Patent Application
20160168235
The present invention relates generally to immunology. More specifically, the instant invention provides transthyretin amyloid-selective and polyreactive catabodies and methods of use thereof.
Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Amyloids are ancient threats. Jawed fish, the first extant organisms with an immune system orthologous to humans, produce TTR with sequence similar to human TTR. The human genome encodes no more than 100,000 non-antibody proteins, including all known enzymes and receptors with specificity for a broad range of ligands. The recombined antibody repertoire derived from the germline variable (V), D and J genes is larger by several orders of magnitude, suggesting the possibility of pre-existing specificity for defined targets in the innate antibody repertoire.
Protein self-aggregation forms amyloids with hydrogen-bonded β-sheets. This may fulfill useful functions, e.g., in bacterial curli and chaplains [6], but there is no known physiological function for age-associated accumulation of misfolded amyloids. Twenty seven misfolded proteins are associated with disease (e.g., TTR, Aβ, tau, α-synuclein, huntingtin, κ/λ light chains), and over 40 diseases are associated with misfolded proteins [1]. In the test-tube, particulate aggregates of amyloidogenic proteins are formed within minutes to days. Oligomeric aggregates of amyloidogenic proteins and subclinical tissue amyloid deposition can be detected decades prior to diagnosis of amyloidosis [7-9]. The soluble oligomers exert toxic effects via various receptors and membrane destabilization [10-12]. Particulate TTR amyloid deposits destroy tissue anatomic integrity. Aging is associated with accelerated protein misfolding. Factors thought to contribute to increased TTR amyloidosis include post-translational oxidation reaction [13] and binding to serum amyloid P component [14] or metals [15]. Localized wildtype TTR amyloidosis in certain anatomic areas is early sign of systemic ATTRwt, which usually appears in the sixth decade of life and becomes increasingly frequent thereafter [16]. Localized ATTRwt is frequent in carpal tunnel syndrome, skeletal dysfunction and lumbar stenosis [17, 18]. About 22-25% of humans over 80 years suffer from systemic ATTRwt, involving amyloidosis in the heart, lung, stomach, kidney, skin or other organs [19]. The rate of disease progression depends on the vulnerability of the affected area. Polyneuropathy and cardiomyopathy are frequent. Other than identifying tissue amyloid by invasive tissue biopsy, no unambiguous diagnosis method is available, and ATTRwt is thought to be an under-diagnosed disease. Several mutations in TTR can cause early onset hereditary ATTRm, including the Val122Ile mutation affecting 3-4% of African Americans [20].
No approved drug is available to treat ATTRwt or ATTRm. The standard of clinical care is replacing the failed organ by transplantation (or in ATTRm, transplantation of the liver, the major TTR synthesis site). Tafamidis, a small molecule that binds the T4-binding site of phyTTR [21], may reduce neuropathy in familial amyloidosis [22] but was not approved in the United States because of questionable efficacy. Anti-sense oligonucleotide and short-interfering RNA approaches that inhibit TTR synthesis were reported [23, 24], but these drugs may interfere with the physiological TTR functions. Inducing dissociation of bound serum amyloid P component (SAP) was suggested as a means to clear TTR amyloid [14]. Catechin, a green tea anti-oxidant, and a doxycycline/tauroursodeoxycholic acid combination were also suggested as treatments [25, 26]. However, it is clear that new and improved methods of treating TTR amyloidosis are needed.
The invention describes novel IgM class antibodies in healthy humans with catalytic activity (catabodies) with a unique specificity for the misfolded form of transthyretin (misTTR) responsible for forming misTTR amyloid deposits. The catabodies dissolved particulate misTTR. Development of specificity in classical IgG antibodies requires immunization with the target antigens. IgGs did not hydrolyze misTTR. The specificity of the misTTR-directed IgM catabodies is an innate property that has evolved by Darwinian evolution. The physiologically folded form of transthyretin was not hydrolyzed by the IgM catabodies. By virtue of the peptide bond hydrolysis and product release reactions, detectable immune complexes were not formed. Also disclosed are human monoclonal misTTR-directed catabodies cloned from B cells; electrophilic misTTR analogs used for preparing B cell lines producing the misTTR-directed catabodies and polyreactive catabodies; and methods to prepare the catabodies and the electrophilic analogs. The sera of aged humans with an established diagnosis of TTR amyloidosis displayed reduced misTTR-directed catabody activity accompanied by increased polyreactive catabody activity, suggesting the potential of the catabody tests for diagnosis and prognosis of developing amyloid disease. Specific catabodies may be conceived as components of a natural defense system that remove pathogenic self-antigens. Supplementing the endogenous catabodies with a misTTR-specific monoclonal catabody holds therapeutic potential.
In accordance with one aspect of the instant invention, isolated catalytic antibody or fragment thereof immunologically specific for misfolded transthyretin are provided. The catalytic antibodies of the instant invention catalyze the hydrolysis of misfolded transthyretin, particularly without hydrolysis of the properly folded transthyretin. In a particular embodiment, the catalytic antibody is an IgM or a fragment thereof. In a particular embodiment, the catalytic antibody is innate. The misfolded transthyretin may comprise soluble oligomers or particulate amyloid formed by one or more misfolded β-sheets. The misfolded transthyretin may be mammalian, particularly human. Compositions comprising at least one catalytic antibody or fragment thereof of the instant invention and at least one pharmaceutically acceptable carrier are also provided.
In accordance with another aspect of the instant invention, methods of inhibiting, treating, and/or preventing transthyretin amyloidosis in a subject are provided. In a particular embodiment, the method comprises administering a composition or catalytic antibody or fragment thereof of the instant invention to the subject. The methods may further comprise the administration of at least one other therapy for the treatment of the amyloidosis.
In accordance with another aspect of the instant invention, methods for detecting the presence of catalytic antibodies in a test sample are provided. In a particular embodiment, the method comprises contacting the test sample with misfolded transthyretin and monitoring the hydrolysis of the misfolded transthyretin (e.g., by detecting the presence of smaller hydrolysis products). The instant invention also provides methods for detecting misfolded transthyretin in a test sample. In another particular embodiment, the method comprises contacting the test sample with a catalytic antibody or fragment thereof of the instant invention and monitoring the hydrolysis of misfolded transthyretin contained in the test sample (e.g., by detecting the presence of smaller hydrolysis products).
In accordance with another aspect of the instant invention, methods of diagnosing amyloidosis caused by transthyretin in a subject are provided. In a particular embodiment, the method comprises detecting the presence of a misfolded transthyretin in a biological sample obtained from a subject. In a particular embodiment, the biological sample is blood. In a particular embodiment, the misfolded transthyretin is detected using the catalytic antibodies or fragments thereof of the instant invention.
In accordance with another aspect of the instant invention, polypeptides or analog thereof comprising the epitope of misfolded transthyretin recognized by the catalytic antibody or fragment thereof of the instant invention are provided. Electrophilic analogs of the polypeptide are also encompassed by the instant invention. Methods for isolating catalytic antibodies that hydrolyze misfolded transthyretin using the polypeptide or analog thereof (e.g., electrophilic analog) are also provided. Methods for regulating the synthesis of catalytic antibodies that bind or hydrolyze misfolded transthyretin in an organism are also provided. In a particular embodiment, the method comprises administering the polypeptide or analog thereof (e.g., electrophilic analog).
In accordance with another aspect of the instant invention, isolated polyreactive catalytic antibodies or fragment thereof which hydrolyze oxidized and intracellular senescent antigens are provided. Methods for isolating catalytic antibodies which hydrolyze oxidized and intracellular senescent antigens are also provided.
Various aspects of the invention are unified by the common feature of unique specificity properties of the catalytic antibodies that derive from the innate immune system and factors regulating innate antibody production.
This invention provides specific catalytic antibodies (catabodies) to misfolded transthyretin and the polyreactive catabodies from which the specific catabodies originate. The present invention indicates that the germline IgM repertoire is a source of catabodies with functionally useful specificity for individual amyloid substrates that has evolved over the Darwinian time frame because of the survival advantage of the amyloid clearing function [5]. Developing antibody specificity generally requires the immunogen-driven acquired immune response. Immune tolerance mostly precludes synthesis of pathogenic autoantibodies to essential self-proteins. Amyloids are pathogenic self-protein assemblies, and anti-amyloid antibodies may be beneficial. Over 27 proteins form misfolded amyloid aggregates with β-sheet character [1]. Transthyretin (TTR) is a blood-borne carrier protein for thyroid hormones and the retinol binding protein-retinol complex [2]. Soluble misfolded TTR (misTTR) aggregates formed as byproducts of alternate folding pathways of wildtype TTR act as seed for deposition of pathological TTR amyloid in the heart, lung, joints and other tissues. With advancing age, humans develop wildtype TTR amyloidosis (formal designation, ATTRwt [3]; also known as “senile systemic amyloidosis” or SSA, a disease usually occurring at age >60 years)[4]. The Val122Ile and Val130Met TTR mutations frequently cause early-onset, hereditary TTR amyloidosis (ATTRm).
Humans produce diverse antibodies by: (a) Germline transmission of multiple variable (V)-domain genes (˜200 V-, D-, J-genes) attached to alternate constant (C)-domain genes (μ, δ, γ, α, ε genes;
The ontogeny of the V-domain noncovalent and proteolytic sites is quite different. The FRs contribute residues that participate directly in the catalytic mechanism. Germline-encoded VL-domains hydrolyze peptide bonds promiscuously with no requirement for high affinity epitope recognition [29]. The extensively mutated CDRs form the classical acquired immunity binding sites of IgGs [30]. The IgM C-domain scaffold improves the integrity of the V-domain catalytic site, indicated by the superior catalytic activity of the same V-domains cloned in the IgM versus IgG scaffold [31]. By definition, germline antibody activities evolved from an ancestral organism. Tracing the evolutionary origin of antibody catalysis is of interest to assess its functional importance in the establishment of immune defense systems, including defense against toxic amyloid self-proteins. There is also a practical justification. Antibodies with the greatest catalytic activity are candidate therapeutic agents. A single catalyst molecule can be reused repeatedly to clear thousands of target protein molecules. The unpaired, single domain human VL constructs with catalytic activity are structurally reminiscent of naturally-occurring single V-domain antibodies found in primordial organisms. The single domain VL constructs are described to catalyze the hydrolysis of amyloid β peptide more efficiently than paired IgG VL-VH domains [32] and single chain Fv fragments (scFv, a surrogate of the antibody binding site) [33].
Both innate and adaptive immunity factors are likely to regulate catabody production. Innate catabody synthesis in the absence of contact or stimulation by an antigen is regulated by the autonomous B cell developmental program in conjunction with numerous antigen-independent environmental factors such as cytokines [34-36]. In addition, the defined innate specificity of B cell receptors (BCRs) for a given antigen epitopes can be lost during BCR sequence maturation driven by other irrelevant antigenic epitopes. Contact of BCRs with innate specificity to a given antigenic epitope, e.g., an amyloid epitope, with that epitope itself is also conceived to regulate catabody synthesis negatively or positively. For example, excess concentrations of an amyloid epitope may induce B cell tolerance and down regulate catabody synthesis, whereas at limiting amyloid epitope concentrations, a stimulatory B cell response can occur.
Knowing changes in the activity of catabodies as a function of aging is of interest. A reduction of a specific catabody activity to an individual amyloid protein may accelerate amyloidosis due to that protein, and an increase of such a catabody activity may retard amyloidosis. In addition, variations in the magnitude of the catabody activity found in different humans without clinically-evident amyloidosis may be a useful predictor for the subsequent appearance of amyloid disease. Monoclonal and polyclonal catabodies hold potential as candidate drugs against amyloidosis. Catabodies utilize nucleophilic mechanism to hydrolyze peptide bonds [30, 37]. Catabodies can be identified and their synthesis can be induced using electrophilic analogs of amyloid proteins. Non-hydrolyzable synthetic electrophilic phosphonates bond covalently with nucleophilic, IgM-class B cell receptors (BCRs) [37]. The synthetic electrophile was placed within several antigenic epitopes of Aβ and gp120, yielding the electrophilic analogs (E-Aβ, E-gp120) that were capable of binding antibodies noncovalently coordinated with covalent binding to nucleophilic sites. E-Aβ and E-gp120 were applied as probes to isolate pre-existing antibodies in the natural antibody repertoire and as immunogens to induce adaptive synthesis of nucleophilic antibodies with specific catabody activity [38, 39]. In addition, immunization with synthetic electrophilic analogs of peptides derived from gp120 induced specific antibody responses.
The functional value of catabodies to amyloids is suggested by previous studies on Aβ. Catalytic IgMs and recombinant VL constructs 5D3 and 2E6 from a human phage library attenuated Aβ oligomer neurotoxicity, reduced the formation of Aβ fibrils and oligomers, and induced disappearance of preformed Aβ aggregates in vitro [33, 40]. Clone 2E6 is a heterodimer of two VL domains. Clone 5D3 is a single domain VL. The catabody constructs hydrolyzed Aβ (His14-Gln15 bond), releasing soluble Aβ fragments [33]. Aβ binding was undetectable. Brain Aβ clearance was shown in 3 different transgenic (Tg) mouse strains expressing human Aβ. Local Aβ removal was evident after intracranially administered catalytic VL2 2E6 in aged 5×FAD mice, a model for parenchymal brain Aβ deposition [41]. Aβ plaque removal was evident after intravenous injection of VL2 2E6 in Tg-SwDI mice. These difficult-to-treat mice express mutated Aβ that forms vascular amyloid, causing microhemorrhages [42, 43]. No increase in cerebral microbleeds was observed in mice following catalyst injection, a common side-effect of Aβ-binding Abs [44-48]. Gene transfer of VL 5D3 in the brain of APPswe/PS1dE9 Tg mice resulted in prolonged expression of VL 5D3 and reduction of Aβ load in the brain. Unlike the report of cerebral microbleeds by gene transfer of an Aβ-binding antibody [49], the catalytic VL did not induce hemorrhages [50, 51].
Innate polyreactive catabodies are detected based on the ability to hydrolyze short peptide substrate without requirement for specific epitope recognition. The ability to recognize multiple, unrelated substrates might result in multiple biological functions for polyreactive catabodies depending on the homeostatic or pathological context. Importantly, the polyreactive catabody subset may fulfill a beneficial role in removal of excess target substrates that exert toxic effects [52, 53], for example, removal of excess bacterial substrates in infections, removal of excess intracellular antigens under conditions of widespread cell death and removal of excess amyloids despite low-affinity amyloid recognition. Moreover, the polyreactive catabodies may represent species with yet to be identified innate specificities directed to broad classes of senescent substrates. For the purpose of the present invention, a “senescent” substrate is defined as a substrate that acquires toxicity due to one or more conformational changes that generate neoepitopes absent in the physiological structure of the substrate. Such toxic neoepitopes can be induced, for example, when a non-toxic physiological substrate is subjected to post-translational chemical modifications. Similarly, neoepitopes can be induced upon release of intracellular substrates into the extracellular environment due to physicochemical deviations between the extracellular and intracellular environments (e.g., redox status, concentration of various metals and chaperones). Removal of such senescent antigens by catabodies will result in beneficial effects.
The antibody of the instant invention may be a naturally occurring antibody or may be a synthetic or modified antibody (e.g., a recombinantly generated antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody; and the like). In a particular embodiment, the antibody is innate and naturally occurring. Once an antibody has been isolated, the sequence of the antibody can be determined by methods known in the art and the antibody can be synthesized recombinantly/synthetically.
In a particular embodiment, the antibody is an antibody fragment. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (e.g., single variable light or heavy chain domain), Fab, Fab′, F(ab′)2, and Fv; and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment. The antibody may comprise at least one purification tag. The antibodies of the instant invention may also be conjugated/linked to other components. For example, the antibodies may be operably linked (e.g., covalently linked, optionally, through a linker) to at least one detectable agent, imaging agent, contrast agent, cytotoxic molecule (e.g., immunotoxin), and/or any other biological compound. In a particular embodiment of the instant invention, the antibody is a monoclonal antibody or fragment thereof.
The antibodies of the instant invention may be further modified. For example, the antibodies may be humanized. In a particular embodiment, the hybrid antibodies (or a portion thereof) are inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light domain and/or variable heavy domain of the antibodies of the instant invention may be inserted into another antibody construct. Methods for recombinantly producing antibodies are well-known in the art. Indeed, commercial vectors for certain antibody and antibody fragment constructs are available. The purity of the antibody molecules of the invention may be assessed using standard methods known to those of skill in the art, including, but not limited to, ELISA, immunohistochemistry, ion-exchange chromatography, affinity chromatography, immobilized metal affinity chromatography (IMAC), size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), western blotting, surface plasmon resonance and mass spectroscopy.
The antibodies or polypeptides of the instant invention ((or composition(s) comprising the same (e.g., with at least one pharmaceutically acceptable carrier)) can be administered by any suitable route, for example, by injection (e.g., for local, direct, or systemic administration), oral, pulmonary, topical, nasal or other modes of administration. The composition may be administered by any suitable means, including parenteral, intramuscular, intravenous, intravascular, intraarterial, intraperitoneal, subcutaneous, topical, inhalatory, transdermal, intrapulmonary, intraareterial, intrarectal, intramuscular, and intranasal administration. In a particular embodiment, the composition is administered to the blood. In general, the pharmaceutically acceptable carrier of the composition is selected from the group of diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. The compositions can include diluents of various buffer content (e.g., Tris HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., Polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The compositions can also be incorporated into particulate preparations of polymeric compounds such as polyesters, polyamino acids, hydrogels, polylactide/glycolide copolymers, ethylenevinylacetate copolymers, polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention (see, e.g., Remington's Pharmaceutical Sciences and Remington: The Science and Practice of Pharmacy). The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized for later reconstitution).
The therapeutic agents described herein will generally be administered to a patient as a pharmaceutical preparation. The term “patient” as used herein refers to human or animal subjects. The compositions of the instant invention may be employed therapeutically or prophylactically, under the guidance of a physician.
The compositions comprising the agent of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). The concentration of agent in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agent to be administered, its use in the pharmaceutical preparation is contemplated.
The dose and dosage regimen of the agent according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the agent is being administered to be treated or prevented and the severity thereof. The physician may also take into account the route of administration, the pharmaceutical carrier, and the agent's biological activity. Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen.
A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment or prevention therapy. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.
Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation or prevention of a particular condition may be determined by dosage concentration curve calculations, as known in the art.
The pharmaceutical preparation comprising the agent may be administered at appropriate intervals until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.
Toxicity and efficacy (e.g., therapeutic, preventative) of the particular formulas described herein can be determined by standard pharmaceutical procedures such as, without limitation, in vitro, in cell cultures, ex vivo, or on experimental animals. The data obtained from these studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon form and route of administration. Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to deliver a therapeutically or prophylactically effective amount.
The following definitions are provided to facilitate an understanding of the present invention:
The term “isolated” may refer to a compound or complex that has been sufficiently separated from other compounds with which it would naturally be associated. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with fundamental activity or ensuing assays, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.
“Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), bulking substance (e.g., lactose, mannitol), excipient, auxilliary agent, filler, disintegrant, lubricating agent, binder, stabilizer, preservative or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized). Suitable pharmaceutical carriers are described, for example, in “Remington's Pharmaceutical Sciences” by E. W. Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.
An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (e.g., single variable light or heavy chain domain), Fab, Fab′, F(ab′)2, and Fv; and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody.
With respect to antibodies, the term “immunologically specific” refers to antibodies that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, a “biological sample” refers to a sample of biological material obtained from a subject, preferably a human subject, including a tissue, a tissue sample, a cell sample, a protein sample, a DNA sample, an RNA sample, and a biological fluid (e.g., blood, serum, plasma, etc.). In a particular embodiment, the biological sample is blood.
As used herein, “diagnose” refers to detecting and identifying a disease or disorder in a subject. The term may also encompass assessing or evaluating the disease or disorder status (progression, regression, stabilization, response to treatment, etc.) in a patient and/or providing a prognosis of the disease or disorder.
As used herein, the term “prognosis” refers to providing information regarding the impact of the presence of a disease or disorder (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting diabetes, and the risk of cardiovascular disease). In other words, the term “prognosis” refers to providing a prediction of the probable course and outcome of a disease/disorder or the likelihood of recovery from the disease/disorder.
The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease or disorder, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.
The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.
Higher organisms possess a structurally diverse innate repertoire of antibody variable (V) domains that was shaped by evolutionary selection pressures imposed by foreign antigens and self-antigens over millions of years. The diversity is supplemented by somatic randomization of the V-domains by acquired immunity processes, which enables selection of V-domains with noncovalent antigen binding activity over a few weeks upon contact with an antigen. Polyreactive catalytic antibodies (catabodies) with a promiscuous peptide bond hydrolytic activity, on the other hand, are produced as an innate property of B cells, requiring no exposure to the antigen [37, 56]. The proteolytic activity was traced to serine protease-like sites in the germline antibody V-regions, suggesting an ancient origin of the catabodies [29, 57-59]. Humans produce catabodies selective for amyloid β peptide (Aβ) [55]. Immune tolerance mechanisms in fetal and adult life are thought to suppress synthesis of autoantibodies to essential self-proteins. When produced, subsets of autoantibodies express autoantigen-selective catalytic activities [40, 60, 61]. Provided catabodies discriminate between individual peptide antigens, they hold potential for specific and permanent destruction of toxic self-antigens.
Many proteins can misfold into the amyloid state [1]. Soluble transthyretin (TTR) is found in large amounts in blood (˜0.3 mg/mL) in its physiological, homotetrameric state (phyTTR) as a carrier protein for thyroid hormones and the retinol-retinol binding protein complex [2]. Misfolded β-sheet containing aggregates are generated from TTR (collectively designated misTTR states, Table 1), a process involving formation of a TTR monomer, destabilization of the phyTTR dimer-dimer interface, and growth of soluble misTTR oligomers into fibrillar amyloid [62-64]. Small misTTR amounts are likely formed continuously as basal byproducts of alternate TTR folding pathways. Initial evidence for the existence of blood-borne misTTR species is available [65]. Dysregulated protein misfolding during normal aging processes causes increased TTR amyloid deposition in several tissues [2]. TTR amyloid deposition in the tenosynovium is associated with carpal tunnel syndrome in middle-aged humans [66]. TTR amyloidosis in the heart, lung, stomach, kidney and other tissues causes organ failure in ATTRwt, a disease affecting >10% humans over age 70 years [2, 4, 16, 67]. In addition, mutations in the TTR gene cause early-onset familial TTR amyloidosis [68]. Amyloid deposits cause architectural and functional tissue damage, and soluble misTTR aggregates exert direct cytotoxic effects. No approved pharmacological treatment for ATTRwt or ATTRm is available, but anti-transthyretin RNAi reagents are undergoing clinical trials [24] and a small molecule drug that interacts with the thyroxine binding site of TTR may help relieve symptoms of polyneuropathy in familial TTR amyloidosis [22]. Reversibly-binding antibodies that can clear amyloids by phagocytosis of the immune complexes were proposed as potential therapeutic reagents [48, 69, 70]. IgM class catabodies produced by healthy humans that hydrolyze misTTR but not phyTTR are described. The catabodies can be conceived as homeostatic mediators that destroy TTR amyloid and its precursor misfolded TTR forms without interfering in the physiological functions of TTR.
arefs [71-73].
Antibodies.
Human studies were approved by the Committee for the Protection of Human Subjects, University of Texas Health Science Center, Houston. Individual or pooled polyclonal IgM, IgG and IgA class antibodies were purified from sera of 12 healthy humans without amyloidosis or autoimmune disease (33±7 years age) by acid elution (pH 2.7) from columns of immobilized anti-human IgM antibodies, Protein G and anti-human IgA antibodies [37, 74]. The antibody preparations were free of detectable non-antibody proteins judged by SDS-electrophoresis and immunoblotting with specific antibodies to IgM, IgG and IgA. For evaluation of aging effects, IgMs were purified from non-aged humans (<35 years age) or aged humans (>70 years) of either gender without amyloidosis or autoimmune disease. The monoclonal IgM (mIgM) panel purified from patients with Waldenström's macroglobulinemia patients was described (our laboratory codes 1800-1804, 1806, 1809-1811, 1813, 1814, 1816-1819, Yvo) [56]. The panel is assumed to be a random collection of mIgMs. There is no known relationship between B cell carcinogenicity and IgM catalytic activity or antigenic specificity. Unfractionated human serum was pooled from 10 healthy humans (36±5 years age). To prepare antibody-free serum, the pooled serum was adsorbed sequentially on the anti-IgM, Protein G and anti-IgA columns (residual IgM, IgA and IgG estimated by ELISA were, respectively, 0.05, 0.007 and 0.003%) [31]. FPLC gel filtration of the unfractionated serum pool (70 μL) was on a Superose-6 column (GE Healthcare, Piscataway, N.J.; flow rate 0.1 mL/min) in 10 mM sodium phosphate, pH 7.4, 137 mM NaCl, 2.7 mM KCl (PBS) containing 0.1 mM CHAPS. The Aβ-hydrolyzing recombinant catalytic antibody fragment (clone 2E6) was purified as described [33]. Nominal molecular mass was computed by comparison with protein markers (a reference 900 kDa mIgM and 1.4-670 kDa markers from Biorad, Hercules, Calif.). Total protein was measured by the microBCA method (ThermoFisher Scientific, Rockford, Ill.). Cell-surface IgMs were analyzed using the purified peripheral blood B cells from a 25 year old human subject without amyloidosis (B cell negative selection kit, Miltenyi, Auburn, Calif.; viability 90-95%; >95% purity determined by staining with phycoerythrin-conjugated mouse antibody to human CD19 (BD Pharmingen, San Jose, Calif.) and flow cytometry.
Aggregated TTR. Wildtype, purified TTR from human plasma (Cell Sciences, Canton, Mass.) was labeled with 125I (125I-TTR) using 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril (ThermoFisher), followed by removal of free 125I by gel filtration (BioSpin-6 column; Bio-Rad). The TTR or 125I-TTR solutions were pre-aggregated in PBS containing 1 mM EDTA (0.4 mg TTR/mL, 28.6 μM; molar TTR concentrations computed from the monomer TTR mass, 14 kDa) by acidification with an equal volume of 200 mM sodium acetate, pH 4.2, 100 mM KCl, 1 mM EDTA, followed by incubation for 5 days at 37° C. [64]. After exchanging the buffer to PBS containing 0.1 mM CHAPS (PBS/CHAPS) with an Ultra-4 centrifugal filter (EMD Millipore, Billerica, Mass.), the aggregated TTR (˜14 μM) was stored in aliquots at −80° C. Non-aggregated TTR is composed of soluble physiological tetramers, and the aggregation reaction produces soluble and particulate misfolded TTR [62-64]. Aggregation was monitored by turbidimetry at 400 nm (10 mm path length; Cary 50 spectrophotometer, Agilent, Santa Clara, Calif.). In addition binding of TTR (100 μL, 0.1 mg/mL) to thioflavin T (ThT) was determined by mixing with ThT (2.5 μL, 0.6 mM in PBS containing 0.1 mM CHAPS and 12% dimethylsulfoxide) and measurement of fluorescence emission (λem 485 nm, λex 440 nm, 600 V PMT voltage; Varian Cary Eclipse fluorimeter). The supernatant and pellet containing the soluble and particulate TTR species, respectively, were separated by centrifugation (17,000×g, 20 min).
The phyTTR and misTTR states were dissociated into monomers (14 kDa) by boiling test samples (10 min) after mixing with a ⅕th volume of 10% SDS in electrophoresis buffer (0.313 M Tris-HCl, pH 6.8, containing 17% 2-mercaptoethanol, 50% glycerol and 0.025% Bromophenol Blue). Electrophoresis was in 16.5% polyacrylamide Criterion gels (Bio-Rad) in 100 mM Tris-Tricine, pH 8.3, containing 0.1% SDS. Protein mass was computed by comparison with standard proteins (14-94 and 1.4-26.6 kDa ladders; Biorad). The phyTTR and misTTR states were distinguished by electrophoresing non-boiled test samples treated with 10% SDS treatment in non-reducing buffer. The 54 kDa phyTTR 4mers dissociate into 1mers by upon boiling in SDS but remain undissociated in SDS at room temperature, whereas SDS treatment at room temperature is sufficient to dissociate misTTR aggregates into 14 kDa 1mers [71-73]. Radioactive TTR bands were detected with a Cyclone Plus Phosphor Imager (PerkinElmer, Waltham, Mass.; 30-120 min exposure, super-sensitive storage screen; minimum detectable radioactivity+3 s.d. values, 500 CPM; linear detection up to 867,000 CPM). % misTTR content of pre-aggregated 125I-TTR was determined by densitometry as: 100×[14 kDa band intensity]/[sum of 14 and 54 kDa band intensities], where the band intensity was in arbitrary volume units (AVU; ImageJ software, NIH Research Services Branch, Bethesda, Md.).
The biotinylated, electrophilic analogs of misTTR (E-misTTR) and phyTTR (E-phyTTR) were prepared as described [39](6.0 and 0.1 mol phosphonateand biotin, respectively, per molmisTTR; 3.9 and 1.5 mol phosphonate groups and biotin, respectively, per mol phyTTR). The starting material for E-misTTR preparation was the particulate misTTR in PBS/CHAPS recovered by centrifugation (30 min, 75,000 g).
Proteolysis.
All tests were done in duplicate or as specified. Non-aggregated or pre-aggregated 125I-TTR (100 nM monomer equivalents) was incubated with purified antibodies in PBS/CHAPS (20 μL). The boiled or non-boiled reaction mixtures were analyzed by SDS-electrophoresis (CPM/lane, 8,930-17,110). Depletion of the 1mer TTR band (14 kDa) and appearance of lower mass products was quantified by densitometry. Antibody hydrolytic activity was computed in units of nM misTTR hydrolyzed/μg antibody/hour ((AnM×(1−[14 kDaAb]/[14 kDaDil]))/(μg Ab in the reaction mixture/Ab treatment time in hours), where: A is the initial misTTR concentration (total TTR concentration×% misTTR content) if non-boiled samples were analyzed or the total TTR concentration (misTTR+phyTTR) if boiled samples were analyzed; Ab denotes antibody; and, [14 kDaAb] and [14 kDaDil] are the 14 kDa band intensities after antibody and diluent treatment, respectively. For data reported in this format, variations of the misTTR hydrolysis rate up to 2.4-fold are predicted because of varying misTTR content in different pre-aggregated TTR preparations (29-70%).
Hydrolysis of non-radiolabeled TTR bands stained with silver was quantified similarly. Protease inhibitors were tested at concentrations sufficient for complete inhibition of metalloproteases (EDTA, 2 mM; 1,10-phenanthroline, 1 mM), cysteine proteases (iodoacetamide, 0.1 mM), acid proteases (pepstatin A, 1 μM) and serine proteases (E-hapten 1, a small molecule phosphonatediester, 0.1 mM)[75]. E-hapten 1 ((diphenylN-[6-(biotinamido)hexanoyl]amino(4-amidinophenyl)methane phosphonate)) is an active site directed inhibitor of nucleophilic catabodies [76]. Alkylation of the free —SH group in TTR was avoided by pre-treating antibodies with iodoacetamide (60 min) and removing this reagent prior to the proteolysis assay (BioSpin-6 gel filtration column). Inhibition of antibody proteolysis by serum amyloid P component (SAP; Calbiochem, Billerica, Mass.) was tested under conditions reported to reduce TTR sensitivity to digestion by human matrix metalloproteinase-9 (MMP9 from human neutrophils, EMD Millipore; pre-aggregated 125I-TTR containing 1 μM misTTR pre-treated with 0.55 μM SAP or control albumin for 2 h in 10 mM Tris HCl, pH 8.0, 138 mM NaCl, 2 mM CaCl2) [77]. MMP9 was activated by incubation for 3 h with 5 mM 4-aminophenylmercuric acetate, and the enzyme was recovered by gel filtration for hydrolysis tests.
Hydrolysis of fluorigenic EAR-AMC (N-t-butoxycarbonyl-O-benzyl-Glu-Ala-Arg-7-amino-4-methylcoumarin) and amyloid β peptide 1-40 (Aβ1-40) was tested as in ref [55]. 125I-Aβ1-42 (Aβ42, 90 μg, EZBiolab, Carmel, Ind.) was radiolabeled as described for TTR, and free 125I was removed using a Sep-Pak Vac C18 cartridge [78]. The fibrillar Aβ42 substrate (2.3×107 CPM/μmol Aβ42) was prepared by co-aggregating 125I-Aβ42 (0.7×109 CPM in 1,1,1,3,3,3-hexafluoro-2-propanol, 0.1 mL) and Aβ42 (1 mg; 5 mM solution in dimethylsulfoxide). The mixture was diluted to 50 μM Aβ42 with PBS, incubated for 92 h (37° C.), and the fibrillar 125I-Aβ42 was collected by centrifugation (17,000×g, 20 min). Following digestion of fibrillar 125I-Aβ1-42 with antibodies (30,000 CPM, 16 h), the reaction mixtures were treated with formic acid (60% v/v) and fractionated on a Superdex peptide gel filtration column (GE Healthcare) in 60% formic acid. Hydrolysis of these biotinylated proteins was measured by SDS-electrophoresis and staining with streptavidin-peroxidase as in ref [56](molar biotin/protein ratio in parentheses): gp120 from HIV strain MN (1.6), S. aureus Protein A (5.0), bovine serum albumin (9.0), extracellular domain of human epidermal growth factor receptor (2.3), bovine thyroglobulin (26.0), human transferrin (5.4), and ovalbumin (1.6).
Binding.
Pre-aggregated 125I-TTR (25,000 cpm; 100 nM TTR, 60% misTTR content) was incubated (16 h) with or without IgMs (130 μg/mL) in 40 μL in PBS/CHAPS. The soluble and particulate fractions were separated by centrifugation (20 min, 17,000 g), the particulate fraction was resuspended in 40 μL PBS/CHAPS, treated separately for 1 h with the immobilized anti-human IgM gel (50 μL settled gel), the mixtures were centrifuged (5 min, 500 g), the gel was washed with 250 μL PBS/CHAPS, and radioactivity in supernatant (unbound 125I-TTR) and gel (bound 125I-TTR) was measured. % bound 125I-TTR was computed as 100×(CPM total binding−CPM nonspecific binding)/CPM 125I-TTR in the initial reaction mixture), where total binding refers to the binding in IgM containing reaction mixtures and nonspecific binding refers to binding in the reactions containing PBS/CHAPS instead of IgMs.
For cell binding studies, large E-misTTR particles were removed by centrifugation (400 g, 10 min), and the E-misTTR did not contain flow cytometrically detectable particles. B cells were pretreated with mouse serum (1% in PBS, 10 min) to saturate Fc-receptors and with Endogenous Avidin/Biotin Blocking kit to saturate cellular biotin as recommended by the manufacturer (Abcam, Cambridge, Mass.). The cells (1.5×105) were incubated with E-phyTTR or E-misTTR (0.3 μM) in PBS for 1 h at 4° C. to minimize B cell receptor internalization (BCR). Competition studies were done by preincubating B cells (1 h, 4° C.) with control diluent or non-biotinylated misTTR, phyTTR or ovalbumin (3 μM). Bound biotinylated probes were detected with streptavidin conjugated to phycoerythrin-Cy7 (PECy7-SA; 8 μg/mL). IgM+ cells were identified with antibody to human IgM conjugated to fluorescein isothiocyanate (FITC; 2 μg/mL, BDPharmingen). Flow cytometry was done using an LSRFortessa X-20 flow cytometer (BD Biosciences, San Jose, Calif.) and FlowJo software (about 10,000 cells/analysis). In control incubations, B cells were stained with FITC-labeled, isotype-matched antibody instead of the anti-IgM antibody or were treated with diluent instead of the biotinylated E-misTTR followed by staining with PECy7-SA. The E-misTTR binding cells were visualized by confocal microscopyas in [37](multiple acquisitions, thickness 0.15 μm; deconvolution by 15 iterations; FITC—λex 488 nm, λem 525 nm; 4,6 diamino-2-phenylindole—λex 350 nm, λem 470 nm; PECy7—λex 488 nm, λem 767 nm).
Amyloid Dissolution.
Pre-aggregated non-radioactive TTR (7.1 μM, 100 μg/mL) was incubated in duplicate with diluent or mIgMs (120 μg/mL) in PBS containing 0.1 mM CHAPS at 37° C. The dissolution reaction was monitored from turbidity or ThT binding measurements.
Statistical Analysis.
Errors are s.d. values. P values were from the unpaired two-tailed Student's t-test. Correlations were determined by the Pearson's two-tailed test.
125I-TTR Substrate.
Wildtype human TTR radiolabeled with 125I (125I-TTR) was applied to determine hydrolysis of the phyTTR tetramers and misTTR aggregates. Non-aggregated 125I-TTR preparations behaved as predicted from studies on phyTTR tetramers [71-73]. The non-aggregated 125I-TTR: (a) did not bind appreciably to Thioflavin-T (ThT;
misTTR but not phyTTR Hydrolysis by Polyclonal IgMs (pIgMs).
The hydrolytic activity of electrophoretically homogeneous polyclonal IgM, IgG and IgA pooled from the blood of humans without TTR amyloidosis was tested. The pIgMs hydrolyzed pre-aggregated 125I-TTR, indicated by depletion of the 1mer TTR band (14 kDa) in SDS-treated, boiled reaction mixtures (
Formation of IgM-misTTR immune complexes by the pooled pIgM (130 μg/ml) was undetectable (binding of soluble and particulate misTTR species, respectively: 1.9±2.4 and 0.7±0.7%). At an equivalent concentration, the same pIgM preparation hydrolyzed 45±1% soluble misTTR and 77±1% particulate misTTR, respectively. The results indicate hydrolysis of misTTR but not phyTTR by IgMs, the first class of antibodies produced by B cells. In contrast, the IgGs and IgAs usually associated with acquired immune responses were devoid of misTTR hydrolytic activity.
Polyclonal IgM Hydrolytic Properties.
Mean misTTR hydrolysis by pIgM preparations from humans >70 years age was only slightly lower than humans <35 years age, and the difference did not reach statistical significance (P=0.101;
pIgM catalyzed misTTR hydrolysis was inhibited by synthetic E-hapten 1, a compound that binds covalently to the nucleophilic sites of serine proteases (
The pIgM hydrolytic activity was saturable with increasing misTTR concentration (Table 3). In addition to misTTR, the pIgMs hydrolyzed small peptide substrates, a reaction that reports the catalytic subsite properties independent of noncovalent epitope binding [37, 52]. The apparent Km for misTTR was 92-fold lower than Glu-Ala-Arg-AMC (EAR-AMC), a small peptide hydrolyzed by IgMs at the Arg-AMC bond, suggesting selective IgM-misTTR recognition (Km approximates Kd, the equilibrium dissociation constant). Diffusional restrictions known to decelerate enzymatic conversion of particulate substrates compared to soluble substrates [85, 86] likely limit the rate of particulate misTTR hydrolysis by the IgMs. The pIgM preparations are a mixture of individual IgM subsets, and misTTR is a mixture of multiple misTTR aggregation states. The apparent kinetic constants, therefore, are average values for hydrolysis of multiple misTTR species by multiple IgMs.
misTTR-Directed B Cells Andmonoclonal IgMs (mIgMs).
The reactivity of mIgMs expressed as BCRs in healthy humans often indicates initial antibody specificity prior to contact with external immunogens. The E-misTTR analog contains electrophilic phosphonates that bind covalently to nucleophilic BCR sites responsible for catalysis, permitting identification of misTTR-selective B cells without loss of the bound probe due to peptide bound hydrolysis. Flow cytometry identified a discrete IgM+ B cell population with saturable binding activity for E-misTTR that was inhibited by competitor misTTR but not the irrelevant protein ovalbumin (
A panel of mIgMs secreted by cancerous B cells from patients with Waldenström's macroglobulinemia without TTR amyloidosis was applied as a model for individual IgMs present in the pIgM preparations. Twelve of 16 mIgMs hydrolyzed pre-aggregated 125I-TTR (Table 2). The hydrolytic rates were highly variable (undetectable to 2.98 nM/μg IgM/h), consistent with the prediction of differing catalysis by individual catabodies with structurally distinct V-domains. Like the pIgMs, the 12 misTTR-hydrolyzing mIgMs hydrolyzed misTTR but not phyTTR, shown by: (a) the depleted 1mer band of pre-aggregated 125I-TTR but not non-aggregated 125I-TTR in boiled reaction mixtures containing; and (b) the depleted 1mer TTR band originating from misTTR and the undepleted 4mer phyTTR band in non-boiled reaction mixtures of pre-aggregated 125I-TTR (
From mutagenesis [30], epitope mapping [87] and protease inhibitor studies [88], the split-site catalysis model was proposed (
Are the Catabodies Polyreactive?
The complementarity determining regions (CDRs) of some polyreactive IgMs produced without immunogen stimulation can bind epitopes with no discernible structural similarity [89], a phenomenon that may originate from CDR flexibility and the induced-fit binding mechanism. Consistent with previous reports [55, 56], the pIgMs and mIgMs did not hydrolyze non-amyloidogenic, non-superantigen proteins from eukaryotic and prokaryotic organisms (
The misTTR-hydrolyzing mIgM Yvo was reported to hydrolyze amyloid 1-40 (Aβ40) and 1-42(Aβ42) peptides [55], a reactivity pattern reminiscent of antibodies that bind the β-strand conformational epitope of amyloids without dependence on the epitope sequence [90, 91]. In contrast, the misTTR hydrolyzing mIgM 1802 did not hydrolyze pre-aggregated Aβ42 (
misTTR Dissolution.
Treatment of a 10-fold molar excess of pre-aggregated TTR with the hydrolytic mIgM 1802 but not non-hydrolytic IgM 1819 resulted in time-dependent reduction of turbidity to the near-basal value of non-aggregated TTR (
Small misTTR amounts are likely produced as normal byproducts of alternate TTR folding pathways. With advancing age, such misTTR species are thought to serve as seeds for pathological TTR amyloid formation. The findings indicate physiological production of misTTR-selective IgM catabodies are a first-line defense function against TTR amyloidosis. The IgMs hydrolyzed soluble and particulate misTTR, but not the non-pathogenic phyTTR tetramer found at micromolar concentrations in blood, indicating selectivity for an aggregation-induced misTTR neoepitope that is absent in phyTTR. The IgMs, therefore, can degrade misTTR with no danger of saturation by the more abundant phyTTR species. There was no evidence for IgM degradation of non-amyloid, non-superantigen proteins [55, 56]. Two misTTR-hydrolyzing mIgMs did not hydrolyze Aβ or microbial superantigens, and other mIgMs recognized these substrates at varying levels. Saturation of oligoreactive IgMs with irrelevant amyloids may limit the misTTR clearance capacity, but this is a threat only in patients with non-TTR amyloid disease. Likewise, superantigens accumulate only during microbial infections, and impairment of misTTR clearance is not predicted in the non-infected state. The selectivity properties, therefore, support a role for the IgMs in defense against TTR amyloidosis.
Further evidence for the beneficial IgM role includes: (a) Pre-aggregated TTR was dissolved completely by a hydrolytic mIgM; (b) misTTR was degraded at IgM concentrations ˜200-fold lower than the blood IgM concentration, and virtually the entire misTTR degradative capacity of serum was attributable to IgMs; (c) the blood IgM concentration exceeds that of non-antibody proteases by several log orders, and pIgMs hydrolyzed misTTR at a rate comparable to a non-antibody protease (MMP-9); (d) the pIgM hydrolytic activity was refractory to serum inhibitors of non-antibody proteases; and (e) pIgM from different healthy adults hydrolyzed misTTR within a narrow rate range, indicating stable maintenance of the misTTR hydrolytic activity under disease-free conditions. The beneficial IgM function extends beyond clearance of blood-borne misTTR. IgMs permeate most anatomic blood-tissue barriers. Lymph from many tissues, a common surrogate for the tissue interstitial fluid, contains IgM in the mg/ml range [97-99], indicating the ability for tissue misTTR clearance.
Notwithstanding the favorable catabody properties, pathological TTR amyloid accumulates in ATTR patients, indicating that the catabody clearance capacity may be overwhelmed by independent age-associated factors that accelerate protein misfolding, e.g., the reaction of amyloidogenic proteins with advanced glycation and lipid peroxidation end-products [100, 101]. In addition one cannot exclude reduced catabody formation as a possible factor in TTR amyloidosis. Age-associated shifts in the structural and functional properties of antibody V-domains are documented [102, 103], individual mIgMs hydrolyzed misTTR at widely variable rates, and aged humans display increased susceptibility to infections [104], A small, non-significant tendency towards reduced misTTR hydrolysis in healthy, aged humans was observed. Future studies on patients with ATTR may be performed to further assess whether immune senescence is a predisposing factor in amyloidosis. In addition, long-term follow-up of humans and mice with genetic antibody deficiencies [105, 106] may be useful to define the contribution of the immune system in defense against amyloidosis. The catalytic function may also be conceived to diminish the inflammatory consequences of antibody-antigen interactions. Reversible binding at the Fab region of conventional antibodies stimulates Fc-region mediated activation of complement and phagocytosis, exemplified by the inflammatory effects of Aβ-binding antibodies [107]. Rapid catabodies form only transient immune complexes and then release the digested antigen products, reducing the probability of harmful immune complex effects.
According to the acquired immunity paradigm, IgGs with specific binding activity directed to individual target epitopes are produced within a few weeks by the immunogen-driven maturation of antibody light and heavy chain variable domain sequences (respectively, VL and VH domains). Acquired immunity rules did not yield catabodies capable of complex peptide bond hydrolytic reactions [108]. Moreover, the rules provide no basis to anticipate the strict catabody selectivity for misTTR. As misTTR-directed IgMs are produced by healthy humans without TTR amyloidosis, they are likely produced constitutively without requirement for acquired immunity processes. Catabody synthesis can occur without immunogen-driven V domain maturation because the serine protease-like nucleophilic sites are encoded by heritable germline V region genes [29, 57-59]. A germline VL gene residue is an essential constituent of the misTTR-hydrolyzing mIgM Yvo catalytic triad (Ser110, IMGT numbering), and the 2 remaining catalytic residues are evidently acquired during V-(D)-J gene recombination, which occurs prior to contact with the immunogen (Arg112 and Glu113 at the VL-J and VHD-J junctions, respectively [109]. Furthermore, the IgMs are the first antibody class produced by B cells, whereas IgGs generated at the terminal acquired immunity stage are poorly hydrolytic. The antibody C domain scaffold can influence antigen binding at the remote V domains [110]. C domain swapping studies indicated loss of catalytic activity at the IgM→IgG class-switch step due to the unfavorable effect of the IgG C domains on the V domain catalytic site [31]. Detailed information about factors regulating IgM constitutive synthesis is not available. However, IgMs are synthesized at stable levels by germ-free animals [111], and their constitutive production may involve an autonomous B cell developmental program and BCR-independent lymphocyte growth stimulators [35].
In adult humans, IgGs are the dominant blood-borne antibodies, found at 5-6 fold greater concentrations than IgMs. Many IgGs express specific antigen binding activity, consistent with maturation of their V-domains by immunogen-driven somatic mutation process. Such antigen-specific IgGs are the basis of protective responses induced by microbes and vaccines, and numerous antigen-binding monoclonal IgGs are approved for therapeutic use [112]. Catalytic IgG preparations from patients with autoimmune disease are reported to hydrolyze self-antigens [40, 113-115], but dysfunctional B cell reactivity in autoimmune disease may contribute to increased catalytic IgG synthesis [40], and the hydrolytic rate of the IgGs is usually lower than IgMs [116, 117]. Monoclonal IgGs from routine immunization protocols display minimal or no catalytic activity, but their dissociated light subunit frequently hydrolyzes peptide bonds [118-120]. Other IgGs generated by healthy humans express low-affinity, polyreactive antigen binding profiles (often designated “natural” IgGs) that may originate from occurrence of class-switch recombination in bystander B cells without sufficient mutations in the V domains required to accomplish specific antigen binding [121, 122]. The polyreactivity was suggested to contribute in the therapeutic effect of pooled IgGs from healthy humans in patients with bacterial infections and certain autoimmune diseases (commonly known as intravenous immune globulin or IVIG preparations) [123]. The catalytic activity of such IVIG preparations is marginal compared to IgMs [74]. Also, the IgMs in the present study display catalytic specificities that are readily distinguishable from the polyreactive IgG binding pattern.
Protein-protein recognition is dictated by sequence-dependent chemical interactions, topographical shape complementarity and local flexibility of the reacting sites. The amyloid-superantigen link is obvious from mIgMs with oligoreactivity for both protein classes, e.g., mIgM Yvo. Comparison of linear sequences did not indicate a homologous epitope that is shared by TTR, Aβ, Protein A and gp120 (
Humans produce no more than 100,000 non-antibody proteins, including all known enzymes and receptors with specificity for a broad range of ligands. The recombined IgM repertoire derived from the germline V, D and J segment configurations is far larger (from germline gene sequences available in IgBLAST, ˜4×109 VL-VH domain pairs derived by recombination of 152 VL with 19 J genes and recombination of 273 VH genes with 34 D and 13 J genes; this estimate excludes further repertoire expansion occurring during the junctional diversification process). The human germline IgM repertoire is a source of catabodies with functionally useful selectivity for individual substrates likely developed by Darwinian immune evolution (
In summary, the physiological production of misTTR-selective catabodies has been shown. Unlike other approaches to suppression of TTR amyloidosis, the catabodies remove misTTR without interfering in the essential functions of the properly-folded TTR state. The misTTR-hydrolyzing IgMs, therefore, hold potential for developing a safe treatment for TTR amyloidosis. Several monoclonal IgMs have been approved in the USA for human tests, and pentamer IgM-containing polyclonal antibodies are approved for human therapy in Europe [135]. Converting pentamer IgM to the monomer IgM state does not compromise the catalytic activity [37], and monomer IgMs can be considered as effective therapeutic reagents. Small amounts of IgM monomers are found physiologically in humans [136], and lower organisms produce larger monomer IgM amounts [54]. In addition, highly catalytic single VL domains and paired VL domains can be engineered [33, 40] as candidate drugs from the misTTR-hydrolyzing IgMs.
The misTTR-selective catabodies fulfill a homeostatic surveillance function for removing small misTTR amounts formed as the byproduct of the TTR folding pathways. The catabodies can also be applied for removing tissue amyloid deposits. Renewable monoclonal catabody sources prepared from the B cells of healthy humans are described. The catabody properties pertinent to the anti-amyloid defense function are also described.
Nucleophilic catalysis is an innate, germline V-gene encoded function. For TTR amyloid, the noncovalent specificity-conferring function is also an innate catabody property [5]. The catalytic activity is best expressed by IgMs produced early in B cell ontogeny [37, 137]. The covalent reactivity of electrophilic phosphonate probes with nucleophilic IgM class BCRs was documented [37]. To select specific monoclonal catabodies, electrophilic E-misTTR that binds B cells from healthy humans specifically was used (see Example 1). This reagent identifies IgM class BCRs that recognize the unique misTTR epitope in coordination with nucleophilic attack on peptide bonds. Following transformation with Epstein-Barr virus (EBV), a screen of the top 30% E-misTTR labeled B cells identified secreted monoclonal IgMs with misTTR-hydrolyzing rates >50-fold superior to the reported rate of IgMs from patients with Waldenström's macroglobulinemia (
To isolate catabodies with the highest possible rate, only the top 1% of E-misTTR labeled B cells are transformed with EBV, ensuring identification of the IgM BCRs with greatest specificity and catalytic activity directed against misTTR. The method consists of preparing B cells expressing IgM class BCRs by routine magnetic bead kits or flow cytometric sorting from human peripheral blood, followed by treatment of cells with the soluble E-misTTR probe (obtained as the supernatant from a mixture of particulate and soluble E-misTTR [5]). Noncovalently bound probe is removed by extensive washing or incubation of the cells with excess misTTR or hapten 1 devoid of the electrophilic group. The covalently bound B cell fraction is transformed with EBV. Secreted IgMs from 20 or more wells containing the cultures cells are screened for ability to hydrolyze 125I-misTTR, 125I-phyTTR (by electrophoresis) and Glu-Ala-Arg-AMC (by fluorimetry). Glu-Ala-Arg-AMC hydrolysis measures overall catalytic efficiency independent of epitope recognition. Other small dipepties, tripeptides and tetrapeptides devoid of an antigenic epitope can also be used to identify polyreactive hydrolytic activity of the antibodies. For the purpose of the instant invention, an antigenic epitope is defined as a peptide epitope composed at a minimum of 5 amino acids. Maintenance of IgM catalytic activity following removal of any trace contaminants by denaturing chromatography is documented [37]. Catalysis assay errors are <15%. The U- and t-tests are used to determine the significance of differences in catalytic activity. Stable cell lines are obtained by cloning the VL/VH cDNA (obtained by the reverse transcriptase-PCR method) adjacent to human κ and μ C-domains (pLC-huCκ, pHC-huCμ vectors) and expressed in NS0 cells as described [96, 138].
The same procedure is applied to isolate polyreactive catabody-secreting B cells, except that the initial B cell selection is conducted with the E-hapten 1 probe containing the electrophilic phosphonate but not an antigenic epitope (
Alternate cellular sources and methods to isolate the desired catabodies are available. For example, IgMs from umbilical cord blood B cells with no or minimally mutated V-genes may express high level catabody activity, as they represent the naïve human antibody repertoire that is minimally biased by memory B cell responses to external immunogens. Conversely, adaptive immunity effects such exposure to naturally-occurring electrophilic analogs of misTTR (NE-misTTR) and other antigens (NE-antigens) may result in amplification of catabody activity in old age or during autoimmune disease processes. Thus, high activity catabodies, including the polyreactive catabodies could be obtained from the cells of old humans, patients with amyloidosis or patient with autoimmune disease. Instead of EBV transformation, the VL and VH cDNA can be rescued from single B cells following selection with the E-misTTR, E-hapten 1 or NE-antigens by the reverse transcriptase-polymerase chain reaction [139], cloned adjacent to human κ and μ C-domains and expressed in a suitable host cell line [31, 140].
In addition to intact catabodies, the unpaired human light chains and unpaired shark or camel single V-domains can be isolated by phage display methods as renewable catabody sources. Unpaired human light chains hydrolyze small peptides rapidly compared to paired IgG VL-VH domains [32]. Jawed fish produce stable single V-domains attached to a heavy chain C-domain. Human light chain and shark single V-domain libraries are available [141, 142]. The libraries displayed on phages and phage selection are done by covalent binding to E-misTTR or E-hapten 1 [33].
In addition, high activity catabodies can be obtained by B cell or phage display selection procedures conducted using the NE-antigens, including NE-misTTR. NE-antigens containing aldehydes and ketones are formed by the reaction of the protein epitopes with advanced glycation/lipid peroxidation end-products (AGEs, LPEs;
Catabodies monoreactive for misTTR can be characterized by standard immunochemistry, molecular biology and enzymology methods. In addition, the novel specificity of properties of polyreactive catabodies pertinent to disease treatment and diagnosis can be revealed from these tests.
Innate Origin and Valency Effects.
Unfractionated culture supernatants and IgMs purified on immobilized anti-human IgM will be tested. The misTTR hydrolytic activity of culture supernatants from the B cell-lines is almost exclusively due to secreted IgMs, with little or no background activity due to cellular proteases. Catabodies from E-misTTR selected cell-lines display superior misTTR hydrolytic activity but not superior Glu-Ala-Arg-AMC hydrolytic activity compared to the E-hapten 1 selected cell-lines. Conversely, polyreactive catabodies from E-hapten 1 selected cell-lines should display superior Glu-Ala-Arg-AMC hydrolytic activity compared to the E-misTTR selected cell lines. The VL/VH domain cDNA of the catabodies and the N-termini of the V-domain proteins is sequenced. Unmutated V-genes will suggest germline-encoded catalytic activity. Replacement/silent mutation ratios that do not exceed the random mutational ratio suggest catabody production without immunogen-driven selection pressures. Over-representation of individual germline VL/VH genes in the panel identifies V-gene(s) encoding innate misTTR specificity. Expression of catalysis by pentamer IgM is confirmed by gel filtration. Monomer IgM is detectable in human serum [136]. The smaller monomers may be the preferred species for potential clinical applications. Monomer IgMs are produced by mild reduction and akylation of the pentamer or expression in an appropriate cell cell line that minimizes oligomerization of the monomers. There was no loss of Glu-Ala-Arg-AMC hydrolytic activity upon pentamer IgM conversion to monomer IgM [31]. Avidity effects due to multivalency are not a factor in solution state assays for substrates such as Glu-Ala-Arg-AMC that are devoid of repeat epitopes but may contribute to the rate for TTR aggregates. misTTR hydrolytic activity of enzymatically-prepared monovalent IgM Fab fragments can also be tested [37]. The IgM C-domains scaffold exerts a favorable effect on the V-domain catalytic site [31]. To explain the failure of IgGs to hydrolyze misTTR [5], the V-domains of an IgMs can be cloned as an IgG, and the hydrolytic activity of the V-domain identical IgM and IgG is compared.
Specificity.
Catabody specificity can be determined as follows: (a) Substrate specificity using wildtype and mutant misTTR species; amyloids formed by proteins unrelated in sequence to TTR; microbial superantigens with known sensitivity to catabodies; non-oxidized extracellular proteins free of known amyloid or superantigen character; oxidized amyloid, oxidized superantigens, and oxidized extracellular proteins free of amyloid or superantigen character; non-oxidized and oxidized intracellular proteins; and (b) Scissile bonds and noncovalent binding epitope; and (c) misTTR amyloid dissolution.
Substrate degradation can be monitored by electrophoresis, gel filtration, HPLC or mass spectroscopy. If required a radioactive label, biotin or a fluorescent label is inserted into the substrate, and immunoblotting with antibodies to various substrate epitopes is applied to visualize depletion of the intact protein and appearance of fragments is analyzed as in previously published hydrolysis assays. Specificity can be tested using: (a) Separately, the soluble and fibrillar misTTR species prepared from wildtype TTR and mutant TTR substrates (e.g., V122I, V30M, L55P, D18G and A25T mutants). The mutants aggregate at varying rates and cause hereditary amyloidosis with differing disease phenotype (neuropathy cardiomyopathy [144] (b) Non-TTR amyloids prepared from human Aβ42 peptide [145], a K-light chain from a multiple myeloma patient [146], α-synuclein[46], atrial natriuretic peptide, islet amyloid polypeptide, and tau [147]; (c) The superantigen proteins HIV gp120 and Staphylococcus Protein A [5]; (d) a panel of 10 or more extracellular proteins without amyloid or superantigen character [5]; (e) the oxidized versions of the aforenamed amyloid proteins, superantigens and extracellular proteins prepared by the reactions with AGEs and LPEs as specified under the section “renewable catabody sources”; and (f) an extract of intracellular antigens obtained by disruption of human cells (e.g., by detergent extraction of neurons or myocytes) with and without prior oxidation with the AGEs and LPEs.
The misTTR epitope recognized noncovalently by catabodies is likely the major determinant of specificity, and individual amino acids at the scissile bonds may also be a contributory factor. Time course studies can reveal whether the initial misTTR products of the catalytic reaction are digested further into smaller products. Multiple cleavage sites suggest the split-site catabody model, in which the catalytic subsite is in register with alternate, spatially-neighboring peptide bonds following initial noncovalent binding [40, 87]. The electrophilic antigen analog binds covalently and specifically to the two catabody subsites (
Essentially the same procedures can be applied to identify the scissile bonds and epitope recognized by the catabodies in other substrate proteins found to be sensitive to catabody hydrolytic activity.
Efficient catabodies do not form stable immune complexes. This can be confirmed by tests of catabody binding to the substrates by routine ELISA and surface plasmon resonance methods
Active Site.
Conventional antigens bind antibody CDRs. The V-domain framework regions (FRs) may have developed amyloid recognition capability early in immune evolution because of the potential survival advantage of an anti-amyloid antibody function [5]. The FRs are well known to contribute amino acids essential for catalysis [30, 57, 58] and noncovalent binding sites for B cell superantigens [92, 93]. The role of the FRs may be confirmed by swapping the FRs of a catabody (e.g., a misTTR-directed catabody with the corresponding FRs of an irrelevant antibody (one FR at-a-time; 8 mutants) using the IgM monomer scaffold, which supports efficient catalysis. This approach has been applied to localize superantigen binding sites [96]. Catalytic rates are measured. A change of Km and maximal velocity (Vmax) indicates, respectively, disruption of the noncovalent binding site and catalytic site. Noncovalent binding cavities and nucleophilic sites may be deduced from V-domain molecular models [95]. The catalytic nucleophile may be identified from the site of the covalent reaction with the E-hapten-1, a validated method for identifying enzymatic nucleophiles by enzymatic digestion and mass spectroscopy [143, 151]. Together, the studies reveal the V-domain residues important for catalysis and noncovalent substrate binding.
misTTR Dissolution.
Treatment with the E-misTTR-selected monoclonal IgM clone 10E10 dissolved particulate misTTR (
Non-electrophilic immunogens do not generally induce catabodies as B cell differentiation is driven by immunogen binding, and catalytic immunogen hydrolysis is not known to be a selection pressure for B cell growth and development into antibody-secreting cells [40, 152]. In contrast, as the misTTR-directed catalytic activity is an innate property, the E-misTTR and NE-misTTR analogs described in the Examples 1 and 2 induce adaptive strengthening of the innate BCR nucleophilicity coordinated with improved noncovalent binding of misTTR. This is supported by previous studies that electrophilic analogs of gp120 induce the synthesis of specific catabodies to gp120 [39]. Application of the E-misTTR and NE-misTTR immunogens is useful for isolating therapeutic catabodies to misTTR. In addition, these immunogens are useful to develop prophylactic and therapeutic vaccines against amyloidosis, both ATTRwt and ATTRm.
The immunogens may be prepared by the incorporation of electrophilic phosphonate groups or carbonyl groups of the AGEs and LPEs (
Catabody synthesis may be accomplished, for example, in groups of 6 mice that receive 4 subcutaneous immunogen administrations at 4 wk intervals. A non-toxic adjuvant (alum/RIBI) may be included to recruit antigen-presenting and T helper (TH) cells. Induction of antibodies may be monitored at 2 week intervals in blood by ELISA using immunogen-coated plates. Antibodies with reversible and covalent binding activity for the immunogen may be distinguished in ELISA plates without and with SDS treatment [39]. IgM and IgG antibodies may be purified from high-titer sera (>1:5000) and covalent binding may be confirmed by electrophoresis. Then, the antibodies may be tested for cleavage of misTTR and physiological TTR tetramers as in Example 1. It is desirable to establish the following properties of polyclonal antibodies: (a) Catalytic specificity using various amyloids and irrelevant proteins; (b) Scissile bonds of wildtype misTTR and hydrolysis rates of mutant misTTRs; (c) Noncovalent binding epitope; and (d) Dissolution of preformed aggregates of wildtype and mutant misTTR preparations.
Individual monoclonal antibodies generally display greater target antigen recognition compared to polyclonal antibody mixtures. Monoclonal catabodies may be prepared using standard hybridoma technology from mice immunized with the E-misTTTR or NE-misTTR immunogens [39]. Monoclonal IgMs/IgGs with immunogen binding activity may be tested for hydrolysis of misTTR as in Example 1, and the catabodies may be characterized for specificity, hydrolytic rate and TTR amyloid dissolution rate as in Examples 1 and 2.
No pharmacologically effective treatment is available for ATTRwt or ATTRm. Dissolution and detoxification of misTTR may be tested using test tube methods. Clearance of misTTR may also be tested in transgenic mice expressing human TTR infused with catabodies.
Examples 1 and 2 describe tests of dissolution of preformed wildtype misTTR and mutant misTTR aggregates (V122I, V30M, L55P, D18G and A25T) by the catabodies. Additional in vitro studies may be done to define the activity profile of the best catabodies with the desired specificity and catalytic properties. Both the physiological pentamer IgM form and the smaller monomer IgM form capable of penetrating various tissues at higher levels may be tested (obtained, respectively from expression in J-chain positive NS0 cells and J-chain negative CHO cells). In addition to verifying dissolution of preformed TTR amyloid, inhibition of formation of wildtype and mutant misTTR aggregates may be measured in the presence of the IgMs by turbiditometry/ThT fluorimetry. The data may be expressed as the IgM concentrations required to achieve 50% inhibition of misTTR amyloids. Dissolution of TTR amyloid may be confirmed by transmission electron microscopy after adsorption to copper grids and staining with uranyl acetate. To confirm recognition of “native” amyloid, catabody dissolution of amyloid deposits in cardiac tissue from ATTRwt patients and ATTRm (V122I, V30M) may be tested. Tissue cryostat sections may be treated with test and control IgMs, followed by staining with Congo red or available monoclonal IgGs that bind TTR specifically and estimation of amyloid deposits by microscopy. The tissue deposits may contain TTR fragments trapped within the amyloid formed by full-length misTTR [153, 154]. The dissolution of the cardiac amyloid deposits found in early-onset ATTRm as well as late-onset ATTRm containing higher and lower full-length protein/fragment ratios, respectively [154], may be tested. Mutant TTR amyloid is known to contain wildtype TTR [153] that is sensitive to the catabodies.
Soluble oligomers of amyloidogenic proteins are toxic [11]. Soluble wildtype and mutant misTTR oligomers may be separated from fibrillar TTR by centrifugation. The toxicity of the soluble misTTR oligomers may be tested using the human myocyte cell line AC16 [12] in the presence of varying test and control IgM concentrations. Cell death may be measured by the resazurin dye reduction assay.
Tg mouse models expressing human wildtype TTR and V30M mutant TTR may be applied as models for testing infused catabodies. Tissue TTR deposits in the transgenic mice expressing wildtype human TTR (Tg-TTR mice) are evident at age 20 months in the kidneys and heart of ˜90% mice [155]. TTR deposition in the Tg-V30MTTR mouse strain in the gut is evident at 11 months and spreads to the heart, lung and intestine at 14 months [156]. Although TTR deposition in both Tg mouse strains is less robust compared to human ATTRwt and ATTRm, these mice are the only in vivo resource for testing drug effects [157].
The following catabodies may be tested in groups of 12 mice each: (a) A misTTR-hydrolyzing pentamer IgM; (b) A misTTR-hydrolyzing monomer IgM; (c) and (d), respectively, a control pentamer IgM and a control monomer IgM devoid of misTTR-hydrolyzing or binding activity (statistical power at P<0.05 and effect size 1.2, 80%). Chimeric antibodies containing the human V domains linked to the murine IgM C-domains may be constructed per published methods [31]. This is necessary to avoid a murine anti-human IgM response in the mouse hosts receiving the test antibodies. The draining lymph from various tissues, a surrogate of the tissue interstitial fluid, contains pentamer IgM at 30-40% of blood IgM concentrations [97, 98]. Several FDA-approved chimeric IgGs containing murine V domains failed to induce an unacceptable anti-V domain response in humans. IgMs were approved for human clinical trials by the FDA [158].
Two types of studies may be conducted. First, aged Tg mice with pre-existing tissue TTR deposits may receive 2 intraperitoneal injections of the test and control catabodies at 2 week intervals (wildtypeTg-TTR mice, 20 months age; mutant Tg-V30MTTR mice, age 14 months). This protocol assesses the catabody therapeutic potential. Second, the catabodies may be injected at monthly intervals from age 1 month through 20 months (wildtypeTg-TTR mice) or 1 month through 14 months (mutant Tg-V30M TTR mice). In both studies, tissue TTR deposits may be visualized by quantitative immunochemistry 7 days after the final antibody injection (sections of kidney, esophagus, stomach and heart). The magnitude of to TTR deposition and the number of mice positive for TTR deposits may be scored on an organ-by-organ basis. Total TTR in tissue extracts and blood may be measured [148]. Inflammatory effects may be evaluated by staining for markers of activated macrophages, complement activation, T cells and B cells. At 12 mice/group, in vivo studies are powered to detect an effect size of 1.2 (80% power).
The half-life and tissue biodistribution of the 125I-labeled catabody injected intravenously in mice may be determined by measuring the radioactivity in blood and various tissues (1 h-14 days)[159]. Catabody molecular integrity in samples may be monitored by electrophoresis. Pentamer IgM half-life in blood is 2-3 days [160].
The “carbonyl stress state” characteristic of inflammation results in formation of oxidized misTTR with enhanced amyloid-forming tendency [13]. Accumulation of toxic oligomers of amyloidogenic proteins and amyloid deposits is detected decades prior to evidence of disease [7-9]. When misTTR formation exceeds the catabody clearance rate, amyloidosis will occur (
Catabodies are produced innately by B cells. Basal and misTTR-induced catabody production is subject to various constitutive (immunogen-independent) and immunogen-induced B cell regulatory processes. The blood of healthy humans subjects <35 years age display variable levels of catabodies directed to various target antigens (
misTTR can act as an immunogen for B cells. Immunogens induce biphasic effects on B cells. Small immunogen amounts enhance antibody synthesis, whereas excess immunogen is a toleragen that saturates the BCR, driving the cells into apoptosis or energy [161, 162]. Exposure to excess misTTR in ATTRwt patients, therefore, may tolerize B cells. misTTR itself or its oxidized variants with electrophilic character may act as toleragens (the NE-misTTR in Example 2). The NE-misTTR is likely to react covalently with nucleophilic BCRs. As the covalent reaction is irreversible, NE-misTTR derivatives will saturate the BCRs efficiently, causing a left-ward shift in the B cell dose response curve compared to a conventional non-covalently-binding immunogen (
The sera of ATTRwt patients displayed reduced catabody activity directed against misTTR, indicating impaired defense against TTR amyloid. Epidemiological details of the ATTRwt patients obtained from the Mayo clinic (provided by Drs. Robert Kyle and Angela Dispenzieri) and the two cohorts of control age-matched healthy humans from the Univ of Texas are provided in Table 4. The reduction of misTTR hydrolysis was reproducibly observed in tests of the purified IgM preparations from ATTRwt patients (
Currently, there is no established non-invasive and cost-effective procedure for diagnosis of ATTRwt, and tissue biopsy followed by immunohistochemical detection of TTR amyloid is the only definitive diagnostic method. The prognostic and diagnostic utility of catabodies may be established from analysis of their relationship with age in healthy humans and the occurrence of amyloidosis in patients with diagnosis of ATTRwt or ATTRm obtained from the biopsy procedures. In addition, it is important to monitor disease severity in individual patients so that their treatment can be tailored appropriately.
Hydrolysis of misTTR and Glu-Ala-Arg-AMC by purified IgM preparations, purified IgG preparations and unfractionated sera may be analyzed. The following groups of subjects may be studied:
Normal Healthy Humans.
Subjects without evidence of amyloid disease or infection belonging to these age groups: (i) 18-30 y; (ii) 30-50 y; (iii) 50-70 y; and (iv) 70-90 y. As an example, subgroups i-iii are composed of 12 subjects each. Subgroup iv is composed of 25 subjects (the larger n value enables valid comparison with ATTR patients below). Catabody activity may be plotted versus age. Comparison between the young 18-35 y and old >70-90 y subgroups provides information about changes in the catabody activity towards the end of the human life span (at P<0.05 and effect size of 1.0, power 80%). As males are more predisposed to ATTRwt compared to females, the catabody associations with gender may be assessed. For valid comparison with the V122I ATTRm patients described below, another subgroup of 25 healthy African-Americans (age 50-80 years) may be included.
Wildtype TTR Amyloidosis Patients with Cardiac Myopathy (ATTRwt Patients).
In addition to the patient cohort reported in
Hereditary Mutant TTR Amyloidosis Patients (ATTRm Patients).
Patients expressing the V122I TTR mutation found in 3-4% African Americans with frequent cardiomyopathy and V30M TTR mutation in Caucasians with frequent polyneuropathy may be studied (n=25 each patients each). The mutations may be determined from the TTR gene sequence and mass-spectrometery in biopsied tissue [164]. Catabody hydrolysis of the Y78:F mutant misTTR responsible for hereditary ATTRm has been verified. The V122I and V30M single mutations are also unlikely to markedly alter the cleavage susceptibility of mutant misTTR species to catabodies. Catabody activities may be compared with age- and gender-matched healthy humans (at P<0.05 and effect size of 0.8, power 80%). The V122I misTTR and V30M misTTR substrates may be tested. The rates of mutant TTR and wildtype TTR by the catabodies may be compared.
All samples from the test human subjects are also analyzed for binding of misTTR and phyTTR. If binding is observed, the correlation with hydrolytic activity may be analyzed. Interference in the assay by blood-borne misTTR is unlikely, as such species are difficult to detect in the blood of ATTRwt patients by routine binding assays [65].
Observations of suppressed misTTR-directed IgM catabodies accompanied by increased Glu-Ala-Arg-AMC cleaving polyreactive catabodies indicate a blood-based ATTRwt diagnostic test. The assay sensitivity, specificity, precision, false positive rate and false negative rate may be computed for the individual and combined assay data as in Table 5. Correlation of the catabody activities with the severity of cardiomyopathy or neuropathy indicates the ability to monitor disease progression.
To rule out non-specific factors, misTTR and Glu-Ala-Arg-AMC hydrolysis by IgM catabodies from control patients with an autoimmune disease (e.g., lupus) and an infection (e.g., HIV infection) may be tested (n=10 or more; both patient cohorts are described) [165-167]. The suppression of misTTR-directed catabodies accompanied by enhanced polyreactive catabodies in ATTRwt contrasts with previous findings on patients with autoimmune disease, who display enhanced antigen-specific hydrolysis and suppressed polyreactive IgG class catabodies [52].
Importantly, accumulation of the naturally-occurring electrophilic antigens in disease can drive antibody class-switching to superantigens and amyloids in disease, an event that is otherwise prohibited in the immune response [167]. Accumulation of NE-misTTR derivatives in ATTRwt and ATTRm patient may result in synthesis of class-switched IgG and IgA antibodies. Proof for this hypothesis was provided by studies on purified IgA class antibodies from ATTRwt patients shown in Table 4. misTTR hydrolysis by pooled purified IgA from these patients was readily detected (2.3 nmol hydrolysis/hour/μg IgA).
Measurements of B cell binding to E-misTTR and E-hapten-1 serve as indices of misTTR selective (
Detection of misTTR as a Disease Marker
The blood of ATTRwt may contain soluble misTTR [65], and healthy humans may also be positive for small amounts of such misTTR species. Owing to their specificity for a neoepitope expressed by misTTR, the catabodies are unique reagents that may permit detection of the blood-borne misTTR species, thereby permitting diagnosis of ATTRwt. Such species are difficult to detect by conventional binding assays. The catabodies detect both soluble misTTR, which act as seeds for amyloid formation, and the TTR amyloid itself (see Example 1). The misTTR species may be detected, for example, by treatment of human blood or another human bodily fluid with a specific anti-misTTR catabody, followed by monitoring of the hydrolytic reaction using conventional antibodies that bind the fragments produced by digestion of the misTTR. Conventional antibodies that bind TTR fragments have been published [148]. The detection of the TTR fragments can be accomplished, for example, using enzyme-linked immunosorbent assays or electrophoresis followed by immunoblotting.
A well-characterized catabody from Example 1 may be employed for detection of misTTR in ATTRwt patients and control age-matched healthy humans described in Example 2. A finding of increased soluble misTTR species in ATTRwt patients using a specific misTTR catabody indicates the ability to use the catabody for ATTRwt diagnosis. The ability of the assay to discriminate between the patient and control groups may be determined from the assay sensitivity, specificity, precision, false positive rate and false negative rate.
AMC, aminomethylcoumarin; AAA-AMC, Ala-Ala-Ala-aminomethylcoumarin; Ag, antigen; AGE, advanced glycation end-product; ATTRm, hereditary TTR amyloidosis caused by mutant TTR; ATTRwt, wildtype TTR amyloidosis; AVU, arbitrary volume units; Aβ, amyloid β peptide; Aβ42, Aβ1-42; Aβ40, Aβ1-40; BCR, B cell receptor; C domain, constant domain; catabody, catalytic antibody; CDR, complementarity determining region; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CPM, count per min; E-Ag, electrophilic antigen; E-hapten, electrophilic hapten; E-misTTR, electrophilic misTTR; E-phyTTR, electrophilic phyTTR; EBV, Epstein-Barr virus; EAR-AMC, Glu-Ala-Arg-aminomethylcoumarin; EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyanate; FR, framework region; LPE, lipid peroxidation end-product; MMP9, matrix metalloproteinase-9; mIgM, monoclonal IgM; misTTR, misfolded TTR; NE-Aβ, naturally-occurring electrophilic Aβ; NE-misTTR, naturally-occurring electrophilic misTTR; Ova, ovalbumin; PBS, phosphate buffered saline; PECy7, phycoerythrin cyanin 7; phyTTR, physiological TTR; pIgM, polyclonal IgM; SAP, serum amyloid P component; SDS, sodium dodecyl sulfate; Tg, transgenic; Tg-TTR, transgenic mice expressing wildtype human TTR; Tg-V30M TTR, transgenic mice expressing mutant V30:M human TTR; ThT, Thioflavin-T; TTR, transthyretin; V domain, variable domain; VH domain; heavy chain V domain; VL, light chain V domain.
Certain of the above experiments were at least partly funded by a grant from the SENS Research Foundation.
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/856,276, filed Jul. 19, 2013. The foregoing application is incorporated by reference herein.
This invention was made with government support under Grant Nos. AG025304 and AG033183 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/47409 | 7/21/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61856276 | Jul 2013 | US |
