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MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however, sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show that manipulating TSC2 at Ser1365 potently regulated activated but not basal mTORC1 signaling in CD8+ T cells. Unlike nonstimulated TSC2-KO cells, CD8+ T cells expressing a phosphosilencing mutant TSC2-S1365A (TSC2-SA) retained normal basal mTORC1 activity. PKC and T cell receptor (TCR) stimulation induced TSC2 S1365 phosphorylation, and preventing this with the SA mutation markedly increased mTORC1 activation and T cell effector function. Consequently, SA CD8+ T cells displayed greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also displayed enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, SA CD8+ T cells used as adoptive cell therapy displayed greater antitumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhanced both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic., (©2022 The Authors; Published by the American Association for Cancer Research.)

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials., (©2021 The Authors; Published by the American Association for Cancer Research.)

PROBODY therapeutics (Pb-Tx) are protease-activatable prodrugs of monoclonal antibodies (mAbs) designed to target tumors where protease activity is elevated while avoiding normal tissue. They are composed of a parental mAb, a mask that inhibits antibody binding to target, and a protease-cleavable substrate between the mask and the mAb. We report a quantitative systems pharmacology model for the rational design and clinical translation of Pb-Tx. The model adequately described monkey pharmacokinetic data following the administration of six anti-CD166 Pb-Tx of varying mask strength and substrate cleavability and captured the trend of decreasing Pb-Tx systemic clearance with increasing mask strength. Projections to humans suggested both higher levels of Pb-Tx in tumor relative to parental mAb and an optimal mask strength for maximizing tumor receptor-mediated uptake. Simulations further suggested the majority of circulating species in humans would be intact/masked Pb-Tx, with no significant flux of cleaved/activated species from tumor to the systemic compartment., (© 2019 CytomX Therapeutics, Inc. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Purpose: Breast cancer is the second leading cause of cancer mortality among women worldwide. The major problem with current treatments is tumor resistance, recurrence, and disease progression. ErbB-2-positive breast tumors are aggressive and frequently become resistant to trastuzumab or lapatinib. We showed previously that Notch-1 is required for trastuzumab resistance in ErbB-2-positive breast cancer., Experimental Design: Here, we sought to elucidate mechanisms by which ErbB-2 attenuates Notch signaling and how this is reversed by trastuzumab or lapatinib., Results: The current study elucidates a novel Notch inhibitory mechanism by which PKCα downstream of ErbB-2 (i) restricts the availability of Jagged-1 at the cell surface to transactivate Notch, (ii) restricts the critical interaction between Jagged-1 and Mindbomb-1, an E3 ligase that is required for Jagged-1 ubiquitinylation and subsequent Notch activation, (iii) reverses trastuzumab resistance in vivo, and (iv) predicts better outcome in women with ErbB-2-positive breast cancer., Conclusions: The clinical impact of these studies is PKCα is potentially a good prognostic marker for low Notch activity and increased trastuzumab sensitivity in ErbB-2-positive breast cancer. Moreover, women with ErbB-2-positive breast tumors expressing high Notch activation and low PKCα expression could be the best candidates for anti-Notch therapy., (©2015 American Association for Cancer Research.)

Antimicrobial resistance (AMR), co-selection phenomenon, and the relationship between reduced susceptibility (RSC) to ciprofloxacin (CIP) and resistance to other antimicrobials in Listeria spp. (n = 103) recovered from food processing environments (FPE) and food were investigated. Resistance of Listeria monocytogenes and other listeriae, respectively, to cefoxitin (FOX; 98% vs. 88%), CIP (7% vs. 4%), clindamycin (CLI; 33% vs. 59%) and tetracycline (6% vs. 8%) was observed, as was RSC to CIP (67% vs. 57%) and CLI (65% vs. 41%). L. monocytogenes also possessed RSC to linezolid (LZD; 6%), rifampicin (2%) and streptomycin (6%), with other listeriae displaying RSC to chloramphenicol (4%). L. monocytogenes serotype 1/2a (90%) isolates were more frequently resistant or possessed RSC to CIP compared to serotype 4b (55%) (p = 0.015). When eight strains were experimentally adapted to high concentrations of CIP, co-selection occurred as MICs to benzalkonium chloride (BAC) increased (n = 5), gentamicin MICs remained the same (n = 6) or increased 2-fold (n = 2), and led to RSC to LZD (n = 1) and resistance to CLI (n = 8). Overall, levels of resistance/RSC to CIP in food chain isolates, particularly 1/2a, are concerning. Further, reduced sensitivity to disparate antimicrobials following CIP exposure highlights the need for increased knowledge of co-selection phenomenon linked with antimicrobial agents., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Patient prognosis in lung cancer largely depends on early diagnosis. The exhaled breath of patients may represent the ideal specimen for future lung cancer screening. However, the clinical applicability of current diagnostic sensor technologies based on signal pattern analysis remains incalculable due to their inability to identify a clear target. To test the robustness of the presence of a so far unknown volatile organic compound in the breath of patients with lung cancer, sniffer dogs were applied. Exhalation samples of 220 volunteers (healthy individuals, confirmed lung cancer or chronic obstructive pulmonary disease (COPD)) were presented to sniffer dogs following a rigid scientific protocol. Patient history, drug administration and clinicopathological data were analysed to identify potential bias or confounders. Lung cancer was identified with an overall sensitivity of 71% and a specificity of 93%. Lung cancer detection was independent from COPD and the presence of tobacco smoke and food odours. Logistic regression identified two drugs as potential confounders. It must be assumed that a robust and specific volatile organic compound (or pattern) is present in the breath of patients with lung cancer. Additional research efforts are required to overcome the current technical limitations of electronic sensor technologies to engineer a clinically applicable screening tool.

The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW(1)TAW(2)K and APPPAW(1)TAW(2)K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C(2)-hexosylated tryptophan (W(1)) and C(2)-hexosylated hydroxytryptophan (W(2)), the latter of which is redox active. The UV absorbance spectrum of W(2) is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion.

The marine mussel Mytilus galloprovincialis is tethered to rocks in the intertidal zone by a holdfast known as the byssus. Functioning as a shock absorber, the byssus is composed of threads, the primary molecular components of which are collagen-containing proteins (preCOLs) that largely dictate the higher order self-assembly and mechanical properties of byssal threads. The threads contain additional matrix components that separate and perhaps lubricate the collagenous microfibrils during deformation in tension. In this study, the thread matrix proteins (TMPs), a glycine-, tyrosine- and asparagine-rich protein family, were shown to possess unique repeated sequence motifs, significant transcriptional heterogeneity and were distributed throughout the byssal thread. Deamidation was shown to occur at a significant rate in a recombinant TMP and in the byssal thread as a function of time. Furthermore, charge heterogeneity presumably due to deamidation was observed in TMPs extracted from threads. The TMPs were localized to the preCOL-containing secretory granules in the collagen gland of the foot and are assumed to provide a viscoelastic matrix around the collagenous fibers in byssal threads.

We previously isolated a pollen-specific gene encoding a pollen tube wall-associated glycoprotein with a globular domain and an extensin domain from maize (mPex1). To evaluate which protein domains might be important for function, we isolated a second monocot gene (mPex2) and a dicot gene (tPex). Each gene encodes a signal sequence, an N-terminal globular domain comprised of a variable region, a leucine-rich repeat (LRR) with an adjacent cysteine-rich region, a transition region and an extensin-like C-terminal domain. The LRRs of the maize and tomato Pex proteins are highly conserved. Although the extensin domains in the maize and tomato proteins vary in length and in amino acid sequence, they are likely to be structurally conserved. Additional putative Pex gene sequences were identified by either GenBank search (Arabidopsis) or PCR (sorghum and potato): all encode conserved LRRs. The presence of a conserved LRR in the known and potential Pex proteins strongly suggests that this motif is involved in the binding of a specific ligand during pollen tube growth. Gene expression studies using RNA and protein blotting as well as promoter-reporter gene fusions in transient and stable transformation indicate that the tomato Pex gene is pollen-specific.