Kay-Gunnar Stubenrauch

Impact of molecular processing in the hinge region of therapeutic IgG4 antibodies on disposition profiles in cynomolgus monkeys

The IgG4 isotype antibody is a potential candidate for immunotherapy when reduced effector functions are desirable. However, antigen binding fragment (Fab) arm exchange leads to functional monovalency with potentially reduced therapeutic efficacy. Mutagenesis studies suggested that the CH3 domain and not the core hinge is dominantly involved in in vivo molecular processing. This work investigated whether stabilization of the core hinge of a therapeutic IgG4 antibody by mutation of Ser228 to Pro (S228P) would be sufficient to prevent in vivo Fab arm exchange. In vitro experiments evaluated the influence of different levels of oxidation-reduction conditions in buffer and serum on Fab arm exchange (swapping) of wild-type (WT) IgG4 and IgG1 and of IgG4 S228P, which included a sterically neutral second mutation (Leu235 replaced by Glu). The objective of single-dose pharmacokinetic experiments in cynomolgus monkeys was to determine whether the mutation reduced IgG4 swapping in vivo. The results indicated that S228P mutation did not completely prevent Fab arm exchange in vitro in buffer under reducing conditions relative to IgG4 WT. The immunoassay findings were confirmed by mass spectrometry measurements. Results of the in vivo studies suggested that the therapeutic IgG4 WT antibody exchanged Fab arms with endogenous cynomolgus monkey IgG4, resulting in bispecific IgG4 antibodies with monovalency for the therapeutic target. In contrast, serum from cynomolgus monkeys dosed with the IgG4 mutant was virtually free of swapped IgG4. In conclusion, the results indicated that IgG4 swapping in vivo was markedly attenuated by S228P mutation.

A high-throughput test to detect C.E.R.A. doping in blood

C.E.R.A., a continuous erythropoietin receptor activator, is a new third-generation erythropoiesis-stimulating agent (ESA) that has recently been linked with abuse in endurance sports. In order to combat this new form of doping, we examined an enzyme-linked immunosorbent assay (ELISA) designed to detect the presence of C.E.R.A. in serum samples. The performance of the assay was evaluated using a pilot excretion study that involved six subjects receiving C.E.R.A. Validation data demonstrated an excellent reproducibility and ensured the applicability of the assay for anti-doping purposes. To maximize the chances of detecting the drug in serum samples, we propose the use of this specific ELISA test as a high-throughput screening method, combined with a classic isoelectric focusing test as a confirmatory assay. This strategy should make C.E.R.A. abuse relatively easy to detect, thereby preventing the future use of this drug as a doping agent.

CD4-anchoring HIV-1 Fusion Inhibitor with Enhanced Potency and in Vivo Stability

In this study, we describe a novel CD4-targeting bifunctional human immunodeficiency virus (HIV-1) fusion inhibitor (CD4-BFFI) that blocks HIV-1 entry by inhibiting both HIV-1 attachment and fusion and is highly potent against both R5 and X4 HIV-1 viruses in various antiviral assays, including peripheral blood mononuclear cell (PBMC) infection assays. Previously, we have reported a CCR5 antibody-based bifunctional HIV-1 fusion inhibitor (BFFI) that was highly active in blocking R5 HIV-1 infection but was ineffective against X4 viruses infecting human PBMCs (Kopetzki, E., Jekle, A., Ji, C., Rao, E., Zhang, J., Fischer, S., Cammack, N., Sankuratri, S., and Heilek, G. (2008) Virology J. 5, 56–65). CD4-BFFI, which consists of two HIV-1 fusion inhibitor (FI) T-651 variant peptides recombinantly fused to the Fc end of a humanized anti-CD4 monoclonal antibody, has demonstrated more than 100-fold greater antiviral activity than T-651 variant or the parental CD4 monoclonal antibody. Mechanistic studies revealed that CD4-BFFI primarily blocks the HIV-1-cell fusion step through its FI peptide moieties. The enhanced antiviral activity of CD4-BFFI is most likely due to avid binding of the bivalent FI peptides as well as the increased local concentration of CD4-BFFI via attachment to the target cell surface receptor CD4. In vivo pharmacokinetic studies demonstrated that CD4-BFFI was stable in monkey blood, and a dose of 10 mg/kg maintained serum concentrations greater than 2,000-fold over the IC90 value for 7 days postdosing. This novel bifunctional inhibitor with improved potency and favorable pharmacokinetic properties may offer a novel approach for HIV-1 therapy.

Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies

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 pharma‐cokinetics 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. FASEB J. 29, 1763‐1779 (2015). www.fasebj.org

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice.

Abstract. Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(ab′)2-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F(ab′)2 fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

First-in-Human Phase I Study of Single-agent Vanucizumab, A First-in-Class Bispecific Anti-Angiopoietin-2/Anti-VEGF-A Antibody, in Adult Patients with Advanced Solid Tumors

Purpose: Vanucizumab is an investigational antiangiogenic, first-in-class, bispecific mAb targeting VEGF-A and angiopoietin-2 (Ang-2). This first-in-human study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of vanucizumab in adults with advanced solid tumors refractory to standard therapies. Experimental Design: Patients received escalating biweekly (3–30 mg/kg) or weekly (10–30 mg/kg) intravenous doses guided by a Bayesian logistic regression model with overdose control. Results: Forty-two patients were treated. One dose-limiting toxicity, a fatal pulmonary hemorrhage from a large centrally located mediastinal mass judged possibly related to vanucizumab, occurred with the 19 mg/kg biweekly dose. Arterial hypertension (59.5%), asthenia (42.9%), and headache (31%) were the most common toxicities. Seventeen (41%) patients experienced treatment-related grade ≥3 toxicities. Toxicity was generally higher with weekly than biweekly dosing. A MTD of vanucizumab was not reached in either schedule. Pharmacokinetics were dose-linear with an elimination half-life of 6–9 days. All patients had reduced plasma levels of free VEGF-A and Ang-2; most had reductions in KTRANS (measured by dynamic contrast-enhanced MRI). Two patients (renal cell and colon cancer) treated with 30 mg/kg achieved confirmed partial responses. Ten patients were without disease progression for ≥6 months. A flat-fixed 2,000 mg biweekly dose (phamacokinetically equivalent to 30 mg/kg biweekly) was recommended for further investigation. Conclusions: Biweekly vanucizumab had an acceptable safety and tolerability profile consistent with single-agent use of selective inhibitors of the VEGF-A and Ang/Tie2 pathway. Vanucizumab modulated its angiogenic targets, impacted tumor vascularity, and demonstrated encouraging antitumor activity in this heterogeneous population. Clin Cancer Res; 24(7); 1536–45. ©2017 AACR.

Abstract 3285: Ang-2-VEGF CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function mediates potent anti-tumoral and anti-angiogenic efficacy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL VEGF-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. In tumors Ang-2 is up-regulated and a bad prognostic factor. Recent data demonstrated that Ang-2 inhibitors, both as a single agent or in combination with chemo- or anti-VEGF therapy mediate anti-tumoral effects. We have recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies (CrossMabs) based on the crossover of the CH1 and CL domains within the Fab region of one half of a bispecific antibody combined with the knobs-into-holes technology to enforce heterodimerization of the Fc portion. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab and recognizing Ang-2 with the other arm based on LC06, a highly Ang-2 selective human IgG1 antibody. The Ang-2-VEGF CrossMAb could be produced with good yields and purity by eukaryotic. Surface Plasmon resonance studies showed that the two different arms of the Ang-2-VEGF CrossMab retained their antigen binding affinity for VEGF-A and Ang-2 and interfered with VEGF-induced HUVEC proliferation and Ang-2 induced Tie2-phosphorylation in a similar manner than the parental antibodies. Crossmab showed very potent tumor growth inhibition in orthotopic (KPL-4) and in subcutaneous xenograft tumors (Colo205). In the orthotopic KPL-4 and the s.c. Colo205 xenograft we observed a strong inhibition of angiogenesis by ex vivo analysis. In the VEGF-induced cornea pocket assay Crossmab resulted in a complete shutdown of angiogenesis. We have generated a bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function simultaneously. Our data indicate that the Ang-2-VEGF CrossMab mediates potent anti-tumoral, anti-metastatic and anti-angiogenic efficacy and represents a promising therapeutic agent for the therapy of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2011-3285

Abstract 2319: Dual inhibition of Ang-2 and VEGF via a novel human bispecific bivalent IgG1 CrossMAb shows potent anti-angiogenic, antitumoral, and antimetastatic efficacy and leads to a reduced side effect profile compared to single therapies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL VEGF-A blockade has been extensively clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling, tumor angiogenesis, and metastasis. In tumors, Ang-2 is up-regulated and associated with poor prognosis. Recent data demonstrated that Ang-2 inhibitors, both as single agents or in combination with chemo- or anti-VEGF therapy, mediate anti-tumor effects. Additionally, it has been shown that the Ang-2/-1/Tie and the VEGF/VEGFR systems act in complementary ways suggesting that dual targeting may be more effective than targeting each pathway alone. Based on bevacizumab and the Ang-2 selective antibody LC06 we have generated a novel human bispecific bivalent IgG1 CrossMab antibody blocking VEGF-A and Ang-2 function simultaneously. Here we show in multiple subcutaneous and orthotopic in vivo models including models (semi-) resistant to anti-VEGF treatment that the systemic application of the Ang-2-VEGF CrossMAb effectively reduces angiogenesis, tumor growth and metastasis. Furthermore, we demonstrate that a highly selective anti-Ang-2 approach has safety related advantages in this model over an unselective treatment with an antibody targeting Ang-1 and Ang-2 simultaneously. Whereas anti-Ang-1/Ang-2 long-term treatment resulted in regression of healthy vessels in the mouse trachea, an anti-Ang-2 selective treatment did not affect the physiological vessels in the trachea of the mice at all. These results imply a clear differentiation between selective Ang-2 and unselective Ang-1/Ang-2 inhibition. Although anti-tumoral efficacy is retained selective Ang-2 inhibition did not lead to a further impairment of healthy vessels compared to anti-VEGF-A treatment only. Finally, we demonstrate a clear disadvantage of Ang-2 monotherapy compared to Ang-2-VEGF dual inhibition due to strong up-regulation of VEGF resulting not only in revascularization and tumor growth, but also in toxicity with macroscopic evidence of histopathological subcapsular peliosis-like changes in the liver. These pathological effects were inhibited by dual inhibition of Ang-2 and VEGF-A. Taken together, our data indicate that Ang-2 and VEGF-A exhibit angiogenic synergy in a mutually compensatory fashion and that their inhibition via the novel Ang-2-VEGF CrossMab mediates potent anti-tumoral, anti-metastatic and anti-angiogenic efficacy. Additionally the CrossMAb is expected to exhibtit a better side effect profile compared to the respective monotherapies and thereby represents a promising therapeutic agent for the therapy of cancer. These data support the investigation of the Ang-2-VEGF CrossMAb in PhI clinical trials scheduled for 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2319. doi:1538-7445.AM2012-2319