Influence of the in vivo half-antibody exchange on the therapeutic efficacy of an IgG4 antibody-drug conjugate

A high number of therapeutic antibodies and their derivates e.g. antibody-drug conjugates (ADC) are under preclinical or clinical evaluation for the treatment of cancer. Most of those ADCs are based on the IgG1 or IgG4 subtype, depending on whether additional effector functions are desired or not. I...

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Bibliographic Details
Main Author: Herbener, Peter
Contributors: Maier, Uwe G. (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2018
Biologie
Subjects:
ADC
Online Access:PDF Full Text
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Summary:A high number of therapeutic antibodies and their derivates e.g. antibody-drug conjugates (ADC) are under preclinical or clinical evaluation for the treatment of cancer. Most of those ADCs are based on the IgG1 or IgG4 subtype, depending on whether additional effector functions are desired or not. In contrast to IgG1, IgG4 is hardly capable to induce antibody-dependent cell-mediated or complement-dependent cytotoxicity and thus IgG4-based ADCs with cytotoxic payloads targeting only proliferating cells may have a preferred safety profile. Another unique property of the IgG4 subtype is the in vivo exchange of half-antibodies, resulting in random bispecific antibodies. BT062 (indatuximab ravtansine) is an ADC composed of an anti-CD138 IgG4 antibody conjugated to the highly cytotoxic maytansin derivate DM4. BT062 is currently evaluated in a clinical trial for the treatment of multiple myeloma. To investigate the influence of IgG4 half-antibody exchange on the functional properties and efficacy of ADCs, the following BT062 model antibodies mimicking the different process-derived antibody species were generated: (I) Wildtyp (WT) nBT062; (II) stable nBT062, comprising S228P and R409K mutations to prevent IgG4 shuffling in vivo; (III) half nBT062, serving as a model of the transistant state as C226S and C229S amino acid substitutions lead to the lack of covalent half antibody dimerization; and (IV) bispecific nBT062-natalizumab monovalently recognizing CD138 and CD49d antigens. All nBT062 variants were produced in FreeStyle CHO-S cells and purified by protein A affinity chromatography. Electrophoresis, western blotting and size exclusion chromatography were used to confirm the purification quality and provide first evidence on the aimed characteristics of each antibody due to the introduced mutations. In vitro analyses on NCI-H929 (CD138+/CD49d+), Ba/F3-hCD138 (CD138+/CD49d-) and Jurkat (CD138-/CD49d+) cells demonstrated nanomolar binding activities of all nBT062 variants towards CD138 followed by antigen-mediated internalization. After successful conjugation with model-corresponding quanities of DM4, resulting ADCs were investigated by an in vitro cytotoxicity assay. All nBT062-DM4 variants were capable to inhibit tumor cell proliferation by picomolar quanitities (IC50: ~80-460 pM). The MAXF 1322 xenograft mouse model was used to directly assess the influence of IgG4 shuffling in vivo. Bispecific nBT062-natalizumab-DM4 was the least potent model demonstrating only a boarderline efficacy even without the presense of human IgG4. WT nBT062-DM4, stable nBT062-DM4 and half nBT062 were highly effective against the tumor cells. At a low dosage, the efficacy of WT nBT062-DM4 and half nBT062-DM4 was reduced by the presence of human IgG4, while stable nBT062-DM4 was hardly affected. Analysis of mouse plasma samples confirmed the formation of bispecific antibodies out of WT nBT062-DM4 and half nBT062-DM4, but no half-antibody exchange was detected within samples of stable nBT062. These data clearly demonstrate an advantage of incorporating half-antibody exchange-preventing mutations into IgG4-based ADCs.
Physical Description:149 pages.
DOI:https://doi.org/10.17192/z2018.0126