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PubMed2026

Target Antigen Identification for Antibody Drug Conjugate Therapy in Biliary Tract Cancer.

BACKGROUND: Data on antibody-drug conjugates (ADCs) target expression prevalence, intertumoral heterogeneity, genomic concordance, and its effect on clinical outcomes is limited in biliary tract cancers (BTC). METHODS: Resected primary BTC specimens, and when available, matched metastatic samples were assembled into tissue microarrays and tested for CLDN18.2, c-MET, Nectin-4, TROP2, and HER2 expression by immunohistochemistry (IHC). A subset underwent targeted next-generation sequencing using MSK-IMPACT (NCT01775072). Exploratory associations of target expression with clinicopathologic parameters, genomic alterations, recurrence-free (RFS), and overall (OS) survival were evaluated. RESULTS: 65 patients with resected BTC and 18 paired metastatic sites were identified-43% extrahepatic cholangiocarcinoma, 40% intrahepatic cholangiocarcinoma, and 17% gallbladder cancer. All evaluated target antigens were expressed; percent positivity and H-score ≥200 were: TROP2 (83%, 26%), c-MET (75%, 26%), Nectin-4 (66%, 35%), and CLDN18.2 (46%, 7.7%). HER2 overexpression occurred in 3.1% of tumors. Overall agreement among paired primary and metastatic samples on calling either positive or negative ranged from 43% to 75% with the highest observed for HER2 [75%; κ=0.29 (95%CI: -0.32 to 0.91)] and TROP2 (71%; κ not available) and lowest for c-MET, CLDN18.2, and Nectin-4. Frequently altered genes included TP53 (36%), SMAD4 (27%), ELF3 (21%). We observed no significant association between target antigen expression with genomics, RFS, or OS. CONCLUSIONS: BTC displays frequent but heterogeneous expression of multiple ADC targets. These hypothesis generating findings suggest inherent complexity of target protein quantification, target threshold determination, and target sampling discordance. Future studies will be required to refine our understanding the utlitiy of ADCs in BTC.

Journal Article↗
PubMed1988

Target cell expression of MHC antigens is not (always) a turn-off signal to natural killer cells.

Recent evidence has demonstrated that the lytic function of natural killer cells might be regulated by potential target cells through the target cells' expression of cell surface components that are able to inhibit the lytic process. Specifically, it has been shown in many target cell systems that the expression of class I MHC proteins by target cells is inversely proportional to their susceptibility to lysis by NK cells. It has been suggested, therefore, that MHC proteins may act as important negative regulatory elements in the ongoing control of NK cell function. Herein, we examined two closely related murine lymphoma cells (ASL1 and ASL1w), both in terms of their susceptibility to lysis by NK cells as well as their expression of both H-2K and H-2D class I MHC proteins. The results of these studies showed that whereas ASL1 and ASL1w cells differed greatly in their susceptibility to NK cell lysis (ASL1 was much more NK resistant than ASL1w), both expressed high levels of H-2K and D proteins. In contrast to what might have been predicted base on reports from other target cell systems, the more NK susceptible ASL1w cells expressed somewhat higher levels of H-2K Ag than did ASL1 cells. These results indicate that expression of H-2 class I proteins by target cells, in and of itself, is not sufficient to inhibit the lytic activity of murine NK cells.

Animals↗
PubMed1989

Tat trans-activates the human immunodeficiency virus through a nascent RNA target.

Expression of the human immunodeficiency virus type 1 (HIV-1) genome is greatly dependent on the viral trans-activator protein Tat. Tat functions through the TAR element, which is represented in both viral DNA and RNA. At present, there is no definitive evidence that determines whether Tat acts through a DNA or RNA form of TAR. We have used an intramolecular mutagenesis approach to change selectively the RNA secondary structure of TAR without affecting its primary sequence. We show that a specific RNA secondary structure for TAR is needed for biological activity. Furthermore, transcripts that only transiently form a native TAR RNA hairpin, which is not maintained in the mature mRNA, are completely trans-activated by Tat, suggesting that TAR is recognized as a nascent RNA.

Animals↗
PubMed1989

Reversal of natural killing susceptibility in target cells expressing transfected class I HLA genes.

A number of studies have suggested that resistance of target cells to natural killing (NK) may be correlated with their level of expression of major histocompatibility complex (MHC) class I antigens. To examine this hypothesis directly, a NK-sensitive class I-deficient human B-cell line was transfected with MHC class I genes. The expression of transfected HLA, but not H-2, class I gene products resulted in loss of susceptibility to human NK-mediated conjugation and cytolysis. Furthermore, this protection did not extend to cytotoxicity mediated by interleukin 2-stimulated human NK effector cells.

B-Lymphocytes↗
PubMed1985

Natural killer cell recognition of target cells expressing different antigens of vesicular stomatitis virus.

Natural killer (NK) cells have the capability of lysing virus-infected, transformed, and embryonal cells, yet the nature of the target structure(s) recognized remains unclear. The availability of well-characterized temperature-sensitive (ts) mutants of vesicular stomatitis virus, defective in expression of individual viral-encoded polypeptides at the nonpermissive temperature (39 degrees C), offered an approach to elucidating NK-cell recognition of virus-infected cells. Target cells were infected with ts mutants in three functions: the viral surface glycoprotein (G protein; ts 045); the matrix (M) protein (ts G31, ts G33), and the polymerase (ts G11). Cells infected with wild-type virus and all ts mutants at the permissive temperature (31 degrees C) were killed by murine spleen cells. Similar to results on cytotoxic T lymphocytes, target cells infected by ts 045 defective in expression of G protein at 39 degrees C were not killed by NK cells. Unexpectedly, cells infected at 39 degrees C with the M-protein mutants also were not killed, although G protein was expressed at the cell surface. Target binding studies indicated that conjugates were not formed by cells infected with the ts mutants at the nonpermissive temperature. That expression of G protein was not sufficient for NK cell-mediated cytotoxicity was established in experiments in which a plasmid (pSVGL) containing the gene for vesicular stomatitis virus G protein was transfected into COS cells. Although G antigen was expressed on the plasma membrane, the cells were not lysed. These results suggest either that recognition of virus-infected cells depends on an appropriate conformation imparted to the viral G protein by association with the M protein or that NK cells can recognize alterations in the structure of the cell membrane induced by insertion of viral M and G molecules.

Animals↗
PubMed1990

Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice.

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.

Animals↗
PubMed1992

Co-localized but target-unrelated expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons after peripheral nerve crush injury.

Expression of vasoactive intestinal polypeptide (VIP) and galanin in dorsal root ganglion (DRG) neurons is known to be induced by peripheral nerve injury. We investigated (1) whether VIP and galanin were co-expressed by DRG neurons and (2) whether such neurons innervated specified peripheral targets (visceral, cutaneous or muscular). An antibody to the 200 kDa neurofilament subunit (NF200) was used as a marker for large type-A cells in the DRG. VIP and galanin were respectively observed in 22% and 67% of DRG neurons at the L5 spinal level after crushing of the sciatic nerve. Most VIP-containing neurons were small type-B cells (about 90%) and approximately 95% of VIP-containing neurons also showed galanin-like immunoreactivity. Galanin was expressed by both large type-A and small type-B cells. Immunocytochemistry combined with a retrograde tracer revealed that about 70-80% of the small type-B cells in each sensory division displayed VIP-like immunoreactivity, and that most of the tracer-labeled neurons also expressed galanin. These findings suggest that the expression VIP and/or galanin in response to peripheral nerve crush injury is a property common to visceral, cutaneous and muscular sensory neurons.

Animals↗
PubMed1990

Generation of hen egg lysozyme-specific and major histocompatibility complex class I-restricted cytolytic T lymphocytes: recognition of cytosolic and secreted antigen expressed by transfected cells.

Syngeneic cells exogenously supplied with hen egg lysozyme (HEL) or endogenously synthesizing HEL were used as antigen-presenting cells to induce major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL). Immunization of C57BL/6 mice followed by repeated stimulation of their splenocytes in vitro with trypsinized HEL peptides led to the generation of CTL lines specific for trypsinized HEL peptides and restricted by H-2K. Immunization of C3H mice with a mixture of soluble native HEL and irradiated syngeneic spleen cells followed by in vitro stimulation of immune spleen cells with soluble HEL could in a few cases result in HEL-specific CTL able to kill syngeneic transfectant L cells secreting HEL (HELs) or expressing cytosol-targeted HEL (HELc). The use of HELs or HELc transfectant L cells as in vivo and in vitro immunogens was a potent way for eliciting HEL-specific polyclonal CTL. These CTL and two CD8+ clones were found to be H-2K restricted and specific for the 1-17 N-terminal HEL peptide. In addition, the anti-HEL CTL could also exhibit a significant cross-reactivity against unsensitized and HEL-untransfected targets expressing the K restriction element. This cross-reactivity was likely due to recognition of unidentified HEL mimicking peptides (self-derived?) presented by the MHC class I (H-2K or H-2K) molecule used as the restriction element for the specific recognition of HEL. The CTL raised after immunization with HELs or HELc transfectant cells were found to recognize both the HELs and HELc transfectant cells even though HEL was not detected in the latter after a 2- or 5-min radiolabeling pulse. Recognition of both HELs and HELc transfectant cells by a given CTL clone suggests that HEL subjected to two separate processing pathways, each depending on the initial subcellular localization, can ensure the generation of similar MHC class I peptide complexes.

Animals↗
PubMed1990

Expression and targeting of intracellular antibodies in mammalian cells.

Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm and led us to ask whether antibodies could be further targeted to the nucleus. Mutations were therefore made in which the leader sequence of the light chain was replaced by the nuclear localization signal of the SV40 large T antigen. Transfectants in which the heavy chain lacking the hydrophobic leader was expressed together with a light chain carrying the nuclear localization signal were selected and a nuclear distribution of the assembled antibody was found. Thus, it should prove possible to target a specific antibody to the cell nucleus with the aim of interfering with the function of a nuclear antigen.

Amino Acid Sequence↗
PubMed1988

AK cells were developed from NK cells during in vitro culture of allogeneic or F1 anti-parental stimulation: functional conversion in recognizing H-2 expression of target cells accompanied by phenotypical conversion.

In vitro sensitization of (CBA x A)F1 spleen cells for 3 days with allogeneic C57BL cells raised the killer activity to the NK-sensitive YAC-1 target. When (A x C57BL)F1 spleen cells were cultured with parental C57BL cells, the lytic activity to YAC-1, P815 and EL-4 targets occurred on Day 6 after the culture. Phenotypical analyses showed that these culture-activated killer (AK) cells were derived from asialo-GM1+Thy-1-NK cells; however, they expressed Thy-1 antigen but not asialo-GM1 antigen at the effector cell level. Generation of the AK cells was not evident in cultures of spleen cells from mice with a neonatally induced tolerance to stimulator antigen and in those from T-cell-depleted mice. The supernatant of allostimulated culture, which contained a low concentration of IL-2, rendered the above cells capable of evoking AK activity. The H-2-reduced target cells were sensitive to NK cells, but less sensitive to AK cells; on the contrary, the H-2 highly expressed cells (interferon-treated cells) were less susceptible to NK cells but highly susceptible to AK cells. Thus, the relation between NK susceptibility and susceptibility to AK cells is inverse. Our study shows that stimulation with lymphokines causes a functional conversion accompanied by a phenotypical conversion of NK cells. With reference to immunosurveillance, these observations lead to the idea that NK and AK cells represent two functionally distinct but complementary systems involved in cell-mediated immunosurveillance.

Animals↗
PubMed2026

PGC1α expression using targeted redox-responsive nanogels protects against prostate cancer in vivo.

Prostate cancer is among the most frequently diagnosed cancers in men in the UK and US. Increasing evidence implicates metabolic dysregulation as a critical driver of disease progression. Central to this process is peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) that promotes oxidative metabolism and mitochondrial biogenesis while inhibiting metastatic programs. This work investigated the therapeutic potential of PGC1α overexpression via mRNA delivery. Here, we report a prostate-specific, targeted disulphide-crosslinked nanogel system for intracellular delivery of mRNA encoding the N-terminal isoform of PGC1α (NT-PGC1α). Functionalization of the NGs with a peptide targeting prostate-specific membrane antigen (PSMA) enabled selective delivery of NT-PGC1α mRNA in PCa cells and 3D spheroid models. We confirmed sustained PGC1α expression, and increased mitochondrial protein content, indicative of enhanced mitochondrial biogenesis. These nanogels, which were prepared in situ using a nanopolymerization technique, exhibited high mRNA loading capacity, low cytotoxicity, and redox-responsive cargo release, enabling controlled cytosolic delivery following intracellular glutathione-mediated degradation. In vivo, systemic administration of the PSMA-targeted NT-PGC1α mRNA-loaded nanogels resulted in tumor-preferential accumulation and significant suppression of xenograft growth (by 73.4% relative to untreated control), with minimal systemic toxicity. This study presents the first example of a prostate-targeted, disulfide-crosslinked nanogel system for mRNA-mediated metabolic reprogramming in prostate cancer, and highlights its promise as a platform for future RNA-based targeted and precision stimuli-responsive cancer therapies.

Male↗
PubMed2016

Matrix stiffness promotes cartilage endplate chondrocyte calcification in disc degeneration via miR-20a targeting ANKH expression.

The mechanical environment is crucial for intervertebral disc degeneration (IDD). However, the mechanisms underlying the regulation of cartilage endplate (CEP) calcification by altered matrix stiffness remain unclear. In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Through a dual luciferase reporter assay, we confirmed that miR-20a directly targets 3'-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP. Thus, our findings demonstrate that the miR-20a/ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD.

3' Untranslated Regions↗
PubMed1990

Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.

Adenoviruses, Human↗
PubMed2026

Expression patterns of potential targets for antibody-directed therapy in metastatic castration-resistant prostate cancer patients.

INTRODUCTION: Survival in metastatic castration-resistant prostate cancer (mCRPC) patients remains limited and treatment is complicated by tumor heterogeneity. As antibody-based therapeutics emerge, identifying actionable antigen targets and patient subgroups most likely to benefit is essential. MATERIALS & METHODS: Gene expression of 62 antibody-targetable proteins was analyzed in 296 mCRPC biopsies. These genes encode proteins targeted by approved or investigational antibody-based cancer therapeutics. Associations between target expression with genomic classifications and transcriptomic subtypes were evaluated. Target expression was also assessed in tumors with low expression of established mCRPC targets. Subgroup-specific targets were validated in an independent cohort and single-cell transcriptomics. RESULTS: Established targets KLK2, FOLH1 (PSMA) and STEAP1 showed the highest median expression across the cohort. Target expression did not correlate with genomic classifications, including homologous recombination deficiency, microsatellite instability, CDK12, TP53, PTEN or AR alterations Target expression did associate with transcriptomic subtypes: CRPC-AR (driven by androgen receptor-signaling) and CRPC-SCL (stem cell-like features, AP-1/YAP/TAZ-driven), displayed the highest expression of multiple targets, including KLK2, FOLH1, and SLC44A4. CRPC-NE (neuroendocrine phenotype) showed heterogeneous expression, with high CD46 expression, whereas CRPC-WNT (Wnt-signaling driven) generally showed low target expression. Notably, CD46 was highly expressed in tumors with low KLK2, FOLH1, and STEAP1 expression, a subgroup associated with poor prognosis. CONCLUSIONS: Although several antibody targets showed broad expression in mCRPC-tumors, expression varied by transcriptomic subtype. Subgroups such as CRPC-WNT expressed fewer targets, suggesting the need for alternative therapeutic strategies. CD46 emerged as a promising target, with wide expression across multiple subtypes, including clinically challenging CRPC-NE and mCRPC tumors lacking expression of established targets.

Humans↗
PubMed1990

Retrovirus vector-targeted inducible expression of human beta-interferon gene to B-cells.

We have introduced the human beta-interferon gene with its promoter region into murine B-cell and fibroblast cell lines via a Moloney murine leukemia virus (M-MuLV) vector and have studied the inducible expression of the beta-interferon gene as a function of the various retroviral vector designs. By deleting the enhancer within the 3' viral long terminal repeat (LTR), inserting the human beta-interferon gene, and varying placement of the immunoglobulin heavy chain enhancer, we were able to construct vectors which yielded proviruses with various cell type-specific regulation. One of the vectors (pT154) led to a greater than 21-fold increase in beta-interferon protein synthesis after viral infection in the two B-cell lines analyzed, while no inducibility was seen in the fibroblast cells. The data show that inducible beta-interferon expression within a MuLV vector was highly dependent on the absence of the viral enhancer region in the long terminal repeat and the orientation of the beta-interferon gene within the proviral transcriptional unit; the insertion of the immunoglobulin enhancer elevated both constitutive and (or) inducible expression of beta-interferon in B-cells but inhibited constitutive expression of this gene in fibroblasts.

Animals↗
PubMed1981

Noncodominant expression of target antigens recognized by human cytotoxic T lymphocytes.

Human CTL that recognized MHC-controlled determinants distinct from HLA-A. B, C, and D/DR antigens were tested in a family with nine siblings. Segregation analysis of positive CML reactions showed strong lysis of target cells of three HLA-identical siblings, inheriting the b and c MHC haplotypes (blc), but not of the parents or siblings inheriting only one of these haplotypes. Some cytotoxicity was seen against parental target cells, although it seemed to be qualitatively distinct from that directed against the b/c targets. Studies using the competitive inhibition technique showed that cells inheriting only the b or c haplotypes were not effective in decreasing the specific cytotoxicity. Furthermore, simultaneous inclusion of inhibitor cells of both the b and c haplotypes did not hinder the specific cytotoxicity. Induction of CTL recognizing this new determinant did not occur when either antigens of the b or c haplotypes were used as MLC stimulating cells, or when antigens of both haplotypes were presented simultaneously, but on separate stimulating cells, in a three-cell MLC. These results suggest that the target determinant recognized by these unusual CTL is complex; it may be formed through interactions of two surface molecules or by genetic complementation yielding a hybrid antigen.

Binding, Competitive↗