25 research outputs found
Structural insights and biomedical potential of IgNAR scaffolds from sharks
Peer reviewedPublisher PD
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
Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies
The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part
of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-
cell lymphomas, the tumor cells express a tumor-speci fi c and functionally active BCR, also
known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to
be demanding since the idiotype differs from patient to patient. Previous studies have
shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic
CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this
study, we evaluated the potential of generating patient-speci fi c binders against the
idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines
SUP-B8, Daudi, and IM-9 were identi fi ed, the variable domains were reformatted and the
resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in
fl uorescence-activated cell sorting (FACS)-based screening of the yeast-displayed
vNAR libraries which resulted after three rounds of screening in the enrichment of
antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and
consequently analyzed for their binding to soluble antigen using biolayer interferometry
(BLI) revealing binding constants in the lower single-digit nanomolar range. These variants
showed speci fi c binding to the parental SUP-B8 cell line con fi rming a similar folding of the
recombinantly expressed proteins compared with the native cell surface-presented BCR.
First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to
induce apoptosis or ADCC/ADCP did not result in a signi fi cant decrease of cell viability.
Here, we report an alternative approach for a personalized B-cell lymphoma therapy
based on the construction of vNAR-Fc antibody-drug conjugates to enable speci fi c killing
of malignant B cells, which may widen the therapeutic window for B-cell
lymphoma therapy
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
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
Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies
The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy
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
Enabling technologies for the generation of high-affinity binders based on immunoglobulin scaffolds
Monoclonal antibodies (mAbs) emerged as one of the most successful and frequently used
biotherapeutics over the last decade. For the discovery of antibodies, currently two approaches are
predominant: Immunization of animals and surface display techniques.
The first investigation in frame of the present cumulative study was focused on the optimization of
yeast surface display by skipping the time-consuming staining of epitope tags required to verify fulllength
presentation of displayed antibody fragments. For this reason a ribosomal skipping sequence
enabling the translation of two separate proteins from one open reading frame was genetically
incorporated between the displayed antibody fragment and a green fluorescent protein (GFP) as
reporter protein. Translation of the protein of interest and the ribosomal skipping sequence (called 2A
peptide) results in release of the N-terminally located antibody fragment from the polypeptide chain
and secretion to the cell surface. The 2A peptide-mediated ribosomal skip re-initiates the translation of
the GFP-encoding mRNA. Consequently, only yeast cells showing GFP fluorescence display the protein
of interest. The combination of this system with the semi-synthetic shark-derived vNAR library
emerged as a setup being efficient as the conventional procedure with the advantages of being more
cost-efficient and less time-consuming.
The second investigation evaluated the potential of yeast surface display (YSD) in combination with
fluorescence-activated cell sorting (FACS) to isolate high-affine and specific antibodies derived from a
chicken immunization campaign. To this end, yeast-displayed recombinant antibody libraries from
splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human
chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap
extension polymerase chain reaction. A plethora of antigen binding scFvs were isolated in a convenient
screening process. The production of target-specific variants as soluble scFv-Fc molecules enabled the
subsequent extensive characterization by confirming high affinity, good specificity, thermostability and
promising aggregation profiles. Furthermore, the biotechnological applicability of binders directed
against both antigens could be demonstrated. For EGFR binders this could be achieved via specific
cellular binding and for hCG-binders in the context of a lateral flow test by utilizing hCG-binding scFvs
as capturing antibodies for pregnancy detection. In summary, the strategy using yeast surface display
emerged as a powerful tool being able to expand the repertoire of display methods for the isolation of
antibodies resulting from chicken immunization campaigns.
The third investigation was focused on the development of a more convenient generation of yeastdisplayed
Fab libraries. The conservative method for Fab library generation relies on using a three-step
protocol including individual heavy- and light chain sub-libraries generated in haploid yeast strains
followed by chain combination using yeast mating. The herein newly developed strategy bases on a
Golden Gate cloning approach resulting in diversities of both heavy and light chain being efficiently
combined on one single plasmid using a bidirectional promoter. The applicability of this system could
be verified by two individual successful screening campaigns. The first investigation enabled the
isolation of high-affine human antibodies resulting from immunized transgenic rats. Furthermore, it
could be proven that the method can also be used to successfully screen and isolate chimeric
chicken/human antibodies after avian immunization. In the end it could be shown that the Golden
Gate based one-step process enables the delivery of libraries and antibodies from heavy- and light
chain diversities with comparable quality to the conventional method while being significantly less
time-consuming and complex.
The last part of this work elucidated the potential usage of shark-derived single chain antibodies
(vNARs) to be used for specific lymphoma cell targeting. It could be shown in previous studies that a
shark-derived semi-synthetic CDR3 loop randomized yeast library is suited pretty well to isolate antiidiotype
binders recognizing exclusively the paratope of mABs. Lymphoma cells might be an attractive
target for anti-idiotype vNAR molecules since B-cell lymphoma cells exhibit tumor-specific and
functionally active B-Cell receptors (BCR). However, targeting BCRs for lymphoma therapy has shown
to be demanding since the BCR differs from patient to patient. For this reason in the presented study
we evaluated the rapid and convenient isolation of specific BCR-binding vNARs derived from the semisynthetic
library. To this end the BCRs of three different lymphoma cell lines Daudi, SUP-B8, and IM-9
were identified, followed by reformatting the variable domains and expression of the resulting BCRs as
monoclonal antibodies. The SUP-B8 BCR was consequently utilized as antigen in a FACS-based
screening of the yeast-displayed vNAR libraries resulting in the enrichment of several antigen-binding
vNARs. Further characterization of the isolated vNARs after reformatting as Fc fusion proteins
confirmed an affine and specific binding to both the soluble recombinantly expressed BCR molecules
and the respective lymphoma cell line. Initial experiments to exploit the specific BCR-binding for BCRclustering
followed by induction of cell apoptosis did not show a significant influence on cell viability.
However, an alternative approach that includes the generation of multivalent vNAR constructs or the
construction of vNAR antibody-drug conjugates might enable a vNAR-induced patient-specific
lymphoma cell killing in the future
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