Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

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A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy

The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil.

The metastasis suppressor KISS1 is reported to be involved in the progression of several solid neoplasias, making it a promising molecular target for controlling their metastasis. The KISS1 sequence contains an N-terminal secretion signal and several dibasic sequences that are proposed to be the proteolytic cleavage sites. We present the first structural characterization of KISS1 by circular dichroism, multi-angle light scattering, small angle X-Ray scattering and NMR spectroscopy. An analysis of the KISS1 backbone NMR chemical shifts does not reveal any preferential conformation and deviation from a random coil ensemble. The backbone 15N transverse relaxation times indicate a mildly reduced mobility for two regions that are rich in bulky residues. The small angle X-ray scattering curve of KISS1 is likewise consistent with a predominantly random coil ensemble, although an ensemble optimization analysis indicates some preference for more extended conformations possibly due to positive charge repulsion between the abundant basic residues. Our results support the hypothesis that KISS1 mostly samples a random coil conformational space, which is consistent with its high susceptibility to proteolysis and the generation of Kisspeptin fragments

p15(PAF) Is an Intrinsically Disordered Protein with Nonrandom Structural Preferences at Sites of Interaction with Other Proteins.

International audienceWe present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15(PAF), showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15(PAF) is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15(PAF) gene is overexpressed in several types of human cancer. The nearly complete NMR backbone assignment of p15(PAF) allowed us to measure 86 N-H(N) residual dipolar couplings. Our residual dipolar coupling analysis reveals nonrandom conformational preferences in distinct regions, including the proliferating-cell-nuclear-antigen-interacting protein motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that targets p15(PAF) for degradation). In accordance with these findings, analysis of the (15)N R2 relaxation rates shows a relatively reduced mobility for the residues in these regions. The agreement between the experimental small angle x-ray scattering curve of p15(PAF) and that computed from a statistical coil ensemble corrected for the presence of local secondary structural elements further validates our structural model for p15(PAF). The coincidence of these transiently structured regions with protein-protein interaction and posttranslational modification sites suggests a possible role for these structures as molecular recognition elements for p15(PAF)

KISS1 residue plots of secondary chemical shifts for backbone and C^β nuclei referenced against their random coil values [33].

<p>The large deviation of C^β for C53 is not due to cisteine oxidation. The measured value of 27.89 ppm indicates a reduced side chain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172507#pone.0172507.ref051" target="_blank">51</a>].</p

KISS1 is monomeric and has little secondary and no tertiary structure in solution.

<p>(A) Size exclusion chromatogram of KISS1 (thin line and axis to the left), and molar mass derived from MALS (thick line and axis to the right). (B) CD spectrum of KISS1 at 25°C. (C) Thermal denaturation as measured by changes in the mean residue molar ellipticity at 222 nm.</p

Amino acid sequence features and backbone dynamics of KISS1.

<p>The sites of proteolytic processing by furin are indicated with arrows F. The site of peptidyl-glycine-α-amidating monooxygenase (PAM) action that converts G122 into an amide is indicated by arrow P. The black line shows the predicted disorder tendency (left axis), the grey line shows the normalized bulkiness along the polypeptide chain (left axis), and the bars show the backbone amide ¹⁵N T₂ relaxation times (right axis). The bulkiness was calculated with the ProtScale tool in the expasy web server (<a href="http://web.expasy.org/protscale/" target="_blank">http://web.expasy.org/protscale/</a>) with a window size of 9.</p

SAXS data and analysis on KISS1.

<p>(A) Logarithmic (top) and Kratky (bottom) representations of SAXS intensity versus momentum transfer <i>s</i> (open circles). (B) Averaged back-calculated curves derived from a statistical random coil ensemble (black line) and EOM selected sub-ensemble (red line). The goodness-of-fit is indicated by χ² values. The bottom panel shows the residuals of the fitting for both conformational ensembles. (C) Distribution of the radius of gyration, Rg, in the random coil ensemble (gray-bars) and EOM selected sub-ensemble, where a shoulder peak around 50 Å contains ca. 30% of all conformers.</p

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy