Merge Two into One: Multispecific Symmetric Antibodies for Cancer Immunotherapy

This work is focused on the generation and characterization of multispecific symmetric antibodies for cancer immunotherapy. In contrast to asymmetric antibodies, where protein engineering technologies need to be applied to ensure the correct pairing of the different polypeptide chains, symmetric antibodies consist of two identical heavy and light chains. Consequently, these molecules exhibit monoclonal antibody (mAb)-like characteristics in terms of established manufacturing and a conventional approval process. Two-in-One antibodies are symmetrical tetravalent IgG-like bispecific antibodies (bsAbs) in which each fragment antigen binding (Fab) addresses two distinct antigens. Simultaneous targeting of two tumor-associated antigens (TAAs) on the same malignant cell offers advantages like increased specificity and reduced immune escape. Affinity optimization can further improve the efficacy of drug candidates and limit adverse effects. The first investigation within this cumulative thesis was dedicated on the generation of a symmetrical Two-in-One antibody targeting epidermal growth factor receptor (EGFR) and programmed cell death ligand-1 (PD-L1), two therapeutic targets upregulated in many solid tumors. To this end, the heavy chain of a chicken-derived anti-PD-L1 common light chain (cLC) antibody was combined with a chicken-derived anti-EGFR light chain yeast surface display (YSD) library, followed by subsequent screening for binding properties towards both antigens. The isolated Two-in-One antibody HCP-LCE simultaneously targeted EGFR and PD-L1 at the same Fab fragment and exhibited favorable biophysical characteristics. BLI measurements and cell-based assays revealed that HCP-LCE inhibited EGFR signaling by binding to EGFR dimerization domain II and blocked the PD-1/PD-L1 interaction. Remarkably, both antigens were addressed with comparatively low binding affinities in the double- to triple-digit nanomolar range, but specific and high-affinity cellular binding properties were demonstrated on EGFR and PD-L1 double positive tumor cells. HCP-LCE represented the first Two-in-One antibody without complementarity-determining region (CDR) engineering, targeting two antigens simultaneously with a single Fab fragment. This approach of library generation paves the way for the further development of Two-in-One antibodies derived from avian immunization with tailor-made binding properties. In a second project, a symmetrical trispecific natural killer (NK) cell engager (NKCE) was generated based on the previously isolated Two-in-One antibody. By the simultaneous targeting of a TAA and a specific marker on the surface of NK cells, the immune function of NK cells to kill tumor cells is harnessed for tumor therapy. For the generation of such an antibody, the cLC technology was applied, an established method to circumvent light chain mispairing in multispecific antibodies. To this end, the light chain of the Two-in-One antibody HCP-LCE was used as cLC for the generation of a chicken-derived anti-CD16a YSD library. The isolated CD16a engaging cLC Fab fragment was fused in a head-to-tail setup with the parental Two-in-One antibody, resulting in a symmetrical trispecific 2+2 antibody that simultaneously bound EGFR, PD-L1 and CD16a with six independent paratopes on four Fabs. The antibody exhibited specific cellular binding on EGFR and PD-L1 double positive tumor cells and induced NK cell-mediated tumor cell killing (ADCC) already at low concentrations. This study pioneers the straightforward generation of trispecific cLC immune cell engager molecules in a 2+2 design, which facilitates subsequent process development due to the symmetrical architecture. The third part of this work focused on the affinity maturation of the Two-in-One antibody for EGFR binding by site-directed mutagenesis and YSD in combination with fluorescence-activated cell sorting (FACS). Individual amino acids of the light chain CDR1 and CDR3 were randomized and the resulting YSD library provided a Two-in-One variant that exhibited a 60-fold improvement in EGFR binding affinity due to the replacement of a single amino acid at position three of the light chain CDR3, while PD-L1 binding was not impaired. AlphaFold2-based modeling predicted that the exchange of the neutral amino acid tyrosine to the acidic amino acid glutamic acid causes the formation of an additional salt bridge between the introduced glutamic acid and an arginine at EGFR position 165. The increase in affinity was demonstrated by BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding studies using flow cytometry and real-time interaction cytometry. This easily adaptable approach provides a generic strategy for the affinity maturation of Two-in-One antibodies

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

Conditional activation of an anti-IgM antibody-drug conjugate for precise B cell lymphoma targeting

Cancerous B cells are almost indistinguishable from their non-malignant counterparts regarding their surface antigen expression. Accordingly, the challenge to be faced consists in elimination of the malignant B cell population while maintaining a functional adaptive immune system. Here, we present an IgM-specific antibody-drug conjugate masked by fusion of the epitope-bearing IgM constant domain. Antibody masking impaired interaction with soluble pentameric as well as cell surface-expressed IgM molecules rendering the antibody cytotoxically inactive. Binding capacity of the anti-IgM antibody drug conjugate was restored upon conditional protease-mediated demasking which consequently enabled target-dependent antibody internalization and subsequent induction of apoptosis in malignant B cells. This easily adaptable approach potentially provides a novel mechanism of clonal B cell lymphoma eradication to the arsenal available for non-Hodgkin's lymphoma treatment

Potent Apoptosis Induction by a Novel Trispecific B7-H3xCD16xTIGIT 2+1 Common Light Chain Natural Killer Cell Engager

Valued for their ability to rapidly kill multiple tumor cells in succession as well as their favorable safety profile, NK cells are of increasing interest in the field of immunotherapy. As their cytotoxic activity is controlled by a complex network of activating and inhibiting receptors, they offer a wide range of possible antigens to modulate their function by antibodies. In this work, we utilized our established common light chain (cLC)-based yeast surface display (YSD) screening procedure to isolate novel B7-H3 and TIGIT binding monoclonal antibodies. The chicken-derived antibodies showed single- to low-double-digit nanomolar affinities and were combined with a previously published CD16-binding Fab in a 2+1 format to generate a potent NK engaging molecule. In a straightforward, easily adjustable apoptosis assay, the construct B7-H3xCD16xTIGIT showed potent apoptosis induction in cancer cells. These results showcase the potential of the TIGIT NK checkpoint in combination with activating receptors to achieve increased cytotoxic activity

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

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

Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted

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

Better safe than sorry: dual targeting antibodies for cancer immunotherapy

Antibody-based therapies are revolutionizing cancer treatment and experience a steady increase from preclinical and clinical pipelines to market share. While the clinical success of monoclonal antibodies is frequently limited by low response rates, treatment resistance and various other factors, multispecific antibodies open up new prospects by addressing tumor complexity as well as immune response actuation potently improving safety and efficacy. Novel antibody approaches involve simultaneous binding of two antigens on one cell implying increased specificity and reduced tumor escape for dual tumor-associated antigen targeting and enhanced and durable cytotoxic effects for dual immune cell-related antigen targeting. This article reviews antibody and cell-based therapeutics for oncology with intrinsic dual targeting of either tumor cells or immune cells. As revealed in various preclinical studies and clinical trials, dual targeting molecules are promising candidates constituting the next generation of antibody drugs for fighting cancer

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