Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig.

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.

Transmembrane TNF-dependent uptake of anti-TNF antibodies.

TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside 'reverse' signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.

Targeting repulsive guidance molecule A to promote regeneration and neuroprotection in multiple sclerosis.

Repulsive guidance molecule A (RGMa) is a potent inhibitor of neuronal regeneration and a regulator of cell death, and it plays a role in multiple sclerosis (MS). In autopsy material from progressive MS patients, RGMa was found in active and chronic lesions, as well as in normal-appearing gray and white matter, and was expressed by cellular meningeal infiltrates. Levels of soluble RGMa in the cerebrospinal fluid were decreased in progressive MS patients successfully treated with intrathecal corticosteroid triamcinolone acetonide (TCA), showing functional improvements. In vitro, RGMa monoclonal antibodies (mAbs) reversed RGMa-mediated neurite outgrowth inhibition and chemorepulsion. In animal models of CNS damage and MS, RGMa antibody stimulated regeneration and remyelination of damaged nerve fibers, accelerated functional recovery, and protected the retinal nerve fiber layer as measured by clinically relevant optic coherence tomography. These data suggest that targeting RGMa is a promising strategy to improve functional recovery in MS patients.

Cutting Edge: DNase II deficiency prevents activation of autoreactive B cells by double-stranded DNA endogenous ligands.

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.