Leukotriene C4 synthase

Details Leukotriene C4 synthase Leukotriene C4 synthase

1994-02-15

2001-03-20

Witz, Jean C. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Reexamination Certificate

active

06204034

ABSTRACT:

BACKGROUND OF THE INVENTION
Leukotrienes (LTs) are products of arachidonic acid metabolism derived through the 5-lipoxygenase pathway. The initial step in LT production involves oxygenation of arachidonic acid to produce 5-hydroperoxyeicosatetranoic acid and a subsequent dehydrase step to produce the epoxide intermediate, LTA
4
, both enzymic steps being catalyzed by 5-lipoxygenase in association with a 5-lipoxygenase activating protein. Two routes of metabolism of LTA
4
lead to the production of biologically active products. LTA
4
hydrolase catalyses the stereoselective hydrolysis of LTA
4
to produce the dihydroxy fatty acid, LTB
4
. LTB
4
interacts with high-affinity receptor sites to induce leukocyte and lymphocyte activation. A second pathway involves conjugation with glutathione to produce the peptidolipid conjugate, LTC
4
, this reaction being catalyzed by LTC
4
synthase. LTC
4
in turn is metabolized to LTD
4
by &ggr;-glutamyl transpeptidase and then to LTE
4
by a dipeptidase. In the human lung LTC
4
, LTD
4
, and LTE
4
all interact with a high affinity LTD
4
receptor.
Further details of the biosynthesis as well as the metabolism of the leukotrienes are to be found in the book
Leukotrienes and Lipoxygenases
, ed. J. Rokach, Elsevier, Amsterdam (1989). The actions of the leukotrienes in living systems and their contribution to various diseases states are also discussed in the book by Rokach.
SUMMARY OF THE INVENTION
LTC
4
synthase has now been identified by photoaffinity labelling and purified to homogeneity. The amino-terminal sequence has been determined from the purified polypeptide.
DETAILED DESCRIPTION
The present invention is related to the purified LTC
4
synthase, which in its enzymically active form has a molecular mass of 38±2 kDa and appears to be a homodimer of two 18 kDa subunits. The enzyme is useful in the identification of specific LTC
4
synthase inhibitors, in the biosynthetic production of LTC
4
, and as an antigen for the production of polyclonal and monoclonal antibodies, which in turn could be used to neutralize the enzyme and thereby prevent the production of the pro-inflammatory LTC
4
.
Monospecific antibodies reactive with LTC
4
synthase are purified from mammalian antisera containing antibodies reactive against the enzyme or are prepared as monoclonal antibodies reactive with the enzyme using the technique of Kohler and Milstein, Nature 256:495-497 (1975). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for LTC
4
synthase. Homogeneous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with LTC
4
synthase, as described herein.
Enzyme specific antibodies are raised by immunizing animals such as mice, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of LTC
4
synthase either with or without an immune adjuvant. Pre-immune serum is collected prior to the first immunization. Each animal receives between about 0.1 &mgr;g and about 1000 &mgr;g of the enzyme associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alumprecipitate, water-in-oil emulsion containing
Corynebacterium paryum
and tRNA. The initial immunization consists of the enzyme in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the enzyme in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 10-14 days after each booster immunization or about biweekly after a single immunization, the animals are bled, the serum collected, aliquoted and stored at about −20° C.
Monoclonal antibodies (mAb) reactive with LTC
4
synthase are prepared by immunizing inbred mice, preferably BALB/c, with the enzyme. The mice are immunized by the IP or SC route with about 0.1 &mgr;g to about 10 &mgr;g, preferably about 1 &mgr;g, of LTC
4
synthase in about 0.5 mL buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 &mgr;g of the enzyme in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11;S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18 and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using the LTC
4
synthase as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in “Tissue Culture Methods and Applications”, Kruse and Paterson, Eds., 276-280, Academic Press, 1973.
Monoclonal antibodies are produced in vivo by injecting pristane primed BALB/c mice, approximately 0.5 mL per mouse, with about 2×10
6
to about 6×10
6
hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
In vitro production of anti-LTC
4
synthase mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques which are well known in the art. Similar assays are used to detect the presence of LTC
4
synthase in body fluids or tissue and cell extracts.
It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for LTC
4
synthase polypeptide fragments or full-length biologically active enzyme.
ENZYMATIC ASSAYS
Measurement of LTC
4
Synthase Activity
LTC
4
synthase activity was measured by the formation of LTC
4
in incubation mixtures containing reduced glutathione (Sigma, St. Louis, Mo.) and LTA
4
(free acid) as determined by reverse-phase HPLC following termination of reactions.
Hydrolysis of LTA
4
Methyl Ester—The methyl ester of leukotriene A
4
was hydrolyzed to the free acid essentially as described by Carrier, D. J. et al., Prostaglandins Leukot. Essent. Fatty Acids,

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