Engineered Human Antibody Constant Domains with Increased Stability

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (T_m = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (T_m = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.Monoclonal antibodies (mAbs)² with high affinity and specificity are now well established therapeutics and invaluable tools for biological research. It appears that their use will continue to expand in both targets and disease indications. However, a fundamental problem for full-size mAbs that limits their applications is their poor penetration into tissues (e.g. solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g. on the HIV envelope glycoprotein) that are accessible by molecules of smaller size. Antibody fragments, e.g. Fabs (∼60 kDa) or single chain Fv fragments (scFvs) (20∼30 kDa), are significantly smaller than full-size antibodies (∼150 kDa), and have been used as imaging reagents and candidate therapeutics. Even smaller fragments of antibodies are of great interest and advantageous for pharmaceutical applications including cancer targeting and imaging.During the last decade a large amount of work has been aimed at developing of small size binders with scaffolds based on various highly stable human and non-human molecules (1–8). A promising direction is the development of binders based on the heavy or light chain variable region of an antibody; these fragments ranging in size from 11 kDa to 15 kDa were called “domain antibodies” or “dAbs” (7, 9). A unique kind of antibodies composed only of heavy chains are naturally formed in camels, dromedaries, and llamas, and their variable regions can also recognize antigens as single domain fragments (10). Not only is the overall size of the dAbs much smaller than that of full-size antibodies but also their paratopes are concentrated over a smaller area so that the dAbs provide the capability of interacting with novel epitopes that are inaccessible to conventional antibodies or antibody fragments with paired light and heavy chain variable domains.The structure of the constant antibody domains is similar to that of the variable domains consisting of β-strands connected mostly with loops or short helices. The second domain of the α, δ, and γ heavy chain constant regions, CH2, is unique in that it exhibits very weak carbohydrate-mediated interchain protein-protein interactions in contrast to the extensive interchain interactions that occur between the other domains. The expression of murine CH2 in bacteria, which does not support glycosylation, results in a monomeric domain (11). It has been hypothesized that the CH2 domain (CH2 of IgG, IgA, and IgD, and CH3 of IgE and IgM) could be used as a scaffold and could offer additional advantages compared with those of dAbs because it contains binding sites or portions of binding sites conferring effector and stability functions (12).It was found previously that an isolated murine CH2 is relatively unstable at physiological temperature with a temperature of 50% unfolding (T_m) slightly higher than 37 °C (11). We have hypothesized that human CH2 would exhibit different stability because of significant differences in the sequence compared with its murine counterpart. Therefore, we have extensively characterized the stability of an isolated unglycosylated single CH2 domain. We found that its stability is significantly higher than the previously reported for the murine CH2. We further increased the stability of the human CH2 by engineering an additional disulfide bond between the A and G strands. One of the newly developed mutants, denoted as m01, exhibited significantly higher stability (T_m = 73.8 °C) than that of wild type CH2. We suggest that both the wild type CH2 and the newly developed mutant, m01, could be used as scaffolds for binders. These results also demonstrate for the first time that the stability of constant antibody domains can be dramatically increased by engineering of an additional disulfide bond. The increase in stability of isolated domains may result in an increase in stability of larger antibody fragments, e.g. Fc, and therefore could have implications as a general method for increasing antibody stability. Thus, it appears that further development of CH2 and its more stable variants as scaffolds could provide new opportunities for identification of potentially useful therapeutics.     

Identification of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> genes whose expression determines changes in the shoot structure

A system was created to obtain and select Arabidopsis thaliana genes whose superexpression causes development of a mutant phenotype. Three morphological mutants (two with a markedly retarded growth and one with a fasciated stem) associated with the superexpression of genes At5g10080, At1g33390, and At5g13760 were generated with the use of this system. Localization, structure, and a possible functional organization of these genes were determined.     

Limitations of Peptide Retro-inverso Isomerization in Molecular Mimicry

A retro-inverso peptide is made up of d-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Despite their limited success as antigenic mimicry, retro-inverso isomers generally fail to emulate the protein-binding activities of their parent peptides of an α-helical nature. In studying the interaction between the tumor suppressor protein p53 and its negative regulator MDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D. (2004) J. Am. Chem. Soc. 126, 16288–16289) made a surprising finding that the retro-inverso isomer of p53(15–29) retained the same binding activity as the wild type peptide as determined by inhibition enzyme-linked immunosorbent assay. The authors attributed the unusual outcome to the ability of the d-peptide to adopt a right-handed helical conformation upon MDM2 binding. Using a battery of biochemical and biophysical tools, we found that retro-inverso isomerization diminished p53 (15,–29) binding to MDM2 or MDMX by 3.2–3.3 kcal/mol. Similar results were replicated with the C-terminal domain of HIV-1 capsid protein (3.0 kcal/mol) and the Src homology 3 domain of Abl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopic as well as x-ray crystallographic studies showed that d-peptide ligands of MDM2 invariably adopted left-handed helical conformations in both free and bound states. Our findings reinforce that the retro-inverso strategy works poorly in molecular mimicry of biologically active helical peptides, due to inherent differences at the secondary and tertiary structure levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structure level.     

Principles of formalizing a quantitative evaluation of the genetic danger of chemical compounds for man

Specific characteristics of the mutagenic effect of chemicals, which must be taken into account in developing the test system to assess the potential genetic risk caused by chemical substances, are considered. The organizational principles of the procedures currently available for testing and ranking chemicals by their mutagenic and carcinogenic hazard to humans are discussed. The use of selective information suggested by Wiener and Shannon as an efficiency measure of testing and estimating the potential genetic hazard of chemical substances is substantiated. The feasibility of this approach was demonstrated by testing the efficiency of the battery of two short-term in vitro tests as an example. It was shown that selective information is able to serve as an integral universal criterion of the efficiency of testing, if either one test or the test battery were used.     

Transgenic tobacco plants expressing Tarin 1 inhibit the growth of Pseudomonas syringae pv. tomato and the development of Spodoptera frugiperda

The protein Tarin 1, from Colocasia esculenta, was expressed in Nicotiana tabacum. Bioassays were done on plants expressing Tarin 1 at different levels using Spodoptera frugiperda larvae, various bacteria and fungi and the root‐knot nematode Meloidogyne javanica. It was found that S. frugiperda larvae fed on transformed plants had retarded and lower pupation, lower accumulated biomass and higher mortality rate than larvae fed on control plants. Also, Tarin 1 was found to inhibit the growth in vitro of Pseudomonas syringae pv. tomato. For Meloidogyne javanica, both relative replication and root damage were greater in control plants than in transformed plants, but the results were not statistically significant. This work illustrates the effects of plants expressing Tarin 1, on the growth and development of insects and bacteria, and shows its potential for pest management.     

Mid-Holocene environmental and human dynamics in northeastern China reconstructed from pollen and archaeological data

A pollen record from the Taishizhuang site (40°21.5′N, 115°49.5′E) located in the transitional forest-steppe zone near the present-day limit of the summer monsoon is used to reconstruct vegetation and climate. Quantitative biome reconstruction suggests that between ca. 5700 and 4400 cal. years B.P. temperate deciduous forest dominated the vegetation cover around the Taishizhuang site. After that time the landscape became more open and the scores of the steppe biome were always higher than those of the temperate deciduous forest except for two oscillations dated to ca. 4000 cal. years B.P. and ca. 3500 cal. years B.P. However, ca. 3400-2100 cal. years B.P. the common vegetation became steppe and the landscape was more open in comparison with the previous time interval. The results of the pollen-based precipitation reconstruction suggest that annual precipitation was ca. 550-750 mm (ca. 100-300 mm higher than present) during the mid-Holocene ‘forest phase’, and ca. 450-650 mm during the following ‘forest-steppe phase’. From ca. 3400 cal. years B.P. during the ‘steppe phase’ annual precipitation was similar to modern values (ca. 300-500 mm). Archaeological records from 100 sites prove the habitation of northeastern China during the prehistoric and early historic periods from ca. 8200 cal. years B.P., but do not provide evidence of the use of wood resources intensive enough to influence the regional vegetation development and to leave traces in the pollen assemblages. Both archaeological and palaeoenvironmental data support the conclusion that changes in pollen composition in northeastern China between 5700 and 2100 cal. years B.P. reflect natural variations in precipitation and not major deforestation caused by humans.     

Population genetic analysis of the association between the BRCA1 and P53 gene polymorphisms and the risk of sporadic breast cancer

Polymorphism of two tumor-suppressor genes, BRCA1 and P53, was examined. DNA was extracted from blood leukocytes of the women affected with breast cancer (N = 151) and of the women with no clinical symptoms of tumor diseases (N = 191). Typing of the polymorphic variants was performed using PCR-RFLP method. It was demonstrated that the genetic structure of the patient group (taking into consideration BRCA1 and P53 polymorphic variants) differed from that of the control group. The group of genotypes, found exclusively among the patients, as well as the group of "resistant" genotypes revealed predominantly among the controls, was described. Detection of the genotype A1A1 B1B1 S1S1 C1C1 F1F1 J2J2, whose frequency in control group was eight times higher than in the patient group, was an additional confirmation of the existence of "resistant" genotypes. These findings point to the association between the combinations of the BRCA1 and P53 allelic variants and the risk of breast cancer.