significant increase in mortality among J20 mice that received 5E2. Because both the J20 mice used here and tau transgenic mice used in prior tau immunotherapy trials are imperfect models of AD our results recommend extensive preclinical testing of anti-tau antibody-based therapies using multiple mouse models and a variety of different anti-tau antibodies.

Aß dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies.

Dimers of Aß (amyloid ß-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aß](DiY) (dityrosine cross-linked Aß). For comparison, we used the Aß monomer and a design dimer cross-linked by replacement of Ser²6 with cystine [AßS26C]2. We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [Aß](DiY) and [AßS26C]2 have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aß monomers. Our results indicate that the link between Aß dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.

Physiological IgM class catalytic antibodies selective for transthyretin amyloid.

Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded ß-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid ß peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.

Transthyretin aggregate-specific antibodies recognize cryptic epitopes on patient-derived amyloid fibrils.

Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses.

Human anti-Aß IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Soluble non-fibrillar assemblies of amyloid-beta (Aß) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aß immunotherapy is a promising and advanced therapeutic strategy, but the precise Aß species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aß conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aß dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aß extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aß's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aß monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aß NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aß NAbs are warranted.

A monoclonal antibody against synthetic Aß dimer assemblies neutralizes brain-derived synaptic plasticity-disrupting Aß.

Diverse lines of evidence indicate that pre-fibrillar, diffusible assemblies of the amyloid ß-protein (Aß) play an important role in Alzheimer's disease pathogenesis. Although the precise molecular identity of these soluble toxins remains unsettled, recent experiments suggest that sodium dodecyl sulfate (SDS)-stable Aß dimers may be the basic building blocks of Alzheimer's disease-associated synaptotoxic assemblies and as such present an attractive target for therapeutic intervention. In the absence of sufficient amounts of highly pure cerebral Aß dimers, we have used synthetic disulfide cross-linked dimers (free of Aß monomer or fibrils) to generate conformation-specific monoclonal antibodies. These dimers aggregate to form kinetically trapped protofibrils, but do not readily form fibrils. We identified two antibodies, 3C6 and 4B5, which preferentially bind assemblies formed from covalent Aß dimers, but do not bind to Aß monomer, amyloid precursor protein, or aggregates formed by other amyloidogenic proteins. Monoclonal antibody 3C6, but not an IgM isotype-matched control antibody, ameliorated the plasticity-disrupting effects of Aß extracted from the aqueous phase of Alzheimer's disease brain, thus suggesting that 3C6 targets pathogenically relevant Aß assemblies. These data prove the usefulness of covalent dimers and their assemblies as immunogens and recommend further investigation of the therapeutic and diagnostic utility of monoclonal antibodies raised to such assemblies.

Amyloid beta-protein dimers rapidly form stable synaptotoxic protofibrils.

Nonfibrillar, water-soluble low-molecular weight assemblies of the amyloid ß-protein (Aß) are believed to play an important role in Alzheimer's disease (AD). Aqueous extracts of human brain contain Aß assemblies that migrate on SDS-polyacrylamide gels and elute from size exclusion as dimers (∼8 kDa) and can block long-term potentiation and impair memory consolidation in the rat. Such species are detected specifically and sensitively in extracts of Alzheimer brain suggesting that SDS-stable dimers may be the basic building blocks of AD-associated synaptotoxic assemblies. Consequently, understanding the structure and properties of Aß dimers is of great interest. In the absence of sufficient brain-derived dimer to facilitate biophysical analysis, we generated synthetic dimers designed to mimic the natural species. For this, Aß(1-40) containing cysteine in place of serine 26 was used to produce disulphide cross-linked dimer, (AßS26C)2. Such dimers had no detectable secondary structure, produced an analytical ultracentrifugation profile consistent for an ∼8.6 kDa protein, and had no effect on hippocampal long-term potentiation (LTP). However, (AßS26C)2 aggregated more rapidly than either AßS26C or wild-type monomers and formed parastable ß-sheet rich, thioflavin T-positive, protofibril-like assemblies. Whereas wild-type Aß aggregated to form typical amyloid fibrils, the protofibril-like structures formed by (AßS26C)2 persisted for prolonged periods and potently inhibited LTP in mouse hippocampus. These data support the idea that Aß dimers may stabilize the formation of fibril intermediates by a process distinct from that available to Aß monomer and that higher molecular weight prefibrillar assemblies are the proximate mediators of Aß toxicity.

Anti-amyloidogenic activity of IgGs contained in normal plasma.

INTRODUCTION: We have previously shown that a subpopulation of naturally occurring human IgGs has therapeutic potential for the amyloid-associated disorders. These molecules cross-react with conformational epitopes on amyloidogenic assemblies, including amyloid beta (Abeta) protein fibrils that are a pathological hallmark of Alzheimer's disease. MATERIALS AND METHODS: Using our europium-linked immunosorbant assay, we established that approximately 95% of 260 screened donor plasma samples had amyloid fibril-reactive IgGs and Abeta conformer-reactive IgGs with minimal binding to Abeta monomers. Anti-amyloidogenic reactivity was diverse and attributed to Abeta targeting multiple fibril-related binding sites and/or variations in multidentate binding. RESULTS AND DISCUSSION: There was no correlation between anti-fibril and anti-oligomer reactivity and donor age (19 to 60 years old) or gender. These findings demonstrate the inherent but diverse anti-amyloidogenic activity of natural IgGs contained in normal plasma. CONCLUSION: Our studies provide support for investigating the clinical significance and physiological function of this novel class of antibodies.

Inherent anti-amyloidogenic activity of human immunoglobulin gamma heavy chains.

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.

Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope.

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.