A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

To harness the potent tumor-killing capacity of T cells for the treatment of CD19⁺ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19⁺ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19⁺ malignancies with an advantageous safety risk profile and anticipated dosing regimen.     

Individualized,discrete event,simulations provide insight into inter- and intra-subject variability of extended-release,drug products

Objective

Develop and validate particular, concrete, and abstract yet plausible in silico mechanistic explanations for large intra- and interindividual variability observed for eleven bioequivalence study participants. Do so in the face of considerable uncertainty about mechanisms.

Methods

We constructed an object-oriented, discrete event model called subject (we use small caps to distinguish computational objects from their biological counterparts). It maps abstractly to a dissolution test system and study subject to whom product was administered orally. A subject comprises four interconnected grid spaces and event mechanisms that map to different physiological features and processes. Drugs move within and between spaces. We followed an established, Iterative Refinement Protocol. Individualized mechanisms were made sufficiently complicated to achieve prespecified Similarity Criteria, but no more so. Within subjects, the dissolution space is linked to both a product-subject Interaction Space and the GI tract. The GI tract and Interaction Space connect to plasma, from which drug is eliminated.

Results

We discovered parameterizations that enabled the eleven subject simulation results to achieve the most stringent Similarity Criteria. Simulated profiles closely resembled those with normal, odd, and double peaks. We observed important subject-by-formulation interactions within subjects.

Conclusion

We hypothesize that there were interactions within bioequivalence study participants corresponding to the subject-by-formulation interactions within subjects. Further progress requires methods to transition currently abstract subject mechanisms iteratively and parsimoniously to be more physiologically realistic. As that objective is achieved, the approach presented is expected to become beneficial to drug development (e.g., controlled release) and to a reduction in the number of subjects needed per study plus faster regulatory review.

     

Competition with Egyptian Geese Alopochen aegyptiaca overrides environmental factors in determining productivity of Black Sparrowhawks Accipiter melanoleucus

We studied a population of Black Sparrowhawks Accipiter melanoleucus that has recently colonized the Cape Peninsula, South Africa. Black Sparrowhawks are winter breeders: over most of their range winters are dry, but the Cape Peninsula has a Mediterranean climate, with most rain falling in winter. The birds bred at an average density of 38 pairs/100 km². Monitoring of 70 breeding attempts by 26 pairs showed that the birds favoured large stands of trees for breeding. Birds breeding in smaller stands were more randomly spaced, dispersion being determined by habitat availability. Parameters related to climate, habitat and aspects of nest-site selection failed to predict the birds' productivity despite the fact that they had recently colonized a novel climatic window. Rather, productivity was best explained by the probability of nest usurpation by highly aggressive Egyptian Geese Alopochen aegyptiaca . Pairs encountering Goose usurpation problems raised an average of 0.76 chicks per breeding attempt, compared with 1.58 chicks for pairs without Geese. The only defence against nest usurpation by Geese appears to be the construction of multiple nests, an option most available to and most exploited by birds breeding in large stands of trees.     

The Effects of the Methylating Agent 1,2-Bis(methylsulfonyl)-1-methylhydrazine on Morphology, DNA Content and Mitochondrial Function of Trypanosoma brucei Subspecies

Repeated exposure of trypanosomes in vitro or in vivo to low concentrations of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine induces a series of moderately synchronous morphological and biochemical changes. Cell division halts and the long-slender bloodstream forms transform to short-stumpy forms via larger intermediate-stage cells which contain approximately double the normal G₂ content of DNA. In common with naturally occurring short-stumpy trypanosomes, drug-induced short-stumpy forms do not infect rodents and when transferred to Cunningham's medium, transform to and replicate as procylics. Furthermore, these short-stumpy forms exhibit α-ketoglutarate supported motility and oxygen consumption, acquire the ability to reduce nitroblue tetrazolium (NADH diaphorase positivity) and appear to be in the G₁ or G₀ stage of the cell cycle based upon DNA content.     

The relative efficacy of repellents against mosquito vectors of disease

Laboratory tests of insect repellents by various different methods showed that An.stephensi Liston was consistently more susceptible than An.gambiae Giles, An.albimanus Wiedemann or An.pulcherrimus Theobald. The six repellents tested were di-ethyl toluamide (deet), di-methyl phthalate (DMP), ethyl-hexanediol, permethrin, citronella and cedarwood oil. Testing systems in which the mosquitoes were presented with a choice gave consistently lower ED50 values than when there was no choice, i.e. the standards of tolerance are not absolute but depend on the options available. In field tests in an experimental hut a curtain with a high dose of di-ethyl toluamide (deet) reduced biting in the hut but had to be re-impregnated frequently. Deet-impregnated anklets gave about 84% protection against Culex quinquefasciatus Say for 80 days after one impregnation, in a trial in which the anklets were brought out of sealed storage and tested for 2 h nightly. Similar protection was found against An.funestus Giles but the protection against An.gambiae s.l., An. coustani Laveran and Mansonia spp. was not as good. There were highly significant differences between the four collectors' mosquito attractiveness but this varied highly significantly between the mosquito species.     

4-Aminotetrolic Acid : New Conformational-restricted Analogue of γ-Aminobutyric Acid

RECENT studies strongly support a role for γ-aminobutyric acid (GABA) as an inhibitory transmitter at certain synapses in the mammalian central nervous system. Structure activity correlations of many GABA analogues implicate both the intramolecular distance between the zwitterionic centres and the rotational freedom of the molecule as important factors governing the synaptic activity of these substances¹. The following observations provide pertinent information about the active conformation(s) of GABA recognized by the receptor. (1) Muscimol, an isoxazole isolated from Amanita muscaria, seems to function as a GABA analogue as its inhibitory action on central neurones is comparable with that of GABA both in potency² and with respect to antagonism by bicuculline³. Molecular orbital calculations suggest that GABA and muscimol can assume similar conformations as zwitterions with the charged centres (N⁺ and 0^?) at least 5 and, more likely, 6 Å apart⁴. (2) The selective GABA antagonist bicuculline exhibits some degree of structural similarity with particular conformations of GABA and muscimol³. (3) X-ray crystallography indicates that GABA exists in a partially folded conformation in the solid state^5,6. (4) A model of the GABA receptor proposes that GABA adopts a folded conformation with a distance of less than 4.4 Å between the charged centres⁷. Observations (1) and (2) suggest extended conformations for GABA, while (3) and (4) suggest folded conformations.     

Effects of community-wide use of lambdacyhalothrin-impregnated bednets on malaria vectors in rural Sierra Leone

Abstract. The effect of community-wide use of bednets treated with lambdacyhalothrin 10mg/m² on the malaria vector Anopheles gambiae (forest form) was evaluated in Sierra Leone. Sixteen similar villages near the town of Bo were randomly allocated either to remain without nets or to receive treated bednets for all inhabitants, with effect from June 1992. Mosquitoes were sampled using human biting catches on verandas, light-trap catch (beside an occupied untreated bednet), window exit-trap catch and pyrethrum spray collections. During the first year of intervention (June 1992 to July 1993) the treated bednets provided personal protection for people sleeping under them, but had very little impact on densities of An. gambiae collected on human bait. The human blood index (HBI) of An.gambiae was not affected (HBI = 99% in villages with and without nets). An.gambiae parous rates were significantly reduced in all intervention villages, but malaria sporozoite rates fell in only some of the villages. These results are intermediate between those obtained from other projects in Tanzania and Burkina Faso, where treated bednets reduced man-biting, parity and sporozoite rates, versus The Gambia where treated bednets had no significant impact on any of these factors. Possible reasons for these contrasted findings are discussed.     

Variation in rates of nitrogen fixation in termites: response to dietary nitrogen in the field and laboratory

Abstract. Termites contribute nitrogen to their habitat through the nitrogenase activity of their bacterial symbionts. Previous studies indicate that high levels of dietary nitrogen suppress nitrogen fixation in termites. We examined the effects of dietary nitrogen on fixation rates in termites in both field and laboratory experiments. Ten field cplonies of Reticulitermes were collected and assayed for nitrogenase activity in July 1993, October 1993, January 1994, and April 1994. The nitrogen content of the wood collected with each colony was determined. There was no correlation between termite nitrogen fixation rates and the amount of nitrogen in their food for any of the four collection periods. In laboratory experiments, nitrogen fixation rates decreased when termites were fed filter paper treated with 2% and 5% ammonium nitrate or a 5% mixture of the amino acids proline, tryptophan and leucine, compared to water-treated controls. By contrast, the nitrogenase activity of termites fed filter paper treated with 2% and 5% ammonium phosphate, a mixture of the amino acids histidine, serine and aspartic acid, or 2% and 5% urea did not differ from the controls. However, nitrogenase activity increased when termites were fed with 2% uric acid. No clear association exists between termite nitrogen fixation and the nitrogen content of their food.     

HANDS-FREE, "POP-UP," ADHESIVE TAPE METHOD FOR MICROBIAL SAMPLING OF MEAT SURFACES

Microbial contamination of food is a concern to both food producers and consumers. For the food production industry surface sampling of foods is one of the simplest ways to monitor the microbial load. The objective of this experiment was to investigate the feasibility of using the less expensive and quicker "Pop-up" tape method instead of the conventional swab/rinse method for the microbial sampling of meat surfaces. An analyst can place the unit on the wrist and then use both hands to lay out all the necessary materials and take the sample with one hand. The "Pop-up" tape method was able to measure microbial loads up to 2.2 log CFU/cm² on meat surfaces. The conventional swab/rinse method was able to measure up to 8.3 log CFU/cm² on meat surfaces. The correlation coefficient (R) between the two methods was 0.91 (n=42). These data show that the "Pop-up" tape method is a viable alternative to other methods for estimating microbial surface contamination.