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NEJM Editorial

Inhibitory antibodies to factor VIII arise in more than 20 percent of patients with hemophilia A who receive replacement therapy with factor VIII protein.1 Since these antibodies profoundly complicate the treatment of hemophilia, understanding how they inhibit factor VIII has important clinical implications. In this issue of the Journal, Lacroix-Desmazes and colleagues report the intriguing finding that inhibitory antibodies cause specific proteolytic degradation of factor VIII.2 They propose that proteolysis underlies the rapid inactivation of the protein when it is administered to patients who have the antibodies. Confirmation of this proposal could provide new insight into hemophilia and its treatment.

The report lands on ground prepared by many studies of catalytic antibodies. There was a time when a clear distinction was drawn between enzymes and antibodies: enzymes could catalyze chemical reactions, but antibodies could not. However, since both were known to bind ligands of similar size, with similar three-dimensional specificity, affinity, and intermolecular interactions, Jencks proposed that it should be possible to design an antibody with catalytic activity at its antigen-binding site with the use of an appropriate antigen.3 The antigen, Jencks suggested, would resemble a transient structure - known as the transition state - formed from the substrate during an enzyme-catalyzed reaction. An early attempt to make such an antibody met with some success.4 Shortly thereafter, a myeloma protein with ester-cleaving activity was identified.5

A great surge in the design of catalytic antibodies began in the mid-1980s. Although catalytic antibodies elicited with transition-state analogues rarely achieve the proficiency of even moderately active enzymes, they catalyze a remarkable number and variety of reactions, including some for which enzymes are not available.6 Many catalytic mechanisms of antibodies have been established by rigorous biochemical, kinetic, and crystallographic structural analysis,7 and mutational analysis has revealed critical amino acids in the antigen-binding, catalytic sites of the antibodies. This work sets a high standard for the characterization of antibody-mediated catalysis.

Recently, it has been discovered that most antibodies, whatever their antigen-binding specificity, can catalyze the formation of hydrogen peroxide from singlet oxygen, a product generated, for example, by the microbicidal activity of phagocytes.8 This catalytic activity probably depends on highly conserved amino acids in the immunoglobulin variable domains. Thus, in contrast with the early distinction between enzymes and antibodies, this work suggests that a catalytic activity - the generation of hydrogen peroxide - is in fact a physiologic function of immunoglobulins that contributes to the killing of invading organisms.

Reports of autoantibodies with proteolytic activity also appeared in the late 1980s. Since those autoantibodies were spontaneously arising polyclonal serum antibodies, they were more difficult to characterize than monoclonal antibodies obtained in response to known immunogens. Strenuous efforts were made to establish that the proteolytic activity was indeed a property of the autoantibody. In many cases, the light-chain variable region alone had the proteolytic activity.9 Some myeloma light-chain proteins also mediated proteolysis, including the cleavage of prothrombin.10 In addition, some proteolytic antibodies were obtained by immunization with a peptide antigen rather than a transition-state analogue. Mutagenesis identified amino acids in the light-chain variable region of such an antibody as critical for proteolysis.11 Whether this property of the variable region arises by chance or by selection based on function is an open question.

How do the data on the degradation of factor VIII fit into this background? Lacroix-Desmazes et al. have shown that factor VIII is degraded when incubated with purified IgG or affinity-purified antibodies against factor VIII from patients who have an inhibitor of factor VIII. The same IgG did not degrade factor IX or albumin. Nevertheless, the characterization of catalysis mediated by antibodies against factor VIII was not carried out to the level achieved for designed catalytic antibodies or catalytic autoantibodies, and there are reasons for caution in assigning the proteolytic activity to the binding site. The authors note the occurrence of spontaneous hydrolysis of recombinant factor VIII in buffer alone and additional, albeit minimal, hydrolysis in the presence of normal immunoglobulin. For these reasons there is a possibility that, instead of having catalytic activity themselves, the antibodies bind to factor VIII and render it more susceptible to whatever produces the hydrolysis in the absence of antibodies. Another reason for caution is that there must be many cleavage sites in recombinant factor VIII to account for the numerous degradation products of the antibody-mediated reactions. If each cleavage depends on an epitope-specific interaction, then serum with such inhibitors of factor VIII must contain a diverse set of antibodies, each with a different binding site but all with a structure required for catalysis. We should anticipate additional steps toward establishing whether diverse binding sites for antibodies against factor VIII have proteolytic activity. The availability of a crystal structure of a factor VIII-Fab complex12 and the results of other studies indicate that monoclonal antibodies against factor VIII and light chains can be obtained for such tests.

In the present study and a previous article,13 the authors have calculated that, at the concentrations of antibody and factor VIII found in plasma, the proteolytic activity may cause infused factor VIII to have a very short half-life in vivo. That conclusion remains to be tested by measurements of in vivo degradation of injected factor VIII in patients with or without proteolytic antibody activity. Since several interactions may combine to neutralize factor VIII activity in patients who have inhibitory antibodies,12 it will be important to determine the extent and mechanism of proteolysis in vivo.

Could this insight into mechanisms affect clinical management? The current options for patients who have inhibitory antibodies to human factor VIII are to administer porcine factor VIII, which differs from the human protein in regions that contribute to antigenicity,12 or to bypass the requirement for factor VIII by the use of recombinant activated factor VII (factor VIIa) or activated prothrombin-complex concentrates.1 A different approach is induction of tolerance by the administration of large doses of recombinant factor VIII or the suppression of immune responsiveness with steroids and immune globulin therapy.1,8

If antibody-mediated proteolytic degradation of factor VIII is an important mechanism of inhibitory action, the question arises whether a specific inhibitor of the proteolysis could usefully prolong the therapeutic function of factor VIII. If there is a diverse set of antibodies with different binding sites, one would have to hope that all of them would be susceptible to a single inhibitor. Ideally, such an inhibitor would block not only proteolysis but also binding of the antibodies to factor VIII. Alternatively, if just one site of factor VIII were critical for the initiation of proteolysis, engineered amino acid substitutions in factor VIII might make that site resistant to cleavage while retaining coagulation activity.

It may also be possible to develop an inhibitor of the degradation of factor VIII if the antibodies are not directly proteolytic but act by enhancing the susceptibility of factor VIII to proteolysis by some other mechanism. If this is the case, however, it will be important to determine whether there is residual inhibition of factor VIII due to antibody binding that prevents the functional interaction of factor VIII with membrane phospholipids and other coagulation proteins. If such binding blocks the functioning of factor VIII without proteolysis, the current options for individualized management will continue to be important. The observations of Lacroix-Desmazes and colleagues point to the experiments that can test these possibilities.

B. David Stollar, M.D.

Tufts University School of Medicine

Boston, MA 02111

References

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2.Lacroix-Desmazes S, Bayry J, Misra N, et al. The prevalence of proteolytic antibodies against factor VIII in hemophilia A. N Engl J Med 2002;346:662-667.[Abstract/Full Text]

3.Jencks WP. Catalysis in chemistry and enzymology. New York: McGraw-Hill, 1969.

4.Raso V, Stollar BD. The antibody-enzyme analogy: comparison of enzymes and antibodies specific for phosphopyridoxyltyrosine. Biochemistry 1975;14:591-599.[Medline]

5.Kohen F, Kim JB, Lindner HR, Eshhar Z, Green B. Monoclonal immunoglobulin G augments hydrolysis of an ester of the homologous hapten: an esterase-like activity of the antibody-containing site? FEBS Lett 1980;111:427-431.[Medline]

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8.Wentworth AD, Jones LH, Wentworth P Jr, Janda KD, Lerner RA. Antibodies have the intrinsic capacity to destroy antigens. Proc Natl Acad Sci U S A 2000;97:10930-10935.[Abstract/Full Text]

9.Sun M, Gao QS, Kirnarskiy L, Rees A, Paul S. Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain. J Mol Biol 1997;271:374-385.[Medline]

10.Thiagarajan P, Dannenbring R, Matsuura K, Tramontano A, Gololobov G, Paul S. Monoclonal antibody light chain with prothrombinase activity. Biochemistry 2000;39:6459-6465.[Medline]

11.Gololobov G, Sun M, Paul S. Innate antibody catalysis. Mol Immunol 1999;36:1215-1222.[Medline]

12.Spiegel PC Jr, Jacquemin M, Saint-Remy JM, Stoddard BL, Pratt KP. Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII. Blood 2001;98:13-19.[Abstract/Full Text]

13.Lacroix-Desmazes S, Moreau A, Sooryanarayana, et al. Catalytic activity of antibodies against factor VIII in patients with hemophilia A. Nat Med 1999;5:1044-1047.[Medline]