Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1995-05-30
2003-04-22
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Lymphokine
C530S351000
Reexamination Certificate
active
06551585
ABSTRACT:
This invention relates to substances used to enhance or induce an immune response to an antigen. In particular, it is directed to the use of substances normally found in the body for such purposes.
Immunization is a critical component of many industrial and therapeutic processes. The capability to raise high titer, high specificity antisera against a selected antigen is important in the manufacture of antibodies for in vitro and in vivo use and for ensuring successful vaccinations. Vaccination is becoming significant outside its well-known role in protecting against infectious diseases. For example, vaccination against self or syngeneic tumors is a developing technology
1
, as is the use of vaccination to control physiological processes such as reproduction and growth by inducing autoimmune responses that antagonize or agonize biological effector molecules in vivo. In all of these new approaches it is important to induce a vigorous immune response to the target antigen, either humoral or cell mediated. This heretofore has been accomplished by administering an adjuvant in connection with the antigen, and in some cases by complexing the antigen with a carrier. Adjuvants and carriers are substances that in themselves share no immune epitopes with the target antigen but which stimulate the immune response to the target antigen. Freund's adjuvant, a mineral oil emulsion, commonly has been used for this purpose, as have a variety of toxic microbial substances such as mycobacterial extracts
59, 60
. Carriers often act as adjuvants as well, but are generally distinguished from adjuvants in that carriers comprise water insoluble macromolecular particulate structures which aggregate the antigen. Typical carriers include aluminum hydroxide, latex particles, bentonite and liposomes.
Many diverse materials have been demonstrated to possess adjuvant activity, and their mechanisms of action are nearly as varied, as is apparent from several reviews on the subject
61,62,63,64
. Adjuvant effects have been attributed to antigen aggregation, antigen depot formation, altered lymphocyte recirculation, stimulation of T lymphocytes, mitogenic effects on B lymphocytes, activation of phospholipase A, inhibition of prostaglandin synthesis, cell membrane alterations, localization of antigens in thymus-dependent areas of lymph nodes, modified antigen processing by macrophages, and stimulation of macrophage replication and activation
65
. It is clear that the biology of adjuvants is quite complex and that a number of hypotheses exist for the mode of action of immune adjuvants.
Monokines such as interleukin-1 have-been implicated as mediators in adjuvant effects. The adjuvant effect of IL-1 has been attributed to its activity as a lymphocyte growth factor
65,66,47
. Certain synthetic adjuvants also are known to be able to induce interferon synthesis
65
and coadministration of antigen and &ggr;-interferon to mice is known to potentiate immune response to the antigen
9
.
Potentially undesirable effects of the heretofore available adjuvants and adjuvant formulations are well known. A list of side effects of adjuvants includes: (1) sensitization to tuberculin or any other antigen used in screening tests for infections; (2) presence in food animals of materials that cannot safely be ingested by humans; (3) inflammatory, granulomatous, necrotizing, or other unacceptable reactions at injection sites most notably as occurs with Freund's complete adjuvant; (4) pyrogenicity; (5) central nervous system effects and untoward behavioral effects; (6) impairment of growth; (7) arthritis; (8) increased vascular permeability and inflammatory reactions in the eye; (9) induction of undesired autoimmune responses and (10) immune suppression for adjuvant epitopes.
Whether the induction of autoimmune responses is undesirable depends upon the therapeutic objective. For example, the induction of autoimmune responses such as allergic encephalomyelitis in a small minority of subjects would be a very undesirable property of an adjuvant for human use. In contrast, the generation of an autoimmune response is frequently the objective in the veterinary field, e.g., suppressing fertility in animals by inducing autoantibodies against luteinizing hormone-releasing hormone, increasing fertility by vaccinating against endogenous inhibin, or increasing growth by eliciting autoantibodies against somatostatin, and is also desirable in inducing an immune response to host tumors.
Vaccination adjuvants are needed which are capable of use so as to be free of the undesirable side effects noted, above. Accordingly, it is an object of the invention herein to enhance the titer and duration of the mammalian immune response, both humoral and cellular, without toxic reactions. This and other objects of the invention will be apparent from the specification as a whole.
Monocytes and lymphocytes are known to produce the cytokines TNF-&agr;
8
and TNF-&bgr; (previously called lymphotoxin)
2,3
, respectively. Their complete primary structures have been determined and the cDNAs of both TNF-&bgr;
4
and TNF-&agr;
5-7,10
have been cloned by recombinant DNA methods and expressed in
E. coli.
In vivo and in vitro studies using the pure TNFs have shown that both TNF-&agr; and TNF-&bgr; possess the unique ability to kill neoplastic tissue selectively, while sparing most normal cells. In addition to their antitumor activity, these proteins mediate a diverse array of biological responses in vitro. Although their true in vivo significance is still unknown, the biologic studies strongly suggest that TNF-&agr; and TNF-&bgr; play an important role in immunomodulatory and inflammoatory responses.
TNF-&agr; and TNF-&bgr; differ significantly in their physical and chemical properties. TNF-&agr; is a 157 residue polypeptide with a molecular weight of 17,000
8
by SDS-PAGE. Under the same conditions, two different forms of TNF-&bgr;, with molecular weights of about 20,000 (148 residues) and 25,000 (171 residues), have been found
2,3
. The 20 kD species is a proteolytic cleavage product of the 25 kD form.
2,3
The molecular weight of TNFs under non-denaturing conditions are very different. Purified human TNF-&agr; has a native molecular weight of 45,000
8
, whereas TNF-&bgr; elutes at a position corresponding to a molecular weight of 60-70,000
2,3
during gel filtration. The isoelectric points (pI) of these cyto-toxic factors have been reported to be in the range of 4.5-6.5. TNF-&bgr; has a pI of 5.8, and 5.3 is the pI determined for TNF-&agr;.
The amino acid sequence of human TNF-&agr; as determined from the protein
6,7,11
or predicted from the nucleotide sequence
6,7,10,11
has been described. Some variations in the protein sequences at the amino terminal end have been observed. The N-terminal protein sequence of the natural human TNF-&agr; purified from HL-60 cells obtained by Wang et al.,
7
has two discrepancies with the sequence reported earlier
8
and that predicted from the cloned cDNA sequence
5,7
. Two out of the three serines in positions 3-5 of the mature protein are missing, and the His-Val sequence in position 15 and 16 has been replaced by Val-Ser-Val-Ser. The reason for this discrepancy is not clear. Two groups
6,11
have reported the N-terminal sequence of recombinant human TNF-&agr; expressed and purified in
E. coli
in which Val-Arg from position 1 and 2 of the natural protein sequence, respectively, are missing, even though the nucleotide codons for these amino acids are present at the corresponding positions in both the genomic
6
and cDNA
11
sequences. These investigations assumed that the N-terminal sequence of human TNF-&agr; was Ser-Ser-Ser-Arg-. . . based on an analogy with the sequence of rabbit TNF-&agr; purified from serum.
TNF-&agr; has been extensively studied and has been found to exert a variety of effects on normal cells. One major indication of an effect of TNF-&agr; on normal tissue stems from studies on cachectin
12,13,14
, a macrophage secreted factor that inhibited the synthesis of lipoprotein lipase in the mouse adipocyte ce
Shepard H. Michael
Talmadge James E.
Carlson Karen Cochrane
Genentech Inc.
Marschang Diane L.
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