Immunomodulation profile of the biosimilar trastuzumab MYL-1401O in a bioequivalence phase I study.

The initial Phase-I single centre, single dose, randomized, double-blind, cross-over study was planned to assess the pharmacokinetic and pharmacodynamic bioequivalence of the trastuzumab biosimilar (MYL-1401O) compared to the reference Herceptin®. Their respective immunomodulation profile presented in this paper involved healthy males receiving a single infusion of both monoclonals, separated by a washout period. Sixty parameters were assessed in total, including serum cytokines, peripheral mononuclear cell (PBMC) subsets, cell activation and response to recall antigens and mitogen, pre- and post- infusion, as well as a cytokine release assay (CRA) at baseline. Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6 h, and a modulation of mononuclear cell subset profile and activation level, notably CD16 + cells. Except for CD8 + T cells, there were no significant differences between Herceptin® and MYL-1401O. In CRA, PBMC stimulated with MYL-1401O or Herceptin® similarly secreted IL-6, TNF-α, IL-1ß, GM-CSF, IFN-γ, and IL-10, but no or low level of IL-2. Interestingly, some observed adverse events correlated with IL-2 and IFN-γ in CRA. MYL-1401O exhibited a very similar immunomodulation profile to Herceptin®, strongly supporting its bioequivalence. This approach may thus be included in a proof-of-concept study. CRA may be used as a predictive assay for the evaluation of clinical monoclonals.

Effect of Lactobacillus paracasei ST11 on a nasal provocation test with grass pollen in allergic rhinitis.

BACKGROUND: Probiotics have been associated with prevention and improvement of symptoms in atopic diseases such as atopic dermatitis. However, few studies exist that document their efficacy for upper airways allergies such as allergic rhinitis. OBJECTIVE: To investigate the effect of short-term oral administration of Lactobacillus paracasei ST11 on a nasal provocation test (NPT) with grass pollen. METHODS: Thirty-one adult volunteers with allergic rhinitis were enrolled in a randomized, double-blind, placebo-controlled study, based on two 4-week cross-over periods of product consumption (ST11-fermented milk vs. placebo), separated by a wash-out period of 6-8 weeks. Objective and subjective clinical parameters of NPT as well as systemic and nasal immunological parameters were compared between the two treatment periods (registration number: NCT 011 50 253). RESULTS: Subjects that received ST11-fermented milk had lower nasal congestion than subjects under placebo (visual analogical scale; P<0.05). Nasal pruritus followed the same trend. However, no significant change in combined nasal reaction threshold was observed between the two periods. IL-5 secretion by peripheral blood mononuclear cells and serum allergen-specific IgG4 were significantly lower in ST11-fermented milk group compared to placebo group. IL-8 and IL-10 secretion followed the same trend. CONCLUSION AND CLINICAL RELEVANCE: Short-term treatment with ST11-fermented milk before NPT significantly improved a clinical marker of NPT (subjective nasal congestion) and down-regulated systemic immune markers (IL-5 from peripheral blood mononuclear cells and serum IgG4). These data strongly suggest that probiotics may down modulate key parameters of allergic rhinitis and warrant future evaluation in seasonal trials.

Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

[Specific interferon-gamma assays: a modern tool for tuberculosis diagnosis]. / Diagnostic moderne de l'infection tuberculeuse: mesure de l'interféron-gamma.

Compared with the tuberculin skin test, the antigen-specific interferon-gamma assays, using a combination of two antigens ESAT-6 and CFP-10, has higher specificity for the diagnosis of latent tuberculosis, better correlation with exposure to M. tuberculosis, no cross-reactivity due to BCG vaccination and less towards nontuberculous mycobacterial infection. Fewer false positive results in uninfected persons avoid the costs of unnecessary therapy and its possible side effects. In low endemic areas, interferon-y assays are useful in addition of diagnostic algorithm for individuals with suspected tuberculosis. Further studies are required to evaluate the utility of the interferon-gamma assays in specialised subgroups of patients (immunocompromised, young children, patients with extrapulmonary disease,...) and as a marker of disease activity.

A synthetic malaria vaccine elicits a potent CD8(+) and CD4(+) T lymphocyte immune response in humans. Implications for vaccination strategies.

We report the first synthetic peptide vaccine eliciting strong CD8(+) and CD4(+) T lymphocyte responses in humans. The vaccine, representing the C-terminal region of the circumsporozoite protein of Plasmodium falciparum (amino acids 282-383) was well tolerated and strong sporozoite-specific antibodies were elicited. In addition, robust lymphocyte proliferation responses were equally elicited with concomitant in vitro production of IFN-gamma, crucial in the elimination of the parasite. Most importantly, we also observed the development of CD8(+) T lymphocyte responses decisive in the immunity to malaria. The latter finding opens new, possibly safer, avenues for vaccination strategies when a CD8(+) T cell response is needed.

Treatment of metastatic renal cell carcinoma with activated autologous macrophages and granulocyte--macrophage colony-stimulating factor.

Fifteen patients with progressive metastatic renal cell carcinoma were treated with granulocyte-macrophage colony-stimulating factor and intravenous infusions of activated autologous macrophages (AAMs). The latter were prepared from leukapheresis-separated mononuclear cells cultured in the presence of granulocyte-macrophage colony-stimulating factor, exposed to gamma interferon, and submitted to elutriation to separate AAMs. Three intravenous injections of AAMs were performed within a 2-week interval. This treatment cycle was repeated once or twice, in cases of tumor response or stabilization. Ninety-seven preparations containing a mean 3 x 10(9) AAMs were administered and usually well tolerated. One partial response, eight stabilizations and six progressions were observed. The median time to progression and median overall survival time after inclusion were 7 and 9 months, respectively. The cells injected did not accumulate substantially in tumor lesions, as shown by scintigraphic imaging of indium-111-labeled AAMs. Thus, combined granulocyte-macrophage colony-stimulating factor and AAM treatment was well tolerated and resulted in transitory stabilization (n = 8) or partial regression (n = 1) in 9 of 15 patients.

MHC class I- and class II-restricted processing and presentation of microencapsulated antigens.

Macrophages were found of having a strong capacity of phagocytosing small size microcapsules (MS) and presenting microencapsulated antigens to either CD4+ and CD8- T cells. The class I-restricted presentation of microencapsulated tetanus toxoid by macrophages requires an intracellular processing which might follow the phagosome-to-cytosol route to enter the classical MHC class I presentation pathway. In contrast, presentation of microencapsulated cytotoxic peptide PbCS252-260 to specific CD8+ T cells has been observed with different APC and is not blocked by cytochalasin D, suggesting that peptide released from MS may directly bind to MHC class I molecules on the cell surface. In the case of MHC class II-restricted T cells, prefixation or treatment of macrophages with chloroquine, brefeldin A and cycloheximide inhibits the presentation of microencapsulated and soluble tetanus toxoid. These findings illustrate the capacity of microencapsulated antigens to enter different presentation pathways and should facilitate the development of subunit vaccines.

Plasmodium falciparum CS C-terminal fragment: preclinical evaluation and phase I clinical studies.

Preclinical evaluation of synthetic peptides corresponding to the C-terminal regions of the circumsporozoite (CS) protein in various Plasmodia showed that these preparations were immunogenic and safe upon injection in various animal models. Additionally, the corresponding peptide from Plasmodium falciparum was widely recognized by sera and PBL obtained from semi-immune adults living in malaria endemic areas. Moreover, the CS C-terminal peptide derived from P. berghei conferred protection upon challenge with live sporozoites in mice. A GLP preparation of the synthetic peptide corresponding to residues 282-383 of the Pf CS, NF-54 strain is currently evaluated in a open, non-randomized, Phase I human trial. Data obtained after the second antigen injection show that the malaria vaccine Pf CS 282-383 is safe, well tolerated and gives rise to high antibody titre, CD4+ and CD8+ lymphocyte responses.

Improving stability and release kinetics of microencapsulated tetanus toxoid by co-encapsulation of additives.

PURPOSE: Tetanus toxoid (Ttxd) encapsulated in polyester microspheres (MS) for single injection immunization have so far given pulsatile in vitro release and strong immune response in animals, but no boosting effect. This has been ascribed to insufficient toxoid stability within the MS exposed to in vivo conditions over a prolonged time period. This study examined the effect of co-encapsulated putative stabilizing additives. METHODS: Two different Ttxd were encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA 50:50) and poly(D,L-lactic acid) (PLA) MS by spray-drying. The influence of co-encapsulated additives on toxoid stability, loading in and release from the MS, was studied by fluorimetry and ELISA. RESULTS: Co-encapsulated albumin, trehalose and gamma-hydroxypropyl cyclodextrin all improved the toxoid encapsulation efficiency in PLGA 50:50 MS. Albumin increased the encapsulation efficiency of antigenic Ttxd by one to two orders of magnitude. Further, with albumin or a mixture of albumin and trehalose ELISA responsive Ttxd was released over 1-2 months following a pulsatile pattern. CONCLUSIONS: Optimized Ttxd containing MS may be valuable for a single-dose vaccine delivery system.

Enhanced immunogenicity of microencapsulated tetanus toxoid with stabilizing agents.

PURPOSE: Antigenic proteins encapsulated in biodegradable polyester microspheres (MS) can slowly denature or aggregate, which results in decreased antigenicity. In this study, we have evaluated the ability of co-encapsulated additives to protect against the loss of tetanus toxoid (TT) antigenicity. METHODS: Antibody responses were analyzed after immunization of mice with TT microencapsulated in the presence of additives (TT-MS-additive). RESULTS: Immunization with TT-MS-additives gave rise to higher responses than those obtained in the absence of additive. BSA, trehalose. Gamma-hydroxypropylcyclodextrin and calcium salts preserved the immunogenicity of the incorporated antigen with the highest efficacy. Sustained responses were obtained with mixtures of fast and slowly releasing TT-MS containing BSA plus trehalose or calcium salts. CONCLUSIONS: The selected additives may stabilize the antigen in MS during storage and rehydration in body fluids. Regulated antigen release from MS-based vaccines permits a reduction of the antigen dose and optimization of single-dose vaccine formulations.

Induction of a cytotoxic T lymphocyte response by immunization with a malaria specific CTL peptide entrapped in biodegradable polymer microspheres.

We have previously reported that biodegradable polymer microspheres (MS) are capable of eliciting strong and long-lasting antibody and T cell proliferative responses for either natural protein antigens or synthetic peptides. In this study, we investigated the possibility of inducing antigen-specific cytotoxic T lymphocyte (CTL) responses in vivo with a short synthetic peptide from the circumsporozoite (CS) protein of Plasmodium berghei (Pb) 252-260 by using different MS formulations. We show that injection of mice with a short CTL epitope microencapsulated in MS or adsorbed on empty MS enhanced a specific CTL response comparable to that obtained with the incomplete Freund's adjuvant (IFA) formulation, indicating that MS are a potent antigen delivery system/immunostimulant for CTL response. These results might be of practical interest for MS preparation and development of subunit vaccines.

Adhesion molecule expression and response to chemotactic agents of human monocyte-derived macrophages.

Human monocyte-derived macrophages have been proposed as agents of anti-tumour immunotherapy. The aim of the present study was to investigate in vitro the properties of these cells likely to control their recruitment to the sites of inflammation and tumours. The expression of adhesion molecules involved in the binding of monocytes to endothelial cells was modified during monocyte-macrophage differentiation, with a significant increase in CD11c, CD14 and intercellular adhesion molecule-1 (ICAM-1). Monocyte-derived macrophages were sensitive to chemoattractants, in particular to the monocyte-specific chemokine monocyte chemotactic protein-1 (MCP-1). They responded by an increased expression of adhesion molecules and were attracted by the cytokine in an under-agarose migration assay. The migration response, however, decreased after days 4-5 of monocyte differentiation into macrophage. In conclusion, human monocyte-derived macrophages show alterations of surface structures involved in the recognition of inflammatory endothelium. This may explain why the cells are poorly recruited to the sites of inflammation and tumours when introduced into the circulation.

Internalization of human macrophage surface antigens induced by monoclonal antibodies.

Drugs intended to be endocytosed by macrophages may be transported by MAbs directed against these cells. Twenty MAbs were investigated for this purpose. The binding of these MAbs to macrophages obtained from a 7 day culture of blood monocytes showed that anti-CD11b and anti-CD14 recognized the highest number of cell surface antigen sites. Further assays determined that anti-CD63, Mo5 and anti-CD33 were the MAbs that induced the strongest modulation of the corresponding antigens, the highest rate being with anti-CD63. Endocytosis of antigen-antibody complexes was evidenced by the presence of MAbs in the cytoplasm. Anti-CD63 MAbs induced the highest internalization in this assay. For most MAbs, however, the density of antigen sites and the intensity of antigen modulation were not predictive of the amount of MAb detected in the cytoplasm.

Fate of mouse macrophages radiolabelled with PKH-95 and injected intravenously.

Mouse macrophages purified by elutriation from thioglycollate-induced peritoneal exudate cells were labelled with indium-111-oxine and injected intravenously into mice. A substantial amount of unbound radioactivity remained in the circulation, suggesting that the radionuclide was not stably bound to the cells. Culture experiments with radiolabelled cells showed that indium-111 was released in the medium. Another cell marker, PKH-95, an iodine-125-labelled aliphatic compound insertable into the cell membrane, bound more stably than indium-111. Five minutes after injection of 125I-PKH-95-labelled macrophages, about 98% of the cells were in a non-circulating pool. It was checked that PKH-95 labelling did not compromise the viability and functions of the macrophages and that autologous erythrocytes and blood mononuclear cells labelled with PKH-95 remained in the circulation after i.v. injection. One hour after injection, 125I-PKH-95-labelled macrophages were distributed mainly in lung (36%), liver (19%) and spleen (5%). Subsequently, radioactivity decreased in the lung while increasing in liver, spleen and in an artificially induced footpad inflammation. The radioactivity accumulation in the inflammation persisted at least for 7 days. It represented a small proportion of radioactivity injected (0.2%) but was trapped very specifically in the inflammation. This raised the hypothesis that macrophages of the non-circulating pool could be released in the circulation and recruited into the inflammation with slow kinetics.

Interactions between human macrophages and tumor cells in three-dimensional cultures.

Human blood mononuclear cells were cultured for 7 days in hydrophobic plastic bags. Macrophages differentiated from monocytes and purified by elutriation were then cocultured with round-shaped aggregates of epithelial cells (spheroids). Spheroids prepared from the SK-MES-1 carcinoma cell line were cultured individually, under constant stirring, in multiwell plates coated with agarose. Macrophage/spheroid interactions were investigated under various experimental conditions. Macrophages activated with interferon gamma aggregated to each other and to spheroids, in contrast to control unactivated macrophages. Histological examination, after staining with a macrophage-specific monoclonal antibody, showed that both control and interferon-gamma-activated macrophages migrated between epithelial tumor cells and infiltrated the spheroids. The addition of anti-ICAM-1 monoclonal antibody inhibited macrophage homotypic aggregation as well as aggregation to and penetration into spheroids. The macrophages did not exert cytolytic effects, as judged by a chromium-51 release assay, but provoked a diminution of tritiated thymidine incorporation by tumor cells. Cytostatic activity was observed with effector: target ratios as low as 1:16, and was maximal (99% at a 1:1 E:T ratio) with macrophages differentiated in the presence of granulocyte/macrophage-colony-stimulating factor. The cytostatic effect was not related to tumor necrosis factor alpha secretion.

Monoclonal antibody AMH152 reacts with human monocytes in culture and with inflammatory macrophages.

Monoclonal antibodies (mAb) raised against human peritoneal macrophages were selected for their non-reactivity with freshly sampled blood cells. One of these mAb, AMH152, initially non-reactive, bound to monocytes after 18 h of culture, a property which was not shared by an unrelated antibody of the same isotype (IgG1). The induction of the expression of the antigen detected by AMH152 on monocytes in culture was not influenced by the addition of serum or by the substrate used, plastic that favoured adhesion or teflon bags. Overnight incubation at 4 degrees C in adhesion conditions did not enable antigen expression. A 1-h treatment with phorbol myristate acetate or formyl-methionyl-leucyl-phenylalanine did not increase AMH152 binding. Culturing monocytes with cycloheximide tended to inhibit antigen expression. These observations suggested that antigen expression represents an active phenomenon, requiring protein synthesis. The antigen recognized by mAb AMH152 could be visualized on sections of formalin-fixed and paraffin-embedded tissues. Macrophages of healthy lymphoid organs and tissues that expressed CD68 antigen failed to bind AMH152. In contrast, chronic inflammatory lesions, like those of sarcoidosis, tuberculosis and cat scratch disease, contained epithelioid and multinucleated giant cells that reacted with AMH152. In serous exudates of cancer metastases, 10-40% of macrophages were also stained. The antigenic material was essentially present at the cell periphery. Thus, mAb AMH152 recognized a surface antigen, detectable on paraffin-embedded tissue sections, and which accompanied differentiation of monocytes into inflammatory cells. The expression of this antigen on monocytes in culture suggests that these cells underwent an activation process, even when maintained for some hours in teflon bags and in a serum-free medium.

Some proteins not belonging to the clotting or to the kallikrein-kinin system altered by kallikrein.

Plasma kallikrein activation occurs frequently during blood drawing and subsequent plasma handling. The purified enzyme was incubated with ceruloplasmin, inter-alpha-trypsin inhibitor and complement factor C4. Proteolysis caused by this enzyme was compared with the degradative effects of plasmin and thrombin. Among these proteins C4 proved to be most easily degraded; its cleavage products can interact with C4-binding protein.

[Proteolytic breakdown of human inter-alpha-trypsin inhibitor by plasmin (author's transl)]. / Etude des fragments de l'inter-alpha-trypsine inhibiteur obtenus par digestion plasminique.

Inter-alpha-trypsin inhibitor was isolated from human plasma and submitted to proteolytic degradation by plasmin. A split product of low molecular weight (18 000 daltons) is obtained by gel filtration or solubilisation in perchloric acid. This fragment reacts with an anti-inter-alpha-trypsin inhibitor immune serum and migrates as beta1 globulins. Its specific activity against trypsin (after absorption of residual plasmin on sepharose lysine) was estimated to be 900 mU1/mg. Thus one molecule of fragment can inhibit one molecule of trypsin. As well with native protein as with its fragment, complexes formed with trypsin can be dissociated by urea or sodium dodecyl sulfate. This fragment is similar to the small molecular weight inhibitors obtained directly by solubilisation in perchloric acid from serum, urine and bronchial secretions.

Distribution of IgG subclasses in commercial and some experimental gamma-globulin preparations.

The IgG subclass distribution was determined in six commercial and in four experimental human gamma-globulin preparations. The concentrations of IgG subclasses were measured in a modified radiommunoassay using subclass-specific antisera. In commercial gamma-globulins, the distribution of the subclasses corresponded roughly to the distribution in normal human serum. A considerable enrichment of the IgG 4 was found in experimental lots prepared either from the ethanol fraction III or from the rivanol-precipitable IgG.

[Preparation of immunoglobulins G,A,M (IgGAM) for therapeutic use. Conditions for enrichment in IgA or in IgM]. / Obtention d'une préparation d'immunoglobulines G,A,M (IgGAM) à usage thérapeutique. Etude des conditions d'enrichissement en IgA ou en IgM

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...