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University of Colorado Denver

Dr. Jeffrey Bennett

Principal Investigator: Dr. Jeffrey Bennett

Project Title 1: AQP4 Autoantibodies and Disease Pathogenesis in Neuromyelitis Optica

NMO is a demyelinating disorder causally linked to autoantibodies against astrocyte water channel aquaporin-4 (AQP4). Multiple lines of evidence support a pathogenic role of AQP4 autoantibodies in NMO. First, NMO-IgG demonstrates high specificity for NMO and NMO spectrum disorders, correlates with the length of spinal cord lesions, and is depleted with salutatory therapies such as plasma exchange. Second, in cultured cells and spinal cord explants, NMO-IgG binding to AQP4 causes complement activation and cytotoxicity. And third, administration of human NMO-IgG or cloned AQP4-specific rAbs to naïve mice or to rats with pre-existing neuroinflammation produces NMO-like pathology. Mechanistically, NMO-IgG binding to AQP4 on CNS astrocytes is thought to initiate a series of inflammatory events,
including antibody-dependent complement activation and cell-mediated cytotoxicity, leukocyte recruitment, cytokine release and demyelination.

Despite our basic understanding of NMO-IgG mediated pathogenesis, key questions remain unanswered. What subsets of B cells produce pathogenic AQP4 autoantibodies during an NMO exacerbation? If AQP4 autoantibodies are pathogenic, why do serum anti-AQP4 titers correlate poorly with disease activity and clinical severity? And, how does a targeted immune response against astrocytes result in glial injury, myelin destruction, and neurodegeneration? The initial work at UC Denver to dissect the humoral immunopathology of NMO has allowed them to develop the necessary resources and technology to address these critical questions and to further basic and clinical translational NMO research.


Project Title 2: Second-Generation Aquaporumabs and NMO Superantibodies for NMO Research and Therapy

The generation of recombinant monoclonal antibodies (rAb) against aquaporin-4 has provided major new opportunities in NMO research and therapeutics. As NMO-rAbs are monoclonal and pure, they have afforded the ability to study NMO pathogenesis mechanisms in the absence of confounding serum factors in order to: (i) measure antibody targeting and pharmacokinetics in animal models; (ii) identify binding epitopes on AQP4; (iii) screen for small-molecule blockers; and (iv) develop aquaporumab blocking antibodies.

The NMO-rAbs generated to date are derived from sequencing clonally expanded plasma blasts in CSF of NMO patients. NMO-rAbs are fully human antibodies. The two NMO-rAbs with highest affinity for binding to AQP4 have dissociation constants of ~15 and 40 nM. Mutations of the Fc region of the antibody have generated antibodies with greatly reduced, though non-zero, CDC and/or ADCC effector functions. The intended use of these ‘aquaporumabs’ is for NMO therapy. Other mutations of the Fc region have generated antibodies with increased (by >10-fold) CDC or ADCC effector function. The intended use of these ‘NMO superantibodies’ is to generate animal models of NMO, which are badly needed to study NMO pathogenesis mechanisms and to develop new therapies.

The major goal of this research is to carry out screening/mutagenesis to produce second-generation aquaporumabs and NMO superantibodies. It has become evident that improvements in the AQP4 binding portion (Fab) and effector function (Fc) region of NMO-rAbs is needed. For aquaporumab therapy, an optimal antibody should have AQP4 binding dissociation constant ~ 0.1-0.5 nM and zero residual effector function. Very tight AQP4 binding affinity is also desired for generation of animal models with NMO superantibodies, as is greatly enhanced (>25-fold) effector function. It is anticipated that, with additional screening / affinity maturation / mutagenesis, NMO-rAbs with the target properties can be generated.

UC Denver NMO Team

  • Jeffrey L. Bennett, M.D., Ph.D., Professor of Neurology and Ophthalmology
  • Greg Owens, Ph.D., Research Professor of Neurology
  • Allana Ritchie, B.S., Professional Research Assistant