A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10–100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.

IntroductionMutations in the ATP-binding cassette (ABC) halftransporters ABCG5 and ABCG8 cause sitosterolemia (1, 2), a rare autosomal recessive disorder characterized by elevated plasma levels of both plant and animal sterols (3,4). Normal individuals absorb less than 5% of dietary sitosterol, the major plant sterol, and efficiently excrete plant sterols into bile (5, 6). Plasma sitosterol concentrations rarely exceed 1 mg/dl, even in individuals with very high intakes of dietary sitosterol. In contrast, sitosterolemic subjects absorb about 15-20% of dietary sitosterol and have a profoundly reduced capacity to excrete sitosterol into bile. Consequently, sitosterol accumulates in the blood and body tissues of patients with this genetic disease (5,(7)(8)(9)(10)(11). A greater fraction of both animal-and plant-derived sterols are absorbed from the diet in affected individuals. The increased fractional absorption and reduced biliary excretion of cholesterol in sitosterolemia results in hypercholesterolemia and premature coronary artery disease (4,11). The phenotype of sitosterolemia suggests that ABCG5 and ABCG8 limit intestinal absorption and promote biliary excretion of neutral sterols.ABC transporters share a common molecular architecture that includes two nucleotide-binding folds and two transmembrane domains containing 6-11 membrane-spanning α-helices (12, 13). ABC transporters are organized as full transporters containing two transmembrane domains and two nucleotide-binding folds, or as half-transporters that form homo-or heterodimers (14). ABCG5 and ABCG8 are members of the G subfamily of ABC transporters, which are predicted to contain a single magnesium-dependent ATP catalytic domain N-terminal to six transmembrane segments (1). Mutations in either ABCG5 or ABCG8 cause an identical phenotype, which is consistent with these two gene products functioning as a heterodimer (1,15), though this has yet to be demonstrated.Most ABC half-transporters reside in intracellular membranes (14). The best-characterized mammalian half transporters are TAP1 and TAP2 (ABCB2 and ABCB3), which are endoplasmic reticulum (ER) resident proteins that transport peptides from the cytosol into the lumen of this compartment (16). All four ABC Mutations in either ATP-binding cassette (ABC) G5 or ABCG8 cause sitosterolemia, an autosomal recessive disorder of sterol trafficking. To determine the site of action of ABCG5 and ABCG8, we expressed recombinant, epitope-tagged mouse ABCG5 and ABCG8 in cultured cells. Both ABCG5 and ABCG8 underwent N-linked glycosylation. When either protein was expressed individually in cells, the N-linked sugars remained sensitive to Endoglycosidase H (Endo H). When ABCG5 and ABCG8 were coexpressed, the attached sugars were Endo H-resistant and neuraminidase-sensitive, indicating that the proteins were transported to the trans-Golgi complex. The mature, glycosylated forms of ABCG5 and ABCG8 coimmunoprecipitated, consistent with heterodimerization of these two proteins. The Endo H-sensitive forms of ABCG5 and A...

SummaryMonoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.

SummaryAnti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes.

The magnitude and quality of T cell responses generated when Ag is targeted to receptors on DC is influenced by both the specific receptor targeted and its distribution among DC subsets. Here we examine the targeting of the model Ag OVA to potential DC targets, including CD11c, CD205, MHC class II, CD40, TLR2 and FccRII/III, using a panel of (Fab' Â OVA) conjugates. In vitro studies identified CD11c, CD205 and MHC class II as superior and comparably effective immunotargets for the delivery of OVA to APC for presentation to T cells. In vivo studies, however, showed a marked advantage of targeting Ag to CD11c for both CD4 (OT-II) and CD8 (OT-I) responses, with robust stimulation after a single, low dose (equivalent to 0.5 lg OVA); in contrast, (anti-CD205 Â OVA) and (anti-MHC class II Â OVA) resulted in markedly less proliferation of both OT-I and OT-II cells. Biodistribution and immunohistochemical studies suggest that the exceptional ability of CD11c to capture Ag in lymphoid tissues may, at least partially, explain its ability to promote T cell responses. These results suggest that targeting antigen via CD11c offers a previously unappreciated strategy for vaccine development which, unlike most targets, delivers robust responses of both CD4 and CD8 T cells.