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B-cell maturation antigen (BCMA) is an ideal target in multiple myeloma (MM) due to highly specific expression in malignant plasma cells. BCMA-directed therapies including antibody drug conjugates, chimeric antigen receptor-T cells and bispecific antibodies (BsAbs) have shown high response rates in MM. WVT078 is an anti-BCMA× anti-CD3 BsAb that binds to BCMA with subnanomolar-affinity. It was selected based on potent T cell activation and anti-MM activity in preclinical models with favorable tolerability in cynomolgus monkey. In the ongoing first-in-human phase I dose-escalation study (NCT04123418), 33 patients received intravenous WVT078 once weekly at escalated dosing. At the active doses of 48-250 µg/kg tested to date (n = 26), the overall response rate (ORR) was 38.5% (90% CI: 22.6-56.4%) and the complete response rate (CRR, stringent complete response + complete response) was 11.5%, (90% CI: 3.2-27.2%). At the highest dose level tested, the ORR was 75% (3 of 4 patients). 26 (78.8%) patients reported at least one Grade ≥3 AE and 16 of these AEs were suspected to be drug related. 20 patients (60.6%) experienced cytokine release syndrome. WVT078 has an acceptable safety profile and shows preliminary evidence of clinical activity at doses tested to date., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Interleukin-22 (IL-22) is a key mediator of inflammatory processes associated with diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. The measurement of this cytokine in human plasma may provide insight into safety, pharmacodynamics and efficacy of drugs targeting inflammatory pathways. However, commonly used immunoassays are not sufficiently sensitive to measure baseline concentrations of IL-22. Here we describe the analytical validation of an ultrasensitive assay for the measurement of IL-22 in human serum using the Erenna® system by Singulex (Alameda, CA). The lower limit of quantification (LLOQ) of the Erenna assay estimated at 0.2pg/mL was sensitive enough to measure IL-22 in all human serum samples tested. The assay ranged from 0.2 to 100.0pg/mL and showed good dilution linearity. The inter-assay and intra-assay imprecision were <9% and <7% CV respectively. The accuracy determined by spiked recovery in serum samples was >86%. In addition, the results using Erenna assay correlated well with those using the IL-22 Quantikine immunoassay (R&D Systems, Minneapolis, MN) with a coefficient R(2) of 0.9285. However the Erenna assay showed an improved sensitivity by approximately 2 logs. These results show that this novel assay offers a significant improvement over previous methods for high-sensitive quantitative measurement of IL-22 in human serum samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is one of four glycoproteins necessary and sufficient for HSV cellular entry. Recently, the crystal structures of HSV-1 gB and vesicular stomatitis virus glycoprotein G were determined. Surprisingly, the two proteins share remarkable structural homology. Both proteins are homotrimeric and center about a long alpha-helix, features reminiscent of class I fusion proteins, such as influenza virus hemagglutinin or paramyxovirus F. However, these structures revealed that G has internal fusion loops, similar to the fusion loops of the class II fusion proteins, and that these loops are structurally conserved in gB. To examine whether these putative fusion loops are important for gB function, we mutated potential membrane-interacting (hydrophobic) residues to charged amino acids. Of most interest were mutant gB proteins that were expressed on the cell surface and were recognized by monoclonal antibodies against conformational epitopes but lacked the ability to function in cell-cell fusion assays. We find that three of the five hydrophobic amino acids targeted in these loops, tryptophan 174, tyrosine 179, and alanine 261, are integral in the function of gB. Our data suggest that they are part of an important functional domain. We hypothesize that two loops in domain 1 of HSV gB function as fusion loops. Our data are further evidence that gB is a viral fusogen and suggest clues as to how gB may function.

Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.

Glycoprotein B (gB) is the most conserved component of the complex cell-entry machinery of herpes viruses. A crystal structure of the gB ectodomain from herpes simplex virus type 1 reveals a multidomain trimer with unexpected homology to glycoprotein G from vesicular stomatitis virus (VSV G). An alpha-helical coiled-coil core relates gB to class I viral membrane fusion glycoproteins; two extended beta hairpins with hydrophobic tips, homologous to fusion peptides in VSV G, relate gB to class II fusion proteins. Members of both classes accomplish fusion through a large-scale conformational change, triggered by a signal from a receptor-binding component. The domain connectivity within a gB monomer would permit such a rearrangement, including long-range translocations linked to viral and cellular membranes.

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Caveolin-1 expression and function were investigated in human colon cancer. Low levels of caveolin-1 mRNA and protein were detected in several colon carcinoma cell lines. Moreover, caveolin-1 protein levels were significantly reduced in human tumor epithelial mucosa (3.6 +/- 1.4-fold) when compared with normal colon mucosa for a majority (10 of 15) of the patients characterized. To directly assess the role of caveolin-1 in tumor development, caveolin-1 was reexpressed in the HT29 and DLD1 colon carcinoma cells, and the resulting HT29-cav-1 or DLD1-cav-1 cells were tested for tumorigenicity in nude mice. In most experiments, tumor formation was either blocked or retarded for HT29-cav-1 cells (10 of 13 mice) and DLD1-cav-1 cells (5 of 7 mice), as compared with both mock-transfected and parental HT29 or DLD1 cells. Interestingly, basal caveolin-1 levels were significantly reduced in HT29-cav-1 and DLD1-cav-1 cells isolated from tumors. Likewise, endogenous caveolin-1 mRNA and protein levels were found to be reduced in NIH-3T3 cells recovered from tumors after injection into nude mice. Thus, reexpression of caveolin-1 in colon carcinoma lines reduced the probability of tumor formation in vivo, and when tumors did develop from either HT29-cav-1, DLD1-cav-1, or NIH-3T3 cells, lower basal levels of caveolin-1 were detected. Finally, evidence was obtained indicating that initial caveolin-1 down-regulation in colon cancer cells need not be an entirely irreversible process because cell survival on selection for either drug resistance or increased metastatic potential correlated with increased caveolin-1 expression levels.