Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch 1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus. However, we show here that tissue-specific inactivation of the gene encoding Notch 1 in immature (CD25+CD44-)T cell precursors does not affect subsequent thymocyte development. Neither steady-state numbers nor the rate of production of CD4+ and CD8+ mature thymocytes is perturbed in the absence of Notch 1. In addition, Notch 1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis. In contrast to earlier reports, these data formally exclude an essential role for Notch 1 in CD4-CD8 lineage commitment, maturation or survival.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8β chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8αβ, but not CD8αα or soluble CD8αβ, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8β endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8β constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2Kd, and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8β, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56lck. In addition, the cytoplasmic portion of CD8β mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8αβ partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56lck in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man 5 -GlcNAc 2 glycans (Man 5 -MR) or non-sialylated complex glycans (SA 0 -MR) did not bind mannosylated glycans, but could recognize SO 4 -3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA 0 -MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands. The mannose receptor (MR)6 was the first member of a family of four mammalian endocytic receptors to be discovered (1). These receptors share the same overall structure: an N-terminal cysteine-rich (CR) domain, followed by a fibronectin type II domain, several C-type lectin domains (CTLD; eight in the case of MR), and transmembrane and cytoplasmic regions (2-4). MR CTLD mediate binding to carbohydrates terminating in Man, Fuc, or GlcNAc (5), and the MR CR domain recognizes sugars terminating in SO 4 -4/3-GalNAc or SO 4 -3-Gal (6, 7). A natural cleavage product of MR can be found in supernatants of MR ϩ cells and in mouse serum (8 -10). In murine primary macrophages, this soluble form of MR (sMR) is generated from the cell-bound form by proteolytic cleavage by a metalloprotease (9). sMR comprises the whole extracellular region of the molecule and binds to both sulfated and mannosylated sugars (11). Our previous studies suggested that multimerization of the CR domain through the interaction of sMR with multivalent ligands for CTLD, such as mannan, leads to enhanced recognition of sulfated carbohydrates (11).Natural endogenous ligands for MR CTLD include lysosomal hydrolases, tissue plasminogen activator, and myeloperoxidase (5) and thyroglobulin (12). Microbial ligands include endotoxin from selected strains of Klebsiella pneumoniae (11); capsular polysaccharide from selected strains of Streptococcus pneumoniae (11), Pneum...

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.

The B cell surface molecule CD22 is a member of the Siglec family. Siglecs possess a conserved membrane‐distal immunoglobulin domain that mediates binding to sialylated glycoproteins or glycolipids. Although the structural basis of sialic acid recognition by Siglecs is quite well understood, the binding properties of the interaction between Siglecs and their native ligands have not been investigated. CD22 binding requires α2–6‐linked sialic acid, which is mostly carried on N‐glycans. One protein that carries such N‐glycans is CD45. In this study we used surface plasmon resonance to perform thermodynamic and kinetic analysis of CD22 binding to native CD45. CD22 bound with a low affinity (Kd 130 μM at 25 °C) and very fast kinetics (koff ⩾18 s–1, calculated kon ⩾1.5×105 M–1s–1). Van't Hoff analysis revealed that binding was enthalpically driven at physiological temperatures, as is typical of most lectin‐carbohydrate interactions. Since there is evidence that CD22 binds preferably to CD45, even though many cell surface proteins carry α2–6‐linked sialic acid, we compared the affinities of CD22 binding to CD45, to CD4 carrying α2–6‐linked sialic acid, and to a synthetic α2–6‐sialoglycoconjugate. The affinities did not differ substantially, suggesting that CD22 binds preferentially to CD45 not because the latter presents higher affinity ligands but because it carries multiple copies of thereof.

BackgroundThe VEGF/VEGFR and the HGF/cMET pathways are key mediators of the interplay of tumor cells and their microenvironment. However, inhibition of VEGF has been shown to produce only limited clinical benefit and inhibition of the activation of cMET by HGF has not translated into clinical benefit in pivotal trials. MP0250, a DARPin® molecule that specifically inhibits both VEGF and HGF has been developed to explore the clinical potential of dual inhibition of these pathways.ResultsMP0250 binding to VEGF and HGF inhibited downstream signalling through VEGFR2 and cMET resulting in inhibition of proliferation of VEGF- and HGF-dependent cells. Antitumor activity was demonstrated in VEGF- and HGF-dependent xenograft and syngeneic models with activity superior to that of individual VEGF- and HGF-blocking DARPin® molecules. Combination therapy studies showed potentiation of the antitumor activity of chemotherapy and immunotherapy agents, including an anti-PD1 antibody.Materials and MethodsPotency of MP0250 was assessed in cellular models and in a variety of xenograft models as monotherapy or in combination with standard-of-care drugs.ConclusionsDual inhibition of VEGF and HGF by MP0250 produced powerful single agent and combination antitumor activity. This, together with increasing understanding of the role of the HGF/cMET pathway in resistance to VEGF (and other agents), supports testing of MP0250 in the clinic.