Grazia Luccarelli

β2-Microglobulin-Free HLA Class I Heavy Chain Epitope Mimicry by Monoclonal Antibody HC-10-Specific Peptide

mAb HC-10 loses its reactivity with HLA class I (HLA-I) H chain (HC) following its association with β2-microglobulin (β2m). Furthermore, the HC-10 defined epitope appears to be involved in the pathogenesis of spondyloarthropathies, because HC-10 reduced their incidence in HLA-B27+β2m°/MHC class II knockout mice. This study has characterized the determinant recognized by HC-10. Panning of a phage display peptide library with HC-10 resulted in isolation of the motif PxxWDR, which could be aligned with P57, W60, D61, and R62 of the first domain of the HLA-I HC allospecificities reactive with HC-10. The 55EGPEYWDR(N/E)T64 (p-1) is the shortest motif-bearing peptide that reacts with HC-10 and inhibits its binding to soluble HLA-B7 HC, irrespective of whether N (p-1a) or E (p-1b) is present at position 63. By contrast, HC-10 did not react with six additional peptides, each bearing motif amino acid substitutions present in HC-10-not-reactive HLA-I allospecificities. The p-1-derived Qp-1, synthesized with the additional conserved Q54, which displays the highest in vitro reactivity with HC-10, was the only one to induce in mice IgG resembling HC-10 in their fine specificity. Mapping of the HC-10-defined determinant suggests that the lack of mAb reactivity with β2m-associated HLA-I HC is caused by blocking by the peptide in the groove of β2m-associated HLA-I HC, though a role of HC conformational changes following its association with β2m cannot be excluded. This information contributes to our understanding of the molecular basis of the antigenic profiles of β2m-free and β2m-associated HLA-I HC and may serve to develop active specific immunotherapy of spondyloarthropathies.

Increased serum levels of β2m‐free HLA class I heavy chain in multiple myeloma

Serum levels of β2‐microglobulin (β2m)‐free HLA class I heavy chain (FHC) in 94 patients with multiple myeloma (MM) were higher than in 29 patients with monoclonal gammopathy of undetermined significance (MGUS) (P = 0.023) and in 97 sex‐ and age‐matched healthy controls (P < 0.0001). Spearman correlation analysis indicated that in MM, FHC correlated with β2m (r = 0.31, P = 0.003) and the percentage of bone marrow plasma cells (BMPC%) (r = 0.36, P = 0.002), whereas β2m, in addition to BMPC% (r = 0.43, P = 0.0003), also correlated with creatinine levels (r = 0.63, P < 0.0001), haemoglobin levels (r = −0.35, P = 0.0007) and patient age (r = 0.34, P < 0.0011). Furthermore, MM patients with poor prognosis (β2m  6 mg/l) displayed higher FHC levels than those with a better prognosis (β2m < 6mg/l) (P < 0.021). At variance from β2m, these levels were not influenced by renal failure, as indicated by the lack of Spearman correlation of FHC with creatinine concentration and of statistical significance between the median FHC concentration of MM patients with creatinine < 176.6 μmol/l and those with creatinine  176.6 μmol/l (P = 0.3). Stratification of patients according to disease activity and stage showed that FHC levels were only statistically different (P = 0.04) for disease activity, whereas β2m and C‐reactive protein were not. Taken together, our data indicate that serum FHC may be a useful disease marker in MM.

Size variants of beta-2-microglobulin-free human leucocyte antigen class I heavy chain make different contributions to its serum increase in multiple myeloma

Summary. We previously showed that serum beta‐2‐microglobulin (β2m)‐free human leucocyte antigen (HLA) class I heavy chain (FHC) levels were increased in MM and correlate with disease activity. The present investigation, carried out in 124 multiple myeloma patients, studied the expression of the three size variants of FHC, namely the 42 kDa intact heavy chain (A variant, AV), released through a shedding process, and the truncated FHC (tFHC) 39 kDa (BV) and 36–35 kDa (CV) released by means of membrane‐type metalloprotease activity. The increase in FHC correlated with a high expression percentage of BV (r = 0·32, P = 0·0002) and tFHC (r = 0·42, P < 0·0001), which could help to discriminate multiple myeloma from monoclonal gammopathy of undetermined significance (tFHC mean ratio = 3·2; Mann–Whitney U‐test, P < 0·0001). tFHC levels highly correlated with other disease activity markers, namely haemoglobin (r = −0·35; Spearmans rank, P = 0·0001), percentage of bone marrow plasma cells (r = 0·4, P < 0·0001) and β2m levels (r = 0·36, P < 0·0001), while only the last barely correlated (r = 0·2, P = 0·03) with AV. Finally, the 0·4, 0·57 and 0·71 mg/l BV, tFHC and (to a lesser extent) FHC cut‐off values divided patients into two groups with different survival curves (P = 0·0005, P = 0·0025 and P = 0·04 respectively). These data are in favour of a correlation between disease aggressiveness and cleavage of these variants by membrane‐type metalloprotease enzymes.

Human CD4 internal antigen anti-idiotypic monoclonal antibody. Immunochemical and sequence analysis

Abstract The mouse mAb2 16D7 recognizes the paratope of the syngeneic anti-human CD4 mAb HP2/6 (mAb1 of our idiotypic cascade) and mimics CD4 in xenogeneic settings in humans. Immunochemical and sequence analyses were performed to define the minimum structural requirement for this mimicry. Binding assay of mAb1 with isolated naive 16D7 H and L chains showed that only the second reacted with mAb1. Specificity was indicated by the lack of reactivity of mAb1 with the L chain of mAb2 14D6, which also recognizes mAb1-paratope. It is likely that the 16D7-L mAb1-specific epitope is “sequence-dependent”, since fully denatured 16D7-L still reacted with mAb1. Sequence analysis of 16D7 and mAb1 showed a high degree of homology of their VH, as both were coded by the same gene family (V/II), whereas CDR3 showed the greatest diversity. Alignment of 16D7-H CDR3 with CD4, however, produced no similarity. In contrast, analyses of the 16D7 VL sequence (XX/V) defined a CDR3 6-mer peptide with a 50% identity (83% of similarity) to the CD4 stretch 218–223. This peptide seems a suitable replacement for 16D7 in active immunotherapy as it did not match any protein fragment retrieved from the n-r database (NCBI) and both the peptide and the corresponding CD4 amino acid stretch are surface accessible. Based on their immunochemical profiles and similarity to CD4, four additional 16D7-derived peptides were designed for synthesis. The data indicate that CD4 mimicry by mAb2 can be obtained at the level of primary structure and provide useful information for the synthesis of peptide(s) with bioactive potential.

SERUM LEVELS OF BETA-2-MICROGLOBULIN-FREE HEAVY CHAIN OF HLA CLASS I ANTIGEN IN HEALTHY INDIVIDUALS : RELATIONSHIP TO THEIR CLASS I ALLOTYPE

An ELISA-based double determinant immunoassay has been established to measure the soluble beta2-microglobulin (beta2m)-free heavy chain (FHC) of the HLA-B, -C (and HLA-A3, -A28 and -A30) class I molecular complex in sera from 212 HLA-typed healthy unrelated individuals. FHC was calculated by means of a standard curve constructed using serial concentrations of beta2m-associated HLA-class I heavy chain (HLA-I)/FHC purified from cultured human lymphoid cell C1R-sB7-supernatant. The mean FHC concentration (+/-SD) was 0.25 mg/l (+/-0.2). Its median concentration did not statistically differ between males and females, though the male/female ratio was greater in the high secretor (FHC >0.45 mg/l; mean + 1SD) than in the low secretor group (FHC < 0.05 mg/l; mean - 1SD). FHC < 0.05 mg/l was statistically (Fishers exact test) associated with HLA-B17 (p = 0.003); FHC > 0.45 mg/l was statistically associated with HLA-B35 (p = 0.003) and -Cw4 (p = 0.002). None of these allele-positive groups showed a mean FHC concentration 1.5 times higher than that of the corresponding allele-negative ones. This allotype-dependent HLA-B and C FHC enhancement was less marked than that previously reported for HLA-I in individuals carrying HLA-A9 (and its splits). These results indicate that FHC could be a more valuable marker when its levels are compared among individuals carrying different allotypes. Moreover the lack of correlation between FHC and HLA-I levels measured in 52 HLA-A3, -A28 or -A30 positive individuals suggests that the two molecules may be regulated by different metabolic pathways and their serum expression may have a different biological significance.

Assessment of safety and the immune response to the CD4 "internal antigen" mouse anti-idiotypic Mab 16D7 in four patients with SLE.

We have analyzed the immune response elicited with the human CD4 internal antigen anti-idiotypic monoclonal antibody 16D7 in four patients with active systemic lupus erythematosus and assessed the safety of the treatment. Patients 1 and 2 received three 2-mg 16D7 subcutaneous (SC) injections at 3-week intervals and mainly developed IgG1, whereas IgG1, IgG3, and IgG4 were detected in the sera of the other two patients (3 and 4) who received the same amount of 16D7 on days 0, 28, and 70. 16D7-F(ab`)2 isotypic determinant-specific antibodies levels, evaluated by sera reactivity with the 16D7-isotype matched anti-idiotypic monoclonal antibody 14D6-F(ab`)2, were low or undetectable in patient 1 and became detectable following the first and second boosters in patient 3 and patients 2 and 4, respectively. The highest level was seen in patients 3 and 4. The focusing pattern (spectrotype) of 16D7 idiotypic-specific antibodies suggested that multiple V genes are involved or many somatic mutations occur. Once established, each patient’s spectrotype remained stable. Although spectrotype were individually distinct, all four patients produced CD4-specific antibodies, indicating that this response crosses the genetic barrier. Disease relapsed after 11 (patient 2), 40 (patients 3 and 4), and 125 (patient 1) weeks. The lack of side effects and the prolonged periods of disease control (33 and 103 weeks after the last booster) warrants an extension of this study.

Absence of streptococcal protein G (PG)‐specific determinant in the Fab region of human IgG2

It has been previously reported that CH1 Fab protein G‐contact site is responsible for the widespread recognition of mouse and human IgG Fab by PG. Here we present evidence that PG binding to F(ab′)2 is restricted, as indicated by the lack of reactivity with PG‐Sepharose columns of a portion of F(ab′)2 fragments obtained by pepsin digestion of human IgG from a commercial immunoglobulin preparation for intravenous use or purified from sera of two healthy blood donors and two patients with polyclonal hypergammaglobulinaemia. Isoelectric focusing showed that F(ab′)2 fragments that did not bind PG focused in a lower pH range compared with those which did. Testing of the Fab fractions with MoAbs to κ and λ light chains or to γ1, γ2 and γ3‐Fab subclass determinants showed that γ2‐F(ab′)2 were mainly found in the PG non‐reactive F(ab′)2 fraction, and that this distribution was not influenced by the L chain isotype. These results indicate that the PG‐specific binding determinant(s) is not expressed in the F(ab′)2 region of most human IgG2.