Membrane-proximal Ig Domain Research Articles

Mechanistic aspects of tapasin-mediated antigen presentation revealed by structure of a tapasin/MHC-I complex

Abstract T cell immunity to viruses and cancers is crucially dependent on the cell surface display of major histocompatibility complex class I (MHC-I) molecules selectively loaded with high affinity peptides. Peptide loading occurs in the ER within a multimolecular assembly known as the peptide loading complex (PLC). A critical component of the PLC is tapasin, a 45 kDa membrane glycoprotein in whose absence the expression and stability of MHC-I are greatly reduced. To determine the mechanistic basis of tapasin-mediated peptide loading, we determined the crystal structures of human tapasin in complex with HLA-B44:05 as well as tapasin complexed to each of two well-characterized antibodies. The tapasin-stabilized peptide receptive state is characterized by distortions of the peptide binding groove, destabilization of the b 2-microglobulin interaction, and rearrangements of the membrane proximal Ig domains, all of which are reversed on high affinity peptide binding. Additionally, the structural footprints of the anti-tapasin antibodies confirm previous functional assays and together with the tapasin/MHC-I complex and mutagenesis data reveal dynamic aspects of tapasin function. Supported by the Intramural Research Program, NIAID, NIH.

A High Sensitivity aCD22 CAR Combined with aCD19 CAR to Generate Dual Targeting CAR T Cells for the Treatment of r/r B-ALL

Background:Anti-CD19 CAR treatment has shown a remarkable response in r/r B-ALL patients, however, a major cause for relapse following CAR administration is the loss of the CD19 antigen. CD22 is a promising alternative target for B-ALL malignancies, although generating an effective CAR to CD22 is challenging due to the size, density and rigidity of this ligand. Furthermore, in the B-ALL setting the CD22 expression levels are known to down regulate in response to selective CAR pressure. This resulted in patients relapsing with lower CD22 density post-treatment (2,839 epitopes/cell) presumably due to the target density falling below the sensitivity threshold for the aCD22 CAR 1. In this study we aim to improve CAR efficacy for the treatment of r/r B-ALL by firstly developing a highly sensitive aCD22 CAR capable of targeting cells that express less than 1000 CD22 molecules per cell. Secondly, we combined this new aCD22 CAR with an aCD19 CAR to generate a dual targeting CD19 and CD22 product via co-transduction for the treatment of paediatric r/r B-ALL as part of an extension cohort of the CARPALL clinical trial (NCT02443831).Results:We screened 18 anti-CD22 binders with a range of affinities (1-36.5nM) and binding epitopes in CAR format for high functional sensitivity to low density targets. We filtered binders for their ability to maintain cytotoxicity, proliferation and cytokine release with low expressing CD22 targets. We identified the optimal aCD22 binder (9A8) which when combined in a CAR structure containing a 41BBz endodomain was capable of lysing target cells transduced to express less than 255 CD22 mols/cell while not affecting CD22 negative targets. Similar to the previously tested aCD22 binder M971, our new 9A8 binder also targets the membrane proximal Ig domain of the CD22 molecule. However unlike M971, 9A8 utilises a conventional 16 amino acid linker between the heavy and light chain to form the scFv and has a lower propensity to tonically signal compared to the M971 CAR. To generate a dual CD19 and CD22 targeting product, the 9A8 aCD22 CAR was co-transduced with the aCD19 CAT19 CAR vector (AUTO1) utilised in the CARPALL clinical trial for the treatment of paediatric r/r B-ALL. In a NSG animal model engrafted with WT CD19+/CD22+ Nalm-6 cells, the dual targeting co-transduced T cells (AUTO1/22) were significantly better at supressing tumour growth compared to single targeting AUTO1 CAR T cells (Figure 1). Furthermore, CAR T cells recovered from these animals showed an equal persistence of both aCD19 CAR and aCD22 CAR. In an effort to recapitulate the loss of CD19 target observed in aCD19 CAR relapsed patients, we challenged our transduced T cells in a CD19-knockout Nalm-6 NSG model (Figure 1). In this setting, single transduced AUTO1 CAR T cells were unable to control tumour growth above the non-transduced T cell controls. In contrast, the dual targeting co-transduced AUTO1/22 CAR T cells significantly supressed tumour growth and showed enrichment of CAR T cells expressing the aCD22 9A8 CAR in the bone marrow.The dual targeting co-transduced AUTO1/22 is currently being evaluated in an extension cohort of the CARPALL clinical trial (NCT02443831) for the treatment of paediatric r/r B-ALL.Conclusion:We engineered a highly sensitive aCD22 9A8 CAR which when combined with the aCD19 CAT19 CAR allows for improved in vivo function and the targeting of CD19 negative, CD22 positive cells. The use of these dual targeting T cells may reduce the incidence of CD19 negative relapses in the r/r B-ALL setting.1. Fry TJ, Shah NN, Orentas RJ, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat. Med. 2018;24(1):20-28. [Display omitted] DisclosuresKokalaki: Autolus Ltd: Current Employment. Grothier: Autolus Ltd: Ended employment in the past 24 months. Manzoor: Autolus Ltd: Ended employment in the past 24 months. Costu: Autolus Ltd: Ended employment in the past 24 months. Srivastava: Autolus Ltd: Current Employment. Jha: Autolus Ltd: Current Employment. Gealy: Autolus Ltd: Current Employment. Stanczuk: Autolus Ltd: Ended employment in the past 24 months. Robson: Autolus Ltd: Current Employment. Taylor: Autolus Ltd: Current Employment. El-Kholy: Autolus Ltd: Current Employment. Baldan: Autolus Ltd: Current Employment. Righi: Autolus Ltd: Current Employment. Sillibourne: Autolus Ltd: Current Employment. Onuoha: Autolus: Ended employment in the past 24 months. Ghorashian: UCLB: Patents & Royalties; Novartis: Honoraria. Cordoba: Autolus Ltd: Current Employment. Amrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Pule: Autolus Ltd: Current Employment.

Cell–cell fusion induced by the Ig3 domain of receptor FGFRL1 in CHO cells

FGFRL1 is a single-pass transmembrane protein with three extracellular Ig domains. When overexpressed in CHO cells or related cell types, it induces cell–cell fusion and formation of large, multinucleated syncytia. For this fusion-promoting activity, only the membrane-proximal Ig domain (Ig3) and the transmembrane domain are required. It does not matter whether the transmembrane domain is derived from FGFRL1 or from another receptor, but the distance of the Ig3 domain to the membrane is crucial. Fusion can be inhibited with soluble recombinant proteins comprising the Ig1–Ig2–Ig3 or the Ig2–Ig3 domains as well as with monoclonal antibodies directed against Ig3. Mutational analysis reveals a hydrophobic site in Ig3 that is required for fusion. If a single amino acid from this site is mutated, fusion is abolished. The site is located on a β-sheet, which is part of a larger β-barrel, as predicted by computer modeling of the 3D structure of FGFRL1. It is possible that this site interacts with a target protein of neighboring cells to trigger cell–cell fusion.

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment.

BackgroundSignal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ.ResultsWe obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/− 0.3 μM).ConclusionThe three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.

Surfactant Protein D (Sp-D) Binds to Membrane-proximal Domain (D3) of Signal Regulatory Protein α (SIRPα), a Site Distant from Binding Domain of CD47, while Also Binding to Analogous Region on Signal Regulatory Protein β (SIRPβ)

Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPβ and the lack of known ligands for the latter, we examined Sp-D binding to SIRPβ. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPβ. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.

Identification of an alternate splice form of tapasin in human melanoma

Assembly of major histocompatibility complex (MHC) class I molecules with peptide in the endoplasmic reticulum requires the assistance of tapasin. Alternative splicing, which is known to regulate many genes, has been reported for tapasin only in the context of mutations. Here, we report on an alternate splice form of tapasin (tpsnΔEx3) derived from a human melanoma cell line that does not appear to be caused by mutations. Excision of exon 3 results in deletion of amino acids 70 to 156 within the beta barrel region, but the membrane proximal Ig domain, the transmembrane domain, and cytoplasmic tail of tapasin are intact. Introduction of tpsnΔEx3 into a tapasin-deficient cell line does not restore MHC class I expression at the cell surface. Similar to a previously described tapasin mutant (tpsnΔN50), tpsnΔEx3 interacts with TAP. Therefore, we used these altered forms of tapasin to test the importance of MHC class I interaction with TAP. In the presence of wild-type tapasin, transfection of tpsnΔN50, but not tpsnΔEx3, reduced MHC class I expression at the cell surface likely due its ability to compete MHC class I molecules from TAP. Together these findings suggest that tumor cells may contain alternate splice forms of tapasin which may regulate MHC class I antigen presentation.

The Coxsackievirus–Adenovirus Receptor Reveals Complex Homophilic and Heterophilic Interactions on Neural Cells

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

Differential intra-endothelial delivery of polymer nanocarriers targeted to distinct PECAM-1 epitopes

Coupling drug carriers to antibodies for targeting endothelial cells (ECs) may improve treatment of vascular and pulmonary diseases. Selecting antibodies that deliver carriers to the cell surface or intracellularly may further optimize specificity of interventions. We studied antibody-directed targeting of nanocarriers to platelet–endothelial cell adhesion molecule (PECAM)-1, an endothelial glycoprotein containing 6 Ig-like extracellular domains. PECAM-1 antibodies bind to ECs without internalization, but ECs internalize by endocytosis nanocarriers carrying multiple copies of anti-PECAM (anti-PECAM/NCs). To determine whether binding and intracellular transport of anti-PECAM/NCs depend on the epitope engaged, we targeted five PECAM-1 epitopes: mAb35, mAb37 and mAb62 (membrane-distal Ig domain 1), mAbGi34 (Ig domains 2/3), and mAb4G6 (membrane-proximal Ig domain 6). The antibodies bound to ECs regardless of the epitope proximity to the plasmalemma, whereas 130 nm diameter nanocarriers only targeted effectively distal domains (mAb4G6/NCs did not bind to ECs). ECs internalized mAb35, mAb62, and mAbGi34 carriers regardless of their size (0.13 to 5 μm diameter), yet they did not internalize mAb37/NCs. After internalization, mAb62/NCs trafficked to lysosomes within 2–3 h, whereas mAb35/NCs had prolonged residence in pre-lysosomal vesicles. Therefore, endothelial binding, endocytosis, and intracellular transport of anti-PECAM/NCs are epitope-specific. This paradigm will guide the design of endothelial drug delivery systems providing specific cellular localizations.

Molecular cloning characterization and expression of porcine immunoreceptor SIRP α

SWC3 is a porcine CD that has been the reference marker of myeloid lineage. It is expressed in every myelomonocytic cell from early bone marrow precursors. We have identified the molecule recognized by anti-SWC3 antibodies as a member of the signal-regulatory proteins (SIRPs) α family. Here, we describe the cloning of a cDNA coding for a porcine SIRP α protein. The sequence is 2470 nucleotides long and contains an open reading frame encoding a 507 amino acid sequence. The predicted polypeptide was composed of a 30 amino acids putative signal peptide, a 342 amino acid extracellular region, a 23 amino acid transmembrane segment and a 112 amino acid cytoplasmic domain. Analysis of the sequence reveals a high degree of homology with known SIRPs in other species, being easily identified the three extracellular Ig type domains and two cytoplasmic ITIM motifs characteristic of this molecule. The gene coding for porcine SIRP α has been mapped to porcine chromosome 17, in a region syntenic to the human chromosome 20 where SIRP genes have been mapped. During the analysis of SIRP gene expression in tissues by RT-PCR, we noticed the existence of a shorter mRNA, and cloned the corresponding cDNA. This coded for a splicing variant of SIRP α that lacked the two membrane proximal Ig domains. In transfection experiments, we have been able to show that anti-SWC3 antibodies recognize both forms of the molecule, mapping the SWC3 epitopes to the N-terminal IgV type domain.

A novel family of diversified immunoregulatory receptors in teleosts is homologous to both mammalian Fc receptors and molecules encoded within the leukocyte receptor complex

Three novel and closely related leukocyte immune-type receptors (IpLITR) have been identified in channel catfish (Ictalurus punctatus). These receptors belong to a large polymorphic and polygenic subset of the Ig superfamily with members located on at least three independently segregating loci. Like mammalian and avian innate immune regulatory receptors, IpLITRs have both putative inhibitory and stimulatory forms, with multiple types coexpressed in various lymphoid tissues and clonal leukocyte cell lines. IpLITRs have an unusual and novel relationship to mammalian and avian innate immune receptors: the membrane distal Ig domains of an individual IpLITR are related to fragment crystallizable receptors (FcRs) and FcR-like proteins, whereas the membrane proximal Ig domains are related to several leukocyte receptor complex encoded receptors. This unique composition of Ig domains within individual receptors supports the hypothesis that functionally and genomically distinct immune receptor families found in tetrapods may have evolved from such ancestral genes by duplication and recombination events. Furthermore, the discovery of a large heterogeneous family of immunoregulatory receptors in teleosts, reminiscent of amphibian, avian, and mammalian Ig-like receptors, suggests that complex innate immune receptor networks have been conserved during vertebrate evolution.Electronic supplementary materialSupplementary material is available for this article at http://dx.doi.org/10.1007/s00251-006-0134-1 and is accessible for authorized users.

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Genomic organization of the human leukocyte immunoglobulin-like receptors within the leukocyte receptor complex on chromosome 19q13.4.

The leukocyte immunoglobulin (Ig)-like receptors (LIRs) comprise a family of cell surface receptors that couple to either activating or inhibitory signals depending on the nature of their transmembrane and cytoplasmic domains. We describe the organization and fine localization of the genes for LIR-1 and LIR-5, which are inhibitory receptors, and LIR-6, which is an activating receptor. The genomic organization of all three genes is highly conserved from the signal peptide through the membrane-proximal Ig domain but diverges thereafter depending on the inhibitory or activating nature of the gene product. The 3' untranslated region of the gene for LIR-6 contains a 37-base pair repeat not present in the LIR-1 or LIR-5 genes. 5' rapid amplification of cDNA ends defined the putative transcription initiation site of the LIR-5 gene, which is TATA-less. A nucleotide substitution in the LIR-5 gene led to loss of an intron present in the 5' untranslated region of the LIR-1 and LIR-6 genes. Differences in the genomic structure of these three LIR genes suggests possible mechanisms for their differential expression in cells of hematopoietic lineage. The three genes are in a region of Chromosome 19q13.4 that is immediately centromeric of the killer cell Ig-like receptor genes and are separated from one another by approximately 20 to 30 kb, suggesting that they arose by gene duplication from a common ancestor.

NK cell receptor gene of the KIR family with two IG domains but highest homology to KIR receptors with three IG domains.

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) cells and some T cells. They recognize polymorphic human HLA class I molecules. Two families of KIRs have been identified and named p58 and p70. The p58 family of genes encode type I membrane proteins with two extracellular immunoglobulin (Ig) domains, while the p70 genes have three Ig domains. We here report the cloning and characterization of a novel KIR cDNA obtained from tumor cell lines with NK reactivity (YT and NK-92). This gene is also expressed in the normal cell line NK 3.3 and in NK cells obtained from some but not all normal donors. The clone, KIR103AS, has an open reading frame consistent with a KIR with two extracellular Ig domains, a transmembrane region and a 114 amino acid long cytoplasmic domain containing a single src homology 2 (SH2) binding motif. The membrane distal Ig domain of KIR103AS has highest homology with the first Ig domain of p70 KIRs and differs significantly from the first Ig domain of p58 KIRs. The second, membrane proximal Ig domain of KIR103AS has similar and high homology with the membrane proximal Ig domains of both p70 and p58 KIRs. The extracellular domains of KIR103AS therefore share characteristic features with both p70 and p58 genes: the domain structure is identical to p58 KIRs but the sequence homology matches closely with p70 KIRs. The putative transmembrane and cytoplasmic domains are distinctly different from all previously reported KIR cDNAs.