The antileukemic efficacy of an immunotoxin composed of a monoclonal anti-Thy-1 antibody disulfide linked to the ribosome-inactivating protein gelonin

Summary We prepared an immunoconjugate consisting of a monoclonal antibody recognizing the Thy-1 antigen and the ribosome-inactivating protein gelonin linked by a disulfide bond. This immunotoxin preparation was judged to contain less than 5% free antibody or gelonin. It was highly toxic in vitro in an antigen-specific fashion to the Thy-1 expressing RADA leukemia of A/J mice. The IC₅₀ of this preparation on RADA in vitro was 10^–12 M, while the IC₅₀ on the Thy-1 negative S1509a fibrosarcoma of A/J mice was 10^–7 M. The toxicity of this immunoconjugate was also measured in a direct proliferation assay and it was found that a 4-h exposure and a 24-h exposure of RADA cells to a 1 nM concentration of immunotoxin killed 90% and 99.9% of cells, respectively. Furthermore, efficacy in vitro was not due to the intrinsic susceptibility of RADA cells to tis type of immunotoxin, as one prepared with gelonin and an antibody recognizing the TL^a determinant on this leukemia had no efficacy in vitro. Clearance of the anti-Thy-1-gelonin immunoconjugate from the circulation of A/J mice after i.v. injection was rapid, especially during the first 8 h after injection, possibly because of binding to Thy-1 expressing tissue. Delivery of immunoconjugate to ascitic tumor in vivo was substantially better if the immunoconjugate was given by i.p. injection, rather than by the i.v. route. When given either i.v. or i.p. at the time of i.p. tumor inoculation in vivo, the anti-Thy-1-gelonin immunotoxin showed potency in an antigen-specific fashion; while this immunoconjugate prolonged survival and frequently cured RADA-inoculated mice, neither anti-Thy-1 antibody, gelonin, a combination of the two, nor immunotoxin of irrelevant specificity had any significant effect on survival. Anti-Thy-1-gelonin also had no effect on survival of A/J mice inoculated i.p. with S1509a. Furthermore, it was determined that a single i.p. dose of anti-Thy-1-gelonin killed 90% to 99% cells in vivo, and that the immunoconjugate was about as effective in this model as either adriamycin or cytoxan.This work was supported by ImmunoGen Inc. and in part by a grant from the National Institutes of Health, CA-14723     

13C-NMR spectroscopy of biflavanoids

The ¹³C-NMR spectra of nine naturally occurring CC linked biflavanoids have been assigned. The signals for the carbon atoms I-6, I-8, II-6, and II-8 appear in the region 90.0 ppm to 105.0 ppm. On the basis of the chemical shifts of these signals and their multiplicities in the off-resonance spectra, it is possible to determine the interflavonoid linkage in biflavanoids, provided that the A ring is involved. The level of oxidation of the ring C can be readily determined by a consideration of the chemical shift value of the carbonyl resonances. The position of the methoxyl substitution can also be inferred.     

A comprehensive interaction map of the human survival of motor neuron (SMN) complex

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.     

Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans‐acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln‐Sm units are displaced by new pICln‐Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis‐assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.     

PI3 Kinase inhibition on TRAIL-induced apoptosis correlates with androgen-sensitivity and p21 expression in prostate cancer cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many types of cancer cells. TRAIL is considered a therapeutic target, therefore, it was of interest to examine molecular mechanisms that may modulate sensitivity to TRAIL signaling in prostate cancer cells. LNCaP cells were found to be relatively resistant to TRAIL induced cell death while PC3 cells were sensitive. PI3-kinase (PI3 K) inhibitors were able to render LNCaP cells sensitive to TRAIL but conferred resistance to PC3 cells. PI3 K inhibitors were associated with an increase in p21^{waf1, cip1} expression in PC3 cells where as p21 decreases in LNCaP cells suggesting that p21 may impart TRAIL resistance. Since androgen receptor (AR) signaling can be modulated by AKT, and p21 is an AR responsive gene, the impact of PI3 K inhibition on TRAIL sensitivity was evaluated in AR transfected PC3 cells (PC3AR). The expression of AR was significantly downregulated by PI3 K inhibition in LNCaP cells, which have an intact AR signaling axis. PC3AR cells expressed higher levels of p21 protein and were relatively resistant to TRAIL compared to control cells. Finally, using adenoviral p21 gene transfer we directly demonstrated that p21 can confer resistance to TRAIL-induced cell death. These results suggest that TRAIL resistance is not regulated simply by a PI3 K/AKT survival pathway associated with inactivating PTEN mutations but may also be modulated by downstream AR responsive targets such as p21. These findings may have significant clinical implications for the utility of TRAIL in the management of prostate cancer.     

Natural history of SLC11 genes in vertebrates: tales from the fish world

Background  

The SLC11A1/Nramp1 and SLC11A2/Nramp2 genes belong to the SLC11/Nramp family of transmembrane divalent metal transporters, with SLC11A1 being associated with resistance to pathogens and SLC11A2 involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the SLC11 gene family have been clearly identified in tetrapods; however SLC11A1 has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the SLC11 genes in teleosts and evaluated if the roles attributed to mammalian SLC11 genes are assured by other fish specific SLC11 gene members.     

Disulfide-linked antibody-maytansinoid conjugates: optimization of in vivo activity by varying the steric hindrance at carbon atoms adjacent to the disulfide linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.