Establishment of Membrane-Bound IgA1-Specific Antibody Possessing Antibody-Dependent Cellular Cytotoxicity Activity.

Therapeutic agents targeting B cells, such as rituximab, have been proven to be effective in several diseases. However, since this therapy targets the whole B cell population, there are still concerns about critical adverse events. Therefore, a new type of antibody that can specifically deplete a particular type of B cell may have the potential to become an efficient and safer therapy. Membrane-bound IgA1 (mIgA1) is expressed on soluble IgA1 (sIgA1) producing B cells and/or their precursor, B cells. Although most of the amino acid sequence in the N-terminal regions of the extracellular domains of mIgA1 and sIgA1 is shared, there is a membrane type-specific region, named the membrane-bound immunoglobulin isotype-specific (migis-α) region, at the membrane-proximal region of mIgA1. We hypothesized that the migis-α region would be a suitable antigen for therapeutic antibodies to target mIgA1-expressing B cells without binding to sIgA1, which may cause undesired adverse effects and poor pharmacokinetics (PK). We established two anti-migis-α monoclonal antibodies (mAbs), KM4641 and KM4644, by immunization of the synthetic peptide corresponding to an migis-α region. These mAbs both showed robust binding to mIgA1-expressing transfectant cells. As we expected, neither mAbs bound to sIgA1 and we found that the mAbs recognized different seven to eight amino acid sequences within the migis-α region. Furthermore, the rat-human chimeric type of both mAbs showed antibody-dependent cellular cytotoxicity against mIgA1-expressing transfectant cells. Taken together, this study showed that established mAbs have therapeutic potential in IgA-related diseases, such as IgA nephropathy.

A Phase 1 Study of KHK4083: A Single-Blind, Randomized, Placebo-Controlled Single-Ascending-Dose Study in Healthy Adults and an Open-Label Multiple-Dose Study in Patients With Ulcerative Colitis.

OX40 plays an essential role in maintaining late T-cell proliferation and survival by suppressing apoptosis and by inducing T-cell memory formation. Here, we report the results of the phase 1 study of KHK4083, a fully human antimonoclonal antibody specific for OX40. In this study, we aimed to assess the safety and tolerability of a single intravenous or subcutaneous administration of KHK4083 compared with placebo in healthy Japanese and Caucasian subjects and determined the pharmacokinetics (PK) and immunogenicity. Also, we assessed the preliminary efficacy and pharmacodynamics of multiple intravenous doses in Japanese patients with moderate to severe ulcerative colitis (UC). Drug-related treatment emergent adverse events occurred in 21 healthy subjects (58.3%) and 5 patients with UC (62.5%) after administration of KHK4083. There were no serious adverse events. The PK profile of a single intravenous dose of 10 mg/kg KHK4083 was similar in healthy Japanese and Caucasian subjects. Of 8 UC patients, a clinical response was observed in 3 patients (37.5%) and clinical remission in 2 patients (25.0%) in week 6. Our study demonstrated the safety and tolerability of single and multiple administrations of KHK4083 in healthy Japanese and Caucasian subjects and Japanese patients with moderate to severe UC. It also indicated favorable pharmacological properties of the drug.

Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.

BACKGROUND: Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). METHODS: Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. RESULTS: Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. CONCLUSION: This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease.

Fucosyltransferase VII improves the function of selectin ligands on cord blood hematopoietic stem cells.

Selectins and their carbohydrate ligands mediate the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow. We have previously shown that ex vivo fucosylation of selectin ligands on HSPCs by α1,3 fucosyltransferase VI (FUT6) leads to improved human cord blood (CB)-HSPC engraftment in non-obese diabetic (NOD)/severe combined immune deficient (SCID) mice. In the present study, we determined whether surface fucosylation with α1,3 fucosyltransferase VII (FUT7), which is primarily expressed by hematopoietic cells, improves the function of selectin ligands on CB-HSPCs in comparison with FUT6. A saturating amount of either FUT6 or FUT7, which generates comparable levels of expression of fucosylated epitopes on CB CD34(+) cells, was used for these experiments. In vitro, FUT7-treated CB CD34(+) cells exhibited greater binding to P- or E-selectin than that of FUT6-treated CB CD34(+) cells under static or physiological flow conditions. In vivo, FUT7 treatment, like FUT6, improved the early engraftment of CB CD34(+) cells in the bone marrow of sublethally irradiated NOD/SCID interleukin (IL)-2Rγ(null) (NSG) mice. FUT7 also exhibited marginally-yet statistically significant-increased engraftment at 4 and 6 weeks after transplantation. In addition, FUT7-treated CB CD34(+) cells exhibited increased homing to the bone marrow of irradiated NSG mice relative to sham-treated cells. These data indicate that FUT7 is effective at improving the function of selectin ligands on CB-HSPCs in vitro and enhancing early engraftment of treated CB-HSPCs in the bone marrow of recipients.

Growth control of genetically modified cells using an antibody/c-Kit chimera.

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs.

Optimizing therapeutic antibody function: progress with Fc domain engineering.

Since the establishment of monoclonal antibody production using hybridoma technology in the mid-1970s, there has been expanding progress and continuous technological improvement in the development of therapeutic antibodies. The initial technological breakthroughs involved reduction of immunogenicity and thus enabled repeated administration. The establishment of chimeric, humanized, and fully human antibodies has led to the great success of several 'second-generation' therapeutic antibodies, such as rituximab, trastuzumab, cetuximab, and bevacizumab. However, there still exists an urgent demand for improvement in the efficacy of the current antibody therapeutics, which is not yet fully satisfactory for patients. Based on the current understanding of the clinical mechanisms of several therapeutic antibodies, many now believe that Fc-mediated functions (e.g. antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neonatal Fc receptor [FcRn]-mediated storage) will improve the clinical outcomes of therapeutic antibodies. The present review focuses on the recent progress in the development of 'Fc engineering,' which dramatically improves (and sometimes silences) Fc-mediated functions. These achievements can be classified into two technological approaches: (i) introducing amino acid mutations and (ii) modifying Fc-linked oligosaccharide structures. The effectiveness of multiple third-generation therapeutic antibodies armed with various engineered Fcs is now ready to be tested in clinical trials.

Enhanced binding affinity for FcgammaRIIIa of fucose-negative antibody is sufficient to induce maximal antibody-dependent cellular cytotoxicity.

Antibody-dependent cellular cytotoxicity (ADCC) is considered to be an important therapeutic function for clinical efficacy of monoclonal antibodies. Recent studies have revealed two methods to increase binding affinity for FcgammaRIIIa and enhance ADCC more efficiently for antibodies: (i) fucose removal from antibody N-linked complex oligosaccharides and (ii) amino acid mutations in the antibody Fc region. In this study, we compare the biological activities of the methods of generating high ADCC antibodies. We used a fucose-negative antibody and two antibodies with sets of mutations, demonstrated previously to optimally enhance ADCC using the chimeric anti-CD20 antibody, rituximab, as the model. Both amino acid mutant antibodies showed a significantly higher affinity for recombinant FcgammaRIIIa than fucose-negative antibody when compared using biosensor analysis. The removal of fucose from the antibodies bearing amino acid mutations exhibited a further enhancement of binding to recombinant FcgammaRIIIa and significantly increased binding to natural killer (NK) cells. Despite the differences manifested in binding for the FcgammaR, ADCCs were indistinguishable between methods and even when the methods were combined. These results indicate that the affinity of binding to FcgammaRIIIa does not predict ADCC beyond a certain threshold and that each method alone is sufficient to induce maximal ADCC of the antibody.