Large-scale Isolation of Highly Pure "Untouched" Regulatory T Cells in a GMP Environment for Adoptive Cell Therapy.

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.

A novel human anti-interleukin-1ß neutralizing monoclonal antibody showing in vivo efficacy.

The pro-inflammatory cytokine interleukin (IL)-1ß is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1ß have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1ß with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a>30-fold increased affinity to human IL-1ß compared with its parent antibody. This anti-human IL-1ß IgG also cross-reacts with mouse and monkey IL-1ß, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1ß pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1ß monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.

Use of human monoclonal antibodies to treat Chikungunya virus infection.

Chikungunya virus (CHIKV) is an alphavirus prevalent in tropical regions. It causes an acute febrile disease that, in elderly individuals and newborns, is often associated with severe complications. We previously reported the isolation and characterization of 2 human monoclonal antibodies neutralizing CHIKV in vitro: 5F10 and 8B10. Here, we tested their efficacy in vivo as prophylactic and therapeutic treatments of CHIKV infection in AGR129 mice. In both settings, 5F10 and 8B10 were able to significantly delay CHIKV-driven lethality. Our results support the development of prophylactic and therapeutic treatments for CHIKV infection, using a combination of 5F10 and 8B10.

Chikungunya virus envelope-specific human monoclonal antibodies with broad neutralization potency.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC(50) of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O'nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host-pathogen interactions and the rational design of vaccines against CHIKV.

Interleukin-1ß produced in response to islet autoantigen presentation differentiates T-helper 17 cells at the expense of regulatory T-cells: Implications for the timing of tolerizing immunotherapy.

OBJECTIVE: The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelB(lo) DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1ß is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1ß production and the response to RelB(lo) DCs in the prediabetic period. RESEARCH DESIGN AND METHODS: We injected RelB(lo) DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro. RESULTS: Tolerizing RelB(lo) DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1ß production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion. CONCLUSIONS: IL-1ß, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1ß/IL-17 checkpoint signals the need for other strategies.

Improvement of collagen-induced arthritis by active immunization against murine IL-1beta peptides designed by molecular modelling.

Interleukin-1beta (IL-1beta) is a crucial cytokine in inflammation processes and has been implicated in the pathogenesis of several chronic inflammatory diseases. Strategies designed to blocking IL-1beta by passive administration of inhibitors (mAbs, IL-1 receptor antagonist) have previously demonstrated efficacy in rheumatoid arthritis (RA). Using molecular modelling, we have defined three murine IL-1beta peptide regions characterized by their close proximity to the receptor. Synthetic peptides corresponding to these regions, in cyclic and linear form, were delivered as immunogens in Swiss mice, resulting in significant levels of autoantibodies directed against the native murine IL-1beta cytokine as determined by ELISA and by an assay for neutralization of IL-1beta biological activity. More importantly, one of the cyclic peptides showed a protective effect against inflammation and articular destruction in DBA/1 mouse collagen-induced arthritis, a model of RA. The high rate of success observed for active immunization against cytokine peptides in vivo suggests that the in silico approach to autoantigen design may be a promising avenue for the development of anti-cytokine immunotherapeutics.

Dominant effects of CCR2-CCR5 haplotypes in HIV-1 disease progression.

Three haplotypes for the CCR2-CCR5 region previously have been shown to affect AIDS progression; however, it is not known if the protective and accelerating effects of the haplotypes are relatively constant throughout infection or exert their effects early or late in HIV type 1 infection. The authors report the relative contributions to AIDS progression of CCR2 64I, CCR5 Delta32, and the CCR5 promoter haplotype +.P1.+ in the GRIV cohort, which included patients representing the extremes of the distribution for AIDS progression: rapid progressors (RP) who developed CD4 T-cell counts of <300/ mm within 3 years after the last HIV-1-seronegative test and slow progressors (SP) who were HIV-1 infected for > or =8 years with CD4 T-cell counts of >500/mm. Comparing the RP with a seroconverter control group including intermediate progressors to AIDS, we observed the early protective effect of CCR5 Delta32 (odds ratio = 0.25; P = 0.007) was similar in strength to the early susceptible effect of CCR5 +.P1.+ (odds ratio = 2.1, P = 0.01). Comparison of the intermediate control group to the SP showed weaker and less significant odd ratios, suggesting that the effect of these factors tended to be stronger on early progression; the tendency towards a disproportionately early effect was significant for CCR5 Delta32 (P = 0.04) but not for CCR5 +.P1.+ (P = 0.12). Follow-up of SP demonstrated that these polymorphisms have little effect after 8 years, because the subset of SP who had progression after study entry had the same genotype distribution as the global population of SP, suggesting that factors other than CCR5 or CCR2 genetic variants must be responsible for the long-term maintenance of nonprogression.

Associations of MHC ancestral haplotypes with resistance/susceptibility to AIDS disease development.

We tested the association of MHC ancestral haplotypes with rapid or slow progression to AIDS by comparing their frequencies in the French genetics of resistance/susceptibility to immunodeficiency virus cohort with that reported in a control French population. Seven ancestral haplotypes were identified in the genetics of resistance/susceptibility to immunodeficiency virus cohort with a frequency >1%. The 8.1 (odds ratio (OR) = 3, p = 0.006), 35.1 (OR = 5.7, p = 0.001), and 44.2 (OR = 3.4, p = 0.007) ancestral haplotypes were associated with rapid progression, whereas the 35.2 (OR = 3.6, p = 0.001), 44.1 (OR = 5.4, p < 10(-4)), and 57.1 (OR = 5.8, p < 10(-4)) ancestral haplotypes were associated with slow progression to AIDS. Although the frequency of each ancestral haplotype is low in the population, the OR were quite higher than those previously obtained for single HLA allele associations, with some p values as low as 10(-4). The analysis of the recombinant fragments of these haplotypes allowed the identification of the MHC regions in the 35.1, 35.2, and 44.2 haplotypes associated with rapid progression to AIDS and the MHC regions of the 44.1 and 57.1 haplotypes associated with slow progression to AIDS. Previous studies have identified single HLA alleles associated with disease progression. Our results on recombinant fragments confirm the direct role of HLA-B35 in rapid progression. Associations with HLA-A29 and -B57 might be due to linkage disequilibrium with other causative genes within the MHC region.