ENTPD1/CD39 is a promising therapeutic target in oncology.

Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells that are essential for maintaining the homeostasis of the immune system, limiting self-reactivity and excessive immune responses against foreign antigens. In cancer, infiltrated Tregs inhibit the effector lymphocytes and create a favorable environment for the growth of the tumor. Although Tregs mediate immunosuppression through multiple, non-redundant, cell-contact dependent and independent mechanisms, a growing body of evidence suggests an important role for the CD39-CD73-adenosine pathway. CD39 ectonucleotidase is the rate-limiting enzyme of a cascade leading to the generation of suppressive adenosine that alters CD4 and CD8 T cell and natural killer cell antitumor activities. Here, we review the recent literature supporting CD39 as a promising therapeutic target in oncology. In vitro and in vivo experiments involving knockout models and surrogate inhibitors of CD39 provide evidence in support of the anticancer activity of CD39 inhibition and predict a favorable safety profile for CD39 inhibitory compounds. In addition, we report the ongoing development of CD39-blocking monoclonal antibodies as potential anticancer drugs. Indeed, CD39 antagonistic antibodies could represent novel therapeutic tools for selectively inhibiting Treg function without depletion, a major limitation of current Treg-targeting strategies.

Pharmacokinetics, foreign protein immune response, cytokine release, and lymphocyte subsets in patients receiving thymoglobuline and immunosuppression.

The pharmacokinetics and immune response to the rabbit IgG of rabbit antihuman thymocyte globulin, Thymoglobuline has been characterized. A cytokine release pattern of TNF alpha and IL-6 but not IL-1 beta and IFN chi has been demonstrated with the first and not subsequent doses. An effect on lymphocyte depletion of peripheral blood with major subset suppression has been shown to last more than the 3-month observation period in patients on a regimen of quadruple sequential immunosuppression.

Evidence for an involvement of the neutrophil integrin lymphocyte function-associated antigen-1 in early failure of heart transplants.

BACKGROUND: The adhesion of neutrophils to the coronary vascular wall contributes to reperfusion injury of cardiac allografts. This phenomenon involves interactions between neutrophil beta 2-integrins (CD11a/CD18 [lymphocyte function-associated antigen-1, or LFA-1], CD11b/CD18 [membrane attack complex-1, or MAC-1], and CD11c/CD18 [p150,95]) and their endothelial ligands. Whereas the roles of the common beta-chain (CD18) and of the alpha-subunit of MAC-1 (CD11b) have been studied extensively, the role of the alpha-subunit of LFA-1 (CD11a) remains less well defined. The objective of this study, therefore, was to assess the effects of CD11a blockade on postischemic function and neutrophil infiltration of cardiac allografts. METHODS AND RESULTS: Twenty-six rat hearts were kept in cold storage for 4 hours, heterotopically transplanted in the abdomen of recipient rats, and reperfused for 1 hour. In 10 hearts, a monoclonal antibody against LFA-1 alpha was given as a single intravenous bolus (100 micrograms) 35 minutes before reperfusion. The control groups consisted of 10 hearts that received saline and 6 hearts treated with an isotype-matched, nonbinding antibody (OKT3) administered at the same dosage and schedule as in the anti-LFA-1 alpha group. Before reperfusion, all hearts were instrumented with an intraventricular balloon-tipped catheter to allow serial isovolumic measurements of left ventricular function during reperfusion, after which myocardial accumulation of neutrophils was measured by myeloperoxidase activity. Postischemic heart rate and diastolic pressure were comparable among groups. However, the best recovery of contractility was achieved with anti-LFA-1 alpha treatment. After 60 minutes of reperfusion, dP/dt values were 1680 +/- 66 mm Hg/s-1, 1733 +/- 25 mm Hg/s-1, and 2550 +/- 95 mm Hg/s-1 in the saline, OKT3, and anti-LFA-1 alpha groups, respectively (P < .0001 between anti-LFA-1 alpha and the two control groups). This correlated with a significant (P < .0001) reduction in myocardial accumulation of neutrophils in the anti-LFA-1 alpha group (3.3 +/- 0.1 versus 7.9 +/- 0.6 and 6.7 +/- 0.3 U/100 mg tissue in the saline and OKT3 groups, respectively). CONCLUSIONS: These results suggest the involvement of the alpha-subunit of LFA-1 (CD11a) in neutrophil-mediated reperfusion injury incurred by transplanted hearts. This finding is clinically relevant in view of the recent development of an anti-LFA-1 alpha monoclonal antibody for human use, the cardioprotective effects of which might thus extend beyond the initially intended prevention of lymphocyte-mediated rejection.

A dose-searching trial of an anti-LFA1 monoclonal antibody in first kidney transplant recipients.

25.3, a mouse IgG1 monoclonal antibody (MoAb), directed at the alpha chain of the LFA1 molecule (CD11a) has been used in prophylaxis of rejection in recipients of cadaveric kidney graft. Promising clinical results have been obtained for both tolerance and efficacy [1]. The aim of this trial was to determine the optimal dosage, base on a pharmacokinetic-pharmacodynamic analysis of the data obtained from the 15 patients included in this dose-searching study. Biological parameters, such as circulating levels and functional inhibition (as detected in an adhesion assay of patient lymphocytes), were measured during and after treatment. A Hill relation was calculated between the effect and the concentration measured and led us to select a 15 mg/day dose for further clinical trials, with a loading dose of 30 mg. An additional group receiving this protocol was submitted to the same calculation, and the results from this last group were in agreement with this previous analysis.

Evaluation of a new preservation solution: Celsior in the isolated rat lung. Paris-Sud University Lung Transplatation Group.

BACKGROUND: We compared Celsior solution, a new and original extracellular preservation solution, with blood-based Wallwork's solution. METHODS: In two groups of isolated rat lungs submitted to 4 hours of cold ischemia, pulmonary arterial and venous resistances, coefficient of filtration, and wet-to-dry lung weight ratio were determined at baseline and after 90 minutes of reperfusion. RESULTS: After ischemia-reperfusion, percentage of increases above the respective baseline coefficient of filtration values were 93% +/- 7% in the Wallwork group and 7% +/- 3% in the Celsior group (p < 0.001 versus Wallwork's solution). CONCLUSIONS: These results show that Celsior consistently prevented the ischemia-reperfusion-induced increase in pulmonary microvascular permeability as compared with Wallwork's solution.

Experimental evaluation of Celsior, a new heart preservation solution.

An original heart preservation solution (Celsior) has been developed, the formulation of which has been designed to fulfil two major objectives: (1) to combine the general principles of hypothermic organ preservation with those specific for the myocardium, and (2) to offer the possibility of being used not only as a storage medium but also as a perfusion fluid during initial donor heart arrest, poststorage graft reimplantation and early reperfusion. The major principles addressed by the Celsior formulation include (1) prevention of cell swelling (by mannitol and lactobionate), (2) prevention of by the Celsior formulation include (1) prevention of cell swelling (by mannitol and lactobionate), (2) prevention of oxygen-derived free radical injury (by reduced glutathione, histidine and mannitol), and (3) prevention of contracture by enhancement of energy production (glutamate) and limitation of calcium overload (high magnesium content, slight degree of acidosis). Two experimental preparations were used: The isolated isovolumic buffer-perfused rat heart model and the heterotopic rabbit heart transplantation model. In isolated heart experiments, hearts were arrested with and stored in Celsior for 5 h at 4 degrees C and subsequently reperfused for 1 h. A similar protocol was used in the transplantation experiments except that the total ischemic time was approximately 1 1/2 h longer (corresponding to 6 h of storage followed by the 25 additional minutes of cold ischemia required for graft implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of lactobionate-enriched cardioplegic solution in preserving compliance of cold-stored heart transplants.

Cardioplegic solutions of the extracellular type are commonly used as storage media for heart transplants. Because this type of formulation was not originally designed for preventing hypothermically induced edema, we assessed the effects of supplementing a standard, extracellular-like cardioplegic solution with the high molecular weight impermeant lactobionate on water content and postischemic compliance of isolated rat hearts. In one series of experiments, hearts were immersed in either a standard cardioplegic solution of the extracellular type or in the same solution supplemented with lactobionate (80 mmol/L). Hearts were then processed for measurements of water content after 4 hours, 6 hours, and 8 hours of storage at 4 degrees C. In a second series of experiments, hearts were stored in the same solutions for 4 hours and 8 hours and subsequently reperfused for 1 hour on a Langendorff column, at which time left ventricular pressure-volume curves were constructed and compared with those obtained during the preischemic perfusion. Lactobionate-treated hearts gained significantly less water than controls after 4 hours and 6 hours of storage, but the difference was no longer significant at the 8-hour time point. In contrast, the treated group yielded a significantly better recovery of compliance after both 4 hours and 8 hours of storage, suggesting that lactobionate might exert protective effects in addition to those caused by its impermeant properties, possibly involving calcium chelation and subsequent limitation of calcium-dependent contracture. Extracellular-type cardioplegic solutions are attractive because a single solution can be used during all phases of the transplantation procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical, behavioral, and electrophysiologic actions of the selective sigma receptor ligand (+)-pentazocine.

Research on the sigma receptor, a binding site associated with drug-induced psychotomimetic behaviors, has been hampered because most sigma agonists also interact with the phencyclidine (PCP) receptor. (+)-Pentazocine, a human psychotogen, is a selective sigma receptor ligand. To demonstrate sigma receptor activities, we studied the behavioral and electrophysiologic actions for (+)-pentazocine. In the behavioral drug discrimination procedure in which rats were trained to discriminate between 2.0 mg/kg (5.59 mumol/kg) (+)-pentazocine and saline, (+)-pentazocine produced dose-related increases in the percentage of trials completed on the (+)-pentazocine lever. At a dose of 1.0 mg/kg (3.29 mumol/kg) (+)-N-allylnormetazocine generalized completely to (+)-pentazocine. By contrast, PCP only partially generalized. In the visual evoked potential test, these compounds produced a significant dose-dependent slowing of the N2 latency. This response was prevented by haloperidol pretreatment. These results demonstrate pharmacologic actions for the selective sigma receptor ligand (+)-pentazocine and suggest some overlapping pharmacologic properties of the sigma and PCP receptor sites despite differences in central nervous system distribution.

Conjugates of elliptinium acetate with mouse monoclonal anti-alpha-fetoprotein antibodies or Fab fragments: in vitro cytotoxic effects upon human hepatoma cell lines.

Elliptinium acetate (EA) is a new anti-cancer compound displaying cytostatic activity against various malignancies including hepatoma. Using 3 hepatoma cell lines, we compared the in vitro activity of doxorubicin (reference drug), of EA and of conjugates made up with this latter drug and monoclonal antibodies (MAbs). The linkage was performed by a direct oxidation method. Specific immunoconjugates were prepared with an anti-alphafetoprotein (AFP) MAb (AF01) or its Fab fragment (Fab AF01). Non-specific conjugates were obtained with an anti-thyroglobulin MAb (TG01) or its Fab (Fab TG01). Direct membrane injury (51Cr-release), DNA and protein synthesis as well as AFP release were investigated for all compounds. Free EA displayed only weak activity on DNA and protein synthesis, at 10-fold higher molar concentration than doxorubicin. Conjugation of EA with whole AF01 allowed significant potentiation of protein synthesis inhibition without affecting the 3 other tests. In contrast, Fab AF01 x EA conjugates displayed a marked effect in the 4 tests; in particular, this conjugate was at least 100 times more efficient than any other compound when tested in the 51Cr-release test. Neither Fab AF01 nor free EA alone or in combination exhibited such an effect. Fab TG01 x EA conjugate was not directly cytotoxic but potentiated inhibition of DNA and protein synthesis between 2- and 10-fold. The mechanism of the direct cytotoxic effect of anti-AFP Fab x EA conjugate, which has never been described in any other immunodrug model, was investigated.

Development of a computerized system for inventory of controlled drug substances.

An automated drug inventory system was developed to facilitate record keeping requirements for controlled drug substances. A hand-held barcode scanner and a computer-accessible top-loading electronic balance are used to identify and weigh samples each time they are removed from or returned to the safe. A computer compares the initial sample weight to its last recorded weight to assure that no discrepancies occurred prior to sample use. Upon return, the computer compares the difference in initial and return tare weights to the stated amount of drug used to assure accuracy of the written inventory record. Unreconciled errors are immediately brought to the attention of the safe custodian. During its first year of operation, 2791 sample out/sample return transactions on 381 drug samples were tracked by the system. The most common errors detected by the system include sample loss of moisture (9), sample absorption of moisture (4), wrong sample used (4), incorrect weight recorded (4), and sample used without log entry (4). Importantly, all errors were immediately identified and reconciled. At the end of each working day, a printout of all daily transactions shows sample use, possible errors, and whether all samples have been returned to the safe. A printout of the total drug inventory is immediately available when called for. This system provides enhanced vigilance and security, and has been readily accepted by all investigators using controlled drug substances in our laboratory.

Antibody recognition of substituted ammonium ions. Modulation by the counterion.

Antibodies directed against elliptinium acetate, a quaternary ammonium compound with antineoplastic activity in man, were obtained in rabbits, after conjugation of the drug with haemocyanin. These antibodies are specific for the quaternary ammonium structure. However, the recognition of the drug can be markedly decreased (ten-fold) by changing the associated counterion. These observations were extended to other ellipticine derivatives that exist in two forms: acetate and chloride. In each case, the recognition of the acetate form was 7-11-fold higher than that of the chloride form. These results could be explained by high-energy strengths existing between the cation and the anion, resulting in a paired-ion antigen. This represents the first identification of antibodies directed to a paired-ion structure, with specificity for both the cation and anion used for immunization. Such results are relevant in the construction of immunoassays for quaternary ammonium compounds.

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group.

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.

The immune response to a synthetic peptide analogous to the 109-145 beta hCG carboxyl-terminus is directed against two major and two minor regions.

The immune response to a 37-amino acid synthetic peptide analogous to the carboxyl-terminal part (109-145) of the human chorionic gonadotropin beta subunit (beta hCG) was studied with monoclonal antibodies selected from 31 cell fusion experiments. Analysis of the immunogenic determinants borne on the synthetic peptide (CTP) showed a prevailing response to two immunodominant regions. The first was located on the 110-116 amino acid sequence of the CTP which is also the most hydrophilic region: 50% of anti-CTP antibodies selected for their high binding to 125I beta hCG were directed to this sequence. A second immunodominant portion was recognized by four antibodies, and comprised amino acids 134 to 139, representing a highly O-glycosylated region on the native protein. Moreover, a unique antibody designated FB13 bound to a region located on the last seven amino acids (139-145) of beta hCG. Finally, a hypothetical conformational determinant was recognized by antibody FB02 within the 121-145 region. Thus, the immune response to CTP was directed against two major and two minor regions. These antigenic determinants were demonstrated to be accessible for antibody binding on both the hCG molecule and its beta subunit. Localization of these epitopes suggests a relationship between the hydrophilicity and the immunological potency of different CTP regions.

Polyclonal antibodies to elliptinium acetate: structure-activity relationships and comparison with human anti-elliptinium IgM.

In order to elucidate the immune-mediated hemolytic disease induced in man by elliptinium acetate, a quaternary ammonium compound with antineoplastic activity, polyclonal antibodies directed against this hapten were raised in rabbits. The coupling step between drug and carrier was performed according to a putative human in vivo hapten conjugation mechanism. Structure-activity relationships of the resulting IgG were compared with the epitope site recognized by human anti-elliptinium IgM by using a panel of twelve elliptinium acetate analogues. Although both antibodies were directed principally against the quaternary ammonium ion, a poor correlation between the cross-reactivity indices was obtained. In fact, it appeared that both antibodies recognized specifically the ammonium group plus different regions of the molecule: the indole ring for human antibodies, the N-alkyl group and its vicinity for rabbit ones. The specificity of the obtained rabbit polyclonal antisera is discussed, with regard to the conjugation mechanism of the drug occurring in man.

Utilization of synthetic peptides for the study of calcitonin and biosynthetic precursors for calcitonin.

By using synthetic peptides and a library of monoclonal anti-peptide antibodies, we have developed a panel of techniques that allow the dissection of circulating immunoreactive calcitonin in the serum. C Cells of the thyroid were found to release both mature calcitonin and biosynthetic intermediates in the circulation. Finally, these products were found to circulate as heterogenous molecular species. A methodology for the standardization of the measurement of calcitonin is proposed in the form of a two-site immunoradiometric assay specific for mature calcitonin.

A radioimmunoassay for the antimalarial drug chloroquine.

We describe a 3H-based RIA for the antimalarial drug chloroquine (CLQ), the most commonly used antimalarial drug. In the assay a monoclonal antibody is used that is directed against 4-amino-7-chloroquinoline conjugated to keyhole limpet hemocyanin by the glutaraldehyde method. Besides CLQ, this antibody also recognizes with good affinity the 4-aminoquinoline homologs, hydroxychloroquine and amodiaquine. No extraction step or sample preparation is required, and the method can detect as little as 10 micrograms/L, the lower concentration in plasma of humans who are taking CLQ as a preventive measure. The between-assay CV is less than or equal to 10%, the within-assay CV less than or equal to 3%. Results correlate with those by liquid chromatography (r2 = 0.96). The speed and simplicity of the RIA method make it useful in evaluating the CLQ concentrations in acutely toxic patients.

Monoclonal antibodies to lipophilic and short-sized haptens: application to the 4-amino-quinoline antimalarial drugs.

Monoclonal antibodies recognizing the 4-amino-7-chloro-quinoline (ACQ) structure, which represents the backbone of the 4-amino-quinoline antimalarial drugs, were obtained in mice, after injection of ACQ coupled to hemocyanin via the glutaraldehyde method. The resulting antibodies show a definite specificity to this hapten, but react better with compounds substituted on the exocyclic amino group in 4. It is postulated that the quinoline ring is not sufficient for the reaction with the antibodies, and that an enlarged structure, which is given by the bridge used to link hapten and carrier, entails an important increase (1000-fold) in the apparent affinity. The striking similarities between this bridge and the lateral chains of the antimalarial drugs are accountable for this enhanced recognition. This result allows us to indicate that in some instances, the bridge-structure of the immunogen should be positively involved in the epitope. This observation may become useful in the conception of immunogens, aiming to obtain antibodies directed against some lipophilic and small-sized haptens.