Current strategies to produce homogeneous antibody-drug conjugates (ADCs) rely on mutations or inefficient conjugation chemistries. Here we present a strategy to produce site-specific ADCs using a highly reactive natural buried lysine embedded in a dual variable domain (DVD) format. This approach is mutation free and drug conjugation proceeds rapidly at neutral pH in a single step without removing any charges. The conjugation chemistry is highly robust, enabling the use of crude DVD for ADC preparation. In addition, this strategy affords the ability to precisely monitor the efficiency of drug conjugation with a catalytic assay. ADCs targeting HER2 were prepared and demonstrated to be highly potent and specific in vitro and in vivo. Furthermore, the modular DVD platform was used to prepare potent and specific ADCs targeting CD138 and CD79B, two clinically established targets overexpressed in multiple myeloma and non-Hodgkin lymphoma, respectively.

Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6 ؉ Rab35 ؉ EHD1 ؉ tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.

The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface expression, intracellular distribution, and survival of pMHC-II in DCs. DCs isolated from March-I-KO mice express very high levels of pMHC-II on the plasma membrane even before DC activation. Although ubiquitination does not affect the kinetics of pMHC-II endocytosis from the surface of DCs, the survival of pMHC-II is enhanced in DCs obtained from March-Ideficient and MHC-II ubiquitination-mutant mice. Using pMHC-IIspecific mAb, we show that immature DCs generate large amounts of pMHC-II that are remarkably stable under conditions in which pMHC-II ubiquitination is blocked. Thus, the cellular distribution and stability of surface pMHC-II in DCs is regulated by ubiquitindependent degradation of internalized pMHC-II.T he initiation of an acquired immune response requires coordinated activation of antigen-specific T cells to provide both immune cell help (in the form of cytokines secreted from CD4 T cells) and immune cell effector function (in the form of cytotoxic CD8 T-cell responses and antigen-specific antibody secretion). This cascade of events is regulated primarily by antigen-presenting cells (APCs) in peripheral tissues that take up foreign antigens, process these antigens into immunogenic peptides, and display these antigenic peptides bound to MHC class II molecules on the APC surface (1). Dendritic cells (DCs) are professional APCs that function to prime naïve T cells. In their resting (or immature) state, DCs are relatively poor stimulators of naïve T cells; however, DC activation by a variety of signals induces a DC maturation cascade that up-regulates expression of peptide-loaded MHC-II complexes (pMHC-II), costimulatory molecules, and chemokine receptors that promote DC migration to secondary lymphoid organs and efficient T-cell activation.Given the central role that pMHC-II expressed on the surface of DCs play in the initiation of immune responses, there is intense interest in understanding the mechanisms leading to immunogenic peptide loading onto MHC-II, pMHC-II transport to the cell surface, and turnover of pMHC-II in DCs. Immature DCs express relatively small amounts of specific pMHC-II on their surface after exposure to antigen (2, 3), and large amounts of MHC-II are retained in intracellular antigen-processing compartments (4). Upon activation of these cells with inflammatory cytokines or Toll-like receptor (TLR) ligands (such as LPS or dsRNA), additional pMHC-II are generated (2, 5), and these pMHC-II are "released" from intracellular stores and traffic to the plasma membrane (6). Curiously, pMHC-II that ar...