HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Potent Apoptosis Induction by a Novel Trispecific B7-H3xCD16xTIGIT 2+1 Common Light Chain Natural Killer Cell Engager.

Valued for their ability to rapidly kill multiple tumor cells in succession as well as their favorable safety profile, NK cells are of increasing interest in the field of immunotherapy. As their cytotoxic activity is controlled by a complex network of activating and inhibiting receptors, they offer a wide range of possible antigens to modulate their function by antibodies. In this work, we utilized our established common light chain (cLC)-based yeast surface display (YSD) screening procedure to isolate novel B7-H3 and TIGIT binding monoclonal antibodies. The chicken-derived antibodies showed single- to low-double-digit nanomolar affinities and were combined with a previously published CD16-binding Fab in a 2+1 format to generate a potent NK engaging molecule. In a straightforward, easily adjustable apoptosis assay, the construct B7-H3xCD16xTIGIT showed potent apoptosis induction in cancer cells. These results showcase the potential of the TIGIT NK checkpoint in combination with activating receptors to achieve increased cytotoxic activity.

A First-in-Human Randomized Study to Assess the Safety, Tolerability, Pharmacokinetics, and Neutralization Profile of Two Investigational Long-Acting Anti-SARS-CoV-2 Monoclonal Antibodies.

INTRODUCTION: COVID-19 remains a significant risk for the immunocompromised given their lower responsiveness to vaccination or infection. Therefore, passive immunity through long-acting monoclonal antibodies (mAbs) offers a needed approach for pre-exposure prophylaxis (PrEP). Our study evaluated safety, anti-SARS-CoV-2 neutralizing activity, nasal penetration, and pharmacokinetics (PK) of two half-life-extended investigational mAbs, AER001 and AER002, providing the first demonstration of upper airway penetration of mAbs with the LS-modification. METHODS: This randomized, double-blind, placebo-controlled phase I study enrolled healthy adults (n = 80) who received two long-acting COVID mAbs (AER001 and AER002), AER002 alone, or placebo. The dose ranged from 100 mg (mg) to 1200 mg per mAb component. The primary objective was to describe the safety and tolerability following intravenous (IV) administration. Secondary objectives were to describe PK, anti-drug antibodies (ADA), neutralization activity levels, and safety evaluation through 6 months of follow-up. RESULTS: The majority (97.6%) of the reported adverse events (AE) post administration were of grade 1 severity. There were no serious adverse events (SAE) or ADAs. AER001 and AER002 successfully achieved an extended half-life of 105 days and 97.5 days, respectively. Participants receiving AER001 and AER002 (300 mg each) or AER002 (300 mg) alone showed 15- and 26-fold higher neutralization levels against D614G and omicron BA.1 than the placebo group 24 h post-administration. Single 300 or 1200 mg IV dose of AER001 and AER002 resulted in nasal mucosa transudation of approximately 2.5% and 2.7%, respectively. CONCLUSION: AER001 and AER002 showed an acceptable safety profile and extended half-life. High serum neutralization activity was observed against D614G and Omicron BA.1 compared to the placebo group. These data support that LS-modified mAbs can achieve durability, safety, potency, and upper airway tissue penetration and will guide the development of the next generation of mAbs for COVID-19 prevention and treatment. TRIAL REGISTRATION: EudraCT Number 2022-001709-35 (COV-2022-001).

Bulk Reformatting of Antibody Fragments Displayed on the Surface of Yeast Cells to Final IgG Format for Mammalian Production.

While yeast surface display (YSD) has gained traction for antibody hit discovery efforts with the first therapeutic YSD-isolated antibody sintilimab approved in 2018, a major drawback that remains is the time-consuming reformatting of monoclonal antibody (mAb) candidates. By using a Golden Gate cloning (GGC)-dependent workflow, the bulk transfer of genetic information can be performed from antibody fragments displayed on yeast cells to a bidirectional mammalian expression vector. Herein, we describe in-depth protocols for the reformatting of mAbs, starting from the generation of Fab fragment libraries in YSD vectors and ending up with IgG molecules in bidirectional mammalian vectors in a consolidated two-pot, two-step procedure.

Cell Line Development Using Targeted Gene Integration into MAR-Rich Landing Pads for Stable Expression of Transgenes.

Integration of a gene of interest (GOI) into the genome of mammalian cells is the first step of cell line development campaigns for the production of biotherapeutics. Besides random integration methods, targeted gene integration approaches have emerged as promising tools over the last few years. In addition to reducing heterogeneity within a pool of recombinant transfectants, this process can also facilitate shorter timelines of the current cell line development process. Herein, we describe protocols for generating host cell lines carrying matrix attachment region (MAR)-rich landing pads (LPs), including BxB1 recombination sites. These LP-containing cell lines allow for site-specific and simultaneous integration of multiple GOIs. The resulting transgene-expressing stable recombinant clones can be used for the production of mono- or multispecific antibodies.

Generation of a symmetrical trispecific NK cell engager based on a two-in-one antibody.

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures.

Targeted Phagocytosis Induction for Cancer Immunotherapy via Bispecific MerTK-Engaging Antibodies.

The Tyro, Axl, and MerTK receptors (TAMRs) play a significant role in the clearance of apoptotic cells. In this work, the spotlight was set on MerTK, as it is one of the prominent TAMRs expressed on the surface of macrophages and dendritic cells. MerTK-specific antibodies were previously isolated from a transgenic rat-derived immune library with suitable biophysical properties. Further characterisation resulted in an agonistic MerTK antibody that led to phospho AKT activation in a dose-dependent manner. In this proof-of-concept study, a MerTK-specific antibody, MerK28, was combined with tandem, biparatopic EGFR-binding VHH camelid antibody domains (7D9G) in different architectures to generate bispecific antibodies with the capacity to bind EGFR and MerTK simultaneously. The bispecific molecules exhibited appropriate binding properties with regard to both targets in their soluble forms as well as to cells, which resulted in the engagement of macrophage-like THP-1 cells with epidermoid carcinoma A431 cells. Furthermore, targeted phagocytosis in co-culture experiments was observed only with the bispecific variants and not the parental MerTK-binding antibody. This work paves the way for the generation of bispecific macrophage-engaging antibodies for targeted phagocytosis harnessing the immune-modulating roles of MerTK in immunotherapy.

TriTECM: A tetrafunctional T-cell engaging antibody with built-in risk mitigation of cytokine release syndrome.

Harnessing the innate power of T cells for therapeutic benefit has seen many shortcomings due to cytotoxicity in the past, but still remains a very attractive mechanism of action for immune-modulating biotherapeutics. With the intent of expanding the therapeutic window for T-cell targeting biotherapeutics, we present an attenuated trispecific T-cell engager (TCE) combined with an anti- interleukin 6 receptor (IL-6R) binding moiety in order to modulate cytokine activity (TriTECM). Overshooting cytokine release, culminating in cytokine release syndrome (CRS), is one of the severest adverse effects observed with T-cell immunotherapies, where the IL-6/IL-6R axis is known to play a pivotal role. By targeting two tumour-associated antigens, epidermal growth factor receptor (EGFR) and programmed death ligand 1 (PD-L1), simultaneously with a bispecific two-in-one antibody, high tumour selectivity together with checkpoint inhibition was achieved. We generated tetrafunctional molecules that contained additional CD3- and IL-6R-binding modules. Ligand competition for both PD-L1 and IL-6R as well as inhibition of both EGF- and IL-6-mediated signalling pathways was observed. Furthermore, TriTECM molecules were able to activate T cells and trigger T-cell-mediated cytotoxicity through CD3-binding in an attenuated fashion. A decrease in pro-inflammatory cytokine interferon γ (IFNγ) after T-cell activation was observed for the TriTECM molecules compared to their respective controls lacking IL-6R binding, hinting at a successful attenuation and potential modulation via IL-6R. As IL-6 is a key player in cytokine release syndrome as well as being implicated in enhancing tumour progression, such molecule designs could reduce side effects and cytotoxicity observed with previous TCEs and widen their therapeutic windows.

Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells.

Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents.

A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization.

Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two-in-One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties.

Antibody Library Screening Using Yeast Biopanning and Fluorescence-Activated Cell Sorting.

Yeast surface display (YSD) emerged as a prominent screening methodology for the isolation of monoclonal antibodies (mAbs) against various antigens. However, phage display remains the gold standard in cell panning-based screenings to isolate mAbs against difficult-to-screen targets, such as G-protein coupled receptors (GPCR) and ion channels. Herein we describe a step-by-step protocol to establish and perform the isolation of mAbs using YSD in a fluorescence-activated cell sorting (FACS)-assisted biopanning manner, yielding a variety of antibodies binding their antigen with high affinity in the natural environment of the cell. Upon mixing antibody-displaying yeast cells with antigen-displaying mammalian cells, complexes are specifically formed and isolated for enrichment of yeast cells encoding binders against the antigen. The utilization of mammalian cells expressing the respective target accounts for accessibility of the epitope and the correct conformation of the antigen. Furthermore, critical characterization methods mandatory for this kind of antibodies are illuminated.

Humanization of Chicken-Derived Antibodies by Yeast Surface Display.

Chicken-derived antibodies emerged as a promising tool for diagnostic and therapeutic usage. Due to the phylogenetic distance between birds and mammals, chicken immunization campaigns with human antigens result in a chicken antibody (IgY) repertoire targeting epitopes not addressed by rodent-derived antibodies. However, this phylogenetic distance accounts for a low homology of IgY molecules to human antibodies, resulting in potential immunogenicity and thus excluding IgYs from therapeutic applications. Herein, we describe a straightforward method to efficiently humanize chicken-derived antibodies by a CDR-grafting-based approach, including a simultaneous randomization of key residues (Vernier residues). Utilizing yeast surface display (YSD) and fluorescence-activated cell sorting (FACS), yeast cells displaying functional humanized scFvs and Fab variants are isolated, and subsequent next-generation sequencing (NGS) enables the identification of humanized antibody variants with restored affinity and beneficial protein characteristics.

Generation of a host cell line containing a MAR-rich landing pad for site-specific integration and expression of transgenes.

In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix-Attachment Region (MAR)-rich landing pads (LPs), which allow for the simultaneous and site-specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR-based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP-containing host cell lines can be used for the site-specific integration of several GOIs and thus, generation of transgene-expressing stable recombinant clones. Transgene expression was shown by site-specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi-specific antibody (bsAb). The genetic stability of the herein described LP-based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR-rich landing pads can be successfully used for stable expression of one or several GOIs.

Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System.

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner.

Treating Bladder Cancer: Engineering of Current and Next Generation Antibody-, Fusion Protein-, mRNA-, Cell- and Viral-Based Therapeutics.

Bladder cancer is a frequent malignancy and has a clinical need for new therapeutic approaches. Antibody and protein technologies came a long way in recent years and new engineering approaches were applied to generate innovative therapeutic entities with novel mechanisms of action. Furthermore, mRNA-based pharmaceuticals recently reached the market and CAR-T cells and viral-based gene therapy remain a major focus of biomedical research. This review focuses on the engineering of biologics, particularly therapeutic antibodies and their application in preclinical development and clinical trials, as well as approved monoclonal antibodies for the treatment of bladder cancer. Besides, newly emerging entities in the realm of bladder cancer like mRNA, gene therapy or cell-based therapeutics are discussed and evaluated. As many discussed molecules exhibit unique mechanisms of action based on innovative protein engineering, they reflect the next generation of cancer drugs. This review will shed light on the engineering strategies applied to develop these next generation treatments and provides deeper insights into their preclinical profiles, clinical stages, and ongoing trials. Furthermore, the distribution and expression of the targeted antigens and the intended mechanisms of action are elucidated.

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture.

Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations.

Isolation of Common Light Chain Antibodies from Immunized Chickens Using Yeast Biopanning and Fluorescence-Activated Cell Sorting.

The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here the authors report the isolation of common light chain (cLC)-based chicken monoclonal antibodies from an anti-epidermal growth factor receptor (EGFR) immune library utilizing yeast surface display in combination with yeast biopanning and fluorescence-activated cell sorting (FACS). For the selection of high-affinity antibodies, a yeast cell library presenting cLC-comprising fragment antigen binding (Fab) fragments is panned against hEGFR-overexpressing A431 cells. The resulting cell-cell-complexes are sorted by FACS resulting in gradual enrichment of EGFR-binding Fabs in three sorting rounds. The isolated antibodies share the same light chain and show high specificity for EGFR, resulting in selective binding to A431 cells with notable EC50 values. All identified antibodies show very good aggregation propensity profiles and thermostabilities. Additionally, epitope binning demonstrates that these cLC antibodies cover a broad epitope space. Isolation of antibodies from immunized chickens by yeast cell biopanning makes an addition to the repertoire of methods for antibody library screening, paving the way for the generation of cLC-based bispecific antibodies against native mammalian receptors.

From cell line development to the formulated drug product: The art of manufacturing therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies and related products have steadily grown to become the dominant product class within the biopharmaceutical market. Production of antibodies requires special precautions to ensure safety and efficacy of the product. In particular, minimizing antibody product heterogeneity is crucial as drug substance variants may impair the activity, efficacy, safety, and pharmacokinetic properties of an antibody, consequently resulting in the failure of a product in pre-clinical and clinical development. This review will cover the manufacturing and formulation challenges and advances of therapeutic monoclonal antibodies, focusing on improved processes to minimize variants and ensure batch-to-batch consistency. Processes put in place by regulatory agencies, such as Quality-by-Design (QbD) and current Good Manufacturing Practices (cGMP), and how their implementation has aided drug development in pharmaceutical companies will be reviewed. Advances in formulation and considerations on the intended use of a therapeutic antibody, including the route of administration and patient compliance, will be discussed.

Humanization of Chicken-Derived scFv Using Yeast Surface Display and NGS Data Mining.

Generation of high-affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken-derived antibodies. To this end, high-affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high-affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high-affinity binders in the scFv and Fab formats. Next-generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high-throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance.

Engineering therapeutic antibodies for patient safety: tackling the immunogenicity problem.

Established monoclonal antibodies (mAbs) allow treatment of cancers, autoimmune diseases and other severe illnesses. Side effects either arise due to interaction with the target protein and its biology or result from of the patient's immune system reacting to the foreign protein. This immunogenic reaction against therapeutic antibodies is dependent on various factors. The presence of non-human sequences can trigger immune responses as well as chemical and post-translational modifications of the antibody. However, even fully human antibodies can induce immune response through T cell epitopes or aggregates. In this review, we briefly describe, how therapeutic antibodies can interact with the patient's immune system and summarize recent advancements in protein engineering and in silico methods to reduce immunogenicity of therapeutic monoclonal antibodies.