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BACKGROUND: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models. METHODS: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology. RESULTS: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies. CONCLUSION: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in retinal and subretinal neovascularization (NV). Increased levels of HIF-1 cause increased expression of vascular endothelial growth factor (VEGF-A) and current therapies for ocular NV focus on neutralizing VEGF-A, but there is mounting evidence that other HIF-1-responsive gene products may also participate. In this study, we tested the effect of a designed ankyrin repeat protein (DARPin) that selectively binds and antagonizes the hypoxia-regulated gene product PDGF-BB in three models of subretinal NV (relevant to neovascular age-related macular degeneration) and compared its effects to a DARPin that selectively antagonizes VEGF-A. Daily intraperitoneal injections of 10 mg/kg of the anti-PDGF-BB DARPin or 1 mg/kg of the anti-VEGF DARPin significantly suppressed subretinal NV from laser-induced rupture of Bruch's membrane. Injections of 1 mg/kg/day of the anti-PDGF-BB DARPin had no significant effect, but when combined with 1 mg/kg/day of the anti-VEGF-A DARPin there was greater suppression than injection of the anti-VEGF-A DARPin alone. In Vldlr−/− mice which spontaneously develop subretinal NV, intraocular injection of 1.85 μg of anti-PDGF-BB or anti-VEGF-A DARPin caused significant suppression of NV and when combined there was greater suppression than with either alone. The two DARPins also showed an additive effect in Tet/opsin/VEGF double transgenic mice, a particularly severe model of subretinal NV and exudative retinal detachment. In addition, intraocular injection of 1.85 μg of anti-PDGF-BB DARPin strongly suppressed ischemia-induced retinal NV, which is relevant to proliferative diabetic retinopathy and retinopathy of prematurity. These data demonstrate that PDGF-BB is another hypoxia-regulated gene product that along with VEGF-A contributes to ocular NV and suppression of both provides an additive effect.

The homozygous deletion of the phenylalanine at position 508 (DeltaPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and DeltaPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the 'folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the DeltaPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against CF.

T-cell engagers (TCEs) direct cytotoxic T-cell response towards tumor cells by binding simultaneously to a tumor-associated antigen (TAA) on target cells and to CD3 on T-cells, thereby forming an artificial immune synapse. They have been shown to be very potent anti-tumor drugs, as exemplified by blinatumomab, an α-CD19 x α-CD3 bispecific. However, the development of TCEs for hematological and solid tumors has been hampered by several factors, amongst them severe toxicity, elicited by on-target/off-tumor recruitment of T-cells and cytokine release syndrome (CRS). In order to overcome this challenge, an anti-CD3 Prodrug DARPin® (CD3-PDD) has been developed, consisting of a mouse cross-reactive EGFR-binder and a CD3-binder, linked via a protease-cleavable linker to an anti-idiotypic anti-CD3 binder (termed blocker hereafter). This α-EGFR x α-CD3 x blocker Prodrug is unable to bind and recruit T-cells in its non-cleaved state, but is designed to become activated in the tumor microenvironment upon cleavage of the linker by tumor-associated proteases. A control Prodrug DARPin® with a non-cleavable linker showed neither tumor cell killing, nor T-cell activation at concentrations >1'000-fold over the EC50 of the active, non-blocked TCE DARPin® in in vitro tumor cell killing and T-cell activation assays. In contrast, a CD3-PDD containing a cleavable linker was partially activated by proteases secreted from the tumor cells (HCT 116). Pre-treatment of the CD3-PDD with recombinant protease prior to the in vitro assay fully activated the molecule, with EC50 values comparable to the active, non-blocked TCE. Next, an in vivo proof-of-principle study was performed in a human colon carcinoma xenograft model (HCT 116) using immunodeficient mice humanized with hematopoietic stem cells (CD34+) and optimized for the presence of human myeloid cells. Due to the mouse cross-reactivity of the EGFR-binder, this animal model allowed to assess both anti-tumor efficacy and safety (therapeutic window). The cleavable CD3-PDD demonstrated a robust anti-tumor activity, similar to the one observed with active, non-blocked TCE. Most importantly, while the active, non-blocked TCE elicited strong toxicity, leading to loss of animals and requiring treatment stop, the cleavable CD3-PDD could be dosed without significant safety findings. In summary, a conditionally activated CD3-PDD shows similar efficacy but none of the toxicity of the active, non-blocked TCE. Our approach therefore holds great promise for the development of future CD3-PDD as therapeutics, enabling the utilization of less tumor-specific targets for highly potent TCEs. Ultimately, the ability of the versatile DARPin® technology to generate tailor-made anti-idiotypic DARPin® molecules can unlock novel therapeutic design spaces, which we are exploring beyond the conditionally activated CD3-PDD format. Citation Format: Andreas Bosshart, Julia Katharina Ahlskog, Aline Eggenschwiler, Dieter Schiegg, Yvonne Grübler, Sandra Wandel, Simon Fontaine, Maria Paladino, Susanne Mangold, Tanja Hospodarsch, Alexandra Neculcea, Chloé Iss, Christel Herzog, Bernd Schlereth. A solution to T-cell engager toxicity: An anti-CD3 Prodrug DARPin (CD3-PDD) shows no toxicity, but potent anti-tumor activity in a humanized mouse model [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 1890.

During the last years, immune-modulating drugs became an important cornerstone in the treatment of cancer patients. In particular, the PD1/PDL-1 and CTLA-4 antagonists revolutionized the field. However, only a limited number of patients benefit from these antagonistic molecules and more combination therapies are on the way to increase the number of patients benefiting from these novel therapies. Among the combinations, drugs that are T-cell or myeloid cell agonists belonging to the TNFR-superfamily show first promising clinical results. However, systemic immune activation bears the risk of severe side effects that will not allow using these powerful drugs at an effective dose. We have developed a new class of DARPin molecules that enable tumor-restricted immune cell activation of TNFR-superfamily agonists in the tumor only, thereby preventing systemic immune-activation. We generated DARPin molecules that bind with high affinity to TNFR-superfamily members (CD134, CD137 and CD40) and DARPin molecules that bind to tumor-specific antigens such as HER2 and EGFR or targets restricted to the tumor stroma compartment like FAP and extra-domain B of fibronectin (ED-B). Using these building blocks from our DARPin toolbox, we constructed a variety of multi-specific molecules consisting of a TNFR-superfamily receptor targeting DARPin molecule and a tumor-localizing DARPin molecule. In reporter cell assays the multi-specific DARPin molecules activate the respective TNF-superfamily receptor only in the presence of a cell expressing the tumor-localizer; e.g. the FAP-CD134 molecule activates CD134 only in the presence of a stromal cell expressing FAP and not in its absence. Moreover, activation of CD134 was dependent on the density of FAP expression on cells, showing that CD134 only becomes activated if a certain level of the tumor-localizing target is expressed. These finding were confirmed in experiments with primary immune cells where we see immune-cell activation only upon binding to the tumor-localizing target. This could be shown for multiple combinations (>8) and supports the concept that multi-specific DARPin molecules are powerful drug-candidates allowing tumor-restricted immune cell activation. Citation Format: Ulrike Fiedler, Christian Reichen, Joanna Taylor, Patricia Schildknecht, Sophie Barsin, Clara Metz, Anja Schlegel, Simon Fontaine, Denis Villemagne, Julia Ahlskog, Yvonne Kaufmann, Alexander Link, Nicolo Rigamonti, Julia Hepp, Michael T. Stumpp. Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox [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 4552.

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