Phase 1a/b study of monoclonal antibody CAEL-101 (11-1F4) in patients with AL amyloidosis.

Systemic immunoglobulin light-chain amyloidosis is characterized by pathologic deposition of immunoglobulin light chains as amyloid fibrils in vital organs, leading to organ impairment and eventual death. That the process is reversible was evidenced in an in vivo experimental model in which fibril-reactive chimeric monoclonal antibody (mAb) 11-1F4 directly targeted human light-chain amyloid deposits and affected their removal via a phagocyte-mediated response. To determine the tolerability and potential amyloidolytic effect of this agent (now designated mAb CAEL-101), we conducted a phase 1a/b study involving 27 patients, most of whom had manifestations of organ involvement. This was an open-label study in which phase 1a patients received mAb CAEL-101 as a single intravenous infusion with escalating dose levels from 0.5 mg/m2 to 500 mg/m2 to establish the maximum tolerated dose (MTD). In phase 1b, the antibody was administered as a graded series of 4 weekly infusions. For both phases, there were no drug-related serious adverse events or dose-limiting toxicities among recipients, and the MTD was not reached. The majority of patients had deep hematologic responses but persistent organ disease prior to treatment. Fifteen of 24 patients (63%) who manifested cardiac, renal, hepatic, gastrointestinal, or soft tissue involvement had a therapeutic response to mAb CAEL-101 as evidenced by serum biomarkers or objective imaging modalities with a median time to response of 3 weeks. Infusions of mAb CAEL-101 were well tolerated and, for the majority, resulted in improved organ function, notably for those with cardiac impairment. This trial was registered at www.clinicaltrials.gov as #NCT02245867.

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.

Clinical, morphologic, and genetic features of renal leukocyte chemotactic factor 2 amyloidosis.

Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a recently described form of amyloidosis that most frequently manifests clinically with progressive renal failure. In a series of 414 cases of amyloidosis, there were 40 cases of ALECT2: the second most common type of renal amyloidosis in this series. This was particularly common in Hispanic patients in the Southwest United States, where more than half of amyloidosis cases were ALECT2. It is possible that this represents a familial amyloidosis as there were two brothers with ALECT2 in our study. Morphologically, there was consistent amyloid deposition in the renal cortex with medullary involvement in only about a third of cases. There were no mutations detected in the LECT2 gene, although all patients tested were homozygous for the G nucleotide in a non-synonymous SNP at position 172. Most patients presented with chronic kidney disease and, on follow-up, showed progression with an average deterioration in renal function of 0.5 ml/min/1.73 m(2) per month. Unfortunately, the etiology of ALECT2 is currently unknown and there is currently no efficacious treatment of the disease.

Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling.

BACKGROUND: The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. METHODS: The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM-transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. RESULTS: ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. CONCLUSIONS: The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior.

Radioimmunodetection of amyloid deposits in patients with AL amyloidosis.

Care of patients with AL amyloidosis currently is limited by the lack of objective means to document disease extent, as well as therapeutic options that expedite removal of pathologic deposits. To address these issues, we have initiated a Phase I Exploratory IND study to determine the biodistribution of the fibril-reactive, amyloidolytic murine IgG1 mAb 11-1F4 labeled with I-124. Patients were infused with less than 1 mg (∼ 74 MBq) of GMP-grade antibody and imaged by PET/CT scan 48 and 120 hours later. Among 9 of 18 subjects, there was striking uptake of the reagent in liver, lymph nodes, bone marrow, intestine, or, unexpectedly, spleen (but not kidneys or heart). Generally, positive or negative results correlated with those obtained immunohistochemically using diagnostic tissue biopsy specimens. Based on these findings, we posit that (124)I-mAb m11-1F4 can be used to identify AL candidates for passive immunotherapy using the chimeric form of the antibody.

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.

Splenic plasma cells can serve as a source of amyloidogenic light chains.

Bone marrow-derived clonal plasma cells, as found in systemic amyloidogenic light chain-associated (AL) amyloidosis, are presumed to be the source of light chains that deposit as fibrils in tissues throughout the body. Paradoxically, people with this disorder, in contrast to multiple myeloma, often have a low percentage of such cells, and it is unknown whether this relatively sparse number can synthesize enough amyloidogenic precursor to form the extensive pathology that occurs. To investigate whether another hematopoietic organ, the spleen, also contains monoclonal light chain-producing plasma cells, we have immunostained such tissue from 26 AL patients with the use of antiplasma cell, antifree kappa and lambda, and anti-V(L) subgroup-specific monoclonal antibodies (mAbs). In 12 cases, there was statistically significant evidence of a monoclonal population bearing the same kappa or lambda isotype as that within the bone marrow and identical to the amyloid. Our studies have shown that the spleen may be another source of amyloidogenic light chains.

Prevalence and morphology of leukocyte chemotactic factor 2-associated amyloid in renal biopsies.

Renal pathologists identify the protein component of renal amyloid deposits by immunohistochemistry using antibodies against known amyloidogenic proteins. The majority of amyloid cases can be categorized by a simple antibody panel that includes immunoglobulin light chains lambda and kappa, and serum amyloid A. In some instances, however, these reagents do not recognize materials that stain with Congo red or yield ambiguous staining results, thus creating a diagnostic dilemma. Chemical analysis of fibrils extracted from such a nonreactive renal biopsy led to the discovery of a previously unknown amyloid formed from leukocyte chemotactic factor 2 (LECT2). Over the past 8 years, we received 285 renal amyloid samples, of which 31 remained unclassified. In an effort to determine whether any of the latter samples were LECT2 related, tandem mass spectrometry was performed. In all, 7 of the 31 cases were identified as an amyloid LECT2 (ALECT2), a finding confirmed immunohistochemically using a LECT2-specific antibody. The deposits strongly stained for Congo red and, in most cases, had distinctive morphological features with diffuse involvement of the interstitium, arteries, and glomeruli. Hence, we believe that ALECT2 represents the third common form of renal amyloidosis.

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.

Leukocyte chemotactic factor 2 (LECT2)-associated renal amyloidosis: a case series.

BACKGROUND: Renal amyloidosis is characterized by the pathologic deposition within glomeruli and/or interstitium of congophilic fibrils, most often composed of either immunoglobulin light chains or serum amyloid A-related protein and, less commonly, mutated forms of apolipoproteins AI or AII, lysozyme, fibrinogen, gelsolin, or transthyretin. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 10 patients with renal amyloidosis who had an amyloidogenic protein that was not identified using routine immunohistochemistry. OUTCOMES: Clinical, pathologic, biochemical, and genetic characteristics. MEASUREMENTS: Tandem mass spectrometry was used to analyze fibrils extracted from sections of formalin-fixed paraffin-embedded amyloid-containing kidney biopsy specimen blocks. RESULTS: Chemical analyses showed peptides corresponding to the carboxy-terminal portion of the leukocyte chemotactic factor 2 (LECT2) molecule. In addition, deposits were immunostained using an anti-human LECT2 monoclonal antibody. Plasma specimens were available from 2 individuals for whom LECT2 concentration in these samples was within the reference range. Additionally, in 4 of the cases analyzed at the molecular level, isolation of genomic DNA and polymerase chain reaction amplification of LECT2-encoding exons showed no mutations. However, all were homozygous for the G allele encoding valine at position 40 in the mature protein, a finding confirmed using restriction enzyme analysis of the polymorphic site. LIMITATIONS: Causality is not addressed. CONCLUSIONS: Based on our studies, we posit that LECT2-associated renal amyloidosis represents a unique and perhaps not uncommon disease, especially in Mexican Americans. The pathogenesis, extent, and prognosis remain to be determined.

Amyloidogenic potential of foie gras.

The human cerebral and systemic amyloidoses and prion-associated spongiform encephalopathies are acquired or inherited protein folding disorders in which normally soluble proteins or peptides are converted into fibrillar aggregates. This is a nucleation-dependent process that can be initiated or accelerated by fibril seeds formed from homologous or heterologous amyloidogenic precursors that serve as an amyloid enhancing factor (AEF) and has pathogenic significance in that disease may be transmitted by oral ingestion or parenteral administration of these conformationally altered components. Except for infected brain tissue, specific dietary sources of AEF have not been identified. Here we report that commercially available duck- or goose-derived foie gras contains birefringent congophilic fibrillar material composed of serum amyloid A-related protein that acted as a potent AEF in a transgenic murine model of secondary (amyloid A protein) amyloidosis. When such mice were injected with or fed amyloid extracted from foie gras, the animals developed extensive systemic pathological deposits. These experimental data provide evidence that an amyloid-containing food product hastened the development of amyloid protein A amyloidosis in a susceptible population. On this basis, we posit that this and perhaps other forms of amyloidosis may be transmissible, akin to the infectious nature of prion-related illnesses.

Odontogenic ameloblast associated protein as a novel biomarker for human breast cancer.

Odontogenic Ameloblast Associated Protein (ODAM) is a protein isolated in ameloblasts during odontogenesis. ODAM expression was identified in breast cancer, but its significance remains unknown. The purpose of this study is to determine if ODAM expression can serve as a prognostic marker and provide information regarding treatment in human breast cancer. Breast cancer patients were identified from our tumor registry from 1993 to 2003. Archived breast cancer tissue from 243 patients (stage 0 = 53, stage I = 51, stage II = 53, stage III = 47, stage IV = 39) was stained using monoclonal antibody for ODAM. Presence or absence of immunostaining was correlated with stage, histologic grade, response to chemotherapy, and survival using chi2 and logistic regression analyses. Tumor nuclear staining for ODAM increased with increasing group stage (P < 0.001). Staining for ODAM did not correlate with histologic grade or chemotherapy (P = 0.558, P = 0.093). Improved outcomes within each stage were noted with ODAM staining, statistically significant for stages 0, I, and II (P < 0.001, P = 0.003, P = 0.003) and underpowered for stages III and IV (P = 0.724, P = 0.059). Survival benefit associated with tumor nuclear staining increased with advancing stage (P < 0.001). These results show that ODAM predicts survival in breast cancer. Research is ongoing to determine ODAM's clinical utility and role in carcinogenesis.

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.

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains.

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

Quantitative tomography of early-onset spontaneous AA amyloidosis in interleukin 6 transgenic mice.

Mice that constitutively express the human interleukin 6 (huIL6) protein from a heritable transgene (H2-L(d)-IL-6) express high levels of the acute-phase reactant, serum amyloid protein A, a liver-derived apoprotein of high-density lipoprotein that is the precursor of AA amyloid. Typically at approximately 5 mo of age B6(C)- Tg(H2-L(d)-IL-6)Kish (H2/huIL-6) animals begin to develop splenic deposits of AA amyloid, which progress to involve the liver, kidney, and vasculature, ultimately resulting in death due to severe systemic AA amyloidosis at 8 to 9 mo of age. These mice provide a robust model in which to study novel therapeutic and diagnostic imaging agents for AA amyloidosis. We recently have noted a change in onset of spontaneous disease, as evidenced by 2 female transgenic mice that were found moribund at only 5 mo of age. Extensive hepatosplenic amyloid deposits in both mice were identified and quantified by single-photon emission computed tomography, which further revealed heterogeneous distribution of radiotracer in the spleen indicating a distinction between amyloid-laden red pulp and the disease-free lymphoid follicles. The AA nature of the deposits was evidenced immunohistochemically and by mass spectrometric analyses of extracted amyloid fibrils. Our studies have documented the manifestation of early-onset, severe, spontaneous AA amyloidosis in 2- to 5-mo-old H2/ huIL-6 mice; we hypothesize that this disease is due to genetic rather than environmental factors.

Characterization of systemic amyloid deposits by mass spectrometry.

The human systemic (noncerebral) amyloidoses represent a heterogeneous group of disorders characterized by the widespread deposition of proteins as fibrils in organs or tissues throughout the body. The unequivocal identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these illnesses. Heretofore, this information was inferred from clinical data, ancillary laboratory tests, and results of immunohistochemical, as well as genetic, analyses. However, to establish definitively the type of amyloid present, the chemical composition of the fibrillar components must be determined. For this purpose, we have developed micro-methods, whereby this information can be obtained by tandem mass spectrometry (MS/MS) using material extracted from formalin-fixed, amyloid-containing tissue biopsy specimens or subcutaneous fat aspirates. The ability to identify precisely the protein nature of the pathologic deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disease.

Micro-imaging of amyloid in mice.

Scintigraphic imaging of radioiodinated serum amyloid P-component is a proven method for the clinical detection of peripheral amyloid deposits (Hawkins et al., 1990). However, the inability to perform comparably high-resolution studies in experimental animal models of amyloid disease has impacted not only basic studies into the pathogenesis of amyloidosis but also in the preclinical in vivo evaluation of potential anti-amyloid therapeutic agents. We have developed microimaging technologies, implemented novel computational methods, and established protocols to generate high-resolution images of amyloid deposits in mice. (125)I-labeled serum amyloid P component (SAP) and an amyloid-fibril reactive murine monoclonal antibody (designated 11-1F4) have been used successfully to acquire high-resolution single photon emission computed tomographic (SPECT) images that, when fused with x-ray computed tomographic (CT) data, have provided precise anatomical localization of secondary (AA) and primary (AL) amyloid deposits in mouse models of these diseases. This chapter will provide detailed protocols for the radioiodination and purification of amyloidophilic proteins and the generation of mouse models of AA and AL amyloidosis. A brief description of the available hardware and the parameters used to acquire high-resolution microSPECT and CT images is presented, and the tools used to perform image reconstruction and visualization that permit the analysis and presentation of image data are discussed. Finally, we provide established methods for measuring organ- and tissue-specific activities with which to corroborate the microSPECT and CT images.

Radioimaging of light chain amyloid with a fibril-reactive monoclonal antibody.

UNLABELLED: Currently, there are no available means in the United States to document objectively the location and extent of amyloid deposits in patients with systemic forms of amyloidosis. To address this limitation, we have developed a novel diagnostic strategy, namely, the use of a radiolabeled fibril-reactive murine monoclonal antibody (mAb) as an amyloid-specific imaging agent. The goal of this study was to determine the pharmacokinetics, biodistribution, and ability of this reagent to target the type of amyloid that is formed from immunoglobulin light chains, that is, AL. METHODS: Subcutaneous tumors (amyloidomas) were induced in BALB/c mice by injection of human AL fibrils. The IgG1 mAb designated 11-1F4 and an isotype-matched control antibody were radioiodinated, and the pharmacokinetics and localization of these reagents were determined from blood and tissue samples. Amyloidoma-bearing animals that received (125)I- or (124)I-labeled antibodies were imaged by whole-body small-animal SPECT/CT or small-animal PET/CT technology, respectively. RESULTS: Radioiodinated mAb 11-1F4 retained immunoreactivity, as evidenced by its subnanomolar affinity for light chains immobilized on 96-well microtiter plates and for beads conjugated with a light chain-related peptide. Additionally, after intravenous administration, the labeled reagents had the expected biologic half-life of murine IgG1, with monoexponential whole-body clearance kinetics. In the amyloidoma mouse model, (125)I-11-1F4 was predominately localized in the tumors, as demonstrated in biodistribution and autoradiographic analyses. The mean uptake of this reagent, that is, the percentage injected dose per gram of tissue, 72 h after injection was significantly higher for amyloid than for skeletal muscle, spleen, kidney, heart, liver, or other tissue samples. Notably, the accumulation within the amyloidomas of (125)I- or (124)I-11-1F4 was readily visible in the fused small-animal SPECT/CT or small-animal PET/CT images, respectively. CONCLUSION: Our studies demonstrate the amyloid-imaging capability of a radiolabeled fibril-reactive mAb and provide the basis for a clinical trial designed to determine its diagnostic potential in patients with AL amyloidosis and other systemic amyloidoses.