Haemophilia management: time to get personal?

Abstract

The possibility of alloimmunization in patients receiving protein replacement therapy depends on (at least) three risk factors, which are necessary concomitantly but insufficient alone. The first is the degree of structural difference between the therapeutic protein and the patient's own endogenous protein, if expressed. Such differences depend on the nature of the disease mutation and the pre-mutation endogenous protein structure as well as on post-translational changes and sequence-engineered alterations in the therapeutic protein. Genetic variations in the recipients' immune systems comprise the second set of risk determinants for deleterious immune responses. For example, the limited repertoire of MHC class II isomers encoded by a given person's collection of HLA genes may or may not be able to present a 'foreign' peptide(s) produced from the therapeutic protein - following its internalization and proteolytic processing - on the surface of their antigen-presenting cells (APCs). The third (and least characterized) variable is the presence or absence of immunologic 'danger signals' during the display of foreign-peptide/MHC-complexes on APCs. A choice between existing therapeutic products or the manufacture of new proteins, which may be less immunogenic in some patients or patient populations, may require prior definition of the first two of these variables. This leads then to the possibility of developing personalized therapies for disorders due to genetic deficiencies in endogenous proteins, such as haemophilia A and B. [Correction made after online publication 11 July 2011: several critical corrections have been made to the abstract].

© 2011 Blackwell Publishing Ltd.

Figures

Figure 1

Missense FVIII proteins expressed by HA patients with inhibitors (based on reference 25). This illustration of FVIII designates the location of 42 different F8 missense mutations, all of which were identified in at least one patient who developed an inhibitor during treatment. In these individuals, the replacement FVIII molecule differed from the endogenous mutant FVIII protein at only one amino acid residue (if recipient and therapeutic haplotypes matched), yet this difference was sufficient to induce alloimmunization. The bottom half of the figure shows 19 non-recurrent, “private” mutations that have been found in only one individual, while the top part shows 23 recurrent mutations that have arisen independently in more than one unrelated family. Among the recurrent mutations, those shown in black indicate that only a single individual with this mutation developed an inhibitor. Those shown in blue indicate that more than one individual developed an inhibitor. Those shown in red indicate that more than one individual developed an inhibitor and that at least one of these alloimmunized patients also developed an autoantibody (presumably through epitope spreading) against endogenous FVIII resulting in an undetectable FVIII:C level.

Figure 2

Nonsynonymous-SNPs defining distinct haplotypes for FVIII proteins. To date, the human F8 gene has been found to contain four common and two less common ns-SNPs whose allelic combinations encode eight distinct wild-type FVIII proteins, only two of which have the amino acid sequences found in recombinant FVIII molecules used clinically [8,13]. Panel A illustrates both F8, with its 26 exons and 25 introns indicated by red triangles and intervening lines, respectively, and the FVIII protein, with the A1, A2, and A3 domains shown in gray, the B domain in blue, and the C1 and C2 domains in yellow; the three acidic regions a1, a2, and ap are shown in black. A region in both the A2 domain (red oval) and the C2 domain (blue oval) are known to be immunodominant B-cell inhibitor epitopes. By sequencing all 26 exons of the F8 gene in 137 unrelated healthy persons from seven racial groups, four common ns-SNPs were identified: one in exon 10 (G1679A), two in exon 14 (A2554G and C3951G), and one in exon 25 (A6940G). These polymorphisms encode the following amino acid substitutions, respectively: histidine for arginine at position 484 (R484H), glycine for arginine at position 776 (R776G), glutamic acid for aspartic acid at position 1241 (D1241E), and valine for methionine at position 2238 (M2238V). The numbering systems used to designate the four ns-SNPs and the amino acid substitutions they encode are based on their nucleotide and residue locations, respectively, in the full-length F8 complementary DNA and the mature circulating form of FVIII. R484H and M2238V are components of the A2 and C2 domains, which are both known to be immunogenic regions of FVIII. Panel B shows eight structurally distinct wild-type FVIII proteins encoded by the naturally occurring allelic combinations (haplotypes) of the F8 nonsynonymous SNPs G1679A, A2554G, C3951G, A6940G, A1229C, and G4007A. Amino acid residues at positions 334 (Q or P), 484 (R or H), 776 (R or G), 1241 (D or E), 1260 (R or K), and 2238 (M or V) are shown. Indicated are the frequencies (f) of FVIII proteins H1–H6 in healthy persons — including some females providing two X-chromosomes for analysis — 86 white (fwhite), 67 black (fblack), and 10 Chinese (fChinese), and also a cohort of 78 black HA patients; FVIII proteins H7 and H8 are based on two novel ns-SNPs, each found in a single patient of this cohort. The last column shows the frequency of inhibitor development in each haplotype group among these black patients, i.e. the number of inhibitor patients divided by the total number of patients with any given haplotype (percentage). In Panel C, the two full-length recombinant FVIII proteins used in replacement therapy, Kogenate (same as Helixate) and Recombinate (same as Advate), contain the same amino acid sequences found in H1 (Q–R–R–D–R–M) and H2 (Q–R–R–E–R–M), respectively. The B-domain deleted recombinant FVIII protein, Refacto (same as Xyntha), does not contain the ns-SNP site differentiating Kogenate and Recombinate (D1241E).

Figure 3

Population-associated allele frequencies at the highly polymorphic DRB1 structural locus (DRB1). Data in this figure are redrawn from information provided in references and . Black and white columns represent the 20 most frequent DRB1 alleles in African Americans (found in between 12.2% to 1.2% of that population) and the 20 most frequent DRB1 alleles in Caucasian Americans (found in between 14.9% to 0.7% of that population), respectively. Most of these alleles are not found exclusively in either population, e.g. DRB1*1503, the most common allele in the black population, is also found in approximately 0.2% of the white population. While these differences in allele frequencies may have arisen through known population genetic mechanisms, racial admixture cannot be excluded. The data show that some alleles are common in persons with either black African or white Caucasian genetic ancestry, whereas others are clearly more common in particular racial groups (e.g. DRB1*1503 in blacks). The 27 alleles shown here represent only a small subset of the more than 800 DRB1 alleles identified in the human population to date.