Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.This work was supported by Wellcome Trust Grant 091157, JDRF International Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre, and the Medical Research Council Cusrow Wadia Fund. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (100140). U.M.N. was the recipient of a Hoffmann-La Roche postdoctoral fellowship.This is thefinal version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S089684111400146

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration

A new generation of protein display scaffolds for molecular recognition

Engineered antibodies and their fragments are invaluable tools for a vast range of biotechnological and pharmaceutical applications. However, they are facing increasing competition from a new generation of protein display scaffolds, specifically selected for binding virtually any target. Some of them have already entered clinical trials. Most of these nonimmunoglobulin proteins are involved in natural binding events and have amazingly diverse origins, frameworks, and functions, including even intrinsic enzyme activity. In many respects, they are superior over antibody-derived affinity molecules and offer an ever-extending arsenal of tools for, e.g., affinity purification, protein microarray technology, bioimaging, enzyme inhibition, and potential drug delivery. As excellent supporting frameworks for the presentation of polypeptide libraries, they can be subjected to powerful in vitro or in vivo selection and evolution strategies, enabling the isolation of high-affinity binding reagents. This article reviews the generation of these novel binding reagents, describing validated and advanced alternative scaffolds as well as the most recent nonimmunoglobulin libraries. Characteristics of these protein scaffolds in terms of structural stability, tolerance to multiple substitutions, ease of expression, and subsequent applications as specific targeting molecules are discussed. Furthermore, this review shows the close linkage between these novel protein tools and the constantly developing display, selection, and evolution strategies using phage display, ribosome display, mRNA display, cell surface display, or IVC (in vitro compartmentalization). Here, we predict the important role of these novel binding reagents as a toolkit for biotechnological and biomedical applications

Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity

Abstract Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fc gamma receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by Fc gamma R binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-gamma and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies