1. Humanized Cobra Venom Factor (hCVF), a Novel Complement Inhibitor.
David C. Fritzinger, Ph.D., and Carl-Wilhelm Vogel, MD, Ph.D.
In this document, we describe the intellectual property developed in my laboratory at the
University of Hawaii Cancer Center. The IP consists of inhibitors of the complement
system that are modified human C3 proteins, in which short C3 sequences have been
replaced by homologous sequences from a C3 analog found in cobra venom, cobra
venom factor (CVF). First, I will provide some background.
The complement system is part of the innate immune system. There are 3 major pathways
of complement activation, the antibody-mediated Classical Pathway, the carbohydrate-
mediated Lectin Pathway, and the Alternative Pathway. All three pathways result
formation of an enzyme that is a C3 convertase, called C3b,Bb. The C3 convertase
activates C3 by removing a 77 amino acid peptide (C3a) from C3, causing a substantial
conformational change in C3. This conformational change exposes several binding sites
for other complement proteins, as well as breaking a high-energy bond in C3, called the
thioester linkage. Breaking this bond allows C3 to covalently bind to cell surfaces. The
C3 activation step acts as the bridge between the three activation pathways and the
terminal complement pathway, in which the Membrane Attack Complex (MAC) is
formed. This occurs because the C3 convertase is also able to activate another protein in
the complement system, C5. The activated form of C5, C5b is then able to bind other
complement components (C6, C7, C8, and C9) to form the Membrane Attack Complex
(MAC). The MAC is able to insert itself into cellular membranes, thereby killing the cell
to which it binds. This is explained in greater detail in the two enclosed reviews.
As stated above, CVF is a protein isolated from the venom of elapid snakes such as
cobras, that is responsible for depleting complement in bite victims. CVF is similar to C3,
both structurally and functionally. CVF and human C3 are structurally similar, being
about 50% identical in sequence (~70% if conservative replacements are counted). The
crystal structures of both proteins have been solved, revealing proteins with very closely
related 3 dimensional structures. Functionally, both proteins are also very similar. Both
are able to form a C3 convertase by binding a second complement protein, factor B,
which is then cleaved by another complement protein, factor D, to form the active
convertase (C3b,Bb and CVF,Bb). Both convertases then act in a similar way to cleave
C3a from C3, resulting in the active form of C3, C3b. While they are similar in function,
differences do exist. Most importantly, CVF forms a convertase that is far more stable
than the C3-containing convertase, with t1/2 of dissociation of 7 hours vs. 1.5 minutes,
respectively. In addition, the CVF-containing convertase is resistant to the action of
complement control proteins. CVF,Bb acts in the fluid phase, while C3b,Bb is attached to
cell surfaces. Finally, while both convertases can also cleave C5, thereby initiating the
terminal pathway of complement, C3b,Bb requires the presence of a second C3b, while
CVF,Bb does not. Because of the unique properties of CVF, injection of CVF into a
mammal (or adding it to animal serum) will result in the exhaustive activation of
complement, thus inactivating several complement components (C3b, Bb) which
prevents complement activation. This feature of CVF has proven valuable, both in
helping to elucidate the complement pathways and for showing the role of complement in
the disease process of a number of diseases.

2. The IP described here was a result of experiments performed in our laboratory to
determine structure/function relationships in C3 and CVF, and to elucidate which
sequences in CVF are needed for the unique properties described above. During this
process, we prepared several C3/CVF hybrid proteins that were able to form active,
stable C3 convertases, thus demonstrating these portions of the C3/CVF sequence are
required for stable convertase formation. In several cases, we were able to define either
single amino acid residues, or very short sequences that were able to increase convertase
stability. Indeed, several of the hybrid proteins we designed form convertases that are
more stable than convertases containing CVF.
Inappropriate activation of complement plays a major role in the disease process of a
number of diseases, including ischemia/reperfusion diseases (Myocardial Infarctions and
stroke), Age-related Macular Degeneration (AMD), Myasthenia Gravis, and Rheumatoid
Arthritis. For this reason, there has been an increased interest in developing complement
inhibitors. Currently, only one complement inhibitor has been approved by the FDA, a
humanized C5 monoclonal antibody (called Soliris) developed by Alexion
Pharmaceuticals, Inc. Soliris has been approved by the FDA for the treatment of two rare
diseases, Peroxysmal nocturnal hemoglobinuria (PNH) and Atypical Hemolytic-Uremic
Syndrome (aHUS). Soliris has become one of the most profitable drugs on the market,
thus showing the potential value of developing inhibitors of complement.
Because of the ability of several proteins we developed to exhaustively activate, thereby
depleting, complement, we decided to investigate the ability of one of the hybrid proteins,
called HC3-1496 or humanized CVF (hCVF) for efficacy in several different animal
models of disease. At the same time, this IP attracted the interest of an entrepreneur who
was interested in starting a biotech company to investigate uses for this promising
intellectual property. We were able to obtain one round of venture capital funding from
Avalon Ventures, which allowed us prepare significant quantities of the company’s lead
protein, and to test its efficacy and toxicity in animal models of several diseases. These
include mouse models of rheumatoid arthritis, myocardial ischemia/reperfusion,
Myasthenia gravis and an in vitro model of PNH. In all cases, hCVF proved to be
efficacious in treating the diseases. hCVF was also tested for toxicity in a non-human
primate, and proved to be virtually non-toxic, even when the animals were injected with a
dose four times that needed for complete complement depletion. These studies are
described in more detail in the reviews referenced below. It should be noted that one of
these papers was an invited review included in a special issue of Molecular Immunology,
published in the summer of 2014 as a companion volume to the 25th
International
Complement Workshop, held in Rio de Janeiro in September, 2014. This article describes
new uses for hCVF and demonstrates that hCVF shows far less immunogenicity in mice
than does CVF.
One important consideration that should be noted is that hCVF is unique as a complement
inhibitor. While most complement inhibitors block activation by binding complement
components stoichiometrically, hCVF forms an enzyme, meaning that each molecule of
hCVF can activate many C3b molecules. Therefore, hCVF can be effective at much
smaller doses than most other complement inhibitors.
While the version of hCVF described here has been shown to be efficacious in a number
of disease models, there are still some improvements that could be made to the protein.

3. For example, while hCVF depletes complement in mammals, the depletion is not as long
lasting as depletion with native CVF. Our working hypothesis is that hCVF is degraded
by two regulators of complement activity, proteins called factor H and factor I. Factor H
binds to C3b and prevents the binding of factor B. In addition, factor H can competitively
bind to C3b,Bb, displacing Bb. Once bound to C3b, factor H serves as a cofactor in the
cleavage of C3b by factor I, which inactivates C3b and prevents it from binding factor B.
Since the crystal structure of the factor H:C3b complex has been elucidated, it should be
possible to design variants of hCVF lacking amino acid residues required for factor H
binding to C3b. We have designed several such variants and will test them once sufficient
funds have been found.
References
Vogel, C-W, Finnegan, PW, Fritzinger, DC. (2014) ”Humanized Cobra Venom Factor:
Structure, Activity, and Therapeutic Efficacy in Preclinical Disease Models.” Mol.
Immunol. 61, 191-203.
Vogel, C.-W., and Fritzinger D.C. (2010) “Cobra venom factor: Structure, function, and
humanization for therapeutic complement depletion.” Toxicon 56, 1198-1222