Engineered human FcγRIIa fusion: A novel strategy to extend serum half-life of therapeutic proteins.

The immunoglobulin G (IgG) molecule has a long circulating serum half-life (~3 weeks) through pH- dependent FcRn binding-mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non-antibody therapeutic proteins, we have evolved the ectodomain of a low-affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2-CH3 interface). High-throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32-fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10-7 M for wild type FcγRIIa and 2.82 × 10-8 M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD-L1 fused with engineered FcγRIIa (PD-L1-2A45.1) was compared with that of PD-L1 fused with wild type FcγRIIa (PD-L1-wild type FcγRIIa) and human PD-L1 in mice. PD-L1-2A45.1 showed 11.7- and 9.7-fold prolonged circulating half-life (t1/2 ) compared to PD-L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUCinf of PD-L1-2A45.1 was two-fold higher compared to that of PD-L1-wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half-life of therapeutic proteins.

Effective clearance of rituximab-resistant tumor cells by breaking the mirror-symmetry of immunoglobulin G and simultaneous binding to CD55 and CD20.

Complement-dependent cytotoxicity (CDC), which eliminates aberrant target cells through the assembly and complex formation of serum complement molecules, is one of the major effector functions of anticancer therapeutic antibodies. In this study, we discovered that breaking the symmetry of natural immunoglobulin G (IgG) antibodies significantly increased the CDC activity of anti-CD20 antibodies. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in a rituximab-resistant cell line generated in-house, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance. Based on these findings, we developed an asymmetric bispecific antibody (SBU-CD55 × CD20) that simultaneously targets both CD55 and CD20 to effectively eliminate rituximab-resistant cancer cells. In various cancer cell lines, including rituximab-resistant lymphoma cells, the SBU-CD55 × CD20 antibody showed significantly higher CDC activity than either anti-CD20 IgG antibody alone or a combination of anti-CD20 IgG antibody and anti-CD55 IgG antibody. Furthermore, the asymmetric bispecific antibody (SBU-CD55 × CD20) exhibited significantly higher CDC activity against rituximab-resistant cancer cells compared to other bispecific antibodies with symmetric features. These results demonstrate that enhancing CDC with an asymmetric CD55-binding bispecific antibody could be a new strategy for developing therapeutics to treat patients with relapsed or refractory cancers.

Cell-free production of functional antibody fragments.

Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 microg of functional Fab was obtained after purification from 1 mL reaction mixture.

Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies.

The Fc region of IgG antibodies is crucial for binding to Fc receptors expressed on the surfaces of various immune leukocytes and eliciting therapeutic effector functions such as clearance of antibody-opsonized tumor cells. Despite abrogated Fc gamma receptor (FcγR) binding and therapeutic effector function in the absence of N-linked glycosylation at Asn297, the aglycosylated Fc region of IgG antibodies has bioprocessing advantages such as the absence of glycan heterogeneity and simple bacterial antibody production. Therefore, these antibodies have been comprehensively engineered as effector functional units for human therapy. In this work, we constructed a huge library of Fc variants with combinations of 25 beneficial mutations that were previously identified to improve binding of glycosylated or aglycosylated Fc regions to human FcγRs in previous studies. High-throughput screening of the resulting library led to the identification of an aglycosylated Fc variant that exhibited almost double the antibody-dependent cell-mediated cytotoxicity than wild-type glycosylated Fc. All mutations in this aglycosylated Fc variant were derived from previously identified beneficial mutations for engineered aglycosylated Fc variants as opposed to glycosylated variants, suggesting that significantly different sets of beneficial mutations are necessary to improve the effector function of aglycosylated Fc.

Engineered aglycosylated full-length IgG Fc variants exhibiting improved FcγRIIIa binding and tumor cell clearance.

FcγRIIIa, which is predominantly expressed on the surface of natural killer cells, plays a key role in antibody-dependent cell-mediated cytotoxicity (ADCC), a major effector function of therapeutic IgG antibodies that results in the death of aberrant cells. Despite the potential uses of aglycosylated IgG antibodies, which can be easily produced in bacteria and do not have complicated glycan heterogeneity issues, they show negligible binding to FcγRIIIa and abolish the activation of immune leukocytes for tumor cell clearance, in sharp contrast to most glycosylated IgG antibodies used in the clinical setting. For directed evolution of aglycosylated Fc variants that bind to FcγRIIIa and, in turn, exert potent ADCC effector function, we randomized the aglycosylated Fc region of full-length IgG expressed on the inner membrane of Escherichia coli. Multiple rounds of high-throughput screening using flow cytometry facilitated the isolation of aglycosylated IgG Fc variants that exhibited higher binding affinity to FcγRIIIa-158V and FcγRIIIa-158F compared with clinical-grade trastuzumab (Herceptin®). The resulting aglycosylated trastuzumab IgG antibody Fc variants could elicit strong peripheral blood mononuclear cell-mediated ADCC without glycosylation in the Fc region.

Dimerization of translationally controlled tumor protein is essential for its cytokine-like activity.

BACKGROUND: Translationally Controlled Tumor Protein (TCTP) found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF). Human recombinant HRF (HrHRF) has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn). METHODS AND FINDINGS: We found that only NH(2)-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH(2)-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP) was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP). Allergenic activity of Del-N11TCTP prompted us to see whether the NH(2)-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice. CONCLUSIONS: Dimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development.

Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display.

A monoclonal antibody to the light chain of botulinum neurotoxin type B (BoNT/B) was generated and its protective activity was evaluated in vivo. A chimeric rabbit/human Fab library was generated using bone marrow and spleen cDNAs of rabbits immunized with the BoNT/B light chain, and three monoclonal antibodies specific to the catalytic domain of BoNT/B were isolated. One of these clones, BCXRH1, was specific to a conformation-dependent epitope, and partially neutralized the BoNT/B complex in vivo.