2013 Research Report (Part 7 of 12): Extracellular Aggregates

Posted by Iain Inkster on January 06, 2014 | SRF Research

The following is an excerpt from SRF's 2013 official research report available in full here.

As part of the degenerative aging process, proteins that normally remain dissolved in bodily fluids become damaged and adopt an abnormally-clumped form called amyloid. Amyloid clumps are toxic and hard for the body to break down. They accumulate as deposits in various organs with advancing age, disrupting organ structure and impairing function. One important amyloid disease of aging is caused by the aggregation of the transporter protein transthyretin (TTR) into amyloid that deposits in many organs, including the heart. The insidious effects of TTR amyloid can start appearing in middle age, progressively becoming sufficient to impair the function of the lungs, the kidneys, and other organs — most particularly the heart. Over 10% of individuals over the age of 70 are seriously affected by TTR amyloid, and the condition becomes near-universal with further aging. TTR amyloid appears to be a major contributory factor to the death of “supercentenarian” — those among us who achieve 110 years or more of life.

Additionally, mutations in the TTR gene produce a protein form that is more easily twisted into amyloid, including a mutation affecting 3-4% of African Americans that causes heart failure. Patients carrying some of these mutations can develop early-onset familial amyloidosis prior to age 30. There is no approved treatment for TTR amyloidosis, and replacement of the failed organ is the only option. The SENS Research Foundation-funded TTR Extracellular Aggregates collaboration is developing antibodies that recognize and remove TTR amyloid deposits from tissues safely. The antibodies could be used for diagnosis and treatment of patients with both age-induced and genetic forms of TTR amyloid.

To generate antibodies that bind TTR, Dr. O’Nuallain is exploiting the acquired immunity paradigm. Such antibodies might be used to identify people with undiagnosed cardiac amyloidosis and possibly also as therapeutic agents. Dr. O’Nuallain immunized three strains of mice with three different immunogens containing TTR aggregates. One group was immunized with either fibrils of the non-mutant protein’s amyloid, or with a mutant TTR rendered into a soluble state. Three other groups of mice, each of a different strain, were immunized with a mixture of both non-mutant TTR fibrils and the resolubilized mutant protein. Exposure to these foreign proteins triggered the mouse immune system to generate novel antibodies that targeted aggregated TTR and not the physiologically-functional form of TTR. B-cells from the immunized mice were then fused with cancerous mouse B-cells to generate twenty ‘immortal’ mouse cell lines, each secreting a unique TTR-reactive monoclonal antibody.

Seven of the resulting antibodies demonstrate diagnostic and therapeutic potential. They bind strongly to patient-derived TTR amyloid without reacting with the physiological TTR form, and they retain their binding activity in the presence of plasma from normal humans. These and additional antibodies from the immunized mice are also being tested for the ability to catalyze the degradation of TTR amyloid in collaboration with Dr. Paul.

Meanwhile, Dr. Paul has been developing a class of catalytic antibodies (catabodies) that break peptide bonds in TTR amyloid, based on innate immunity principles. In previous research, Dr. Paul identified catabodies naturally produced by young and old humans that specifically degrade the beta-amyloid peptide clumps found in the brain of patients with Alzheimer’s disease. With funding from SENS Research Foundation, he has identified catabodies that target TTR amyloid. These newly-discovered molecules are members of the immunoglobulin M (IgM) class of antibodies that are synthesized as part of the first-line, innate defense against dangerous substances detected by the immune system (see Figure 1).

 

Figure 1: Immunoglobulin M (IgM) Structure.
Image by Artur Jan Fijalkowski, licensed under the
Creative Commons Attribution-Share Alike 2.5
Generic license.


These catabodies completely dissolved TTR amyloid in the test-tube without degrading the physiological TTR form or off-target proteins. Probing the catabodies showed that their degrading power derived from a serine protease type of enzyme mechanism, with no dependence on other blood enzymes or phagocytic cells required by conventional antibodies for effective target removal. Moreover, the catabodies did not form stable immune complexes that exert harmful side-effects. Immunoglobulins G (IgGs), the class of antibodies usually associated with acquired immunity, were unable to degrade TTR amyloid. From these findings, Dr. Paul’s group theorizes that production of catabodies to TTR amyloid is an ancestral immune function that humans use as a first-line surveillance strategy to delay age-associated amyloidosis.

Supplementing the natural defense function of catabodies with an externally-provided catabody that is specific for the TTR amyloid form holds potential for a safe and efficacious treatment. The catabody can be delivered intravenously or by gene therapy. Both delivery methods have been validated for the Alzheimer beta-amyloid target in animal models by Dr. Paul and his collaborators.

Catabodies offer several potential advantages over the conventional amyloid binding antibodies that have been the focus until now in developing immunotherapies for amyloid disease. The catabodies actually destroy their targets, rather than merely binding to them. A single catabody molecule can be reused thousands of times to degrade molecule after molecule of the amyloid. In contrast, conventional IgG antibodies offer only a “one-shot” capability, and some of them may rely on phagocytic cells that ingest the IgG-target protein immune complex.

Because catabodies remove the amyloid independent of phagocytic cells, they do not carry the risk of inflammation and vascular damage caused by conventional antibodies. By minimizing the amount of catabody needed to remove large amyloid quantities, the cost of catabody therapy should be lower than immunotherapy regimens that rely on conventional monoclonal antibodies. Delivering catabodies via a gene therapy approach could offer the additional advantage of making repeat catabody administrations unnecessary.

SENS Research Foundation is now funding further work to isolate and optimize a cell line producing a specific catabody that clears TTR amyloid, thus providing a renewable source of the candidate therapeutic catabody. Dr. Paul's team is employing mechanism-based TTR amyloid analogs to selectively trap the best catabody from a large antibody library (see Figure 2). Various genetic engineering technologies to optimize the catabody activity are available to the team, including combinatorial pairing of the light and heavy chain IgM variable domains, and use of alternate constant domains to maximize catalytic activity and catabody half-life in the blood.

In 2013, the collaborative team hopes to identify lead antibodies and catabodies suitable for diagnosis and therapy of TTR amyloidosis. Further evaluation will entail testing in a transgenic mouse model of TTR amyloidosis. If these antibodies and catabodies prove themselves to be efficacious and safe in animal models, then the ground is laid for clinical trials to treat age-associated and familial amyloidosis in humans.

 
Figure 2: Native catabodies appear to have evolved as an innate, physiological defense
mechanism against the development of amyloidosis in humans.
Dr. Paul and colleagues are
working to harness the power of acquired immunity by immunization with mechanism-based
electrophilic amyloids that stimulate the cells responsible for producing the catabodies.
Image via Sudhir Paul, redrawn by Anne Corwin.
 
 
End of part 7
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