Clinical landscape of LAG-3-targeted therapy.

Lymphocyte-activated gene 3 (LAG-3) is a cell surface inhibitory receptor and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. LAG-3 is considered a next-generation immune checkpoint of clinical importance, right next to programmed cell death protein 1 (PD-1) and cytotoxic T-cell lymphocyte antigen-4 (CTLA-4). Indeed, it is the third inhibitory receptor to be exploited in human anticancer immunotherapies. Several LAG-3-antagonistic immunotherapies are being evaluated at various stages of preclinical and clinical development. In addition, combination therapies blocking LAG-3 together with other immune checkpoints are also being evaluated at preclinical and clinical levels. Indeed, the co-blockade of LAG-3 with PD-1 is demonstrating encouraging results. A new generation of bispecific PD-1/LAG-3-blocking agents have also shown strong capacities to specifically target PD-1+ LAG-3+ highly dysfunctional T cells and enhance their proliferation and effector activities. Here we identify and classify preclinical and clinical trials conducted involving LAG-3 as a target through an extensive bibliographic research. The current understanding of LAG-3 clinical applications is summarized, and most of the publically available data up to date regarding LAG-3-targeted therapy preclinical and clinical research and development are reviewed and discussed.

[Dendritic cells in cancer immunotherapy]. / Inmunoterapia genética con células dendríticas para el tratamiento del cáncer.

Since the beginning of the 20th century, biomedical scientists have tried to take advantage of the natural anti-cancer activities of the immune system. However, all the scientific and medical efforts dedicated to this have not resulted in the expected success. In fact, classical antineoplastic treatments such as surgery, radio and chemotherapy are still first line treatments. Even so, there is a quantity of experimental evidence demonstrating that cancer cells are immunogenic. However, the effective activation of anti-cancer T cell responses closely depends on an efficient antigen presentation carried out by professional antigen presenting cells such as DC. Although there are a number of strategies to strengthen antigen presentation by DC, anti-cancer immunotherapy is not as effective as we would expect according to preclinical data accumulated in recent decades. We do not aim to make an exhaustive review of DC immunotherapy here, which is an extensive research subject already dealt with in many specialised reviews. Instead, we present the experimental approaches undertaken by our group over the last decade, by modifying DC to improve their anti-tumour capacities.

Inmunoterapia genética con células dendríticas para el tratamiento del cáncer / Dendritic cells in cancer immunotherapy

Desde comienzos del siglo XX, los científicos han intentado aprovechar las actividades naturales del sistema inmunológico para curar el cáncer. Sin embargo, las inmunoterapias no han dado el resultado clínico que podría haberse esperado. De hecho, lo tratamientos anti-neoplásicos clásicos como la cirugía, la radioterapia y la quimioterapia siguen consistiendo en la primera línea de tratamiento. Aun así, existe un gran número de evidencias experimentales sobre la inmunogenicidad de las células cancerosas. Sin embargo, la activación efectiva de las respuestas T anti-cancerosas depende estrechamente de la presentación eficiente de antígenos tumorales por parte de células presentadoras de antígeno profesionales, como las células dendríticas (dendritic cells, DC). Aunque se han desarrollado un gran número de estrategias para reforzar las funciones de presentación de antígeno de las DC, la inmunoterapia como tratamiento anti-neoplásico todavía no es tan efectiva como esperaríamos de acuerdo con los resultados obtenidos en modelos preclínicos durante las últimas décadas. En este trabajo no pretendemos revisar exhaustivamente la inmunoterapia con DC, un campo ampliamente extenso y tratado en otras revisiones especializadas. Aquí se exponen la experiencias que nuestro grupo ha llevado a cabo durante la última década modificando genéticamente a las DC para mejorar su eficacia anti-tumoral (AU)

Since the beginning of the 20th century, biomedical scientists have tried to take advantage of the natural anti-cancer activities of the immune system. However, all the scientific and medical efforts dedicated to this have not resulted in the expected success. In fact, classical antineoplastic treatments such as surgery, radio and chemotherapy are still first line treatments. Even so, there is a quantity of experimental evidence demonstrating that cancer cells are immunogenic. However, the effective activation of anti-cancer T cell responses closely depends on an efficient antigen presentation carried out by professional antigen presenting cells such as DC. Although there are a number of strategies to strengthen antigen presentation by DC, anti-cancer immunotherapy is not as effective as we would expect according to preclinical data accumulated in recent decades. We do not aim to make an exhaustive review of DC immunotherapy here, which is an extensive research subject already dealt with in many specialised reviews. Instead, we present the experimental approaches undertaken by our group over the last decade, by modifying DC to improve their antitumour capacities (AU)

Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder.

Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (T(m)) of 39.3+/-1.0 and 37.1+/-0.7 degrees C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 degrees C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (Delta T(m max)=8 degrees C, half-maximal effective ligand concentration, AC(50)=3 microM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in T(m) of approximately 16 degrees C, followed by MeCbl at approximately 13 degrees C, each evaluated at 50 microM cofactor. The other cobalamins stabilized in the order (CN)(2)Cbi>OHCbl>CNCbl. Interestingly, the AC(50)'s for AdoCbl, MeCbl, (CN)(2)Cbi and OHCbl were similar and ranged from 1-3 microM, which compares well with the K(d) of 6 microM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (Delta T(m max)=4 degrees C). The dose-response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC(50) of 7 microM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC(50) for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.

Formation of Pseudorabies virus glycoprotein E/I complex in baculovirus recombinant system.

Glycoproteins E (gE) and I (gI) of Pseudorabies virus (PRV) form a non-covalently bound complex to which a number of functions have been attributed. The gE/gI complex formation was studied using a series of full-length and truncated forms of gE and gI expressed in baculovirus recombinant system. Both glycoproteins were truncated by stepwise removal of their C-terminal parts and their ability to form the complex was studied by radioimmunoprecipitation. It was found that N-terminal domains of gE and gI containing first 122 and 106 aa, respectively, were sufficient for the complex formation.

Characterization of the RNA-binding activity of VP3, a major structural protein of Infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is the agent of an immune-depressive disease affecting the poultry industry worldwide. Infection of IBDV leads to expression of five mature virus-encoded proteins. Proteolytic processing of the virus-encoded polyprotein generates VP3 which coats the inner surface of the IBDV capsid. In this report, we describe the characterization of the RNA-binding activity of VP3. For these studies, the VP3 coding region was fused to a histidine tag and expressed in insect cells using a recombinant baculovirus. The histidine-tagged VP3 was affinity-purified and used to study its ability to bind RNA molecules using three complementary methods: (i) Northwestern blotting; (ii) binding of VP3 protein-RNA complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. The results demonstrated that VP3 efficiently bound ssRNA and dsRNA. Under the experimental conditions used in this study, the formation of VP3-RNA complexes did not depend upon the presence of specific RNA sequences. A series of histidine-tagged VP3 deletion mutants spanning the whole VP3 coding region were generated. The use of these mutants revealed that the VP3 RNA-binding domain layed in a highly conserved 69 aa stretch close to the N-terminus of the protein.

An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs.

Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.

[Expression of genes encoding animal herpesvirus glycoproteins in baculovirus]. / Ekspresja genów kodujacych glikoproteiny herpeswirusów zwierzecych w systemie bakulowirusowym.

Baculovirus system has been used for the expression of genes of herpesvirus glycoproteins. In this system highly glycosylated proteins are obtained. The glycoproteins may find potential use as vaccines and in diagnosis of pseudorabies.

Differentiation of Aujeszky's disease virus strains isolated in Poland using DNA biotinylated probes.

The aim of this study was to compare 17 different Aujeszky's disease virus (ADV) isolates from clinical outbreaks of AD by using DNA biotinylated probes. All isolates were collected in Poland between 1984 and 1991. The restriction fragment pattern (RFP) analysis done by hybridization to NIA-3 DNA biotinylated probe indicated that all Polish ADV field strains can be classified as type I of Suid herpesvirus 1. Hybridization with BamHI fragment 7 and gI gene biotinylated probes revealed an unusual heterogeneity of BamHI fragment 7 in almost 50% of strains isolated in Poland. The nature of the molecular changes in this fragment will be discussed.