Monoclonal versus polyclonal anti-D in the treatment of ITP

Abstract

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by low numbers of platelets generally due to the production of anti-platelet antibodies. One effective treatment for ITP patients who express the RhD antigen on their red blood cells has been the use of blood donor-derived pooled polyclonal anti-D. Although anti-D has served us well, it needs to be replaced with a recombinant product. While the mechanism of action of anti-D in ITP remains highly speculative, this has not thwarted attempts to replace anti-D with a monoclonal product. Although a single attempt at a monoclonal antibody was not successful in the 1990s for the treatment of ITP, more recent efforts in mouse models of ITP and ITP patients now show that monoclonal antibodies can be successful in ITP. These studies also finally help substantiate the concept that it is unlikely that contaminants in the original donor-derived preparations mediate the major ameliorative activity of anti-D in ITP.

Keywords::

• anti-D
• autoimmune disease
• donor-derived blood products
• haemolytic disease of the fetus and newborn
• HDFN
• IgG
• IgG carbohydrates
• ITP
• platelets
• Rozrolimupab

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by the production of antiplatelet antibodies which can induce thrombocytopenia. One effective treatment for patients with ITP who are RhD antigen positive has been polyclonal anti-D. Greater than 70% of ITP patients treated with anti-D have responded well to therapy [1] but unfortunately we do not as of yet have a clear understanding as to how anti-D works in ITP [2].

Another clinical situation where anti-D is of great benefit is in the prevention of hemolytic disease of the fetus and newborn (HDFN) [3]. Again, there are more questions than answers when it comes to understanding how anti-D works to prevent HDFN [3]. However, if one considers the work that has gone into understanding how anti-D (or antibodies with anti-D like activity) work in ITP versus HDFN, it is quite possible that the product works by different mechanisms in these situations [2,4,5]. In contrast to ITP where multiple treatment options exist, the use of anti-D in HDFN is the only option to prevent this disease and in many countries donor-derived anti-D is conserved for preventing HDFN.

1. Polyclonal versus monoclonal anti-D

The development of a recombinant replacement for anti-D in both ITP and HDFN is highly desirable. Several different monoclonal anti-D antibodies have been applied to the prevention of red cell immunization as related to HDFN with mixed results [3]. Many of the monoclonal antibodies seemed to decrease red cell immunization successfully but often not as well as anti-D itself while some other antibodies actually had the opposite effect and exacerbated the immune response [3]. Unfortunately in the case of HDFN, we do not at present have enough mechanistic information to be able to predict which monoclonal anti-D antibodies would work based on in vitro testing. Clearly, a better understanding of how polyclonal anti-D prevents immunization to red cells in the setting of HDFN is needed.

In the case of ITP, a single monoclonal anti-D antibody was evaluated in seven adult chronic ITP patients [6]. Although the antibody bound to red cells and induced a degree of anemia comparable to that of polyclonal anti-D, the therapeutic results were disappointing with a marginal response (at best) in six of the seven patients and only a transient response in one patient with mild disease. After the publication of this manuscript several letters to the editor appeared which criticized or commented on the concept of trying an anti-D monoclonal antibody in ITP [7,8]. One letter indicated that polyclonal anti-D was contaminated with antibodies specific for Fc γ receptors which themselves were postulated to directly block Fc receptor function and increase platelet counts in ITP. The second letter asserted that antibodies to HLA antigens contaminated anti-D and these anti-HLA antibodies were what mediated anti-D's effects in ITP. While the original study and the two letters that followed were entirely reasonable and appropriate for the time, doubt had been cast on whether a monoclonal anti-D could actually increase platelet counts in ITP.

In the decade that followed, a murine model of passive ITP became available [9] and monoclonal antibodies to a variety of different cellular and soluble antigens were examined to determine if in fact monoclonal antibodies could potentially increase platelet counts in this new model of ITP [10-12]. Although antibodies to the murine equivalent of RhD are not available, there are a number of monoclonal antibodies to other murine red cell antigens. Some of these were tested in the murine ITP model as a surrogate for anti-D and some red cell specific monoclonal antibodies could in fact raise platelet counts in murine ITP [10]. A potential mechanism of action was identified which suggested that the antibodies likely worked in a manner consistent with the down modulation, inactivation or neutralization of the major Fc receptor involved in mediating the thrombocytopenia (FcγRIII) [11]. This down modulation in FcγRIII was not observed with another donor-derived immunoglobulin used to treat ITP (intravenous immunoglobulin; IVIg) indicating that the mechanisms of the two donor-derived polyclonal therapeutics were likely fundamentally different [11]. In human patients receiving anti-D, Cooper also showed that the spectrum of cytokines produced in patients receiving anti-D versus IVIg were different [13]. Today, we do not as yet have a confirmed mechanism of action for polyclonal anti-D (nor IVIg) and thus the successful development of any particular monoclonal anti-D antibody for human patients with ITP could be considered a risky venture.

2. A monoclonal anti-D blend is born

To circumvent some of the issues described above, the Danish biotech company Symphogen manufactured a new therapeutic which is a mixture of 25 different monoclonal antibodies to the RhD antigen (reviewed in [14]). This new product is called Rozrolimupab and is comprised of 25 recombinant human IgG1 antibodies derived from eight different individuals recognizing multiple epitopes on the RhD antigen. This therapeutic was tested in a phase I/II dose escalation study in adult patients with primary ITP [15]. The product was reasonably well tolerated in most patients and it significantly elevated platelet counts in a clear majority of patients at higher dosages [14-16]. The success of this new recombinant reagent demonstrates that good therapeutic efficacy can be obtained in ITP with, at the very least, a monoclonal antibody blend.

There are some important positive attributes of Rozrolimupab (Table 1); it can be manufactured without the need of blood or plasma donors, there are no issues of ‘paid plasma donors' versus volunteer donors, the therapeutic can be made in an unlimited supply, it theoretically should be consistent from batch to batch, and there is the ability to refine the product if desired (e.g., removal of inactive or antagonistic antibodies in the product, or even removal of antibodies that may contribute to adverse events). What could be considered interesting about the product is that Rozrolimupab only consists of IgG1 antibodies whereas polyclonal anti-D contains all IgG subclasses but is predominantly IgG1 followed by IgG3 and much lower amounts of IgG2 and IgG4 [3]. As IgG2, IgG3 and IgG4 antibodies are not represented in Rozrolimupab yet this product increased platelet counts in the majority of ITP patients treated, this indicates that these other isotypes are dispensable for the effects seen. Whether Rozrolimupab would have higher (or lower) activity if other IgG isotypes were represented in the product will never likely be known.

Table 1. Polyclonal anti-D vs Rozrolimupab.

Polyclonal anti-D	Rozrolimupab
Plasma-derived blood product	Synthetic product made from CHO cells grown in vitro
Supply dependent on suitable donors	Donor-independent
Product shortages possible	Shortages not anticipated
Batch-to-batch variations due to the plasma donor pool	Batch-to-batch variation should be in line with other recombinant products
Potential contamination with emerging blood-borne pathogens can be a concern	Blood borne pathogens not anticipated*
Proven track record of therapeutic efficacy	Efficacy demonstrated in one published study

*In vitro cultured cells have requirements for growth factors (e.g., insulin) which can be made by recombinant means or derived from human or animal sources. Even when the growth factors are made by recombinant technology (as is currently the case with Rozrolimupab) there always exists the possibility that a foreign agent can enter the production process at the raw materials stage.

3. A final thought worth considering?

In the early days of donor-derived anti-D, virtually all of the plasma donors would have been previously pregnant RhD negative women who it been naturally immunized by pregnancy with an Rh positive fetus or newborn. However, the tremendous success of anti-D in preventing HDFN has meant that the number of these donors is diminishing over time. These donors are being replaced with volunteer RhD negative males who are purposely immunized with RhD positive red cells.

One of the attributes of a true polyclonal immune response is that the carbohydrate structures on the IgG molecules would be oriented towards inflammatory or anti-inflammatory activity depending on the nature of the immune response [17]. Monoclonal antibodies in contrast have a carbohydrate structure that is characteristic of the cell line used for its manufacture [17]. Because IgG carbohydrate structures, particularly galactosylation and sialylation change during pregnancy and have been associated with improvement in rheumatoid arthritis [18], one could also speculate that these changes could also occur with the anti-D produced as a result of an incompatible pregnancy and this could contribute to the rarity (∼ 0.02%) of severe reactions seen with earlier versions of polyclonal anti-D [19]. Even during the period 2007 – 2009 where it is likely that an increased percentage of intentionally immunized male plasma donors would be used, the estimated percentage of severe reactions was still low at an estimated incidence of 0.04% [19].

Whether or not monoclonal anti-D antibodies, or anti-D antibody blends such as Rozrolimupab will have a similar safety profile only time will tell. Of course the advantage of a monoclonal antibody is that there is the potential of engineering the carbohydrates to be anti-inflammatory and perhaps the opportunity to even make a better product down the road.

Declaration of interest

The author has patents on monoclonal antibodies to treat ITP. Supported by a grant from the CBS/CIHR partnership fund to AHL; FRN6897.