Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer

Data from an ongoing clinical radioimmunoscintigraphy trial indicate that^99mTc-labeled monoclonal antibody (mAb) E48 is highly capable of selectively targeting squamous cell carcinoma of the head and neck (HNSCC). The percentage of the injected dose per gram of tumor tissue was found to be high, rendering mAbE48 a promising candidate mAb for therapeutic purposes. We now describe the construction of a chimeric (moouse/human) mAb E48 by recombinant DNA technology. The genes encoding the variable domains of the heavy and light chain were cloned and ligated into experession vectors containing the human 1 heavy-chain gene and the human k lightchain gene respectively. Biological properties of the resulting chimeric mAb E48 were compared to the murine form in vitro and in vivo. The reactivities of chimeric (c)mAb and murine (m)mAb E48 with HNSCC, as assessed by immunohistochemical staining as well as immuno-blotting were shown to be similar. The affinity constant appeared to be 0.9×10¹⁰ M^–1 and 1.6×10¹⁰ M^–1 for the mmAb and cmAb respectively. The biodistribution of both antibodies was tested by simultaneous injection into nude mice bearing human HNSCC xenografts. cmAb E48 was found to be cleared more rapidly from the blood than mmAb E48, resulting in a 30% lower tumor uptake but similar tumor to non-tumor ratios, 3 days after injection. Moreover, it was shown that cmAb E48 is highly capable of lysing HNSCC targets in ADCC assays in vitro, whereas the mmAb appeared to be almost incative. These data indicate that cmAb E48 has potential as a targeting agent for the eradication of HNSCC in man.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Intraspecific variation of spectral sensitivity in threespine stickleback (Gasterosteus aculeatus) from different photic regimes

To examine the influence of the spectral characteristics of underwater light on spectral sensitivity of the ON and OFF visual pathways, compound action potential recordings were made from retinal ganglion cells of threespine stickleback from different photic regimes. In fish from a red-shifted photic regime (P₅₀ 680 nm for downwelling light at 1m), peak sensitivity of both the ON and OFF pathways was limited to long wavelength light (_max 600–620). In contrast, the ON pathway of fish from a comparatively blue-shifted (P₅₀ 566 nm) photic regime exhibited sensitivity to medium (_max 540–560) and long (_max 600 nm) wavelengths, while the OFF pathway exhibited peak sensitivity to only medium (_max 540 nm) wavelength light. In a third population, where the the ambient light is moderately red-shifted (P₅₀ 629 nm), the ON pathway once again exhibited only a long wavelength sensitivity peak at 620 nm, while the OFF pathway exhibited sensitivity to both medium (_max 560 nm) and long (_max 600–620 nm) wavelength light. These findings suggest that the photic environment plays an integral role in shaping spectral sensitivity of the ON and OFF pathways.     

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity againstp-nitrophenyl acetate

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.Abbreviations used MBP myelin basic protein - MW molecular weight - kDa kilo Dalton - octyl-POE n-octylpolydisperse oligooxyethylene - CHAPS 3-3-cholamidopropyl dimethylammonio-1-propane-sulfonate - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - G 3707 heptaoxyethylene lauryl ether - TWEEN-20 polyoxyethylenesorbitan-monolaurat - EDTA ethylenediaminetetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)     

内皮衍生舒张因子对缺血（缺氧）,再灌注（复氧）心肌的保护作用

血管内皮产生的内皮衍生舒张因子（ｅｎｄｏｔｈｅｌｉｕｍ－ｄｅｒｉｖｅｄ ｒｅｌａｘｉｎｇ ｆａｃｔｏｒ,ＥＤＲＦ）即一氧化氮（ｎｉｔｒｉｃ ｏｘｉｄｅ,ＮＯ）本工作分别在大鼠Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流模型和培养的大鼠心肌细胞上观察了ＮＯ、ＮＯ的前体物质Ｌ－精氨酸（Ｌ－Ａｒｇ）、ＮＯ的前体物质Ｌ－精氨酸（Ｌ－Ａｒｇ）、ＮＯ的合成阻断剂Ｌ－硝基精氨酸（Ｌ－ＮＮＡ）对心肌缺血（缺氧）再灌注（复氧     

Long timestep dynamics of peptides by the dynamics driver approach

Previous experience with the Langevin/implicit-Euler scheme for dynamics (“LI”) on model systems (butane, water) has shown that LI is numerically stable for timesteps in the 5–20 fs range but quenches high-frequency modes. To explore applications to polypeptides, we apply LI to model systems (several dipeptides, a tetrapeptide, and a 13-residue oligoalanine) and also develop a new dynamics driver approach (“DA”). The DA scheme, based on LI, addresses the important issue of proper sampling, which is unlikely to be solved by small-time step integration methods or implicit methods with intrinsic damping at room temperature, such as LI. Equilibrium averages, time-dependent molecular properties, and sampling trends at room temperature are reported for both LI and DA dynamics simulations, which are then compared to those generated by a standard explicit discretization of the Langevin equation with a 1 fs timestep. We find that LI's quenching effects are severe on both the fast and slow (due to vibrational coupling) frequency modes of all-atom polypeptides and lead to more restricted dynamics at moderate timesteps (40 fs). The DA approach empirically counteracts these damping effects by adding random atomic perturbations to the coordinates at each step (before the minimization of a dynamics function). By restricting the energetic fluctuations and controlling the kinetic energy, we are able with a 60 fs timestep to generate continuous trajectories that sample more of the relevant conformational space and also reproduce reasonably Boltzmann statistics. Although the timescale for transition may be accelerated by the DA approach, the transitional. information obtained for the alanine dipeptide and the tetrapeptide is consistent with that obtained by several other theoretical approaches that focus specifically on the determination of pathways. While the trajectory for oligoalanine by the explicit scheme over the nanosecond timeframe remains in the vicinity of the full α_R-helix starting structure, and a high-temperature (6000°K) MD trajectory departs slowly from the a helical structure, the DA-generated trajectory for the same CPU time exhibits unfolding and refolding and reveals a range of conformations with an intermediate helix content. Significantly, this range of states is more consistent with spectroscopic experiments on small peptides, as well as the cooperative two-state model for helix–coil transition. The good, near-Boltzmann statistics reported for the smaller systems above, in combination with the interesting oligoalanine results, suggest that DA is a promising tool for efficiently exploring conformational spaces of biomolecules and exploring folding/unfolding processes of polypeptides. © 1995 Wiley-Liss, Inc.     

Atomic and residue hydrophilicity in the context of folded protein structures

Water-protein interactions drive protein folding, stabilize the folded structure, and influence molecular recognition and catalysis. We analyzed the closest protein contacts of 10,837 water molecules in crystallographic structures to define a specific hydrophilicity scale reflecting specific rather than bulk solvent interactions. The tendencies of different atom and residue types to be the nearest protein neighbors of bound water molecules correlated with other hydrophobicity scales, verified the relevance of crystallographically determined water positions, and provided a direct experimental measure of water affinity in the context of the folded protein. This specific hydrophilicity was highly correlated with hydrogen-bonding capacity, and correlated better with experimental than computationally derived measures of partitioning between aqueous and organic phases. Atoms with related chemistry clustered with respect to the number of bound water molecules. Neutral and negatively charged oxygen atoms were the most hydrophilic, followed by positively-charged then neutral nitrogen atoms, followed by carbon and sulfur atoms. Agreement between observed side-chain specific hydrophilicity values and values derived from the atomic hydrophilicity scale showed that hydrophilicity values can be synthesized for different functional groups, such as unusual side or main chains, discontinuous epitopes, and drug molecules. Two methods of atomic hydrophilicity analysis provided a measure of complementarity in the interfaces of trypsin:pancreatic trypsin inhibitor and HIV protease:U-75875 inhibitor complexes. © 1995 Wiley-Liss, Inc.     

Progress of 1D protein structure prediction at last

Accuracy of predicting protein secondary structure and solvent accessibility from sequence information has been improved significantly by using information contained in multiple sequence alignments as input to a neural 'network system. For the Asilomar meeting, predictions for 13 proteins were generated automatically using the publicly available prediction method PHD. The results confirm the estimate of 72% three-state prediction accuracy. The fairly accurate predictions of secondary structure segments made the tool useful as a starting point for modeling of higher dimensional aspects of protein structure. © 1995 Wiley-Liss, Inc.     

Chemical shifts and three-dimensional protein structures

Summary During the past three years it has become possible to compute ab initio the ¹³C, ¹⁵N and ¹⁹F NMR chemical shifts of many sites in native proteins. Chemical shifts are beginning to become a useful supplement to more established methods of solution structure determination, and may find utility in solid-state analysis as well. From ¹³C NMR, information on , and torsions can be obtained, permitting both assignment verification, and structure refinement and prediction. For ¹⁵N, both torsional and hydrogen-bonding effects are important, while for ¹⁹F, chemical shifts are primarily indicators of the local charge field. Chemical shift calculations are still slow, but shielding hypersurfaces — the shift as a function of the dihedral angles that define the molecular conformation — are becoming accessible. Over the next few years, theoretical and computer hardware improvements will enable more routine use of chemical shifts in structural studies, including the study of metal-ligand interactions, the analysis of drug and substrate binding and catalysis, the study of folding/unfolding pathways, as well as the characterization of conformational substates. Rather than simply being a necessary prerequisite for multidimensional NMR, chemical shifts and chemical shift non-equivalence due to folding are now beginning to be useful for structural characterization.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].