CA1340437C - Interleukin 2 receptor and applications thereof - Google Patents
Interleukin 2 receptor and applications thereofInfo
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- CA1340437C CA1340437C CA000570319A CA570319A CA1340437C CA 1340437 C CA1340437 C CA 1340437C CA 000570319 A CA000570319 A CA 000570319A CA 570319 A CA570319 A CA 570319A CA 1340437 C CA1340437 C CA 1340437C
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- lymphokine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention is related to the field of receptor molecules and complexes. More particularly, the present invention is related to a new polypeptide receptor for interleukin-2 having a molecular weight of about 70-75,000, which is a component of the high affinity IL-2 receptor, antibodies against this new polypeptide, and recombinant interleukins capable of binding to the new receptor. Various applications of the p70-75 receptor, the anti-p70-75 antibodies and IL-2W1 and IL2W2 have also been described.
Description
1340~37 4 Technical Field The present invention is related to the field of 6 receptor molecules and complexes. More particularly, the 7 present invention is related to a new polypeptide 8 receptor for interleukin-2 (IL-2) having a molecular 9 weight of about 70-75,000, which is also a component of the high affinity IL-2 receptor. The invention further 11 relates to antibodies against this new polypeptide and 12 recombinant interleukins which have binding affinity for 13 the new receptor.
14 State of the Art There are at least two categories of cells which 16 respond to interleukin-2 (IL-2). The first category 17 includes those types of cells which must express high 18 affinity IL-2 receptors and express the Tac antigen 19 (defined by anti-Tac monoclonal antibody, denoted herein as p55) for interaction with IL-2 and are designated 21 herein as "Tac-positive" cells. An example of a 22 Tac-positive cell is the activated T cell.
23 The second category includes those types of cells 24 which do not express high affinity IL-2 receptors or . ~. . .. . .
1 p55, but interact with IL-2 by expressing a novel 2 glycoprotein, designated herein as "p70-75", which is 3 distinct and different from p55. Examples of p55 4 negative p70-75 expressing cells are resting large granular lymphocytes (LGL), natural killer (NK) and 6 precursors of lymphokine-activated-killer (LAK) cells.
7 A review of the structure, function and role of 8 IL-2 receptors can be found in Waldmann (Science, 232:
9 727-732, 1986; Cell Immunol., 99:53-60, 1986) and Greene and Leonard (Ann. Rev. Immunol., 4:69-95, 1986).
11 The discovery of p70-75 and elucidation of its 12 nature, function and use in biological systems is the 13 sub~ect matter of the present invention.
It is, therefore, an object of the present 16 invention to provide a new p70-75 peptide having binding 17 affinity for IL-2 at an epitopic site different from that 18 of the p55.
19 It is a further object of the present invention to provide antibodies having specific binding affinity for 21 p70-75.
22 It is another ob;ect of the present invention to 23 provide an altered IL-2 molecule, designated "IL-2W1", 24 having epitopes required for binding with the p70-75 but devoid of epitopes required for binding with the p55.
26 It is a still further object of the present 27 invention to provide altered IL-2 molecule, designated 28 "IL-2W2", having epitopes required for binding with the 29 p55 peptide but devoid of epitopes required for binding with the p70-75 peptide.
., ~
1 It is an additional object of the present 2 invention to provide a method of producing LAK cells by 3 reacting LGL cells with IL-2W1.
4 It is yet another object of the present invention to provide a method of destroying LAK-susceptible cells 6 by contacting said LAK-susceptible cells with LAK cells 7 produced by reacting LGL cells with IL2-W1.
8 A still further object of the present invention is 9 to provide a pharmaceutical composition comprising an effective amount of LAK cells, produced by reacting LGL
11 cells with IL2-Wl, to destroy LAK-susceptible cells, and 12 a pharmaceutically acceptable carrier. This combination 13 may optionally further contain IL2-W1 in an amount 14 sufficient to maintain sustained killer activity of LAK
cells.
16 Various other objects and advantages of the 17 present invention will become apparent from the Detailed 18 Description of the Invention.
19 BRIEF DESCRIPTION OF T~E DRAWINGS
These and other objects, features and many of the 21 attendant advantages of the invention will be better 22 understood upon a reading of the following detailed 23 description when considered in connection with the 24 accompanying drawings wherein:
Figure 1 is a schematic representation of a 26 multichain model of IL-2 receptor in which an 27 independently existing p55 or p70-75 represent low and 28 intermediate affinity receptors, respectively, whereas 29 high affinity receptors are present when both p55 and p70-75 are associated in a receptor complex. Cell lines, 31 as shown on the right that express only the p55 and low 32 affinity receptors, do not respond to the IL-2.
1~0437 1 Activated T cells as shown in the center express both p55 2 and p70-75 and manifest high affinity receptors. Such 3 high affinity receptors are required for certain T cell 4 functions. Certain cells such as lymphokine activated killer precursor cells and natural killer cells express 6 only the p70-75 peptide as shown on the left and 7 manifest intermediate affinity receptors. Such cell 8 types can respond to IL-2 utilizing the p70-75 IL-2 9 binding peptide alone.
DETAILED DESCRIPTION OF THE INVENTION
11 The above and various other ob;ects and advantages 12 of the present invention are achieved by a polypeptide 13 characterized by a molecular weight of about 70-75,000 14 and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin 2.
16 Unless defined otherwise, all technical and 17 scientific terms used herein have the same meaning as 18 commonly understood by one of ordinary skill in the art l9 to which this invention belongs. Although any methods and materials similar or equivalent to those described 21 herein can be used in the practice or testing of the 22 present invention, the preferred methods and materials 23 are now described.
Of course, apart from being distinct from the p55, 26 the p70-75 of the present invention has the following 27 additional properties.
28 (a) A mouse monoclonal antibody L34 that is 29 directed toward the region of amino acids 1 to 25 of IL-2 precipitated unbound IL-2 and IL-2 bound to p55, but did 31 not precipitate IL-2 bound to p70-75 suggesting that 32 amino acids 1-25 are the part of IL-2 bound by p70-75.
.. ......
1 In a parallel study, a polyclonal rabbit antiserum 2 directed toward amino acids 30-55 of IL-2 precipitated 3 IL-2 and IL-2 bound to p70-75 but did not precipitate 4 IL-2 bound to Tac peptide.
These results suggest that amino acids 1-25 of 6 IL-2 are involved in the binding to p70-75.
7 (b) LGL cells obtained from normal human 8 peripheral blood express the p70-75 peptide alone, that 9 is this cell population expresses an IL-2 receptor tP70-75), which is not recognized by anti-Tac monoclonal 11 antibody.
12 (c) LGL leukemic cells manifest only p70-75 and 13 not p55.
14 (d) Even in Tac-negative but IL-2 dependent cell line (such as LGL leukemia cells), it was discovered that 16 IL-2 can provide Tac-inducing signal through the p70-75 17 peptide by upregulation so that transduction, both at the 18 mRNA and protein level, occurs resulting in newly 19 expressed p55 in the initially Tac-negative cells.
Without being bound to any theory, such results from 21 Tac-negative cells lead to the postulation that once the 22 p55 is produced, p55 then associates with the p70-75 23 peptide, the resulting combination of p55 plus p70-75 24 forming the high-affinity IL-2 receptor which in turn is then readily inhibited ~y anti-Tac antibody.
26 Preferred materials and methods and various 27 experiments which support some of the findings noted 28 above are described in Sharon, et al. (Science, 234:
29 859-863, 1986) and Tsudo, et al. (Proc. Natl. Acad. Sci.
USA, 83:9694-9698, 1986).
.. ..
1340~37 1 Preparation of monoclonal antibodies against the p75 2 peptide.
3 Two approaches are used to prepare monoclonal 4 antibodies against the p70-75 peptide. (a) Cell lines MLA 144 or HUT 78 that express the p70-75 peptide are 6 injected into 8 week old female Balb/c mouse on two 7 occasions with a three week interval. 1 x 106 viable 8 cells in 0.2 ml of RPMI 1640 culture media are injected.
9 Cell fusion is performed at three days after the second injection. Spleen cell suspension are obtained by 11 teasing of the spleen and lysis of red blood cells with 12 standard ACK lysing buffer (155mM N~4Cl, lOmM KHC03, 13 O.lmM Na2-EDTA and distilled water). Cell fusions are 14 carried out according to the standard procedure of Kohler and Milstein (Nature, 256:495, 1975). 150 million spleen 16 cells are fused with 3 x 107 NSl mouse myeloma cells 17 using 30~ polyethylene glycol (MW 1,000, Backers, 18 Philipsburg, N.J.) dissolved in Delbecco's Modified 19 Eagle's Media. Cells a-e resuspended in medium and are distributed into flat bottom microtiter plates where they 21 are selected by feeding with HAT media containing 0.1 mM
22 hypoxanthine, 0.4 uM aminopterin, 16 mM thymidine.
23 Fifteen days after cell fusion the culture supernatants 24 from the wells are tested for antibody activity by an ELISA technique to determine reactivity aginst cell lines 26 (e.g. MLA 144 and HUT 78) that express the p70-75 and 27 non-reactivity against cell lines (e.g. MT-l) that do not 28 express p55. After screening, hybridoma cultures that 29 showed an appropriate pattern of reactivity, are expanded and cloned by a limiting dilution method. Fourteen days 31 later when hybridoma clones are expanded, the 32 supernatants are tested again for their reactivity 33 against the same target cells. Two specific systems are ~ 7 ~ 1~40~37 1 used to define blocking and non-blocking antibodies to 2 p70-75. Blocking antibodies are defined by their ability 3 to inhibit the binding of radioactive IL-2 to cell lines 4 such as MLA 144 that express p70-75 but not p55.
Non-blocking antibodies are defined by their ability to 6 precipitate radiolabelled IL-2 crosslinked to p70-75. Of 7 course, radiolabelled IL-2 can be easily prepared by 8 routine standard procedures well known in the art.
9 (b) Xn the second procedure anti-idiotype antibodies to antibodies that are directed toward IL-2 11 are prepared. In such studies, the immunogen is an 12 antibody to the part of the IL-2 molecule that is 13 involved in IL-2 binding to the p70-75. Such anti-IL-2 14 antibodies are defined by their ability to bind to unbound IL-2 or IL-2 bound to p55 but are unreactive to 16 IL-2 bound by crosslinking to p70-75. Antibodies to the 17 idiotype of such antibodies toward IL-2 also crossreact 18 with the part of the p70-75 IL-2 binding receptor 19 involved in IL-2 binding. For such studies, the procedure for the production of a monoclonal antibody 21 vide supra is utilized with the exception that use is 22 made of the monoclonal antibody directed toward IL-2 as 23 the immunogen in lieu of the cell lines expressing 24 p70-75.
It should be noted that various modifications of 26 the p70-75 antibodies such as conjugating the p70-75 27 antibodies with cytotoxic agents (for instance with ricin 28 A or salmonella toxin or cytotoxic radionuclides and the 29 like) to impart cytotoxic properties to the antibodies are easily ac:hieved by routine and standard methods well 31 known in the art. Apart form being useful in the 32 purification of p70-75, antibodies of the present 33 invention, including the cytotoxic antibodies, are useful 34 in neutralizing or killing p70-75 expressing cells such - 8 - 13~0437 1 as p70-75 expressing leukemias and those conditions (such 2 as autoimmune disease or organ allograft protocols) which 3 arise due to the abnormal interaction or pathophysiology 4 caused by the expression of p70-75.
Isolation and Purification of p70-75 6 -. The p70-75 is obtained from any of the 7 Tac-negative, IL-2 responsive cell lines, a few examples 8 of which have been mentioned herein above. Anti-p70-75 9 and a control monoclonal UPC10 (a monoclonal non-reactive with T cells) are used to generate immunoaffinity 11 columns. Imunoaffinity columns are prepared by coupling 12 purified anti-p70-75 and UPC10 monoclonal antibodies to 13 cyanogen bromide activated sepharose. HUT-78 cells are 14 grown in 10% fetal bovine serum/RPMI media collected by ultrafiltration washed twice in phosphate buffered saline 16 and solubized in an extraction buffer containing lOmM
17 Tris pH 7.4, 0.15 M sodium chloride 100 micrograms ml~
18 phenylmethyl sulfonylfluoride (PMSF) 1% Triton-X-100.
19 Extracts are centrifuged and the top lipid layer is discarded. The remaining supernatant is passed over a 21 UPClO column four times and then over an anti-p70-75 22 column three times. The anti-p70-75 column is washed 23 with extraction buffer, then with lOmM Tris pH 7.5, 0.5M
24 NaCl, 1% Triton-X-100, then with lOmM Tris pH 7.5, 0.2%
triton-X-100 and then with lOmM of Tris pH 7.5. The 26 anti-p70-75 column is then eluted with 2.5~ (v/v) acetic 27 acid. The eluted protein is lyophilized, resuspended in 28 phosphate buffered saline and an aliquot is analyzed on 29 SDS-polyacrylamide gels under reducing conditions: Gels are silver stained to assess the yield and purity.
* Trade-mark '~D
9 13~37 1 Preparation of Altered IL-2 2 IL-2W1 molecules that bind to p70-75 alone and 3 IL-2W2 molecules that bind to p55 alone are generated by 4 constructing mutants by site directed mutagenesis. For both IL-2 analogs 18 bases long oligonucleotide chains 6 are syntheslzed using commercially available DNA
7 synthesizers~ The oligonucleotides are complementary to 8 the DNA of the IL-2 gene that has been cloned and is 9 available with the exception that they contain 1-2 mismatches. In the presence of the DNA primer and the 11 four nucleotide precursors of the DNA, the slightly 12 mismatched short DNA segments can function as primers for 13 DNA extention when added to the circular single stranded 14 chains of M13 phage. The resulting complete double stranded DNA will be perfectly complementary except for 16 the mismatched bases at the primer site. After entry 17 into E. coli, DNA replication generates two types of 18 daughter DNA strands; wild type strands and mutant l9 strands, th~ latter containing changes imposed by the mismatched primer. The strands can be easily 21 distinguished by the way they bind the corresponding 22 oligonucleotides. Wild type daughter DNAs bind more 23 strongly to wild type oligonucleotide while the short DNA
24 segment that primes the mutant sequences will bind best to the mutated progeny strands that are to be selected.
26 To generate IL-2Wl that binds to p70-75 but not to the 27 p55, mutations are generated in the peptide region 28 involved in binding to p55. In a complementary study, 29 IL-2W2 that binds to p55 but not to the p70-75 peptide is generated using a slightly mismatched oligonucleotide 31 that has a mismatch at the bases encoding the amino acids 32 involved ln binding to p70-75 but not at the site 33 involved in binding to p55.
13~0~37 1 Activation o~ NK or LAK cells 2 To activate natural killer (NK) and lymphokine 3 activated ki].ler (LAK) cells without activating T
4 lymphocytes, IL-2Wl is selectively and advantageously employed. At: present lymphokine activated killer cells 6 are generated by incubating peripheral blood mononuclear 7 cells with natural IL-2. The activated cells are then 8 readministered to the patient along with daily 9 intravenous doses of IL-2. Certain cancer patients, especially those with renal tumors and melanoma, respond 11 to this therapy; however, there is marked toxicity 12 associated with this approach. A part of this toxicity 13 is considered to be due to the activation of T cells 14 following the interaction of native IL-2, and secretion by these T cells of toxic lymphokines. The present 16 invention for the first time provides means for producing 17 LAK cells without inducing the production of toxic 18 lymphokines. In accordance with the present invention, 19 IL-2Wl, which interacts only with the p70-75, is substituted in vitro and/or in vivo for natural IL-2 for 21 the generation and maintenance of LAK cells. IL-2Wl 22 binds to the p70-75 which is the receptor for IL-2 on 23 large granular lymphocytes (the precursors of natural 24 killer and lymphokine activated killer cells) and is sufficient for IL-2 activation of these cells. In 26 contrast, many T cell functions depend on the 27 simultaneous binding of IL-2 to the p55 and p70-75 IL-2 28 binding peptides of the high affinity receptor complex.
29 Since IL-2Wl does not bind to the Tac peptide, it does not activate T-cells at the concentrations used. Thus, 31 IL-2Wl activates the large granular lymphocytes to become 32 effective natural killer and lymphokine activated killer 33 cells without simultaneously activating T lymphocytes to 34 become activated cells that synthesize 13~0~37 1 toxic lymphokines. Thus, with this recombinant 2 lymphokine IL-2W1 in contrast to natural IL-2, the 3 desired lymphokine activated killer cells to destroy the 4 tumor cell populations are activated without activating T
lymphocytes and their toxic lymphokine.
6 It is understood that the examples and embodiments 7 described herein are for illustrative purposes only and 8 that various modifications or changes in light thereof 9 will be suggested to persons skilled in the art and are to be included within the spirit and purview of this 11 application and scope of the appended claims.
14 State of the Art There are at least two categories of cells which 16 respond to interleukin-2 (IL-2). The first category 17 includes those types of cells which must express high 18 affinity IL-2 receptors and express the Tac antigen 19 (defined by anti-Tac monoclonal antibody, denoted herein as p55) for interaction with IL-2 and are designated 21 herein as "Tac-positive" cells. An example of a 22 Tac-positive cell is the activated T cell.
23 The second category includes those types of cells 24 which do not express high affinity IL-2 receptors or . ~. . .. . .
1 p55, but interact with IL-2 by expressing a novel 2 glycoprotein, designated herein as "p70-75", which is 3 distinct and different from p55. Examples of p55 4 negative p70-75 expressing cells are resting large granular lymphocytes (LGL), natural killer (NK) and 6 precursors of lymphokine-activated-killer (LAK) cells.
7 A review of the structure, function and role of 8 IL-2 receptors can be found in Waldmann (Science, 232:
9 727-732, 1986; Cell Immunol., 99:53-60, 1986) and Greene and Leonard (Ann. Rev. Immunol., 4:69-95, 1986).
11 The discovery of p70-75 and elucidation of its 12 nature, function and use in biological systems is the 13 sub~ect matter of the present invention.
It is, therefore, an object of the present 16 invention to provide a new p70-75 peptide having binding 17 affinity for IL-2 at an epitopic site different from that 18 of the p55.
19 It is a further object of the present invention to provide antibodies having specific binding affinity for 21 p70-75.
22 It is another ob;ect of the present invention to 23 provide an altered IL-2 molecule, designated "IL-2W1", 24 having epitopes required for binding with the p70-75 but devoid of epitopes required for binding with the p55.
26 It is a still further object of the present 27 invention to provide altered IL-2 molecule, designated 28 "IL-2W2", having epitopes required for binding with the 29 p55 peptide but devoid of epitopes required for binding with the p70-75 peptide.
., ~
1 It is an additional object of the present 2 invention to provide a method of producing LAK cells by 3 reacting LGL cells with IL-2W1.
4 It is yet another object of the present invention to provide a method of destroying LAK-susceptible cells 6 by contacting said LAK-susceptible cells with LAK cells 7 produced by reacting LGL cells with IL2-W1.
8 A still further object of the present invention is 9 to provide a pharmaceutical composition comprising an effective amount of LAK cells, produced by reacting LGL
11 cells with IL2-Wl, to destroy LAK-susceptible cells, and 12 a pharmaceutically acceptable carrier. This combination 13 may optionally further contain IL2-W1 in an amount 14 sufficient to maintain sustained killer activity of LAK
cells.
16 Various other objects and advantages of the 17 present invention will become apparent from the Detailed 18 Description of the Invention.
19 BRIEF DESCRIPTION OF T~E DRAWINGS
These and other objects, features and many of the 21 attendant advantages of the invention will be better 22 understood upon a reading of the following detailed 23 description when considered in connection with the 24 accompanying drawings wherein:
Figure 1 is a schematic representation of a 26 multichain model of IL-2 receptor in which an 27 independently existing p55 or p70-75 represent low and 28 intermediate affinity receptors, respectively, whereas 29 high affinity receptors are present when both p55 and p70-75 are associated in a receptor complex. Cell lines, 31 as shown on the right that express only the p55 and low 32 affinity receptors, do not respond to the IL-2.
1~0437 1 Activated T cells as shown in the center express both p55 2 and p70-75 and manifest high affinity receptors. Such 3 high affinity receptors are required for certain T cell 4 functions. Certain cells such as lymphokine activated killer precursor cells and natural killer cells express 6 only the p70-75 peptide as shown on the left and 7 manifest intermediate affinity receptors. Such cell 8 types can respond to IL-2 utilizing the p70-75 IL-2 9 binding peptide alone.
DETAILED DESCRIPTION OF THE INVENTION
11 The above and various other ob;ects and advantages 12 of the present invention are achieved by a polypeptide 13 characterized by a molecular weight of about 70-75,000 14 and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin 2.
16 Unless defined otherwise, all technical and 17 scientific terms used herein have the same meaning as 18 commonly understood by one of ordinary skill in the art l9 to which this invention belongs. Although any methods and materials similar or equivalent to those described 21 herein can be used in the practice or testing of the 22 present invention, the preferred methods and materials 23 are now described.
Of course, apart from being distinct from the p55, 26 the p70-75 of the present invention has the following 27 additional properties.
28 (a) A mouse monoclonal antibody L34 that is 29 directed toward the region of amino acids 1 to 25 of IL-2 precipitated unbound IL-2 and IL-2 bound to p55, but did 31 not precipitate IL-2 bound to p70-75 suggesting that 32 amino acids 1-25 are the part of IL-2 bound by p70-75.
.. ......
1 In a parallel study, a polyclonal rabbit antiserum 2 directed toward amino acids 30-55 of IL-2 precipitated 3 IL-2 and IL-2 bound to p70-75 but did not precipitate 4 IL-2 bound to Tac peptide.
These results suggest that amino acids 1-25 of 6 IL-2 are involved in the binding to p70-75.
7 (b) LGL cells obtained from normal human 8 peripheral blood express the p70-75 peptide alone, that 9 is this cell population expresses an IL-2 receptor tP70-75), which is not recognized by anti-Tac monoclonal 11 antibody.
12 (c) LGL leukemic cells manifest only p70-75 and 13 not p55.
14 (d) Even in Tac-negative but IL-2 dependent cell line (such as LGL leukemia cells), it was discovered that 16 IL-2 can provide Tac-inducing signal through the p70-75 17 peptide by upregulation so that transduction, both at the 18 mRNA and protein level, occurs resulting in newly 19 expressed p55 in the initially Tac-negative cells.
Without being bound to any theory, such results from 21 Tac-negative cells lead to the postulation that once the 22 p55 is produced, p55 then associates with the p70-75 23 peptide, the resulting combination of p55 plus p70-75 24 forming the high-affinity IL-2 receptor which in turn is then readily inhibited ~y anti-Tac antibody.
26 Preferred materials and methods and various 27 experiments which support some of the findings noted 28 above are described in Sharon, et al. (Science, 234:
29 859-863, 1986) and Tsudo, et al. (Proc. Natl. Acad. Sci.
USA, 83:9694-9698, 1986).
.. ..
1340~37 1 Preparation of monoclonal antibodies against the p75 2 peptide.
3 Two approaches are used to prepare monoclonal 4 antibodies against the p70-75 peptide. (a) Cell lines MLA 144 or HUT 78 that express the p70-75 peptide are 6 injected into 8 week old female Balb/c mouse on two 7 occasions with a three week interval. 1 x 106 viable 8 cells in 0.2 ml of RPMI 1640 culture media are injected.
9 Cell fusion is performed at three days after the second injection. Spleen cell suspension are obtained by 11 teasing of the spleen and lysis of red blood cells with 12 standard ACK lysing buffer (155mM N~4Cl, lOmM KHC03, 13 O.lmM Na2-EDTA and distilled water). Cell fusions are 14 carried out according to the standard procedure of Kohler and Milstein (Nature, 256:495, 1975). 150 million spleen 16 cells are fused with 3 x 107 NSl mouse myeloma cells 17 using 30~ polyethylene glycol (MW 1,000, Backers, 18 Philipsburg, N.J.) dissolved in Delbecco's Modified 19 Eagle's Media. Cells a-e resuspended in medium and are distributed into flat bottom microtiter plates where they 21 are selected by feeding with HAT media containing 0.1 mM
22 hypoxanthine, 0.4 uM aminopterin, 16 mM thymidine.
23 Fifteen days after cell fusion the culture supernatants 24 from the wells are tested for antibody activity by an ELISA technique to determine reactivity aginst cell lines 26 (e.g. MLA 144 and HUT 78) that express the p70-75 and 27 non-reactivity against cell lines (e.g. MT-l) that do not 28 express p55. After screening, hybridoma cultures that 29 showed an appropriate pattern of reactivity, are expanded and cloned by a limiting dilution method. Fourteen days 31 later when hybridoma clones are expanded, the 32 supernatants are tested again for their reactivity 33 against the same target cells. Two specific systems are ~ 7 ~ 1~40~37 1 used to define blocking and non-blocking antibodies to 2 p70-75. Blocking antibodies are defined by their ability 3 to inhibit the binding of radioactive IL-2 to cell lines 4 such as MLA 144 that express p70-75 but not p55.
Non-blocking antibodies are defined by their ability to 6 precipitate radiolabelled IL-2 crosslinked to p70-75. Of 7 course, radiolabelled IL-2 can be easily prepared by 8 routine standard procedures well known in the art.
9 (b) Xn the second procedure anti-idiotype antibodies to antibodies that are directed toward IL-2 11 are prepared. In such studies, the immunogen is an 12 antibody to the part of the IL-2 molecule that is 13 involved in IL-2 binding to the p70-75. Such anti-IL-2 14 antibodies are defined by their ability to bind to unbound IL-2 or IL-2 bound to p55 but are unreactive to 16 IL-2 bound by crosslinking to p70-75. Antibodies to the 17 idiotype of such antibodies toward IL-2 also crossreact 18 with the part of the p70-75 IL-2 binding receptor 19 involved in IL-2 binding. For such studies, the procedure for the production of a monoclonal antibody 21 vide supra is utilized with the exception that use is 22 made of the monoclonal antibody directed toward IL-2 as 23 the immunogen in lieu of the cell lines expressing 24 p70-75.
It should be noted that various modifications of 26 the p70-75 antibodies such as conjugating the p70-75 27 antibodies with cytotoxic agents (for instance with ricin 28 A or salmonella toxin or cytotoxic radionuclides and the 29 like) to impart cytotoxic properties to the antibodies are easily ac:hieved by routine and standard methods well 31 known in the art. Apart form being useful in the 32 purification of p70-75, antibodies of the present 33 invention, including the cytotoxic antibodies, are useful 34 in neutralizing or killing p70-75 expressing cells such - 8 - 13~0437 1 as p70-75 expressing leukemias and those conditions (such 2 as autoimmune disease or organ allograft protocols) which 3 arise due to the abnormal interaction or pathophysiology 4 caused by the expression of p70-75.
Isolation and Purification of p70-75 6 -. The p70-75 is obtained from any of the 7 Tac-negative, IL-2 responsive cell lines, a few examples 8 of which have been mentioned herein above. Anti-p70-75 9 and a control monoclonal UPC10 (a monoclonal non-reactive with T cells) are used to generate immunoaffinity 11 columns. Imunoaffinity columns are prepared by coupling 12 purified anti-p70-75 and UPC10 monoclonal antibodies to 13 cyanogen bromide activated sepharose. HUT-78 cells are 14 grown in 10% fetal bovine serum/RPMI media collected by ultrafiltration washed twice in phosphate buffered saline 16 and solubized in an extraction buffer containing lOmM
17 Tris pH 7.4, 0.15 M sodium chloride 100 micrograms ml~
18 phenylmethyl sulfonylfluoride (PMSF) 1% Triton-X-100.
19 Extracts are centrifuged and the top lipid layer is discarded. The remaining supernatant is passed over a 21 UPClO column four times and then over an anti-p70-75 22 column three times. The anti-p70-75 column is washed 23 with extraction buffer, then with lOmM Tris pH 7.5, 0.5M
24 NaCl, 1% Triton-X-100, then with lOmM Tris pH 7.5, 0.2%
triton-X-100 and then with lOmM of Tris pH 7.5. The 26 anti-p70-75 column is then eluted with 2.5~ (v/v) acetic 27 acid. The eluted protein is lyophilized, resuspended in 28 phosphate buffered saline and an aliquot is analyzed on 29 SDS-polyacrylamide gels under reducing conditions: Gels are silver stained to assess the yield and purity.
* Trade-mark '~D
9 13~37 1 Preparation of Altered IL-2 2 IL-2W1 molecules that bind to p70-75 alone and 3 IL-2W2 molecules that bind to p55 alone are generated by 4 constructing mutants by site directed mutagenesis. For both IL-2 analogs 18 bases long oligonucleotide chains 6 are syntheslzed using commercially available DNA
7 synthesizers~ The oligonucleotides are complementary to 8 the DNA of the IL-2 gene that has been cloned and is 9 available with the exception that they contain 1-2 mismatches. In the presence of the DNA primer and the 11 four nucleotide precursors of the DNA, the slightly 12 mismatched short DNA segments can function as primers for 13 DNA extention when added to the circular single stranded 14 chains of M13 phage. The resulting complete double stranded DNA will be perfectly complementary except for 16 the mismatched bases at the primer site. After entry 17 into E. coli, DNA replication generates two types of 18 daughter DNA strands; wild type strands and mutant l9 strands, th~ latter containing changes imposed by the mismatched primer. The strands can be easily 21 distinguished by the way they bind the corresponding 22 oligonucleotides. Wild type daughter DNAs bind more 23 strongly to wild type oligonucleotide while the short DNA
24 segment that primes the mutant sequences will bind best to the mutated progeny strands that are to be selected.
26 To generate IL-2Wl that binds to p70-75 but not to the 27 p55, mutations are generated in the peptide region 28 involved in binding to p55. In a complementary study, 29 IL-2W2 that binds to p55 but not to the p70-75 peptide is generated using a slightly mismatched oligonucleotide 31 that has a mismatch at the bases encoding the amino acids 32 involved ln binding to p70-75 but not at the site 33 involved in binding to p55.
13~0~37 1 Activation o~ NK or LAK cells 2 To activate natural killer (NK) and lymphokine 3 activated ki].ler (LAK) cells without activating T
4 lymphocytes, IL-2Wl is selectively and advantageously employed. At: present lymphokine activated killer cells 6 are generated by incubating peripheral blood mononuclear 7 cells with natural IL-2. The activated cells are then 8 readministered to the patient along with daily 9 intravenous doses of IL-2. Certain cancer patients, especially those with renal tumors and melanoma, respond 11 to this therapy; however, there is marked toxicity 12 associated with this approach. A part of this toxicity 13 is considered to be due to the activation of T cells 14 following the interaction of native IL-2, and secretion by these T cells of toxic lymphokines. The present 16 invention for the first time provides means for producing 17 LAK cells without inducing the production of toxic 18 lymphokines. In accordance with the present invention, 19 IL-2Wl, which interacts only with the p70-75, is substituted in vitro and/or in vivo for natural IL-2 for 21 the generation and maintenance of LAK cells. IL-2Wl 22 binds to the p70-75 which is the receptor for IL-2 on 23 large granular lymphocytes (the precursors of natural 24 killer and lymphokine activated killer cells) and is sufficient for IL-2 activation of these cells. In 26 contrast, many T cell functions depend on the 27 simultaneous binding of IL-2 to the p55 and p70-75 IL-2 28 binding peptides of the high affinity receptor complex.
29 Since IL-2Wl does not bind to the Tac peptide, it does not activate T-cells at the concentrations used. Thus, 31 IL-2Wl activates the large granular lymphocytes to become 32 effective natural killer and lymphokine activated killer 33 cells without simultaneously activating T lymphocytes to 34 become activated cells that synthesize 13~0~37 1 toxic lymphokines. Thus, with this recombinant 2 lymphokine IL-2W1 in contrast to natural IL-2, the 3 desired lymphokine activated killer cells to destroy the 4 tumor cell populations are activated without activating T
lymphocytes and their toxic lymphokine.
6 It is understood that the examples and embodiments 7 described herein are for illustrative purposes only and 8 that various modifications or changes in light thereof 9 will be suggested to persons skilled in the art and are to be included within the spirit and purview of this 11 application and scope of the appended claims.
Claims (20)
1. A polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
2. The polypeptide of claim 1 being a subunit of high affinity Interleukin-2 receptor complex.
3. A method for destroying lymphokine-activated killer-sensitive cells in vitro comprising contacting lymphokine-activated killer sensitive cells with an effective amount of the lymphokine-activated killer cells produced by the interaction of lymphocytes that express the peptide of claim 1 with lymphokine IL-2W1 and then further contacting the lymphokine-activated killer-sensitive cells with a fortifying amount of IL-2W1 to destroy the lymphokine-activated killer-sensitive cells.
4. A recombinant lymphokine analog of IL-2 having an epitope for binding a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
5. A recombinant lymphokine analog of IL-2 having an epitope for binding a p55 tac antigen polypeptide and being devoid of an epitope for binding a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
6. A recombinant lymphokine analog of IL- 2 which is IL-2W1
7. A recombinant lymphokine analog of IL-2 which is IL-2W2.
8. A pharmaceutical composition comprising an effective amount of lymphokine activated killer cells produced by reacting large granular lymphocytes with IL-2W1 and a pharmaceutically acceptable carrier.
9. The composition of claim 8 further comprising an activating amount of IL-2W1.
10. An antibody or a fragment thereof having specific binding affinity for a polypeptide said polypeptide being characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
11. The antibody of claim 10 being conjugated with a cytotoxic agent.
12 The antibody of claim 11 where in the cytotoxic agent is a toxin or a radionuclide.
13. A method for neutralizing or killing p70-75 expressing cells in vitro, comprising reacting p70-75 expressing cells with an effective amount of anti-p70-75 antibody to neutralize or kill p70-75 expressing cells, the p70-75 being a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
14. The method of claim 13 wherein said antibody is conjugated with a cytotoxic ligand.
15. Use of a composition comprising an effective amount of the lymphokine-activated killer cells produced by the interaction of lymphocytes that express the peptide of claim 1 with lymphokine IL-2W1 and a fortifying amount of IL-2W1 to destroy lymphokine-activated killer-sensitive cells.
16. Use of an effective amount of anti-p70-75 antibody to neutralize or kill p70-75 expressing cells, the p70-75 being a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
17. Use of an effective amount of anti-p70-75 antibody conjugated with a cytotoxic ligand to neutralize or destroy p70-75 expressing cells, the p70-75 being a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
18. A use of lymphokine-activated killer cells and a fortifying amount of IL-2W1 for the manufacture of a medication for the therapeutic killing of lymphokine-activated killer-sensitive cells.
19. A use of anti-p70-75 antibody in the manufacture of a medication for the therapeutic effect of neutralizing or killing p70-75 expressing cells, the p70-75 being a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
20. A use of anti-p70-75 antibody further conjugated with a cytotoxic ligand in the manufacture of a medication for the therapeutic neutralizing or killing of p70-75 expressing cells, the p70-75 being a polypeptide characterized by a molecular weight of about 70-75,000 under a reducing state as determined by SDS-polyacrylamide gel and having a specific binding affinity for interleukin-2 (IL-2) at an epitopic site different from that of a p55 tac antigen, and which binds specifically to an epitope contributed to by amino acids 1-25 of interleukin-2 and found expressed on resting large granular lymphocytes where other IL-2 receptors are absent.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US165,302 | 1980-07-02 | ||
| US6698987A | 1987-06-29 | 1987-06-29 | |
| US066,989 | 1987-06-29 | ||
| US16530288A | 1988-03-03 | 1988-03-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1340437C true CA1340437C (en) | 1999-03-16 |
Family
ID=26747386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000570319A Expired - Lifetime CA1340437C (en) | 1987-06-29 | 1988-06-21 | Interleukin 2 receptor and applications thereof |
Country Status (4)
| Country | Link |
|---|---|
| AU (1) | AU1992388A (en) |
| CA (1) | CA1340437C (en) |
| IL (1) | IL86710A0 (en) |
| WO (1) | WO1989000168A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0386289A1 (en) * | 1989-03-07 | 1990-09-12 | Taniguchi, Tadatsugu, Dr. | Recombinant interleukin-2 receptor |
| CA2011450C (en) * | 1989-03-07 | 2002-06-04 | Tadatsugu Taniguchi | Recombinant b-chain of the il-2 receptor |
| EP0408790A1 (en) * | 1989-07-20 | 1991-01-23 | Taniguchi, Tadatsugu, Dr. | Recombinant protein receptor |
| KR0172123B1 (en) * | 1989-03-07 | 1999-02-01 | 게르하르트 후버; 루돌프 호프만 | Recombinant protein receptor |
| US5679160A (en) * | 1995-06-07 | 1997-10-21 | Nd Industries, Inc. | Apparatus for coating threaded fasteners |
| US5807743A (en) * | 1996-12-03 | 1998-09-15 | Ribozyme Pharmaceuticals, Inc. | Interleukin-2 receptor gamma-chain ribozymes |
-
1988
- 1988-05-27 AU AU19923/88A patent/AU1992388A/en not_active Abandoned
- 1988-05-27 WO PCT/US1988/001806 patent/WO1989000168A1/en unknown
- 1988-06-12 IL IL86710A patent/IL86710A0/en unknown
- 1988-06-21 CA CA000570319A patent/CA1340437C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| WO1989000168A1 (en) | 1989-01-12 |
| AU1992388A (en) | 1989-01-30 |
| IL86710A0 (en) | 1988-11-30 |
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