Edited by Karen G. FlemingReceptor protein tyrosine phosphatases (RPTPs) play critical regulatory roles in mammalian signal transduction. However, the structural basis for the regulation of their catalytic activity is not fully understood, and RPTPs are generally not therapeutically targetable. This knowledge gap is partially due to the lack of known natural ligands or selective agonists of RPTPs. Contrary to what is known from structure-function studies of receptor tyrosine kinases (RTKs), RPTP activities have been reported to be suppressed by dimerization, which may prevent RPTPs from accessing their RTK substrates. We report here that homodimerization of protein tyrosine phosphatase receptor J (PTPRJ, also known as DEP-1) is regulated by specific transmembrane (TM) residues. We found that disrupting these interactions destabilizes homodimerization of full-length PTPRJ in cells, reduces the phosphorylation of the known PTPRJ substrate epidermal growth factor receptor (EGFR) and of other downstream signaling effectors, antagonizes EGFR-driven cell phenotypes, and promotes substrate access. We demonstrate these observations in human cancer cells using mutational studies and identified a peptide that binds to the PTPRJ TM domain and represents the first example of an allosteric agonist of RPTPs. The results of our study provide fundamental structural and functional insights into how PTPRJ activity is tuned by TM interactions in cells. Our findings also open up opportunities for developing peptide-based agents that could be used as tools to probe RPTPs' signaling mechanisms or to manage cancers driven by RTK signaling.
A growing class of immunotherapeutic agents work by redirecting components of the immune system to recognize specific markers on the surface of cancer cells and initiate a selective immune response. However, such immunotherapeutic modalities will remain confined to a relatively small subgroup of patients until two major hurdles are overcome: (1) the specific targeting of cancer cells relative to healthy cells, and (2) the lack of common targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidic microenvironment of solid tumors to selectively graft the surface of cancer cells with immuno-engager epitopes for directed destruction by components of the immune system. Specifically, conjugates were assembled using an antigen that recruit antibodies present in human serum, and the pH(Low) Insertion Peptide (pHLIP), a unique peptide that selectively target tumors in vivo by anchoring onto cancer cell surfaces in a pHdependent manner. We established that conjugates can recruit antibodies from human serum to the surface of cancer cells, and induce complementdependent and antibody-dependent cellular cytotoxicity by peripheral blood mononuclear cells and also an engineered NK cell line. These results suggest that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent the problem of narrow windows of selectivity..
Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge‐based design of cancer‐targeting molecular tools, such as pH‐low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca2+, and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond‐timescale all‐atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide‐lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH‐induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+‐induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+‐dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid‐coupled regulation of protein‐membrane insertion by divalent cations.
Monoclonal antibodies are an efficacious therapy against SARS-CoV-2. However, rapid viral mutagenesis, led to escape from most of these therapies, outlining the need for an antibody cocktail with a broad neutralizing potency. Using an unbiased interrogation of the memory B cell repertoire of convalescent COVID-19 patients, we identified human antibodies with broad antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants of concern, including Delta, Omicron BA.1 and BA.2. Here, we describe an antibody cocktail IMM-BCP-01, that consists of three patient-derived broadly neutralizing antibodies directed at non-overlapping surfaces on the SARS-CoV-2 spike protein. Two antibodies, IMM20184 and IMM20190, directly blocked Spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its cryptic epitope on the outer surface of RBD, altered the conformation of the Spike Trimer, promoting release of Spike monomers. These antibodies decreased Omicron SARS-CoV-2 infection in the lungs of Syrian golden hamsters in vivo, and potently induced antiviral effector response in vitro, including phagocytosis, ADCC, and complement pathway activation. Our pre-clinical data demonstrated that the three antibody cocktail IMM-BCP-01 could be a promising means for preventing or treating infection of SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2, in susceptible individuals.
Immune checkpoint inhibitors that overcome T cell suppressive mechanisms in tumors have revolutionized the treatment of cancer but are only efficacious in a small subset of patients. Targeting suppressive mechanisms acting on innate immune cells could significantly improve the incidence of clinical response by facilitating a multi-lineage response against the tumor involving both adaptive and innate immune systems. Here, we show that intra-tumoral interleukin (IL)-38 expression is a feature of a large frequency of head and neck, lung and cervical squamous cancers and correlates with reduced immune cell numbers. We generated IMM20324, an antibody that binds human and mouse IL-38 proteins and inhibits the binding of IL-38 to its putative receptors, interleukin 1 receptor accessory protein-like 1 (IL1RAPL) and IL-36R. In vivo , IMM20324 demonstrated a good safety profile, delayed tumor growth in a subset of mice in an EMT6 syngeneic model of breast cancer, and significantly inhibited tumor expansion in a B16.F10 melanoma model. Notably, IMM20324 treatment resulted in the prevention of tumor growth following re-implantation of tumor cells, indicating the induction of immunological memory. Furthermore, exposure of IMM20324 correlated with decreased tumor volume and increased levels of intra-tumoral chemokines. Together, our data suggest that IL-38 is expressed in a high frequency of cancer patients and allows tumor cells to suppress anti-tumor immunity. Blockade of IL-38 activity using IMM20324 can re-activate immunostimulatory mechanisms in the tumor microenvironment leading to immune infiltration, the generation of tumor-specific memory and abrogation of tumor growth.
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