Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility.

Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40.

Special Issue "Antibody Engineering for Cancer Immunotherapy".

Since the approval of Rituximab in the late 1990s, the first chimeric monoclonal antibody for the treatment of non-Hodgkin lymphoma, antibody engineering for cancer immunotherapy has become a rapidly growing field, with almost 50 antibody therapeutics approved in the USA and EU and hundreds undergoing testing in clinical trials [...

Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity.

Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored.

Complex Interplay between Epitope Specificity and Isotype Dictates the Biological Activity of Anti-human CD40 Antibodies.

Anti-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.

Influence of immunoglobulin isotype on therapeutic antibody function.

Monoclonal antibody (mAb) therapeutics are revolutionizing cancer treatment; however, not all tumors respond, and agent optimization is essential to improve outcome. It has become clear over recent years that isotype choice is vital to therapeutic success with agents that work through different mechanisms, direct tumor targeting, agonistic receptor engagement, or receptor-ligand blockade, having contrasting requirements. Here we summarize how isotype dictates mAb activity and discuss ways in which this information can be used for the development of enhanced therapeutics.

Conformation of the human immunoglobulin G2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies.

Monoclonal 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.

FcγRIIB as a key determinant of agonistic antibody efficacy.

Fc gamma Receptor (FcγR) IIB (CD32B) is an immunoreceptor tyrosine inhibitory motif (ITIM)-bearing Fc receptor that is involved in abrogating the signalling and function delivered from other receptors; archetypally those arising from other, activatory, FcγR and from the B cell receptor (BCR) for antigen. In the context of immunotherapy, it has convincingly been shown to limit a variety of clinically important therapeutic monoclonal antibodies (mAb) such as rituximab and trastuzumab in preclinical models. However, recent exploration of so-called immunomodulatory mAb, for example agonist mAb directed against various members of the TNFR super-family, has cast new light on the ability of FcγRIIB to regulate immune responses and immunotherapy.

Fcγ receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization.

Immunomodulatory mAbs, led by the anti-CTLA4 mAb ipilimumab, are an exciting new class of drugs capable of promoting anticancer immunity and providing durable control of some tumors. Close analysis of a number of agents has revealed a critical yet variable role for Fcγ receptors in their efficacy. In this article, we reveal that agonistic anti-CD40 mAbs have an absolute requirement for cross-linking by inhibitory FcγRIIB when used systemically to treat established BCL1 syngeneic lymphoma, and therapy is lost when using a mouse IgG2a mAb not cross-linked by FcγRIIB.

FcγRΙΙB controls the potency of agonistic anti-TNFR mAbs.

Isotype plays a crucial role in therapeutic monoclonal antibody (mAb) function, mediated in large part through differences in Fcγ receptor (FcγR) interaction. Monoclonal Abs such as rituximab and alemtuzumab, which bind target cells directly, are designed for efficient recruitment of immune effector cells through their activatory FcγR engagement to mediate maximal target cell killing. In this setting, binding to inhibitory FcγRIIB is thought to inhibit function, making mAbs with high activatory/inhibitory (A/I) FcγR binding ratios, such as mouse IgG2a and human IgG1, the first choice for this role.

Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody.

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.

Ligation of CD11c during vaccination promotes germinal centre induction and robust humoral responses without adjuvant.

In this study, we investigated the mouse dendritic cell (DC) receptor, complement receptor 4 (CR4; CD11c/CD18), as an immunotarget for triggering humoral immunity. Comparison of antibody titres generated against a panel of 13 anti-antigen-presenting cell receptor monoclonal antibodies, with or without conjugated ovalbumin (OVA), revealed uniquely rapid and robust responses following CR4 targeting, with antibody titres approaching 1 : 100 000 7 days after a single dose of antigen. Furthermore, using just 100 ng OVA conjugated to anti-CD11c Fab', we generated anti-OVA titres greater than those produced by a 100-fold higher dose of OVA in complete Freund's adjuvant at day 28.

CD11c provides an effective immunotarget for the generation of both CD4 and CD8 T cell responses.

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 FcgammaRII/III, using a panel of (Fab' x 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.

The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association.

Human hepatic lipase (hHL) mainly exists cell surface bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL.