Concentrations of infliximab and anti-drug antibodies in relation to clinical response in patients with rheumatoid arthritis.

OBJECTIVE: The efficacy of anti-tumour necrosis factor-α (anti-TNF-α) treatment with infliximab (IFX) may be reduced by the development of anti-drug antibodies (ADAs). This study evaluated drug concentration and the presence of ADAs, relative to response, in rheumatoid arthritis (RA) patients treated with IFX. METHOD: Ninety-four RA patients were consecutively included and assessed for disease activity at baseline, and after 14, and 30 or 52 weeks. Serum IFX concentration and ADAs were analysed using in-house enzyme-linked immunosorbent assays. ADA analysis was based on binding to TNF-α-coated plates, with the lower detection limit set at mean + 2 sd of controls. RESULTS: At 14 and 52 weeks, 74.5% of the patients had moderate to good response. Good responders had significantly higher IFX concentrations than moderate and poor responders at 52 weeks (6.6 ± 1.4 µg/mL vs 3.6 ± 1.3 µg/mL and 2.6 ± 1.6 µg/mL, respectively). An IFX concentration ≥4.66 µg/mL at 14 weeks yielded a moderate to good response at 30/52 weeks, with 91.3% specificity and 39.3% sensitivity. Eleven patients dropped out owing to lack of efficacy and eight owing to side effects; three with IFX concentration ≤ 0.5 µg/mL were ADA positive. At an IFX concentration ≤ 0.5 µg/mL, 43.8% and 30.1% at 14 and 52 weeks, respectively, were ADA positive. None of the good responders had ADAs. CONCLUSION: One-quarter of patients had an IFX concentration ≤ 0.5 µg/mL but only 11.7% had ADAs. High IFX concentration was related to a good response, suggesting that the lack of response could be due to a lack of IFX, rather than to the presence of ADAs.

Genetic variations in the serotonin 5-HT2A receptor gene (HTR2A) are associated with rheumatoid arthritis.

OBJECTIVES: To analyse the association between the genetic polymorphisms within the HTR2A gene for the serotonin receptor and rheumatoid arthritis (RA). METHODS: HTR2A gene polymorphisms were analysed in patients with RA and controls from two study populations using PCR based restriction endonuclease mapping or TaqMan allelic discrimination with more than 4000 individuals included in the current study. RESULTS: At the discovery stage we detected significant differences in frequency of rs6313 (T102C polymorphism) between the patients with RA and controls (p = 0.006). Following validation with an extended set of single nucleotide polymorphisms (SNPs) and number of DNA samples, a trend in associations in allelic model for SNPs rs6314, rs1328674, rs6313 and rs6311 (p = 0.006, 0.002, 0.006, 0.009) was seen, although it was lost after correction for multi-comparison for all but rs1328674 (empirical p value = 0.021). However, haplotype frequency analysis based on these four SNPs showed significantly low representation of TCTT combination in patients with RA in comparison with controls (3.6% and 5.6%, p<0.001 on chi(2) test, empirical p = 0.004 after 100 000 permutations) and a significantly higher frequency of CTCC combination in patients with RA in comparison with controls (3.6% and 2.2%, p = 0.002 on chi(2) test, empirical p = 0.022 after 100 000 permutations). CONCLUSIONS: In our study, genetic polymorphisms at the HTR2A gene are associated with susceptibility for RA, suggesting possible links between the serotonergic system and development of the disease.

Polymorphism of genes related to cardiovascular disease in patients with rheumatoid arthritis.

OBJECTIVE: To analyze candidate genes, related to cardiovascular disease (CVD) in general, and potentially involved in the inflammatory process, in RA patients from northern Sweden. METHODS: Four hundred and sixty-seven individuals (345 females; 122 males) with RA (ACR criteria), having a mean age of 61.8 +/- 13.0 years and mean disease duration of 16.2 +/- 12.1 years, were consecutively recruited and followed-up for 3 years. The prevalence of CVD, [(ischemic heart disease (IHD), deep vein thromboses/pulmonary embolism (DVT/PE) and/or stroke/TIA] and hypertension was registered. Candidate genes encoding for Beta-fibrinogen (G-455A), Factor XIIIA (Val34Leu), plasminogen activator inhibitor type-1 (PAI-1 4G/5G), and tumor necrosis factor receptor (TNFR)II (M196R) were analysed. Controls (n = 672) were randomly selected according to age and gender from the Medical Biobank of Northern Sweden. Polymorphisms were genotyped using a TaqMan 9700HT and the 5'nuclease allelic discrimination assay. RESULTS: The genotypes, carriers and alleles did not differ in distribution between patients and controls. Carriage of the TNFRII R variant was more frequent among patients with hypertension (p = 0.018). The genotype distribution of PAI-1 in patients with IHD differed significantly (p = 0.002) because carriage of 4G was more frequent (p = 0.024). Combined carriage of TNFRII 196R variant and Beta-fibrinogen-455A was a stronger predictor for hypertension than each genotype separately. The distribution of FXIIIA genotypes deviated significantly in RA patients with DVT/PE (p = 0.028) with an increased frequency of the Leu34 variant. CONCLUSION: The unusual alleles of TNFRII, PAI-1 and FXIIIA were associated with CVD in RA patients. The combination of several of the rare types further increased the predictive values for CVD.

Oestrogen receptor {alpha} gene polymorphisms in systemic lupus erythematosus.

OBJECTIVE: To analyse associations of two oestrogen receptor alpha (ORalpha) gene polymorphisms in 260 patients with SLE from northern Sweden. The two polymorphisms, PvuII T/C and the XbaI A/G, are located in the first intron of the ORalpha gene. METHODS: All patients fulfilling at least four of the ACR criteria for SLE were consecutively recruited during one year. The SLEDAI score and SLICC damage index were recorded. 670 individuals from the same geographical area served as controls. DNA from the patients and controls was extracted and genotyped using the 5' nuclease assay with an ABI PRISM 7900HT instrument. The genotype/phenotype relationships were calculated using SPSS. RESULTS: The unusual PvuII C allele was associated with malar rash and the unusual XbaI G allele with photosensitivity (p = 0.001, OR = 2.53, 95% CI = 1.43 to 4.47 and p = 0.007, OR = 2.12, 95% CI = 1.22 to 3.66, respectively). The common XbaI AA genotype was associated with serositis (p = 0.013, OR = 1.92, 95% CI = 1.15 to 3.22). Based on the SLICC damage index associations of the common TT genotype and AA genotype with cognitive impairment were identified (p = 0.018, OR = 2.47, 95% CI = 1.17 to 5.25 and p = 0.018, OR = 2.75, 95% CI = 1.19 to 6.38 respectively). There was also an association of the XbaI AA genotype with the angina/coronary artery bypass variable (p = 0.042, OR = 2.58, 95% CI = 1.03 to 6.43). Of the variables describing disease severity and duration it was found that carriers of the unusual PvuII C allele showed a later onset of SLE (p = 0.02) and carriers of the unusual XbaI G allele a lower SLICC damage index. CONCLUSIONS: The unusual PvuII C and XbaI G alleles were associated with a milder form of SLE characterised by skin manifestations, later onset, and less organ damage.

Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

Epitope specificity of 30 monoclonal antibodies against cytokeratin antigens: the ISOBM TD5-1 Workshop.

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins. i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology. All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270-400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340-365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270-429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322-342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311-335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346-367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356-370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients' blood for tumor marker evaluation.

Endogenous anti-tumor antibody responses in nude mice.

BACKGROUND: Nude mice with xenografted human tumors is the most exploited animal model used to elucidate the efficacy of experimental radioimmunolocalization and radioimmunotherapy. These animals accept transplants and are generally considered immunologically inert with regard to cell-mediated and humoral immune responses against the tumors. METHODS: Nude control mice and mice carrying human HeLa Hep-2 tumor xenografts were studied for appearance of endogenous antibodies following inoculation with tumor cells. The titers of these antibodies were investigated by isotype-specific enzyme-linked immunosorbent assay (ELISA) technologies, fluorescence-activated cell sorter analysis (FACS), BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden) technology, and immunofluorescence. RESULTS: The HeLa Hep-2 cell line was found to be immunogenic in all investigated animals by means of ELISA, FACS, and BIAcore evaluations as well as by immunofluorescence against both tested antigens, placental alkaline phosphatase and cytokeratin 8. Predominantly immunoglobulin M antibodies were induced, but immunoglobulin G isotypes could also be identified. Sera from these tumor-bearing mice were used for immunohistochemistry of the tumor cells. The antibodies seemed to be of low affinity and may be displaced by high-affinity monoclonal antibodies used in radioimmunotargeting. CONCLUSIONS: Nude mice bearing tumor xenografts produce significant amounts of antibodies against these two human tumor-derived antigens. These endogenous antibodies may influence targeting of radiolabeled antibodies. They also have the potential to interfere with the pharmacokinetics of labeled or nonlabeled idiotypic antibodies during experimental immunolocalization.

Twenty years with monoclonal antibodies: State of the art--Where do we go?

In this review, we have selected some parameters with the potential to improve the efficacy of RIL and RIT. Focus has partially been on the behaviour of radiolabelled antibodies in vivo in relation to properties and amounts of both target antigen and the antibodies used. If, out of the 28 factors listed in Table 1, some should be given preference in future work, it is our opinion that after the initial saturation of the tumour site a rapid decrease in redundant antibody is of significant importance. Furthermore, quantitative aspects of both antigens and antibodies should be more carefully evaluated when possible. By combining several of the listed approaches toward increasing efficiency, a more extensive use of RIL and RIT could be expected in the future.

Secondary antibodies as tools to improve tumor to non tumor ratio at radioimmunolocalisation and radioimmunotherapy.

One way of selectively improving the efficiency of radioimmunolocalization and radioimmunotherapy is to eliminate redundant, circulating, non-targeting radiolabeled antibodies after saturation of the target sites. Secondary antibodies of different types have been proposed as clearing agents for such purposes. The conceptually different approaches of the 'secondary antibody' strategy including its advantages and limitations are discussed. This mini-review also presents a model describing the kinetics of the components (the antigen, the primary and secondary antibodies) and approaches required to improve the efficacy of both radioimmunolocalization and radioimmunotherapy.

Use of anticytokeratin monoclonal anti-idiotypic antibodies to improve tumor:nontumor ratio in experimental radioimmunolocalization.

A syngeneic, high-affinity, anti-idiotypic monoclonal antibody (MAb; alpha TS1) raised against an anticytokeratin monoclonal antibody (TS1) was evaluated as a second antibody to promote the rapid clearance of radiolabeled TS1 from the blood during experimental radioimmunolocalization. By using a novel biosensor technology (BIAcore), association rate dissociation rate, and affinity constants between the idiotype and the anti-idiotype could be determined. The in vivo results in nude mice carrying HeLa Hep 2 tumors demonstrate the possibility of selectively regulating the amount of the idiotypic 125I-labeled circulating MAb by in vivo injection of this high-affinity, anti-idiotypic antibody. Injection of the anti-idiotype in a molar ratio of 0.75 to the idiotype cleared the blood pool from circulating radiolabeled idiotype within 24 h, with a concomitant rapid excretion of 125I in urine. The total amount of remaining radioactivity in the animals decreased to 15-20% during these 24 h, with the tumors still retaining 60-65% of their initial radioactivity. This approach, using syngeneic primary and secondary MAbs with minimized immunogenicity, significantly improves the tumor:nontumor ratio, thus improving efficiency in experimental radioimmunolocalization and radioimmunotherapy, leaving the endogenous antibody repertoire of the host unaffected.