Chimeric 101F (ch101F) is a mouse-human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422-438 spanning antigenic site IV, V, VI was analysed. Residues 423-436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.
Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.
There are a number of proteins whose active forms are non-covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild-type (wt) proteins. We have used tumor necrosis factor-α (TNF-α) to establish that it is possible to use mass spectrometry to monitor the non-covalent solution structure of therapeutically relevant proteins and correlate the results with binding data. Mass spectrometry is shown to be able to directly monitor the state of the solution complexes to within 5 Da errors mass accuracy of theoretical mass at 50 kDa, as well as to resolve homocomplex from heterocomplex. Furthermore, it was determined that perturbation of the TNF-α complex, at or below pH 4.0, results in monomers that cannot reform into the multimeric complex, and the resulting protein solution can no longer bind to an anti-TNF-α antibody. Dissociation and re-association of the trimer was possible with the use of dimethyl sulfoxide at pH 5.5 and allowed for the resulting detection of both homotrimer and heterotrimer in solution with no impact on antibody binding. This work demonstrates that mass spectrometric techniques offer a means to monitor native solution interactions of non-covalent complexes and to differentiate multiple complexes from each other in solution. This method has applicability in the biopharmaceutical arena for monitoring engineering non-covalent drug complexes for the purpose of altering biological activity.
In general, megakaryocytic myelosis is nowadays considered to be a separate disease entity, one of the myeloproliferative syndromes. Morphologically there are localised or diffuse proliferations of usually large pleomorphic megakaryocytes and immature atypical megakaryocytes up to megakaryoblasts in the bone marrow, in the sense of a haemoblastosis. In the course of the disease megakaryocytic splenomegaly develops. A sarcomatous form (megakaryoblastoma, megakaryo-sarcoma) is rare. Megakaryocytic myelosis may arise from chronic meyloid leukaemia or polycythaemia vera, rarely as a transitional stage to an acute myeloblastic leukaemia or megakaryoblastic leukemia in the sense of a blast crisis. The mature form of the disease, which has an age peak at 59 years and is not sex-linked, often takes a course over years with increasing splenomegaly, anaemia, moderate leucocytosis and usually marked thrombocytosis (average value of 720 X 10(9)/1). Life threatening complications are haemorrhages, thromboembolism and increased frequency of infections due to antibody deficiency in the advanced stage.
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