Thies Rösner

An anti-EGFR IgA that displays improved pharmacokinetics and myeloid effector cell engagement in vivo

Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two N-glycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab- and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoprotein-receptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcαRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in non-FcαRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo.

An IgG3 switch variant of rituximab mediates enhanced complement‐dependent cytotoxicity against tumour cells with low CD20 expression levels

Breccia, M., Salaroli, A., Loglisci, G., Finsinger, P., Serrao, A. & Alimena, G. (2012) MDS-specific comorbidity index is useful to identify myelodysplastic patients who can have better outcome with 5-azacitidine. Haematologica, 97, e2. Dayyani, F., Conley, A.P., Strom, S.S., Stevenson, W., Cortes, J.E., Borthakur, G., Faderl, S., O’Brien, S., Pierce, S., Kantarjian, H. & Garcia-Manero, G. (2010) Cause of death in patients with lower-risk myelodysplastic syndrome. Cancer, 116, 2174–2179. Della Porta, M.G., Malcovati, L., Strupp, C., Ambaglio, I., Kuendgen, A., Zipperer, E., Travaglino, E., Invernizzi, R., Pascutto, C., Lazzarino, M., Germing, U. & Cazzola, M. (2011) Risk stratification based on both disease status and extra-hematologic comorbidities in patients with myelodysplastic syndrome. Haematologica, 96, 441–449. Fenaux, P., Mufti, G.J., Hellstrom-Lindberg, E., Santini, V., Finelli, C., Giagounidis, A., Schoch, R., Gattermann, N., Sanz, G., List, A., Gore, S.D., Seymour, J.F., Bennett, J.M., Byrd, J., Backstrom, J., Zimmerman, L., McKenzie, D., Beach, C. & Silverman, L.R. (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncology, 10, 223–232. Follo, M.Y., Finelli, C., Mongiorgi, S., Clissa, C., Bosi, C., Testoni, N., Chiarini, F., Ramazzotti, G., Baccarani, M., Martelli, A.M., Manzoli, L., Martinelli, G. & Cocco, L. (2009) Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to Azacitidine in high-risk MDS. Proceedings of the National Academy of Sciences U S A, 106, 16811 –16816. Itzykson, R., Th epot, S., Quesnel, B., Dreyfus, F., Beyne-Rauzy, O., Turlure, P., Vey, N., Recher, C., Dartigeas, C., Legros, L., Delaunay, J., Salanoubat, C., Visanica, S., Stamatoullas, A., Isnard, F., Marfaing-Koka, A., de Botton, S., Chelghoum, Y., Taksin, A.L., Plantier, I., Ame, S., Boehrer, S., Gardin, C., Beach, C.L., Ad es, L. & Fenaux, P. (2011) Prognostic factors for response and overall survival in 282 patients with higher-risk myelodysplastic syndromes treated with azacitidine. Blood, 117, 403–411. Naqvi, K., Garcia-Manero, G., Sardesai, S., Oh, J., Vigil, C.E., Pierce, S., Lei, X., Shan, J., Kantarjian, H.M. & Suarez-Almazor, M.E. (2011) Association of comorbidities with overall survival in myelodysplastic syndrome: development of a prognostic model. Journal of Clinical Oncology, 29, 2240–2246. Ozbalak, M., Cetiner, M., Bekoz, H., Atesoglu, E.B., Ar, C., Salihoglu, A., Tuzuner, N. & Ferhanoglu, B. (2012) Azacitidine has limited activity in ‘real life’ patients with MDS and AML: a single centre experience. Hematology Oncology, 30, 76–81. Piccirillo, J.F., Costas, I., Claybour, P., Borah, A.J., Grove, L. & Jeffe, D. (2003) The measurement of comorbidity by cancer registries. Journal of Registry Management, 30, 8–14.

Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing Tumor Cells through the Alternative Pathway Amplification Loop

Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti–EGFR-IgG1 did not promote CML of investigated target cells, anti–EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti–EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti–EGFR-IgG3 promoted strong C1q and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti–EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti–EGFR-IgG3–triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti–EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway.

A Complement-Optimized EGFR Antibody Improves Cytotoxic Functions of Polymorphonuclear Cells against Tumor Cells

Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)–targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3–dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.

An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity

Background: Engineering of the antibodys fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. Methods and Results: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. Conclusion: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

Effector mechanisms of IgA antibodies against CD20 include recruitment of myeloid cells for antibody‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity

Children, Department of Biochemistry, University of Toronto, Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Division of Haematology/Oncology, Department of Paediatrics, CHU Sainte-Justine, Montreal, QC, and Child Health Evaluative Sciences, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada E-mail: manuel.carcao@sickkids.ca

Monoclonal Antibodies against Epidermal Growth Factor Receptor Acquire an Ability To Kill Tumor Cells through Complement Activation by Mutations That Selectively Facilitate the Hexamerization of IgG on Opsonized Cells

Triggering of the complement cascade induces tumor cell lysis via complement-dependent cytotoxicity (CDC) and attracts and activates cytotoxic cells. It therefore represents an attractive mechanism for mAb in cancer immunotherapy development. The classical complement pathway is initiated by IgG molecules that have assembled into ordered hexamers after binding their Ag on the tumor cell surface. The requirements for CDC are further impacted by factors such as Ab epitope, valency, and affinity. Thus, mAb against well-validated solid tumor targets, such as the epidermal growth factor receptor (EGFR) that effectively induces complement activation and CDC, are highly sought after. The potency of complement activation by IgG Abs can be increased via several strategies. We identified single-point mutations in the Fc domain (e.g., E345K or E430G) enhancing Fc:Fc interactions, hexamer formation, and CDC after Ab binds cell-surface Ag. We show that EGFR Abs directed against clinically relevant epitopes can be converted into mAb with unprecedented CDC activity. Alternative strategies rely on increasing the affinity of monomeric IgG for C1q by introduction of a quadruple mutation at the C1q binding site or via generation of an IgG1/IgG3 chimera. In this study we show that selective enhancement of C1q binding via avidity modulation is superior to the unattended increase in C1q binding via affinity approaches, particularly for target cells with reduced EGFR expression levels. Improving Fc:Fc interactions of Ag-bound IgG therefore represents a highly promising and novel approach for potentiating the anti-tumor activity of therapeutic mAb against EGFR and potentially other tumor targets.

Fc Engineering of Antibodies and Antibody Derivatives by Primary Sequence Alteration and Their Functional Characterization

Therapeutic antibodies used in the treatment of cancer patients are able to mediate diverse effector mechanisms. Dependent on tumor entity, localization, and tumor burden different effector mechanisms may contribute to the in vivo antitumor activity to a variable degree. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been suggested as being important for the in vivo activity of therapeutic antibodies like rituximab or trastuzumab. In recent years, several strategies have been pursued to further optimize the cytotoxic potential of monoclonal antibodies by modifying their Fc part (Fc engineering) with the ultimate goal to enhance antibody therapy.Since Fc engineering approaches are applicable to any Fc-containing molecule, strategies to enhance CDC or ADCC activity of full antibodies or scFv-Fc fusion proteins by altering the primary Fc sequence are described.

Fc Glyco- and Fc Protein-Engineering: Design of Antibody Variants with Improved ADCC and CDC Activity

Monoclonal antibodies are established treatment options in cancer therapy. However, not all patients benefit from antibody therapy. Basic research and findings from clinical trials revealed that certain Fc-mediated effector mechanisms triggered by monoclonal antibodies are essential for efficient antitumor activity. Today, next-generation monoclonal antibodies can be designed displaying tailor-made improved effector functions. The introduction of Fc-engineering technologies offers the potential to fine-tune Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDC). Fc-engineered antibodies hopefully will overcome some limitations of current forms of antibody therapy.

Abstract 4601: Mechanisms of action for therapeutic antibody variants of human IgG3 isotype: Enhancing the CDC activity of cetuximab and rituximab

Objectives. Human IgG3 differs from other antibody isotypes by its extended hinge region and by its higher affinity for C1q. The ability of complement to promote lysis of antibody-opsonized cells is well-established and may represent a main mechanism in antibody mediated tumor therapy. The aim of this project was to investigate effector mechanisms of tumor- directed IgG3 antibodies in more detail. Experimental procedures. Therapeutic antibodies against EGFR (cetuximab, 225) and CD20 (rituximab, C2B8) were produced as IgG1 and IgG3 isotype variants by co-transfecting CHO cells with the respective de novo synthesized VH and VL domains fused to the respective heavy chain containing vectors. A Fab fragment of C2B8-IgG3 was crystallized and analyzed by X-ray structure analysis at PETRA III (DESY, Hamburg, Germany). Based on the resolved structure selected mutations were introduced into the CH1 domain of IgG3 aiming to improve or abolish CDC. Purified antibodies were assessed for complement fixation in ELISA and on tumor cells. Complement- and antibody-dependent cellular cytotoxicity (CDC and ADCC) activities were measured using 51chromium release assays. The significance of complement regulatory factors on target cells was investigated by siRNA mediated knock-down. A peritoneal tumor model was used to investigate the in vivo efficacy of IgG3 in comparison to IgG1 antibodies. Results. Switching isotypes from IgG1 to IgG3 did neither affect target antigen binding nor the proliferation inhibition by EGFR antibodies. Unexpectedly, the IgG3 antibodies were not very active in ADCC, in spite of their strong FcγRIII (CD16) binding affinity. However, for target cells expressing low antigen levels, the IgG3 isotype improved CDC against CD20 and enabled CDC against EGFR expressing tumor cells. The complement-regulatory factor CD55 in particular mitigated the IgG3 induced CDC activity. Structural comparison of Fab fragments of IgG1 and IgG3 demonstrated significant differences in the CH1 domains of the respective heavy chains, which contain the disulfide bond to the light chain and also harbor the docking interface for the complement factors C3b and C4b. Preliminary results from an peritoneal tumor model demonstrated that 225-IgG3 was effective in killing EGFR- expressing cells. This in vivo activity was abolished by cobra venom factor treatment. Conclusion. Switching the isotype of a therapeutic IgG1 antibody to IgG3 represents a promising approach to enable complement-mediated killing of tumor cells. Protein engineering of the C3b/C4b interaction interface in the CH1 domain may constitute a novel strategy to modulate the CDC activity against tumor target cells. Together, these approaches may enhance the CDC efficacy of CD20- and EGFR- directed antibodies. Citation Format: Ralf Schwanbeck, Anna Skof, Thies Rosner, Marco Jansen, Anina Rauchle, Anna Kretschmer, Axel Scheidig, Jeanette Leusen, Stefanie Derer, Thomas Valerius. Mechanisms of action for therapeutic antibody variants of human IgG3 isotype: Enhancing the CDC activity of cetuximab and rituximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4601. doi:10.1158/1538-7445.AM2017-4601