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Background Anti-PD1 and anti-PD-L1 are effective checkpoints inhibitors to stimulate the immune system against many types of cancers. The flip side of these immunotherapies is the generation of immune-related adverse events (irAE), which can theoretically affect all organs including muscle tissue. Immune-related creatine phosphokinase (irCPK) increase is associated with immune related myositis. The meaning of CPK monitoring in clinical practice remains poorly understood. In this observational study, all the grade ≥ 2 increased CPK occurring in patients treated with anti-PD-1 or anti-PD-L1 were investigated to determine their clinical significance. Methods All consecutive pts treated with anti-PD1 or anti-PD-L1, in monotherapy or in combine therapy with other agents, monitored by routine CPK in the Drug Development Department (DITEP) and in the Dermatology Unit of Gustave Roussy between January 2016 and December 2018 were retrospectively reviewed. Patients CPK monitoring level were extracted from biological chart and all the patients with grade ≥ 2 increased CPK were selected for the analysis. Results Among the 1151 patients treated with anti PD1/PD-L1, 53 (4.6%) had grade ≥ 2 increased CPK regardless of immune causality and 31 (2.7%) had a grade ≥ 2 irCPK increase. Among the 53 pts with CPK increase, mean age was 54 years old, tumor types were skin (n = 21), bladder and urothelial (n = 5), head and neck (n = 4) and others (n = 23). CPK increase was not related to the treatment in 22 (41%) pts, was immune-related to anti-PD1/PDL1 and not clinically significant in 19 (36%) pts, was immune-related with muscular adverse events in 10 (19%) pts, including 2 myositis and 2 acute polyradiculoneuritis; and was immune-related with myocarditis in 2 (4%) pts. The mean of CPK maximal peak was significantly higher in patients with immune-related cardiac or muscular adverse event that in the asymptomatic CPK increase pts (1271 UI/L vs. 771 UI/L, p-value = 0.02). Conclusions The frequency of grade ≥ 2 irCPK increase is estimated at 2.7% of patients treated with anti-PD1 or anti-PD-L1. For patients treated with anti PD1/PD-L1, regular CPK monitoring appears as clinically relevant in the early detection of immune-related cardiac and muscular adverse events. Legal entity responsible for the study Jean-Marie Michot. Funding Has not received any funding. Disclosure C. Baldini: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. P. Romano-Martin: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. P. Vuagnat: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. S. Champiat: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. A. Varga: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. A. Hollebecque: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. C. Robert: Advisory / Consultancy: Roche, BMS, MSD, Amgen, Novartis. C. Massard: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. O. Lambotte: Advisory / Consultancy: BMS, MSD, AstraZeneca, LFB, CSL Behring, Janssen. J. Michot: Advisory / Consultancy: Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro. All other authors have declared no conflicts of interest.

The retinoblastoma tumour-suppressor protein Rb inhibits cell proliferation by repressing a subset of genes that are controlled by the E2F family of transcription factors and which are involved in progression from the G1 to the S phase of the cell cycle. Rb, which is recruited to target promoters by E2F1, represses transcription by masking the E2F1 transactivation domain and by inhibiting surrounding enhancer elements, an active repression that could be crucial for the proper control of progression through the cell cycle. Some transcriptional regulators act by acetylating or deacetylating the tails protruding from the core histones, thereby modulating the local structure of chromatin: for example, some transcriptional repressors function through the recruitment of histone deacetylases. We show here that the histone deacetylase HDAC1 physically interacts and cooperates with Rb. In HDAC1, the sequence involved is an LXCXE motif, similar to that used by viral transforming proteins to contact Rb. Our results strongly suggest that the Rb/HDAC1 complex is a key element in the control of cell proliferation and differentiation and that it is a likely target for transforming viruses.

In humans, intermediate basic proteins HPI1 and HPI2 are considered as common precursors of the P2 protamine family, according to data provided by structural studies of these proteins. The occurrence and fate of proteins HPI1 and HPI2 were investigated in nuclei of human spermatids and spermatozoa by means of immunoelectron microscopy. A specific polyclonal antibody against a synthetic peptide overlapping the N-terminus of HPI1 and HPI2 was prepared and used to detect these proteins on sections of testis and ejaculated sperm. A quantitative analysis of labelling density was performed on micrographs using an interactive image analysis system. The first signs of labelling of intermediate basic proteins appeared in spermatid nuclei at steps 4-5 of spermiogenesis, i.e. during the chromatin condensation process. The nuclear labelling density strongly increased in elongating spermatids (steps 5 and 6) and then sharply decreased from step 6 to step 8 of spermiogenesis. However, weak labelling persisted in the nuclei of mature spermatids and ejaculated spermatozoa. The present results show that the intermediate basic proteins HPI1 and HPI2 are synthesized in large amounts in human spermatids during elongation phase and disappear almost totally in mature spermatids when deposition of protamines is completed in condensed nuclei.

Recently, a new member of the insulin gene superfamily termed insulin-like 4 (INSL4) was identified in the human placenta and uterus. The present study investigated whether placenta translates INSL4 mRNA into a putative peptide named early placenta insulin-like (EPIL). Among antibodies elicited against the C chain of pro-EPIL, one antibody (AB7381) was specifically directed against the C chain 59-88 portion, and among those elicited against the A and B chains of EPIL, one antibody (Ab1661) was directed against the A chain 115-139 and the B chain 23-52 portions. Immunohistochemistry based on antibody 7381 to pro-EPIL and antibody 1661 to EPIL demonstrated that the cytotrophoblast from early placenta preferentially expresses the pro-EPIL peptide, whereas the EPIL peptide is expressed by both the cytotrophoblast and the syncytiotrophoblast. At term, the pro-EPIL peptide was detected in villous cytotrophoblast cells, whereas the EPIL peptide was not detected. Moreover, in vitro experiments performed on term placenta showed that the steady state levels of INLS-4 mRNA in the cytotrophoblast are 10 times (one log unit) lower than in the differentiated villous syncytiotrophoblast cells. Taken together, these findings reveal that expression of EPIL peptides in the villous cytotrophoblast is different from that displayed by the syncytiotrophoblast. Finally, these data are the first demonstration that INSL4 mRNA are translated into pro-EPIL and EPIL peptides.

Results are presented on a peptide fragment (1013-1056) from human DNA topoisomerase II alpha. This was selected using the procedure of Lupas et al. (Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) for its potential to adopt a stable coiled-coil structure. The same theoretical treatment rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling, L. A., and Perry, W. M. (1989) Mol. Endocrinol. 3, 603-604) as a possible core for leucine-zipper formation. Our experimental studies combine cross-linking and CD analysis. Cross-linking establishes that the 1013-1056 fragment forms a stable homodimer in solution. Effects of increasing peptide concentration on CD spectra confirm that only the 1013-1056 fragment can undergo a coiled-coil stabilization from an isolated alpha-helix. Unfolding experiments further show that the coiled-coil is more stable in guanidium chloride than in urea. Values of -6.8 and -7.4 kcal/mol for the dimerization free energy are determined by thermal and urea unfolding, respectively. These are strikingly similar to the value recently found for the dissociation/reassociation of the entire yeast topoisomerase II from sedimentation equilibrium experiments (Lamhasni, S., Larsen, A. K., Barray, M., Monnot, M., Delain, E., and Fermandjian, S. (1995) Biochemistry 34, 3632-3639), although their significance relatively to topoisomerase II undoubtedly requires further analysis.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

ObjectiveThe progression of medullary thyroid cancer is difficult to assess with imaging modalities; we studied the interest of calcitonin and carcinoembryonic antigen (CEA) doubling times and of Ki-67 labeling and mitotic index (MI).Patients and methodsFifty-five consecutive medullary thyroid carcinoma (MTC) patients with elevated calcitonin levels underwent repeated imaging studies in order to assess tumor burden and progression status. We looked for relationships between tumor burden and levels of calcitonin and CEA and between progression status according to the response evaluation criteria in solid tumors (RECIST) and calcitonin and CEA doubling times, and Ki-67 labeling and MI.ResultsThe calcitonin and CEA levels were correlated with tumor burden. Ten patients with calcitonin levels below 816 pg/ml had no imaged tumor foci. Among the 45 patients with imaged tumor foci, 19 had stable disease and 26 had progressive disease, according to the RECIST. The calcitonin and CEA doubling times were strongly related to disease progression, with very few overlaps: 94% of patients with doubling times shorter than 25 months had progressive disease and 86% of patients with doubling times longer than 24 months had stable disease. Ki-67 labeling and MI were not significantly associated with disease progression.ConclusionFor MTC patients, the doubling times of both calcitonin and CEA are efficient tools for assessing tumor progression.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

Background: Serum thyroglobulin (Tg) is the marker of differentiated thyroid cancer after initial treatment and TSH stimulation increases its sensitivity for the diagnosis of recurrent disease. Aim: The goal of the study is to compare the diagnostic values of seven methods for serum Tg measurement for detecting recurrent disease both during L-T4 treatment and after TSH stimulation. Methods: Thyroid cancer patients who had no evidence of persistent disease after initial treatment (total thyroidectomy and radioiodine ablation) were studied at 3 months on L-T4 treatment (Tg1) and then at 9–12 months after withdrawal or recombinant human TSH stimulation (Tg2). Sera with anti-Tg antibodies or with an abnormal recovery test result were excluded from Tg analysis with the corresponding assay. The results of serum Tg determination were compared to the clinical status of the patient at the end of follow-up. Results: Thirty recurrences were detected among 944 patients. A control 131I total body scan had a low sensitivity, a low specificity, and a low clinical impact. Assuming a common cutoff for all Tg assays at 0.9 ng/ml, sensitivity ranged from 19–40% and 68–76% and specificity ranged from 92–97% and 81–91% for Tg 1 and Tg2, respectively. Using assays with a functional sensitivity at 0.2–0.3 ng/ml, sensitivity was 54–63% and specificity was 89% for Tg1. Using the two methods with a lowest functional sensitivity at 0.02 and 0.11 ng/ml resulted in a higher sensitivity for Tg1 (81% and 78%), but at the expense of a loss of specificity (42% and 63%); finally, for these two methods, using an optimized functional sensitivity according to receiver operating characteristic curves at 0.22 and 0.27 ng/ml resulted in a sensitivity at 65% and specificity at 85–87% for Tg1. Conclusion: Using an assay with a lower functional sensitivity may give an earlier indication of the presence of Tg in the serum on L-T4 treatment and may be used to study the trend in serum Tg without performing any TSH stimulation. Serum Tg determination obtained after TSH stimulation still permits a more reliable assessment of cure and patient’s reassurance.

International audience; The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701-748 segment homologous to the human topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)talpha(4) conformation, hereafter termed alpha(2)HTH module (helix turn helix (HTH), alpha(3)talpha(4)). The crystal structure of gyrase A has suggested a model wherein HTH is involved in both the enzyme dimerization and the binding to DNA. These two properties were investigated in solution, using the recombinant alpha(2)HTH module of human topoisomerase II alpha and its synthetic components HTH, alpha(4), alpha(3) and turn. The homology-based structure model of human alpha(2)HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 A. Circular dichroism spectra showed that the helical content of human alpha(2)HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that alpha(2)HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that alpha(2)HTH, HTH and alpha(4), but not alpha(3), bind efficiently to DNA (dissociation constants of 3.10(-7) M for alpha(2)HTH and alpha(4), of 3.10(-6) M for HTH and of only 1.10(-5) M for alpha(3)). Correlatively, alpha(2)HTH, alpha(4) and HTH, but not alpha(3), were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that alpha(4) is the DNA recognition helix. All suggests that the alpha(2)HTH module once separated from the whole protein conserves a compact conformation, integral to specific dimerization and DNA recognition. The module may thus be used for the search of drugs efficient in hindering topoisomerase II dimerization or binding to DNA.

International audience; The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (

International audience; The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix alpha4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the alpha4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (Kd of 1.5 nm). Data corroborate previous in vivo results obtained with mutated viruses.

International audience; Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.

Monospecific antibodies were raised against a synthetic peptide K159 (SQGVVESMNKELKKIIGQVRDQAEH-LKTA) reproducing the segment 147–175 of HIV-1 integrase (IN). Synthesis of substituted and truncated analogs ofK159 led us to identify the functional epitope reacting with antibodies within the C-terminal portion 163–175 ofK159. Conformational studies combining secondary structure predictions, CD and NMR spectroscopy together withELISA assays, showed that the greater is the propensity of the epitope for helix formation the higher is therecognition by anti-K159. Both the antibodies and the antigenic peptide K159 exhibited inhibitory activities againstIN. In contrast, neither P159, a Pro-containing analog of K159 that presents a kink around proline but with intactepitope conformation, nor the truncated analogs encompassing the epitope, were inhibitors of IN. While the activityof antibodies is restricted to recognition of the sole epitope portion, that of the antigenic K159 likely requiresinteractions of the peptide with the whole 147–175 segment in the protein [Sourgen F., Maroun, R.G., Fre`re, V.,Bouziane, A., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765–773]. Actually, of alltested peptides only K159 was found to fulfill conditions of minimal number of helical heptads to achieve theformation of a stable coiled-coil structure with the IN 147–175 segment. The binding of antibodies and of theantigenic peptide to this segment of IN hampers the binding of IN to its DNA substrates in filter-binding assays. Thisappears to be the main effect leading to inhibition of integration. Quantitative analysis of filter-binding assay curvesindicates that two antibody molecules react with IN implying that the enzyme is dimeric within these experimentalconditions. Together, present data provide an insight into the structure–function relationship for the 147–175peptide domain of the enzyme. They also strongly suggest that the functional enzyme is dimeric. Results could helpto assess models for binding of peptide fragments to IN and to develop stronger inhibitors. Moreover, K159antibodies when expressed in vivo might exhibit useful inhibitory properties.Keywords: antibodies, conformational aspects, HIV integrase, inhibition, peptides.The human immunodeficiency virus (HIV) replication cycle isinitiated by integration of the viral DNA into the cell genome[1,2]. Integrase (IN) [2–4] encoded by the virus pol genecatalyses integration sequentially through the deletion of twonucleotides at the conserved 3

We have previously shown that a synthetic peptide (dL) consisting of amino acids 1013-1056 of human alpha topoisomerase II adopted an alpha-helix structure and formed a stable dimer coiled-coil in solution [Frère, V., Sourgen. F., Monnot, M., Troalen, F.Fermandjian, S. (1995) J. Biol. Chem. 270, 17502-17507]. Here we studied two peptides, dP and dLshort, which are related to dL but which have a double substitution Leu1026--Pro, Leu1037--Pro and a deletion of the 15 C-terminal residues, respectively. The peptides were studied for their ability to form alpha-helix structures, coiled coils, and to inhibit topoisomerase II activity. In combining circular dichroism spectra with AGADIR prediction for helix structures, we demonstrated that the dLshort peptide, like its parent dL peptide, adopts an alpha-helix structure and can autoassociate into coiled-coils, while dP is completely devoid of such properties. Remarkably, only the dL and dLshort peptides act as good inhibitors of topoisomerase II in various in vitro assays. However, the dLshort peptide has a stronger helix potential and behaves as a much more potent inhibitor (5 microM versus 200 microM) compared to the dL peptide. All these data strongly suggest that the greater inhibitory effect demonstrated by the dLshort peptide is related to its higher ability to form a stable amphiphilic helix, which in turn better recognizes its homologous helical segment in topoisomerase II. Finally, we propose that the dL and the dLshort peptides could interfere with the enzymatic activity of topoisomersase II in modifying its autoassociation or translocation properties. Such peptides may serve as useful models for developing simpler and more specific inhibitors of topoisomerase II.

Measurement of serum luteinizing hormone (hLH) is important for the detection and follow-up of patients with pathological processes of the reproductive axis. Detection of the uncombined form of the β-subunit of hLH, or free hLHβ, has proved to be of clinical interest in the recognition of gonadotroph adenomas. As no monoclonal antibody specific for the free hLHβ is at present available, we elicited monoclonal antibodies using free hLHβ as an immunogen. An antibody, named BLH01, was selected for its specific binding to the free β-subunit, and its antibody-binding site was characterized at the molecular level, emphasizing the importance of amino acid residues located between the disulfide-bonded Cys93 and Cys100. This region has been demonstrated as being particularly critical for the specific binding of lutropic hormones to their receptors. Topographic assignment of the epitope recognized by BLH01 was then achieved by cross-matching studies based on a library of antibodies directed to hLHβ, and the location of some epitopes on the three-dimensional model of the β-subunit is proposed. A two-site immunoassay based on BLH01 as capture antibody was then developed. This assay, using BLH01, may constitute a simple, sensitive and highly specific procedure for assessing the clinical usefulness of measuring free hLHβ, particularly for the diagnosis and follow-up of patients presenting with pituitary adenomas. Journal of Endocrinology (1996) 151, 251–258

International audience; The monoclonal antibody, HT13 recognizes human choriogonadotropin (CG) bound to the extracellular domain of its receptor, but not to the full-length receptor. The HT13 epitope is located in the regions of residues 15-17 and 73-75 of the human CG alpha-subunit. Only one synthetic peptide, lutropin (LH)/CG-receptor-(481-497)-peptide (EL2 peptide), which spans the second putative extracellular loop of the LH/CG-receptor endodomain, prevents recognition of human CG by HT13 mAb. EL2 peptide decreases hormone-induced cAMP production, but not high-affinity binding. An anti-EL2 serum also displays the capacity to inhibit human CG-stimulated cAMP production. These results suggest that the second extracellular loop of the receptor is in contact with the HT13 epitope of human CG alpha-subunit and is involved in signal transduction. A relative orientation of the hormone versus the endodomain is proposed.

Integration of the human immunodeficiency virus (HIV-1) DNA into the host genome is catalysed by a virus-encoded protein integrase. Here, we report some of the structural and functional properties of two synthetic peptides: integraw(147 - 175)-peptide reproducing the residues 147 - 175 (SQGVVESMNKELKl59KIIGQVRDQAEHLKTAY) of the HIV-1 integrase, and [Pro1591 integrase-( 147- 175)-peptide where the lysine 159 is substituted for a proline. Circular dichroism revealed that both peptides are mostly under unordered conformation in aqueous solution, contrasting with the a-helix exhibited by residues 147- 175 in the protein crystal structure. In a weak a-helix-promoting environment, integrase-(147- 175)peptide self-associated into stable coiled-coil oligomers, while [Pro1591 integrase-( 147 - 175)-peptide did not. This property was further confirmed by cross-linking experiments. In our in vitro experiments, only integrase-(147 - 175)-peptide was able to reduce the integration activity of the enzyme. We propose that the inhibitory activity shown by integrase-(147- 175)-peptide is dependent on its ability to bind to its counterpart in integrase through a peptide-protein coiled-coil structure disturbing the catalytic properties of the enzyme.

Lutropin (LH), follitropin (FSH), thyrotropin (TSH) and choriogonadotropin (CG) are assembled of two non-covalently alpha (alpha) and beta (beta) subunits. We studied the discontinuous antigenic regions recognized by a monoclonal anti-hLH antibody designated as LH05 which binds to hLH, hCG and hTSH and does not cross-react with either the free subunits or hFSH. LH05 and an antibody designated HT13, recognizing an epitope partly comprizing the alpha64-76 region, did not bind simultaneously to hCG. Furthermore, LH05 was unable to combine with an anti-peptide antibody (LHP03) directed to residues 43-52 of hLHbeta. Thus, LH05 recognizes an epitope partly overlapping with those recognized by HT13 and LHP03. Using various hybrid molecules, we showed that the human alpha-subunit plays a critical role in the assembly of the epitope that, in contrast, contains amino acid residues conserved in the various beta-subunit of several species. Together, our results suggest that the amino acids Leu49-Pro50, Tyr59-Arg60 and Leu86-Ser87 in the hLHbeta and the alpha64-76 region are probably included in the epitope recognized by LH05 which appeared to be not accessible on the CG/LH receptor.

To investigate the epitopes present on the beta-subunit of the human lutropin (hLHbeta) and their topographical relationship at the surface of the molecule, we produced two monoclonal antipeptide antibodies, designated LHP03 and LHP04, capable of binding to the radiolabeled 125I-hLHbeta and directed to the 43-52 and 110-117 regions of the hLHbeta, respectively. Analysis of the accessibility of the epitopes on hLH and on the beta-subunit of human chorionic gonadotropin (hCGbeta), equine LH (eLHbeta) and ovine LH (oLHbeta) indicated that: (i) LHP03 binds to both the free hLHbeta subunit and dimeric hLH whereas LHP04 binds preferentially to the free hLHbeta, (ii) LHP03 recognizes weakly the hCGbeta and oLHbeta in comparison to hLHbeta and (iii) LHP04 binds oLHbeta as well as hLHbeta but does not bind to hCGbeta and eLHbeta. The topographical relationship of epitopes recognized by LHP03 and monoclonal antibodies recognizing dimer specific epitopes on hLH allowed us to localize discontinuous antigenic sites that overlaps or are located outside the hHLbeta(43-52) region. Together, our results demonstrated that the hHLbeta(43-52) portion is accessible on both the free hLHbeta subunit and hLH whereas the COOH-terminal portion, hHLbeta(110-117), is probably buried at the alpha/beta interface of the hormone.

The production of human chorionic gonadotropin (hCG) and its free alpha subunit (hCGα) and beta subunit (hCGβ) by nongonadal neoplasms is controversial. Following the initial report (1), numerous studies have provided indirect evidence for the production of hCG, hCG-like substances, or free subunits by nongonadal cancers (2–8). Indeed, between 1973 and 1985, 105 studies reported immunoreactive hCG in sera of nontrophoblastic tumor patients; between 1962 and 1986, 107 investigations showed the immunocytochemical localization of hCG in various tissue (9, 10). However, these results were questionable, since most investigations used polyclonal and monoclonal antibodies lacking the required specificity. One of the major problems was that most antibodies cross-reacted with human luteinizing hormone (hLH) (6, 11, 12). More recently developed assays based on monoclonal antibodies continued to display low, but significant, cross-reactivity with hLH (13–17). More importantly, most assays were unable to distinguish between the intact hCG molecule and the free hCGβ-subunit. Indeed, the observed cross-reactivity of intact hCG varied between 3% and 100% in radioimmunoassays used for the detection of free hCGβ. It was much lower, but still significant (0.2% to 4%), with most immunoradiometric assays (m-IRMAs) for free hCGβ based on monoclonal antibodies (13–15, 18–20). Finally, the nomenclature for hCGβ assays is ambiguous, since the same designation was given to at least two types of assays with widely different specificities, including those measuring only the free hCGβ-subunit and those measuring both intact hCG and the free hCGβ-subunit (21, 22). Similarly, the nomenclature for hCG assays may be misinterpreted, since these assays also measure the free hCGβ-subunit in addition to hCG (23).

We analyzed the status of retinoblastoma and p53 genes in 10 human hepatoma cell lines. Polyclonal anti-peptide antibodies generated against peptides homologous to COOH-terminal and leucine-zipper domains of the retinoblastoma protein allowed us to identify two cell lines (Hep 3B and FOCUS) with abnormal expression. The same cell lines have both lacked p53 expression. In contrast to the retinoblastoma gene, the expression of the p53 gene was abnormal in six additional cell lines. Indeed, only the Hep G2 hepatoblastoma cell line (and its derivative Hep G2/2215) appeared to have normal p53 and retinoblastoma gene expression. Our studies indicate that p53 abnormalities are common but retinoblastoma gene aberrations are rare in human hepatoma cell lines.

The present study was designed to characterize monoclonal antibodies (mAbs) specific for the free beta-subunit of hCG (free hCG beta), to develop two-site immunoradiometric assays (m-IRMAs) specific for free hCG beta, and to study the reactivities of various molecular forms of hCG beta in these assays. We attempted first to delineate the antigenic regions present specifically on the free hCG beta by studying the binding pattern of seven mAbs directed preferentially to hCG beta, designated FBT11, P8E, P10F, HB2, P5D, P5H, and INN-64. Competitive inhibition experiments performed by RIA demonstrated the specificity of these mAbs for the free hCG beta as noncross-reacting with the beta-subunit of human (h) LH beta. m-IRMAs were used to analyze the arrangement of epitopes on hCG beta. Experiments performed with the seven mAbs used either as capture antibodies or radiolabeled indicators confirmed the specificity of the seven mAbs for the free hCG beta, and that mAbs FBT11, P8E, and P10F bound to equine LH beta (eLH beta), but did not bind to a fragment of hCG beta called the beta-core fragment (beta CF). These antibodies defined an antigenic domain identified as A. In contrast, mAb HB2 bound neither to eLH beta (e beta) nor to beta CF and was directed to domain B (e beta negative, beta CF negative). Finally, mAbs P5D, P5H, and INN-64 bound to beta CF, but did not bind to eLH beta, and defined a third domain identified as C (e beta negative, beta CF positive). Collectively, these results demonstrate that at least six antigenic domains are present on the free hCG beta and that a limited set of amino acids was shared among these domains; domains A, B, and C are present only on the free beta-subunit, while three other domains recognized by mAbs FB19, FBT10, and 518B7 are present on both free hCG beta and hCG. Thus, most of the surface of the hCG beta appears to be antigenic and accessible to antibody binding. Three different m-IRMAs specific for free hCG beta were then constructed using either mAb FBT11 (domain A) or HB2 (domain B). The study of the reactivities of various molecular forms of hCG beta in these assays demonstrated that the recognition of hCG beta forms nicked at position 43 (beta 43), 44, and/or 51 (beta 44/51) varied between assays.(ABSTRACT TRUNCATED AT 400 WORDS)

To study antigenic sites present in the beta-subunit of human thyrotropin (hTSH), we produced site-specific antibodies directed against synthetic peptides analogous to the 1-18, 44-59, and 85-112 regions of the thyrotropin beta-subunit. The hTSH beta(1-18) peptide-carrier conjugate elicited antisera capable of binding to both radiolabeled hTSH and its beta-subunit whereas antibodies elicited against the hTSH beta(44-59) peptide-carrier conjugate bound only to the peptide. Thus, the NH2-terminal region of hTSH beta appears to be accessible at the surface of the hormone whereas the hTSH beta(44-59) region may be poorly accessible. Two monoclonal antipeptide antibodies that bound to 125I-hTSH beta, designated as TS01 and TS02, were selected after immunization with the hTSH beta(85-112) peptide-carrier conjugate. The antigenic site recognized by TS01 was located on the eight COOH-terminal(105-112) amino acid residues. TS02 antibody bound to an antigenic region included within Cys95 and Cys105. Both antigenic sites appeared to be more accessible on the free hTSH beta than on the hormone. Immunoblots performed on various preparations containing TSH revealed that TS02 antibody detected the beta-subunit from both the human and bovine species but not the rat TSH beta. Under reducing conditions, a low molecular weight material was identified in hTSH beta, likely caused by intrachain nicking.

Recent observations based on immunohistochemistry and RT-PCR demonstrate that early placenta insulin-like peptide (EPIL), encoded by the INSL4 gene, is present in the placenta during gestation. In the present study, we report on the development of a specific immunoassay, entirely based on two monoclonal anti-peptide antibodies (mAbs), and its use for the detection of pro-EPIL forms in biological fluids during pregnancy. One mAb directed against the C-connecting peptide was used to capture pro-EPIL forms and their binding was revealed by a radiolabeled anti-A chain mAb as the indicator. A composite synthetic peptide, encompassing the C- and A-domains, was utilized as the standard. Under these experimental conditions, the assay displays a sensitivity limit of 2 ng/mL. Pro-EPIL molecular forms were detected in both amniotic fluid and maternal serum of pregnant women. At 12 to 16 weeks of pregnancy, the pro-EPIL level was higher in amniotic fluid (246 +/- 50.8 ng/mL) than in maternal serum (5 +/- 2.0 ng/mL). As gestation advanced, so the concentration of pro-EPIL forms decreased in amniotic fluid while its level increased in maternal serum. Interestingly, in amniotic fluid, the pattern of pro-EPIL concentration during pregnancy is very similar to that observed for human chorionic gonadotropin (hCG) and its free subunits. The pattern of serum pro-EPIL concentration is similar to that of the free alpha-subunit. Together with our previous immunohistochemical observations, these results indicate that pro-EPIL is preferentially secreted by cytotrophoblasts in amniotic fluid and that the biosynthesis of hCG subunits and EPIL may be regulated by common pathways. Overall, our observations strongly suggest that EPIL may play a critical physiological role during embryonic and foetal development.

The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the alpha or beta-subunit. Under reducing conditions, immunoblotting with antibodies directed to the beta-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111-145 region. Using microsequencing techniques, we found that the fragment had a NH2 terminal portion corresponding to the sequence of the beta-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48-145 portion of the beta-subunit and is probably a cleavage product of the native protein with intrachain nicking.

In an attempt to localize topographic domains specific to native human chorionic gonadotropin (hCG), we studied the discontinuous antigenic regions recognized by a monoclonal anti-hCG antibody designated as C8 which binds only to hCG and does not cross-react with either the free hCG-alpha and hCG-beta subunits or other glycoprotein hormones. Using two-site monoclonal immunoradiometric assays (M-IRMAs), we found that C8 antibody and an anti-peptide antibody (FB12) directed to residues 110-116 of hCG-beta did not bind simultaneously to hCG. This observation suggested that C8 binds to residues of hCG-beta included either in the antibody-binding region of FB12 or in close proximity to amino acids 110-116. To further delineate the regions of hCG-beta recognized by C8, we carried out hapten inhibition experiments with synthetic peptides corresponding to various regions of hCG-beta. The peptide corresponding to residues 109-122 and subpeptides (111-122 or 112-122) inhibited the binding of 125I-hCG to C8, whereas weak inhibition was observed with subpeptide 113-122. By studying the binding of C8 to the 1-112 disulfide-bonded part of hCG-beta (hCG-beta core) recombined with hCG-alpha, we were able to confirm that C8 binds to a region including or near to Asp112. M-IRMAs showed that C8 does not bind to the recombinant molecule lacking residues 113-145 of hCG. Taken together, these results indicate that a limited number of residues located on hCG-beta near to Asp112, and most likely the sequence Asp111-Asp112-Pro113, are included in the discontinuous antigenic region recognized by C8. We then attempted to localize residues of hCG-alpha that constitute another part of the determinant which bound to C8. Six synthetic peptides corresponding to various regions of hCG-alpha did not inhibit binding of 125I-hCG to C8. In contrast, M-IRMAs demonstrated that C8 is capable of binding recombinant products composed of the hCG-beta subunit and the alpha subunits from human, equine, and porcine species. These results indicate that C8 recognizes a region of the alpha subunit highly conserved in these three species. Finally, we determined that the discontinuous regions recognized by C8 are partially accessible on the CG/LH-receptor complex.

Drug-dependent antibodies were investigated in patients treated with elliptinium acetate, a cytostatic drug with activity in advanced breast cancer. Retrospective analysis of 83 patients, receiving weekly intravenous elliptinium, showed a high incidence of anti-elliptinium antibodies (20%). Hemolysis occurred among antibody-positive patients, apparently related to the antibody titer. The predictability of anti-elliptinium antibodies for hemolysis and the schedule dependency of antibody development was examined prospectively. Among 42 patients treated weekly for at least three courses, 40% developed antibodies. Of 30 patients receiving elliptinium daily for three days every three weeks, none developed either antibodies or hemolysis. Only antibody positive patients, with titers greater than or equal to 32 were at risk for hemolysis. The possible mechanisms are discussed.

Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.

We have recently derived from human fetal blood (25 wks) a series of cloned cell lines that were selected for their ability to kill the conventional natural killer (NK) target cell K562. It was found that a fraction of these clones express CD3 proteins but not the monomorphic Ti alpha beta determinant recognized by WT31 antibody. One interleukin-2-dependent CD3+ WT31- clone, termed F6C7, was used for immunization of mice to generate monoclonal antibodies directed at a potentially novel recognition receptor. It was shown that F6C7 cells, which transcribe Ti beta but not Ti alpha genes, surface-express a clonotypic structure, termed NKFi. Immunoprecipitations performed with anti-NKFi monoclonal antibody (mAb) indicated that the corresponding molecule is resolved in SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of relative molecular mass approximately 85,000 (Mr approximately 85K). After reduction, a major band was detected at 44K and a faint band was present at 41K. The present study was designed to characterize this structure. It was found that NKFi represents either two 44K disulphide-linked gamma (TCR) chains, or possibly one gamma chain associated to an additional undetected molecule, and that the 41K material corresponds to a partially glycosylated fraction of the gamma protein. Anti-NKFi mAb both induces a specific autocrine proliferative response and blocks cytotoxic function, demonstrating that gamma chains serve as functional receptor structures on subpopulations of normal human lymphocytes.

Immune-mediated hemolytic disease is a phenomenon rarely encountered with cancer chemotherapeutic agents. Elliptinium, a tetracyclic ammonium compound used in breast and kidney cancer, can induce antibodies that may result in clinical hemolysis. This study reports the characterization of the elliptinium haptenic determinant by use of two different methodologies: a hemagglutination test and a radioimmunoassay. Binding of 12 analogues or derivatives of elliptinium was also studied. Good correlation between the two methods was obtained, indicating that the determinant is most likely located on the quaternary ammonium-containing ring. Furthermore, the hydrophilicity of the drug appears to be an important factor in the antibody reaction. The mechanism of the binding of elliptinium to its antibodies is discussed.