Fc Variants Research Articles

Fc-engineered antibodies promote neutrophil-dependent control of Mycobacterium tuberculosis

Mounting evidence indicates that antibodies can contribute towards control of tuberculosis (TB). However, the underlying mechanisms of humoral immune protection and whether antibodies can be exploited in therapeutic strategies to combat TB are relatively understudied. Here we engineered the receptor-binding Fc (fragment crystallizable) region of an antibody recognizing the Mycobacterium tuberculosis (Mtb) capsule, to define antibody Fc-mediated mechanism(s) of Mtb restriction. We generated 52 Fc variants that either promote or inhibit specific antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole-blood model of infection. While there is likely no singular Fc profile that universally drives control of Mtb, here we found that several Fc-engineered antibodies drove Mtb restriction in a neutrophil-dependent manner. Single-cell RNA sequencing analysis showed that a restrictive Fc-engineered antibody promoted neutrophil survival and expression of cell-intrinsic antimicrobial programs. These data show the potential of Fc-engineered antibodies as therapeutics able to harness the protective functions of neutrophils to promote control of TB.

Targeting TNFRSF25 by agonistic antibodies and multimeric TL1A proteins co-stimulated CD8+ T cells and inhibited tumor growth

BackgroundTumor necrosis factor receptor superfamily 25 (TNFRSF25) is a T-cell co-stimulatory receptor. Expression of its ligand, TNF-like cytokine 1A (TL1A), on mouse tumor cells has been shown to promote tumor...

Fc engineering by monoclonal mammalian cell display for improved affinity and selectivity towards FcγRs.

Fc optimization can significantly enhance therapeutic efficacy of monoclonal antibodies. However, existing Fc engineering approaches are sub-optimal with noted limitations, such as inappropriate glycosylation, polyclonal libraries, and utilizing fragment but not full-length IgG display. Applying cell cycle arrested recombinase-mediated cassette exchange, this study constructed high-quality monoclonal Fc libraries in CHO cells, displayed full-length IgG on cell surface, and preformed ratiometric fluorescence activated cell sorting (FACS) with the antigen and individual FcγRs. Identified Fc variants were quantitatively evaluated by flow cytometry, ELISA, kinetic and steady-state binding affinity measurements, and cytotoxicity assays. An error-prone Fc library focusing on the hinge-CH2 region was constructed in CHO cells with a functional diversity of 7.5×106. Panels of novel Fc variants with enhanced affinity and selectivity for FcγRs were isolated. Particularly, clone 2a-10 (G236E/K288R/K290W/K320M) showed increased binding strength towards FcγRIIa-131R and 131H allotypes with kinetic dissociation constants (KD-K) of 140nM and 220nM, respectively, while reduced binding strength towards FcγRIIb compared to WT Fc; clone 2b-1 (K222I/V302E/L328F/K334E) had KD-K of 180nM towards FcγRIIb; clone 3a-2 (P247L/K248E/K334I) exhibited KD-K of 190nM and 100nM towards FcγRIIIa-176F and 176V allotypes, respectively, and improved potency of 2.0ng/ml in ADCC assays. Key mutation hotspots were identified, including P247 for FcγRIIIa, K290 for FcγRIIa, and K334 for FcγRIIb bindings. Discovery of Fc variants with enhanced affinity and selectivity towards individual FcγR and the identification of novel mutation hotspots provide valuable insights for further Fc optimization and serve as a foundation for advancing antibody therapeutics development.

Mixed IgG Fc immune complexes exhibit blended binding profiles and refine FcR affinity estimates

Immunoglobulin G (IgG) antibodies coordinate immune effector responses by interacting with effector cells via fragment crystallizable γ (Fcγ) receptors. The IgG Fc domain directs effector responses through subclass and glycosylation variation. Although each Fc variant has been extensively characterized in isolation, during immune responses, IgG is almost always produced in Fc mixtures. How this influences effector responses has not been examined. Here, we measure Fcγ receptor binding to mixed Fc immune complexes. Binding of these mixtures falls along a continuum between pure cases and quantitatively matches a mechanistic model, except for several low-affinity interactions mostly involving IgG2. We find that the binding model provides refined estimates of their affinities. Finally, we demonstrate that the model predicts effector cell-elicited platelet depletion in humanized mice. Contrary to previous views, IgG2 exhibits appreciable binding through avidity, though it is insufficient to induce effector responses. Overall, this work demonstrates a quantitative framework for modeling mixed IgG Fc-effector cell regulation.

Complement Activation by an Anti-Dengue/Zika Antibody with Impaired Fcγ Receptor Binding Provides Strong Efficacy and Abrogates Risk of Antibody-Dependent Enhancement.

To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.

Abstract 2964: Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042

Abstract Tumor-associated macrophages (TAMs) are the largest immune cell population in many cancers and play a key role in establishing the immunosuppressive tumor microenvironment (TME) that enables tumor progression. However, TAMs are phenotypically plastic and have the potential to be reprogrammed into immunostimulatory cells that enhance innate and adaptive anti-tumor immunity. To this end, we developed BDC-3042, an agonistic antibody targeting an immune-activating receptor expressed on TAMs known as Dectin-2 (CLEC6A). Dectin-2 is a C-type lectin receptor (CLR) known best for its role in pathogen recognition and induction of protective immune responses against fungi and other microbes. We previously demonstrated that Dectin-2 agonism with natural ligands stimulates pro-inflammatory cytokine secretion and antigen presentation by TAMs, resulting in robust CD8+ T cell-mediated anti-tumor immunity in syngeneic mouse models. Here we present our preclinical studies demonstrating the therapeutic potential of the Dectin-2 agonistic antibody, BDC-3042, as a novel TAM-directed immunotherapy for diverse human cancers. BDC-3042 exhibits strong binding to Dectin-2-expressing macrophages generated in vitro and to primary human TAMs from a range of solid tumor types. In contrast, BDC-3042 binds weakly to peripheral monocytes and minimally to other immune cells in blood and tumor tissues. Macrophages exposed to cytokines and growth factors commonly found in the TME exhibit increased Dectin-2 expression and BDC-3042 binding. Treatment with BDC-3042 activates in vitro-generated macrophages and primary TAMs to produce an array of pro-inflammatory cytokines and chemokines associated with anti-tumor immunity. Consistent with low target expression, BDC-3042 elicits little to no activation of peripheral monocytes or cells in whole blood. BDC-3042 activity is dependent on both Dectin-2 and FcγRs, as indicated through studies utilizing Fc variants with enhanced or attenuated effector function. In mice with humanized immune systems, BDC-3042 elicits activation of TAMs, as evidenced by modulation of key activation markers. Systemically administered BDC-3042 mediates anti-tumor efficacy as a monotherapy, and combination with checkpoint blockade therapy enhances anti-tumor efficacy. The data presented demonstrate the therapeutic potential of targeting Dectin-2 expressed by TAMs with the agonistic antibody BDC-3042 as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Fang Xiao, Po Y. Ho, Jess L. Nolin, Rishali K. Gadkari, Laughing Bear Torrez, David T. Omstead, Katelynn A. McEachin, Jason Ptacek, Rachel Grgich, Cecelia I. Pearson, Stefan Chun, Cindy L. Kreder, Karla A. Henning, Han K. Kim, Lu Xu, Steven J. Chapin, Michael N. Alonso, Shelley E. Ackerman. Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2964.

Harnessing Fc/FcRn Affinity Data from Patents with Different Machine Learning Methods.

Monoclonal antibodies are biopharmaceuticals with a very long half-life due to the binding of their Fc portion to the neonatal receptor (FcRn), a pharmacokinetic property that can be further improved through engineering of the Fc portion, as demonstrated by the approval of several new drugs. Many Fc variants with increased binding to FcRn have been found using different methods, such as structure-guided design, random mutagenesis, or a combination of both, and are described in the literature as well as in patents. Our hypothesis is that this material could be subjected to a machine learning approach in order to generate new variants with similar properties. We therefore compiled 1323 Fc variants affecting the affinity for FcRn, which were disclosed in twenty patents. These data were used to train several algorithms, with two different models, in order to predict the affinity for FcRn of new randomly generated Fc variants. To determine which algorithm was the most robust, we first assessed the correlation between measured and predicted affinity in a 10-fold cross-validation test. We then generated variants by in silico random mutagenesis and compared the prediction made by the different algorithms. As a final validation, we produced variants, not described in any patents, and compared the predicted affinity with the experimental binding affinities measured by surface plasmon resonance (SPR). The best mean absolute error (MAE) between predicted and experimental values was obtained with a support vector regressor (SVR) using six features and trained on 1251 examples. With this setting, the error on the log(KD) was less than 0.17. The obtained results show that such an approach could be used to find new variants with better half-life properties that are different from those already extensively used in therapeutic antibody development.

Engineered Human Antibody with Improved Endothelin Receptor Type A Binding Affinity, Developability, and Serum Persistence Exhibits Excellent Antitumor Potency

Endothelin receptor A (ETA), a class A G protein-coupled receptor (GPCR), is a promising tumor-associated antigen due to its close association with the progression and metastasis of many types of cancer, such as colorectal, breast, lung, ovarian, and prostate cancer. However, only small-molecule drugs have been developed as ETA antagonists with anticancer effects. In a previous study, we identified an antibody (AG8) with highly selective binding to human ETA through screening of a human naïve immune antibody library. Although both in vitro and in vivo experiments indicated that the identified AG8 had anticancer effects, there is a need for improvement in biochemical and physicochemical properties such as the ETA binding affinity, thermostability, and productivity. In this study, we engineered the framework regions of AG8 and isolated an anti-ETA antibody (MJF1) exhibiting significantly improved thermostability and ETA binding affinity. Subsequently, our previously isolated PFc29, an Fc variant with an enhanced pH-dependent human FcRn binding profile, was introduced to MJF1, and the resulting Fc-engineered anti-ETA antibody (MJF1-PFc29) inhibited the proliferation of tumor cells comparably to MJF1 and showed a 4.2-fold increased serum half-life in human FcRn transgenic mice. Moreover, MJF1-PFc29 elicited higher tumor growth inhibition in colorectal cancer xenograft mice compared to MJF1. Our results demonstrate that the engineered human anti-ETA antibody MJF1-PFc29 has great therapeutic potential and high antitumor potency against various types of cancers including colorectal cancer.

Monoclonal Antibody Pharmacokinetics in Cynomolgus Monkeys Following Subcutaneous Administration: Physiologically Based Model Predictions from Physiochemical Properties.

An integrated physiologically based modeling framework is presented for predicting pharmacokinetics and bioavailability of subcutaneously administered monoclonal antibodies in cynomolgus monkeys, based on in silico structure-derived metrics characterizing antibody size, overall charge, local charge, and hydrophobicity. The model accounts for antibody-specific differences in pinocytosis, transcapillary transport, local lymphatic uptake, and pre-systemic degradation at the subcutaneous injection site and reliably predicts the pharmacokinetics of five different wild-type mAbs and their Fc variants following intravenous and subcutaneous administration. Significant associations were found between subcutaneous injection site degradation rate and the antibody's local positive charge of its complementarity-determining region (R = 0.56, p = 0.0012), antibody pinocytosis rate and its overall positive charge (R = 0.59, p = 0.00063), and antibody paracellular transport and its overall charge together with hydrophobicity (R = 0.63, p = 0.00096). Based on these results, population simulations were performed to predict the relationship between bioavailability and antibody local positive charge. In addition, model simulations were conducted to calculate the relative contribution of absorption pathways (lymphatic and blood), pre-systemic degradation pathways (interstitial and lysosomal), and the influence of injection site lymph flow on antibody bioavailability and pharmacokinetics. The proposed physiologically based modeling framework integrates fundamental mechanisms governing antibody subcutaneous absorption and disposition, with structured-based physiochemical properties, to predict antibody bioavailability and pharmacokinetics in vivo.

An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies

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.

Residue interaction network and molecular dynamics simulation study on the binding of S239D/I332E Fc variant with enhanced affinity to FcγRIIIa receptor

Engineering of Fc has been adapted as an efficient method for enhanced or reduced affinity towards Fc receptors in the development of therapeutic antibodies. S239D/I332E mutation of Fc induces approximately two logs greater affinity to the FcγRIIIa receptor and has been extensively employed in various Fc engineering studies. It is known that the mutation gives rise to the formation of salt bridges between the mutated residues of Fc and FcγRIIIa, but the overall effect of the mutation in the binding interface of the Fc-FcγRIIIa complex is still unclear. In this study, the molecular interactions in the binding interface of mutant Fc and FcγRIIIa were analyzed and compared with those of wild-type Fc binding through residue interaction network (RIN) analysis and molecular dynamics (MD) simulation. RIN analysis identified specific molecular interactions and Hub residues in the interfaces, and their numbers were increased by introducing the mutation, with maintaining most of the molecular interactions in the wild-type complex. MD simulation study revealed that the numbers of stable electrostatic interactions and stable Hub residues in the mutant complex were higher than those in the wild-type complex. The introduced mutations were shown to form further charge-charge attractive interactions in addition to the identified salt bridges without generating any repulsive interactions. These results are expected to provide further structural insight into Fc variants’ design based on the S239D/I332E mutation.

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood. However, limited information regarding the effect of these mutations or isotype difference on physicochemical properties (PCP), developability, and manufacturability of therapeutics bearing these different Fc regions is available. In this report, we systematically characterize the effects of the mutations and IgG4 isotype on conformation stability, colloidal stability, solubility, and storage stability at accelerated conditions in two buffer systems using six Fc variants. Our results provide a basis for selecting appropriate Fc region during development of IgG or Ig-based therapeutics and predicting effect of the mutations on CMC development process.

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc–FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants Kd,7.4/Kd,6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human FcγRIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acid-Fc at pH 7.4, based on EC50 ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.

Yeast Mating as a Tool for Highly Effective Discovery and Engineering of Antibodies via Display Methodologies.

Yeast surface display (YSD) is a powerful methodology for discovery and engineering of antibodies, and the yeast mating has been used to overcome low transformation efficiency of yeast in antibody library generation. We developed an optimized method of yeast mating for generating a large, combinatorial antibody fragment library and heterodimeric protein library by cellular fusion between two haploid cells carrying different library each other. This method allows for increased diversity in screening of target-specific fragment antigen-binding (Fab) antibodies as well as in the development of heterodimeric Fc variants for bi-specific antibody generation and T-cell receptor (TCR). Here we describe the efficient isolation of human antibodies against the activated GTP-bound form of the oncogenic Ras mutant (KRasG12D-GTP) by sequential isolation of their heavy chains (HCs) followed by combination with light chains (LCs) via the yeast mating process. This strategy facilitates guided selection of the antigen-specific HC with either a fixed functional LC, which has cytosol penetrating ability, or an LC library to generate the Fab. It also allows for deeper exploration of a sequence space with fixed diversity, leading to a higher probability of successful isolation of human antibodies with high specificity and affinity.

Engineering IgG1 Fc Domains That Activate the Complement System.

Fc-mediated effector functions are important for the clearance of pathologic cells by therapeutic IgG antibodies through two mechanisms: via the activation of the classical complement pathway and through the binding to Fcγ receptors (FcγRs) which mediate clearance of targeted cells by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by effector cells such as macrophages, NK cells, and other leukocytes subsets. Complement activation results in direct cell killing through the formation of the membrane attack complex (MAC, complement-dependent cytotoxicity or CDC) and in the deposition of complement opsonins on pathogen surfaces. The latter are recognized by complement receptors on effector cells in turn triggering complement-dependent cell cytotoxicity and phagocytosis (CDCC and CDCP, respectively). Little is known about the role of CDCC and CDCP on therapeutic antibody function because on the one hand, IgG isotype antibodies bind to both FcγR and C1q to activate the complement pathway, and on the other, immune cells express complement receptor as well as FcγRs. We engineered IgG1 Fc domains that bind with high affinity to C1q but have very little or no binding to FcγR. To this end, we employed display of IgG in E. coli (which lack protein glycosylation machinery) for the screening of very large libraries (>2×109) of randomly mutated human Fc domains to isolate Fc variants that bind to C1q. Herein we introduce and describe the method.

In vivo pharmacokinetic enhancement of monomeric Fc and monovalent bispecific designs through structural guidance

In a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.

Abstract 1882: Clinical approach and biomarker strategy for HFB301001, a novel OX40 agonistic antibody

Abstract Agonist OX40 antibodies have shown promising pre-clinical activities, but their clinical activities have been limited thus far. Several reasons may account for this limited clinical activity, including sub-optimal antibody design, dose selection, and lack of a biomarker strategy for indication selection and patient enrichment. Previous clinical trials selected doses that maximized receptor occupancy, but some patients responded at lower doses, indicating nuances to choosing the correct therapeutic doses for an agonist antibody. In addition, preclinical work has demonstrated a so-called “hook” effect whereby agonist activity decreases at higher concentrations, which further emphasizes the need to develop a novel anti-OX40 therapeutic antibody that addresses the previously encountered challenges. HFB301001 is a novel human IgG1 agonist antibody that binds to a unique epitope on OX40. This allows for agonistic activity that does not compete with the endogenous OX40 ligand. Relative to other clinical stage OX40 antibodies, HFB301001 has reduced OX40 downregulation following co-stimulation of T cells, and it has demonstrated superior in vivo anti-tumor activity and pharmacodynamic immune modulation in a human OX40 knock-in mouse model. HFB301001 is well tolerated in cynomolgus monkeys. To progress into clinical studies, we have determined human dose projections for clinical evaluation of HFB301001 using PKPD modeling, serum exposure in non-human primates, antitumor efficacy in mouse models and immune cell pharmacodynamics. We also took advantage of Fc variants to delineate the relative contributions of Treg depletion versus enhancing T cell activity by agonism to efficacy of HFB301001. To further enhance probability of success (POS) in clinical studies, we are applying our single-cell Drug Intelligent Science (DIS™) platform to rationally identify cancer indications and to define novel predictive response biomarkers. We have used single-cell profiling to identify unique tumor-infiltrating T cell signatures that may help identify patients more likely to response to HFB301001 treatment, inform indication selection, and establish a patient stratification biomarker strategy. Finally, we show here our phase I trial design for HFB301001 that implements these findings. In conclusion, HFB301001 is a highly differentiated therapeutic antibody which is well positioned to enter a global, multi-center Phase I clinical trial to explore optimal biologically active dose and evaluate predictive biomarker hypotheses. Here, we present results supporting the rationale for indication selection, biomarker identification, dose selection, and phase I clinical trial design. Citation Format: Ross Fulton, Jinping Gan, Yun-Yueh Lu, Julianna Crivello, Zachery Duda, Zhiyuan Wang, Rebecca Silver, Alexandra Staskus, Charina Ortega, Sami Ellouze, Carine George, Sophie Foulon, Wenhua Xu, Xing Cai, Joyce Pi, Dean Lee, Monika Manne, Ruina Jin, Yuan Wang, Hongkai Zhang, Nicola Beltraminelli, Francisco Adrian, Robert Petit, Liang Schweizer, Andreas Raue. Clinical approach and biomarker strategy for HFB301001, a novel OX40 agonistic antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1882.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent invivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

Recent Achievements and Challenges in Prolonging the Serum Half-Lives of Therapeutic IgG Antibodies Through Fc Engineering.

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.

Generation of enzymatically competent SARS-CoV-2 decoy receptor ACE2-Fc in glycoengineered Nicotiana benthamiana.

Human angiotensin‐converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS‐CoV‐2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2‐fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2‐Fc variant is glycosylated with mainly complex human‐type N‐glycans and functional with regard to enzyme activity, affinity to the SARS‐CoV‐2 receptor‐binding domain, and wild‐type virus neutralization.