Monoclonal antibodies (mAbs) are the fastest growing class of biopharmaceuticals reflecting their diverse applications in research and the clinic. The correct glycosylation of mAbs is required to elicit effector functions such as complement-dependent and antibody-dependent cell-mediated cytotoxicity, although these may be undesirable for the treatment of certain chronic diseases. To gain insight into the properties of glycan-deficient mAbs, we generated and characterized six different aglycosylated human IgG1 mAbs (carrying the N297A mutation) and compared them to their glycosylated counterparts. We found no differences in solubility or heterogeneity, and all mAbs the remained stable in stress tests at 4 and 37 °C. Surface plasmon resonance spectroscopy showed no differences in binding affinity, and the in vivo terminal serum half-life and plasma clearance were similar in rats. However, differential scanning calorimetry revealed that the aglycosylated mAbs contained a less stable C(H)2 domain and they were also significantly more susceptible to pH-induced aggregation. We conclude that aglycosylated mAbs are functionally equivalent to their glycosylated counterparts and could be particularly suitable for certain therapeutic applications, such as the treatment of chronic diseases.

Despite recent progress in the treatment of B-cell malignancies, patients are still in need of innovative therapeutic approaches. CXCR5 is a chemokine receptor expressed in a majority of B-cell malignancies including diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), follicular lymphoma and chronic lymphocytic leukemia. Evaluation of tumor biopsies from relapsed DLBCL patients shows that CXCR5 staining remains high, suggesting altogether that it could be a relevant target to explore for the treatment of non-Hodgkin lymphoma. BAY-924 is a novel first-in-class antibody drug conjugate (ADC) consisting of a humanized anti-CXCR5 IgG1 antibody (Ab) linked to a potent proprietary kinesin spindle protein inhibitor (KSPi). Of importance, the structure of the ADC is optimized for a specific metabolism, matching the KSPi mode of action and enabling a maximal retention of the payload within the tumor cells (Lerchen HG et al.; Angew. Chem. Int. Ed, 2018). Surface plasmon resonance assay showed a high binding affinity of the Ab to CXCR5 (2.5 nM). Affinities of 0.8 to 10 nM were measured by flow cytometry for the ADC in different CXCR5+ lymphoma cell lines. In vitro, BAY 924 had high and selective anti-proliferative activity in a panel of tumor cell lines with different levels of CXCR5 expression (<0.03 - 2 nM IC50). Efficient internalization and lysosomal co-localization of the Ab was observed in a variety of cell lines including the CXCR5+ MCL REC-1 cells. In vivo, BAY-924 was highly active in several CXCR5+ lymphoma models with a specific accumulation of the payload in tumor versus liver, spleen and kidney and almost undetectable levels in plasma. In the REC-1 model implanted subcutaneously (SC) in mice and treated at large tumor size (500 mm3), long lasting tumor regression was observed after 2 intravenous injections of BAY 0924 at 10 mg/kg, Q7D, whereas the model was insensitive to ibrutinib, a current standard of care (SoC) for the MCL indication. In the advanced ABC DLBCL model OCI-LY1 (SC), a single injection of BAY-924 at 10 mg/kg induced complete responses in 10/10 mice (up to day 95 post-treatment). In this model, head-to-head comparison showed superior activity of BAY-924 compared to the SoCs rituximab (R)-CHOP, R/bendamustine and R/lenalidomide. Also, BAY-924 induced potent antitumor effect with a 4% ΔT/ΔC (day 55) in the ABC DLBCL OCI-Ly3-2b model, expressing weak to moderate levels of CXCR5 in vivo. Given its unique structure, and based on supportive data from other projects, it is anticipated that BAY-924 shows a favorable safety profile, due to the high stability of the ADC and a non-cell permeable free payload which is trapped inside the tumor cells. Overall, these results support further development of BAY-924 as an innovative approach for the treatment of CXCR5+ non-Hodgkin lymphoma. Citation Format: Sarah Johannes, Stefanie Hammer, Stephan Maersch, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Hannah Joerissen, Oliver von Ahsen, Christoph Schatz, Simone Greven, Christoph Mahlert, Dominik Mumberg, Pascale Lejeune. Preclinical characterization of BAY-924, a first in class ADC targeting CXCR5-positive B-cell malignancies, with a KSP inhibitor as novel payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4825.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) independent from cell proliferation. This opens up the possibility to target slowly growing tumors as well as resting antigen-positive tumor cells in addition to highly proliferative tumors. We developed a novel structural class of NAMPT inhibitors as ADC payloads to complement the currently available effector chemistries. An SAR-driven approach supported by available structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffold and linker led to highly potent effector chemistries which were conjugated to anti-C4.4a (LYPD3) or anti-B7H3 (CD276) antibodies and tested on antigen-positive and -negative cancer cell lines derived from solid and hematological tumor indications. Furthermore, tuning of the hydrophilicity of the linker and the conjugation method ensured low aggregation of the NAMPT inhibitor ADCs. Pharmacokinetic studies were performed in human plasma to assess the stability of the linkage of the NAMPT inhibitor payload to the antibody. Moreover, permeability studies of the payload metabolites helped to evaluate potential by-stander effects of the ADCs. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile versus the corresponding isotype control ADCs. In depth in vitro and in vivo studies on the internalization and the metabolism allowed analysis of the intracellular fate of the payload metabolites and revealed the formation of the phosphoribosylated catabolite adducts in C4.4a expressing A549-cells. Taken together, we hereby present the development of a new NAMPT inhibitor-based payload class applicable for conjugation to diverse antibodies with a good technical profile and high potency and selectivity in antigen-positive cancer models. Citation Format: Niels Böhnke, Markus Berger, Nils Griebenow, Anja Giese, Judith Günther, Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Rudolf Beier, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Antje Rottmann, Michael Erkelenz, Naomi Barak, Ulf Bömer, Dominik Mumber, Bertolt Kreft, Lars Linden, Carl Friedrich Nising, Hilmar Weinmann. Identification and optimization of a novel NAMPT inhibitor-based ADC payload class for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2907.

Inhibitors of kinesin spindle protein (KSP/Eg5) have raised great interest because of their high antitumor potency which, however, could not be transferred into highly efficient clinical regimens due to dose limiting side effects such as neutropenia. We have developed a new, highly potent pyrrole subclass of KSPis as a novel payload class in ADCs. To increase the therapeutic window, a tailor-made effector chemistry has been designed. The tumor associated protease legumain (LGMN) is utilized for ADC cleavage to provide active metabolites with high and long-lasting exposure in tumors, thus matching the KSPi mode of action. LGMN is a lysosomal asparaginyl endopeptidase overexpressed in solid tumors which is associated with invasion, metastasis and poor survival. In our studies, we show an efficient LGMN mediated linker cleavage and payload release from different ADCs. In addition, the unique LGMN substrate sequence allows for high cleavage specificity and discrimination versus other proteases such as elastase, which has been associated with an undesired extracellular cleavage of e.g. vc-MMAE ADCs resulting in neutropenia. Variations of the linker chemistry were performed to retain high potency of the KSPi-ADCs against tumor cells while decreasing the activation of KSPi-ADCs in cells of healthy organs such as the liver. From this approach, highly potent KSPi-ADCs with improved tumor/organ ratios of active metabolites were obtained. In conclusion, ADC metabolism under legumain control is a versatile strategy to provide KSPi-ADCs with high potency and an improved safety profile. Citation Format: Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Anette Sommer, Sandra Berndt, Anne-Sophie Rebstock, Sarah Johannes, Leo Marx, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Hilmar Weinmann. KSPi-ADCs: ADCs with novel kinesin spindle protein inhibitor payloads and a tailor-made linker chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 228.

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