ALBERT

Description: Following [R] binding, CD20 antigen redistributes into lipid raft domains (LRD) and initiates signaling events leading to apoptosis in malignant B-cells. In addition, the clustering of CD20 receptors into LRD results in a relative increase in the antigen density within a given area of the cell membrane, thus facilitating [R]-mediated complement mediated cytotoxicity (CMC) and/or antibody dependent cellular cytotoxicity (ADCC). Combinations of monoclonal antibodies (mAbs) directed against unique tumor-associated targets can potentially alter the reorganization of LRD and modify their anti-tumor activity. Our main objective was to study the effects of [A] upon [R]-mediated anti-tumor effects. For ADCC/CMC studies, 51Cr-labeled NHL cells (Raji, DHL-4, DHL-10, Karpas422 or Ramos cells) were exposed to concurrent [A+R], sequential [R→A] or [A→R] prior to the addition of peripheral blood mononuclear cells (effector:target ratio of 40:1) or human serum, respectively. Trastuzumab [I] served as isotype control. Following a 6-hour period incubation, supernatant was harvested and % lysis calculated. In addition, LRD of 1x108 Raji cells exposed to similar mAb-combinations were extracted by sucrose gradient ultracentrifugation. Reorganization of CD20 into LRD was determined by Western blotting. For in vivo studies six to 8 week old SCID mice were inoculated by tail vein injection (iv) with 1x106 Raji cells (day 0). After tumor engraftment (Day +7), animals were divided in seven cohorts to receive 8 doses of vehicle control, [R], [A], [I], alternating doses of [R+A], or sequential dosing of [A→R] or [R→A]. MAb were administered IV at 5mg/kg/dose. The end point of the study was overall survival. Statistical analysis was performed with Kaplan-Meier survival curves and P values calculated by log rank test. In vitro exposure to [A] prior to [R] therapy resulted a 30 to 50% decrease in rituximab-mediated ADCC. In addition, exposure of Raji cells to [A] led to a decrease in the amount of CD20 reorganized into LRD following rituximab exposure. No decrease in [R]-mediated ADCC or reorganization of CD20 into LRD was observed when [A] was administered following [R] therapy. In vivo studies demonstrated a better anti-tumor activity in animals treated with alternating doses of [R + A], when compared to [R→A], [A→R], [R] or [A] therapy. The median survival for mice treated with combination mAb therapy [R+A] was 90 days, compared to 43 and 70 days for those treated with [R→A] and [A→R] respectively. Combination therapy with [R+A] resulted in a statistically significant longer survival when compared to [R], [A], or [I] (P

Description: The CD40 antigen is involved in cell survival and differentiation of B-cells and is uniformly expressed on chronic lymphocytic leukemia (CLL) cells. The CD40/CD40L interaction stimulates B-cells, dendritic cells and monocytes to proliferate, differentiate, up regulate co-stimulatory molecules and increase antigen presentation. While activation of CD40 can protect CLL cells against early fludarabine-induced apoptosis, these cells become sensitive to delayed death by extrinsic pathway apoptosis. (Blood, 105: 3193–8, 2005). SGN-40 is a humanized anti-CD40 antibody entering clinical trials and has been reported to have weak agonistic properties following CD40 ligation. To pursue rational clinical development of SGN-40, we studied the effects of this antibody in fresh, non-cryopreserved primary CLL cells. These studies included classic antibody mediated killing mechanisms and evidence of both CLL cell activation and protection against early fludarabine-mediated apoptosis. CLL cells treated with SGN-40 (10 mcg/ml) for 2 hours (hrs) in the presence of human serum promoted no complement mediated cytoxicity (CDC) in 8 pts tested. Direct SGN-40 induced apoptosis of human CLL cells with or without anti-Fc IgG cross-linking at 24, 48 and 72 hrs was not increased over that observed with the isotype control antibody trastuzumab in 8 pts studied. In contrast, SGN-40 induced antibody dependent cellular cytotoxicity (ADCC) against CLL cells an average of 12% (±11.39 SD, range 2–32%) killing at 4 hrs (effector to target cell ratio 25:1) in 6 pts tested. The SGN-40 induced ADCC against CLL cells were similar to that observed with alemtuzumab (average 19%, SD 6.9, range10–30%) or rituximab (average 18%, SD 12.48, range 8–42.5%). SGN-40 also mediated death in Raji and 697 lymphoblastic lymphoma cell lines via ADCC. Similar to reports by others with CD40 ligand, SGN-40 mediated activation was noted with modest up-regulation of CD80 and HLA-DR at 48hrs. When administered prior to fludarabine, SGN-40 also protected against death in 5 consecutive samples, although this was less than observed with CD40 ligand transfected HeLa cells, consistent with incomplete CD40 activation. Concurrent administration of SGN-40 and fludarabine did not protect from drug-mediated apoptosis. In conclusion, these findings suggest that SGN-40 has dual property of mediating cytotoxic effect by ADCC and partial CD40 activation. Development of SGN-40 as a therapeutic agent in CLL is justified and future studies by our group are focusing on enhancing SGN-40 mediated ADCC against CLL cells and potentially designing combination studies with SGN-40 to exploit this agent’s ability to engage the CD40/CD40L network.

Description: The acquirement of resistance to rituximab has been observed in lymphoma patients. To further define the molecular basis for rituximab-resistance we have developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 structure at the protein and gene level, membrane reorganization, and signaling events following rituximab exposure. RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or concurrently with human complement (4RH). Functional assays were performed to demonstrate decrease in rituximab-associated CMC and ADCC. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Sequencing of the CD20 gene from Raji parental and RRCL was performed to determine differences. Following rituximab treatment, lipid raft domains were extracted from Raji and RRCL to determine changes in polarization. Redistribution of CD20 antigen were evaluated by Western blotting. No significant changes were observed in the expression of the external domain of CD20 antigen between rituximab-sensitive and RRCL as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. The CD20 gene sequence was found to be identical between Raji cells and RRCL, suggesting a post-transcriptional regulatory mechanism is responsible for the changes in the structure of CD20. Redistribution of CD20 into the lipid raft domains was more evident in Raji parental cells when compared to RRCL, and a decrease in p38 activation was observed following exposure of RRCL to rituximab. In conclusion, our data strongly suggests that the acquirement of rituximab-resistance is associated with significant changes in the structure of the internal domain of the CD20 antigen. The abnormal production of truncated forms of the CD20 antigen, while not affecting external rituximab binding resulted in changes in the redistribution of CD20 antigen into lipid raft domains and a decrease in signaling events. Post-transcriptional events leading to the acquirement of truncated forms of CD20 may impair signaling activation, thus contributing to rituximab-resistance seen in RRCL.

Description: Sorghum is grown the world over for both human and animal consumption. Recently, the increase in grain production has slowed; simultaneously, its marketability has declined. Grain size is one of the most important determinants of grain yield and market price in India. One important factor responsible for the decline in consumption of sorghum in India is the inferior quality of rainy season produce. Consumers prefer post rainy season sorghums as the grains are bold, round and lustrous in appearance. Any improvement in the quality of rainy season sorghum grain would help it to fetch higher market prices.In the present study, the genetics of important grain quality characteristics such as grain size, grain shape and lustre were studied using two crosses based on elite sorghum lines 463B and AKMS 14B, and a germplasm line, IS 17600, during 2001 at the National Research Centre for Sorghum, Hyderabad, India. Generation mean analysis and frequency distribution studies revealed that grain size is governed by dominant genes that are polygenic in nature. Predominance of dominance and epistatic interactions in both crosses indicates that selection for higher grain size would be more effective if the dominance and epistatic effects are first reduced by a few generations of selfing. Biparental mating is suggested for developing homozygous bold grain lines. Round grain shape is governed by a single dominant gene and grain lustre by two complementary recessive genes. The study suggested that developing a sorghum hybrid with bold, round grain is feasible provided either of the parents has bold and/or round grain. However, for the hybrid to be lustrous, both parents need to be lustrous and homozygous for the alleles conferring grain lustre at a common locus. With the possibility of development of sorghum hybrids with bold, round and lustrous grain, there is scope for improvement in consumer preference of rainy season sorghum grain.

Description: SUMMARYForage sorghum is an important component of the fodder supply chain in the arid and semi-arid regions of the world because of its high productivity, ability to utilize water efficiently and adaptability to a wide range of climatic conditions. Identification of high-yielding stable genotypes (G) across environments (E) is challenging because of the complex G × E interactions (GEI). In the present study, the performance of 16 forage sorghum genotypes over seven locations across the rainy seasons of 2010 and 2011 was investigated using GGE biplot analysis. Analysis of variance revealed the existence of significant GEI for fodder yield and all eight associated phenotypic traits. Location accounted for a higher proportion of the variation (0·72–0·91), while genotype contributed only 0·06–0·21 of total variation in different traits. Genotype-by-location interactions contributed 0·02–0·13 of total variation. Promising genotypes for fodder yield and each of the associated traits could be identified effectively using a graphical biplot approach. The majority of test locations were highly correlated. A ‘Which-won-where’ study partitioned the test locations into two mega-environments (MEs): ME1 was represented by five locations with COFS 29 as the best genotype, while ME2 had two locations with S 541 as the best genotype. The existence of two MEs suggested a need for location-specific breeding. Genotype-by-trait biplots indicated that improvement for forage yield could be achieved through indirect selection for plant height, leaf number and early vigour.

Description: SUMMARYTwelve sorghum lines resistant to sorghum shoot fly were evaluated for their combining ability for shoot fly resistance and traits associated with resistance, using three male sterile lines in two environments. Using a completely randomized block design with three replications, 36 hybrids and 15 parental genotypes were raised. Considerable genetic variation was observed for all the traits studied. Non-additive gene effects played an important role in governing glossiness, seedling vigour and proportion of plants with deadhearts. For trichome density, both additive and non-additive gene actions were important. Among the lines evaluated, those identified to be good combiners were SFCR 1047 for seedling vigour, deadheart proportion and trichome density, RSE 03 for glossiness, deadheart proportion at 21 DAE and trichome density, and SPSFR 94032 for seedling vigour and shoot fly eggs per plant. Genetic diversity and cluster analysis grouped the 15 parents (12 resistant and 3 susceptible parents) into five clusters. Utilization of the resistant lines belonging to different clusters in improving shoot fly resistance in sorghum is discussed.