Crystallizable Domain Research Articles

Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants

Molecular conjugation to antibodies has emerged as a growing strategy to combine the mechanistic activities of the attached molecule with the specificity of antibodies. A variety of technologies have been applied for molecular conjugation; however, these approaches face several limitations, including disruption of antibody structure, destabilization of the antibody, and/or heterogeneous conjugation patterns. Collectively, these challenges lead to reduced yield, purity, and function of conjugated antibodies. While glycoengineering strategies have largely been applied to study protein glycosylation and manipulate cellular metabolism, these approaches also harbor great potential to enhance the production and performance of protein therapeutics. Here, we devise a novel glycoengineering workflow for the development of site-specific antibody conjugates. This approach combines metabolic glycoengineering using azido-sugar analogs with newly installed N-linked glycosylation sites in the antibody constant domain to achieve specific conjugation to the antibody via the introduced N-glycans. Our technique allows facile and efficient manufacturing of well-defined antibody conjugates without need for complex or destructive chemistries. Moreover, introduction of conjugation sites in the antibody fragment crystallizable (Fc) domain renders this approach widely applicable and target agnostic. Our platform can accommodate up to 3 conjugation sites in tandem, and the extent of conjugation can be tuned through use of different sugar analogs or production in different cell lines. We demonstrated that our platform is compatible with various use-cases, including fluorescent labeling, antibody-drug conjugation, and targeted gene delivery. Overall, this study introduces a versatile and effective yet strikingly simple approach to produce antibody conjugates for research, industrial, and medical applications.

Impact of site-specific conjugation strategies on the pharmacokinetics of antibody conjugated radiotherapeutics

Antibody radionuclide conjugates are an emerging modality for targeted imaging and potent therapy of disseminated disease. Coupling of radionuclides to monoclonal antibodies (mAbs) is typically achieved by applying non-site-specific labelling techniques. With the ambition of reducing variability, increasing labelling efficacy and stability, several site-specific conjugation strategies have been developed in recent years for toxin- and fluorophore-mAb conjugates. In this study, we studied two site-specific labelling strategies for the conjugation of the macrocyclic chelating agent, DOTA, to the anti-Leucine Rich Repeat Containing 15 (LRRC15) mAb DUNP19. Specifically, one approach utilized a DOTA-bearing peptide (FcIII) with a strong affinity for the fragment crystallizable (Fc) domain of the human IgG1 of DUNP19 (DUNP19LF-FcIII-DOTASS), while the other leveraged a chemo-enzymatic technique to substitute the N-linked bi-antennary oligosaccharides in the human IgG1 Fc domain with DOTA (DUNP19LF-gly-DOTASS). To assess if these methods impact the antibody's binding properties and targeting efficacy, comparative in vitro and in vivo studies of the generated DUNP19-conjugates were performed. While the LRRC15 binding of both radioimmunoconjugates remained intact, the conjugation methods had different impacts on their abilities to interact with FcRn and FcγRs. In vitro assessments of DUNP19LF-FcIII-DOTASS and DUNP19LF-gly-DOTASS demonstrated markedly decreased affinity for FcRn and FcγRIIIa (CD16), respectively. DUNP19LF-FcIII-DOTASS demonstrated increased blood and tissue kinetics in vivo, confirming loss of FcRn binding. While the ablated FcγR interaction of DUNP19LF-gly-DOTASS had no immediate impact on in vivo biodistribution, reduced immunotherapeutic effect can be expected in future studies as a result of reduced NK-cells interaction. In conclusion, our findings underscore the necessity for meticulous consideration and evaluation of mAb labelling strategies, extending beyond mere conjugation efficiency and radiolabeling yields. Notably, site-specific labelling methods were found to significantly influence the immunological impact of Fc interactions. Therefore, it is of paramount importance to consider the intended diagnostic or therapeutic application of the construct and to adopt conjugation strategies that ensure the preservation of critical pharmacological properties and functionality of the antibody in use.

Enhancing Fc-mediated effector functions of monoclonal antibodies: The example of HexaBodies.

Since the approval of the CD20-targeting monoclonal antibody (mAb) rituximab for the treatment of lymphoma in 1997, mAb therapy has significantly transformed cancer treatment. With over 90 FDA-approved mAbs for the treatment of various hematological and solid cancers, modern cancer treatment relies heavily on these therapies. The overwhelming success of mAbs as cancer therapeutics is attributed to their broad applicability, high safety profile, and precise targeting of cancer-associated surface antigens. Furthermore, mAbs can induce various anti-tumor cytotoxic effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), all of which are mediated via their fragment crystallizable (Fc) domain. Over the past decades, these effector mechanisms have been substantially improved through Fc domain engineering. In this review, we will outline the different approaches to enhance Fc effector functions via Fc engineering of mAbs, with a specific emphasis on the so-called "HexaBody" technology, which is designed to enhance the hexamerization of mAbs on the target cell surface, thereby inducing greater complement activation, CDC, and receptor clustering. The review will summarize the development, preclinical, and clinical testing of several HexaBodies designed for the treatment of B-cell malignancies, as well as the potential use of the HexaBody technology beyond Fc-mediated effector functions.

A unique serum IgG glycosylation signature predicts development of Crohn's disease and is associated with pathogenic antibodies to mannose glycan.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gut. There is growing evidence in Crohn's disease (CD) of the existence of a preclinical period characterized by immunological changes preceding symptom onset that starts years before diagnosis. Gaining insight into this preclinical phase will allow disease prediction and prevention. Analysis of preclinical serum samples, up to 6 years before IBD diagnosis (from the PREDICTS cohort), revealed the identification of a unique glycosylation signature on circulating antibodies (IgGs) characterized by lower galactosylation levels of the IgG fragment crystallizable (Fc) domain that remained stable until disease diagnosis. This specific IgG2 Fc glycan trait correlated with increased levels of antimicrobial antibodies, specifically anti-Saccharomyces cerevisiae (ASCA), pinpointing a glycome-ASCA hub detected in serum that predates by years the development of CD. Mechanistically, we demonstrated that this agalactosylated glycoform of ASCA IgG, detected in the preclinical phase, elicits a proinflammatory immune pathway through the activation and reprogramming of innate immune cells, such as dendritic cells and natural killer cells, via an FcγR-dependent mechanism, triggering NF-κB and CARD9 signaling and leading to inflammasome activation. This proinflammatory role of ASCA was demonstrated to be dependent on mannose glycan recognition and galactosylation levels in the IgG Fc domain. The pathogenic properties of (anti-mannose) ASCA IgG were validated in vivo. Adoptive transfer of antibodies to mannan (ASCA) to recipient wild-type mice resulted in increased susceptibility to intestinal inflammation that was recovered in recipient FcγR-deficient mice. Here we identify a glycosylation signature in circulating IgGs that precedes CD onset and pinpoint a specific glycome-ASCA pathway as a central player in the initiation of inflammation many years before CD diagnosis. This pathogenic glyco-hub may constitute a promising new serum biomarker for CD prediction and a potential target for disease prevention.

Reprogramming human B cells with custom heavy-chain antibodies.

The immunoglobulin locus of B cells can be reprogrammed by genome editing to produce custom or non-natural antibodies that are not induced by immunization. However, current strategies for antibody reprogramming require complex expression cassettes and do not allow for customization of the constant region of the antibody. Here we show that human B cells can be edited at the immunoglobulin heavy-chain locus to express heavy-chain-only antibodies that support alterations to both the fragment crystallizable domain and the antigen-binding domain, which can be based on both antibody and non-antibody components. Using the envelope protein (Env) from the human immunodeficiency virus as a model antigen, we show that B cells edited to express heavy-chain antibodies to Env support the regulated expression of B cell receptors and antibodies through alternative splicing and that the cells respond to the Env antigen in a tonsil organoid model of immunization. This strategy allows for the reprogramming of human B cells to retain the potential for in vivo amplification while producing molecules with flexibility of composition beyond that of standard antibodies.

Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding Rapidly Characterize the Structural Polydispersity and Stability of an Fc-Fusion Protein.

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.

Virion-surface display of a chimeric immunoglobulin Fc domain facilitating uptake by antigen-presenting cells

Antigen-presenting cells (APCs) play an important role in virus infection control by bridging innate and adaptive immune responses. Macrophages and dendritic cells (DCs) possess various surface receptors to recognize/internalize antigens, and antibody binding can enhance pathogen-opsonizing uptake by these APCs via interaction of antibody fragment crystallizable (Fc) domains with Fc receptors, evoking profound pathogen control in certain settings. Here, we examined phagocytosis-enhancing potential of Fc domains directly oriented on a retroviral virion/virus-like particle (VLP) surface. We generated an expression vector coding a murine Fc fragment fused to the transmembrane region (TM) of a retroviral envelope protein, deriving expression of the Fc-TM fusion protein on the transfected cell surface and production of virions incorporating the chimeric Fc upon co-transfection. Incubation of Fc-displaying simian immunodeficiency virus (SIV) with murine J774 macrophages and bone marrow-derived DCs derived Fc receptor-dependent enhanced uptake, being visualized by imaging cytometry. Alternative preparation of a murine leukemia virus (MLV) backbone-based Fc-displaying VLP loading an influenza virus hemagglutinin (HA) antigen resulted in enhanced HA internalization by macrophages, stating antigen compatibility of the design. Results show that the Fc-TM fusion molecule can be displayed on certain viruses/VLPs and may be utilized as a molecular adjuvant to facilitate APC antigen uptake.

Antibody-displaying extracellular vesicles for targeted cancer therapy

Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody–drug conjugates.

Abstract 2722: CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms

Abstract Many therapeutic monoclonal antibodies mediate the immune effector functions through the engagement of their fragment crystallizable (Fc) domain and Fcγ receptors on the immune cells. However, these functions are limited due to the low affinity of their interactions and the requirement of abundancy of antigens for the avidity effects. Recently, mutation-based Fc engineered antibodies showed their enhanced immune effector functions mostly through the NK cell-mediated ADCC. To overcome the limited improvement of current Fc engineering, we developed an innovated platform, Twin Fc-Immune Cell Engager (Twin Fc-ICE) composed of a single Fab domain and two Fc domain with drug-like properties. Here we demonstrated the superior efficacy of CTN001, a first Twin Fc-ICE, in in vitro and in vivo Her2+ tumor models. CTN001 bound to various types of Fcγ receptor-expressing immune cells with stronger affinity, supported by its better in vitro affinity to all types of Fcγ receptors than wild type and Fc-engineered antibodies. To evaluate the pharmacological efficacies, CTN001 was tested for its functions of in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) and in vivo efficacy with Her2+ xenograft models. In Her2+ models, both CTN001 and trastuzumab induced ADCC-mediated effects, but CTN001 showed better anti-tumor cytotoxicity through ADCP and CDC than trastuzumab. Importantly, CTN001 also showed cytotoxic effects on Her2-low tumor cells. Consistently, CTN001 showed better anti-tumor efficacy than trastuzumab or its combination with pertuzumab in Her2+ tumor xenograft model and, surprisingly, even in Her2-low or resistant models. These results suggest that CTN001 can provide new therapeutic options for treating Her2-low patients differentiated from antibody-drug conjugates (ADCs), and that the Twin Fc-ICE can be an innovative platform to overcome limitations of existing therapeutics. Citation Format: Deokjae Lee, Sujin Yoon, Jiseon Bae, Minju Kim, Ki-June Lee, Eunmi Kim, Hyunkyu Park, Eun S. Choi, Gi-Hyeok Yang. CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2722.

STAR-0215 is a Novel, Long-Acting Monoclonal Antibody Inhibitor of Plasma Kallikrein for the Potential Treatment of Hereditary Angioedema.

Hereditary angioedema (HAE) is a rare autosomal dominant disorder caused by a deficiency in functional C1 esterase inhibitor (C1-INH), a serpin family protein that blocks the activity of plasma kallikrein. Insufficient inhibition of plasma kallikrein results in the overproduction of bradykinin, a vasoactive inflammatory mediator that produces both pain and unpredictable swelling during HAE attacks, with potentially life-threatening consequences. We describe the generation of STAR-0215, a humanized immunoglobulin G1 (IgG1) antibody with a long circulating half-life (t1/2) that potently inhibits plasma kallikrein activity, with a >1000-fold lower affinity for prekallikrein and no measurable inhibitory activity against other serine proteases. The high specificity and inhibitory effect of STAR-0215 is demonstrated through a unique allosteric mechanism involving N-terminal catalytic domain binding, destabilization of the activation domain, and reversion of the active site to the inactive zymogen state. The YTE (M252Y/S254T/T256E) modified fragment crystallizable (Fc) domain of STAR-0215 enhances pH-dependent neonatal Fc receptor (FcRn) binding, resulting in a prolonged t1/2 in vivo (⁓34 days in cynomolgus monkeys) compared to antibodies without this modification. A single subcutaneous dose of STAR-0215 ({greater than or equal to} 100 mg) was predicted to be active in patients for 3 months or longer, based on simulations using a minimal physiologically based pharmacokinetic (mPBPK) model. These data indicate that STAR-0215, a highly potent and specific antibody against plasma kallikrein with extended t1/2, is a potential agent for long-term preventative HAE therapy administered every 3 months or less frequently. Significance Statement STAR-0215 is a YTE-modified immunoglobulin G1 monoclonal antibody with a novel binding mechanism that specifically and potently inhibits the enzymatic activity of plasma kallikrein and prevents the generation of bradykinin. It has been designed to be a long-lasting prophylactic treatment to prevent attacks of HAE and to decrease the burden of disease and the burden of treatment for people with HAE.

Mapping Antibody Domain Exposure on Nanoparticle Surfaces Using DNA-PAINT.

Decorating nanoparticles with antibodies (Ab) is a key strategy for targeted drug delivery and imaging. For this purpose, the orientation of the antibody on the nanoparticle is crucial to maximize fragment antibody-binding (Fab) exposure and thus antigen binding. Moreover, the exposure of the fragment crystallizable (Fc) domain may lead to the engagement of immune cells through one of the Fc receptors. Therefore, the choice of the chemistry for nanoparticle-antibody conjugation is key for the biological performance, and methods have been developed for orientation-selective functionalization. Despite the importance of this issue, there is a lack of direct methods to quantify the antibodies' orientation on the nanoparticle's surface. Here, we present a generic methodology that enables for multiplexed, simultaneous imaging of both Fab and Fc exposure on the surface of nanoparticles, based on super-resolution microscopy. Fab-specific Protein M and Fc-specific Protein G probes were conjugated to single stranded DNAs and two-color DNA-PAINT imaging was performed. Hereby, we quantitatively addressed the number of sites per particle and highlight the heterogeneity in the Ab orientation and compared the results with a geometrical computational model to validate data interpretation. Moreover, super-resolution microscopy can resolve particle size, allowing the study of how particle dimensions affect antibody coverage. We show that different conjugation strategies modulate the Fab and Fc exposure which can be tuned depending on the application of choice. Finally, we explored the biomedical importance of antibody domain exposure in antibody dependent cell mediated phagocytosis (ADCP). This method can be used universally to characterize antibody-conjugated nanoparticles, improving the understanding of relationships between structure and targeting capacities in targeted nanomedicine.

Towards a Rational Design of Antibody-Recruiting Molecules through a Computational Microscopy View of their Interactions with the Target Antibody.

Antibody recruiting molecules (ARMs) are an innovative class of chimeric molecules, consisting of an antibody-binding ligand (ABL) and a target-binding ligand (TBL). ARMs mediate ternary complex formation between a target cell of interest for elimination and endogenous antibodies that are present in human serum. Clustering of fragment crystallizable (Fc) domains on the surface of antibody-bound cells mediate destruction of the target cell by innate immune effector mechanisms. ARMs are typically designed by conjugating small molecule haptens to a (macro)molecular scaffold, without considering the structure of the respective anti-hapten antibody. Here we report on a computational molecular modeling methodology that allows for studying the close contacts between ARMs and the anti-hapten antibody, considering (1) the spacer length between ABL and TBL; (2) the number of ABL and TBL, and (3) the molecular scaffold onto which these are positioned. Our model predicts the difference in binding modes of the ternary complex and predicts which ARMs are optimal recruiters. Avidity measurements of the ARM-antibody complex and ARM-mediated antibody recruitment to cell surfaces in vitro confirmed these computational modeling predictions. This kind of multiscale molecular modelling holds potential for design of drug molecules that rely on antibody binding for their mechanism of action.

IgG1 and IgG4 antibodies sample initial structure dependent local conformational states and exhibit non-identical Fab dynamics

We have investigated the dynamics of two upgamma-immunoglobulin molecules, IgG1 and IgG4, using long all atom molecular dynamics simulations. We first show that the de novo structures of IgG1 and IgG4 predicted using AlphaFold, with no interactions between the fragment crystallizable (Fc) domain and the antigen fragment binding domain (Fab), eventually relaxes to a state with persistent Fc–Fab interactions that mirrors experimentally resolved structures. We quantified the conformational space sampled by antibody trajectories spawned from six different initial structures and show that the individual trajectories only sample states bound by a local minimum and display very little mixing in their conformational states. Furthermore, the dynamics of the individual Fab domains are strongly dependent on the initial crystal structure and isotype. In all conditions, we observe non-identical dynamics between the Fab arms in an antibody. For a six-bead coarse grained model, we show that non-covalent Fc–Fab interactions can modulate the stiffnesses associated with Fc–Fab distances, angles, and dihedral angles by up to three orders of magnitude. Our results clearly illustrate the inherent complexities in studying antibody dynamics and highlight the need to include non-identical Fab dynamics as an inherent feature in computational models of therapeutic antibodies.

Integrating ultra-high-performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in-depth characterization of complex fusion proteins.

Fc-fusion proteins represent a successful class of biopharmaceutical products, which combine the tailored pharmacological properties of biological ligands with the multiple functions of the fragment crystallizable domain of immunoglobulins. There is great diversity in terms of possible biological ligands creating highly diverse structures, therefore the analytical characterization of fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. Employing etanercept analogues as studied fusion proteins, the Orbitrap mass analyzer with ultra-high performance liquid chromatography (UHPLC-MS) and imaged capillary isoelectric focusing (icIEF) were utilized for the in-depth fusion protein characterization. The amino acid sequence coverage, peptide mapping, and post-translational modifications of etanercept analogues were analyzed by UHPLC-MS. The post-translational modification results were complemented by imaged capillary isoelectric focusing to produce quality research on etanercept analogues. The developed workflow integrating UHPLC-MS and icIEF provided an innovative strategy for characterizing complex fusion proteins in the process of quality control and manufacturing.

A Review of Margetuximab-Based Therapies in Patients with HER2-Positive Metastatic Breast Cancer.

Breast cancer (BC) is the most commonly diagnosed cancer globally, with high mortality rates. Targeted drug therapies have revolutionized cancer treatment. For example, treatment with human epidermal receptor 2 (HER2) antagonists has markedly improved the prognosis of patients with HER2-positive BC (HER2 + BC). However, HER2+ metastatic BC (MBC) remains prevalent owing to its resistance to conventional anti-HER2 drugs. Therefore, novel agents are needed to overcome the limitations of existing cancer treatments and to enhance the progression-free and overall survival rates. Progress has been made by optimizing the fragment crystallizable (Fc) domain of trastuzumab, an IgG1 monoclonal, chimeric anti-HER2 antibody, to develop margetuximab. The modified Fc domain of margetuximab enhances its binding affinity to CD16A and decreases its binding affinity to CD32B, thereby promoting its antitumor activity. This review summarizes studies on the efficacy of margetuximab, discusses its utility as an anti-HER2 monoclonal antibody drug for the treatment of HER2 + BC, and presents the latest advances in the treatment of BC. This review provides insights into the clinical implication of margetuximab in HER2 + MBC treatment.

O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS

Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics.

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.

Immunotherapeutic effects of recombinant colorectal cancer antigen produced in tomato fruits

The production of pharmacological vaccines in plants has been an important goal in the field of plant biotechnology. GA733-2, the protein that is also known as colorectal carcinoma (CRC)-associated antigen, is a strong candidate to produce a colorectal cancer vaccine. Tomato is the one of the major targets for production of an edible vaccine, as tomato is a fruit consumed in fresh form. It also contains high content of vitamins that aid activation of immune response. In order to develop an edible colorectal cancer vaccine, the transgene rGA733-Fc that encodes a fusion protein of GA733-2, the fragment crystallizable (Fc) domain, and the ER retention motif (rGA733-Fc) was introduced into tomato plants (Solanumlycopersicum cv. Micro-Tom). The transgenic plants producing rGA733-Fc (rGA733-FcOX) protein were screened based on stable integration of transgene expression cassette and expression level of rGA733-Fc protein. Further glycosylation pattern analysis revealed that plant derived rGA733-Fc protein contains an oligomannose glycan structure, which is a typical glycosylation pattern found on ER-processing proteins. The red fruits of rGA733-FcOX transgenic tomato plants containing approximately 270 ng/g FW of rGA733-Fc protein were orally administered to C57BL/6 mice. Oral administration of tomato fruits of the rGA733-Fc expressing transgenic plants delayed colorectal cancer growth and stimulated immune responses compared to oral administration of tomato fruits of the h-Fc expressing transgenic plants in the C57BL/6J mice. This is the first study showing the possibility of producing an edible colorectal cancer vaccine using tomato plants. This research would be helpful for development of plant-derived cancer edible vaccines.

Drug Conjugation via Maleimide-Thiol Chemistry Does Not Affect Targeting Properties of Cysteine-Containing Anti-FGFR1 Peptibodies.

With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody–peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells’ proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide–thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol–maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody–drug conjugates.

Dual checkpoint targeting of B7-H3 and PD-1 with enoblituzumab and pembrolizumab in advanced solid tumors: interim results from a multicenter phase I/II trial

BackgroundAvailability of checkpoint inhibitors has created a paradigm shift in the management of patients with solid tumors. Despite this, most patients do not respond to immunotherapy, and there is considerable...