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

Carrara, Stefania C / Bogen, Jan P / Fiebig, David / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2681, Page(s) 291–311

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Animals ; Saccharomyces cerevisiae/metabolism ; Peptide Library ; Antibodies, Monoclonal/genetics ; Antibodies, Monoclonal/metabolism ; Immunoglobulin Fab Fragments ; Immunoglobulin G ; Genetic Vectors ; Mammals/genetics
Chemical Substances	Peptide Library ; Antibodies, Monoclonal ; Immunoglobulin Fab Fragments ; Immunoglobulin G
Language	English
Publishing date	2023-07-05
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3279-6_16
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Carrara, Stefania C / Harwardt, Julia / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Frontiers in immunology

2022 Volume 13, Page(s) 1051875

Abstract: 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 ... ...

Abstract	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
MeSH term(s)	Humans ; Cytokine Release Syndrome/etiology ; B7-H1 Antigen ; Interleukin-6/metabolism ; T-Lymphocytes ; Antilymphocyte Serum ; Cytokines
Chemical Substances	B7-H1 Antigen ; Interleukin-6 ; Antilymphocyte Serum ; Cytokines
Language	English
Publishing date	2022-11-10
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2022.1051875
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Article ; Online: Targeted Phagocytosis Induction for Cancer Immunotherapy via Bispecific MerTK-Engaging Antibodies.

Carrara, Stefania C / Bogen, Jan P / Fiebig, David / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

International journal of molecular sciences

2022 Volume 23, Issue 24

Abstract: 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- ... ...

Abstract	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.
Language	English
Publishing date	2022-12-10
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms232415673
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Article ; Online: Antibody Library Screening Using Yeast Biopanning and Fluorescence-Activated Cell Sorting.

Carrara, Stefania C / Bogen, Jan P / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2491, Page(s) 177–193

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Animals ; Antibodies, Monoclonal/chemistry ; Antigens ; Bioprospecting ; Cell Surface Display Techniques ; Flow Cytometry/methods ; Mammals ; Peptide Library ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Antibodies, Monoclonal ; Antigens ; Peptide Library
Language	English
Publishing date	2022-04-28
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2285-8_10
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Harwardt, Julia / Carrara, Stefania C / Bogen, Jan P / Schoenfeld, Katrin / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Frontiers in immunology

2023 Volume 14, Page(s) 1170042

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	B7-H1 Antigen/metabolism ; Killer Cells, Natural ; Antibodies/metabolism ; ErbB Receptors/metabolism
Chemical Substances	B7-H1 Antigen ; Antibodies ; ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2023-04-04
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2023.1170042
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Book ; Online ; Thesis: Generation of Multispecific Antibodies with Immune Cell Modulating Functions

Carrara, Stefania C. [Verfasser] / Kolmar, Harald [Akademischer Betreuer] / Hock, Björn [Akademischer Betreuer] / Peipp, Matthias [Akademischer Betreuer]

2023

Author's details	Stefania C. Carrara ; Harald Kolmar, Björn Hock, Matthias Peipp
Keywords	Medizin, Gesundheit ; Medicine, Health
Subject code	sg610
Language	English
Publisher	Universitäts- und Landesbibliothek
Publishing place	Darmstadt
Document type	Book ; Online ; Thesis
Database	Digital theses on the web

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Article ; Online: Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System.

Carrara, Stefania C / Fiebig, David / Bogen, Jan P / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Antibodies (Basel, Switzerland)

2021 Volume 10, Issue 2

Abstract: 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 ... ...

Abstract	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 (
Language	English
Publishing date	2021-05-13
Publishing country	Switzerland
Document type	Journal Article
ISSN	2073-4468
ISSN (online)	2073-4468
DOI	10.3390/antib10020018
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Article ; Online: Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture.

Bogen, Jan P / Carrara, Stefania C / Fiebig, David / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Frontiers in immunology

2021 Volume 12, Page(s) 669496

Abstract: 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. ...

Abstract	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.
MeSH term(s)	Animals ; Antibodies, Bispecific/immunology ; Antibodies, Bispecific/pharmacology ; Antibodies, Monoclonal, Humanized/immunology ; Antibodies, Monoclonal, Humanized/pharmacology ; Antibody Specificity ; B7-H1 Antigen/antagonists & inhibitors ; B7-H1 Antigen/immunology ; B7-H1 Antigen/metabolism ; Cell Line, Tumor ; Chickens ; Cytotoxicity, Immunologic/drug effects ; Drug Design ; Epitopes ; ErbB Receptors/antagonists & inhibitors ; ErbB Receptors/immunology ; ErbB Receptors/metabolism ; Immune Checkpoint Inhibitors/immunology ; Immune Checkpoint Inhibitors/pharmacology ; Immunization ; Immunoglobulin Light Chains/immunology ; Immunoglobulin Light Chains/pharmacology ; Killer Cells, Natural/drug effects ; Killer Cells, Natural/immunology ; Killer Cells, Natural/metabolism ; Receptors, IgG/antagonists & inhibitors ; Receptors, IgG/immunology ; Receptors, IgG/metabolism ; Skin Neoplasms/drug therapy ; Skin Neoplasms/immunology ; Skin Neoplasms/metabolism ; Skin Neoplasms/pathology
Chemical Substances	Antibodies, Bispecific ; Antibodies, Monoclonal, Humanized ; B7-H1 Antigen ; CD274 protein, human ; Epitopes ; FCGR3A protein, human ; Immune Checkpoint Inhibitors ; Immunoglobulin Light Chains ; Receptors, IgG ; EGFR protein, human (EC 2.7.10.1) ; ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2021-05-10
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.669496
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Article ; Online: A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization.

Harwardt, Julia / Bogen, Jan P / Carrara, Stefania C / Ulitzka, Michael / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Frontiers in immunology

2022 Volume 13, Page(s) 888838

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Antibodies, Bispecific ; B7-H1 Antigen/metabolism ; Chickens ; ErbB Receptors/metabolism ; Immunization
Chemical Substances	Antibodies, Bispecific ; B7-H1 Antigen ; ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2022-04-11
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2022.888838
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Fiebig, David / Bogen, Jan P / Carrara, Stefania C / Deweid, Lukas / Zielonka, Stefan / Grzeschik, Julius / Hock, Björn / Kolmar, Harald

Frontiers in bioengineering and biotechnology

2022 Volume 10, Page(s) 794389

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2022-05-10
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2719493-0
ISSN	2296-4185
ISSN	2296-4185
DOI	10.3389/fbioe.2022.794389
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