A robust enzyme-linked immunosorbent assay to measure serum ramucirumab concentrations.

Aim: Ramucirumab, an anti-VEGFR2 monoclonal antibody, has been approved for the treatment of metastatic gastric and colorectal cancer. An assay measuring ramucirumab serum concentrations was needed to investigate its pharmacokinetics and concentration-response relationship. Results: An ELISA was developed and validated according to the international guidelines for ligand-binding assays. Ramucirumab calibration standards ranged from 0.125 to 40 mg/l. Low, middle and high quality controls were spiked at 0.2, 4 and 8 mg/l, respectively. The limits of quantification were established to be 0.125 and 10 mg/l for LLOQ and ULOQ, respectively. No cross-reactivity with anti-VEGF or anti-EGFR was detected. Conclusion: This in-house-developed ELISA is sensitive, accurate, reproducible and suitable for pharmacokinetic studies of ramucirumab.

Monoclonal Antibodies Targeting the IL-17/IL-17RA Axis: An Opportunity to Improve the Efficiency of Anti-VEGF Therapy in Fighting Metastatic Colorectal Cancer?

Colorectal cancer is a major problem for public health worldwide because of its frequency and its severity. Many efforts have been carried to target the vascular endothelial growth factor (VEGF) pathway, one of the main promoters of pathological angiogenesis. Therapeutic monoclonal antibodies against VEGF have emerged as essential biopharmaceuticals for the advanced stages of the disease, in association with appropriate backbone chemotherapy. Unfortunately, after an initial benefit for the patients, resistance invariably develops. These mechanisms of resistance are largely studied and recent publications indicate that the interleukin (IL)-17/IL-17 receptor (IL-17R)A axis could be a key player in the pathological progression. In this mini review, we present evidence for IL-17A/IL-17RA axis targeting in colorectal cancer to improve efficiency of anti-VEGF therapy and to implement a new therapeutic strategy.

Influence of FcγRIIIA genetic polymorphism on T-lymphocyte depletion induced by rabbit antithymocyte globulins in kidney transplant patients.

INTRODUCTION: Polyclonal antithymocyte globulins (ATG) have been used in transplantation for several decades, but the sources of the interindividual variability of their effect are poorly understood. An influence of the FCGR3A-158V/F genetic polymorphism on the horse ATG concentration-effect relationship was reported in kidney transplant patients. The objective of the present study was to confirm the influence of the FCGR3A polymorphism on the extent of lymphocyte depletion in kidney transplant patients treated with rabbit antithymocyte globulin (r-ATG). MATERIALS AND METHODS: Of the 194 transplant patients treated with r-ATG between 1998 and 2002 in our institution, 69 patients were eligible and included in this retrospective study. Biomarkers of response were CD3 and CD4 counts. Dose-effect data were analyzed using a population approach, and a two-compartment turnover model with stimulation of lymphocyte 'output'. Since r-ATG concentrations were not available, a K-PD model was used. The influence of FCGR3A genotype on estimated parameters was investigated. RESULTS: The r-ATG infusion rate leading to a 50% stimulation of CD3+ output (EDK(50)), which is inversely related to patient sensitivity to r-ATG treatment, decreased with the number of V alleles (P=0.0016). CONCLUSION: The genetic polymorphism of FCGR3A influences r-ATG effect on CD3 count in kidney transplant patients, those with the V allele being more sensitive to antilymphocyte serum. These results also suggest that r-ATG act, at least in part, by antibody-dependent cellular cytotoxicity.

Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies.

The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman (®) technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs' distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

FCGR2C genotyping by pyrosequencing reveals linkage disequilibrium with FCGR3A V158F and FCGR2A H131R polymorphisms in a Caucasian population.

The FCGR3A-V158F and FCGR2A-H131R polymorphisms are associated with clinical responses to therapeutic mAbs and with immune thrombocytopenic purpura (ITP). The FCGR2C-ORF/STOP polymorphism, controlling FcγRIIC expression on natural killer cells and therefore FcγRIIC-mediated antibody dependent cell-mediated cytotoxicity, is also associated with ITP. Using a new pyrosequencing assay to determine this polymorphism in a control population, we observed the expected allele frequencies (ORF:12.6%) and percentages of individuals with a single copy (10.0%) or 3 copies (12.1%) of FCGR2C, or with at least one FCGR2C-ORF allele (20.1%). No association of FCGR2C copy number variations with the FCGR3A-V158F or FCGR2A-H131R genotype was detected. More importantly, our results demonstrate a strong and a weaker linkage disequilibrium associating the FCGR2C-ORF allele with the FCGR3A-158V and the FCGR2A-131H allele, respectively.

VEGF polymorphisms are associated with an increasing risk of developing renal cell carcinoma.

PURPOSE: Vascular endothelial cell growth factor is studied in different malignant tumors as a key endothelial cell mitogen. Many single nucleotide polymorphisms in the VEGF gene have been described. We compared VEGF gene polymorphisms between a control group and a renal cancer group. MATERIALS AND METHODS: This study was performed in 202 control, white, healthy blood donors (control group) and in 51 consecutive patients with renal cell carcinoma. We studied VEGF genotype polymorphisms at positions -2549, -460, -1154, +405 and +936 using polymerase chain restriction fragment length polymorphism, and looked for correlations with clinical data. RESULTS: No association was found between VEGF gene polymorphism and renal cell carcinoma prognostic parameters. However, in contrast as observed for controls and other polymorphisms the patient group displayed a heterozygote excess (p = 0.0179, 35.9% more than that expected) at the -460 polymorphism. Comparing the control group and the renal cell carcinoma group we detected a significantly increased risk of renal cell carcinoma in subjects with the C-460T polymorphism. T carrier genotypes and the T allele increased the risk of renal cell carcinoma with an OR of 14.15 (95% CI 1.900-105.41, p = 0.0017) and 2.14 (95% CI 1.34-3.419, p = 0.0018), respectively. The genotype at the -2549 polymorphism exhibited a nonsignificant trend for increased risk but the D allele was significantly associated with increased risk (p = 0.0305). CONCLUSIONS: Our results suggest that the -460 polymorphism is a risk factor for renal cancer. An individual screening test could be proposed for high risk populations.

[Neonatal Fc receptor, key control of immunoglobulins biodistribution]. / FcRn, un récepteur d'IgG aux multiples facettes.

In 1969, Brambell, while studying the long serum half-life of IgG and their ability to cross the materno-foetal barrier, attributed these two properties to the existence of a specific Fc receptor, which was later denominated FcRn for neonatal Fc receptor. The resolution of its structure revealed that it is a MHC class-I-like molecule. FcRn is able to load IgG and albumin in a pH-dependent manner. It acts as an intracellular transport protein and as such is controling the serum half-life of these proteins (apical recycling of IgG and albumin in endothelial cells), IgG biodistribution (apical to basolateral and basolateral to apical transport of IgG in epithelial and endothelial cells) and it may also contribute to phagocytosis. FcRn is thus a key partner in the pharmacokinetics of therapeutic antibodies, opening interesting prospects for optimisation of their use.

IgG1 heavy chain-coding gene polymorphism (G1m allotypes) and development of antibodies-to-infliximab.

OBJECTIVE: The chimeric anti-tumor necrosis factor-alpha antibody infliximab is known to induce antibodies-to-infliximab (ATI) in some treated patients. Immunogenicity in murine variable domains is expected; however, constant domains of its human heavy gamma1 chain may also be implicated as it expresses G1m1 and G1m17 allotypes. This allelic form may be immunogenic in patients that are homozygous for the G1m3 allotype commonly expressed in Caucasoid populations. METHODS: As G1m allotypic divergence may explain the presence of ATI or may influence their concentration, a genotyping method was developed and validated to determine antithetical (i.e. mutually exclusive) G1m3 and G1m17 allotypes (amino acid 120 of CH1 according to the international ImMunoGeneTics information system unique numbering) at the IGHG1 gene level (CH1 359g/a nucleotide polymorphism). Two hundred forty-five blood donors and 118 previously described patients suffering from Crohn's disease, treated with infliximab, and having developed ATI in 73 of them, were genotyped. RESULTS: The IGHG1 CH1 359g/a polymorphism does not depart from the Hardy-Weinberg equilibrium in the control population, and allele frequencies were similar in controls and patients. No association was found between the patient G1m allotypes and the presence of ATI or their concentration. It remains possible that anti-Gm1 antibodies are not well detected by the enzyme-linked immunosorbent assays used for ATI detection and/or that the G1m allotypes are minor antigens on IgG1. CONCLUSION: The IGHG1 polymorphism does not seem to play a major role in the induction of ATI. Further analyses will be required to determine whether it is also the case for humanized or fully human antibodies bearing the same G1m allotypes.

Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20.

Clinical studies have shown a large interindividual variability in rituximab exposure and its significant influence on clinical response in patients receiving similar doses of antibody. The aim of this study was to evaluate the influence of tumor burden on dose-concentration-response relationships of rituximab. Murine lymphoma cells (EL4, 8 x 10(3)), transduced with human CD20 cDNA and transfected with luciferase plasmid (EL4-huCD20-Luc), were intravenously injected into C57BL/6J mice. Tumor burden detection, dissemination, and progression were evaluated quantitatively by in vivo bioluminescence imaging. Different doses of rituximab (6, 12, 20, or 40 mg/kg) were infused 13 days after lymphoma cell inoculation, and rituximab serum concentrations were measured by enzyme-linked immunosorbent assay. Without rituximab, all mice developed disseminated lymphoma and died within 30 days, whereas a significant dose-response relationship was observed in mice receiving rituximab. The 20-mg/kg dose was adequate to study interindividual variability in response because 23% of mice were cured, 59% had partial response, and 18% had disease progression. Rituximab concentrations were inversely correlated with tumor burden; mice with low tumor burden had high rituximab concentrations. Furthermore, rituximab exposure influenced response and survival. Finally, using a pharmacokinetic-pharmacodynamic model, we demonstrated that tumor burden significantly influenced rituximab efficacy.

Fc gamma RIIIa expression is not increased on natural killer cells expressing the Fc gamma RIIIa-158V allotype.

The presence of a valine (V) versus a phenylanaline (F) at position 158 of Fc gamma RIIIa/CD16a improves the affinity for IgG and is associated with higher therapeutic response to rituximab. Increased CD16 expression on natural killer (NK) cells from donors with the VV or VF versus FF genotype has recently been reported. We indeed observed higher binding of the anti-CD16 monoclonal antibody (mAb) 3G8 on NK cells from V carriers (VV = VF > FF). However, the binding of two other anti-CD16 mAbs, LNK16 and DJ130c, decreased with the number of V allele (VV < VF < FF). CD16 transcript levels were independent on the genotype. Rituximab binding to NK cells from V carriers was higher than its binding to FF NK cells at low concentrations (10 and 100 microg/mL). However, the difference was nearly completely abolished at saturating concentrations (>or=1,000 microg/mL). Finally, nearly 100% of CD16-expressing NK cells displayed a complete down-modulation of the receptor after optimal engagement by plate-bound 3G8, whatever the genotype. By contrast, the percentages of NK cells down-modulating CD16 after competitive engagement of the receptor by plate-bound rituximab increased with the number of V allele (FF, 18.2 +/- 8.6%; VF, 32.0 +/- 4.9%; and VV, 42.4 +/- 9.9%). These results are in discrepancy with the expected increased competition that would result from an increased expression of CD16 on VV and VF NK cells. We conclude that increased binding and functional and clinical responses associated with the high-affinity Fc gamma RIIIa-158V are unrelated to an increased expression of this allotype.

Interindividual variability in the concentration-effect relationship of antilymphocyte globulins - a possible influence of FcgammaRIIIa genetic polymorphism.

AIMS: Polyclonal antilymphocyte globulins (ALGs) are currently used in transplantation, but the sources of interindividual variability of their effect are poorly understood. No pharmacokinetic-pharmacodynamic (PK-PD) study of ALG is available. Moreover, the genetic polymorphism of FcgammaRIIIa, a receptor for the Fc portion of immunoglobulins involved in antibody-dependent cellular cytotoxicity (ADCC), may influence their concentration-effect relationship. METHODS: Fourteen kidney transplant patients treated by horse ALG were included in a prospective, noncomparative study. A population two-compartment PK model including a time dependence of the central volume of distribution was developed. Total lymphocyte count was used as biomarker of effect. Concentration-effect data were described using a physiological indirect response model, combining concentration-dependent and -independent inhibitions of lymphocyte input into the circulation. In addition, six kidney transplant patients in whom ALG concentrations were not available were included retrospectively. All patients were genotyped for FCGR3A. RESULTS: Both the PK and the PK-PD model described the data satisfactorily and showed high interindividual variability. Asymptotic T(1/2)-alpha and T(1/2)-beta-values were 1.3 and 25 days, respectively. The concentration of ALG leading to a 50% inhibition of lymphocyte input (IC(50)) was lower in FCGR3A-V carriers than in FCGR3A-F/F patients (383 +/- 199 vs. 593 +/- 209 mg l(-1), P = 0.008). CONCLUSIONS: This is the first description of the ALG effect on lymphocyte count using PK-PD modelling. Our results show that part of the variability in their concentration-effect relationship may be explained by FcgammaRIIIa genetic polymorphism and therefore that horse ALG may deplete lymphocytes by ADCC.

[Target antigens for therapeutic antibodies in oncology: many candidates, few successes]. / Antigènes-cibles d'anticorps thérapeutiques en cancérologie: beaucoup de candidats, peu d'élus.

Targeted therapies, especially monoclonal antibodies, have reached an increasing importance in oncology. High-throughput techniques have allowed the identification of numerous transcripts, proteins, or non-protein antigens, which have generated the concept of immunome. This epitope library constitutes a huge reservoir of candidate antigens susceptible to become some say the target of an antibody for passive immunotherapy. However, the conception and development of a therapeutic antibody represent a very important investment, both in terms of human power and finance, such that there is a requirement for an early identification of the best candidates among the potential target antigens. Among multiple criteria, the function of the antigen is crucial when it has been identified. A receptor antigen can be targeted by an agonistic or an antagonistic antibody, according to what is sought. When the antigen function is unknown, a therapeutic antibody can be useful, for instance through induction of apoptosis or through accrual of immuno-competent cells, via its Fc portion (complement-dependent cytotoxicity or antibody-dependent cytotoxicity). Other antibody features, unrelated to its function, can also be exploited, such as its internalisation or its translocation in membrane lipid rafts. The expression of the target antigen may also be crucial, in terms of localisation and levels, as is its tumour specificity, which can influence the efficacy and toxicity of the targeting antibody. The multiplicity of the factors to be taken into account and the complexity of the mechanism of action of therapeutic antibodies renders the choice of a target antigen a hazardous bet. Very often, this is only when the clinical efficacy of a targeting antibody is demonstrated that the antigen can be considered as a good target.

Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment.

Due to their exquisite specificity for a given epitope on the target antigen, recombinant monoclonal antibodies (rmAb) can deliver "targeted therapy" in oncology. This review focuses on the structural bases of "antigen specificity" to aid clinical researchers and pharmacologists in managing these new drugs. The fine structure of the Fv (Fragment variable) module (combination of VH and VL domains) from the five unconjugated antibodies currently approved for cancer treatment, namely rituximab, cetuximab, alemtuzumab, trastuzumab and bevacizumab, is presented and analysed. Co-crystal and functional studies are reviewed to define rmAb residues contributing to antigen binding site (paratope)-epitope interfaces. The genetic origin of these recombinant monoclonal antibodies, determined through the IMGT/3Dstructure-DB database and IMGT/V-QUEST (http://imgt.cines.fr), is presented, allowing the evaluation of homologies between antibodies and their closest germline human counterparts and hence their possible immunogenicity. Overall, the IMGT standards appear as a first and crucial step in the evaluation of recombinant antibodies.

Relevance, advantages and limitations of animal models used in the development of monoclonal antibodies for cancer treatment.

Antibody humanisation through recombinant DNA technology was a key step in allowing monoclonal antibodies (mAbs) to reach the clinic, particularly for the treatment of cancer. As a consequence, they are less adapted to animal studies, although these studies continue to be important tools to study antibody distribution and action at the level of a whole organism. Moreover, preclinical studies in animals are mandatory before the approval of biologics license applications for mAbs by the U.S. Food and Drug Administration (FDA) or European Agency for the Evaluation of Medicinal Products (EMEA). Different parameters should be taken in consideration before starting animal experiments with recombinant mAbs, including antibody cross-reactivity, immunogenicity, pharmacokinetics, and possible interactions with the host immune system. The various interspecies differences are reviewed and discussed in light of the pharmacological properties expected in patients. In doing so, this article aims to provide a critical review of the animal models used in preclinical studies of mAbs for cancer treatment. In particular, their relevance, advantages and limitations will be discussed.

CXCL12 polymorphism and malignant cell dissemination/tissue infiltration in acute myeloid leukemia.

Stromal cell-derived factor 1 (SDF-1), a chemokine abundantly produced by the bone marrow microenvironment, and its receptor CXCR4 have crucial roles in malignant cell trafficking. In acute myeloid leukemia (AML), blasts invade the bloodstream and may localize in extramedullar sites, with variations from one patient to another. We hypothesized that a polymorphism in the SDF-1 coding gene (CXCL12 G801A) could influence blast dissemination and tissue infiltration in AML. CXCL12 G801A polymorphism was determined in 86 adult patients and 100 healthy volunteers. The allelic status and CXCR4 expression on bone marrow blasts were analyzed in relation to peripheral blood blast (PBB) counts and frequency of extramedullar tumor sites. 801A carrier status (801G/A, 801A/A) was found to be associated with a higher PBB count compared with 801G/G homozygous patients (P=0.031) and higher frequency of extramedullar tumor sites (odds ratio 2.92, 95% confidence interval 1.18-7.21, P=0.018). Moreover, the PBB count was correlated with CXCR4 expression (correlation coefficient 0.546, P=0.001) when considering 801A carriers. In conclusion, a polymorphism in the SDF-1 gene is shown for the first time to be associated with the clinical presentation of a malignant hematological disease and more generally with the risk of distant tissue infiltration by tumor cells.

No association between C-reactive protein gene polymorphisms and decrease of C-reactive protein serum concentration after infliximab treatment in Crohn's disease.

We recently showed an association between the FCGR3A V/F polymorphism and the biological response [assessed on the basis of a C-reactive protein (CRP) concentration decrease] to infliximab in Crohn's disease. The CRP and FCGR3A genes are located on the same 1q23 locus. The present study aimed: (i) to exclude a linkage disequilibrium (LD) between the two genes and (ii) to study the association between CRP polymorphisms and the response to infliximab, particularly the decrease in CRP after treatment, in Crohn's disease patients. FCGR3A (V/F) polymorphism and three CRP polymorphisms (-717G/A, 1444C/T, CRP 4A/G) were determined in 206 healthy blood donors and 189 Crohn's disease patients who had received infliximab for either refractory luminal or fistulizing Crohn's disease. Clinical response was defined as complete, partial or absent according to the same definition as in controlled trials. The biological response was defined on the basis of CRP decrease. There was no LD between CRP and FCGR3A in healthy donors or Crohn's disease patients. CRP polymorphisms had no impact on CRP decrease after infliximab. The proportions of Crohn's disease having a positive clinical or biological response were not statistically different among the various genotypes of CRP polymorphisms. There was no LD between CRP and FCGR3A polymorphisms. CRP polymorphisms were not associated with the response to infliximab in Crohn's disease.

Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis. Functional analysis of the promoter identifies a calcium sensitive region required for basal activity.

To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5' region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5' end of the xMGP promoter revealed a minimal activating element in the sequence from -70 to -36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from -180 to -36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from -70 to -36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression.