G-protein–coupled receptors (GPCRs) are one of the most attractive therapeutic target classes because of their critical roles in intracellular signaling and their clinical relevance to a variety of diseases, including cancer, infection and inflammation. However, high conformational variability, the small exposed area of extracellular epitopes and difficulty in the preparation of GPCR antigens have delayed both the isolation of therapeutic anti-GPCR antibodies as well as studies on the structure, function and biochemical mechanisms of GPCRs. To overcome the challenges in generating highly specific anti-GPCR antibodies with enhanced efficacy and safety, various forms of antigens have been successfully designed and employed for screening with newly emerged systems based on laboratory animal immunization and high-throughput-directed evolution.

Association of FcRn molecules to the Fc region of IgG in acidified endosomes and subsequent dissociation of the interaction in neutral pH serum enables IgG molecules to be recycled for prolonged serum persistence after internalization by endothelial cells, rather than being degraded in the serum and in the lysosomes inside the cells. Exploiting this intracellular trafficking and recycling mechanism, many researchers have engineered the Fc region to further extend the serum half-lives of therapeutic antibodies by optimizing the pH-dependent IgG Fc–FcRn interaction, and have generated various Fc variants exhibiting significantly improved circulating half-lives of therapeutic IgG antibodies. In order to estimate pharmacokinetic profiles of IgG Fc variants in human serum, not only a variety of in vitro techniques to determine the equilibrium binding constants and instantaneous rate constants for pH-dependent FcRn binding, but also diverse in vivo animal models including wild-type mouse, human FcRn transgenic mouse (Tg32 and Tg276), humanized mouse (Scarlet), or cynomolgus monkey have been harnessed. Currently, multiple IgG Fc variants that have been validated for their prolonged therapeutic potency in preclinical models have been successfully entered into human clinical trials for cancer, infectious diseases, and autoimmune diseases.

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.

Background Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens. Results We devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2. Conclusions We constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics. Electronic supplementary material The online version of this article (10.1186/s13036-019-0188-x) contains supplementary material, which is available to authorized users.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.