In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.
Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.
neered factor IX variants with improved pharmacokinetics and subcutaneous efficacy. J Thromb Haemost 2013; 11: 1699-706.Summary. Background: The rapid clearance of factor IX (FIX) necessitates frequent intravenous administration to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration would be a preferred route of administration but is limited by bioavailability. Objectives: To improve the pharmacokinetics (PK) and bioavailability of FIX, a screen was performed to identify positions for the introduction of novel glycosylation sites with maximal effect on PK and maintenance of coagulation activity. Methods: Two hundred fifty-one variants, each containing one additional N-linked glycosylation site, were screened in vitro, and the PK profiles of selected variants mapping to spatially distinct regions of FIX were evaluated in mice. Optimal variants were combined, and their PK and efficacy were determined in mice with hemophilia B. Results: Variants that mapped to spatially distinct regions of the FIX structure exhibited different degrees of improved PK and enabled selection of optimized sites while minimizing the loss of FIX activity. Combining the most effective N-glycan sites in the same FIX molecule resulted in further improvements in PK. An optimized variant containing three novel N-glycan sites (at amino acids 103, 151, and 228), and the activity enhancing 338A variant had double the specific activity of wild-type FIX, exhibited 4.5-fold reduced clearance and 2.4-fold increased subcutaneous bioavailability, and was efficacious at a fivefold lower mass dose than wildtype FIX after subcutaneous injection in a bleeding model in mice with hemophilia B. Conclusions: Glycoengineering was used to significantly improve the subcutaneous PK and efficacy of FIX and may have advantages for subcutaneous dosing.
have ownership interest as shares in Bayer AG; Zurit Levine and Ofer Levy have ownership interest as shares in Compugen Ltd.
The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening.
Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation‐specific antibody against ATβ, TPP2009, was made to investigate ATβ in hemostasis. TPP2009 bound specifically to heparin–ATβ and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATβ in hemostasis. Summary BackgroundAntithrombin III (AT)β is an isoform of AT that lacks the post‐translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. ObjectivesTo characterize a conformation‐specific antibody against ATβ and begin to investigate the role of ATβ in maintaining hemostasis. MethodsSurface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin‐bound ATβ (ATβ*H) by the use of phage display. ResultsSPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATβ*H and glycosaminoglycan‐associated ATβ on endothelial cells. In diluted prothrombin and activated factor X (FXa)‐induced clotting assays, TPP2009 dose‐dependently reduced the anticoagulant effect of heparin in non‐hemophilic and FVIII‐deficient human plasma, with half‐maximal effective concentrations (EC50) of 10.5 nm and 4.7 nm, respectively. In AT‐deficient human plasma, TPP2009 dose‐dependently inhibited the effects of exogenously added ATβ and heparin. In purified systems with ATβ and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose‐dependently reversed the effects of heparin in rabbit (EC50, 25.7 nm) and cynomolgus monkey (EC50, 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function‐neutralizing antibodies. ConclusionsTPP2009 specifically targets a unique conformational epitope on ATβ*H and blocks ATβ‐mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATβ in hemostasis.
CEACAM6 (CD66c) was previously shown to act as a novel immune checkpoint regulator suppressing the activity of effector T cells against tumors (Witzens-Harig et al., Blood 2013). CEACAM6 is a GPI-linked protein that is strongly expressed at the tumor cell surface in multiple cancer indications such as non-small cell lung adenocarcinoma (NSCLC), colorectal carcinoma (CRC), gastric adenocarcinoma and pancreatic cancer. In general, elevated CEACAM6 expression is associated with advanced tumor stages and poor prognosis. In vitro experiments showed that engagement of T-cells with CEACAM6, either expressed on tumor cells or presented on beads, resulted in suppression of TCR-mediated T-cell activation and ZAP70 phosphorylation. Based on these findings, we hypothesized that antibodies targeting CEACAM6 may be employed to enhance T-cell responses against CEACAM6-expressing cancers. Here we report the generation and characterization of BAY 1834942, a humanized monoclonal antibody selectively blocking the inhibitory impact of CEACAM6 on human T cells. There is no rodent ortholog of CEACAM6 precluding in vivo efficacy studies. In tumor cell / T cell co-culture systems, BAY 1834942 increased secretion of T-cell cytokines and effector molecules (e.g. IFNγ, TNFα, IL-2, granzyme B) and resulted in improved tumor cell killing. The effects of BAY 1834942 were dose-dependent, only observed in the context of CEACAM6-expressing tumor cells and could be reproduced in experiments using tumor cell lines and T-cell preparations from different sources, including T cells derived from tumor infiltrating lymphocytes from pancreatic cancer. BAY 1834942 is cross-reactive with the cynomolgus CEACAM6 ortholog and was well-tolerated in monkey toxicology studies. In summary, BAY 1834942 is a novel checkpoint inhibitor with potential for the treatment of patients with CEACAM6 expressing cancers, both as single agent and in combination with other checkpoint inhibitors. First-in-man trials are expected to commence in 2018. Citation Format: Joerg Willuda, Mark Trautwein, Jessica Pinkert, Wolf-Dietrich Doecke, Hans-Henning Boehm, Florian Wessel, Yingzi Ge, Eva Maria Gutierrez, Joerg Weiske, Christoph Freiberg, Uwe Gritzan, Julian Glueck, Dieter Zopf, Sven Golfier, Oliver von Ahsen, Ruprecht Zierz, Sabine Wittemer-Rump, Heiner Apeler, Ziegelbauer Karl, Rienk Offringa, Bertolt Kreft, Beckhove Philipp. BAY 1834942 is an immunotherapeutic antibody blocking the novel immune checkpoint regulator CEACAM6 (CD66c) [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 1771.
ILDR2 is a type I transmembrane protein belonging to the B7 family of immunomodulatory receptors with 98 % sequence identity between the extracellular domains of human and murine orthologues. The exact physiological role of ILDR2 is still unclear, but it has been implicated in the development of type 2 diabetes and the formation of tri-cellular junctions. Our detailed analyses reveal that ILDR2 is expressed in kidney, testis, liver, and lymph node fibroblastic reticular cells (FRC). The latter is particularly interesting since FRC belong to the CD45- stromal population, a specialized cell subset located in the T cell zone. These cells are essential for recruitment of naïve T cells and activated dendritic cells to the lymph node and have been reported to exhibit immuno-regulatory properties. Due to its structural similarity to members of the B7 family and due to its expression in FRCs, we speculated that ILDR2 might play a role e.g. in T cell priming and the initiation of antigen-specific T cell responses. In line with this, we and our collaborators from Compugen were able to show that an ILDR2 -Fc fusion protein is able to a) bind to activated (but not naïve) T cells, b) suppress TCR-stimulated cytokine secretion and c) exhibit immunomodulatory effects in several models of autoimmune diseases, i.e. multiple sclerosis, rheumatoid arthritis, and type 1 diabetes. As a consequence, we set out to generate antibodies against this novel immuno-oncology target and describe here for the first time the characterization of BAY 1905254, a human/mouse cross-reactive IgG2 antibody blocking the immunosuppressive activity of ILDR2. BAY 1905254 specifically binds to ILDR2 but not to the closely related family members ILDR1 and ILDR3/LSR. It enhances antigen-specific T cell proliferation in vivo in an ovalbumin vaccination model with OT-I transgenic T cells. BAY 1905254 exhibits anti-tumor activity in different syngeneic mouse models with efficacy correlating with increasing mutational load. Furthermore, additive/synergistic anti-tumor effects can be observed in combination with either an anti-PD-L1 Ab, an immunogenic cell death inducing chemotherapeutic (Docetaxel) or tumor antigen immunization. Ex vivo analysis reveal that BAY 1905254 treatment results in increased intra-tumoral IFN-y levels and enhanced infiltration of CD45+ cells, e.g. of CD8α+ dendritic cells (DCs). Interestingly, this DC population is known to play a crucial role in cross-presentation, thus, in the initiation of CD8+ T cell responses. In summary, BAY 1905254 is a function-blocking Ab able to counteract the immuno-suppressive function of the newly discovered immuno-oncology target ILDR2. The Ab exhibits in vivo efficacy both in monotherapy as well as in combination with various other approaches, and it is planned to advance BAY 1905254 into FiM trials in 2018. Citation Format: Julia Huetter, Uwe Gritzan, Ilona Gutcher, Sven Golfier, Wolf-Dietrich Doecke, Merlin Verena Luetke-Eversloh, Helge Roider, John Hunter, Andrew Pow, Spencer Liang, Zurit Levine, Ofer Levy, Ilan Vaaknin, Bertolt Kreft, Lars Roese. Discovery and preclinical characterization of BAY 1905254 a novel immune checkpoint inhibitor for cancer immunotherapy targeting the immunoglobulin-like domain containing receptor 2 (ILDR2) [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 2778.
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