How to get rid of inhibitors

Antibodies to Factor VIII (FVIII) develop in the context of allo- and auto-immunization. Haemophilia patients recognize FVIII as an alloantigen and produce specific antibodies, some of which are endowed with the capacity to inhibit the function of FVIII. Acquired inhibitors are observed in the setting of both systemic autoimmunity, or as the result of a break in tolerance caused by a pathogenic event or medical intervention. The distinction between these three situations is important for the understanding of the mechanisms by which inhibitors are formed and for subsequent therapeutic approaches.

This does not mean that these three categories of FVIII inhibitors inactivate FVIII by distinct mechanisms. Inactivation depends more on the epitope specificity, namely the region of FVIII to which antibodies bind. There is no indication that antibody specificity differs between allo- and autoantibodies. Thus, antibodies can inhibit the function of FVIII by competing with the binding to an essential partner, be it Factor IX, Factor X or phospholipids. Inhibition of the binding of FVIII to von Willebrand factor accelerates the catabolism of FVIII. In addition, some inhibitors directly degrade FVIII by proteolytic cleavage. Let us not forget that inhibitors represent just one part of the immune response to FVIII, i.e. antibodies to functional epitopes of FVIII.

Inhibitors are produced by B cells that are instructed to differentiate into plasmocytes. It should be understood that the bone marrow of any healthy individual is producing millions of new B cells every day. Such B cells acquire specificity through random rearrangement of their variable chains, which means that thousands of B cells with capacity to recognize FVIII are produced daily. Several mechanisms do keep this potential immune response at bay: elimination of the most reactive B cells within the bone marrow, after the B cells have been offered a chance to escape deletion by editing or revising their receptors, induction of anergy in the periphery, deletion or passive death because of absence of stimulation.

Although the specificity of inhibitors appearing under various clinical contexts does not differ, B cells producing such antibodies are qualitatively heterogeneous. Acquired inhibitors appearing in the context of systemic autoimmunity are part of a general propensity to produce autoantibodies. Those observed after for instance surgery or pregnancy, are produced under specific and temporary circumstances. In contrast, inhibitors observed as response towards replacement therapy have all reasons to undergo every maturation step towards long-term production of high affinity inhibitors. We should therefore keep in mind that the B-cell response to FVIII is very much heterogeneous. Even in a given clinical situation, B cells reacting to FVIII are in fact a mixture of cells in different numbers, at different stages of phenotypic maturation and of affinity. This has immediate consequences on the design of therapy aiming at inducing tolerance.

From a pragmatical point of view, the question is whether or not the B-cell response will evolve towards memorization. Without memorization, B cells will shortly disappear, as we see with transient inhibitors, observed for instance when a chemically altered FVIII preparation is administered to patients for a short period of time. When memorization is acquired, then the chance is high that the production of inhibitors will be maintained.

The reasons as to why memorization is important are three-fold. First, activation of memory B cells into plasmocytes means that antibody-producing cells locate in the bone marrow and in the spleen, in ‘niches’ in which they become less accessible to therapy. Second, a plasmocyte down-regulates surface receptors, rendering it even less susceptible to interact with its environment. Plasmocytes survive for weeks if not months, just producing antibodies. Third, memory B cells are highly activable cells, even in the absence of cognate antigen. This is because of the presence of surface receptors, called Toll-like receptors (TLR), which can turn cells to activation through the binding of ligands derived from microorganisms. This could explain why, for instance, some haemophilia patients continue to produce high titres of inhibitors while not being exposed to FVIII, sometimes for years.

From the above, it can be concluded that memory B cells represent the optimal target for tolerance induction. It is likely that the conventional therapy for tolerance induction, which consists of infusion of FVIII at high doses, and for prolonged periods of time, has a significant impact on memory B cells, by inducing apoptosis. However, by the same token, FVIII infusion has the potential to recruit newly formed B cells emerging from the bone marrow and still in germline configuration. So what is still the therapy of choice for inhibitors, tolerance induction, is in fact a double-edged sword. Whether or not variants of tolerance induction in which doses administered, time of treatment initiation, age and other commonly invoked parameters would lead to improvement therapy is debatable.

We have chosen to target memory B cells making use of the fact that their receptor (BCR) offers unique determinants, called idiotypes, easily recognizable by anti-idiotypic antibodies. Thus, starting from human monoclonal antibodies derived from patients with inhibitors, is has been possible to produce a number of anti-idiotypic antibodies. Surprisingly, from this angle of view, the anti-FVIII immune response seems to be much less diverse than anticipated, thanks to the finding in which we have made rapid progress towards a possible therapy of inhibitors. In addition, a significant proportion of FVIII inhibitors obtained from patients – and it is seemingly not the case for mouse antibodies to human FVIII – are able to bind and inhibit FVIII while in germline configuration. Preliminary evidence indicates that this is a characteristic of anti-C2 antibodies, which could provide an explanation for the clinical observation that anti-C2 inhibitors are readily eliminated by FVIII infusion, while antibodies of alternative specificity are more resistant.

Much remains to be learned if we wish to develop therapeutic strategies on the basis of specific elimination of memory B cells through idiotypic interaction. We are currently analysing this by generating B-cell lines transfected with viral constructs allowing surface expression of specific anti-FVIII receptors. On the other hand, a transgenic mouse strain has recently been created, which is expressing a human B cell receptor. This strain is now evaluated for defining the circumstances under which antibodies are produced, the conditions under which B cells can be deleted in vivo, as well as central mechanisms by which tolerance to FVIII is established. It should also help us to understand what can be expected in terms of B-cell regulation by anti-idiotypic antibodies.

We, however, strongly believe that a thorough understanding is required, of the signalling pathways that are activated upon BCR recognition by either FVIII (or derivatives) or by anti-idiotypic antibodies (or derivatives). The signal provided on the surface of specific B cells can lead to various cell fates, including deletion by apoptosis.

It should not be forgotten that T lymphocytes are required to produce antibodies. T cells drive the process of B-cell maturation and transformation into plasmocytes, and enforce the process of somatic hypermutation. The latter relies on activation of an error-prone polymerase introducing random mutations into the antibody (or B-cell receptor) variable parts, followed by a selection leading to higher affinity. This is not to deny the fact that some, especially short-term inhibitors could be formed in the absence of T cells. It is known that a maturation process in the absence of T cells can occur for B cells, leading to full differentiation. This kind of ‘short-circuit’ depends on activation of a set of transcription factors such as Pax5 or Bimp1, which control the fate (as well as the ontogeny) of B cells.

T cells help the maturation of B cells by recognition of either FVIII-derived epitopes or idiotype-derived epitopes presented in the context of major histocompatibility (MHC) class II determinants. In theory, it should be possible to interfere with the T-cell arm of the anti-FVIII response for therapeutic purposes. However, much less knowledge is available with regard to specific T cells. The size of the T-cell repertoire at birth is large enough to respond to any conceivable antigen, even glycolipids. It follows that there are literally armies of T cells with capacity to react to FVIII with variable affinities, and the difficulty lies in identifying those that are relevant for the production of inhibitors. It does not make much sense attempting to switch off activation of every T cell with FVIII specificity.

We made use of patients with single amino acid FVIII substitution to identify relevant T cells and various characteristics of such T cells have been delineated, thanks to a meticulous cloning strategy. As expected, many T cells can be activated by a small stretch of amino acids, illustrating the size of the T-cell repertoire. Such clones have also allowed us to determine that the same peptide could be presented to T cells by various MHC class II molecules, establishing the promiscuous nature of T-cell epitopes and, consequently, the limited chances of identifying a relationship between expression of some MHC class II alleles and the risk of producing inhibitors.

Whether specific T cells will offer an opportunity to prevent or suppress the production of FVIII inhibitors is debatable. Early work had identified several mechanisms by which T cells could be induced into anergy or even deleted by overstimulation. It is unfortunate that following the re-discovery of suppressor T cells, nowadays called regulatory T cells (Tregs), there is virtually no study being undertaken on anything else but such Tregs.

A clear distinction should be made between natural Tregs, actively selected in the thymus and which function to prevent autoimmunity and associated inflammation in a non-specific context. Obtaining antigen-specific Tregs represents a big obstacle to surmount before envisioning therapeutic applications. Such cells are difficult to grow in vitro, not to mention our current inability to expand natural Tregs in vivo. The second category of Tregs is made of a heterogeneous cell population having in common to be elicited in the periphery in the presence of a cognate antigen and cytokines such as transforming growth factor (TGF)-beta. The best defined of these adaptive Treg populations is the Tr1 or Tr1-like Treg, again somewhat difficult to expand in vitro and in vivo, although significant progress has been recently achieved to this aim. Therapeutic applications will, however, as for natural Tregs, first require gaining better skills in purifying and characterizing such adaptive Tregs.

However, Tregs are only one part of the T-cell story. It is nowadays possible to identify alternative populations of T cells with potentially useful properties therapy-wise. This requires the identification of phenotypic markers, cytokines produced, including transcription factors usage, and of genetic imprinting. With these markers in hand, it becomes much easier to define the circumstances under which such T cells are elicited, in vitro and in vivo, for expansion and purification. We recently identified a population of antigen-specific T cells presenting properties combining those of regulatory T cells and of effector T cells. We hope that these findings will provide another opportunity to relegate inhibitors to past history.

More than ever, clinicians and scientists should work together very much in the interest of haemophilia patients waiting for a cure without drawbacks, the prevailing one still being the formation of inhibitors. And this, whatever the future, is gene replacement therapy or ‘improved’ FVIII molecules.