RESEARCH ARTICLES Biotechnology Relationship Between Potential Aggregation-Prone Regions and HLA-DR-Binding T-Cell Immune Epitopes: Implications for Rational Design of Novel and Follow-on Therapeutic Antibodies SANDEEP KUMAR, 1 MARK A. MITCHELL, 2 BONITA RUP, 3 SATISH K. SINGH 1 1 Biotherapeutics Pharmaceutical Sciences Research and Development, Pfizer Inc., Chesterfield, Missouri 63017 2 Pharmaceutical Sciences Business Technology, Pfizer Inc., St. Louis, Missouri 63141 3 Protein Bioanalytics, Pfizer Inc., Andover, Massachusetts 08100 Received 26 January 2012; revised 21 March 2012; accepted 6 April 2012 Published online 22 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23169 ABSTRACT: Aggregation and unwanted immunogenicity are hurdles to avoid in success- ful commercial development of antibody-based therapeutics. In this article, the relationship between aggregation-prone regions (APRs), capable of forming cross-$ motifs/amyloid fibrils, and major histocompatibility complex class II-restricted human leukocyte antigen (HLA)-DR- binding T-cell immune epitopes (TcIEs) is analyzed using amino acid sequences of 25 therapeutic antibodies, 55 TcIEs recognized by T-regulatory cells (tregitopes), 1000 randomly generated 15- residue-long peptides, 2257 human self-TcIEs (autoantigens), and 11 peptides in HLA-peptide cocrystal structures. Sequence analyses from these diverse sources consistently show a high level of correlation between APRs and TcIEs: approximately one-third of TcIEs contain APRs, but the majority of APRs occur within TcIE regions (TcIERs). Tregitopes also contain APRs. Most APR-containing TcIERs can bind multiple HLA-DR alleles, suggesting that aggregation- driven adverse immune responses could impact a broad segment of patient population. This article has identified common molecular sequence—structure loci that potentially contribute toward both manufacturability and safety profiles of the therapeutic antibodies, thereby laying a foundation for simultaneous optimization of these attributes in novel and follow-on can- didates. Incidence of APRs within TcIERs is not special to biotherapeutics, self-TcIEs from human proteins, involved in various diseases, also contain predicted APRs and experimentally proven amyloid-fibril-forming peptide sequence portions. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2686–2701, 2012 Keywords: mAbs; immune epitopes; bioinformatics; drug; protein aggregation; proteins; computer-aided drug design; Biopharamaceutics Abbreviations used: APR, aggregation-prone region; mAb, monoclonal antibody; IgG, immunoglobulin G; IgM, immunoglob- ulin M; LC, light chain; HC, heavy chain; CDR, complementarity determining region; Fab, Fragment antigen binding; Fc, Fragment crystallizable; ADA, anti-drug antibody; APC, antigen-presenting cell; MHC, major histocompatibility complex; TcIE, MHC class II- restricted HLA-DR-binding T-cell immune epitope; TcIER, TcIE region; tregitope, regulatory T-cell immune epitope; T H , T-helper; T Reg , T-regulatory; TCR, T-cell receptor, HLA, human leukocyte antigen; HLA-DR, HLA molecules from DR locus; APR + HLA-DR + , peptides that contain APR(s) and HLA-DR allele(s)-binding TcIE; APR + HLA-DR − , peptides that contain APR(s) but not HLA-DR allele(s)-binding TcIE; APR − HLA-DR + , peptides that do not con- tain APR(s) but contain HLA-DR allele(s)-binding TcIE; APR + , peptides that contain APR(s); HLA-DR + , peptides that contain HLA-DR allele(s)-binding TcIE. Additional Supporting Information may be found in the online version of this article. Supporting Information INTRODUCTION The potential of therapeutic monoclonal antibodies (mAbs) to elicit undesirable immune responses con- tinues to be a significant concern, even with human- ized or fully human mAb therapeutics. 1–3 Immune re- sponses against biotherapeutic drugs can vary from formation of low levels of low-affinity transient im- munoglobulin M (IgM) anti-drug antibodies (ADAs) Correspondence to: Sandeep Kumar (Telephone: +314-274- 0176; Fax: +314-274-7601; E-mail: Sandeep.Kumar@Pfizer.com) Sandeep Kumar and Mark A. Mitchell contributed equally to this article. Journal of Pharmaceutical Sciences, Vol. 101, 2686–2701 (2012) © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association 2686 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012