Ulrike Reiff

RG7116, a therapeutic antibody that binds the inactive HER3 receptor and is optimized for immune effector activation

The EGF receptor (EGFR) HER3 is emerging as an attractive cancer therapeutic target due to its central position in the HER receptor signaling network. HER3 amplifies phosphoinositide 3-kinase (PI3K)-driven tumorigenesis and its upregulation in response to other anti-HER therapies has been implicated in resistance to them. Here, we report the development and characterization of RG7116, a novel anti-HER3 monoclonal antibody (mAb) designed to block HER3 activation, downregulate HER3, and mediate enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) via glycoengineering of the Fc moiety. Biochemical studies and X-ray crystallography revealed that RG7116 bound potently and selectively to domain 1 of human HER3. Heregulin binding was prevented by RG7116 at concentrations more than 1 nmol/L as was nearly complete inhibition of HER3 heterodimerization and phosphorylation, thereby preventing downstream AKT phosphorylation. In vivo RG7116 treatment inhibited xenograft tumor growth up to 90% relative to controls in a manner accompanied by downregulation of cell surface HER3. RG7116 efficacy was further enhanced in combination with anti-EGFR (RG7160) or anti-HER2 (pertuzumab) mAbs. Furthermore, the ADCC potency of RG7116 was enhanced compared with the nonglycoengineered parental antibody, both in vitro and in orthotopic tumor xenograft models, where an increased median survival was documented. ADCC degree achieved in vitro correlated with HER3 expression levels on tumor cells. In summary, the combination of strong signaling inhibition and enhanced ADCC capability rendered RG7116 a highly potent HER3-targeting agent suitable for clinical development.

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

Candidate Biomarkers of Response to an Experimental Cancer Drug Identified through a Large-scale RNA Interference Genetic Screen

Purpose: A major impediment in the optimal selection of cancer patients for the most effective therapy is the lack of suitable biomarkers that foretell the response of a patient to a given drug. In the present study, we have used large-scale RNA interference–based genetic screens to find candidate biomarkers of resistance to a new acyl sulfonamide derivative, R3200. This compound inhibits the proliferation of tumor cells in vitro and in vivo, but its mechanism of action is unknown. Experimental Design: We used a large-scale RNA interference genetic screen to identify modulators of the efficacy of R3200. We searched for genes whose suppression in an in vitro cell system could cause resistance to the anticancer effects of R3200. Results: We report here that knockdown of either RBX1 or DDB1 causes resistance to the anticancer effects of R3200, raising the possibility that these two genes may have utility as biomarkers of response to this drug in a clinical setting. Interestingly, both RBX1 and DDB1 are part of an E3 ubiquitin ligase complex. Conclusions: We propose that suppression of the activity of a RBX1 and DDB1-containing E3 ligase complex leads to the stabilization of certain proteins, the increased abundance of which is in turn responsible for resistance to R3200. Moreover, our data suggest that RBX1 and DDB1 could potentially be developed into biomarkers of resistance to acyl sulfonamide–based cancer drugs. This will require clinical validation in a series of patients treated with R3200. (Clin Cancer Res 2009;15(18):5811–9)

Abstract 2508: GE-huMab-HER3, a novel humanized, glycoengineered HER3 antibody with enhanced ADCC and superior preclinical in vitro and in vivo efficacy

HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. HER3 is a kinase dead receptor, but by forming heterodimers with other HER family receptors, HER3 works as amplifier for PI3 kinase driven tumorigenesis. It has been reported that tumors treated with EGFR-, HER2- or cMET-targeted therapies can escape via HER3 activation or upregulation. HER3 is expressed in a large variety of tumors for example in non-small cell lung cancer (NSCLC), head and neck, colorectal, gastric, pancreatic, breast, ovarian and prostate cancer. Anti-HER3 antibodies can work via various mechanisms including: (1) blocking ligand (HRGs) binding to the receptor, (2) blocking heterodimerization with other HER family members (HER1, 2 and 4), (3) downregulation of the receptor from the cell surface, and (4) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). The first three mechanisms lead to inhibition of HER3 phosphorylation and downstream signaling thereby resulting in tumor cell growth inhibition, while ADCC is a mechanism of direct target cell killing triggered by cross-linking of Fc receptors on immune effector cells (e.g. NK cells, macrophages). GE-huMab-HER3 is a novel humanized and glycoengineered IgG1 antibody that binds to HER3 with high affinity. This antibody prevents ligand binding and receptor heterodimerization thereby blocking receptor phosphorylation. In various tumor xenograft models treatment with this antibody leads to substantial tumor growth inhibition. E.g. GE-huMab-HER3 treatment achieved >50% tumor growth inhibition in 10 out of 17 NSCLC models and in some cases even resulted in complete tumor remission. However, these xenograft experiments only reveal part of this antibody9s therapeutic potential. A unique feature of GE-huMab-HER3 that differentiates it from other anti-HER3 antibodies, including AMG 888 and MM-121, is its ability to bind to human FcgRIIIa on immune effector cells with a 50-fold higher affinity than standard IgG1 antibodies, a property conferred by the engineered glycosylation of the antibody Fc region. Consequently, GE-huMab-HER3 exhibits superior potency and efficacy in ADCC, as shown in vitro using recombinant A549 cells and in vivo by its Fc mediated greater anti-tumor effect in A549 orthotopic mouse models compared to a non-glyco-engineered variant of the antibody, WT-huMab-HER3. The combination of strong signaling inhibition and enhanced ADCC capability renders GE-huMabHER3 a highly potent HER3-targeting agent. Phase I clinical testing of this promising novel compound is ongoing. 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 2508. doi:1538-7445.AM2012-2508

GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies

ABSTRACT The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification.