Expression liabilities in a four-chain bispecific molecule.

Multispecific antibodies, often composed of three to five polypeptide chains, have become increasingly relevant in the development of biotherapeutics. These molecules have mechanisms of action that include redirecting T cells to tumors and blocking multiple pathogenic mediators simultaneously. One of the major challenges for asymmetric multispecific antibodies is generating a high proportion of the correctly paired antibody during production. To understand the causes and effects of chain mispairing impurities in a difficult to express multispecific hetero-IgG, we investigated consequences of individual and pairwise chain expression in mammalian transient expression hosts. We found that one of the two light chains (LC) was not secretion competent when transfected individually or cotransfected with the noncognate heavy chain (HC). Overexpression of this secretion impaired LC reduced cell growth while inducing endoplasmic reticulum stress and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. The majority of this LC was observed as monomer with incomplete intrachain disulfide bonds when expressed individually. Russell bodies (RB) were induced when this LC was co-expressed with the cognate HC. Moreover, one HC paired promiscuously with noncognate LC. These results identify the causes for the low product quality observed from stable cell lines expressing this heteroIgG and suggest mitigation strategies to improve overall process productivity of the correctly paired multispecific antibody. The approach described here provides a general strategy for identifying the molecular and cellular liabilities associated with difficult to express multispecific antibodies.

Rational selection of building blocks for the assembly of bispecific antibodies.

Bispecific antibodies, engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases. However, these molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary assembly of these chains is critical. Here, we describe Chain Selectivity Assessment (CSA), a high-throughput method to rationally select parental monoclonal antibodies (mAbs) to make bispecific antibodies requiring correct heavy/light chain pairing. By deploying CSA, we have successfully identified mAbs that exhibit a native preference toward cognate chain pairing that enables the production of hetero-IgGs without additional engineering. Furthermore, CSA also identified rare light chains (LCs) that permit positive binding of the non-cognate arm in the common LC hetero-IgGs, also without engineering. This rational selection of parental mAbs with favorable developability characteristics is critical to the successful development of bispecific molecules with optimal manufacturability properties.

Reduced TCR-dependent activation through citrullination of a T-cell epitope enhances Th17 development by disruption of the STAT3/5 balance.

Citrullination is a post-translational modification of arginine that commonly occurs in inflammatory tissues. Because T-cell receptor (TCR) signal quantity and quality can regulate T-cell differentiation, citrullination within a T-cell epitope has potential implications for T-cell effector function. Here, we investigated how citrullination of an immunedominant T-cell epitope affected Th17 development. Murine naïve CD4(+) T cells with a transgenic TCR recognising p89-103 of the G1 domain of aggrecan (agg) were co-cultured with syngeneic bone marrow-derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro-Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL-2, expressed lower levels of the IL-2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL-2 blockade in native p89-103-primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post-translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th-cell development in inflammatory disorders.

BIITE: A Tool to Determine HLA Class II Epitopes from T Cell ELISpot Data.

Activation of CD4+ T cells requires the recognition of peptides that are presented by HLA class II molecules and can be assessed experimentally using the ELISpot assay. However, even given an individual's HLA class II genotype, identifying which class II molecule is responsible for a positive ELISpot response to a given peptide is not trivial. The two main difficulties are the number of HLA class II molecules that can potentially be formed in a single individual (3-14) and the lack of clear peptide binding motifs for class II molecules. Here, we present a Bayesian framework to interpret ELISpot data (BIITE: Bayesian Immunogenicity Inference Tool for ELISpot); specifically BIITE identifies which HLA-II:peptide combination(s) are immunogenic based on cohort ELISpot data. We apply BIITE to two ELISpot datasets and explore the expected performance using simulations. We show this method can reach high accuracies, depending on the cohort size and the success rate of the ELISpot assay within the cohort.

Unconventional T-cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage.

It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II-peptide complex termed type A and type B, and that the two conformers of the same peptide-MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T-cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T-cell recognized the same core epitope. The results are consistent with the generation of aggrecan-specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan-induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage.

Ubiquitin C-terminal hydrolase 1: A novel functional marker for liver myofibroblasts and a therapeutic target in chronic liver disease.

BACKGROUND & AIMS: Ubiquitination is a reversible protein modification involved in the major cellular processes that define cell phenotype and behaviour. Ubiquitin modifications are removed by a large family of proteases named deubiquitinases. The role of deubiquitinases in hepatic stellate cell (HSC) activation and their contribution to fibrogenesis are poorly defined. We have identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation, determined its function in activated HSC and its potential as a therapeutic target for fibrosis. METHODS: Deubiquitinase expression was determined in day 0 and day 10 HSC. Increased UCHL1 expression was confirmed in human HSC and in an alcoholic liver disease (ALD) patient liver. The importance of UCHL1 in hepatic fibrosis was investigated in CCl4 and bile duct ligation injured mice using a pharmacological inhibitor (LDN 57444). The effects of UCHL1 inhibition on HSC proliferation were confirmed by Western blot and 3H thymidine incorporation. RESULTS: Here we report that pharmacological inhibition of UCHL1 blocks progression of established fibrosis in CCl4 injured mice. UCHL1 siRNA knockdown, LDN 57444 treatment, or HSC isolated from UCHL1(-/-) mice show attenuated proliferation in response to the mitogen, platelet-derived growth factor. Additionally, we observed changes in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb) in the absence of UCHL1 highlighting a potential mechanism for the reduced proliferative response. CONCLUSIONS: UCHL1 expression is highly upregulated upon HSC activation and is involved in the regulation of HSC proliferation. This study highlights therapeutic opportunities for pharmacological targeting of UCHL1 in chronic liver disease.

CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.

Antigen-specific B lymphocytes acquire proteoglycan aggrecan from cartilage extracellular matrix resulting in antigen presentation and CD4+ T-cell activation.

The majority of studies examining antigen-presenting cell (APC) function have focused on the capture and presentation of antigens released from pathogens or damaged cells. However, antigen-specific B cells are also capable of efficiently extracting antigens that are either tethered to, or integrally part of the plasma membrane of various target cells. In this study we show that B cells are also highly efficient at extracting integral components of the extracellular matrix (ECM) for subsequent presentation. In particular we demonstrate that B cells specific for aggrecan, an integral component of cartilage ECM, acquire this rheumatoid arthritis candidate autoantigen in both a B-cell-receptor-dependent and a contact-dependent manner. We also demonstrate that the subsequent presentation of aggregan from ECM leads to CD4(+) T-cell activation and effector cell formation. Recent studies have identified B-cell-mediated antigen presentation as essential for the development of autoimmunity, but a unique role for B cells compared with other APC has yet to be defined. Our findings lead us to propose that the acquisition of ECM-derived autoantigens represents a mechanism that defines the APC requirement for B cells in the development of autoimmunity.

Matrix metalloproteinase 13 expression in response to double-stranded RNA in human chondrocytes.

OBJECTIVE: To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA). METHODS: Differential expression of PRRs was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively. RESULTS: The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid-inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain-like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation-associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C). CONCLUSION: Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation.

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.

Transduction of vertebrate cells with Spodoptera exigua multiple nucleopolyhedrovirus F protein-pseudotyped gp64-null Autographa californica multiple nucleopolyhedrovirus.

Budded virions of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can enter a variety of non-host cells. The capacity of GP64, AcMNPV's endogenous envelope fusion protein, and SeF, the fusion protein from a gp64(-) baculovirus, to mediate baculovirus entry into vertebrate cells was examined by comparing the transduction efficiencies of engineered AcMNPV variants with either of the two envelope proteins into 17 vertebrate cell lines. At an m.o.i. of 500, GP64-expressing viruses transduced all cell lines with varying efficiencies. Transduction efficiencies of SeF-pseudotyped gp64-null AcMNPV into all cell lines were lower than those of GP64-expressing viruses, and were undetectable in seven cell lines. At an m.o.i. of 50, transduction of all mammalian cell lines transducible by the SeF-pseudotyped gp64-null AcMNPV at an m.o.i. of 500 was no longer detectable. An amplifiable SeF-pseudotyped gp64-null AcMNPV vector with greatly reduced tropism for vertebrate cells may have applications in engineering AcMNPV for targeted transduction.

Bartonella henselae in captive and hunter-harvested beluga (Delphinapterus leucas).

Previously, we reported the isolation of Bartonella henselae from the blood of harbor porpoises (Phocoena phocoena) and loggerhead sea turtles (Caretta caretta) from the North Carolina coast. Hematologic, pathologic, and microbiologic findings surrounding the death of a juvenile captive beluga in Vancouver initiated an outbreak investigation designed to define the molecular prevalence of Bartonella infection in belugas. Using polymerase chain reaction analyses targeting the intergenic spacer region (ITS), two B. henselae ITS strains were identified in 78% of captive and free-ranging hunter-harvested belugas. These findings may have public health implications and may influence aquarium management procedures for captive marine mammals.

Mechanisms of major histocompatibility complex class II-restricted processing and presentation of the V antigen of Yersinia pestis.

We mapped mouse CD4 T-cell epitopes located in three structurally distinct regions of the V antigen of Yersinia pestis. T-cell hybridomas specific for epitopes from each region were generated to study the mechanisms of processing and presentation of V antigen by bone-marrow-derived macrophages. All three epitopes required uptake and/or processing from V antigen as well as presentation to T cells by newly synthesized major histocompatibility complex (MHC) class II molecules over a time period of 3-4 hr. Sensitivity to inhibitors showed a dependence on low pH and cysteine, serine and metalloproteinase, but not aspartic proteinase, activity. The data indicate that immunodominant epitopes from all three structural regions of V antigen were presented preferentially by the classical MHC class II-restricted presentation pathway. The requirement for processing by the co-ordinated activity of several enzyme families is consistent with the buried location of the epitopes in each region of V antigen. Understanding the structure-function relationship of multiple immunodominant epitopes of candidate subunit vaccines is necessary to inform choice of adjuvants for vaccine delivery. In the case of V antigen, adjuvants designed to target it to lysosomes are likely to induce optimal responses to multiple protective T-cell epitopes.

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes.

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice.

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII259-273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII259-273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.

The impact of glycosylation on HLA-DR1-restricted T cell recognition of type II collagen in a mouse model.

OBJECTIVE: Type II collagen (CII) is a candidate autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of CII could alter intracellular antigen processing, leading to the development of autoimmune T cell responses. To address this possibility, we studied the intracellular processing of CII for presentation of the arthritogenic glycosylated epitope CII(259-273) to CD4 T cells in macrophages from HLA-DR1-transgenic mice. METHODS: HLA-DR1-transgenic mice were generated on a class II major histocompatibility complex-deficient background, and T cell hybridomas specific for the glycosylated and nonglycosylated epitope CII(259-273) were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype of proteinases involved. RESULTS: We showed that the glycosylated CII(259-273) epitope required more extensive processing than did the nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in the processing of CII for antigen presentation, since these compartments contained 1) enzyme activity that generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide-receptive HLA-DR1 molecules, and 4) peptide/HLA-DR1 complexes that could directly activate T cell hybridomas. Degradation of CII by dense fractions occurred optimally at pH 4.5 and was abrogated by inhibitors of serine and cysteine proteinases. CONCLUSION: Processing of the arthritogenic glycosylated CII(259-273) epitope, which is implicated in the induction of autoimmune arthritis, is more stringently regulated than is processing of the nonglycosylated form of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel therapeutic targets for the treatment of RA.

Major histocompatibility class II molecules prevent destructive processing of exogenous peptides at the cell surface of macrophages for presentation to CD4 T cells.

We studied factors affecting major histocompatibility complex class II (MHC-II)-restricted presentation of exogenous peptides at the surface of macrophages. We have previously shown that peptide presentation is modulated by surface-associated proteolytic enzymes, and in this report the role of the binding of MHC-II molecules in preventing proteolysis of exogenous synthetic peptides was addressed. Two peptides containing CD4 T-cell epitopes were incubated with fixed macrophages expressing binding and non-binding MHC-II, and supernatants were analysed by high-performance liquid chromatography and mass spectrometry to monitor peptide degradation. The proportion of full-length peptides that were degraded and the number of peptide fragments increased when non-binding macrophages were used, leading to reduction in peptide presentation. When MHC-II molecules expressed on the surface of fixed macrophages were blocked with monoclonal antibody and incubated with peptides and the supernatants were transferred to fixed macrophages, a significant reduction in peptide presentation was observed. Peptide presentation was up-regulated at pH 5.5 compared to neutral pH, and the latter was found to be the pH optimum of the proteolytic activity of the surface enzymes involved in the degradation of exogenous peptides and proteins. The data suggest that MHC-II alleles that bind peptides protect them from degradation at the antigen-presenting cell surface for presentation to CD4 T cells and we argue that this mechanism could be particularly pronounced at sites of inflammation.

Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers.

Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change, htrA mutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91phox-/- mice deficient in NADPH oxidase, as was a S. typhimurium SPI-2 mutant known to be virulent in gp9lphox-/- mice. Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9lphox-/- mice, with more than 60% reduction in cell numbers 2.5h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9lphox-/- mice, whereas an aroA live vaccine strain had no effect on either normal or gp9lphox-/- macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.