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Figure S1: Characterization of binding properties and thermal stability of Fynomer C12; Figure S2: Results of binding studies with C12-pertuzumab bispecific FynomAbs; Figure S3: Additional in vitro tumour growth inhibition studies with COVA208 on more HER2 positive cancer cell lines; Figure S4: Additional data on the induction of apoptosis in NCI-N87 cells by COVA208: dose-response curve of Caspase 3/7 activity, and Tunel assay; Figure S5: Expression data (heat map) of signalling mediators in response to treatment with COVA208, pertuzumab, or trastuzumab (RPPA analysis); Supplementary Table 1 listing expression data of 167 signaling proteins (phospho- and total proteins) in response to COVA208, pertuzumab or trastuzumab treatment of NCI-N87 cells in vitro.

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3–derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Mol Cancer Ther; 13(8); 2030–9. ©2014 AACR.

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition.

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3–derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Mol Cancer Ther; 13(8); 2030–9. ©2014 AACR.

CD3 bispecific therapies retargeting T cells to tumors have recently demonstrated striking activity in patients. Several CD3 bispecific antibodies directed against various tumor targets are currently being investigated in the clinic across different tumors. One limitation of these therapies is the risk of target-related toxicity due to low-level expression of tumor antigen in normal tissue. In this study we have engineered a bispecific CD3/HER2 FynomAb, COVA420, which redirects T cells with high potency and selectivity to tumor cells with high HER2 expression in vitro and in vivo. COVA420 activity depends on high HER2 density as no activity was observed on cells with lower HER2 levels as found in human normal tissue. These results suggest that COVA420 may spare normal tissue expressing low levels of HER2 while still having uncompromised efficacy on tumor cells with high HER2 expression. This concept may be applied to other cancer antigens that otherwise cannot be targeted by T cell redirecting approaches, and may therefore expand the applicability of CD3 bispecific FynomAbs to a larger number of solid tumors.

Acute myeloid leukemia (AML) is an aggressive blood cancer with neoplastic infiltration of leukemic blasts in blood, bone marrow and viscera. Current treatment options have limited efficacy, and the 5-year survival rate is only 27%. CD3 bispecific antibodies that re-direct T cells towards the tumor have shown promising efficacy in hematological malignancies. The CD3/CD19 bispecific antibody blinatumomab has recently been approved by the FDA for the treatment of B-cell acute lymphocytic leukemia (ALL) (Kantarjian H et al (2017) NEJM). We evaluated COVA4231, a CD3/CD33 bispecific FynomAb, preclinically as therapeutic candidate for AML. CD33 is a cell surface receptor expressed on blasts of the majority of AML patients (Walter RB (2014) Expert Opin Ther Targets). COVA4231 was constructed by fusing the CD33-specific Fynomer D5 - a small (7 kDa) globular protein derived from the human Fyn SH3 domain with engineered affinity for CD33 - to a CD3-specific antibody with a novel silent IgG1 Fc. COVA4231 induced T cell activation and cytokine release in a CD33-dependent manner, and elicited potent T cell mediated cytotoxicity of CD33-expressing target cells in vitro with EC50 in the range of 4 - 29 pM (E:T cell ratio of 2:1, KG-1 or MOLM-13 as target cells). COVA4231 showed potent activity against 13 out of 15 primary AML blast samples ex vivo at E:T cell ratio of 1:1. The in vivo efficacy of COVA4231 was investigated in a subcutaneous HL-60 leukemia xenograft model in NSG mice in the presence of human T cells. COVA4231 was highly active across a wide dose range (0.05 - 5 mg/kg) and prevented tumor outgrowth in all treated mice. COVA4231 demonstrated an IgG1-like pharmacokinetic profile in mice with a terminal half-life 15.8 days (total drug), providing the opportunity to avoid continuous intravenous infusion protocols required for established CD3 bispecific formats (e.g. (scFv)2). In conclusion, COVA4231 is a highly active therapeutic candidate in vitro, in vivo and ex vivo, has IgG-like pharmacokinetics and is a promising therapeutic candidate for further preclinical and clinical development. Citation Format: Kristina Klupsch, Vanessa Baeriswyl, Roland Scholz, Joana Dannenberg, Roger Santimaria, David Senn, Elena Kage, Adrian Zumsteg, Isabella Attinger-Toller, Ulrike von der Bey, Susann König-Friedrich, Fanny Dupuy, Wibke Lembke, Clara Albani, Severin Wendelspiess, Lucijana Dinkel, Chelsea J. Gudgeon, Roland B. Walter, Julian Bertschinger, Simon Brack. COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1787.

The cellular generation of toxic metabolites and subsequent detoxification failure can cause the uncontrolled accumulation of these metabolites in cells, leading to cellular dysfunction. Amyloid-beta protein (A beta), a normal metabolite of neurons, tends to form toxic oligomeric structures that cause neurodegeneration. It is unclear how healthy neurons control the levels of intracellular oligomeric A beta in order to avoid neurodegeneration. Using immunochemical and biochemical studies, we show that the A beta-binding serine protease Omi is a stress-relieving heat-shock protein that protects neurons against neurotoxic oligomeric A beta. Through its PDZ domain, Omi binds preferentially to neurotoxic oligomeric forms of A beta rather than non-toxic monomeric forms to detoxify oligomeric A beta by disaggregation. This specific interaction leads not only to mutual detoxification of the pro-apoptotic activity of Omi and A beta-induced neurotoxicity, but also to a reduction of neurotoxic-A beta accumulation. The neuroprotective role of Omi is further supported by its upregulation during normal neurogenesis and neuronal maturation in mice, which could be in response to the increase in the generation of oligomeric A beta during these processes. These findings provide novel and important insights into the detoxification pathway of intraneuronal oligomeric A beta in mammals and the protective roles of Omi in neurodegeneration, suggesting a novel therapeutic target in neurodegenerative diseases.

Summary Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na+/Ca2+ exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson's disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinson's disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinson's disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinson's disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.

The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.

The protease inhibitor Ucf-101 induces cellular responses independently of its known target, HtrA2/Omi

Cellular stress responses can be activated following functional defects in organelles such as mitochondria and the endoplasmic reticulum. Mitochondrial dysfunction caused by loss of the serine protease HtrA2 leads to a progressive movement disorder in mice and has been linked to parkinsonian neurodegeneration in humans. Here, we demonstrate that loss of HtrA2 results in transcriptional upregulation of nuclear genes characteristic of the integrated stress response, including the transcription factor CHOP, selectively in the brain. We also show that loss of HtrA2 results in the accumulation of unfolded proteins in the mitochondria, defective mitochondrial respiration and enhanced production of reactive oxygen species that contribute to the induction of CHOP expression and to neuronal cell death. CHOP expression is also significantly increased in Parkinson's disease patients' brain tissue. We therefore propose that this brain-specific transcriptional response to stress may be important in the advance of neurodegenerative diseases.

The fourth author's name appears incorrectly in the author byline and in the citation. It should read: Andrey Y. Abramov, and the citation should read: Wood-Kaczmar A, Gandhi S, Yao Z, Abramov AY, Miljan EA, et al. (2008) PINK1 Is Necessary for Long Term Survival and Mitochondrial Function in Human Dopaminergic Neurons. PLoS ONE 3(6): e2455. doi:10.1371/journal.pone.0002455

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra- and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor-limiting conditions. E4-ORF1 mimics growth factor signaling by activating PI3-kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4-ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4-ORF4 protein phosphatase 2A-binding domain. We also show that mTOR activation is required for efficient S-phase entry, independently of E2F activation, in adenovirus-infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2 targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. Based on the approved antibody pertuzumab we have created a panel of bispecific FynomAbs which target two epitopes on HER2. The activity of the HER2 targeting FynomAbs was found to depend on the FynomAb architecture, i.e. the spatial arrangement of the binding sites of antibody and the Fynomer. The most potent of these FynomAbs, termed COVA208, demonstrated superior tumor cell growth inhibition in vitro compared to pertuzumab and trastuzumab. COVA208 was characterized in detail and showed an increased ability to induce rapid HER2-internalization and apoptosis in vitro. Moreover, it elicited a stronger inhibition of downstream HER2 signaling which was accompanied by a reduction of HER2, HER3 and EGFR levels in vitro and in vivo. The therapeutic potential of COVA208 has been demonstrated in vivo in four different HER2 mouse models, where COVA208 exhibited excellent anti-tumor activity. Importantly, COVA208 demonstrated superior activity in vivo compared to trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Citation Format: Babette Schade, Simon Brack, Isabella Attinger-Toller, Kristina Klupsch, Richard Woods, Helen Hachemi, Ulrike von der Bey, Susann Koönig-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2 targeting FynomAb with superior anti-tumor activity and novel mode of action. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2014-658

There is increasing evidence that T cells are able to control tumor growth and increase survival of cancer patients. However, tumor-specific T cell responses are difficult to mount and sustain in cancer patients, and are limited by numerous immune escape mechanisms of tumor cells. A promising approach in the immunotherapy of cancer is to engage T cells to target tumor cells using bispecific therapeutics targeting a tumor-associated surface antigen and CD3e on T cells. Such therapeutics elicit T cell mediated lysis of tumor cells independent of T cell specificity. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. We have generated a novel bispecific FynomAb which can simultaneously bind HER2 on tumor cells and CD3 on T cells. The bispecific HER2/CD3 targeting FynomAb COVA420 potently redirected T cells to HER2 expressing tumor cells showing picomolar tumor cell lysis activity. We present for the first time that a tailored architecture of the FynomAb leads to optimal tumor cell killing properties. The activity of COVA420 was found to be highly specific, as no lysis of cells was observed in the absence of HER2 expression. In addition, COVA420 demonstrated an antibody-like pharmacokinetic profile in mice. We anticipate that the increased half-life of T cell recruiting FynomAbs compared to other bispecific formats translates into a significant benefit for patients, since it circumvents the need for continuous infusion and prolongs the intervals between successive treatments. In summary, bispecific T cell recruiting FynomAbs represent a novel platform technology to redirect T cells to tumor cells with optimal biophysical properties, long-half lives and tailored architectures. Citation Format: Ulrich Wuellner, Fabian Buller, Kristina Klupsch, Simon Brack, Irene Zbinden, Roger Santimaria, Isabella Attinger-Toller, Susann König-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2/CD3 targeting FynomAb with excellent tumor killing and favorable pharmacokinetic properties. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2014-656

3066 Background: A promising approach in the immunotherapy of cancer is to recruit T cells to attack tumor cells using bispecific therapeutics targeting a tumor-associated surface antigen and CD3 o...

Age-related macular degeneration (AMD), a neurodegenerative disease of the central retina, is the leading cause of blindness in industrialized countries1 and among the elderly throughout the world.2 Although the exact pathogenesis of AMD remains unclear, the current pathophysiologic concept points to the critical role of cumulative oxidative damage to the retinal pigment epithelium (RPE).3,4 The RPE not only participates in photoreceptor metabolism by transporting nutrients from the choroid into the retina, but also removes waste products from the retina, thereby acting as a metabolic gatekeeper between the photoreceptors and choriocapillaris.5 These cells are involved in maintaining retinal homeostasis by phagocytizing the discs shed from the photoreceptor outer segments.6 Deliberate disruption of these processes in experimental animal models has been shown to result in retinal degeneration,7 and defects in the RPE contribute to the initiation and progression of AMD.8,9 Oxidative damage due to an imbalance between the generation and elimination of reactive oxygen species (ROS) is a major cause of RPE damage in AMD.3,4 High oxygen tension in the macula, exposure to light, and the RPE-mediated processes of phagocytosis and lipid peroxidation increase ROS generation in the RPE.10 Accordingly, oxidative stress may promote AMD pathogenesis by interfering with RPE function, decreasing RPE junctional integrity, enhancing RPE expression of proinflammatory and proangiogenic cytokines, and/or promoting RPE apoptosis.5,11,12 Mitochondria is a major producer of ROS and is particularly prone to ROS injury.13 HtrA2/Omi belongs to the family of high temperature requirement protein A (HtrA) serine proteases conserved from bacteria to humans.14,15 Another member of the family, HtrA1, has been reported to possess several single-nucleotide polymorphisms that are associated with increased risk of AMD.16–21 HtrA1 is a stress-inducible serine protease that has dual functions in both extracellular protein degradation and intracellular growth or survival. It may therefore play a role in angiogenesis or RPE and photoreceptor atrophy in AMD.22–24 Human HtrA2/Omi has been described both as a chaperone protein and as a serine protease responsible for cleavage of denatured proteins at elevated temperatures. HtrA2 is expressed as a 45-kDa precursor protein that is cleaved on post-translational translocation to the mitochondria to generate the mature 36-kDa protein. The mature protein is then localized to the mitochondrial intermembrane space, where it remains until subsequent translocation to the cytosol in response to apoptotic stimuli.25 Once in the cytosol, HtrA2/Omi promotes cell death by two different mechanisms. It can either bind to an inhibitor of apoptosis proteins (IAPs) via its amino terminal, reaper-related motif, and thus, induce caspase activity, or it can mediate caspase-independent death through its own protease activity.26–29 In this study, we sought to determine the role of HrtA2/Omi in oxidatively damaged RPE cells and to assess HtrA2/Omi expressions in the retina of the Ccl2−/−/Cx3cr1−/− murine model showing oxidative stress-related retinal degeneration and HtrA2/Omi−/− mice.

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.