Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
1998-06-10
2001-10-09
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S070100, C435S070200, C435S320100, C435S326000, C435S328000, C435S339000, C424S141100, C424S147100, C424S133100, C530S387100, C530S808000
Reexamination Certificate
active
06300104
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to secretory Ig molecules produced by a single cultured cell. The invention also relates to methods of producing and using the secretory Ig molecules.
BACKGROUND OF THE INVENTION
Secretory IgA (sIgA) is found in external secretions such as colostrum, respiratory and intestinal mucin, saliva, tears and genitourinary tract mucin and is often the first line of defense against infectious agents.
Monoclonal antibodies, specific for different diseases are available to combat infection. However, these monoclonal antibodies are predominantly of the IgG and IgM subclasses, which can be injected into a patient after an infection has been contracted. Monoclonal IgA would be a preferred agent and could be used for treatment and to prevent an infection before it enters the body of the host. Currently available monoclonal IgA is of limited therapeutic use since stable, secretory forms can only be produced in limited amounts and the non-secretory forms are unstable with relatively short half-lives in vivo.
IgA occurs in various polymeric forms including monomers (H
2
L
2
), dimers (H
4
L
4
) and even higher multimers (H
2n
L
2n
). In addition to heavy and light chains, the polymeric forms of IgA also usually contain J chains. The heavy, light and J chains are all produced by a lymphoid cell. Secretory IgA found at the mucosal surface also contains a secretory component (SC) which is attached during transport of the IgA across the epithelial lining of mucosal and exocrine glands into external secretions.
In vivo, sIgA is the product of two different cell types, the plasma cell and the epithelial cell. Plasma cells synthesize and assemble &agr; H-chains and L chains with J chains into polymeric IgA. The polymeric IgA secreted by the plasma cell binds to a polymeric Ig receptor (pIgR) expressed on the basolateral surface of the mucosal epithelium. The IgA-pIgR complex is transcytosed to the apical surface. During transit, a disulfide bond is formed between the IgA and the pIgR. At the apical surface, the IgA molecule is released by proteolytic cleavage of the receptor. This cleavage results in a fragment, approximately 70,000 molecular weight, being retained on the IgA molecule. This fragment is the SC fragment, which is attached by disulfide bonds to the IgA molecule. The IgA-SC complex is thereby released into external secretions.
Passive administration of IgA could provide protection against a wide range of pathogens including bacteria and viruses such as HIV and respiratory syncytial virus. Hybridoma produced IgA antibodies applied directly to mucosal surfaces or transported into external secretions after injection into blood are protective, but have been found to be rapidly degraded (Mazanec et al.,
J. Virol
. 61 2624, 1987; Mazanec et al.,
J. Immunol
. 142 4275, 1989; Renegar et al.,
J. Immunol
. 146 1972, 1991). In vitro, sIgA is more resistant to proteases than serum IgA (Brown et al.,
J. Clin. Invest
. 49 1374, 1970; Lindh,
J. Immunol
. 114 284, 1975) suggesting that sIgA would be a more effective molecule for therapeutic use. However, co-culture systems containing hybridomas and polarized monolayers of epithelial cells (Hirt et al.,
Cell
74 245-255, 1993) and in vitro mixing of purified polymeric IgA (pIgA) and SC (Lullau et al.,
J. Biol. Chem
. 271 16300,1996) have succeeded in producing only analytical quantities of sIgA.
Methods to purify large quantities of dimeric IgA (dIgA) and SC have been developed and noncovalent association of dIgA and SC has been shown by mixing dIgA and SC. However, the formation of disulfide bonds between dIgA and SC in vitro was inefficient. While the initial association between pIgA and SC is noncovalent, subsequent covalent association between IgA and SC requires cellular enzymes.
Nicotiana tabacum
plants producing sIgA have been produced by successive sexual crossing of four transgenic
Nicotiana tabacum
plants producing: murine &kgr; L chain; a hybrid Ig H chain containing an &agr; chain with an additional IgG CH
2
domain; murine J chain; and rabbit SC. (Ma et al.,
Science
268 716-719, 1995). Though the assembly of sIgA in plants has been demonstrated, plant cells attach different sugar residues to proteins than do mammalian cells. This difference in glycosylation patterns may influence the biological properties of sIgA in vivo. In addition, the SC bound to IgA in the plant cells has been shown to be only 50 kDa, which is about 15-20 kDa lower than the expected molecular weight. These results suggest the SC fragment had undergone proteolytic degradation.
There is a need for a method of converting IgA produced in cell cultures, to sIgA which is more stable and more resistant to proteolytic attack. This sIgA should be able to be produced in amounts which make commercial production of the antibody for therapeutic use practical.
SUMMARY OF THE INVENTION
The invention provides a method for producing secretory Ig (sIg) molecules. The method permits the production of large quantities of sIg in a form which is stable and resistant to proteolysis. In addition, the method does not require the use of more than one cell type to produce the sIg. In one embodiment, the method comprises transfecting a cell producing an Ig with a polynucleotide encoding secretory component (SC) to form SC transfected Ig producing cells. The method can further comprise collecting, and optionally, purifying, a supernatant produced by the cell.
The secretory Ig and SC can be derived from the same species or from different species. In one embodiment, the cell endogenously produces Ig, while in an alternative embodiment, the cell is genetically modified to produce Ig. In one embodiment, the SC comprises the amino acid sequence shown in SEQ ID NO:4 or a congener thereof.
The cell can be a mammalian, avian, insect, bacterial or yeast cell. Examples of mammalian cells include, but are not limited to, human, rabbit, rodent (e.g., mouse, rat) and bovine cells. In preferred embodiments, the cell is a myeloma cell, chinese hamster ovary (CHO) cell, L cell, COS cell, fibroblast, MDCK cell, HT29 cell or a T84 cell.
The Ig molecule can be an IgA, IgM, IgG, IgD or IgE. Preferably, the Ig molecule is an IgA. The Ig molecule can be a domain-modified Ig molecule. Examples of domain-modified Ig molecules include, but are not limited to, an IgA molecule having the C
H
2 domain of an IgG molecule, or an IgG molecule having the tailpiece of an IgM molecule. The Ig molecule can be modified by site-directed mutagenesis.
The invention provides a secretory Ig molecule produced by the method of the invention. In a preferred embodiment, the secretory Ig molecule is a secretory IgA.
The invention also provides a pharmaceutical composition comprising a secretory Ig molecule produced by the method of the invention and, optionally, a pharmaceutically acceptable carrier. In a preferred embodiment, the secretory Ig molecule is a secretory IgA.
The invention additionally provides a method of preventing infection in a subject comprising administering a secretory Ig molecule or composition of the invention to the subject. The subject can be a mammal, bird or fish. In one embodiment, the subject is a human. In one embodiment, the infection to be prevented is systemic or at a mucosal surface. The infection can be a bacterial, viral, mycoplasmal, mycobacterial, yeast or parasitic infection. Examples of viral infections include, but are not limited to, a human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), herpes simplex virus (HSV), flu virus or cold virus infection.
Also provided is a method of treating an infection in a subject comprising administering a secretory Ig molecule or composition of the invention to the subject. The subject can be a mammal or bird. In one embodiment, the subject is a human. In one embodiment, the infection to be prevented is systemic or at a mucosal surface. The infection can be a bacterial, viral, mycoplasmal, mycobacterial, yeast or parasitic infection. Examples of viral infections include, but are not limit
Chintalacharuvu Kote R.
Morrison Sherie L.
Allen Marianne P.
Gates & Cooper LLP
The Regents of the University of California
Zeman Mary K.
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