Generation of Multispecific Antibodies with Immune Cell Modulating Functions

Within the field of biologics, monoclonal antibodies have ruled the market with blockbuster and top selling drugs over the last decades. With the advancements in technological discoveries, lightspeed progress has been achieved to discover novel antibodies and mechanisms of action to address unmet needs and indications. Particularly, display technologies, in vitro systems, and high/ultra-high-throughput technologies have shown monumental progress to ensure the rapid discovery of novel biological entities. In the first study presented herein, the focus laid on the generation of a bidirectional plasmid for recombinant antibody production in mammalian cells to facilitate native antibody folding and post-translational modifications. Conventional approaches for transient antibody production utilise co-transfection of heavy and light chain genes encoded on separate plasmids. Here, a single plasmid under the control of two independent promoters, constructed in a bidirectional fashion, is used. This study assessed promoter combinations resulting in the best antibody yields of two U.S. Food and Drug Administration (FDA)-approved antibodies, durvalumab and avelumab. By comparing promoters with varying strengths (CMV, minCMV, EF-1α and enhanced CMV), gene expression of heavy and light chain genes and subsequent IgG1 yields gave rise to the 2xeCMV combination, consisting of two mirrored eCMV cassettes controlling the expression of the light and heavy chains individually in each direction. This combination effectuated the most promising mRNA synthesis for both chains in two regularly used mammalian cell lines, human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO) cells, and the highest yields after IgG quantification, comparable to the conventional co-transfection method. By substituting the co-transfection approach with this bidirectional plasmid, lower plasmid preparation efforts are required and further facilitates the handling of a higher number of mAb candidates simultaneously. In the second study, the described bidirectional plasmid was put into practise by generating a Fab-presenting yeast surface display (YSD) library from immunised OmniRats. After screening of antibody formats via fluorescence-activated cell sorting (FACS), reformation of single candidates into their final IgG format is required, rapidly converting itself into a cumbersome step, and often resulting in the bottleneck to proceed with further characterisation. Within this study, a novel workflow based on Golden Gate Cloning (GGC) was established, allowing the bulk reformatting of antibody candidates after YSD FACS screening. By using an OmniRat-derived Fab library against MerTK, two screening rounds of YSD were performed by FACS. Subsequently, the antibody-encoding genes were transferred into a Mammalian_Destination (MD) vector, which contained a partial hinge-CH2-CH3 sequence, resulting in a full-length heavy chain after GGC with Esp3I. In order to produce the full-length variants, the yeast-specific Gal1,10 promoter was exchanged for the described promoter cassette combinations from the first study, 2xeCMV, by a final GGC step with BbsI. After assembly, the resulting MD vector contained the variable domains from the second sorting round with the respective constant domains required for the production of full-length IgG molecules. Next generation sequencing (NGS) of the screening rounds confirmed that the entire VH family diversity was covered in the resulting clones after bulk reformatting. Ten candidates were subsequently transiently expressed in mammalian cells and characterised for target binding and biophysical properties. This workflow presented a two-pot, two-step, PCR-free method to transition from YSD to a mammalian expression vector, eliminating any unwanted polymerase-introduced mutations and allowing for bulk cloning of yeast display-enriched antibody fragments. By this procedure, heavy and light chain pairing is conserved, contrary to other reformatting approaches, and paves the way to accelerate antibody hit discovery campaigns with YSD. Furthermore, this platform is malleable to other antibody formats and immunisation hosts, such as single chain variable fragments (scFvs) and chickens, and has the potential to be developed for bispecific or multispecific antibodies. Next-generation antibodies, including bi- and multispecific antibodies, have been set under the spotlight for their ability to combine multiple modes of action simultaneously and result in higher efficacy, where monoclonal antibodies are lacking. A special class of such are immune cell engagers which target immune cells and tumour-associated antigens (TAAs) to create an immune synapse. Depending on the effector cell being targeted, specialised killing mechanisms are triggered to efficiently kill the targeted cells. Macrophage engagers are aimed at forcing targeted phagocytosis of the engaged cell type and have typically targeted the CD47/SIRPα axis up to date, known as the “do not eat me” signal. Nevertheless, targeting CD47 lacks specificity due to its ubiquitous expression pattern. On the other hand, T-cell engagers (TCEs) result in very specialised signals by targeting CD3 on T cells and additional TAAs. The hyperactivation of T cells results in a feedback loop through the activation of macrophages and the over-release of cytokines, resulting in cytokine storms or cytokine release syndrome (CRS). If left untreated, these can provoke life-threatening conditions. Thus, macrophage engagers and TCEs require novel cell-specific targets and widening of their therapeutic windows for restored patient alleviation. In the third study within this cumulative thesis, the first bispecific macrophage engager targeting the receptor tyrosine kinase MerTK and epidermal growth factor receptor (EGFR) is presented. From the 10 antibody candidates derived in the second study, one candidate displayed agonistic properties, detected by the dose-dependent activation of the downstream signalling molecule phospho AKT (pAKT). MerTK’s overexpression on macrophages and tumour-associated macrophages within the tumour microenvironment (TME) lays the foundation to generate macrophage-engaging bispecific antibodies for targeted phagocytosis of tumour cells. Therefore, tandem biparatopic EGFR-binding VHH molecules (termed 7D9G) were combined in different architectures to generate bispecific molecules. By using the Knob-into-Hole technology, a bispecific with a MerTK-binding Fab arm and an EGFR-binding tandem VHH arm were generated, abolishing the agonistic properties of the parental MerTK mAb. On the other hand, a tetravalent bispecific antibody was generated by fusing the tandem VHHs to the C-terminus of the CH3 domain, resulting in intact MerTK-binding Fabs. The bispecific antibodies were able to bind both targets simultaneously in their soluble form and engage macrophages with EGFR+ tumour cells. Furthermore, they were able to compete with the binding site of EGF and therefore inhibit EGF-mediated signalling transduction by inhibiting pAKT. EGFR domain mapping of 7D9G by YSD resulted in binding to domain III of the extracellular EGFR domain, confirming its ligand-inhibiting abilities. Moreover, the bispecific antibodies resulted in targeted phagocytosis of EGFR+ tumour cells by macrophage-like THP-1 cells. This work represents the first bispecific macrophage-engager targeting MerTK for immuno-oncology applications by harnessing its expression and role in the tumour microenvironment to selectively phagocytise tumour cells. In the last study presented here, a trispecific T-cell engager and cytokine release modulating antibody (TriTECM) was generated. In brief, a tetravalent, bispecific two-in-one antibody binding EGFR and PD-L1 simultaneously with a single Fab arm was combined with anti-CD3 and anti-IL-6R scFvs, derived from foralumab or sarilumab, respectively. By testing two TriTECM architectures varying mainly in the anti-CD3 scFv positioning and valency of IL-6R binding, tetraspecific molecules were generated with multiple mechanisms of action. Firstly, increased tumour specificity was ensured by targeting EGFR and PD-L1 with a low nanomolar on-cell affinity. Checkpoint inhibition by blockage of the PD-1/PD-L1 axis was mediated by binding to PD-L1. T-cell engagement and subsequent T-cell-mediated cytotoxicity was attenuated, resulting in reduced pro-inflammatory cytokine release. And lastly, inhibition of the IL-6/IL-6R pathway can modulate cytokine storms after T-cell activation. The attenuation of CD3 binding could allow existing CD3-binders to be used, that were previously shown to result in cytotoxicity. With cytokine release still putting obstacles in the way of novel immune cell engagers, TriTECM designs represent a novel class of therapeutics with the potential to inertly modulate over-activated immune responses and widen the therapeutic index of T-cell-engaging therapeutics

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

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

Estimating diagnostic accuracy of tests for latent tuberculosis infection without a gold standard among healthcare workers

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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, chickenderived 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

Study of the Application of CCUS in a WtE Italian Plant

Municipal Solid Waste (MSW) contains materials of biogenic and non-biogenic origin. When incinerated, the biogenic component produces CO2, which does not lead to an increase in atmospheric CO2 levels. For WtE plants operating on MSW with a significant biogenic component, Carbon Capture Utilization and Storage (CCUS) can provide a path to negative CO2 emissions by producing energy and managing locally produced waste. This work focuses on the study of a process for treating a flue gas stream from WtE in an Italian context, i.e. the incinerator plant located in Como, to remove CO2 that is, then, planned to be utilized. The CO2 capture process is based on chemical absorption by two different amine solvents: MonoEthanolAmine and Piperazine. The design of the CO2 removal section has been carried out specifically for the considered flue gas to be treated, containing about 7 mol % CO2, by selecting the main process parameters (e.g., absorber packing height, regenerator packing height, lean loading, gas inlet temperature, solvent inlet temperature, regenerator pressure) in order to optimize the reboiler duty and the water requirement. The performances of the two processes have been compared for the same 90 % removal of CO2

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

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

Is IP-10 an Accurate Marker for Detecting M. tuberculosis-Specific Response in HIV-Infected Persons?

The suboptimal sensitivity of Interferon (IFN)-γ-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis (TB). The objective of this study was to evaluate whether interferon-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-2 and interleukin (IL)-2 can be useful biomarkers for evaluating a specific response to RD1 antigens associated to active TB disease in HIV-infected individuals.The study was carried out in India, the country with the highest TB burden in the world. Sixty-six HIV-infected individuals were prospectively enrolled, 28 with active-pulmonary-TB and 38 without. The whole blood assay based on RD1-selected peptides (experimental test) and QuantiFERON-TB Gold In tube (QFT-IT) was performed. Plasma was harvested at day-1-post-culture and soluble factors were evaluated by ELISA. The results indicate that by detecting IP-10, the sensitivity of the experimental test and QFT-antigen (75% and 85.7% respectively) for active TB was higher compared to the same assays based on IFN-γ (42.9% and 60.7% respectively) and was not influenced by the ability to respond to the mitogen. By detecting IP-10, the specificity of the experimental test and QFT-antigen (57.9% and 13.2% respectively) for active TB was lower than what was reported for the same assays using IFN-γ-detection (78.9% and 68.4% respectively). On the other side, in vitro IL-2 and MCP-2 responses were not significantly associated with active TB.HIV infection does not impair RD1-specific response detected by IP-10, while it significantly decreases IFN-γ-mediated responses. At the moment it is unclear whether higher detection is related to higher sensitivity or lower specificity of the assay. Further studies in high and low TB endemic countries are needed to elucidate this