Eden Reichard

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Immune receptor inhibition through enforced phosphatase recruitment.

Nature. 2020 10;586(7831):779-784

Authors: Fernandes RA, Su L, Nishiga Y, Ren J, Bhuiyan AM, Cheng N, Kuo CJ, Picton LK, Ohtsuki S, Majzner RG, Rietberg SP, Mackall CL, Yin Q, Ali LR, Yang X, Savvides CS, Sage J, Dougan M, Garcia KC

Abstract
Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

PMID: 33087934 [PubMed - indexed for MEDLINE]

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Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1.

J Am Chem Soc. 2021 Jan 04;:

Authors: Cotton AD, Nguyen DP, Gramespacher JA, Seiple IB, Wells JA

Abstract
Targeted protein degradation has emerged as a new paradigm to manipulate cellular proteostasis. Proteolysis-targeting chimeras (PROTACs) are bifunctional small molecules that recruit an E3 ligase to a target protein of interest, promoting its ubiquitination and subsequent degradation. Here, we report the development of antibody-based PROTACs (AbTACs), fully recombinant bispecific antibodies that recruit membrane-bound E3 ligases for the degradation of cell-surface proteins. We show that an AbTAC can induce the lysosomal degradation of programmed death-ligand 1 by recruitment of the membrane-bound E3 ligase RNF43. AbTACs represent a new archetype within the PROTAC field to target cell-surface proteins with fully recombinant biological molecules.

PMID: 33395526 [PubMed - as supplied by publisher]

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PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

EMBO J. 2020 01 15;39(2):e103637

Authors: Wiede F, Lu KH, Du X, Liang S, Hochheiser K, Dodd GT, Goh PK, Kearney C, Meyran D, Beavis PA, Henderson MA, Park SL, Waithman J, Zhang S, Zhang ZY, Oliaro J, Gebhardt T, Darcy PK, Tiganis T

Abstract
Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

PMID: 31803974 [PubMed - indexed for MEDLINE]

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The receptor PTPRU is a redox sensitive pseudophosphatase.

Nat Commun. 2020 Jun 26;11(1):3219

Authors: Hay IM, Fearnley GW, Rios P, Köhn M, Sharpe HJ, Deane JE

Abstract
The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels. Most human RPTPs possess an extracellular receptor domain and tandem intracellular phosphatase domains: comprising an active membrane proximal (D1) domain and an inactive distal (D2) pseudophosphatase domain. Here we demonstrate that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. The PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and we show that this is due to multiple structural rearrangements that destabilise the active site pocket and block the catalytic cysteine. Upon oxidation, this cysteine forms an intramolecular disulphide bond with a vicinal "backdoor" cysteine, a process thought to reversibly inactivate related phosphatases. Importantly, despite the absence of catalytic activity, PTPRU binds substrates of related phosphatases strongly suggesting that this pseudophosphatase functions in tyrosine phosphorylation by competing with active phosphatases for the binding of substrates.

PMID: 32591542 [PubMed - as supplied by publisher]

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Protein tyrosine phosphatase receptor type R (PTPRR) antagonizes the Wnt signaling pathway in ovarian cancer by dephosphorylating and inactivating β-catenin.

J Biol Chem. 2019 11 29;294(48):18306-18323

Authors: Wang Y, Cao J, Liu W, Zhang J, Wang Z, Zhang Y, Hou L, Chen S, Hao P, Zhang L, Zhuang M, Yu Y, Li D, Fan G

Abstract
Despite a lack of mutations, accumulating evidence supports an important role for the Wnt/β-catenin pathway in ovarian tumorigenesis. However, the molecular mechanism that contributes to the aberrant activation of the Wnt signaling cascade in ovarian cancer has not been fully elucidated. Here, we found that protein tyrosine phosphatase receptor type R (PTPRR) suppressed the activation of the Wnt/β-catenin pathway in ovarian cancer. We performed an shRNA-based biochemical screen, which identified PTPRR as being responsible for tyrosine dephosphorylation of β-catenin on Tyr-142, a key site controlling the transcriptional activity of β-catenin. Of note, PTPRR was down-regulated in ovarian cancers, and ectopic PTPRR re-expression delayed ovarian cancer cell growth both in vitro and in vivo Using a proximity-based tagging system and RNA-Seq analysis, we identified a signaling nexus that includes PTPRR, α-catenin, β-catenin, E-cadherin, and AT-rich interaction domain 3C (ARID3C) in ovarian cancer. Immunohistochemistry staining of human samples further suggested that PTPRR expression is inversely correlated with disease prognosis. Collectively, our findings indicate that PTPRR functions as a tumor suppressor in ovarian cancer by dephosphorylating and inactivating β-catenin. These results suggest that PTPRR expression might have utility as a prognostic marker for predicting overall survival.

PMID: 31653698 [PubMed - indexed for MEDLINE]

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A head-to-toe dimerization has physiological relevance for ligand-induced inactivation of protein tyrosine receptor type Z.

J Biol Chem. 2019 10 11;294(41):14953-14965

Authors: Fujikawa A, Sugawara H, Tanga N, Ishii K, Kuboyama K, Uchiyama S, Suzuki R, Noda M

Abstract
Protein-tyrosine phosphatase (PTPase) receptor type Z (PTPRZ) has two receptor isoforms, PTPRZ-A and -B, containing tandem intracellular PTP-D1 and -D2 domains, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular PTPRZ region leads to inactivation of its PTPase activity, thereby facilitating oligodendrocyte precursor cell (OPC) differentiation and myelination in the central nervous system. However, the mechanisms responsible for PTN-induced PTPRZ inactivation remain unclear. We herein report that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) shows a "head-to-toe"-type dimer conformation, with D2 masking the catalytic site of D1. MS analyses revealed that PTPRZ-ICR proteins remain in monomer-dimer equilibrium in aqueous solution and that a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bind to the monomer form in a 1:1 ratio. A D2 deletion (ΔD2) or dimer interface mutation (DDKK) disrupted dimer formation, but SCB4380 binding was maintained. Similar to WT PTPRZ-B, monomer-biased PTPRZ-B-ΔD2 and PTPRZ-B-DDKK variants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these variants were not suppressed by PTN treatment, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. Of note, the PTN treatment did not enhance OPC differentiation in primary cultured glial cells from ΔD2 or PTPase-inactive PTPRZ-B (CS) mutant knock-in mice. Our results thus indicate that PTN-induced PTPRZ inactivation results from dimer formation of the intracellular tandem PTP domains in a head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation in vivo.

PMID: 31416834 [PubMed - indexed for MEDLINE]

Cell‐specific cytosolic delivery of proteins is highly challenging. Herein, we demonstrate that mutants of anthrax‐derived protective antigen (mPA) can be expressed as fusions to scFvs for targeted delivery of potent protein therapeutics to the cellular cytosol. Targeting of EGFR or carcinoembryonic antigen with this strategy led to selective delivery to and death of pancreatic cancer cells.

Abstract

The nontoxic, anthrax protective antigen/lethal factor N‐terminal domain (PA/LF_N) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LF_N to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single‐chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer‐relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA−scFv fusions enable cell‐specific delivery of diphtheria toxin catalytic domain and Ras/Rap1‐specific endopeptidase to pancreatic cancer cells. Importantly, mPA−scFv fusion‐based treatments display potent cell‐specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

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PTP1B markedly promotes breast cancer progression and is regulated by miR-193a-3p.

FEBS J. 2019 03;286(6):1136-1153

Authors: Yu M, Liu Z, Liu Y, Zhou X, Sun F, Liu Y, Li L, Hua S, Zhao Y, Gao H, Zhu Z, Na M, Zhang Q, Yang R, Zhang J, Yao Y, Chen X

Abstract
The protein tyrosine phosphatase PTP1B, which is encoded by PTPN1, is a ubiquitously expressed nonreceptor protein tyrosine phosphatase. PTP1B has long been known to negatively regulate insulin and leptin receptor signalling. Recently, it was reported to be aberrantly expressed in cancer cells and to function as an important oncogene. In this study, we found that PTP1B protein levels are dramatically increased in breast cancer (BC) tissues and that PTP1B promotes the proliferation, and suppresses the apoptosis, of both HER2-positive and triple-negative BC cell lines. Bioinformatics analysis identified that the miRNA, miR-193a-3p, might potentially target PTP1B. We demonstrate that miR-193a-3p regulates PTP1B in BC cells and that it regulates the proliferation and apoptosis of BC cells by targeting PTP1B, both in vitro and in vivo. In conclusion, this study confirms that PTP1B acts as an oncogene in BC and demonstrates that miR-193a-3p can serve as a tumour suppressor gene in BC by targeting PTP1B.

PMID: 30548198 [PubMed - indexed for MEDLINE]

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FS222, a CD137/PD-L1 tetravalent bispecific antibody exhibits low toxicity and anti-tumor activity in colorectal cancer models.

Clin Cancer Res. 2020 Apr 28;:

Authors: Lakins MA, Koers A, Giambalvo R, Munoz-Olaya J, Hughes R, Goodman E, Marshall S, Wollerton F, Batey S, Gliddon D, Tuna M, Brewis N

Abstract
PURPOSE: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for relapsing or refractory cancer patients. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1 to induce T cell activation to eradicate tumours without the current toxicity and efficacy limitations seen clinically.
EXPERIMENTAL DESIGN: A bispecific antibody (FS222) was developed by engineering CD137 binding sites into the Fc region of a PD-L1 IgG1 mAb. T cell activation by FS222 was investigated using multiple in vitro assays. The anti-tumour efficacy, survival benefit, pharmacodynamics and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumour models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 was investigated in a non-human primate dose-range finding study.
RESULTS: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T cell activation across CD8+ T cell activation assays in a PD-L1-dependent manner with FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumour growth inhibition and survival benefit, concomitant with CD8+ T cell activation, in CT26 and MC38 syngeneic mouse tumour models.
CONCLUSIONS: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumour microenvironment, FS222 has the potential to leverage a focused, potent and safe immune response augmenting the PD-(L)1 axis blockade.

PMID: 32345647 [PubMed - as supplied by publisher]

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Antibody-PROTAC conjugates enable HER2-dependent targeted protein degradation of BRD4.

ACS Chem Biol. 2020 Apr 27;:

Authors: Maneiro M, Forte N, Shchepinova MM, Kounde CS, Chudasama V, Baker JR, Tate EW

Abstract
Targeting protein degradation with Proteolysis-Targeting Chimeras (PROTACs) is an area of great current interest in drug discovery. Nevertheless, although the high effectiveness of PROTACs against a wide variety of targets has been established, most degraders reported to date display limited intrinsic tissue selectivity, and do not discriminate between cells of different types. Here we describe a strategy for selec-tive protein degradation in a specific cell type. We report the design and synthesis of a trastuzumab-PROTAC conjugate (Ab PROTAC 3) in which E3 ligase-directed degrader activity is caged with an antibody linker which can be hydrolyzed following antibody-PROTAC internal-ization, releasing the active PROTAC and inducing catalytic protein degradation. We show that 3 selectively targets bromodomain-containing protein 4 (BRD4) for degradation only in HER2 positive breast cancer cell lines, whilst sparing HER2 negative cells. Using live cell confocal microscopy, we show internalization and lysosomal trafficking of the conjugate specifically in HER2 positive cells, leading to release of active PROTAC in quantities sufficient to induce potent BRD4 degradation. These studies demonstrate proof-of-concept for tissue-specific BRD4 degradation, overcoming limitations of PROTAC selectivity, with significant potential for application to novel targets.

PMID: 32338867 [PubMed - as supplied by publisher]

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Regulation of PTP1B activation through disruption of redox-complex formation.

Nat Chem Biol. 2020 02;16(2):122-125

Authors: Londhe AD, Bergeron A, Curley SM, Zhang F, Rivera KD, Kannan A, Coulis G, Rizvi SHM, Kim SJ, Pappin DJ, Tonks NK, Linhardt RJ, Boivin B

Abstract
We have identified a molecular interaction between the reversibly oxidized form of protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ζ that regulates PTP1B activity. Destabilizing the transient interaction between 14-3-3ζ and PTP1B prevented PTP1B inactivation by reactive oxygen species and decreased epidermal growth factor receptor phosphorylation. Our data suggest that destabilizing the interaction between 14-3-3ζ and the reversibly oxidized and inactive form of PTP1B may establish a path to PTP1B activation in cells.

PMID: 31873221 [PubMed - indexed for MEDLINE]

Getting in your own way . We report fusion proteins consisting of a HER2 binding domain and antibody‐binding domain. Concomitant treatment with a mixture of fusions increased antibody recruitment and antibody‐dependent cellular cytotoxicity (ADCC) for some mixtures. For a fusion containing a Sac7d IgG‐recruiting domain, IgG recruitment is observed, but not ADCC, likely due to inhibition of CD16 binding.

Abstract

We report fusion proteins designed to bind spatially distinct epitopes on the extracellular portion of HER2, a breast cancer biomarker and established therapeutic target, and recruit IgG (either anti‐His₆ or serum IgG) to the cell surface. When the proteins were incubated with anti‐His₆ antibody and various concentrations of a single HER2‐binding protein His₆ fusion, we observed interference and a decrease in antibody recruitment at HER2‐binding protein concentrations exceeding ∼30 nM. In contrast, concomitant treatment with two or three distinct HER2‐binding protein His₆ fusions, and anti‐His₆, results in increased antibody recruitment, even at relatively high HER2‐binding protein concentration. In some instances, increased antibody recruitment leads to increased antibody‐dependent cellular cytotoxicity (ADCC) activity. While a fusion protein consisting of a HER2‐binding nanobody and Sac7d, a protein evolved to recognize the Fc domain of IgG, binds IgG from serum, antibody recruitment does not lead to ADCC activity. Rationales for these disparities are provided. Collectively, our findings have implications for the design of efficacious targeted immunotherapeutic biologics, and ensembles thereof.

Reprogramming the tumor microenvironment to increase immune-mediated responses is currently of intense interest. Patients with immune-infiltrated “hot” tumors demonstrate higher treatment response rates and improved survival. However, only the minority of tumors are hot, and a limited proportion of patients benefit from immunotherapies. Innovative approaches that make tumors hot can...

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CD148 Serves as a Prognostic Marker of Gastric Cancer and Hinders Tumor Progression by Dephosphorylating EGFR.

J Cancer. 2020;11(9):2667-2678

Authors: Sun Y, Li S, Yu W, Chen C, Liu T, Li L, Zhang D, Zhao Z, Gao J, Wang X, Shi D, Liu L

Abstract
CD148 is a member of the receptor-type protein tyrosine phosphatase family encoded by the PTPRJ gene and has controversial impacts on cancers. In this study, we investigated the clinical significance of CD148 in gastric cancer and the possible mechanisms. Suppressed CD148 expression indicated adverse pathological features and poor outcomes in gastric cancer patients. CD148 overexpression impeded tumor proliferation, motility, and invasiveness, while CD148 knock-down or knockout promoted the ability of gastric cancer cells to grow and metastasize in vitro and in vivo. Mechanistically, CD148 negatively regulated EGFR phosphorylation of multiple tyrosine residues, including Y1173, Y1068, and Y1092, and remarkably inhibited downstream PI3K/AKT and MEK/ERK pathways. In silico analysis revealed that gene deletions or missense/truncated mutations of PTPRJ gene rarely occurred in gastric cancers. Instead, a 3' UTR-specific methylation might regulate CD148 expression, and the potential regulators were TET2 and TET3. Collectively, our results suggest that CD148 is a convincing prognostic marker as well as a potential therapeutic target for gastric cancer.

PMID: 32201537 [PubMed]

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Structural basis of ligand binding modes at the human formyl peptide receptor 2.

Nat Commun. 2020 Mar 05;11(1):1208

Authors: Chen T, Xiong M, Zong X, Ge Y, Zhang H, Wang M, Won Han G, Yi C, Ma L, Ye RD, Xu Y, Zhao Q, Wu B

Abstract
The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family.

PMID: 32139677 [PubMed - in process]

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GP78 Cooperates with Dual-Specificity Phosphatase 1 To Stimulate Epidermal Growth Factor Receptor-Mediated Extracellular Signal-Regulated Kinase Signaling.

Mol Cell Biol. 2019 06 01;39(11):

Authors: Kho DH, Uddin MH, Chatterjee M, Vogt A, Raz A, Wu GS

Abstract
GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.

PMID: 31061093 [PubMed - indexed for MEDLINE]