Nonclinical Safety Assessment of CFZ533, a Fc-Silent Anti-CD40 Antibody, in Cynomolgus Monkeys.

CFZ533 is a pathway blocking, nondepleting anti-CD40 antibody that is in clinical development for inhibition of transplant organ rejection and therapy for autoimmune diseases. A 26-week GLP toxicity study in sexually mature Cynomolgus monkeys was conducted in order to support chronic application of CFZ533. CFZ533 was subcutaneously administered at doses up to 150 mg/kg/week and was safe and generally well tolerated. CFZ533 showed no adverse effects for cardiovascular, respiratory, and neurobehavioral endpoints, and no changes were observed for blood lymphocyte and platelet counts or blood coagulation markers. In line with the nondepleting nature of CFZ533, CD20+ B cells in the blood were only marginally reduced. A complete suppression of germinal center (GC) development in lymph nodes and spleen was the most prominent result of post-mortem histological investigations. This was corroborated by an abrogated T-dependent antibody response (TDAR) to the antigen Keyhole Limpet Hemocyanin (KLH) as well as an absence of anti-drug antibodies (ADAs) in the absence of B cell depletion as seen with immunophenotyping and histology. When serum levels of CFZ533 in recovery animals dropped levels necessary for full CD40 occupancy on B cells, all animals were able to mount a TDAR to KLH. All histological changes also reverted to normal appearance after recovery. In summary, CFZ533 was shown to be well tolerated and safe in the 26-week toxicity study with a distinct pharmacodynamic profile in histology and immune function.

Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity.

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly with DC matured with pro-inflammatory cytokines and prostaglandin E2. We studied the safety and efficacy of vaccination with monocyte-derived DC matured with a cocktail of prophylactic vaccines that contain clinical-grade Toll-like receptor ligands (BCG, Typhim, Act-HIB) and prostaglandin E2 (VAC-DC). Stage III and IV melanoma patients were vaccinated via intranodal injection (12 patients) or combined intradermal/intravenous injection (16 patients) with VAC-DC loaded with keyhole limpet hemocyanin (KLH) and mRNA encoding tumor antigens gp100 and tyrosinase. Tumor antigen-specific T cell responses were monitored in blood and skin-test infiltrating-lymphocyte cultures. Almost all patients mounted prophylactic vaccine- or KLH-specific immune responses. Both after intranodal injection and after intradermal/intravenous injection, tumor antigen-specific immune responses were detected, which coincide with longer overall survival in stage IV melanoma patients. VAC-DC induce local and systemic CTC grade 2 and 3 toxicity, which is most likely caused by BCG in the maturation cocktail. The side effects were self-limiting or resolved upon a short period of systemic steroid therapy. We conclude that VAC-DC can induce functional tumor-specific responses. Unfortunately, toxicity observed after vaccination precludes the general application of VAC-DC, since in DC maturated with prophylactic vaccines BCG appears to be essential in the maturation cocktail.

Problemas bioéticos en la investigación de nuevas vacunas: ¿obedecen a razones de salud pública? / Bioethical problems in researching new vaccines: Do they respond to public health reasons?

Los problemas éticos de las investigaciones sobre vacunas han crecido en las últimas décadas en frecuencia y magnitud debido a la posición dominante de la industria farmacéutica en el desarrollo de esos estudios. Las tradicionales cuestiones de seguridad y eficacia se han visto agravadas por el conflicto de intereses introducido por la competencia comercial en un mercado a escala global de miles de millones de dólares. La integridad profesional de los investigadores, la responsabilidad moral de los patrocinadores, y la regulación y control por parte de los Estados nacionales, se muestra cuestionada en varios ejemplos. Los resultados de estos cambios son las amenazas a la protección de los derechos de las personas incluidas en estas investigaciones y el discutible progreso que resulta para la salud pública.

The ethical problems in vaccine research have grown in frequency and magnitude in last decades, due to the dominant place of the pharmaceutical industry in the development of such studies. Traditional issues of security and efficacy have been aggravated by the conflicts of interests introduced by commercial competition in a global market worth billions of dollars. We present here a few examples in which the professional integrity of researchers, the moral responsibility of sponsors, and the public regulation and control by national States are put into question. The consequences of these changes represent serious threats to the rights of people included in these studies as well as disputable progress for public health.

Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide.

To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, a synthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugated to GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximal conjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultant pFMDV-GNP conjugates were then injected into BALB/c mice. Immunization with pFMDV-keyhole limpet hemocyanin (pFMDV-KLH) conjugate was also performed as a control. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titers against both pFMDV and the carriers were measured. For the pFMDV-GNP immunization, specific antibodies against the synthetic peptide were detected in the sera of mice injected with 2, 5, 8, 12, and 17 nm pFMDV-GNP conjugates. Maximal antibody binding was noted for GNPs of diameter 8-17 nm. The pFMDV-GNPs induced a three-fold increase in the antibody response compared to the response to pFMDV-KLH. However, sera from either immunized mouse group did not exhibit an antibody response to GNPs, while the sera from pFMDV-KLH-immunized mice presented high levels of binding activity against KLH. Additionally, the uptake of pFMDV-GNP in the spleen was examined by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). The quantity of GNPs that accumulated in the spleen correlated to the magnitude of the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. Furthermore, we established that GNPs ranging from 8 to 17 nm in diameter may be ideal for eliciting a focused antibody response against a synthetic pFMDV peptide.

Female mice respond differently to costly foraging versus food restriction.

Experimental manipulation of foraging costs per food reward can be used to study the plasticity of physiological systems involved in energy metabolism. This approach is useful for understanding adaptations to natural variation in food availability. Earlier studies have shown that animals foraging on a fixed reward schedule decrease energy intake and expenditure. However, the extent to which these changes depend on decreased food intake or increased foraging costs per se has never been tested. We manipulated foraging costs per food reward in female Hsd:ICR(CD-1) laboratory mice, comparing animals faced with low (L) and high (H) foraging costs to non-foraging animals receiving a food restriction (R) matched to the intake of H animals. Mice in the H group ran as much as L mice did but ate significantly less. They concurrently reduced daily energy expenditure and resting metabolic rate, decreased the size of major metabolic organs and utilized body fat stores; mass-specific resting metabolic rate did not differ between groups. We found evidence that these alterations in energy balance may carry fitness costs. As a secondary response to our experimental treatment, H females and, eventually, some R females ceased to show signs of estrous cyclicity. Surprisingly, results of an immune challenge with keyhole limpet hemocyanin showed that primary immune response did not differ between L and H groups, and was actually higher in R mice. Our results demonstrate that high foraging costs per se--the combination of high activity and low food intake--have pronounced physiological effects in female mice.

Assessment of the immunocontraceptive effect of a zona pellucida 3 peptide antigen in wild mice.

Immunizing laboratory mice against a short peptide to mouse zona pellucida protein 3 (mZP3; amino acids 328-342) reduces fertility in some strains. This antigen was therefore tested to see if it is suitable for use in an immunocontraceptive vaccine to control wild mice. Mouse zona pellucida protein 3 peptide conjugated to a carrier protein (keyhole limpet hemocyanin) was considerably more immunogenic and effective in reducing fertility in wild mice when compared with inbred BALB/c mice. Fertility of the immunized wild mice was reduced by over 50% compared with controls, whereas BALB/c mice showed no reduction. Variation in the responses between individual animals to mZP3 peptide was observed and infertility correlated to the presence of cross-reacting antibodies to native zona pellucida in wild, but not BALB/c, mice.

Development of immunoassays for biomonitoring of hexamethylene diisocyanate exposure.

Hexamethylene diisocyanate (HDI) is used widely to manufacture polyurethanes for paints and coatings. It is an irritant and a chemical asthmagen. The U.S. Occupational Safety and Health Administration time-weighted average permissible exposure limit is 5 ppb and the ceiling limit is 20 ppb. We sought to develop a sensitive and specific immuno-bioassay to supplement workplace air monitoring and detect recent HDI exposure. For this, we produced rabbit antiserum to HDI-adducted keyhole limpet hemocyanin (HDI-KLH). The specificity of the antiserum was demonstrated by its reaction with a variety of HDI-conjugated proteins and the absence of reactions with conjugates of other diisocyanates, namely toluene diisocyanate and diphenyl methylene diisocyanate. Four immunoassays were developed and compared for their ability to detect decreasing quantities of HDI-adducted human serum albumin (HSA) containing 2 mol HDI adduct per mol HSA (HDI(2)-HSA) as determined by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. The sensitivities of some of the assays are within the range (0.82-45 nM) of current analytic methods. A Western analysis procedure has a sensitivity of 600 nM HDI adduct on HSA. ELISA inhibition assay, in which microtiter plates are coated with the HDI(2)-HSA antigen, has a sensitivity of 300 nM HDI adduct. An immunoblot assay has a sensitivity of 9 nM HDI adduct. The most sensitive bioassay (1.8 nM HDI adduct) is a three-antibody sandwich ELISA in which wells of microtiter plates are coated with the IgG fraction of the anti-HDI-KLH antisera. Compared with analytic methods for HDI biomonitoring, the immunoassays are faster and less costly and accommodate numerous samples simultaneously. The assays have the potential to affect industrial biomonitoring programs significantly.

Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines.

Dendritic cells (DCs) are potent antigen-presenting cells and play a pivotal role in T cell-mediated immunity. DCs have been shown to induce strong antitumor immune responses in vitro and in vivo, and their efficacy is being investigated in clinical trials. Compared with vaccination strategies directed against a single tumor antigen, tumor-cell lysate as the source of antigen offers the potential advantage of inducing a broad T-cell response against multiple known, as well as unknown, tumor-associated antigens expressed by the individual tumor. We used pancreatic carcinoma cell lines to develop an in vitro model for monitoring T-cell responses induced by lysate-pulsed DCs. Monocyte-derived DCs of HLA-A2(+) donors were pulsed with lysate generated from the HLA-A2(+) pancreatic carcinoma cell line Panc-1. In some experiments, the immunogenic protein keyhole limpet hemocyanin (KLH) was added to the lysate. Subsequently, the antigen-loaded DCs were activated with tumor necrosis factor-alpha and prostaglandin E(2). Autologous mononuclear cells were cocultured with DCs in the presence of low-dose interleukin (IL)-2 and IL-7 and were restimulated weekly with new DCs. High levels of IL-12 and IFN-gamma could be detected in the supernatants, indicating a T-helper type 1-type immune response. This cytokine profile was associated with the expression of the activation marker CD69 on both T helper and CTLs and with an antigen-induced proliferative T-cell response. After 4 weeks, CTL-mediated cytotoxicity was assessed. Tumor cell lysis was specific for Panc-1 tumor cells and was MHC class I-restricted. Cytokine secretion, CD69 expression of T cells, and antigen-induced T-cell proliferation correlated with the cytotoxic activity and were more pronounced when KLH was added to the lysate. This is the first study to show that T cells specific for pancreatic carcinoma cells can be generated in vitro by lysate-pulsed DCs and that the T-cell response can be enhanced by KLH. This in vitro model can be applied to compare different strategies in the development of DC-based tumor vaccines.

Metabolic costs of mounting an antigen-stimulated immune response in adult and aged C57BL/6J mice.

Animals must balance their energy budget despite seasonal changes in both energy availability and physiological expenditures. Immunity, in addition to growth, thermoregulation, and cellular maintenance, requires substantial energy to maintain function, although few studies have directly tested the energetic cost of immunity. The present study assessed the metabolic costs of an antibody response. Adult and aged male C5BL/6J mice were implanted with either empty Silastic capsules or capsules filled with melatonin and injected with either saline or keyhole limpet hemocyanin (KLH). O2 consumption was monitored periodically throughout antibody production using indirect calorimetry. KLH-injected mice mounted significant immunoglobulin G (IgG) responses and consumed more O2 compared with animals injected with saline. Melatonin treatment increased O2 consumption in mice injected with saline but suppressed the increased metabolic rate associated with an immune response in KLH-injected animals. Melatonin had no effect on immune response to KLH. Adult and aged mice did not differ in antibody response or metabolic activity. Aged mice appear unable to maintain sufficient heat production despite comparable O2 production to adult mice. These results suggest that mounting an immune response requires significant energy and therefore requires using resources that could otherwise be allocated to other physiological processes. Energetic trade-offs are likely when energy demands are high (e.g., during winter, pregnancy, or lactation). Melatonin appears to play an adaptive role in coordinating reproductive, immunologic, and energetic processes.

Assessment of humoral immune response in mink (Mustela vison): antibody production and detection.

A method for investigating the humoral immune response in mink (Mustela vison) was developed between October 1993 and March 1994. Protein A, 1:8000 dilution, had a high affinity for mink immunoglobulin, while anti-ferret (Mustela putorius) antibody, 1:200 dilution, had a weaker affinity. Four adult mink were immunized with a hapten, dinitrophenol (DNP), conjugated to a large carrier protein, keyhole limpet hemocyanin (KLH), and received two boosters at 3-week intervals. This provoked a strong T-lymphocyte dependent humoral immune response. An indirect enzyme linked immunosorbent assay (ELISA) was used to quantify the antibody produced. All mink had undetectable anti-DNP-KLH antibody in the pre-immune sera, with antibody levels increasing post-immunization, and peaking after the first or second booster.

Assessment of a functional role of auto-anti-idiotypes in idiotype dominance.

We have used a well-defined idiotypic system, the cross-reactive idiotype of A strain (CRIA) (Ab1) idiotype generated in A/J mice injected with arsonate coupled to keyhole limpet hemocyanin (ARS-KLH), to determine the frequency of precursors for auto-anti-idiotypic antibodies (auto-Ab2) in naive and immunized A/J mice by limiting dilution analysis after polyclonal activation by lipopolysaccharide. In naive animals, the precursor frequencies of auto-Ab2 B cells were below the limit of sensitivity of the technique in the majority of A/J mice, and could be detected in only 20% of the animals. Upon immunization with ARS-KLH, a large increase in auto-Ab2 precursor frequency was observed. This shift in frequency was not found when A/J mice were injected with KLH alone, or when BALB/c mice, which do not express the CRIA idiotype, were injected with ARS-KLH. To study the functional role of the auto-Ab2 B cells, we injected neonatal A/J mice with polyclonal rabbit Ab3 antibodies directed against a recurrent idiotype of auto-Ab2. Thereafter, these mice were injected with ARS-KLH. Although the anti-arsonate response level was normal, the CRIA Ab1 expression was reduced tenfold. Thus, the suppression of auto-Ab2 affects Ab1 dominance. We further show that the presence of maternal Ab1 can strongly modify the immune response of the offspring by inducing higher levels of the idiotype after immunization. Furthermore, IgM anti-arsonate antibodies were detected before immunization with antigen. From these data, we conclude that the affinity of antigen alone cannot explain the dominance of CRIA. Network selection is important in the shaping of the available repertoire.

An in vitro micromethod for the quantitative assessment of central demyelination.

We report the development of a simple and reliable method for the study of demyelination in vitro based on the measurement of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. Using only small quantities of myelin (equivalent to 100 micrograms of myelin protein) the system was tested under conditions that are believed to approximate those found at the site of an inflammatory demyelinating lesion. Treatment with a combination of trypsin, phospholipase A2, and lysophosphatidylcholine was used to evaluate the method. This microsystem has the potential not only for testing the myelinotoxicity of soluble factors but also for investigating the involvement of inflammatory cells in the demyelinating process. Myelin degradation by elicited peritoneal macrophages could be demonstrated at relatively high densities of these cells. Nylon wool purified lymph node T cells from myelin basic protein-primed SJL/J mice, after selective expansion with antigen and interleukin 2, failed to induce any significant myelin breakdown unless a limited number of syngeneic activated macrophages were also present. T cells from mice that had been inoculated with keyhole limpet haemocyanin failed to show any effect. The advantages of this technique over other in vitro systems are that it enables the study of demyelination using syngeneic sources of myelin and defined cell populations.