Selection of bispecific antibodies with optimal developability using FcRn­Ph­HPLC as an optimized FcRn affinity chromatography method.

A challenge when developing therapeutic antibodies is the identification of candidates with favorable pharmacokinetics (PK) early in development. A key determinant of immunoglobulin (IgG) serum half­life in vivo is the efficiency of pH-dependent binding to the neonatal Fc receptor (FcRn). Numerous studies have proposed techniques to assess FcRn binding of IgG-based therapeutics in vitro, enabling prediction of serum half-life prior to clinical assessment. FcRn high-performance liquid chromatography (HPLC) assays FcRn binding of therapeutic IgGs across a pH gradient, allowing the correlation of IgG column retention time to the half­life of a therapeutic IgG in vivo. However, as FcRn retention time cannot be directly compared to an in vivo parameter, modifications to FcRn-HPLC are required to enable interpretation of the data within a physiological context, to provide more accurate estimations of serum half-life. This study presents an important modification to this method, FcRn-pH-HPLC, which reproducibly measures FcRn dissociation pH, allowing correlation with previously established half-lives of therapeutic antibodies. Furthermore, the influence of incorporating various antibody modifications, binding modules, and their orientations within IgGs and bispecifics on FcRn dissociation pH was evaluated using antibodies from the redirected optimized cell killing (ROCK®) platform. Target and effector antigen-binding domain sequences, their presentation format and orientation within a bispecific antibody alter FcRn retention; tested Fc domain modifications and incorporating stabilizing disulfide bonds had minimal effect. This study may inform the generation of mono-, bi- and multi-specific antibodies with tailored half-lives based on FcRn binding properties in vitro, to differentiate antibody-based therapeutic candidates with optimal developability.

Cryopreservation of Natural Killer Cells Pre-Complexed with Innate Cell Engagers.

Innate cell engager (ICE®) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the destruction of tumor cells. Pre-complexing of ICE® constructs with adoptive NK cells is a novel approach to enhance NK cell activity. The suitability of such complexes for cryopreservation, whilst retaining the biological activity and specificity, may enable the development of off-the-shelf NK cell products. This study investigates the binding affinity of ICE® constructs targeting EpCAM and NK cell receptors CD16A, NKG2D, or NKp46 to the corresponding antigens, the ICE® antitumor activity, and feasibility of cryopreservation. Cell surface retention assays using primary NK cells confirmed a substantially slower ICE® construct dissociation kinetics compared with control molecules, suggesting the formation of durable complexes independently of the CD16A polymorphism. The high-affinity NK cell and EpCAM/CD16A ICE® complexes were superior to those engaging NKG2D or NKp46 receptors when tested for the NK-cell-mediated elimination of EpCAM-expressing tumor cells. Moreover, the potency and efficacy of these complexes were unaffected after a single freeze-thaw cycle. CD16A-selective ICE® drug candidates complexed with NK cells hold promise as novel cryopreserved off-the-shelf NK cell products with chimeric antigen receptor-like NK cell properties, capable of effective depletion of tumor cells.

Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

10 Minutes Frontal 40 Hz tACS-Effects on Working Memory Tested by Luck-Vogel Task.

Working memory is a cognitive process that involves short-term active maintenance, flexible updating, and processing of goal- or task-relevant information. All frequency bands are involved in working memory. The activities of the theta and gamma frequency bands in the frontoparietal network are highly involved in working memory processes; theta oscillations play a role in the temporal organization of working memory items, and gamma oscillations influence the maintenance of information in working memory. Transcranial alternating current stimulation (tACS) results in frequency-specific modulation of endogenous oscillations and has shown promising results in cognitive neuroscience. The electrophysiological and behavioral changes induced by the modulation of endogenous gamma frequency in the prefrontal cortex using tACS have not been extensively studied in the context of working memory. Therefore, we aimed to investigate the effects of frontal gamma-tACS on working memory outcomes. We hypothesized that a 10-min gamma tACS administered over the frontal cortex would significantly improve working memory outcomes. Young healthy participants performed Luck-Vogel cognitive behavioral tasks with simultaneous pre- and post-intervention EEG recording (Sham versus 40 Hz tACS). Data from forty-one participants: sham (15 participants) and tACS (26 participants), were used for the statistical and behavioral analysis. The relative changes in behavioral outcomes and EEG due to the intervention were analyzed. The results show that tACS caused an increase in the power spectral density in the high beta and low gamma EEG bands and a decrease in left-right coherence. On the other hand, tACS had no significant effect on success rates and response times. Conclusion: 10 min of frontal 40 Hz tACS was not sufficient to produce detectable behavioral effects on working memory, whereas electrophysiological changes were evident. The limitations of the current stimulation protocol and future directions are discussed in detail in the following sections.

Preclinical evaluation of AFM24, a novel CD16A-specific innate immune cell engager targeting EGFR-positive tumors.

Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG1-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with KD values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype.

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity.

Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.

Recombinant Antibodies to Arm Cytotoxic Lymphocytes in Cancer Immunotherapy.

Immunotherapy has the potential to support and expand the body's own armamentarium of immune effector functions, which have been circumvented during malignant transformation and establishment of cancer and is presently considered to be the most promising treatment option for cancer patients. Recombinant antibody technologies have led to a multitude of novel antibody formats, which are in clinical development and hold great promise for future therapies. Among these formats, bispecific antibodies are extremely versatile due to their high efficacy to recruit and activate anti-tumoral immune effector cells, their excellent safety profile, and the opportunity for use in combination with cellular therapies. This review article summarizes the latest developments in cancer immunotherapy using immuno-engagers for recruiting T cells and NK cells to the tumor site. In addition to antibody formats, malignant cell targets, and immune cell targets, opportunities for combination therapies, including check point inhibitors, cytokines and adoptive transfer of immune cells, will be summarized and discussed.

Highly Specific and Effective Targeting of EGFRvIII-Positive Tumors with TandAb Antibodies.

To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving KDs in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. These antibodies exhibited an exquisite specificity for a distinguished epitope in the N-terminal portion of EGFRvIII, as shown on recombinant antigen in Western Blot, SPR, and ELISA, as well as on antigen-expressing cells in FACS assays, and did not bind to the wild-type EGFR. High-affinity EGFRvIII/CD3 TandAbs were most potent in killing assays, displaying cytotoxicity toward EGFRvIII-expressing CHO, F98 glioma, or human DK-MG cells with EC50 values in the range of 1-10 pM in vitro. They also demonstrated dose-dependent growth control in vivo in an EGFRvIII-positive subcutaneous xenograft tumor model. Together with the tumor-exclusive expression of EGFRvIII, the EGFRvIII/CD3 TandAbs' high specificity and strictly target-dependent activation with no off-target activity provide an opportunity to target tumor cells and spare normal tissues, thereby reducing the side effects associated with other anti-EGFR therapies. In summary, EGFRvIII/CD3 TandAbs are highly attractive therapeutic antibody candidates for selective immunotherapy of EGFRvIII-positive tumors.

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening.

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a "swiss army knife solution" for today and future high throughput antibody drug screenings.

Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells.

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.