Potent Therapeutic Activity Against Peritoneal Dissemination and Malignant Ascites by the Novel Anti-Folate Receptor Alpha Antibody KHK2805.

Many ovarian cancer patients often show peritoneal metastasis with malignant ascites. However, unmet medical needs remain regarding controlling these symptoms after tumors become resistant to chemotherapies. We developed KHK2805, a novel anti-folate receptor α (FOLR1) humanized antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The primary aim of the present study was to evaluate whether the anti-tumor activity of KHK2805 was sufficient for therapeutic application against peritoneal dissemination and malignant ascites of platinum-resistant ovarian cancer in preclinical models. Here, both the ADCC and CDC of KHK2805 were evaluated in ovarian cancer cell lines and patient-derived samples. The anti-tumor activity of KHK2805 was evaluated in a SCID mouse model of platinum-resistant peritoneal dissemination. As results, KHK2805 showed specific binding to FOLR1 with high affinity at a novel epitope. KHK2805 exerted potent ADCC and CDC against ovarian cancer cell lines. Furthermore, primary platinum-resistant malignant ascites cells were susceptible to autologous ADCC with KHK2805. Patient-derived sera and malignant ascites induced CDC of KHK2805. KHK2805 significantly reduced the total tumor burden and amount of ascites in SCID mice with peritoneal dissemination and significantly prolonged their survival. In addition, the parental rat antibody strongly stained serous and clear cell-type ovarian tumors by immunohistochemistry. Overall, KHK2805 showed cytotoxicity against both ovarian cancer cell lines and patient-derived cells. These translational study findings suggest that KHK2805 may be promising as a novel therapeutic agent for platinum-resistant ovarian cancer with peritoneal dissemination and malignant ascites.

Correction: Generation and Improvement of Effector Function of a Novel Broadly Reactive and Protective Monoclonal Antibody against Pneumococcal Surface Protein A of Streptococcus pneumoniae.

[This corrects the article DOI: 10.1371/journal.pone.0154616.].

Generation and Improvement of Effector Function of a Novel Broadly Reactive and Protective Monoclonal Antibody against Pneumococcal Surface Protein A of Streptococcus pneumoniae.

A proof-of-concept study evaluating the potential of Streptococcus pneumoniae Pneumococcal Surface Protein A (PspA) as a passive immunization target was conducted. We describe the generation and isolation of several broadly reactive mouse anti-PspA monoclonal antibodies (mAbs). MAb 140H1 displayed (i) 98% strain coverage, (ii) activity in complement deposition and opsonophagocytic killing (OPK) assays, which are thought to predict the in vivo efficacy of anti-pneumococcal mAbs, (iii) efficacy in mouse sepsis models both alone and in combination with standard-of-care antibiotics, and (iv) therapeutic activity in a mouse pneumonia model. Moreover, we demonstrate that antibody engineering can significantly enhance anti-PspA mAb effector function. We believe that PspA has promising potential as a target for the therapy of invasive pneumococcal disease by mAbs, which could be used alone or in conjunction with standard-of-care antibiotics.

Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system.

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.

Novel anti-Tn single-chain Fv-Fc fusion proteins derived from immunized phage library and antibody Fc domain.

Tn[GalNAc(α1-3)-Ser/Thr] antigen, a tumor-associated carbohydrate antigen, is highly expressed in various tumors and an attractive candidate for cancer immunotherapy. The generation of an anti-Tn antibody is a first step toward the construction of new anticancer molecules. However, because of the simple and small conformation of the Tn molecule, it is difficult to generate an anti-Tn antibody for therapeutic use by conventional hybridoma technology. The purpose of this study was to isolate anti-Tn single-chain antibody fragments (scFv) by phage display technology from a novel immunised library, to attach an antibody constant region (Fc) and to convert them to scFv-Fc fusion proteins. The scFv-Fcs obtained here showed strict specificity against the Tn antigen and also showed antibody-dependent cellular cytotoxicity. These results suggest a potential use of this antibody generating method by phage display and indicate the potential of Fc-fusion proteins as therapeutic candidates.

Engineered therapeutic antibodies with improved effector functions.

In the past decade, more than 20 therapeutic antibodies have been approved for clinical use and many others are now at the clinical and preclinical stage of development. Fragment crystallizable (Fc)-dependent antibody functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and a long half-life, have been suggested as important clinical mechanisms of therapeutic antibodies. These functions are primarily triggered through direct interaction of the Fc domain with its corresponding receptors: FcgammaRIIIa for ADCC, C1q for CDC, and neonatal Fc receptor for prolongation of the clearance rate. However, current antibody therapy still faces the critical issues of insufficient efficacy and the high cost of the therapeutic agents. A possible solution to these issues could be to engineer antibody molecules to enhance their antitumor activity, leading to improved therapeutic outcomes and reduced doses. Here, we review advanced Fc engineering approaches for the enhancement of effector functions, some of which are now ready for evaluation of their effectiveness 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.