Therapeutic control of B cell activation via recruitment of Fcγ receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold

Objective

To exploit the physiologic Fcγ receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual‐affinity retargeting (DART) molecule, for therapeutic applications.

Methods

DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the β‐chain of the invariant signal‐transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen‐induced arthritis (CIA) model.

Results

DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B‐mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice.

Conclusion

This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.

     

“DO ONE,GET TWO”: dual venous drainage of the radial forearm free flap by a single venous anastomosis

Oral and Maxillofacial Surgery - The radial forearm free flap (RFFF) remains a workhorse in microsurgical reconstruction. Its failure is primarily due to problems with venous drainage; for this...     

Risk factors for cryptogenic and idiopathic partial epilepsy: a community-based case-control study in Copparo,Italy

OBJECTIVES: The etiology of epilepsy remains unknown in most cases. We sought to investigate the role of some pre-, peri- and postnatal factors in the etiology of idiopathic and cryptogenetic partial epilepsy. METHODS: We carried out a community-based case-control study using the incidence cohort of epileptic patients living in the district of Copparo, in the province of Ferrara, Italy. The study was performed in 55 cases and 165 controls. A standardized questionnaire was used to collect information in face-to-face interviews. RESULTS: Multivariate logistic regression analysis found that a personal history of febrile convulsions [odds ratio (OR) = 4.01, 95% confidence interval (CI) = 1.3-19.1] and a family history of seizures in first-degree relatives (OR = 4.5, 95% CI = 1.8-18.6) were independent risk factors for the condition under study. We failed to demonstrate an association between partial epilepsy and previously suggested perinatal risk factors. CONCLUSION: The findings of this study further support the hypothesis of a genetic propensity for seizures, which may be expressed early by the occurrence of febrile convulsions.     

Identification of a novel DNase I hypersensitive site within the far upstream region of the human HLA-DRA gene

The class II products of the major histocompatibility complex have a distribution restricted to certain tissues and cells. For instance, they are constitutively expressed by B lymphocytes, but not by resting T lymphocytes. In this study, we report the identification of a novel DNase I hypersensitive site within a putative regulatory region of the human HLA-DRA gene, the so-called far upstream region. This hypersensitive site was present in the genome of the DRalpha-positive human B-lymphoid Raji cell line, and absent in the DRalpha-negative T-lymphoid Jurkat cell line. In addition, this hypersensitive site was also present in transgenic B lymphocytes isolated from the murine transgenic line TG 53, carrying a single integrated copy of the human HLA-DRA gene per haploid genome. The correlation between DRA expression and the presence of this far upstream hypersensitive site suggests novel long distance chromatin remodeling mechanisms possibly shared by human and murine class II genes.     

Monoclonal antibody 76F distinguishes IA-2 from IA-2beta and overlaps an autoantibody epitope

IA-2 and IA-2beta are highly related proteins that are autoantigens in type 1 diabetes, and provide a model for developing reagents and assays that distinguish similar proteins with unique autoantibody epitopes. Monoclonal antibodies (mAb) to IA-2 and IA-2beta were prepared and tested for their ability to bind to the related proteins and their ability to compete for specific autoantibody epitope binding by sera from patients with type 1 diabetes. Monoclonal antibodies that specifically bound IA-2 (76F) or bound both IA-2 and IA-2beta (A9) were isolated and characterized. 76F mAb recognized IA-2 of human, rat and mouse origin in native and denatured forms and had an epitope specificity for residues 626-630 (FEYQD) which are found in the juxtamembrane (JM) region of human and mouse IA-2, but not IA-2beta. This region overlaps with the autoantibody epitope JM2. Binding to the 76F monoclonal antibody was specifically inhibited by sera with antibodies to the JM2 epitope but not with antibodies to the adjacent JM1 epitope, indicating that unique epitopes can be distinguished by this approach. 76F mAb has the unique property to distinguish between the two closely related autoantigens IA-2 and IA-2beta by targeting an IA-2 specific epitope of the juxtamembrane region. The findings define an approach to develop assays for specific antibody epitope measurements which may be relevant for disease prognosis and monitoring intervention therapies.