David C. Fritzinger

Depletion of the C3 component of complement enhances the ability of rituximab-coated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model

Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)-cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.

Cobra venom factor: Structure, function, and humanization for therapeutic complement depletion.

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. This manuscript reviews the structure and function of CVF, how it interacts with the complement system, the structural and functional homology to complement component C3, and the use of CVF as an experimental tool to decomplement laboratory animals to study the functions of complement in host defense and immune response as well as in the pathogenesis of diseases. This manuscript also reviews the recent progress in using the homology between CVF and C3 to study C3 structure and function, and to develop human C3 derivatives with the complement-depleting function of CVF. These human C3 derivatives represent humanized CVF, and are a conceptually different concept for pharmacological intervention of the complement system, therapeutic complement depletion. The use of humanized CVF for therapeutic complement depletion in several pre-clinical models of human diseases is also reviewed.

Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex.

Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short‐lived protease complexes are formed through pro‐convertases, which for the alternative pathway consist of the complement component C3b and the pro‐enzyme factor B (FB). Here, we present the crystal structure at 2.2‐Å resolution, small‐angle X‐ray scattering and electron microscopy (EM) data of the pro‐convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro‐peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal‐ion dependent adhesion site of the Von Willebrand factor A‐type domain. A possible dynamic equilibrium between a ‘loading’ and ‘activation’ state of the pro‐convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.

Structure and Function of Cobra Venom Factor, the Complement-Activating Protein in Cobra Venom

Cobra Venom Factor (CVF) is an unusual venom component known to be present in the venom of the cobra species Naja, Ophiophagus, and Hemachatus of the Elapidae family (1). CVF is not a toxin in the classical sense. As a matter of fact, the purified molecule is not toxic. It specifically interacts with components of the serum complement system, leading to complement activation which in turn leads to the consumption of complement activity.

Humanized cobra venom factor decreases myocardial ischemia–reperfusion injury

Cobra venom factor (CVF) is a complement activating protein in cobra venom, which functionally resembles C3b, and has been used for decades for decomplementation of serum to investigate the role of complement in many model systems of disease. The use of CVF for clinical practice is considered impractical because of immunogenicity issues. Humanization of CVF was recently demonstrated to yield a potent CVF-like molecule. In the present study, we demonstrate that mice treated with recombinant humanized CVF (HC3-1496) are protected from myocardial ischemia-reperfusion (MI/R) injuries with resultant preservation of cardiac function. Also, C3 deposition in the myocardium following MI/R was not observed following treatment with HC3-1496. HC3-1496 led to complement activation and depletion of C3, but preserved C5 titers. These data suggest, unlike CVF, HC3-1496 does not form a C5 convertase in the mouse, similar to recent studies in human sera/plasma. These results suggest that humanized CVF (HC3-1496) protects the ischemic myocardium from reperfusion injuries induced by complement activation and represents a novel anti-complement therapy for potential clinical use.

Humanized cobra venom factor: structure, activity, and therapeutic efficacy in preclinical disease models.

The complement system is an integral component of both innate and adaptive immunity. However, complement is also a pathogenetic factor in many diseases. The development of agents for therapeutic complement inhibition is the topic of intense investigations by many investigators. We have developed a distinctly different therapeutic approach: complement depletion rather than inhibition. This approach is based on cobra venom factor (CVF), a C3 analog known to be able to safely deplete complement. This manuscript will briefly review the structure and activity of CVF, along with its similarities and differences to C3. Exploiting the knowledge of the structure/function relationship of CVF and C3, we created derivatives of human C3 which display the CVF-like activity of depleting complement, referred to as humanized CVF (hCVF). This review describes the structure and activity of hCVF, including the important property of not cleaving C5. The efficacy of hCVF for therapeutic complement depletion in nine preclinical models diseases with complement pathology is reviewed, including reperfusion injury, age-related macular degeneration (AMD), paroxysmal nocturnal hemoglobinuria (PNH), and immunogenicity of Factor VIII in hemophilia A. Complement depletion is characterized by the absence of toxicity, even after intra-arterial injection into the pulmonary artery of primates. No immunogenicity has been observed.

Derivatives of Human Complement Component C3 for Therapeutic Complement Depletion: A Novel Class of Therapeutic Agents

To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with less immunogenicity, we have prepared human C3/CVF hybrid proteins, in which the C-terminus of the alpha-chain of human C3 is exchanged with homologous regions of the C-terminus of the beta-chain of CVF. We show that these hybrid proteins are able to deplete complement, both in vitro and in vivo. One hybrid protein, HC3-1496, is shown to be effective in reducing complement-mediated damage in two disease models in mice, collagen-induced arthritis and myocardial ischemia/reperfusion injury. Human C3/CVF hybrid proteins represent a novel class ofbiologicals as potential therapeutic agents in many diseases where complement is involved in the pathogenesis.

Complement Depletion with Humanized Cobra Venom Factor in a Mouse Model of Age-Related Macular Degeneration

The effect of complement depletion with humanized cobra venom factor (CVF) on retinal lesion development/neovascularization was determined in a mouse model of wet age-related macular degeneration (AMD). Mice were treated with the humanized CVF protein HC3-1496 prior to, and once daily for 28 days after laser coagulation surgery of the retina. CVF transgenic mice exhibiting permanently low levels of serum complement activity and PBS-treated mice served as positive and negative controls, respectively. Fluorescein isothiocyanate (FITC)-dextran funduscopy after laser surgery indicated the presence of lesions in all mice that underwent laser surgery. In HC3-1496-treated mice as well as CVF transgenic mice smaller lesions were seen after 8 days. Measurement of lesion sizes by histopathological examination of eyes after 28 days revealed a significant reduction of lesion area and volume in both HC3-1496-treated animals and CVF transgenic animals compared to PBS-treated control animals. Systemic complement depletion with a complement depletor, such as the humanized CVF protein HC3-1496, represents a promising therapeutic concept for patients with wet AMD.

Complement depletion with humanised cobra venom factor: efficacy in preclinical models of vascular diseases.

The complement system is an intrinsic part of the immune system and has important functions in both innate and adaptive immunity. On the other hand, inadvertent or misdirected complement activation is also involved in the pathogenesis of many diseases, contributing solely or significantly to tissue injury and disease development. Multiple approaches to develop pharmacological agents to inhibit complement are currently being pursued. We have developed a conceptually different approach of not inhibiting but depleting complement, based on the complement-depleting activities of cobra venom factor (CVF), a non-toxic cobra venom component with structural and functional homology to complement component C3. We developed a humanised version of CVF by creating human complement component C3 derivatives with complement-depleting activities of CVF (humanised CVF) as a promising therapeutic agent for diseases with complement pathogenesis. Here we review the beneficial therapeutic effect of humanised CVF in several murine models of vascular diseases such as reperfusion injury.

Hybrid proteins of cobra venom Factor and cobra C3: Tools to identify functionally important regions in cobra venom Factor

Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally and functionally highly homologous to complement component C3. CVF, like C3b, the activated form of C3, forms a bimolecular complex with Factor B in serum, called C3/C5 convertase, an enzyme which activates complement components C3 and C5. Despite the high degree of homology, the two C3/C5 convertases exhibit significant functional differences. The most important difference is that the convertase formed with CVF (CVF,Bb) is physico-chemically far more stable than the convertase formed with C3b (C3b,Bb). In addition, the CVF,Bb convertase and CVF are completely resistant to inactivation by the complement regulatory proteins Factor H and Factor I. Furthermore, the CVF,Bb enzyme shows efficient C5-cleaving activity in fluid phase. In contrast, the C3b,Bb enzyme is essentially devoid of fluid-phase C5-cleaving activity. By taking advantage of the high degree of sequence identity at both the amino acid (85%) and DNA levels (93%) between CVF and cobra C3, we created hybrid proteins of CVF and cobra C3 where sections, or only a few amino acids, of the CVF sequence were replaced with the homologous amino acid sequence of cobra C3. In a first set of experiments, we created five hybrid proteins, termed H1 through H5, where the cobra C3 substitutions collectively spanned the entire length of the CVF protein. We also created three additional hybrid proteins where only four or five amino acid residues in CVF were exchanged with the corresponding amino acid residues from cobra C3. Collectively, these hybrid proteins, representing loss-of-function mutants of CVF, allowed the identification of regions and individual amino acid residues important for the CVF-specific functions. The results include the observation that the CVF β-chain is crucially important for forming a stable convertase, whereas the CVF α-chain appears to harbor no CVF-specific functions. Furthermore, the CVF γ-chain is additionally important for the fluid-phase C5-cleaving activity of CVF,Bb. Interestingly, the structural changes in the individual hybrid proteins differentially affected the molecular functions of the CVF,Bb enzyme such as convertase formation, C3 cleavage, and C5 cleavage.