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Title:  Antibodies against human IL-12
United States Patent: 
7,026,456
Issued: 
April 11, 2006
Inventors:
 Gately; Maurice Kent (Parsippany, NJ); Presky; David Howard (Glen Ridge, NJ)
Assignee: 
Hoffman-La Roche, Inc. (Nutley, NJ)
Appl. No.: 
652282
Filed: 
August 30, 2000


 

Woodbury College's Master of Science in Law


Abstract

The present invention relates to novel p75 heterodimer specific anti-human IL-12 antibodies that are characterized by a higher potency and greater efficacy in neutralizing human IL-12 bioactivity than known heterodimer specific IL-12 monoclonal antibodies. The heterodimer specific antibodies recognize one or more epitopes of the human IL-12 p75 heterodimer, but do not bind to the p40 subunit alone. The heterodimer specific IL-12 antibodies neutralize rhesus monkey IL-12 bioactivity with a potency similar to their potency for neutralizing human IL-12 bioactivity making them useful IL-12 antagonists for in vivo studies in the rhesus monkey.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been found that when IL-12 antibodies are produced from mammals deficient in the gene encoding the p35 IL-12 subunit and/or the gene encoding the p40 IL-12 subunit, IL-12 antibodies are obtained which selectively immunologically react with epitopes of the p75 heterodimer of IL-12, and are identified by their ability to selectively immunologically react with the p75 heterodimer of human IL-12, but not immunologically react with the p40 subunit alone.

Unlike previously known IL-12 p75 antibodies, antibodies which substantially neutralize the bioactivity of human IL-12, i.e., neutralize at least about 90% bioactivity of human IL-12, are produced by the methods described herein. In addition, IL-12 p75 heterodimer specific antibodies of the present invention cross react with rhesus monkey IL-12.

The IL-12 antibodies described herein neutralize at least about 90% bioactivity of human IL-12 by inhibiting at least about 90% IL-12 induced PHA-activated human lymphoblast proliferation at concentrations of at least about 0.5 μg/ml, and/or inhibiting at least about 90% of IL-12 stimulated IFN-γ production by PHA-activated human lymphoblasts at concentrations of at least about 0.5 μg/ml. Furthermore, the antibodies described herein have been shown to specifically inhibit IL-12-induced, but not IL-2-induced, proliferation of PHA-activated human lymphoblasts. PHA-activated lymphoblasts are prepared as follows. Peripheral blood mononuclear cells (PBMC) were isolated (Gately et al., J. Natl. Cancer Inst., 69:1245 (1982)) and stimulated with 0.1% PHA-P (Difco Labs., Detroit, Mich.). After 3 days, the cultures were split 1:1 with fresh medium and recombinant 50 U/ml human IL-2 (provided by Dr. F. Khan, Hoffmann-La Roche Inc.) as described in Gately, M. K., Chizzonite, R. and Presky, D. H., Measurement of human and mouse interleukin 12, Current Protocols in Immunology, vol. 1. J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., John Wiley & Sons, Inc., New York, 1995, pp. 6.16.1-6.16.15. The PHA-activated lymphoblasts were used after an additional one day incubation period.

In accordance with the present invention, the IL-12 antibodies are identified for their ability to selectively bind the epitope presented by the p75 heterodimer, but not immunologically react with any epitope presented by the p40 subunit. This selectivity is defined by the fact that the IL-12 antibodies of this invention will react, at a certain minimal concentration, with an epitope solely presented by a given amount of the p75 heterodimer but will not react at that concentration with an epitope presented by the p40 subunit of that same given amount of this p75 heterodimer. In this way the antibodies of this invention have a higher affinity for an epitope solely presented by the p75 heterodimer than any epitope presented by the p40 subunit. Any conventional assay for identifying selective binding of the antibodies to the p75 heterodimer can be used. Generally, in such an assay, antibodies are incubated with human IL-12 p75 heterodimer to determine if the antibodies bind the p75 heterodimer. The antibodies are also incubated with human IL-12 p75 heterodimer in the presence and absence of the p40 subunit to determine if the presence of the p40 subunit blocks antibody binding or capture of the p75 heterodimer. For example, competitive immunoprecipitation assays (see Example 7 herein) can be used to demonstrate that the antibodies described herein selectively immunologically react with the p75 heterodimer of human IL-12, but are not immunologically reactive with the p40 subunit alone.

In accordance with the present invention, the IL-12 antibodies described herein are produced through the use of knock-out mammals. The knock-out mammals are deficient in the gene encoding the p35 subunit and/or the gene encoding the IL-12 p40 subunit and thus, do not express the IL-12 p75 heterodimer. When immunized with the IL-12 p75 heterodimer, the IL-12 p35 subunit deficient and/or the IL-12 p40 subunit deficient knock-out mammal recognizes the IL-12 p75 heterodimer as foreign and produces antibodies thereto. Preferably, the knock-out mammal is a mouse. In accordance with the present invention knock-out mammals are produced by methods that have been described in the art. Knock-out mammals can be produced by conventional means such as by mutation of the gene encoding the p35 IL-12 subunit and/or the p40 IL-12 subunit. For example, mice carrying a mutation in the IL-12 p35 subunit gene can be produced as described by Mattner, F., et al., Eur. J. Immunol., 26:1553-1559 (1996). Mice carrying a mutation in the IL-12 p40 subunit gene can be produced as described by Magram, J., et al., Immunity, 4: 471-481(1996).

In accordance with the present invention, polyclonal and monoclonal antibodies that selectively immunologically react with the p75 heterodimer of human IL-12 are produced from activated cells of the aforementioned knock-out mammal by any conventional means known in the art. Generally, the antibodies are produced by (a) immunizing a "knock-out" mammal deficient in a gene encoding the p35 subunit and/or the p40 subunit with human p75 heterodimer to produce antibodies; (b) obtaining antibodies from the immunized mammal; and (c) screening the antibodies for their ability to selectively bind the epitope presented by the p75 heterodimer to obtain the selectively binding antibodies.

The IL-12 monoclonal antibodies of the present invention which selectively immunologically react with the human IL-12 p75 heterodimer are generally produced by a method including the following steps:

  • (1) immunizing a knock-out mammal, such as, for example, a mouse deficient in the gene encoding the IL-12 p35 subunit and/or IL-12 p40 subunit, with human IL-12 p75 heterodimer;
  • (2) selecting cells from the immunized knock-out mammal that have been activated to express antibodies against IL-12, such as, splenocytes or lymph node cells;
  • (3) fusing the harvested cells to myeloma cells to form hybridoma cells;
  • (4) selecting hybridoma cells which secrete antibodies that recognize human IL-12, for example, by testing hybridoma conditioned medium for the presence of anti-human IL-12 antibodies, for example, through the use of ELISA or immunoprecipitation assays employing labeled or unlabeled human IL-12; and
  • (5) determining if the antibodies are p75 heterodimer specific by demonstrating that the antibodies immunologically react with an epitope of the p75 IL-12 heterodimer, but are not immunologically reactive with any epitope of the p40 subunit, by incubating the antibodies with human IL-12 p75 heterodimer to determine if the antibodies bind the p75 heterodimer, and then incubating the antibodies with human IL-12 p75 heterodimer in the presence and absence of the p40 subunit to determine if the presence of the p40 subunit blocks antibody binding or capture of the p75 heterodimer. For example, competitive immunoprecipitation assays (see Example 7 herein) can be used to demonstrate that the antibodies described herein selectively immunologically react with the p75 heterodimer of human IL-12, but are not immunologically reactive with the p40 subunit alone.

The method for producing the p75 heterodimer specific IL-12 monoclonal antibodies of the present invention can further comprise the step of determining the ability of the heterodimer specific IL-12 antibodies to inhibit both human and rhesus monkey IL-12 bioactivity in any in vitro or in vivo assay system for IL-12 bioactivity, such as assays for determining IL-12-stimulated proliferation of activated lymphocytes, IL-12-stimulated production of IFN-γ, or IL-12-stimulated enhancement of cytolytic activity.

The anti-human IL-12 antibodies of the present invention can be isolated to substantially pure form by standard methods known in the art, such as, for example, ammonium sulfate precipitation, affinity chromatography, or ion exchange chromatography.

Variations of the method for obtaining the antibodies of the present invention are also encompassed within the present invention. Methods known in the art such as, for example, Western blotting, competitive immunoprecipitation assays, or cross-blocking binding assays can be used to determine if the antibodies are p75 heterodimer specific.

In addition to mice, mammals such as rats and rabbits deficient in the IL-12 p35 subunit gene and/or IL-12 p40 subunit gene, can be immunized with the IL-12 p75 heterodimer to produce the antibodies described herein. The deficiency or mutation in the IL-12 p35 subunit gene and/or IL-12 p40 subunit gene can be any deficiency or mutation that results in lack of expression of IL-12 p75 heterodimer. Furthermore, any conventional method for obtaining mammalian cells carrying a mutation in the IL-12 p35 subunit gene and/or IL-12 p40 subunit gene which results in IL-12 p75 deficient phenotype can be used.

In accordance with the present invention, activated mammalian cells expressing antibodies to the human IL-12 p75 heterodimer can be obtained by immunizing a mouse or other mammal with natural human IL-12 or recombinant IL-12. Natural human IL-12 and recombinant human IL-12 can be prepared by any conventional technique known in the art, such as the techniques provided in the examples herein.

Suitable myeloma cell lines, i.e., fusion partners, for use in producing the hybridomas that secrete the IL-12 antibodies of the present invention include myeloma cell lines well known in the art, such as, for example, SP 2/0 and NS/O cell lines. SP2/0 mouse myeloma cells are preferred. Preferably, the myeloma fusion partner and the mammalian cell activated against the IL-12 p75 heterodimer are derived from the same species.

Hybridoma cells producing the antibodies of the present invention can be selected and isolated by any conventional methods known in the art. Preferably, myeloma cells and lymphocytes activated against the IL-12 p75 heterodimer are cultured together in media containing a selection agent capable of killing the myeloma cells but not the lymphocytes. Hybridomas are formed from myeloma cells that fuse with the lymphocytes activated against the IL-12 p75 heterodimer. Such hybridoma cells are capable of growing in the media containing the selection agent because DNA of the lymphocytes supplies to the myeloma cell line the necessary gene encoding an enzyme that prevents the toxic effects of the selection agent by allowing an alternate metabolic pathway to replace the metabolic pathway blocked by the selection agent. Any unfused lymphocytes die because they are not transformed and have short, finite lifetimes in vitro. In accordance with the present invention a suitable selection agent for use in selecting out hybridoma cells is aminopterin. A preferred medium for cultivating the hybridoma cells is Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS (Hyclone), 100 Units/ml penicillin G (Bio Whittaker), 100 μg/ml streptomycin (Bio Whittaker), 250 ng/ml Fungizone (Bio Whittaker), 2 mM glutamine (Bio Whittaker), 100 μg/ml gentamicin sulfate (Bio Whittaker), 50 μM 2-mercaptoethanol (BioRad), 100 μM hypoxanthine (Sigma), 400 nM aminopterin (Sigma), 16 μM thymidine (Sigma), and 2.5% P388D1 supernatant (produced as described by Nordan, R. P., et al., J. Immunol., 139:813 (1987)).

The potency of the IL-12 antibodies of the present invention is determined with respect to the concentration of IL-12 antibodies at which 50% of maximal inhibition of IL-12 bioactivity occurs as measured by IL-12-stimulated human lymphoblast proliferation or IFN-γ production assays. The anti-human IL-12 antibodies of the present invention exhibit higher potency than previously characterized heterodimer specific IL-12 antibodies. In addition, the anti-human antibodies of the present invention exhibit greater efficacy, as measured by the extent of maximal inhibition of IL-12-stimulated lymphocyte proliferation or IFN-γ production, than previously characterized heterodimer specific IL-12 antibodies.

The potency and efficacy of the antibodies described herein can be determined by any conventional assay known in the art, such as, for example, IL-12 induced lymphoblast proliferation assays or IFN-γ synthesis assays.

In accordance with the present invention, any conventional method known in the art can be used to determine inhibition of human IL-12 stimulated lymphoblast proliferation by the IL-12 antibodies. In general, human lymphocytes can be activated by a number of methods, including treatment with mitogenic lectins, e.g. phytohemagglutinin A (PHA), or other activating agents, alone or in combination, such as cytokines, phobol esters and ionophores, antibodies directed against cell surface molecules, or any other method which will lead to activation of the lymphocytes. The activated lymphocytes are then incubated with and without IL-12 in the absence or presence of the antibodies, and the rate of lymphocyte proliferation is measured by determining the rate of DNA synthesis by measuring the incorporation of 3H-thymidine into DNA, by counting the number of cells present after various periods of treatment, or any other method that can be used to monitor the rate of cellular proliferation. Inhibition of proliferation is determined by comparing lymphocyte proliferation at a defined concentration of IL-12 in the absence and presence of various concentrations of anti-IL-12 antibodies.

In a standard lymphocyte proliferation assay, inhibition of human IL-12 stimulated PHA-activated human lymphoblast proliferation is determined with respect to levels of human IL-12 stimulated PHA-activated human lymphoblast proliferation without any added antibodies and background levels of PHA-activated human lymphoblast proliferation, i.e., proliferation in the absence of both IL-12 and antibodies. In general, IL-12-stimulated levels of proliferation yield about 10,000-80,000 cpm in our standard human lymphocyte proliferation assay, with background levels of proliferation yielding about 5,000-20,000 cpm. Due to the inherent variability between batches of stimulated PHA-activated human lymphoblasts, only assays in which the ratio of stimulated proliferation to background proliferation (i.e. the stimulation index) was equal to or greater than 3 are considered valid for the measurement of IL-12-stimulated proliferation.

In accordance with the present invention, any conventional method for determining inhibition of IFN-γ production by the IL-12 antibodies can be used. For example, activated human lymphocytes, prepared as described herein, or activated human peripheral blood mononuclear cells (PBMC), prepared by treating whole blood or isolated PBMC with mitogenic agents including lectins, cytokines, phobol esters, ionophores, or antibodies directed against cell surface molecules, alone or in combination, or by any other method which will lead to the production of activated human PBMC, are incubated with or without IL-12 and various other agents, e.g. IL-2 and/or IL-1 β, in the absence and presence of the antibodies. IFN-γ production is then determined, e.g. by sampling the culture medium and determining the concentration of IFN-γ by ELISA or any other method that can quantitatively measure IFN-γ. Inhibition of IFN-γ production is determined by comparing IFN-γ production at a defined concentration of IL-12 in the absence and presence of various concentrations of anti-IL-12 antibodies.

In a standard IFN-γ synthesis assay inhibition of IFN-γ is determined with respect to IL-12-stimulated IFN-γ production and background levels of IFN-γ production, i.e., IFN-γ synthesis in the presence or absence of IL-12. In general, IL-12-stimulated levels of IFN-γ production are about 7-220 ng/ml, with background levels of production yielding about 1-3 ng/ml.

The antibodies herein neutralize rhesus monkey IL-12 bioactivity with a potency similar to their potency for inhibiting human IL-12 bioactivity, making them useful IL-12 antagonists for in vivo studies in the rhesus monkey. The increased potency and efficacy of these anti-human IL-12 antibodies and their cross reactivity with rhesus monkey IL-12 make them excellent candidates for designing effective IL-12 antagonists for use in humans.

In particular, the present invention provides four antibodies, 5F2, 16F2, 16G2 and 20E11 to the p75 heterodimer of human IL-12 which are produced by hybridomas having ATCC designation numbers HB-12446, HB-12447, HB-12449, and HB-12448, respectively. These hybridomas were deposited on Dec. 11, 1997, with the ATCC (American Type Culture Collection), 10801 University Boulevard, Manassas, Va. 20110-2209, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and comply with the criteria set forth in 37 C.F.R. 1.801-1.809 regarding availability and permanency of deposits. However, the present invention is not limited to these four antibodies. Any antibodies having the characteristics described herein are encompassed.

FIG. 6 provides the nucleotide sequence (SEQ ID NO: 1) encoding a portion of the heavy chain variable region of the p75 heterodimer specific 16G2 antibody and the amino acid sequence (SEQ ID NO: 2) deduced from this nucleotide sequence. The nucleotide sequence (SEQ ID NO: 3) encoding a portion of the heavy chain variable region of the p75 heterodimer specific 20E11 antibody and the amino acid sequence (SEQ ID NO: 4) deduced from this nucleotide sequence (SEQ ID NO: 3) is provided in FIG. 7. It will be understood by those skilled in the art that conservative amino acid changes can be made in the constant regions of the heterodimer specific IL-12 antibodies herein without significantly affecting the antigen binding specificity/affinity. Heterodimer specific IL-12 antibodies containing amino acid changes in the variable framework regions or complementary determining regions can be expected to have a greater effect on antigen binding specificity/affinity.

The IL-12 antibodies of the present invention can be complete antibodies including two full length heavy chains and two full length light chains. Alternatively, the IL-12 antibodies can be constructs such as single chain antibodies or "mini" antibodies that retain binding activity to one or more epitopes of the IL-12 p75 heterodimer. Such constructs can be prepared by methods known in the art such as, for example, the PCR mediated cloning and assembly of single chain antibodies for expression in E. coli (as described in Antibody Engineering, The practical approach series, J. McCafferty, H. R. Hoogenboom, and D. J. Chiswell, editors, Oxford University Press, 1996). In this type of construct, the variable portions of the heavy and light chains of an antibody molecule are PCR amplified from cDNA. The resulting amplicons are then assembled, e.g. in a second PCR step, through a linker DNA that encodes a flexible protein linker composed of the amino acids GLY and SER. This linker allows the variable heavy and light chain portions to fold in such a way that the antigen binding pocket is regenerated and antigen is bound with affinities often comparable to the parent full-length dimeric immunoglobulin molecule.

The IL-12 antibodies of the present invention are useful antagonists for controlling diseases with pathologies that are mediated through immune mechanisms, particularly, diseases associated with increased IL-12 bioactivity that results in aberrant Th1-type helper cell activity. In accordance with the present invention, the IL-12 antibodies are used for treating autoimmune disorders in humans or other mammals, such as, for example, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes mellitus, and inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis. The antibodies described herein can also be used to treat other disease conditions which have been shown to benefit from the administration of IL-12 antibodies including, for example, transplantation/graft-versus-host disease and septic shock.

The dose ranges for the administration of the IL-12 antibodies herein may be determined by those of ordinary skill in the art without undue experimentation. In general, appropriate dosages are those which are large enough to produce the desired effect, i.e., neutralizing at least 90% IL-12 bioactivity. However, the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of disease in the patient, counter indications, if any, immune tolerance and other such variables, to be adjusted by the individual physician.

The IL-12 antibodies may be administered parenterally by injection or by gradual perfusion over time. They can be administered intravenously, intramuscularly, or subcutaneously. Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

Preferred dosages of the IL-12 antibodies of the present invention are from about 0.1 mg/kg to about 10 mg/kg, two to three times per week. However, the dosage and dosage schedule for administration of the IL-12 antibodies herein may vary depending on the individual to be treated, the antibody administered, and the variables discussed above. In accordance with the present invention, the IL-12 antibodies may be administered alone or in combination with other therapeutically active agents.

The anti-human IL-12 antibodies described herein may be humanized to form antibodies that possess the same or substantially similar affinity for the IL-12 p75 heterodimer as mammalian anti-human IL-12 antibodies, but are substantially non-immunogenic in humans. For example, a humanized IL-12 antibody in accordance with the present invention can include heavy and light chain framework regions of human antibodies. Preferably, the amino acid sequences of the humanized antibody framework regions are from about 60% to 95% identical to the donor framework regions. The humanized antibodies may be produced by recombinant techniques well known in the art. Methods for producing humanized immunoglobulins are described in U.S. Pat. No. 5,530,101, the disclosure of which is incorporated herein by reference.
 


Claim 1 of 10 Claims

1. A monoclonal antibody to human IL-12 which consists of a p35 subunit and a p40 subunit forming a p75 heterodimer, wherein said monoclonal antibody

(a) immunologically reacts with an epitope presented by the p75 heterodimer of human IL-12, but is not immunologically reactive with any epitope presented by said p40 subunit; and

(b) neutralizes at least about 90% of the bioactivity of human IL-12, wherein the antibody neutralizes at least about 90% bioactivity of human IL-12 by inhibiting IL-12 stimulated PHA-activated human lymphoblast proliferation wherein the concentration of said antibody is 0.5μ g/ml and the concentration of said human IL-12 is 0.25 ng/ml.

____________________________________________
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