Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Patent
1993-10-15
1996-02-27
Patterson, Jr., Charles L.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
530327, C07K 708
Patent
active
054949998
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a synthetic antigen which may be used in assaying for, purifying or inducing CDw52 antibody.
The CAMPATH-1 family of monoclonal antibodies recognise an antigen expressed on the majority of human lymphocytes and monocytes [1-3, "CAMPATH" is a Registered Trade Mark]. At the fourth leucocyte workshop these antibodies were given the provisional designation CDw52 [4]. The antigen is an unusually good target for complement-mediated attack [1,5]. For this reason the IgM antibody, CAMPATH-1M, has been widely used for removal of T lymphocytes from donor bone marrow to prevent graft-versus-host disease [6,7].
The CDw52 antigen is expressed in most cases of lymphoid malignancy [8,9]. Serotherapy of lymphoma and leukaemia with CAMPATH-1 antibodies has therefore been attempted. The rat IgG2b, CAMPATH-1G, which activates both complement and cell-mediated killing, was found to be rather effective in this setting [9,10]. Recently, a human IgG1 antibody (CAMPATH-1H) with the same specificity has been constructed by genetic engineering [11] and this could be administered for a longer period and produced even better clinical results [12].
It is apparent that not all differentiation or tumour associated antigens are equally good targets for serotherapy and the reasons why the CAMPATH-1 antigen is so good are not yet clear. Its abundant expression [5] and lack of modulation [2] are probably relevant factors but knowledge of its structure would give use helpful clues.
About 50% of the CAMPATH-1 antigen could be removed from peripheral blood lymphocytes by treatment with glycosylphosphatidylinositol (GPI)-specific phospholipase C (from B. thuringiensis) [3]. This shows that at least some of the antigen is anchored by GPI and possibly all of it since a similar partial resistance has been observed with other GPI-linked antigens [13,14].
The CAMPATH-1 antigen can be extracted from cell homogenates into the aqueous phase of a chloroform:methanol:water system [3] and it can be detected by Western blotting as a broad band of apparent molecular weight 21-28 kD. Treatment with N-glycanase reduces the apparent molecular weight to about 6 kD but the antigenicity is not diminished. The molecule is resistant to treatment with narrow specificity proteases but treatment with broad specificity proteases reduces its apparent molecular weight substantially without affecting antigenicity. However, the antigen is very sensitive to treatment with mild alkali [3].
Antigen extracted from human spleens with chloroform/methanol could be further purified by affinity chromatography using the CAMPATH-1 antibodies. We have now used this purified antigen as the starting point for studies leading to the characterisation of the primary structure of the peptide backbone of CDw52 antigen. A synthetic peptide having the amino acid sequence of the peptide backbone of CDw52 antigen, or an antigenic fragment thereof, may be used to assay for, purify or induce CDw52 antibody.
Accordingly, the present invention provides a peptide having the amino acid sequence as set forth in SEQ ID NO: 1 to 7 or an antigenic fragment of the said peptide. The antigenic fragment may be from 2 to 6 amino acid residues long, for example, 2,3,4,5 or 6 amino acid residues long.
The peptide or antigenic fragment constitute a synthetic antigen. The peptide or fragment thereof may be chemically synthesised from single amino acids and/or preformed peptides of two or more amino acid residues. Solid phase or solution methods may be employed.
The synthetic antigen may be used in a simple and reliable assay for concentration. Many types of assay may be used, for example the antigen may be coupled to plastic microtitire plates and the binding of antibody may be determined using an enzyme-labelled antiglobulin. An assay based on synthetic antigen may be useful for quality control tests during the production of antibody and also for measuring the levels of CAMPATH-1 antibodies in patients serum.
The invention therefore further provides a method of assaying CDw52 antibody in
Hale Geoffrey
Tone Masahide
Xia Meng-Qi
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