RESUMEN
ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non-small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).
Asunto(s)
Inmunoconjugados , Proteínas ADAM , Línea Celular Tumoral , Xenoinjertos , Humanos , Inmunoconjugados/química , Proteínas de la Membrana/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.
Asunto(s)
Antígenos B7/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoconjugados/farmacología , Neoplasias/tratamiento farmacológico , Animales , Antígenos B7/genética , Antígenos B7/metabolismo , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas , Técnicas de Silenciamiento del Gen , Humanos , Inhibidores de Puntos de Control Inmunológico/química , Inhibidores de Puntos de Control Inmunológico/aislamiento & purificación , Inmunoconjugados/química , Inmunoconjugados/aislamiento & purificación , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multispecific antibodies bind two or more different antigens and enable new therapeutic applications that cannot be replicated with conventional monoclonal antibodies, such as bridging different cells or bringing soluble proteins in close proximity. The DART and TRIDENT platforms enable the engineering of such antibodies. A DART molecule combines two independent antigen-binding sites in a stabilized, diabody-like structure. A DART molecule can be expressed with or without an Fc domain and thus can be tailored to have a long or short half-life in vivo and to induce or ablate effector function. Linking two DART units or a DART unit and a Fab domain (the latter structure is called TRIDENT format) via an Fc domain creates a monospecific, bispecific, trispecific, or tetraspecific molecule with up to tetravalent targeting of antigens. This article focuses on the design of DART and TRIDENT molecules that target two or three different antigens. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Design and generation of expression plasmids encoding DART and TRIDENT molecules Basic Protocol 2: Expression of DART and TRIDENT molecules by transient transfection of CHO cells Basic Protocol 3: Purification of DART and TRIDENT molecules from CHO cell supernatants.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Ingeniería Genética/métodos , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos/genética , Células CHO , Técnicas de Cultivo de Célula , Cricetulus , Humanos , Fragmentos Fc de Inmunoglobulinas/genéticaRESUMEN
PURPOSE: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo. PROCEDURES: The transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²5I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²4I- or 64Cu-scFv-Fc as tracers. RESULTS: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice. CONCLUSIONS: The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.
Asunto(s)
Antígeno Carcinoembrionario/genética , Endocitosis , Genes Reporteros/genética , Imagen Molecular/métodos , Animales , Antígeno Carcinoembrionario/metabolismo , Femenino , Citometría de Flujo , Humanos , Fragmentos de Inmunoglobulinas , Inmunohistoquímica , Células Jurkat , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones , Proteínas Recombinantes/metabolismo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Linfocitos B/patología , Linfoma de Burkitt/terapia , Región Variable de Inmunoglobulina/inmunología , Leucocitos Mononucleares/inmunología , Linfoma de Células B/patología , Receptores de IgG/inmunología , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/farmacocinética , Linfocitos B/inmunología , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Proteínas Ligadas a GPI , Humanos , Linfoma de Células B/inmunología , Ratones , Ratones Desnudos , Ratones Transgénicos , Trasplante de Neoplasias , Estabilidad Proteica , Receptores de IgG/genética , Trasplante HeterólogoRESUMEN
The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.
Asunto(s)
Anticuerpos/química , Biomarcadores de Tumor/análisis , Inmunoconjugados/química , Neoplasias/diagnóstico , Puntos Cuánticos , Animales , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Compuestos de Cadmio/química , Línea Celular Tumoral , Humanos , Inmunoconjugados/inmunología , Luminiscencia , Ratones , Polietilenglicoles/química , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Compuestos de Selenio/química , Sulfuros/química , Compuestos de Zinc/químicaRESUMEN
Hepatitis C virus (HCV) causes chronic infection in humans leading to liver cirrhosis and hepatocellular carcinoma. rRNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis, and changes in Pol I transcription rate are associated with profound alterations in the growth rate of the cell. Because rRNA synthesis is intimately linked to cell growth and frequently up-regulated in many cancers, we hypothesized that HCV might have the ability to activate rRNA synthesis in infected cells. We show here that rRNA promoter-mediated transcription is significantly (10- to 12-fold) activated in human liver-derived cells following infection with type 2 JFH-1 HCV or transfection with the subgenomic type 1 HCV replicon. Further analysis revealed that HCV nonstructural protein 5A (NS5A) was responsible for activation of rRNA transcription. Both the NH(2)-terminal amphipathic helix and the polyproline motifs of NS5A seem to be essential for rRNA transcription activation. The NS5A-dependent activation of rRNA transcription seems to be due to hyperphosphorylation and consequent activation of upstream binding factor (UBF), a Pol I DNA binding transcription factor. We further show that hyperphosphorylation of UBF occurs as a result of up-regulation of both cyclin D1 and cyclin-dependent kinase 4 by the HCV NS5A polypeptide. These results suggest that the endoplasmic reticulum-associated NS5A is able to transduce signals into the nucleoplasm via UBF hyperphosphorylation leading to rRNA transcription activation. These results could, at least in part, explain a mechanism by which HCV contributes to transformation of liver cells.
Asunto(s)
Ciclina D1/fisiología , Hepacivirus/fisiología , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Ribosómico/biosíntesis , Transcripción Genética , Células Cultivadas , Ciclina D1/análisis , Quinasa 4 Dependiente de la Ciclina/análisis , ADN Polimerasa I/genética , Humanos , Fosforilación , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/fisiologíaRESUMEN
PURPOSE: Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. METHODS: To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human Fc gamma RIIb receptor. The NA3-Fc gamma RIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. RESULTS: Flow cytometry identified Jurkat clones stably expressing NA3-Fc gamma RIIb at low, medium, and high levels. High and medium NA3-Fc gamma RIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA (124)I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 +/- 0.2, 15.2 +/- 1.3, and 4.6 +/- 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 +/- 0.8%ID/g. CONCLUSION: The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo.
Asunto(s)
Antígeno Carcinoembrionario/análisis , Antígeno Carcinoembrionario/genética , Genes Reporteros , Proteínas Recombinantes/genética , Animales , Antígeno Carcinoembrionario/inmunología , Línea Celular , Proteínas Ligadas a GPI , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Isoantígenos/genética , Ratones , Neoplasias/diagnóstico por imagen , Neoplasias/genética , Neoplasias/metabolismo , Tomografía de Emisión de Positrones , Receptores de IgG/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tomografía Computarizada por Rayos XRESUMEN
Small, engineered antibody fragments such as diabodies (50 kDa noncovalent dimers of single-chain Fv fragments) are useful alternatives to their larger antibody counterparts. However, due to their size, they are more susceptible to disruption of their antigen binding sites when modified using random conjugation techniques. Previous work has demonstrated the utility of a C-terminal cysteine modification for site-specific radiolabeling of an anti-CEA diabody, resulting in the creation of a cys-diabody (CysDb). In the present work, the adaptability of the CysDb system was explored by creating two additional CysDbs: one specific for CD20 and one for HER2. Purified CysDbs of both specificities demonstrated behavior consistent with stable, covalent dimers harboring a readily reducible disulfide bond. Each CysDb was site-specifically conjugated to three different fluorophores for optical detection: the large fluorescent proteins phycoerythrin (PE) and allophycocyanin (APC), and the small fluorescent molecule Alexa Fluor488. Fluorophore-conjugated CysDbs bound specifically to their targets in both antigen systems and with each different fluorescent tag as determined by flow cytometry. In vitro specific antigen binding was observed in the presence of a mixture of specific and nonspecifically conjugated CysDbs. Conjugates retained both specificity and fluorescence, demonstrating the successful expansion of the CysDb repertoire to new targets and to new site-specific conjugation possibilities.
Asunto(s)
Antígenos CD20/inmunología , Cisteína/metabolismo , Colorantes Fluorescentes/química , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/metabolismo , Receptor ErbB-2/inmunología , Compuestos de Sulfhidrilo/química , Animales , Sitios de Unión , Línea Celular Tumoral , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Sensibilidad y EspecificidadRESUMEN
Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immunohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Biotinilación/métodos , Ligasas de Carbono-Nitrógeno/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Anticuerpos Monoclonales/genética , Ligasas de Carbono-Nitrógeno/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor. This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP. The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared. It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.
Asunto(s)
Kluyveromyces/enzimología , UDPglucosa 4-Epimerasa/química , Secuencia de Aminoácidos , Catálisis , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Dimerización , Cinética , Kluyveromyces/crecimiento & desarrollo , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Desnaturalización Proteica , Espectrometría de Fluorescencia , Especificidad por Sustrato , UDPglucosa 4-Epimerasa/antagonistas & inhibidores , UDPglucosa 4-Epimerasa/metabolismo , Urea/química , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Monofosfato/metabolismoRESUMEN
A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.
Asunto(s)
Hepacivirus/genética , Biosíntesis de Proteínas/genética , Virus ARN/genética , Ribosomas/fisiología , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Humanos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribosomas/genética , Antígeno SS-BRESUMEN
La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal "La motif" which is required for efficient interaction of La with IRNA and viral 5' untranslated region (5'-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5'-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5'-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.