Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-02-03
1998-07-28
Saunders, David
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 435352, 435362, C12N 1509
Patent
active
057861765
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a recombinant CDw52 antigen which may be used in assaying for, purifying or inducing anti-CDw52 antibodies.
The CAMPATH-1 family of monoclonal antibodies recognise an antigen expressed on the majority of human lymphocytes and monocytes (CAMPATH is a Registered Trade Mark of The Wellcome Foundation Limited). At the Fourth Leucocyte Workshop these antibodies were given the provisional designation CDw52. The CDw52 antigen is an unusually good target for complement-mediated attack and 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.
The CDw52 antigen is expressed in most cases of lymphoid malignancy and 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, has been found to be rather effective in this context. Recently, a human IgG1 antibody (CAMPATH-1H) with the same specificity has been constructed by recombinant DNA technology and this can be administered for a longer period and produces even better clinical results (see Riechmann et al., Nature, 332, 323-327, (1988) and Hale et al., The Lancet, 2, 1394-1399 (1988)). Campath-1H has proved to be effective for lymphocyte depletion in vivo and is being tested for the treatment of lymphoma, transplant rejection and various autoimmune diseases.
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 (about 5.times.10.sup.5 molecules per lymphocyte) and lack of modulation are probably relevant factors but do not provide a complete explanation since even small (sub-saturating) amounts of antibody are effective compared with other specificities.
Hale et al., (Tissue Antigens, 35, 118 (1990)) reported that 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). 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.
Hale et al., also reported that the CAMPATH-1 antigen can be extracted from cell homogenates into the aqueous phase of a chloroform:methanol:water system 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 destroying antigenicity. However, the antigen is very sensitive to treatment with mild alkali.
Xia et al., (Eur. J. Immunol. 21, 1677-1684 (1991)) purified the CAMPATH-1 (CDw52) antigen from human spleen and found the antigenic epitope to be heat stable but sensitive to mild alkali treatment. Experiments with phosphatidylinositol-specific phospholipase C also indicated that it is anchored by a GPI anchor. An N-terminal sequence of 11 amino acids was determined, followed by an abrupt stop. Using short overlapping mixed oligonucleotide primers, cDNA synthesized from the mRNA of a human B cell line was amplified by the polymerase chain reaction. The product was used to isolate CDNA clones and the full amino acid sequence of the CAMPATH-1 antigen was deduced.
The amino acid sequence was found to consist of 37 amino acid residues plus a 24-residue signal peptide. It has all the features expected for a GPI-anchored membrane protein except that the predicted mature protein is remarkably short, comprising no more than 18 residues and possibly as few as 12 (depending on the GPI linkage site). Potential attachment sites for carbohydrate are present and it is shown that the
REFERENCES:
patent: 5354665 (1994-10-01), Wallner et al.
patent: 5494999 (1996-02-01), Hale et al.
M-Q Xia et al, European Journal of Immunology, 21, 1677-1684, 1991.
G. Mave et al, Tissue Antigens, 35, 118-127, 1990.
L. Riechmann et al, Nature, 332, 323-327, 1988.
A.R. Carroll et al, Molecular Immunology, 29, 821-827, 1992.
Hale Christine
Hale Geoffrey
Tite John
Tone Masahide
Waldmann Herman
British Technology Group Limited
Saunders David
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