Heterodimeric Molecules Research Articles

Toll-like receptors and integrins crosstalk.

Immune system recognizes invading microbes at both pathogen and antigen levels. Toll-like receptors (TLRs) play a key role in the first-line defense against pathogens. Major functions of TLRs include cytokine and chemokine production. TLRs share common downstream signaling pathways with other receptors. The crosstalk revolving around TLRs is rather significant and complex, underscoring the intricate nature of immune system. The profiles of produced cytokines and chemokines via TLRs can be affected by other receptors. Integrins are critical heterodimeric adhesion molecules expressed on many different cells. There are studies describing synergetic or inhibitory interplay between TLRs and integrins. Thus, we reviewed the crosstalk between TLRs and integrins. Understanding the nature of the crosstalk could allow us to modulate TLR functions via integrins.

Presence of HLA-DQ2 and HLA-DQ8 /DR4 celiac disease predisposing alleles in tested group of patients in Bosnia and Herzegovina

Celiac disease (CD) is an autoimmune disease characterized by gluten intolerance. The main cause of this immune - mediated enteropathy is gluten, a protein mainly present in wheat, rye, barley and spelt. The predisposition to celiac disease is determined by HLA class II genes encoding MHC II heterodimer molecules, more specifically HLA-DQ2 and HLA-DQ8. Up to date the exact number of people suffering from celiac disease in Bosnia and Herzegovina is still not known because there is no public registry for this disease. The aim of this study was to evaluate the HLA-DQ2 and HLA-DQ8/DR4 presence in tested group of patients (n = 23) referred to Polyclinic Atrijum in Sarajevo in period from August 2022. to August 2023. HLA-DQ2 and DQ8/DR4 allele identification was performed using Real-time PCR technique. According to the obtained results a total of 26% (n=6) of tested patients were positive for HLA-DQ2 and HLA-DQ8/DR4 alleles. Two of the patients were positive for HLA-DQ2 alleles and four patients were positive for HLA-DQ8/DR4 alleles. To our knowledge this is the first study evaluating the presence of HLA-DQ2 and HLA-DQ8 genotypes in tested population in Bosnia and Herzegovina.

A focus on dominant negative variants in a series of 170 heterozygous FXI-deficient patients.

Dominant-negative effects have been described for 10F11 variants in the literature. The current study aimed at identifying putative dominant-negative F11 variants. This research consisted in a retrospective analysis of routine laboratory data. In a series of 170patients with moderate/mild factorXI (FXI) deficiencies, we identified heterozygous carriers of previously reported dominant-negative variants (p.Ser243Phe, p.Cys416Tyr, and p.Gly418Val) with FXI activities inconsistent with a dominant-negative effect. Our findings also do not support a dominant-negative effect of p.Gly418Ala. We also identified a set of patients carrying heterozygous variants, among which five out of 11 are novel, with FXI activities suggesting a dominant-negative effect (p.His53Tyr, p.Cys110Gly, p.Cys140Tyr, p.Glu245Lys, p.Trp246Cys, p.Glu315Lys, p.Ile421Thr, p.Trp425Cys, p.Glu565Lys, p.Thr593Met, and p.Trp617Ter). However, for all but two of these variants, individuals with close to half normal FXI coagulant activity (FXI:C) were identified, indicating an inconstant dominant effect. Our data show that for some F11 variants recognized has having dominant-negative effects, such effects actually do not occur in many individuals. The present data suggest that for these patients, the intracellular quality control mechanisms eliminate the variant monomeric polypeptide before homodimer assembly, thereby allowing only the wild-type homodimer to assemble and resulting in half normal activities. In contrast, in patients with markedly decreased activities, some mutant polypeptides might escape this first quality control. In turn, assembly of heterodimeric molecules as well as mutant homodimers would result in activities closer to 1:4 of FXI:C normal range.

New chemistry in unusual bacteria displays drug-like activity

Genetics revealed a rare soil bacteria may produce bioactive molecules. We discovered two new dimer molecules produced by this organism: one of which had an unusual heterodimer structure. Our assays showed these dimers had increased bioactivity against cultured cancer cells. The heterodimer’s activity was very strong, suggesting that natural-made heterodimeric molecules can be used as templates for new pharmaceuticals.

Combination of GD2-directed bispecific trifunctional antibody therapy with Pd-1 immune checkpoint blockade induces anti-neuroblastoma immunity in a syngeneic mouse model.

Despite advances in treating high-risk neuroblastoma, 50-60% of patients still suffer relapse, necessitating new treatment options. Bispecific trifunctional antibodies (trAbs) are a promising new class of immunotherapy. TrAbs are heterodimeric IgG-like molecules that bind CD3 and a tumor-associated antigen simultaneously, whereby inducing a TCR-independent anti-cancer T cell response. Moreover, via their functional Fc region they recruit and activate cells of the innate immune system like antigen-presenting cells potentially enhancing induction of adaptive tumor-specific immune responses. We used the SUREK trAb, which is bispecific for GD2 and murine Cd3. Tumor-blind trAb and the monoclonal ch14.18 antibody were used as controls. A co-culture model of murine dendritic cells (DCs), T cells and a neuroblastoma cell line was established to evaluate the cytotoxic effect and the T cell effector function in vitro. Expression of immune checkpoint molecules on tumor-infiltrating T cells and the induction of an anti-neuroblastoma immune response using a combination of whole cell vaccination and trAb therapy was investigated in a syngeneic immunocompetent neuroblastoma mouse model (NXS2 in A/J background). Finally, vaccinated mice were assessed for the presence of neuroblastoma-directed antibodies. We show that SUREK trAb-mediated effective killing of NXS2 cells in vitro was strictly dependent on the combined presence of DCs and T cells. Using a syngeneic neuroblastoma mouse model, we showed that vaccination with irradiated tumor cells combined with SUREK trAb treatment significantly prolonged survival of tumor challenged mice and partially prevent tumor outgrowth compared to tumor vaccination alone. Treatment led to upregulation of programmed cell death protein 1 (Pd-1) on tumor infiltrating T cells and combination with anti-Pd-1 checkpoint inhibition enhanced the NXS2-directed humoral immune response. Here, we provide first preclinical evidence that a tumor vaccination combined with SUREK trAb therapy induces an endogenous anti-neuroblastoma immune response reducing tumor recurrence. Furthermore, a combination with anti-Pd-1 immune checkpoint blockade might even further improve this promising immunotherapeutic concept in order to prevent relapse in high-risk neuroblastoma patients.

The Role of Integrin Beta-1 in the Chemoresistance of Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive disease that has a poor prognosis with a survival rate of 30-40%. The bone marrow microenvironment provides protection to AML cells from chemotherapy and thus confers cell adhesion-mediated drug resistance (CAM-DR) and promotes relapse. Integrins are key anchor molecules in CAM-DR of leukemia cells; they are heterodimeric adhesion molecules on the cell surface consisting of an alpha and a beta unit. Upregulated integrin alpha 4 and 6 expression is associated with poor survival in leukemias. We hypothesize that the targeting of integrin beta 1 (ITGB1, β1, or CD29) will also affect the integrin alpha units as they are heterodimer partners. This could be a superior approach to overcome CAM-DR in AML than just targeting the individual alpha units. METHODS: ITGB1 gene was knocked out via CRISPR-Cas9 using 2 guide RNAs in AML cell lines and expression of integrin subunits was profiled by flow cytometry. AML cell lines were treated in vitro with the anti-human ITGB1 monoclonal antibody OS2966 in combination with Ara-C chemotherapy and we determined effects on cell viability by flow cytometry and on cell signaling using RNA-sequencing. Using human patient-derived AML xenograft mouse models, we tested if blockade of ITGB1 combined with chemotherapy can overcome CAM-DR in vivo. Mice were treated with control total Ig (10 mg/kg), OS2966 (10 mg/kg), total Ig + Ara-C (600 mg/kg), or OS2966 + Ara-C twice a week for two weeks. RESULTS: Analysis of patient-derived AML cells at diagnosis and relapse, and of AML cell lines, showed high expression of ITGB1 and varying levels of other alpha and beta units. Loss-of-function studies of ITGB1 using CRISPR/Cas9-mediated knockout of ITGB1 in MLL-rearranged and FLT3-ITD+ AML cell lines MV4-11 and MOLM-13, and in AML cell line U937, revealed that surface expression of integrin alphas 3, 4, 5 and 6 were markedly decreased as determined by flow cytometry analysis. ITGB2 expression was high in all 3 AML cell lines and not affected by ITGB1 deletion. Remarkably, ITGB3 was downregulated upon ITGB1 deletion in 2 out of 3 AML cell lines, whereas the 3rd AML cell line did not express any ITGB3. As a result of the ITGB1 deletion, AML cells showed less adhesion to murine bone marrow stroma (OP9) cells and to extracellular matrix proteins. We found that inhibition of surface ITGB1 on human AML using OS2966 de-adhered the AML cells from OP9 cells. Moreover, ITGB1 blockade using OS2966 synergized with Ara-C in vitro and significantly decreased cell viability compared to single agent controls in human AML cell lines U937 and MV4-11. RNA sequencing data suggest that the NFkB pathway is significantly dysregulated in the combination treatment Ara-C and OS2966 compared to Ara-C alone. To test ITGB1 inhibition in vivo, a patient-derived AML sample, HEM279 (Fig. 1A), and AML cell line, U937 (Fig. 1B), were engrafted into NSG-SGM3 mice. Leukemic mice were treated with single agents including control total Ig, OS2966, or Ara-C; and with total Ig in combination with Ara-C or OS2966. In both xenograft models, the combination treatment of OS2966 with Ara-C significantly prolonged survival of leukemic mice compared to Ara-C only: HEM279: MST 115 +/- 15.4 days vs 73.1 +/- 5.2 days post-AML injection; p=0.0005; U937: MST 170 days vs 32 days post-AML injection; p=0.0004; 50% of the mice engrafted with U937 and treated with OS2966 with Ara-C survived until day 290 post-AML injection, which is when the experiment was terminated. CONCLUSIONS: Integrin alpha subunit expression is dependent on ITGB1 expression, supporting our hypothesis that targeting integrin beta 1 affects alpha integrin units. ITGB1 inhibition with OS2966 combined with chemotherapy significantly decreases cell viability of AML cell lines in vitro and prolongs survival of xenograft mice in vivo. Our findings will allow for better understanding of the role of ITGB1 in drug resistance of AML and of ITGB1 as a preclinical target in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract LB221: Development of PD-L1-targeted sialidase as a novel cancer immunotherapeutic approach

Abstract Sialoglycans have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. We have previously reported that Bi-Sialidase - an engineered human sialidase-Fc fusion - potentiates antitumor immune response by cleaving terminal sialic acids from sialoglycans (desialylating) on tumor cells and immune cells. Furthermore, we have shown that a tumor-targeted sialidase, a heterodimeric molecule consisting of one chain of sialidase-Fc and a second chain of a HER2-targeting antibody (trastuzumab), leads to more efficient desialylation of tumor cells than the non-targeted Bi-Sialidase and demonstrates antitumor activity in trastuzumab-resistant and low HER2-expressing tumor models. To evaluate the potential of targeted desialylation of both tumor cells and immune cells, we designed and characterized a PD-L1-targeted sialidase in preclinical models, since PD-L1 is expressed on both tumor cells and immune cells. Furthermore, the design of the PD-L1-targeted sialidase enables the combinatorial blockade of two orthogonal immune checkpoint pathways, inhibiting both the PD-1/PD-L1 axis and immunosuppressive sialoglycans. We generated a humanized anti-human PD-L1 antibody with comparable PD-1/PD-L1 blockade potency to the existing anti-PD-L1 drugs, atezolizumab and avelumab. Subsequently, we constructed a bifunctional heterodimeric molecule, consisting of one chain of sialidase-Fc of a third generation of engineered human sialidase (Neu 2) and a second chain of the in-house generated anti-PD-L1 antibody. The PD-L1-targeted sialidase maintained its potency for inhibiting the PD-1/PD-L1 axis as compared to its parental anti-PD-L1 antibody, and demonstrated improved desialylation of PD-L1-expressing tumor cells and immune cells in vitro. We tested the PD-L1-targeted sialidase in transgenic mouse tumor models that express human PD-1 and PD-L1 replacing their murine counterparts, since the parental PD-L1 antibody doesn’t cross-react with the mouse antigen. In the transgenic human PD-L1-expressing mouse colon carcinoma CT26 subcutaneous tumor model, the PD-L1-targeted sialidase exhibited enhanced efficacy compared to the Bi-Sialidase or the anti-PD-L1 antibody. Furthermore, the PD-L1-targeted sialidase demonstrated a dose-dependent tumor growth inhibition and modulation of immune cell infiltration. In conclusion, these results suggested that PD-L1-targeted sialidase offers a promising cancer immunotherapeutic approach, which simultaneously inhibits immunosuppressive sialoglycans and the PD-1/PD-L1 axis through targeted delivery of engineered human sialidase to PD-L1-expressing tumor cells and immune cells. Citation Format: Jenny Che, Lihui Xu, Wayne Gatlin, Robert LeBlanc, Lizhi Cao, James Broderick, Li Peng. Development of PD-L1-targeted sialidase as a novel cancer immunotherapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB221.

Abstract 1299: Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells

Abstract Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. Here, we report a novel heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology comprising extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin (IL)-15/IL-15 receptor α complex. This fusion complex exhibited TGF-β neutralizing activity in vitro and sequestered plasma TGF-β in vivo. Subcutaneous administration of HCW9218 was well tolerated in mice, with a half-life sufficient to provide long lasting biological activity. HCW9218 enhanced metabolic and cytotoxic activities of immune cells (CD8+ T cells, NK cells) and reduced tumor progression in models of chemotherapy induced senescence (TIS, docetaxel for B16F10 tumors and gemcitabine plus nab-paclitaxel for SW1990 tumor models) and non-TIS in vivo. Mechanistically, HCW9218 treatment not only affected the immunosuppressive tumor microenvironment but also enhanced CD8+ T cells and NK cells infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immune-depletion studies showed that HCW9218-activated NK cells played a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhanced efficacy of tumor antigen-specific and anti-PDL-1 antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment lowered senescence associated secretory phenotype (SASP) factors in off-target tissues in chemotherapy treated tumor-bearing mice. Thus, HCW9218 may serve as a novel therapeutic (monotherapy or in combination with chemotherapy) to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors. HCW9218 has been cleared by the U.S. Food and Drug Administration to proceed for evaluation in a first-inhuman Phase 1b clinical trial in patients with advanced pancreatic cancer. Citation Format: Pallavi Chaturvedi, Varghese George, Bai Liu, Niraj Shrestha, Meng Wang, Micheal Dee, Xiaoyun Zhu, Jack Egan, Jin-an Jiao, Catherine Spanoudis, Jilan Xing, Victor Gallo, Christian Echeverri, Lijing You, Lin Kong, Gabriela Muniz, Peter Rhode, Hing Wong. Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1299.

Investigating the potential binding partners of H2-O to further elucidate its role in MHCII antigen presentation

Abstract The production of neutralizing antibodies in response to viral pathogens requires CD4+ T cell help. For this to occur, pathogen-derived peptides must be loaded onto class II major histocompatibility complex (MHC II) molecules in endosomal/lysosomal compartments and trafficked to the surface of antigen presenting cells. The αβ heterodimeric nonclassical MHCII molecules, H2-M and H2-O, modulate peptide loading. While H2-M directly catalyzes MHCII peptide loading, H2-O is a negative regulator of MHCII antigen presentation. Current dogma supports that H2-O is an MHCII structural mimic that exerts control over peptide loading via direct competition with MHCII for H2-M-binding. Work done in our labs, however, suggests this current model is incomplete and suggests the involvement of other, yet undefined, factors. I/LnJ mice, which are resistant to retroviruses through the production of potent neutralizing antibodies, inherit an H2-Ob allele that encodes nonfunctional H2-O. I/LnJ H2-O binds to but does not inhibit H2-M, directly challenging current dogma. Compared with H2-Oβ from retrovirus-susceptible mice, I/LnJ H2-Oβ contains three unique amino acid (AA) substitutions in the conserved immunoglobulin domain which we have found to be critical for the H2-O inhibitory activity of H2-M. We hypothesize that altered H2-O function in I/LnJ mice is due to an inability to interact with essential binding partners. We recently generated B6J mice in which we have knocked-in these three AA substitutions. This novel tool will be exploited to discover potential binding partners of variant H2-Oβ and elucidate the mechanism by which H2-O activity is modulated.

Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern.

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

αVβ3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells.

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.

Discovery of an unprecedented benz[α]anthraquinone-type heterodimer from a rare actinomycete Amycolatopsis sp. HCa1

The angucylines are a family of aromatic polyketides featuring a tetracyclic benz[a]anthraquinone skeleton. This class of polycyclic aromatic polyketides are exclusively associated with actinomycetes and can undergo many modifications such as oxidation, ring cleavage, glycosylation and dimerization. Here we report the discovery of a new ether-linked benz[a]anthraquinone heterodimer, named mycolatone (1), from a grasshopper-derived actinomycete, Amycolatopsis sp. HCa1. The structure of mycolatone (1) was determined by comprehensive two-dimensional NMR analysis, high-resolution electrospray ionization mass spectrometry and biogenetic consideration. This new heterodimeric molecule is structurally derived from the dimerization of two tetracyclic angucylines, 2-hydroxy-5-O-methyltetragomycin and PD116779, through an ether bond between C-8 and C-8′. This new structural feature enrich the structural diversity of angucylines. Additionally, the surface tension activity and cytotoxic activities of 1 against human cervical cancer cell line (Hela), human gastric adenocarcinoma cell line (SGC-7901) and human lung adenocarcinoma cell line (SPC-A-1) were evaluated.

Bifunctional TGF-β trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ Tcells invitro and invivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-β neutralizing activity invitro and sequesters plasma TGF-β invivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ Tcells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.

Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

Guidelines To Predict Binding Poses of Antibody–Integrin Complexes

Integrins are cell adhesion receptors that transmit bidirectional signals across the plasma membrane. They are noncovalently linked heterodimeric molecules consisting of two subunits and act as biomarkers in several pathologies. Thus, according to the increase of therapeutic antibody production, some efforts have been applied to produce anti-integrin antibodies. Here, we purposed to evaluate methods of generation and identification of the binding pose of integrin–antibody complexes, through protein–protein docking and molecular dynamics simulations, and propose a strategy to assure the confidence of the final model and avoid false-positive poses. The results show that ClusPro and GRAMM-X were the best programs to generate the native pose of integrin–antibody complexes. Furthermore, we were able to recover and to ensure that the selected pose is the native one by using a simple rule. All complexes from ClusPro in which the first model had the lowest energy, at least 5% more negative than the second one, were correctly predicted. Therefore, our methodology seems to be efficient to avoid misranking of wrong poses for integrin–antibody complexes. In cases where the rule is inconclusive, we proposed the use of heated molecular dynamics to identify the native pose characterized by RMSDi <0.5 nm. We believe that the set of methods presented here helps in the rational design of anti-integrin antibodies, giving some insights on the development of new biopharmaceuticals.

Pivotal role of CD103 in the development of psoriasiform dermatitis

The integrin αE known as CD103 binds integrin β7 to form the complete heterodimeric integrin molecule αEβ7. CD103 is mainly expressed by lymphocytes within epithelial tissues of intestine, lung, and skin as well as subsets of mucosal and dermal conventional dendritic cells (cDCs). CD103 has been originally implicated in the attachment of lymphocytes to epithelium in the gut and skin through the interaction with E-cadherin expressed on intestinal epithelial cells, keratinocytes, and Langerhans cells (LCs). However, an impact of CD103 on the cutaneous immune responses and the development of inflammatory skin diseases remains elusive. Here, we report that CD103 regulates the development of psoriasiform dermatitis through the control of the function of cDCs. Deficiency in CD103 exacerbates psoriasiform dermatitis, accompanied by excessive epidermal hyperplasia and infiltration of inflammatory leukocytes. Furthermore, deficiency in CD103 not only accelerates the production of proinflammatory cytokines in psoriatic lesions but also promotes the generation of lymphocytes producing interleukin (IL)-17 in the skin-draining peripheral lymph nodes (PLNs). Under the deficiency in CD103, cDCs localized in PLNs enhance cytokine production following activation. Thus, our findings reveal a pivotal role for CD103 in the control of the function of cDCs to regulate cutaneous inflammation in psoriasiform dermatitis.

Monomeric Targeted Protein Degraders.

The discovery and development of targeted protein degraders have become important areas of research in the field of medicinal chemistry. Inducing degradation of a target protein presents several advantages relative to simple inhibition including a potential for extended duration of action and more profound pharmacology. While engineered heterodimeric molecules have recently been a major focus within industry and academia, this Perspective highlights examples of targeted protein degradation observed for smaller, monomeric molecules. Methods and tools for evaluating protein degradation as well as a discussion of physical properties of monomeric vs engineered heterodimeric degraders are presented.

Genetic variants in HLA-DP/DQ contribute to risk of acute myeloid leukemia: A case-control study in Chinese

Human leukocyte antigens (HLA) are heterodimeric cell surface molecules that bind short peptides derived from non-self and self proteins. Accumulative evidence showed that specific alleles of HLA class II were associated with the susceptibility to malignant tumors including acute leukemia. In this study, we investigated the association between four single nucleotide polymorphisms (SNPs) at HLA-DP/DQ and acute myeloid leukemia (AML) risk. We genotyped four SNPs in HLA-DP (rs3077 G > A and rs9277535 G > A) and HLA-DQ (rs2856718 A > G and rs7453920 G > A) in a case-control study of 545 AML cases and 1034 cancer-free controls using Taqman allelic discrimination assay. The associations between these SNPs and AML risk were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from multivariate logistic regression analysis. We found significant associations of the variant alleles in HLA-DP (rs3077 and rs9277535) and HLA-DQ rs7453920 with increased AML risk (adjusted OR = 1.29, 95%CI = 1.10-1.51for rs3077 in additive model; adjusted OR = 1.29, 95%CI = 1.11–1.51 for rs9277535 in additive model; adjusted OR = 3.18, 95%CI = 1.86–5.46 for rs7453920 in recessive model). When combining the effects of rs3077, rs9277535 and rs7453920, we found that AML risk was significantly increased with the increasing number of variant alleles of the three SNPs in a dose-dependent manner (P for trend < 0.001). Besides, we found multiplicative interaction between rs3077 and age (≤45 years old and > 45 years old; P = 0.046). In conclusion, HLA-DP and HLA-DQ loci are candidate susceptibility regions for AML in Han Chinese.

Association of HLA-DRB1 genotype with younger age onset and elder age onset rheumatoid arthritis in Japanese populations.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint destructions and human leukocyte antigen (HLA)-DRB1 is an important genetic risk factor for RA and influences the phenotype of RA. The clinical features of elder age onset RA (EORA) were known to be different from those of younger age onset RA (YORA). Previous studies reported the different association pattern of DRB1 alleles with YORA or EORA. The associations of DRB1 genotype with these RA subsets remained almost unknown. We investigated the genotype association of DRB1 with YORA or EORA in Japanese populations.HLA genotyping was performed in Japanese RA patients and the association of allele or genotype carrier frequencies were analyzed.The genotype frequency of DRB104:05/DRB104:06 (P = .0204, OR 7.69, 95%CI 1.39-42.72), DRB104:05/DRB112:01 (P = .0050, OR 5.53, 95%CI 1.71-17.88), and DRB104:05/DRB115:01 (P = .0124, OR 3.34, 95%CI 1.39-8.02) in YORA was higher than EORA. However, the frequencies of DRB101:01/DRB104:05 in YORA was tended to be lower than EORA (P = .0784, OR 0.14, 95%CI 0.01-2.42). The gene dosage effect of the shared epitope alleles was detected in EORA, but not in YORA. Trans-complementing DQ heterodimer molecules, formed by DQA1 and DQB1 of the haplotypes with and without shared epitope alleles, might explain the higher genotype frequencies of "shared epitope /not shared epitope". Linear regression analyses showed the primary role of DQB104:01 allele for the age at onset of RA.This is the first report for the associations of DRB1 genotype with YORA or EORA in the Japanese population and the differential distribution of the genotypes was noted between these RA subsets. The involvement of DQ molecules for the age at onset of RA was suggested.

HLA-DQA1 and HLA-DQB1 Alleles, Conferring Susceptibility to Celiac Disease and Type 1 Diabetes, are More Expressed Than Non-Predisposing Alleles and are Coordinately Regulated.

HLA DQA1*05 and DQB1*02 alleles encoding the DQ2.5 molecule and HLA DQA1*03 and DQB1*03 alleles encoding DQ8 molecules are strongly associated with celiac disease (CD) and type 1 diabetes (T1D), two common autoimmune diseases (AD). We previously demonstrated that DQ2.5 genes showed a higher expression with respect to non-CD associated alleles in heterozygous DQ2.5 positive (HLA DR1/DR3) antigen presenting cells (APC) of CD patients. This differential expression affected the level of the encoded DQ2.5 molecules on the APC surface and established the strength of gluten-specific CD4+ T cells response. Here, we expanded the expression analysis of risk alleles in patients affected by T1D or by T1D and CD comorbidity. In agreement with previous findings, we found that DQ2.5 and DQ8 risk alleles are more expressed than non-associated alleles also in T1D patients and favor the self-antigen presentation. To investigate the mechanism causing the high expression of risk alleles, we focused on HLA DQA1*05 and DQB1*02 alleles and, by ectopic expression of a single mRNA, we modified the quantitative equilibrium among the two transcripts. After transfection of DR7/DR14 B-LCL with HLA-DQA1*05 cDNA, we observed an overexpression of the endogenous DQB1*02 allele. The DQ2.5 heterodimer synthesized was functional and able to present gluten antigens to cognate CD4+ T cells. Our results indicated that the high expression of alpha and beta transcripts, encoding for the DQ2.5 heterodimeric molecules, was strictly coordinated by a mechanism acting at a transcriptional level. These findings suggested that, in addition to the predisposing HLA-DQ genotype, also the expression of risk alleles contributed to the establishment of autoimmunity.