Cytokines have the potential to reinvigorate the immune response against tumors, address shortcomings of checkpoint inhibition and expand the reach of immunotherapy. Yet, development efforts with recombinant cytokines, their engineered muteins and fusion molecules have encountered challenges. We are developing a novel therapeutic modality using bispecific antibodies we refer to as Amplify•R. These antibodies engage the naturally present endogenous cytokines in vivo, enhance persistence of the bound cytokine, while regulating and redirecting its therapeutic effect to target cells of interest. We hypothesize that this modality will overcome limitations such as systemic toxicity, increased immunogenicity, and manufacturing challenges often associated with traditional recombinant cytokine treatment approaches. As a proof of concept, we have designed a panel of bispecific antibodies capable of co-engaging the T and NK cell stimulating cytokine, IL-15, and the immune checkpoint, PD-1. The bispecific antibodies can bind IL-15 and selectively present it to its cognate receptor on PD-1+ cells. In reporter cell-based assays, the panel of antibodies were able to mediate IL-15 signaling in a controlled manner. Further, these bispecific antibodies were as efficient at PD-1 signal blockade as a clinical benchmark. The ability of the bispecific antibodies to stimulate IL-15 dependent STAT5 phosphorylation in human peripheral blood mononuclear cells (PBMC) was tested. In a dose-dependent manner, the bispecific antibodies were able to selectively stimulate pSTAT5 activity in PD-1+ T cells versus NK cells, whereas IL-15 alone stimulated greater pSTAT5 activity in NK cells compared to T cells. The ability of the bispecific antibodies to induce cell proliferation was determined by culturing PBMC in the presence of antibodies complexed with IL-15. After 4 days of culture, a dose dependent expression of Ki67 was detected in CD8+ T cells cultured with bispecific antibodies while they failed to stimulate Ki67 expression in NK cells. These in vitro results demonstrate redirection of IL-15 activity towards PD-1 expressing T cells, and away from NK cells. We have further explored the Amplify•R antibody effect in vivo. Using C57BL/6 mice engineered with human PD1, engrafted with the MC38 colorectal cancer cell line humanized for PD-L1, we observed significantly more efficient control of tumor growth with the bispecific antibodies in the presence of IL-15 relative to pembrolizumab alone or in combination with IL-15. In addition, upon repeated dosing, CD8+ T cells were preferentially expanded over CD4+ and NK cells in the Amplify•R antibody treated animals. In summary, we show that the Amplify•R modality of endogenous cytokine engagement and redirection may be a viable approach in clinic, capable of overcoming limitations encountered with traditional cytokine treatment. Surjit Dixit, Mark Fogg, Stacey Tom-Yew, Harsh Pratap, Vivian Li, Abhishek Mukhopadhyay, Jason Baardsnes, Yuneivy Cepero Donates, David de Graaf. Bispecific antibody-based redirection of endogenous IL-15 to PD-1 positive cells enhances antitumor activity over checkpoint inhibition alone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB137.

Protein sidechain conformation prediction, or packing, is a key step in many in silico protein modeling and design tasks. Popular protein packing methods typically rely on approximated energy functions and complex algorithms to search dense rotamer libraries. Inspired by the recent success of deep learning in protein modeling tasks, we present ZymePackNet, a graph neural network based protein packing tool that does not require a rotamer library, scoring functions or a search algorithm. We train regression models using protein crystal structures represented as graphs, which are employed sequentially to “germinate” the sidechain starting from atoms anchoring the protein backbone to the sidechains’ termini, followed by an iterative refinement stage. ZymePackNet is fast and accurate compared to state-of-the-art protein packing methods. We validate our model on three native backbone datasets achieving a mean average error of 16.6°, 24.1°, 42.1°, and 53.0° for sidechain dihedral angles ( χ 1 to χ 4 ). ZymePackNet captures complex physical interactions such as π stacking without explicitly accounting for it in the model; such effects are currently lacking in the energy terms used in traditional packing tools. Contact abmukho@vt.edu Supplementary information Supplementary data are available at Bioinformatics online.

As immunological selection for escape mutants continues to give rise to future SARS-CoV-2 variants, novel universal therapeutic strategies against ACE2-dependent viruses are needed. Here we present an IgM-based decavalent ACE2 decoy that has variant-agnostic efficacy. In immuno-, pseudovirus, and live virus assays, IgM ACE2 decoy had potency comparable or superior to leading SARS-CoV-2 IgG-based mAb therapeutics evaluated in the clinic, which were variant-sensitive in their potency. We found that increased ACE2 valency translated into increased apparent affinity for spike protein and superior potency in biological assays when decavalent IgM ACE2 was compared to tetravalent, bivalent, and monovalent ACE2 decoys. Furthermore, a single intranasal dose of IgM ACE2 decoy at 1 mg/kg conferred therapeutic benefit against SARS-CoV-2 Delta variant infection in a hamster model. Taken together, this engineered IgM ACE2 decoy represents a SARS-CoV-2 variant-agnostic therapeutic that leverages avidity to drive enhanced target binding, viral neutralization, and in vivo respiratory protection against SARS-CoV-2.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Many T-cell engagers (TCE) and immuno-oncology biologics have limited efficacy in the clinic due to narrow therapeutic windows, checkpoint upregulation, and emergence of resistance mechanisms over time. The PROTECTTM (PROgrammed Tumor Engagement & Checkpoint/Costimulation Targeting) platform is designed to tackle these challenges by combining a masking domain that, when cleaved, provides additional immune-modulatory properties. The PROTECTTM mask consists of a PD1-PDL1 protein pair that sterically hinders CD3 binding in the periphery. The PD-L1 moiety is fused to the anti-CD3 antibody via a linker sequence containing a protease cleavage site. Once cleaved, the resultant molecule is a trispecific antibody providing TCE activity, checkpoint inhibition, and additional differentiated functionality. Previous studies employing pan T cell cytotoxicity assays showed the PROTECTTM mask increased the therapeutic window by 400-fold. We have now expanded our in vitro evaluation to include more relevant PBMC systems showing a greater expansion of the therapeutic window. In vivo treatment of established tumours with the unmasked trispecific results in complete and durable anti-tumour responses. Here we extend those in vivo studies (in context of established tumour models) to evaluate unmasking and pharmacokinetic properties. We further evaluated the unique and differentiated mechanism of action of the cleaved trispecific to not only enhance engagement of effector T-cells with tumor cells through avidity driven cis-engagement of a tumor associated antigen (TAA) and PD-L1 co-expressed on tumor cells, but to also enhance co-engagement of a TAA with effector T cells co-expressing PD-L1. In addition, the ability of the cleaved trispecific to bridge autologous T cells with DCs results in enhanced T cell activation and proliferation. Finally, TCR dependent signaling assays confirmed the ability of the cleaved trispecific to overcome PD1/PDL1 checkpoint activity. Taken together, the PROTECTTM platform integrates a masking and immune-modulatory technology that has the potential to widen the therapeutic window of CD3 engagers. Citation Format: Anna von Rossum, Genevieve Desjardins, Nichole Escalante, Wingkie Wong, Bryant Harbourne, Janessa Li, Begonia Silva Moreno, Prajwal Raghunatha, Richard Kunze, Madeline Fung, Florian Heinkel, Harsh Pratap, Kevin Haworth, Eric Escobar-Cabrera, Brandon Clavette, Surjit Dixit, Nina Weisser, Thomas Spreter von Kreudenstein. PROTECTTM, a novel trispecific antibody masking platform with integrated immune modulation displays unique activity and differentiated modes of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2926.

Functional biologics design is a multi-objective optimization problem often with competing design objectives. We report on a novel deep learning based protein sequence prediction framework, ZymeSwapNet, that can be customized to handle a wide range of quantifiable design objectives, a current limitation of traditional protein design methods. We train a simple convolutional neural network (1D-CNN) on nonredundant curated protein crystal structures, using a set of geometric and topological features that describes a local protein environment, to predict the likelihood of each amino acid type for residue sites in the design region. While the model can be directly used to rank templates derived from mutagenesis campaigns, we extend the scope by developing a sequence/mutation generator that optimizes the desired multivariate distribution using a Monte-Carlo sampling. Using a case study – the design of a stable heterodimeric Fc (HetFc) antibody domain – we show that we can further include a Metropolis criterion to bias the sampling to enhance features such as the heterodimeric binding specificity, in addition to original sampling objective of enhancing stability. We demonstrate that ZymeSwapNet can generate stable HetFc designs, within minutes that had taken several rounds of rational structure and physical force-field based modeling attempts.

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4′) and two thymine (T3, T13′, or T3′, T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9′, 12, 12′ and the thymine residues T10, 10′, 11, 11′, in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3′, 13, 13′) in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A → G or C), 11 (T → A or G) and 12 (A → G, T or C).

Abstract Many novel immune-oncology biologics are limited in clinical utility by narrow therapeutic windows. One strategy to overcome this limitation relies on the engineering of ‘masks' that block antibody paratopes outside of the tumor microenvironment (TME). The PROTECT (PROgrammed Tumor Engagement & Checkpoint/Costimulation Targeting) platform is designed to employ the orthogonal mechanistic features of a multispecific design to increase the therapeutic window by limiting exposure and activity in peripheral tissues while focusing activities to the tumor. In particular, we aim to bring TME-specific activity and enhanced immune modulation in a single transferable, conditionally active design. To achieve this, we engineered the N-termini of antibody heavy and light chains with the fusion of IgV domains of commonly targeted immunomodulatory pairs, such as PD-1 and PD-L1, to sterically preclude binding of the antibody paratopes to their target tumor antigens. We demonstrate that this approach can effectively mask the antibody binding and activity for targets by 10-1000 fold, with recovery of binding and anti-tumor activity upon release of the immunomodulatory mask once proteolytically cleaved by a tumor-specific protease such as urokinase plasminogen activator (uPA). In addition, we also demonstrate that we can selectively cleave and remove one half of the immunomodulatory pair (e.g. PD-L1) from the antibody thereby creating an antibody fused to only PD-1. The resulting bispecific antibody can now co-engage both the tumor antigen (TA) and its counterpart checkpoint inhibitor (PD-L1) to confer additional antitumor activities. As a proof of concept, we showed that for an engineered anti-CD3/HER2 bispecific T-cell engager (TCE), incorporation of the PD-L1/PD-1 PROTECT design creates a conditionally activated anti-CD3/PD-L1/HER2 tri-specific TCE that has (i) masked CD3 engagement with a reduction of the EC50 by 2 orders of magnitude, and (ii) enhanced T-cell dependent cytotoxicity over the parent anti-CD3/HER2 TCE by an order of magnitude once activated by uPA. Importantly, the enhanced activity of the tri-specific TCE is greater than the combination of the parental TCE with the anti-PD-L1 monoclonal antibody atezolizumab. We have also demonstrated that the PROTECT platform can be combined with anti-TA antibody to introduce a masking effect while increasing antibody-dependent cell cytotoxicity (ADCC) activity upon co-engagement of TA and PD-L1, again synergistic relative to the combination of anti-TA antibody and atezolizumab. Taken together, the PROTECT platform represents a novel approach to integrate immune modulation while limiting off-tumor activities for the development of conditional multispecific antibodies with potentially enhanced therapeutic window and activity. Citation Format: Surjit Dixit, Florian Heinkel, Anna Von Rossum, Harsh Pratap, Sifa Arrafi, Javairia Rahim, Purva Bhojane, Liz Stangle, Leisa Stenberg, Gesa Volkers, Eric Escobar-Cabrera, Thomas Spreter. PROTECT, a novel antibody platform for integrating tumor-specific immune modulation and enhancing the therapeutic window of targeted multispecific biologics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 924.

Abstract HER2-directed therapies have improved clinical outcomes for many patients with HER2-positive breast and gastric cancer. Despite these successes, there remains a need to develop improved HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. Zanidatamab (ZW25) is a humanized, bispecific, immunoglobulin (Ig) G1-like antibody directed against the juxtamembrane extracellular domain (ECD4) and the dimerization domain (ECD2) of human epidermal growth factor receptor 2 (HER2), the same domains targeted by trastuzumab (T) and pertuzumab (P), respectively. Data from the ongoing phase 1 study (NCT02892123) demonstrate that zanidatamab is well tolerated and has single agent activity in patients with advanced HER2-expressing cancers that have progressed after standard of care (SOC) therapies, including HER2-targeted agents such as T, P, and trastuzumab emtansine.1,2 We have previously shown that the unique design and bispecific binding of zanidatamab results in multiple mechanisms of action including increased antibody binding density, potent effector function, improved receptor internalization and HER2 downregulation relative to T.3 To better understand the mechanism by which zanidatamab differentiates itself from T, P and T+P, we recently expanded our mechanistic evaluations including cell surface HER2 aggregation, complement-dependant cytotoxicity (CDC) and inhibition of both tumor cell growth and intracellular signaling. Single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM) was used to map HER2 receptor distribution and quantitate the size, density, and frequency of receptor clusters induced by antibody binding. In vitro assessments were performed in a panel of HER2-expressing cell lines using standard assays including CDC with human complement serum and inhibition of both tumor cell growth and intracellular signaling. Using dSTORM, we observed that zanidatamab binding resulted in enhanced HER2 aggregation and distinct HER2 capping on the tumor cell surface compared to T, P or T+P. Evaluation of CDC activity in HER2-overexpressing tumor cells demonstrated that zanidatamab, but not T, P or T+P, elicited CDC suggesting that the enhanced HER2 aggregation and capping on the tumor cell surface provides high avidity docking sites to which C1 binds and is activated. Zanidatamab showed further differentiation in the inhibition of both tumor growth and intracellular signaling of HER2-overexpressing cells compared to T, P and T+P. Zanidatamab has novel cell surface binding and additional mechanisms of action compared to T, P and T+P. Zanidatamab is actively being evaluated in clinical trials in multiple HER2-expressing solid tumors, including a registration-enabling clinical trial in HER2 gene amplified biliary tract cancer (NCT04466891). Citation Format: Nina E. Weisser, Grant Wickman, Libin Abraham, Jason O'Toole, Bryant Harbourne, Joy Guedia, Chi Wing Cheng, Peter Chan, Duncan Browman, Michael R. Gold, Neil Josephson, Surjit Dixit, Gerry Rowse. The bispecific antibody zanidatamab's (ZW25's) unique mechanisms of action and durable anti-tumor activity in HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1005.

Abstract IL-12 is a cytokine produced by antigen-presenting cells that increases T cell proliferation, IFN gamma mediated Th1 effector functions and T and NK cell cytotoxicity. While these effects generate potent anti-tumor immunity in mouse models, the high toxicity of IL-12 in cancer patients has limited its clinical utility. Potency attenuation and intratumoral cytokine localization may improve IL-12 tolerability, but these approaches can reduce efficacy or be limited by intratumoral delivery or tumor antigen expression. To improve both tolerability and efficacy of IL-12, we engineered IL-12Fc fusions with anti-IL-12 antibodies to block IL-12 potency. Using linkers designed to be cleaved by highly active intratumoral proteases, blocking antibodies are released specifically in the tumor microenvironment, thereby increasing intratumoral IL-12 activity. Single chain IL-12 was fused to one C-termini of an Azymetric™ Fc heterodimer. To the other Fc C-termini, an anti-IL-12 scFv was fused via a protease-cleavable linker in order to block IL-12 activity. In addition to antibody blockade, modifications of IL-12Fc fusions were used to further limit IL-12Fc activity. In vitro potency was determined by CD8T cell IFN gamma release. Anti-IL-12 scFv cleavage in the presence of recombinant enzyme or human tumor material was assessed by reducing CE-SDS and LC/MS, respectively. All IL-12Fc fusions were produced with favorable biophysical characteristics. Modified IL-12Fc molecules had up to 500x reduced potency, while antibody blocked IL-12Fc molecules had up to 100,000x reduced potency compared to IL-12Fc control. In vitro cleavage of the anti-IL-12 scFv from IL-12Fc recovered potency to that of the corresponding non-masked IL-12Fc molecules. Reducing CE-SDS confirmed that anti-IL-12 scFvs were cleaved from IL-12Fc in the presence of recombinant enzyme. Cleavage of anti-IL-12 scFvs also occurred when variants were incubated in pancreatic tumor cell supernatant or human tumor tissue lysate. These results indicate that antibody blockade and re-activation by protease cleavage is a promising strategy to localize the activity of IL-12 to the tumor microenvironment while potentially limiting off-tumor toxicities. Further modifying antibody blocked IL-12 has the potential to better fine tune therapeutic index. We are pursuing tumor-specific IL-12 fusions for clinical application in tumors with high intratumoral protease activity. Citation Format: Jennifer Leah Bishop, Ryan Blackler, Gesa Volkers, Maya Poffenberger, Irene Yu, Joel Smith, Akram Khodabandehloo, Sifa Arrafi, Desmond Lau, Liz Stangle, Leisa Stenberg, Patricia Zwierzchowski, Iulia Dude, David Douda, Grant Wickman, Jeff Proctor, Gerry Rowse, Laurence Madera, Genevieve Desjardins, Nicole Afacan, Stuart Barnscher, David Mills, Thomas Spreter, Surjit Dixit. Increasing the therapeutic index of IL12 by engineering for tumor specific protease activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1788.

Abstract Zanidatamab (ZW25) is a biparatopic antibody that simultaneously binds two distinct epitopes of the oncogenic cell surface receptor HER2. Zanidatamab is currently being evaluated in global Phase 1, Phase 2 and registration-enabling clinical trials as a potential new treatment for patients with HER2-expressing cancers, including biliary tract, gastroesophageal adenocarcinomas, breast, and other tumor types. The extracellular domain of the HER2 receptor comprises four domains with zanidatamab binding to epitopes on domain 2 and the membrane proximal domain 4. Domain 2 and domain 4 of HER2 are also respectively targeted by the antibodies pertuzumab and trastuzumab, currently approved for clinical use in a subset of HER2 overexpressing cancers. The unique engineering and HER2-engagement of zanidatamab results in multiple mechanisms of action, including dual HER2 signal blockade, increased antibody binding, receptor clustering, and removal of HER2 from the cell surface, and potent effector function. Preclinical work shows that zanidatamab can exhibit differentiated activity over trastuzumab or combination of trastuzumab and pertuzumab but the precise molecular mechanism by which zanidatamab differentiates itself from these approved agents remains unclear. Structural modeling of zanidatamab in complex with HER2 suggests that unlike trastuzumab and pertuzumab, steric features of zanidatamab induce a complex of this antibody with HER2 comprised of alternating chains of the antibody and HER2 molecules, with each copy of zanidatamab bridging two HER2 molecules and vice-versa. In this study we report on the spatial distribution of cell surface HER2 molecules and the effect of antibody-induced receptor reorganization on prototypical cancer cells with high and low expression levels of this receptor. We employ super-resolution single-molecule microscopy to map the receptor distribution below the diffraction limit of traditional optical imaging techniques. The use of Direct Stochastic Optical Reconstruction Microscopy (dSTORM) allows us to quantitatively differentiate properties such as cluster size, frequency and receptor density induced by the antibodies. We demonstrate that the unique geometry of HER2 engagement achieved by zanidatamab results in the induction of strikingly aggregated HER2 receptor cluster we refer to as “capping” on the cell surface. This effect induced by zanidatamab is notably distinct relative to the receptor reorganization observed with trastuzumab or combination of trastuzumab and pertuzumab which typically induce multiple smaller microclusters on the cell surface. We observe differences in the lifetime and persistence of these capped configurations induced by zanidatamab, leading us to believe that these cell surface reorganization events are likely coupled to events such as internalization and receptor down regulation. Citation Format: Surjit Dixit, Libin Abraham, Nina Weiser, Michael R. Gold. Super-resolution imaging studies of zanidatamab: Providing insights into its bispecific mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1032.

As biologics have become a mainstay in the development of novel therapies, protein engineering tools to expand on their structural advantages, namely specificity, affinity, and valency are of interest. Antibodies have dominated this field as the preferred scaffold for biologics development while there has been limited exploration into the use of albumin with its unique physiological characteristics as a platform for biologics design. There has been a great deal of interest to create bispecific and more complex multivalent molecules to build on the advantages offered by protein-based therapeutics relative to small molecules. Here, we explore the use of human serum albumin (HSA) as a scaffold for the design of multispecific biologics. In particular, we describe a structure-guided approach to the design of split HSA molecules we refer to as AlbuCORE, that effectively and spontaneously forms a native albumin-like molecule, but in a heterodimeric state upon co-expression. We show that the split AlbuCORE designs allow the creation of novel fusion entities with unique alternate geometries. We also show that, apart from these AlbuCORE fusion entities, there is an opportunity to explore their albumin-like small hydrophobic molecule carrying capacity as a drug conjugate in these designs.

Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been described in the literature to knockout effector function, to date all of them have involved the use of a specific antibody subtype (e.g., IgG2 or IgG4), or symmetric mutations in the lower hinge or CH2 domain of traditional homodimeric monospecific antibodies. In the context of a heterodimeric Fc, we describe novel asymmetric Fc mutations with reduced or silenced effector function in this article. These heteromultimeric designs contain asymmetric charged mutations in the lower hinge and the CH2 domain of the Fc. Surface plasmon resonance showed that the designed mutations display much reduced binding to all of the Fc gamma receptors and C1q. Ex vivo ADCC and CDC assays showed a consistent reduction in activity. Differential scanning calorimetry showed increased thermal stability for some of the designs. Finally, the asymmetric nature of the introduced charged mutations allowed for separation of homodimeric impurities by ion exchange chromatography, providing, as an added benefit, a purification strategy for the production of bispecific antibodies with reduced or silenced effector function.