<p>Supplementary Figure S1. A, Structure of the PRLR and HER2 ADCs. B, Upon 24h incubation, PRLR, but not HER2 antibody accumulated in low pH vesicles of T47D cells. T47D cells were grown in 96-well optical plates.</p>
Antibodies directed against the epidermal growth factor receptor (EGFR) offer a potentially powerful therapeutic approach against cancers driven by the EGFR pathway. EGFR antibodies are believed to halt cell surface activation by blocking ligand-induced receptor tyrosine kinase activation, i.e., ligand binding, a change in conformation, or the monomer–dimer transition. In this work, we demonstrate that wild-type EGFR and the truncated de2–7-EGFR (tumor-associated mutant) formed unliganded homo-oligomers and examined the effects of two clinically relevant antibodies on the conformation and quaternary state of these ligand-free EGFR oligomers on the surface of cells. The EGFR antibodies were mAb528, a ligand-blocking antibody that binds domain III, and mAb806, a conformationally sensitive antibody that binds near the dimer interface in domain II. We used a model cellular system, BaF/3 cells, with GFP-tagged receptors in the absence of interference from secreted ligands or other erbB receptor members. Different antibody-mediated effects (conformational transition, receptor cross-linking, or receptor dissociation) were distinguished by combining two complementary biophysical techniques: image correlation spectroscopy (submicrometer scale clustering) and homo-Forster resonance energy transfer (association and/or conformation on a 1–10 nm scale). mAb528 cross-linked EGFR into an inactive EGFR dimer of dimers but had no effect when added to de2–7-EGFR oligomers. mAb806 had a minor effect on EGFR dimers as expected from its poor binding to a conformationally shielded epitope on wtEGFR but bound de2–7-EGFR oligomers, causing a conformational change in the intracellular C-terminal GFP-tagged tail. The combination of the two antibodies had synergistic effects, increasing the level of cross-linking of de2–7-EGFR, but did not lead to enhanced cross-linking of EGFR. The results reveal new modes of receptor–antibody interactions for EGFR and de2–7-EGFR.
Abstract Detection and quantification of immune cell subsets and changes in the tumor immune microenvironment would be a powerful tool to correlated to response in cancer immunotherapy. Currently, biopsies used to monitor the tumor microenvironment suffer from sampling error, restricted sampling over time and to inaccessible tissues/organs, and do not reflect discordance across tumor lesions of the same patient. Immuno-PET detection of CD8-expressing T cells is a promising translational imaging approach to assess CD8+ tumor-infiltrating lymphocytes (TILs) pre- and post-therapy. We have developed a fully human anti-CD8 antibody that was radiolabeled with the positron emitting radionuclide Zirconium-89 (89Zr) using the bifunctional chelator p-SCN-Bn-Desferoxamine (DFO). CD8-genetically humanized immunocompetent mice were used to validate dose dependent targeting of 89Zr-CD8 to lymphoid tissues. Next, two models were developed to monitor the response of the T cell activating bispecific antibody REGN1979 (CD3xCD20). In the first model designed to lack a CD8 antigen sink, Raji lymphoma cells were co-implanted with human peripheral blood mononuclear cells (hPBMCs) in immune deficient NSG mice and treated with REGN1979. Here, 89Zr-CD8 detected a two-fold increase in CD8+ TILs as quantified by flow cytometry compared to control antibody treated mice. To determine if 89Zr-CD8 could detect changes in CD8+ TILs in the presence of increased circulating and splenic T cells, a different model was developed. Raji lymphoma cells were implanted subcutaneously and hPBMCs were implanted intraperitoneally in immune deficient SRG-15 mice (hSIRP KI, Rag2 KO, IL2Rg KO, hIL-15 KI). Due to the humanization of IL-15, T cells from the implanted hPBMCs rapidly expanded and produced a CD8 antigen sink. In the SRG-15 model, total tumor uptake of 89Zr-CD8 was lower and spleen uptake higher than in the NSG model due to the presence of the antigen sink. These SGR-15 mice treated with REGN1979 also demonstrated an increase in CD8+ TILs by flow cytometry that was specifically detected with 89Zr-CD8. This work demonstrates that 89Zr-CD8 can specifically detect CD8+ T cells in lymphoid tissues of CD8 humanized mice and therapy-induced alterations of CD8+ TILs. Importantly, the detection of CD8+ TILs occurs in both the presence and absence of an antigen sink. This work supports the clinical translation of 89Zr-CD8 immuno-PET to investigate its utility for predicting and monitoring response in patients undergoing immunotherapy. Citation Format: Richard Tavare, Makenzie Danton, Jason T. Giurleo, Marcus P. Kelly, Sosina Makonnen, Carlos Hickey, Tomas C. Arnold, Dangshe Ma, William C. Olson, Gavin Thurston, Jessica R. Kirshner. CD8 immuno-PET using a fully human mAb can detect increases in tumor-infiltrating T cells by a T cell activating immunotherapy [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 1131.
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