Impact of Single-chain Fv Antibody Fragment Affinity on Nanoparticle Targeting of Epidermal Growth Factor Receptor-expressing Tumor Cells

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Abstract

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3–18-fold improvement in affinity (KD = 15–88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD = 264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.

Introduction

A wide range of tumors over-express epidermal growth factor receptor (EGFR), including breast, lung, colorectal, and brain cancers.1., 2. EGFR (vIII), a truncated form of EGFR, is found in glioblastomas,3., 4. but not in normal tissues, making it plausible to target tumors expressing this variant with a greater degree of specificity. Monoclonal antibodies targeting the extracellular domain (ECD) of EGFR and small-molecule inhibitors of tyrosine kinase activity have been evaluated in clinical trials and approved for clinical use.5., 6. While these antibodies have demonstrated clinically important response rates, the percentage of patients with metastatic disease who responded, and the duration of their responses, is modest.7., 8., 9. An alternative approach that could show greater efficacy consists of using antibodies to target chemotherapeutic agents or toxins specifically to tumor cells over-expressing EGFR or EGFR (vIII). Internalization, not simply binding, is a known requisite for optimal activity of many such drug delivery strategies.10 Methods for the generation of internalizing antibodies have expanded with the availability of display technologies.11., 12., 13., 14., 15.. For example, internalizing human antibodies against ErbB2 and EGFR have been generated by direct selection of non-immune phage antibody libraries16., 17. on live cells over-expressing ErbB2 or EGFR.13., 14.

Liposomal and immunoliposomal drug delivery have resulted in an improved therapeutic index for a variety of small-molecule therapeutic drugs.10., 18., 19., 20. An anti-EGFR immunoliposome constructed with a high-affinity anti-EGFR antigen-binding fragment (Fab)-targeting ligand derived from Cetuximab (C225 IgG, Imclone) demonstrated efficient drug delivery and activity in cell culture,21and in in vivo tumor xenograft models.22 While C225 binds EGFR with high affinity (KD = 0.5 nM), human antibody fragments isolated from non-immune phage libraries typically have considerably lower affinities.16., 23. It is possible to increase antibody affinity significantly using molecular evolution and display technologies;24., 25. however, it has not been determined whether intrinsic antibody affinity has any substantial effect on cellular uptake of any nanoparticles, including immunoliposomes.

To determine the impact of intrinsic affinity on the cellular binding and uptake of tumor-targeting immunoliposomal nanoparticles, we first evolved an EGFR scFv antibody fragment (C10) to generate a panel of genetically related mutant scFv with monovalent affinities ranging from 264 nM to 0.9 nM. We then compared the cellular binding, uptake, and cytotoxic activities of immunoliposomes constructed using scFv of various affinities in the presence and in the absence of the natural ligand for EGFR. The results clarify the relationship between intrinsic antibody fragment affinity, receptor density, and nanoparticle binding, uptake, and cytotoxicity.

Section snippets

Generation of a library of anti-EGFR C10 scFv mutants

For affinity maturation, we used the internalizing EGFR scFv antibody C10 generated by selection of a non-immune human phage antibody library on EGFR over-expressing tumor cells.14 This scFv bound recombinant EGFR by ELISA, bound EGFR-expressing A431 cells with a KD of 217 nM, and was rapidly endocytosed into EGFR-expressing cells.14 To generate a library of C10 scFv mutants, the C10 scFv gene was cloned into the yeast display vector pYD2 for display with a C-terminal SV5 epitope tag.26 The C10

Discussion

Antibodies and antibody fragments are being utilized increasingly in the treatment of cancer.33 “Naked” antibodies, including trastuzumab (HER2),34 cetuximab (EGFR),35 bevacizumab (VEGF),36 alemtuzumab (CD52),37 and rituximab (CD20)38 are already approved for use in oncology, and are an important component of clinical treatment strategies for various cancers. These antibodies act by using a variety of mechanisms to induce cytotoxic effects including activation of immune responses via

Cell lines, media, antibodies and recombinant EGFR-ECD

Yeast strain EBY100 (GAL1-AGA1::URA3 ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL) was grown in YPD medium, and EBY100 transfected with expression vector pYD226 was selected on SD-CAA medium†. The Aga2p scFv fusion was expressed on the yeast surface by induction in SG-CAA medium (SD-CAA medium with glucose replaced by galactose) at 20 °C for 24∼48 h as described.28

Acknowledgements

This work was supported by National Cancer Institute Specialized Programs of Research Excellence (SPORE) in Breast Cancer (P50-CA58207). D.C.D. was supported, in part, by a New Investigator Award from the California Breast Cancer Research Program of the University of California, grant number 7KB-0066. We thank Dr Jianlong Lou and Richard Tsai for their valuable assistance with yeast display, Anne-Laure Goenaga with fluorescent microscopy, Trudy Poon with the statistical analysis, and Helen

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