Protein sequences of all recombinant fusion proteins described in the manuscript

Time-lapse fluorescence microscopy of CMV-pp65-specific T cell mediated killing of MCSP+ tumor cells (Colo38) incubated with 15nM of EBV-pMHCI-IgG as control. Constructs are indicated in the respective panel. T cells are stained in red (PKH-26, Sigma), tumor cells in green (CMFDA, LifeTechnologies). Effector to target ratio was 3:1. Real-time is indicated in the lower left corner in h:m:s:ms (h=hours, m=minutes, s=seconds, ms=milliseconds), scale bar is shown in the lower right corner 0-50 µm (µm=micrometre).

Time-lapse fluorescence microscopy of CMV-pp65-specific T cell mediated killing of MCSP+ tumor cells (Colo38) incubated with 15nM of CMV-pMHCI-IgG. Constructs are indicated in the respective panel. T cells are stained in red (PKH-26, Sigma), tumor cells in green (CMFDA, LifeTechnologies). Effector to target ratio was 3:1. Real-time is indicated in the lower left corner in h:m:s:ms (h=hours, m=minutes, s=seconds, ms=milliseconds), scale bar is shown in the lower right corner 0-50 µm (µm=micrometre).

Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.

Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.

A transcellular shuttle system was generated for the delivery of non-covalently linked payloads across blood-brain barrier (BBB) endothelial cells. Transcytosis-enabling shuttles are composed of bispecific antibodies (bsAbs) that simultaneously bind transferrin receptor (TfR) and haptens such as digoxigenin or biocytinamide. Haptenylated payloads are attached to these vehicles via non-covalent hapten-antibody complexation. This enables targeting to and internalization into human BBB-derived microvascular endothelial hCMEC/D3 cells. In contrast to other shuttles, this system does not require special affinities or formats of their TfR-binding moieties for transcytosis and subsequent release. Non-covalent payload complexation to bsAb is flexible and robust, works for a multitude of payloads and enables separation of payloads from shuttles during transcytosis. Released payloads can enter the brain without connected bsAb entities, minimizing potential interference with distribution or functionality. Intracellular separation of shuttle and payload and recycling to cell surfaces may also enable recharging of the cell-bound BBB shuttle with payload for subsequent (merry-go-round) transport cycles.

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Title: A new class of bifunctional major histocompatibility class I antibody fusion molecules to redirect CD8 T cells Authors: Martina Schmittnaegel, Eike Hoffmann, Sabine Imhof-Jung, Cornelia Fischer, Georg Drabner, Guy Georges, Christian Klein, Hendrik Knoetgen Authors' Affiliations: ROCHE Pharma Research and Early Development: 1 Large Molecule Research, ROCHE Innovation Center Munich, Germany; 2 Discovery Oncology, ROCHE Innovation Center Zurich, Switzerland; 3 Therapeutic Modalities, ROCHE Innovation Center Basel, Switzerland; 4 Current Affiliation: Swiss Federal Institute of Technology Lausanne (EPFL), The Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland Corresponding Author: Hendrik Knoetgen, ROCHE Pharma Research and Early Development: Therapeutic Modalities, ROCHE Innovation Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland Phone: +41 616821959, mailto:hendrik.knoetgen@roche.com Disclosure of Potential Conflicts of Interest: All authors are or have been employees of the F. Hoffmann-La Roche AG, Switzerland or Roche Diagnostics GmbH, Germany. H. Knoetgen, S. Imhof-Jung and M. Schmittnaegel have ownership interest (including patents). Running Title: Bifunctional MHC I antibody fusions

Abstract Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide–MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8+ T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide–MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130–42. ©2016 AACR.

Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a universal coupling position (52(+2)) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95-100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.-Dengl, S., Hoffmann, E., Grote, M., Wagner, C., Mundigl, O., Georges, G., Thorey, I., Stubenrauch, K.-G., Bujotzek, A., Josel, H.-P., Dziadek, S., Benz, J., Brinkmann, U. Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies.

Abstract Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppression of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen–specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI–IgG). Here, we show that tumor antigen–expressing cancer cells, which are decorated with pMHCI–IgGs containing a HLA-A*0201 molecule associated with a CMV-derived peptide, are specifically eliminated through engagement of antigen-specific CD8+ T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8+ T cells act without additional expansion, preactivation, or provision of costimulatory signals. Elimination of tumor cells is induced at similar concentrations and with similar time kinetics as those seen with bispecific T-cell engagers (BiTE). However, while BiTE-like reagents indiscriminately activate T cells through binding to the T-cell receptor complex, pMHCI–IgGs selectively engage antigen-specific, constantly renewable, differentiated effector cytotoxic T lymphocytes to tumor cells, thereby representing a novel class of anticancer immunotherapeutics with potentially improved safety and efficacy profiles. Cancer Immunol Res; 3(7); 764–76. ©2015 AACR.

We applied noncovalent complexes of digoxigenin (Dig) binding antibodies with digoxigeninylated peptide derivatives to modulate their pharmacokinetic properties. A peptide derivative which activates the Y2R receptor was selectively mono-digoxigeninylated by reacting a NHS-Dig derivative with an ε-amino group of lysine 2. This position tolerates modifications without destroying receptor binding and functionality of the peptide. Dig-peptide derivatives can be loaded onto Dig-binding IgGs in a simple and robust reaction, thereby generating peptide-IgG complexes in a defined two to one molar ratio. This indicates that each antibody arm becomes occupied by one haptenylated peptide. In vitro receptor binding and signaling assays showed that Dig-peptides as well as the peptide-antibody complexes retain better potency than the corresponding pegylated peptides. In vivo analyses revealed prolonged serum half-life of antibody-complexed peptides compared to unmodified peptides. Thus, complexes are of sufficient stability for PK modulation. We observed more prolonged weight reduction in a murine diet-induced obesity (DIO) model with antibody-complexed peptides compared to unmodified peptides. We conclude that antibody-hapten complexation can be applied to modulate the PK of haptenylated peptides and in consequence improve the therapeutic efficacy of therapeutic peptides.

Abstract MHC (major histocompatibility complex) class I-restricted CD8 cytotoxic T cells recognize tumor cells or virus infected cells as foreign and consequently initiate a cascade of events resulting in their destruction. An immunotherapeutic strategy based on antibody-mediated targeting of virus-derived peptide-MHC class I complexes to tumor cells aiming at the subsequent elimination of these tumor cells by peptide-specific cytotoxic T cells has been proposed a few years ago. A crucial limitation hampering advances of this therapeutic concept was the recombinant expression of MHC class I fused full IgG immunoglobulins. We have identified novel recombinant fusion formats that allow the expression of full-length peptide-MHC class I IgG fusion molecules. These formats contain a single recombinant human pMHC class I complex consisting of a viral peptide (CMV or EBV derived), beta-2-microglobulin and HLA heavy chain (A*0201, lacking the transmembrane domain) fused to one of the two immunoglobulin heavy chains of a complete antibody molecule. These fusions can be expressed at high levels in standard mammalian expression systems overcoming several former technical hurdles (e.g. prokaryotic expression, low refolding yield, chemical coupling of components). By flow cytometry we show that the new fusion proteins successfully deliver virus-peptide complexed MHC class I molecules to tumor cells. The fusion proteins are potent CD8 T cell recruiters for human donor derived specific T cells or human PBMCs from chronically infected donors. A low frequency of virus peptide specific CD8 T cells in PBMCs (0.25 to 3.8 % of all CD8 positive T cells) effectively triggers in a peptide specific manner the killing of tumor cells at sub-nanomolar concentrations (cytotoxic T cell assay using real time analysis with xCelligence confirmed by classical LDH release). No unspecific activation of T cells was observed even at high concentrations, indicating a favorable safety profile. Confocal time-lapse microscopy showed T cell synapse formation on the tumor cells and serial killing by the T cells. We experimentally compare the killing of tumor cells of the MHC class I fused antibodies with bispecific anti-CD3 mediated T cell recruiters and characterized the virus specific T cells from human donors in detail. Our results show for the first time that recombinant pMHC class I antibody fusions in our novel full human IgG format can be expressed with a significant yield in common mammalian production cell lines. The redirection of MHC class I mediated recruitment of pre-existing virus specific CD8 T cells from human PBMCs is potent mechanism to attack tumor cells with a low risk of unspecific T cell activation making the concept now suitable as a therapeutic option. Citation Format: Martina Schmittnaegel, Eike Hoffmann, Olaf Mundigl, Gerhard Niederfellner, Klaus Bosslet, Pablo Umana, Victor Levitsky, Christian Klein, Hendrik Knoetgen. Novel MHC class I antibody fusions for cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B69.