Discovery of functionally distinct anti-C7 monoclonal antibodies and stratification of anti-nicotinic AChR positive Myasthenia Gravis patients.

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies.

The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity.

BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

The N-Terminal Region of Fibrillin-1 Mediates a Bipartite Interaction with LTBP1.

Fibrillin-1 (FBN1) mutations associated with Marfan syndrome lead to an increase in transforming growth factor ß (TGF-ß) activation in connective tissues resulting in pathogenic changes including aortic dilatation and dissection. Since FBN1 binds latent TGF-ß binding proteins (LTBPs), the major reservoir of TGF-ß in the extracellular matrix (ECM), we investigated the structural basis for the FBN1/LTBP1 interaction. We present the structure of a four-domain FBN1 fragment, EGF2-EGF3-Hyb1-cbEGF1 (FBN1E2cbEGF1), which reveals a near-linear domain organization. Binding studies demonstrate a bipartite interaction between a C-terminal LTBP1 fragment and FBN1E2cbEGF1, which lies adjacent to the latency-associated propeptide (LAP)/TGF-ß binding site of LTBP1. Modeling of the binding interface suggests that, rather than interacting along the longitudinal axis, LTBP1 anchors itself to FBN1 using two independent epitopes. As part of this mechanism, a flexible pivot adjacent to the FBN1/LTBP1 binding site allows LTBP1 to make contacts with different ECM networks while presumably facilitating a force-induced/traction-based TGF-ß activation mechanism.

¹H, ¹³C and ¹5N resonance assignments for the fibrillin-1 EGF2-EGF3-hybrid1-cbEGF1 four-domain fragment.

Fibrillins are large extracellular glycoproteins that form the principal component of microfibrils. These perform a vital structural function in the extracellular matrix of many tissues. Fibrillins have also been implicated in mediating a number of protein-protein interactions, some of which may be significant in regulating growth factors such as transforming growth factor ß. Here we present the backbone and side-chain (1)H, (13)C and (15)N assignments for a 19 kDa protein fragment derived from the N-terminus of human fibrillin-1, encompassing four domains in total. These domains include the second and third epidermal growth factor-like (EGF) domains, the first hybrid domain (hyb1), and the first calcium-binding EGF domain of fibrillin-1. This region of fibrillin-1 is of particular interest as the hyb1 domain has been suggested to play a role in microfibril assembly, as well as several other protein-protein interactions.

Rfam: Wikipedia, clans and the "decimal" release.

The Rfam database aims to catalogue non-coding RNAs through the use of sequence alignments and statistical profile models known as covariance models. In this contribution, we discuss the pros and cons of using the online encyclopedia, Wikipedia, as a source of community-derived annotation. We discuss the addition of groupings of related RNA families into clans and new developments to the website. Rfam is available on the Web at http://rfam.sanger.ac.uk.