Novel NKG2D-directed bispecific antibodies enhance antibody-mediated killing of malignant B cells by NK cells and T cells.

The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential.

Evaluation of the Neutralizing Antibody STE90-C11 against SARS-CoV-2 Delta Infection and Its Recognition of Other Variants of Concerns.

As of now, the COVID-19 pandemic has spread to over 770 million confirmed cases and caused approximately 7 million deaths. While several vaccines and monoclonal antibodies (mAb) have been developed and deployed, natural selection against immune recognition of viral antigens by antibodies has fueled the evolution of new emerging variants and limited the immune protection by vaccines and mAb. To optimize the efficiency of mAb, it is imperative to understand how they neutralize the variants of concern (VoCs) and to investigate the mutations responsible for immune escape. In this study, we show the in vitro neutralizing effects of a previously described monoclonal antibody (STE90-C11) against the SARS-CoV-2 Delta variant (B.1.617.2) and its in vivo effects in therapeutic and prophylactic settings. We also show that the Omicron variant avoids recognition by this mAb. To define which mutations are responsible for the escape in the Omicron variant, we used a library of pseudovirus mutants carrying each of the mutations present in the Omicron VoC individually. We show that either 501Y or 417K point mutations were sufficient for the escape of Omicron recognition by STE90-C11. To test how escape mutations act against a combination of antibodies, we tested the same library against bispecific antibodies, recognizing two discrete regions of the spike antigen. While Omicron escaped the control by the bispecific antibodies, the same antibodies controlled all mutants with individual mutations.

Antibody Affinity and Stability Maturation by Error-Prone PCR.

Human antibodies are the most important class of biologicals, and antibodies - human and nonhuman - are indispensable as research agents and for diagnostic assays. When generating antibodies, they sometimes show the desired specificity profile but lack sufficient affinity for the desired application. In this article, a phage display-based method and protocol to increase the affinity of recombinant antibody fragments is given.The given protocol starts with the construction of a mutated antibody gene library by error-prone PCR. Subsequently, the selection of high-affinity variants is performed by panning on immobilized antigen with washing conditions optimized for off-rate-dependent selection. A screening ELISA protocol to identify antibodies with improved affinity and an additional protocol to select antibodies with improved thermal stability is described.

Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library.

A panel of potent neutralizing antibodies are protective against orthopoxvirus (OPXV) infections. For the development of OPXV-specific recombinant human single-chain antibodies (scFvs), the IgG repertoire of four vaccinated donors was amplified from peripheral B-lymphocytes. The resulting library consisted of ≥4 × 108 independent colonies. The immuno-screening against vaccinia virus (VACV) Elstree revealed a predominant selection of scFv clones specifically binding to the D8 protein. The scFv-1.2.2.H9 was engineered into larger human scFv-Fc-1.2.2.H9 and IgG1-1.2.2.H9 formats to improve the binding affinity and to add effector functions within the human immune response. Similar binding kinetics were calculated for scFv-1.2.2.H9 and scFv-Fc-1.2.2.H9 (1.61 nM and 7.685 nM, respectively), whereas, for IgG1-1.2.2.H9, the Michaelis-Menten kinetics revealed an increased affinity of 43.8 pM. None of the purified recombinant 1.2.2.H9 formats were able to neutralize VACV Elstree in vitro. After addition of 1% human complement, the neutralization of ≥50% of VACV Elstree was achieved with 0.0776 µM scFv-Fc-1.2.2.H9 and 0.01324 µM IgG1-1.2.2.H9, respectively. In an in vivo passive immunization NMRI mouse model, 100 µg purified scFv-1.2.2.H9 and the IgG1-1.2.2.H9 partially protected against the challenge with 4 LD50 VACV Munich 1, as 3/6 mice survived. In contrast, in the scFv-Fc-1.2.2.H9 group, only one mouse survived the challenge.

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy.

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.

Effects of a Multimerized Recombinant Autoantibody Against Amyloid-ß.

Alzheimer's disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved. As promising approach passive immunization strategies have thereby come forth. In this study, we focused on naturally occurring autoantibodies against the AD-associated peptide amyloid-ß. These antibodies have already reported to show beneficial functions in vitro and in mouse models of AD. However, their availability is limited due to their low abundance in peripheral blood. In a recent study, we were able to generate four recombinant antibodies against amyloid-ß. In the present study, we tested these antibodies in ELISA and SPR assays for their binding behavior and by aggregation- and phagocytosis assays as functional evidences to characterize their amyloid-ß-related neutralizing and clearance abilities. Further ex vivo assay on organotypic hippocampal slice cultures gave first evidence of microglial activation and inflammatory features. The tested recombinant antibodies in IgG format showed, in comparison to naturally occurring autoantibodies against amyloid-ß, insufficient binding capacities and -affinities. However, after conversion of one antibody into a single chain format multimerization of the scFv-Fc construct, the investigated binding capacity and -affinity showed improvements. Further functional assays predict a protective effect of this antibody. Although, all four recombinant antibodies showed binding to amyloid-ß, promising features were only detectable after conversion into a multimeric format. The multimeric scFv-Fc antibody exhibited thereby strong impact on amyloid-ß clearance and inhibition of oligomerization.

COR-101, ein menschlicher Antikörper gegen COVID-19.

COR-101 is a fully human, Fc silenced IgG that was discovered by antibody phage display. It reduced the SARS-CoV-2 virus load in the lung by more than 99 percent in Hamster models and led to much faster recovery. Its mode of action has been elucidated by solving the atomic structure of its interaction with SARS-CoV-2. The antibody competes with ACE2 binding by blocking a large area of the SARS-CoV-2 spike protein.

A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.

In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. We show that single chain variable fragment (scFv) libraries with adequate qualities can readily be cloned in a 'scar-less' manner and that the isolation of antigen-specific antibodies from immunized chickens is feasible within three selection rounds. Moreover, we demonstrate the general applicability of this method by rapidly constructing and panning VHH single domain antibody phage display libraries from immunized Llama repertoires.

Antibody Phage Display: Antibody Selection in Solution Using Biotinylated Antigens.

Antibody phage display is the most used in vitro technology to generate recombinant, mainly human, antibodies as tools for research, for diagnostic assays, and for therapeutics. Up to now (autumn 2018), eleven FDA/EMA-approved therapeutic antibodies were developed using phage display, including the world best-selling antibody adalimumab.A key to generate successfully human antibodies in vitro is the choice of the most appropriate antibody selection method, for our goal. In this book chapter, we describe the antibody selection process (panning) in solution and its advantages over panning on immobilized antigens. Detailed protocols on the panning procedure and the screening of monoclonal binders are given.

Parallelized Microscale Expression of Soluble scFv.

Antibody phage display is a key technology to generate recombinant, mainly human, antibodies for diagnostic and therapy, but also as tools for basic research. After antibody selection by "panning," a crucial step is the screening of monoclonal binders to isolate those which show antigen specificity. For this screening procedure, a highly parallelized approach to produce soluble antibody fragments in microtiter plates is essential. In this chapter, we give the protocol for the parallelized microscale production of scFvs for the screening procedure or further assays.

Regulatory T cells engineered with a novel insulin-specific chimeric antigen receptor as a candidate immunotherapy for type 1 diabetes.

Adoptive immunotherapy with ex vivo expanded, polyspecific regulatory T cells (Tregs) is a promising treatment for graft-versus-host disease. Animal transplantation models used by us and others have demonstrated that the adoptive transfer of allospecific Tregs offers greater protection from graft rejection than that of polyclonal Tregs. This finding is in contrast to those of autoimmune models, where adoptive transfer of polyspecific Tregs had very limited effects, while antigen-specific Tregs were promising. However, antigen-specific Tregs in autoimmunity cannot be isolated in sufficient numbers. Chimeric antigen receptors (CARs) can modify T cells and redirect their specificity toward needed antigens and are currently clinically used in leukemia patients. A major benefit of CAR technology is its "off-the-shelf" usability in a translational setting in contrast to major histocompatibility complex (MHC)-restricted T cell receptors. We used CAR technology to redirect T cell specificity toward insulin and redirect T effector cells (Teffs) to Tregs by Foxp3 transduction. Our data demonstrate that our converted, insulin-specific CAR Tregs (cTregs) were functional stable, suppressive and long-lived in vivo. This is a proof of concept for both redirection of T cell specificity and conversion of Teffs to cTregs.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

Construction of Human Immune and Naive scFv Libraries.

Antibody phage display is the most commonly used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. The prerequisite for successful generation of antibodies using phage display is the construction of high-quality antibody gene libraries. Here, we give the detailed methods for the construction of human immune and naive scFv gene libraries.

Parallelized Antibody Selection in Microtiter Plates.

The most common in vitro technology to generate human antibodies is phage display. This technology is a key technology to select recombinant antibodies for the use as research tools, in diagnostic tests, and for the development of therapeutics.In this review, the high-throughput compatible selection of antibodies (scFv) in microtiter plates is described. The given detailed protocols allow the antibody selection ("panning"), screening and identification of monoclonal antibodies in less than 1 week.

Phage Display and Selections on Cells.

Antibody identification by phage display on protein or peptide targets is well established and many protocols are available. But there are many targets that cannot be expressed recombinantly or, like peptides, do not reflect correct folding of the protein. Most of these targets are cell surface receptors. Here, we describe a protocol for a panning strategy on cells to obtain specific binders to cell surface receptors. A depletion step is included to prevent enrichment of antibodies that bind to unwanted targets. Each step of the protocol is explained and variations of this protocol are given.

Antibody Affinity and Stability Maturation by Error-Prone PCR.

Antibodies are the fastest growing class of pharmaceutical proteins and essential tools for research and diagnostics. Often antibodies do show a desirable specificity profile but lack sufficient affinity for the desired application. Here, we describe a method to increase the affinity of recombinant antibody fragments based on the construction of mutagenized phage display libraries.After the construction of a mutated antibody gene library by error-prone PCR, selection of high-affinity variants is either performed by panning in solution or on immobilized antigen with washing conditions optimized for off-rate-dependent selection. An additional screening protocol to identify antibodies with improved thermal stability is described.

Designing Human Antibodies by Phage Display.

With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by phage display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.

Generation of Recombinant Antibodies against the beta-(1,6)-Branched beta-(1,3)-D-Glucan Schizophyllan from Immunized Mice via Phage Display.

beta-(1,6)-Branched beta-(1,3)-D-glucans like schizophyllan from the basidiomycete Schizophyllum commune excite various immunostimulatory effects and have been clinically tested as adjuvants. Some of the glucans are also applicable in food or petrol industry due to their viscosity and temperature stability in aqueous solution. Antibodies against these glucans could be used as tool for analysis of glucan preparations or for further research of its bioactivity. Therefore, an immune phage display library was constructed from mice immunized with schizophyllan. Three recombinant monoclonal antibodies were isolated from this library by affinity selection (panning) on schizophyllan. The half-maximal effective concentration (EC50) values for those antibodies varied between 16.4 ng mL-1 and 21.3 ng mL-1. The clones showed binding specificity not only for schizophyllan but also for other beta-(1,6)-branched beta-(1,3)-D-glucans of similar macromolecular structure. Denaturation of the secondary structure led to a reduced antibody binding, indicating an epitope requiring the correct conformation of the triple helical structure of the glucans.