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1.
MAbs ; 16(1): 2406548, 2024.
Article in English | MEDLINE | ID: mdl-39304998

ABSTRACT

In this work, we report the discovery of potent anti-epidermal growth factor receptor (EGFR) allosteric heavy-chain antibodies by combining camelid immunization and fluorescence-activated cell sorting (FACS). After immunization and yeast surface display library construction, allosteric clones were obtained by introducing the labeled EGF Fc fusion protein as an additional criterion for FACS. This sorting method enabled the identification of 11 heavy-chain antibodies that did not compete with the orthosteric ligand EGF for the binding to EGFR. These antibodies bind to a triple-negative breast cancer cell line expressing EGFR with affinities in the picomolar to nanomolar range. Those camelid-derived antibodies also exhibit interesting properties by modulating EGFR affinity for EGF. Moreover, they are also able to inhibit EGF-induced downstream signaling pathways. In particular, we identified one clone that is more potent than the approved blocking antibody cetuximab in inhibiting both PI3K/AKT and MAPK/ERK pathways. Our results suggest that allosteric antibodies may be potential new modalities for therapeutics.


Subject(s)
ErbB Receptors , Humans , ErbB Receptors/immunology , ErbB Receptors/antagonists & inhibitors , Animals , Allosteric Regulation/drug effects , Cell Line, Tumor , Camelids, New World/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/immunology , Signal Transduction/drug effects , Signal Transduction/immunology , Cetuximab/pharmacology , Cetuximab/immunology , Cetuximab/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Flow Cytometry
2.
MAbs ; 16(1): 2315640, 2024.
Article in English | MEDLINE | ID: mdl-38372053

ABSTRACT

Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.


Subject(s)
Epidermal Growth Factor , Killer Cells, Natural , Cetuximab/metabolism , Epidermal Growth Factor/metabolism , Binding Sites, Antibody , ErbB Receptors/metabolism , Epitopes/metabolism
3.
MAbs ; 15(1): 2236265, 2023.
Article in English | MEDLINE | ID: mdl-37469014

ABSTRACT

Here, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rß. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.


Subject(s)
Antibodies, Bispecific , Single-Domain Antibodies , Interleukin-18 , Leukocytes, Mononuclear , Binding Sites, Antibody
4.
Methods Mol Biol ; 2681: 47-60, 2023.
Article in English | MEDLINE | ID: mdl-37405642

ABSTRACT

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.


Subject(s)
Bacteriophages , Single-Chain Antibodies , Peptide Library , Cell Surface Display Techniques/methods , Recombinant Proteins/genetics , Immunoglobulin Light Chains/genetics , Antibodies/genetics , Bacteriophages/genetics , Immunoglobulin Fragments/genetics , Single-Chain Antibodies/genetics , Cloning, Molecular
5.
Methods Mol Biol ; 2681: 213-229, 2023.
Article in English | MEDLINE | ID: mdl-37405650

ABSTRACT

Classical yeast surface display (YSD) antibody immune libraries are generated by a separate amplification of heavy- and light-chain antibody variable regions (VH and VL, respectively) and subsequent random recombination during the molecular cloning procedure. However, each B cell receptor comprises a unique VH-VL combination, which has been selected and affinity matured in vivo for optimal stability and antigen binding. Thus, the native variable chain pairing is important for the functioning and biophysical properties of the respective antibody. Herein, we present a method for the amplification of cognate VH-VL sequences, compatible with both next-generation sequencing (NGS) and YSD library cloning. We employ a single B cell encapsulation in water-in-oil droplets, followed by a one-pot reverse transcription overlap extension PCR (RT-OE-PCR), resulting in a paired VH-VL repertoire from more than a million B cells in a single day.


Subject(s)
Antibodies , Reverse Transcription , Polymerase Chain Reaction , Gene Library , Cloning, Molecular
6.
J Immunol ; 209(9): 1724-1735, 2022 11 01.
Article in English | MEDLINE | ID: mdl-36104113

ABSTRACT

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell-mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.


Subject(s)
Cytokines , Natural Cytotoxicity Triggering Receptor 3 , Humans , B7 Antigens/metabolism , Cell Death , Cetuximab/pharmacology , Epitopes , ErbB Receptors , Killer Cells, Natural , Ligands , Natural Cytotoxicity Triggering Receptor 3/metabolism
7.
Biochim Biophys Acta Gen Subj ; 1866(7): 130155, 2022 07.
Article in English | MEDLINE | ID: mdl-35469978

ABSTRACT

BACKGROUND: Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation. METHODS: We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the CH2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines. RESULTS: Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines. CONCLUSION: By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs. GENERAL SIGNIFICANCE: Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques.


Subject(s)
Antineoplastic Agents , Immunoconjugates , Antineoplastic Agents/pharmacology , Cysteine/chemistry , Immunoconjugates/chemistry , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Mass Spectrometry , Trastuzumab/pharmacology
8.
MAbs ; 14(1): 2018960, 2022.
Article in English | MEDLINE | ID: mdl-35014603

ABSTRACT

Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs), with several in clinical development and one product, amivantamab, approved by the Food and Drug Administration. In this study, we expand the cFAE-toolbox by incorporating VHH-modules at the C-termini of DB-IgGs, termed DB-VHHs. This approach enables the combinatorial generation of tri- and tetraspecific molecules with flexible valencies in a straightforward fashion. Using cFAE, a variety of multispecific molecules was produced and assessed for manufacturability and physicochemical characteristics. In addition, we were able to generate DB-VHHs that efficiently triggered natural killer cell mediated lysis of tumor cells, demonstrating the utility of this format for potential therapeutic applications.


Subject(s)
Antibodies, Bispecific , Antibody Specificity/genetics , Immunoglobulin Fab Fragments , Protein Engineering , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics
9.
Front Immunol ; 12: 742418, 2021.
Article in English | MEDLINE | ID: mdl-34759924

ABSTRACT

In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.


Subject(s)
Antibodies/immunology , Cytotoxicity, Immunologic/immunology , Killer Cells, Natural/immunology , Animals , Antibody Affinity , Cattle , Complementarity Determining Regions , ErbB Receptors/immunology , Humans , Protein Engineering/methods
10.
Oncoimmunology ; 10(1): 1958590, 2021.
Article in English | MEDLINE | ID: mdl-34484871

ABSTRACT

Avelumab is an IgG1 anti-programmed death ligand 1 (anti-PD-L1) monoclonal antibody that has been approved as a monotherapy for metastatic Merkel cell carcinoma and advanced urothelial carcinoma, and in combination with axitinib for advanced renal cell carcinoma. Avelumab is cleared faster and has a shorter half-life than other anti-PD-L1 antibodies, such as atezolizumab and durvalumab, but the mechanisms underlying these differences are unknown. IgG antibodies can be cleared through receptor-mediated endocytosis after binding of the antibody Fab region to target proteins, or via Fcγ receptor (FcγR)-mediated endocytosis. Unlike other approved anti-PD-L1 antibodies, avelumab has a native Fc region that retains FcγR binding capability. We hypothesized that the rapid clearance of avelumab might be due to the synergistic effect of both FcγR-mediated and PD-L1 target-mediated internalization. To investigate this, we performed in vitro and in vivo studies that compared engineered variants of avelumab and atezolizumab to determine mechanisms of cellular internalization. We found that both FcγR and PD-L1 binding contribute to avelumab internalization. While FcγR binding was the dominant mechanism of avelumab internalization in vitro, with CD64 acting as the most important FcγR, studies in mice and cynomolgus monkeys showed that both FcγR and PD-L1 contribute to avelumab elimination, with PD-L1 binding playing a greater role. These studies suggest that the rapid internalization of avelumab might be due to simultaneous binding of both PD-L1 and FcγR in trans. Our findings also provide a basis to alter the clearance and half-life of monoclonal antibodies in therapeutic development.


Subject(s)
Carcinoma, Transitional Cell , Skin Neoplasms , Urinary Bladder Neoplasms , Animals , Antibodies, Monoclonal, Humanized , B7-H1 Antigen , Humans , Mice , Receptors, IgG
11.
J Immunol ; 206(1): 225-236, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33268483

ABSTRACT

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.


Subject(s)
B7 Antigens/metabolism , Immunotherapy/methods , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 3/metabolism , Neoplasms/immunology , Antibodies, Bispecific/genetics , Antibodies, Bispecific/metabolism , B7 Antigens/genetics , Cell Line, Tumor , Cetuximab/genetics , Cytokines/metabolism , Cytotoxicity, Immunologic , ErbB Receptors/immunology , ErbB Receptors/metabolism , Genetic Engineering , Humans , Immunoglobulin Fab Fragments/genetics , Inflammation Mediators/metabolism , Killer Cells, Natural/transplantation , Lymphocyte Activation , Natural Cytotoxicity Triggering Receptor 3/immunology , Neoplasms/therapy , Protein Binding , Signal Transduction
12.
MAbs ; 12(1): 1812210, 2020.
Article in English | MEDLINE | ID: mdl-32887531

ABSTRACT

Here, we report the characterization of a VHH-derived IgG-like bi- and trispecific antibody platform that essentially relies on the replacement of the VH and VL regions of a conventional antibody by two independently functioning VHH domains. Consequently, a VHH is engrafted onto constant region CH1 while the other VHH-based paratope is engrafted on the constant region of the light chain, Cκ or Cλ, resulting in a tetravalent bispecific IgG-like molecule. Combined with a heavy chain heterodimerization technique, this platform allows facile engineering of bi- and trispecific antibodies with flexible valencies. We demonstrate the general applicability of this generic platform approach and elaborate on the limitations of specific formats.


Subject(s)
Antibodies, Bispecific , Immunoglobulin G , Immunoglobulin Heavy Chains , Immunoglobulin Variable Region , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , Cell Line, Tumor , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics
13.
MAbs ; 12(1): 1731938, 2020.
Article in English | MEDLINE | ID: mdl-32151188

ABSTRACT

Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.


Subject(s)
Antibodies, Bispecific/analysis , High-Throughput Screening Assays/methods , Inteins , Animals , Humans , Protein Engineering/methods
14.
Mol Biotechnol ; 62(4): 228-239, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31981039

ABSTRACT

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.


Subject(s)
Cell Surface Display Techniques/methods , Single-Chain Antibodies/genetics , Single-Domain Antibodies/genetics , Animals , Antibodies/immunology , Antibodies/isolation & purification , Bacteriophages/genetics , Camelids, New World , Chickens , Deoxyribonucleases, Type II Site-Specific , ErbB Receptors/immunology , Escherichia coli , Single-Chain Antibodies/immunology , Single-Domain Antibodies/immunology
15.
Methods Mol Biol ; 2070: 173-189, 2020.
Article in English | MEDLINE | ID: mdl-31625096

ABSTRACT

In addition to conventional hetero-tetrameric antibodies, the adaptive immune repertoire of camelids comprises the so-called heavy chain-only antibodies devoid of light chains. Consequently, antigen binding is mediated solely by the variable domain of the heavy chain, referred to as VHH. In recent years, these single-domain moieties emerged as promising tools for biotechnological and biomedical applications. In this chapter, we describe the generation of VHH antibody yeast surface display libraries from immunized Alpacas and Lamas as well as the facile isolation of antigen-specific molecules in a convenient fluorescence-activated cell sorting (FACS)-based selection process.


Subject(s)
Antibody Specificity/genetics , Camelids, New World , Flow Cytometry , Peptide Library , Saccharomyces cerevisiae , Single-Chain Antibodies , Animals , Antigens/chemistry , Antigens/immunology , Camelids, New World/genetics , Camelids, New World/immunology , Immunization , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology
16.
Biol Chem ; 400(3): 383-393, 2019 02 25.
Article in English | MEDLINE | ID: mdl-30465712

ABSTRACT

Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.


Subject(s)
Immunoglobulin Heavy Chains/analysis , Immunoglobulin Light Chains/analysis , Saccharomyces cerevisiae/chemistry , Animals , Chickens , Cloning, Molecular , Humans , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Peptide Library , Protein Engineering , Saccharomyces cerevisiae/immunology , Surface Properties
17.
Methods Mol Biol ; 1827: 145-161, 2018.
Article in English | MEDLINE | ID: mdl-30196496

ABSTRACT

Yeast surface display is a versatile platform technology for antibody discovery. Nevertheless, the construction of antibody Fab libraries typically is a tedious multistep process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Here, we present a focused one-step Golden Gate cloning approach for the generation of yeast surface display Fab libraries that allows for simultaneous introduction of heavy-chain and light-chain variable regions into one single display vector. Thereby, the overall time as well as the materials needed for library generation can be reduced significantly.


Subject(s)
Antibodies/metabolism , Cell Surface Display Techniques/methods , Immunoglobulin Fab Fragments/metabolism , Peptide Library , Saccharomyces cerevisiae/metabolism , Base Sequence , Flow Cytometry , Genetic Vectors/metabolism , Humans , Immunoglobulin Variable Region/chemistry , Protein Domains , Transformation, Genetic , Trastuzumab/chemistry
18.
Mol Biotechnol ; 60(10): 727-735, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30076531

ABSTRACT

In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species' cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting. We show that the applied screening stringencies translate directly into binding properties of isolated human antibody variants.


Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/isolation & purification , Cell Separation/methods , Flow Cytometry/methods , Saccharomyces cerevisiae/growth & development , Animals , Antibodies, Monoclonal/metabolism , Antibody Affinity , Cell Line , Cell Surface Display Techniques , Cloning, Molecular , Female , Humans , Male , Peptide Library , Rats , Rats, Transgenic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
19.
Microb Cell Fact ; 17(1): 3, 2018 Jan 09.
Article in English | MEDLINE | ID: mdl-29316915

ABSTRACT

BACKGROUND: Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. RESULTS: Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. CONCLUSION: We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.


Subject(s)
Cloning, Molecular/methods , Immunoglobulin Fab Fragments/isolation & purification , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Antibodies/metabolism , Antibody Affinity , Flow Cytometry , Immunoglobulin Fab Fragments/immunology , Peptide Library , Promoter Regions, Genetic , Surface Properties
20.
Protein Eng Des Sel ; 30(9): 685-696, 2017 09 01.
Article in English | MEDLINE | ID: mdl-28981885

ABSTRACT

Targeting two unique antigens with a single bispecific antibody is an attractive approach with potential broad therapeutic applicability. However, the production of heterodimeric bispecific antibodies (bsAbs) presents a challenge, requiring the co-expression and accurate pairing of two distinct heavy and light chain units. Several undesirable by-products can be formed in the production process, including heavy chain homodimers and non-cognate light chain pairings. Although additional downstream purification methods exist, they are often time consuming and restrict practical large-scale production. In this study, we identify and validate novel Fab interface mutations that increase cognate light chain pairing efficiencies within heterodimeric bsAbs. Importantly, the variable domains remain unaltered as interface mutations were restricted to the CH1 and CL domains. We performed several biochemical assays to demonstrate that the novel engineered interfaces do not adversely impact bispecific antibody expression, stability, affinity and biological function. The designs reported here can easily be applied in a generic manner to use existing antibodies as building blocks for bsAbs which will help to accelerate the identification and production of next generation bispecific antibody therapeutics.


Subject(s)
Antibodies, Bispecific/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Light Chains/chemistry , Animals , Antibodies, Bispecific/biosynthesis , Antibodies, Bispecific/genetics , CHO Cells , Cricetulus , Gene Expression , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Models, Molecular , Mutation , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
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