Tuning the potency and selectivity of ImmTAC molecules by affinity modulation.

T-cell-engaging bispecifics have great clinical potential for the treatment of cancer and infectious diseases. The binding affinity and kinetics of a bispecific molecule for both target and T-cell CD3 have substantial effects on potency and specificity, but the rules governing these relationships are not fully understood. Using immune mobilizing monoclonal TCRs against cancer (ImmTAC) molecules as a model, we explored the impact of altering affinity for target and CD3 on the potency and specificity of the redirected T-cell response. This class of bispecifics binds specific target peptides presented by human leukocyte antigen on the cell surface via an affinity-enhanced T-cell receptor and can redirect T-cell activation with an anti-CD3 effector moiety. The data reveal that combining a strong affinity TCR with an intermediate affinity anti-CD3 results in optimal T-cell activation, while strong affinity of both targeting and effector domains significantly reduces maximum cytokine release. Moreover, by optimizing the affinity of both parts of the molecule, it is possible to improve the selectivity. These results could be effectively modelled based on kinetic proofreading with limited signalling. This model explained the experimental observation that strong binding at both ends of the molecules leads to reduced activity, through very stable target-bispecific-effector complexes leading to CD3 entering a non-signalling dark state. These findings have important implications for the design of anti-CD3-based bispecifics with optimal biophysical parameters for both activity and specificity.

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.

Regulation of the Bioavailability of TGF-ß and TGF-ß-Related Proteins.

The bioavailability of members of the transforming growth factor ß (TGF-ß) family is controlled by a number of mechanisms. Bona fide TGF-ß is sequestered into the matrix in a latent state and must be activated before it can bind to its receptors. Here, we review the molecules and mechanisms that regulate the bioavailability of TGF-ß and compare these mechanisms with those used to regulate other TGF-ß family members. We also assess the physiological significance of various latent TGF-ß activators, as well as other extracellular modulators of TGF-ß family signaling, by examining the available in vivo data from knockout mouse models and other biological systems.

Latent TGF-ß-binding proteins.

The LTBPs (or latent transforming growth factor ß binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFß by covalently binding the TGFß propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFß precursor in the trans-golgi network but LAP and TGFß remain strongly bound through non-covalent interactions. LAP, TGFß, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFß latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFß activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFß regulation, TGFß-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.

Function of latent TGFß binding protein 4 and fibulin 5 in elastogenesis and lung development.

Mice deficient in Latent TGFß Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFß2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFß1 or TGFß3 did not improve lung septation indicating that the TGFß isoform elevated in Ltbp4S(-/-) lungs is TGFß2. Expression of a form of Ltbp4 that could not bind latent TGFß did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFß complexes. Therefore, the change in TGFß-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGFß2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly.

NMR spectroscopic and bioinformatic analyses of the LTBP1 C-terminus reveal a highly dynamic domain organisation.

Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFß and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface. (15)N relaxation studies further demonstrate that the three interdomain regions act as flexible linkers, allowing a wide range of motion between the well-structured domains. This work is consistent with the LTBP1 C-terminus adopting a flexible "knotted rope" structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFß activation.

¹H, ¹³C and ¹5N assignments of the four N-terminal domains of human fibrillin-1.

Fibrillins are extracellular, disulphide-rich glycoproteins that form 10-12 nm diameter microfibrils in connective tissues. They are found in the majority of higher animals, from jellyfish to humans. Fibrillin microfibrils confer properties of elasticity and strength on connective tissue and regulate growth factor availability in the extracellular matrix (ECM). Mutations in FBN1, the human gene encoding the fibrillin-1 isoform, are linked to several inherited connective tissue disorders. The fibrillin-1 N-terminus forms many functionally-important interactions, both with other fibrillin molecules and various ECM components. In particular, the first four domains, the fibrillin unique N-terminal (FUN) and three epidermal growth factor (EGF)-like domains (FUN-EGF3), are implicated in microfibril assembly and growth factor sequestration. The structure of these domains, which comprise 134 residues, is unknown. We have produced a recombinant fragment corresponding to this region of human fibrillin-1. Here, we report (1)H, (13)C and (15)N resonance assignments of the FUN-EGF3 fragment. Assignments will facilitate structure determination, analysis of interdomain dynamics and the mapping of interaction surfaces.

Backbone ¹H, ¹³C and ¹5N resonance assignment of the C-terminal EGF-cbEGF pair of LTBP1 and flanking residues.

Latent TGFß binding protein 1 (LTBP1) is a large extracellular protein that has been shown to bind covalently to the propeptide of TGFß cytokines and form a large latent complex, which is then incapable of binding TGFß receptors. LTBP1 has also been demonstrated to interact with a number of insoluble extracellular matrix components, such as fibrillin, which may play a role in TGFß regulation. Here we present the backbone (1)H, (13)C and (15)N assignments for two EGF domains of human LTBP1, and flanking regions, together forming a 12 kDa protein fragment at the C-terminus of LTBP1. This region is of particular interest as it is postulated to be involved in interactions with fibrillin microfibrils.

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.

Structure of the fibrillin-1 N-terminal domains suggests that heparan sulfate regulates the early stages of microfibril assembly.

The human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly.

Unchaining the beast; insights from structural and evolutionary studies on TGFß secretion, sequestration, and activation.

TGFß is secreted in a latent state and must be "activated" by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFß mobilisation in vivo. Here we review the process of TGFß secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFß is secreted, sequestered and released. This allows the separation of more ancient TGFß activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms.

Dissecting the fibrillin microfibril: structural insights into organization and function.

Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to interact with itself and other cell matrix components. This review summarizes our current knowledge of microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to microfibril dynamics, and how this relates to microfibril function in health and disease.

Dispensable residues in the active site of the cytochrome c biogenesis protein CcmH.

CcmH functions in the assembly of c-type cytochromes in the Escherichia coli periplasm. The conserved cysteine pair in the N-terminal of its two membrane-anchored periplasmic domains is thought to reduce the CXXCH motif of cytochromes c. The recent structure of Pseudomonas aeruginosa CcmH identified conserved residues that might be functionally important. We replaced with alanine the active-site cysteines of E. coli CcmH, as well as R42, S54, R63, and tested the effects on cytochrome c production anaerobically and aerobically. Unexpectedly, replacement of the conserved non-cysteine active-site residues had little effect, whilst the cysteines were required under aerobic, but not anaerobic, conditions. We confirmed that removal of the C-terminal tetratricopeptide-like domain does not, surprisingly, abolish assembly of cytochromes c.

Cytochrome c assembly: a tale of ever increasing variation and mystery?

Formation of cytochromes c requires a deceptively simple post-translational modification, the formation of two thioether bonds (or rarely one) between the thiol groups of two cysteine residues found in a CXXCH motif (with some occasional variations) and the vinyl groups of heme. There are three partially characterised systems for facilitating this post-translational modification; within these systems there is also variation. In addition, there are clear indications for two other distinct systems. Here some of the current issues in understanding the systems are analysed.