Effective anti-tumor immunity in humans has been associated with the presence of T cells directed at cancer neoantigens1, which are T cell epitopes with tumor-specific expression arising from non-silent somatic mutations. They are highly immunogenic because they are not expressed in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumor immune response2, their systematic discovery and evaluation only became feasible with the recent availability of massively-parallel sequencing for detection of all coding mutations within tumors, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous HLA molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T cell populations and induce a broader repertoire of new T cell specificities in cancer patients, tipping the intra-tumoral balance in favor of enhanced tumor control. Here we demonstrate the feasibility, safety and immunogenicity of a vaccine that targets up to 20 predicted personal tumor neoantigens. Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%), respectively, of the 97 unique neoantigens used across patients. These T cells discriminated mutated from wildtype antigens, and in some cases, directly recognized autologous tumor. Of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination, while 2 with progressive disease were subsequently treated with anti-PD-1 therapy and experienced complete tumor regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint therapies.

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent crosslinkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.Lysyl oxidase (LO; EC 1.4.3.13) catalyzes the post-translational modification of elastin and collagen, by oxidizing selected lysine residues within these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde. Subsequent spontaneous reactions of the peptidyl aldehydes yield covalent cross-linkages (1). LO is synthesized as a 46-kDa preproenzyme by fibrogenic cells. After signal peptide cleavage and N-glycosylation, the resulting 50-kDa N-glycosylated proenzyme is secreted (2) and proteolytically processed in the extracellular space to a mature enzyme of 31 Ϯ 1 kDa (3).Although initiation of the cross-linking of elastin and collagen is a critical function of LO, there is evidence that it may have additional biological roles. The mature enzyme isolated from bovine aorta is chemotactic for monocytes and lymphocytes in assays in vitro, with the chemotactic effect requiring a functional active site (4). Moreover, LO expression is negligibly low in several neoplastically transformed cell lines (5), including fibroblasts transformed with the Ha-ras oncogene (6, 7). It is of particular interest in this regard that a murine ras recision gene, the expression of which appears to suppress the tumorigenic effect of Ha-ras, encodes LO (6-9). The basis of this apparent effect of LO has yet to be understood. However, a recent report notes that transfection of revertants derived from ras-transformed NIH 3T3 cells with LO antisense but not LO sense constructs induced a change in the relatively ''loose'' chromatin packing state of the revertants to the tighter chromatin packing state of the original transformants (10). These results raise the possibility that LO may directly or indirectly exert effects on nuclear components. In the present study, we provide evidence that LO occurs and catalytically functions within the nuclei of fibrogenic cells. EXPERIMENTAL PROCEDURESCell Culture. Neonatal rat aorta smooth muscle cells (NRASMCs), explanted from 2-to 3-day-old rat pups as described (11, 12), were used in first passage at or just prior to confluency. Swiss 3T3 fibroblasts (American Type Culture Collection) were used at confluency after 3-4 days of culture in passages 9-12. Cells were cultured in DMEM containing 3.7 g of NaHCO 3 per l, 100 units of penicillin per ml, 100 g of streptomycin per ml, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (GIBCO͞BRL), and 10% fetal bovine serum ...

Lysyl oxidase (EC 1.4.3.13) is unique among the mammalian copper amine oxidases by catalyzing a critical post-translational modification essential to the biogenesis of connective tissue matrices. This enzyme initiates covalent cross-linking between and within the molecular units of elastin and of collagen by oxidizing peptidyl lysine in these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde (1, 2). The peptidyl aldehyde can then condense with neighboring ⑀-amino groups or peptidyl aldehydes to form the covalent cross-linkages found in fibrillar collagen and elastin. Lysyl oxidase contains a tightly bound copper cofactor as well as a covalently bound carbonyl prosthetic group recently identified as lysine tyrosylquinone (3).Lysyl oxidase catalyzes primary amine oxidation through a ping pong bi ter kinetic mechanism (4, 5). Following initial Schiff base formation with the LTQ 1 cofactor, the bound substrate undergoes rate-limiting, general base-facilitated ␣-proton abstraction (6). Electrons migrating from the resulting carbanion reduce the carbonyl cofactor, followed by hydrolysis of the product imine intermediate to release the aldehyde product. The reduced enzyme, retaining the amino function of the substrate, is reoxidized by molecular oxygen to produce hydrogen peroxide and ammonia, regenerating the oxidized enzyme and completing the catalytic cycle.The role of lysyl oxidase in the growth and repair of connective tissues has been well documented. Markedly increased levels of LO activity are observed in a variety of fibrotic diseases in which excess collagen is deposited in the affected tissues, as in models of atherosclerosis, hypertension, and liver and pulmonary fibrosis (2). The possibility that the development of fibrosis may be restricted by the specific suppression of lysyl oxidase activity has stimulated the search for selective and potent inhibitors of this enzyme. These efforts have identified mechanism-based and ground-state inhibitors, including ␤-substituted haloethylamines (7), benzylamines substituted with electronegative para-substituents (8), and 1,2-diamines (9), each of which appear to inhibit as adducts of the carbonyl cofactor.In the present report, we describe our observations that homocysteine thiolactone and the oxygen and selenium lactone analogues of this compound are active site-directed, irreversible inhibitors of lysyl oxidase. The selenium and sulfur lactones are the most potent of these and are selective for lysyl oxidase, among mechanistically similar copper-dependent mammalian amine oxidases tested in the present study. Notably, HCTL occurs in mammalian systems as a metabolic by-product of methyl transfer from S-adenosylhomocysteine. Moreover, the accumulation of HCTL has been suggested to be related to mechanisms of carcinogenesis and atherogenesis (10 -12), whereas it has also been shown to thiolate proteins, including low density lipoproteins in vitro (13,14). Elucidation of interactions of these compounds with lysyl oxidase should increase options for the design of antifib...

Lysyl oxidase (LO), a secreted protein, was recently identified within the nuclei of vascular smooth-muscle cells (SMC) and 3T3 fibroblasts. A possible pathway by which LO can enter cell nuclei was explored in the present study. SMC were incubated with purified 32-kDa bovine aorta LO that had been fluorescently labeled with rhodamine (TRITC-LO). TRITC-LO entered the cytosol and then rapidly concentrated within the nuclei of preconfluent cultures of these cells, whereas carbonic anhydrase, a protein of similar molecular weight and similarly labeled, did not enter the cells under these conditions. LO that had been reductively methylated at lysine residues with [(14)C]HCHO was also taken up into the cytosolic and nuclear compartments. Intracellular uptake and intracellular distribution were not altered by inhibiting LO activity with beta-aminopropionitrile. An excess of native LO but not of carbonic anhydrase competitively inhibited the uptake of the isotopically labeled enzyme. Thus, once secreted and proteolytically processed, mature LO can enter the cells and concentrate within nuclei in a manner that appears to be specific and independent of its catalytic activity.

In this Letter, the 'Data availability' section in the Methods should state 'WES and RNA-seq data are deposited in dbGaP (https://www.ncbi.nlm. nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001451.v1.p1). All other data are available from the corresponding author upon reasonable request.' instead of 'All data are available from the corresponding author upon reasonable request'. In addition, the 'Competing interests' statement should include: 'C.J.W. is subject to a conflict of interest management plan for the reported studies because of her competing financial interests in Neon Therapeutics. Under this plan, C.J.W. may not access identifiable human subjects' data nor otherwise participate directly in the IRBapproved protocol reported herein. C.J.W. 's contributions to the overall program strategy and data analyses occurred on a de-identified basis. ' These errors have been corrected in the online versions of the Letter.