A first-in-human study of the anti-LAG-3 antibody favezelimab plus pembrolizumab in previously treated, advanced microsatellite stable colorectal cancer.

BACKGROUND: Treatment options are limited for participants with microsatellite stable (MSS) metastatic colorectal cancer (mCRC) that progressed after two or more prior therapies. Studies have shown that blockade of both lymphocyte-activation gene 3 (LAG-3) and programmed cell death protein 1 (PD-1) can improve antitumor activity. Here, we evaluate the antitumor activity of the LAG-3 antibody favezelimab alone or in combination with pembrolizumab in participants with MSS mCRC. PATIENTS AND METHODS: Eligible participants with MSS PD-1/programmed death-ligand 1 (PD-L1) treatment-naive mCRC that progressed on two or more prior therapies received 800 mg favezelimab, 800 mg favezelimab plus 200 mg pembrolizumab, or 800 mg favezelimab/200 mg pembrolizumab co-formulation, every 3 weeks. The primary endpoint was safety, the secondary endpoint was objective response rate (ORR), and exploratory endpoints included duration of response, progression-free survival (PFS), and overall survival (OS). RESULTS: At the data cut-off date of 23 October 2020, a total of 20 participants received favezelimab alone, 89 received favezelimab plus pembrolizumab (including as favezelimab/pembrolizumab co-formulation); 48 had PD-L1 combined positive score (CPS) ≥1 tumors. At this interim analysis median follow-up was 5.8 months with favezelimab and 6.2 with favezelimab plus pembrolizumab. Treatment-related adverse events (TRAEs) were 65% with favezelimab and 65.2% with favezelimab plus pembrolizumab. Grade ≥3 TRAEs were 15% with favezelimab and 20% with favezelimab plus pembrolizumab. No grade 5 TRAEs occurred. Common TRAEs (≥15%) included fatigue (20.0%), nausea (15.0%) with favezelimab, and fatigue (16.9%) with favezelimab plus pembrolizumab. Confirmed ORR was 6.3% with favezelimab plus pembrolizumab, with median duration of response of 10.6 months (range 5.6-12.7 months), median OS of 8.3 months (95% confidence interval 5.5-12.9 months), and median PFS of 2.1 months (1.9-2.2 months). In an exploratory analysis of PD-L1 CPS ≥1 tumors, the confirmed ORR was 11.1%, median OS was 12.7 months (4.5 to not reached), and median PFS was 2.2 months (1.8-4.2 months) with favezelimab plus pembrolizumab. CONCLUSIONS: Favezelimab with or without pembrolizumab had a manageable safety profile, with no treatment-related deaths. Promising antitumor activity was observed with combination therapy, particularly in participants with PD-L1 CPS ≥1 tumors.

Assessing the Impact of Tissue Target Concentration Data on Uncertainty in In Vivo Target Coverage Predictions.

Understanding pharmacological target coverage is fundamental in drug discovery and development as it helps establish a sequence of research activities, from laboratory objectives to clinical doses. To this end, we evaluated the impact of tissue target concentration data on the level of confidence in tissue coverage predictions using a site of action (SoA) model for antibodies. By fitting the model to increasing amounts of synthetic tissue data and comparing the uncertainty in SoA coverage predictions, we confirmed that, in general, uncertainty decreases with longitudinal tissue data. Furthermore, a global sensitivity analysis showed that coverage is sensitive to experimentally identifiable parameters, such as baseline target concentration in plasma and target turnover half-life and fixing them reduces uncertainty in coverage predictions. Overall, our computational analysis indicates that measurement of baseline tissue target concentration reduces the uncertainty in coverage predictions and identifies target-related parameters that greatly impact the confidence in coverage predictions.

A novel pathway for the conversion of homocysteine to methionine in eukaryotes.

Activation of amino acid homocysteine was compared with that of methionine in rabbit crude liver extracts and purified multi-enzyme complex of aminoacyl-tRNA synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacetic-acid insoluble materials in the absence of protein synthesis. Homocysteine synthetase activity was found in the crude extract and in the purified multi-enzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five times higher than the activation of homocysteine. There was a partial loss of Hcy-tRNA synthetase activity in the purified multi-enzyme complex. Preliminary reconstitution experiments indicated a requirement for an additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [14C]homocysteine, revealed radioactivity in homocysteine, methionine and homocysteine thiolactone, indicating a conversion of tRNA-attached homocysteine to methionine. Total tRNA was separated on a benzoylated cellulose column into a fraction enriched in initiator tRNA and a methionine-accepting, but initiator tRNA-deficient, fraction. Homocysteine-accepting activity was present only in the initiator tRNA-enriched fraction. Based on the above data we propose that homocysteine activation in reticulocyte lysates, reported previously, also occurs in liver. Activated homocysteine is attached to initiator tRNA and then converted to methionine by a methylating enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.

Kinetics of ribosomal protein S6 phosphorylation by HepG-2 cells in response to insulin.

The effect of insulin on the phosphorylation of ribosomal protein S6 was studied in a human liver cell line (HepG-2), using [32P] inorganic phosphate. Increased rate of protein S6 phosphorylation was detected 8 min following the addition of insulin to serum starved cells. Maximum enhancement of phosphorylation was observed at 80 nM insulin. Minimum level of insulin required to produce measurable increase of S6 phosphorylation was 20 nM. Radioactivity of protein S6 increased most in the native subunit and polysome fractions. Significant increase in radioactivity of this protein was not observed in the monosome fraction during the first 30 min of insulin stimulation. Increase in the specific radioactivity of native 40S subunit was higher than that of polysomes. These results suggest that phosphorylation takes place in the subunit compartment and moves preferentially into the polysomes.

A precaution when preparing very large plasmids by alkaline lysis procedure.

The extractabilities of plasmids of different sizes by the sodium lauryl sulfate (SDS)-alkali procedure were compared using either sodium acetate or potassium acetate buffer as the neutralizing agent. There was a selective loss of large plasmids (above 100 kb) when the potassium salt was used. When N-lauryl sarcosine instead of SDS was used as the detergent, no loss of large plasmids occurred in the presence of potassium salt. A comparison of the kinetics of precipitate formation with sodium acetate and potassium acetate indicated that the rate and the amount of lauryl sulfate precipitated were lower with the sodium salt. It is suggested that faster precipitation of lauryl sulfate with potassium acetate leads to trapping of large denatured plasmids that cannot renature as fast as the small ones.

Translation of globin RNA spliced in vitro.

Pre-mRNA (precursor mRNA) enriched for globin-gene transcription products was prepared from murine erythroleukaemia cells induced to differentiate with dimethyl sulphoxide. The pre-mRNA was prepared from nuclear RNA by oligo(dT)-cellulose chromatography followed by sucrose-gradient centrifugation. The pre-mRNA thus obtained was utilized as the substrate for a splicing assay based on sequential splicing and translation reactions. Small nuclear ribonucleoprotein particles, which were shown to inhibit translation, were removed by an intermediate high-speed centrifugation step. The assay developed is based on a three-step procedure: (1) incubation of pre-mRNA with HeLa-cell nuclear extract in the presence of ATP; (2) incorporation of 3H-labelled amino acid into proteins by wheat-germ extract by using RNA from step 1; and (3) immunoprecipitation of globin chains with rabbit anti-(mouse globin) antibody. No incorporation of label into immune complexes was observed in the absence of HeLa-cell extract or ATP. Preincubation with these components was unnecessary for the incorporation of label into immune complexes when mature cytoplasmic RNA was used as a template for protein synthesis. The assay was further validated by fluorography after polyacrylamide-gel electrophoresis, when the only band detectable corresponded to globin chains. These results demonstrate, for the first time, translational activity of transcripts spliced in vitro. The current assay can be utilized with a few micrograms of pre-mRNA and may have potential for further characterization of the splicing system.

Total occlusion of the left main coronary artery in chronic stable angina pectoris.

We present the clinical and angiographic profile of three patients with class I stable angina pectoris. All had strong coronary risk factors, and stress testing was positive in stage one of the Bruce protocol. Coronary angiography revealed total occlusion of the left main coronary artery (LMCA), and aortocoronary bypass surgery was performed. Thus, total LMCA occlusion may be an unexpected angiographic finding in patients with class I angina.

Quadrivalvular rheumatic heart disease.

Three cases of chronic rheumatic heart disease with involvement of all four valves are presented. The involvement of tricuspid and pulmonary valves was suspected clinically and was confirmed by two-dimensional echo, Doppler, hemodynamic and angiographic findings. These findings were also verified surgically and histopathologically in 2 cases. One of the cases died after cardiac catheterization; the other 2 cases were treated surgically with success.

Supernumerary right coronary artery.

A rare case of a patient with supernumerary right coronary artery in whom the two vessels arose from the right coronary sinus from two separate ostia adjacent to each other is presented. The smaller vessel gave off the sinoatrial nodal branch and the posterior descending artery whereas the larger one gave off the conus branch, the right ventricular branches, and continued as acute marginal branch. This is the first case report in the English literature.

Pulmonary atresia with ventricular septal defect in adult patients.

Hemodynamic data and angiograms of 15 adult patients with pulmonary atresia and ventricular septal defect were reviewed to assess the pulmonary circulation and other associated features. The most common variety of pulmonary atresia was that of pulmonary valve, main pulmonary artery, and the confluence of pulmonary arteries (6 cases, 40%). The collateral vessels to the lungs were well developed in all cases; selective injections into the collateral vessels were of great value in their delineation. The left ventricle was well developed in 11 cases (73.3%). Congestive heart failure was seen in 8 (53.3%), tricuspid regurgitation in 10 (66.7%), and aortic regurgitation in 7 (46.7%) cases. The long survival in these patients was related to the favorable anatomy of central pulmonary arteries (12 cases, 80%) and adequate pulmonary collateral circulation.

Regulation of pancreatic islet cell replication: an inquiry into the controversy regarding the effects of steroid hormones.

The controversial issue of the effects of prednisolone and 17 beta-estradiol on replication of fetal rat pancreatic islets in culture was studied using 32P and [3H]thymidine as probes for studying DNA synthesis. DNA synthesis was not affected by the steroid hormones, as was evident from the rate of incorporation of 32P into total DNA. Decreased incorporation of [3H]thymidine into DNA found in islets treated with either of these steroids seemed to reflect an inhibitory effect of these hormones on thymidine kinase, leading to decreased phosphorylation of labeled thymidine. In addition, the hormones stimulated the activity of thymidylate synthetase, thus enhancing the endogenous synthesis of thymidine and thereby diluting the specific activity of the [3H]thymidine added to the cultured islets. Further support for a lack of inhibition of growth of islet cells treated with steroid hormones was provided by the observation that prednisolone increased uridine kinase activity and RNA biosynthesis, both of which may participate in the growth of cells preceding mitosis and (the latter) in protein hormone biosynthesis.

Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin.

Binding of guanosine nucleotides to purified native and ADP-ribosylated wheat germ EF-2 was measured. Both forms of EF-2 bound [3H]GDP to the same extent. [3H]GDP binding to native but not to ADP-ribosylated EF-2 was reduced in the presence of GTP and ribosomes. Binding of [gamma-32P]GTP to EF-2 was significantly reduced upon ADP-ribosylation. ADP-ribosylation almost abolished both the stimulatory effect of ribosomes on GTP binding to EF-2 and the ability of EF-2 to form a high-affinity complex with GuoPP(CH2)P and ribosomes. Low-affinity complex formation between EF-2 X GDP and ribosomes was not influenced by ADP-ribosylation. The results indicate that the inhibition of the elongation process caused by the toxin is probably due to the inability of modified EF-2 to exchange GDP with GTP.

Culture negative infective endocarditis.

Twenty cases of culture negative infective endocarditis admitted to the Cardiology Department of Green Lane Hospital from 1959 to 1980 out of a total of 265 cases (7.5%), were analysed retrospectively. Cases were included only when adequate proof of endocarditis was available at surgery or postmortem. Indiscriminate use of antibiotics before taking blood cultures was the most common association with failure to obtain positive cultures, seen in 16 of the 20 patients described. Failure to obtain positive cultures in four cases was attributed to inadequate bacteriologic techniques before 1967. Where no antibiotics were given prior to collecting blood cultures and bacteriologic techniques were adequate, proven culture negative endocarditis was virtually unknown. When antibiotics have been given, repeated blood cultures are recommended following withdrawal of antibiotic for at least four days.

Effect of protein synthesis inhibitors on the fidelity of translation in eukaryotic systems.

Factors influencing the accuracy of poly(U)-directed poly(Phe) synthesis in a wheat germ and in a reticulocyte system were studied. Addition of preformed phenylalanyl-tRNA, as well as increasing the ratio of poly(U) to ribosomes, significantly enhanced the poly(Phe) synthesis and concurrently reduced the misincorporation of leucine. The protein synthesis inhibitors cycloheximide, abrin and ricin had little or no effect on the misreading when the system was supplemented with 100 microM phenylalanyl-tRNA, but they reduced the relatively high error rate observed when the poly(U) system was not supplemented with the cognate substrate. Raising the incubation temperature enhanced the accuracy to the same extent whether or not ricin was present i.e., at widely different rates of elongation. The results show that the translational accuracy is not linked to the elongation rate as such. Translational inhibitors affect the fidelity by influencing the kinetics of the system. In systems containing limiting concentrations of cognate substrate, translational inhibitors will cause an increase in the limiting aminoacyl-tRNA species and thereby increase fidelity.