WO1998011917A1

WO1998011917A1 - Hiv-1 therapy with il-2 and anti-tnf antibody

Info

Publication number: WO1998011917A1
Application number: PCT/US1997/016116
Authority: WO
Inventors: Robert E. Walker; Richard V. Mccloskey; James N. Woody
Original assignee: Centocor, Inc.; THE GOVERNMENT OF THE UNITED STATES, represented by the DEPARTMENT OF HEALTH AND HUMAN SERVICES, NATIONAL INSTITUTES OF HEALTH
Priority date: 1996-09-19
Filing date: 1997-09-17
Publication date: 1998-03-26
Also published as: AU4413097A

Abstract

Methods for treating and/or preventing acquired immunodeficiency disease and/or human immunodeficiency virus infection in an individual by co-administering of interleukin-2 and an anti-tumor necrosis factor chimeric antibody are disclosed. Also disclosed is a composition comprising interleukin-2 and an anti-tumor necrosis factor chimeric antibody.

Description

H V-1 THERAPY WITH IL-2 AND ANTI-TNF ANTIBODY

Government Rights

This invention was made with United States Government support. The United States Government has certain rights in this invention.

Background of the Invention

Interleukin-2 (IL-2) has been shown to reverse some of the serious immunologic abnormalities characteristic of human immunodeficiency virus (HIV) infection, especially the depletion of CD4+ thymus-derived (T) lymphocytes (Kovacs et al . , N. Engl . J. Med. 332 (9) : 567-575 (1995)). However, clinical application has been limited by the induction of systemic toxicities in normal tissues.

Summary of the Invention

The present invention pertains to the inhibition of the biological activity of tumor necrosis factor alpha (TNFα in individuals with HIV infection to ameliorate some aspects of the systemic toxicities associated with IL-2- based immunotherapy regimens. The present invention also pertains to the treatment of patients with HIV infection with a combination of anti-TNF chimeric antibody and IL-2 to obtain superior alleviation of symptoms.

A method is provided herein to treat and/or prevent HIV infection and/or acquired immunodeficiency syndrome (AIDS) in an individual comprising co-administering IL-2 and an anti -tumor necrosis factor chimeric antibody or fragment thereof to the individual in therapeutically effective amounts. A method is also provided herein for treating and/or preventing recurrence of HIV infection and/or AIDS in an individual comprising co-administering IL-2 and an anti-TNF chimeric antibody or a fragment thereof to the individual in therapeutically effective amounts .

A further embodiment of the invention relates to compositions comprising IL-2 and an anti-TNF chimeric antibody or fragment thereof .

Detailed Description of the Invention

Serious immunologic abnormalities are characteristic in individuals with HIV infection and/or AIDS. For example, as compared with healthy controls, patients with HIV infection and/or AIDS have decreased levels of CD4+ T lymphocytes .

IL-2 has been shown to increase CD4+ T cells in HIV infected patients. However, HIV infected patients undergoing immunotherapy with IL-2 experience multiple side effects, including capillary leak, severe influenza- like symptoms, hepatic and renal dysfunction, thrombocytopenia, and neutropenia (Kovacs et al . , N. Engl . J. Med. 332(9) -567-575 (1995)). The incidence and severity of these side effects are directly related to the amount of IL-2 administered.

AIDS and AIDS-related complex (ARC) patients undergoing immunotherapy with IL-2 also experience numerous toxicities, including fever with rigors, systolic hypotension, rash, gastrointestinal symptoms, central nervous system side effects, including lethargy, arthralgias, headache, malaise, leukopenia and hepatic dysfunction (Volberding et al . , AIDS Res . Hum . Retro . 3:115-124 (1987); Flad el al . , Lymph . Res . 5:S171-S176 (1986) ) .

A transient increase in plasma viremia is also associated with short term continuous infusions of IL-2.

In addition, although I -2 increases CD4+ T cell levels in patients with HIV infection, it also increases the production of tumor necrosis factor alpha (TNFα) which, in excessive levels, is known to cause painful inflammatory and immunological responses.

As such, in patients with HIV infection, TNFα blockade ameliorates some aspects of the systemic toxicities and viremia burst associated with IL-2 immunotherapy, thereby reducing the incidence and severity of side effects . This result suggests that IL-2 toxicities can be controlled through effective long term TNF blockade. Amelioration of IL-2 toxicities permits administration of IL-2 in higher dosages than would otherwise be tolerated, thereby increasing therapeutic efficacy. Amelioration of I -2 toxicities also permits facilitated administration of IL-2 in therapeutic doses, thereby accelerating the treatment regimen.

The treatment of patients with HIV infection with a combination of anti-TNF chimeric antibody and IL-2 leads to superior alleviation of symptoms.

The present invention is directed to a method for treating and/or preventing HIV infection and/or AIDS in an individual comprising co-administering IL-2 and an anti-TNF antibody or fragment thereof to the individual in therapeutically effective amounts. The anti-TNF antibody and IL-2 can be administered simultaneously or sequentially. The anti-TNF antibody and IL-2 can each be administered in single or multiple doses. Multiple anti- TNF antibodies can be co-administered with IL-2. Other therapeutic regimens and agents can be used in combination with the therapeutic co-administration of anti-TNF antibodies and IL-2.

The invention further relates to a method for treating and/or preventing recurrence of HIV infection and/or AIDS in an individual comprising co-administering IL-2 and an anti-TNF antibody or fragment thereof to the individual in therapeutically effective amounts. The terms "recurrence", "flare-up" or "relapse" are defined to encompass the reappearance of one or more symptoms of the disease state.

In a further embodiment, the invention relates to compositions comprising interleukin-2 and an anti-tumor necrosis factor antibody or fragment thereof . The compositions of the present invention are useful for treating and/or preventing HIV infection and/or AIDS in an individual .

As used herein, an "anti-tumor necrosis factor antibody" decreases, blocks, inhibits, abrogates or interferes with TNF activity in vivo . Anti-TNF antibodies useful in the methods and compositions of the present invention include monoclonal, chimeric, humanized, resurfaced and recombinant antibodies and fragments thereof which are characterized by high affinity binding to TNF and low toxicity (including human anti-murine antibody (HAMA) and/or human anti -chimeric antibody (HACA) response) . In particular, an antibody where the individual components, such as the variable region, constant region and framework, individually and/or collectively possess low immunogenicity is preferred in the present invention. The antibodies which can be used in the invention are preferably characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other properties, may contribute to the therapeutic results achieved.

An example of a monoclonal antibody useful in the methods and compositions of the present invention is murine monoclonal antibody (mAb) A2 and antibodies which will competitively inhibit in vivo the binding to human TNFα of anti-TNFα murine mAb A2 or an antibody having substantially the same specific binding characteristics, as well as fragments and regions thereof. Murine monoclonal antibody A2 and chimeric derivatives thereof, such as cA2 , are described in U.S. Application No. 08/192,093 (filed February 4, 1994), U.S. Application No. 08/192,102 (filed February 4, 1994), U.S. Application No. 08/192,861 (filed February 4, 1994), U.S. Application No. 08/324,799 (filed October 18, 1994), and Le, J. et al . , International

Publication No. WO 92/16553 (published October 1, 1992), which references are entirely incorporated herein by reference. A second example of a monoclonal antibody useful in the methods and compositions of the present invention is murine mAb 195 and antibodies which will competitively inhibit in vivo the binding to human TNFo; of anti-TNFα^* murine 195 or an antibody having substantially the same specific binding characteristics, as well as fragments and regions thereof. Other monoclonal antibodies useful in the methods and compositions of the present invention include murine mAb 114 and murine mAb 199 and antibodies which will competitively inhibit in vivo the binding to human TNFαf of anti-TNFα murine mAb 114 or mAb 199 or an antibody having substantially the same specific binding characteristics of mAb 114 or mAb 199, as well as fragments and regions thereof . Murine monoclonal antibodies 114, 195 and 199 and the method for producing them are described by Mδller, A. et al . (U.S. Patent No. 5,231,024 and CytoJcine 2(3) :162-169 (1990)), the teachings of which are entirely incorporated herein by reference. Preferred methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow, et al . , An tibodies : A Labora tory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988); Colligan et al . , eds . , Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York (1992, 1993); Kozbor et al . , Immunol. Today 4:72-79 (1983); Ausubel et al . , eds. Current Protocols in Mol ecular Biology, Wiley Interscience, New York (1987, 1992, 1993); and Muller, Meth . Enzymol . 52:589-601 (1983), which references are entirely incorporated herein by reference.

Additional examples of monoclonal anti-TNF antibodies that can be used in the present invention are described in the art (see, e.g., U.S. Application No. 07/943,852 (filed September 11, 1992); Rathjen et al . , International Publication No. WO 91/02078 (published February 21, 1991) ; Rubin et a . , EPO Patent Publication 0218868 (published April 22, 1987); Yone et al . , EPO Patent Publication 0288088 (October 26, 1988); Liang, et al . , Biochem .

Biophys . Res . Comm . 137:847-854 (1986); Meager, et al . , Hybridoma 5:305-311 (1987); Fendly et al . , Hybridoma 5:359-369 (1987); Bringman, et al . , Hybridoma 5:489-507 (1987); Hirai, et al . , J^". Immunol. et . 95:57-62 (1987); Moller, et al . , Cytokine 2:162-169 (1990), which references are entirely incorporated herein by reference) .

Chimeric antibodies are immunoglobulin molecules characterized by two or more segments or portions derived from different animal species. Generally, the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as a murine mAb, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, a variable region with low immunogenicity is selected and combined with a human constant region which also has low immunogenicity, the combination also preferably having low immunogenicity. "Low" immunogenicity is defined herein as raising significant HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (Elliott et al . , Lancet 344:1125-1127 (1994), incorporated herein by reference).

As used herein, the term "chimeric antibody" includes monovalent, divalent or polyvalent immunoglobulins . A monovalent chimeric antibody is a dimer (HL) ) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric antibody is a tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge . A polyvalent chimeric antibody can also be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, or μ chain) .

Antibodies comprise individual heavy (H) and/or light (L) immunoglobulin chains. A chimeric H chain comprises an antigen binding region derived from the H chain of a non-human antibody specific for TNF, which is linked to at least a portion of a human H chain C region (CH) , such as CHI or CH2. A chimeric L chain comprises an antigen binding region derived from the L chain of a non-human antibody specific for TNF, linked to at least a portion of a human L chain C region (CL) .

Chimeric antibodies and methods for their production have been described in the art (Morrison et al . , Proc. Natl . Acad. Sci . USA 81:6851-6855 (1984); Boulianne et al . , Na ture 312:643-646 (1984); Νeuberger et al . , Na ture 314:268-270 (1985); Taniguchi et al . , European Patent Application No. 171496 (published February 19, 1985) ; Morrison et al . , European Patent Application No. 173494 (published March 5, 1986); Neuberger et al . , PCT

Application No. WO 86/01533, (published March 13, 1986); Kudo et al . , European Patent Application No. 184187 (published June 11, 1986); Morrison et al . , European Patent Application No. 173494 (published March 5, 1986) ; Sahagan et al . , J. Immunol . 137:1066-1074 (1986); Robinson et al . , International Publication No. PCT/US86/02269 (published May 7, 1987); Liu et a . , Proc . Natl . Acad . Sci . USA 84:3439-3443 (1987); Sun et al . , Proc . Na tl . Acad . Sci . USA 84:214-218 (1987); Better et al . , Science 240:1041-1043 (1988) ; and Harlow and Lane, Antibodies : A Labora tory Manual , Cold Spring Harbor Laboratory, New York, 1988) . These references are entirely incorporated herein by reference .

The anti-TNF chimeric antibody can comprise, for example, two light chains and two heavy chains, each of the chains comprising at least part of a human constant region and at least part of a variable (V) region of non-human origin having specificity to human TNF, said antibody binding with high affinity to an inhibiting and/or neutralizing epitope of human TNF, such as the antibody cA2. The antibody also includes a fragment or a derivative of such an antibody, such as one or more portions of the antibody chain, such as the heavy chain constant or variable regions, or the light chain constant or variable regions.

Humanizing and resurfacing the antibody can further reduce the immunogenicity of the antibody. See, for example, Winter (U.S. Patent No. 5,225,539 and EP 239,400 Bl) , Padlan et al . (EP 519,596 Al) and Pedersen et al . (EP 592,106 Al) . These references are incorporated herein by reference.

Preferred antibodies useful in the methods and compositions of the present invention are high affinity human-murine chimeric anti-TNF antibodies, and fragments or regions thereof, that have potent inhibiting and/or neutralizing activity in vivo against human TNFo; . Such antibodies and chimeric antibodies can include those generated by immunization using purified recombinant TNFα or peptide fragments thereof comprising one or more epitopes.

An example of such a chimeric antibody is cA2 and antibodies which will competitively inhibit in vivo the binding to human TNFα of anti -TNFα murine mAb A2 , chimeric mAb cA2 , or an antibody having substantially the same specific binding characteristics, as well as fragments and regions thereof. Chimeric mAb cA2 has been described, for example, in U.S. Application No. 08/192,093 (filed February 4, 1994), U.S. Application No. 08/192,102 (filed February 4, 1994), U.S. Application No. 08/192,861 (filed February 4, 1994), and U.S. Application No. 08/324,799 (filed October 18, 1994), and by Le, J. et al . (International Publication No. WO 92/16553 (published October 1, 1992)); Knight, D.M. et al . (Mol . Immunol . 30:1443-1453 (1993)); and Siegel, S.A. et al . ( Cytokine 7(l):15-25 (1995)). These references are entirely incorporated herein by reference.

Chimeric A2 anti-TNF consists of the antigen binding variable region of the high-affinity neutralizing mouse anti-human TNF IgGl antibody, designated A2, and the constant regions of a human IgGl, kappa immunoglobulin. The human IgGl Fc region improves allogeneic antibody effector function, increases the circulating serum half -life and decreases the immunogenicity of the antibody. The avidity and epitope specificity of the chimeric A2 is derived from the variable region of the murine A2.

Chimeric A2 neutralizes the cytotoxic effect of both natural and recombinant human TNF in a dose dependent manner. From binding assays of cA2 and recombinant human TNF, the affinity constant of cA2 was calculated to be 1.

Preferred methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow, et al . , Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al . , eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley

Interscience, New York, (1992, 1993); Kozbor et al . , Immunol . Today 4:72-79 (1983); Ausubel et al . , eds. Current Protocols in Molecular Biology, Wiley Interscience, New York (1987, 1992, 1993); and Muller, Λfeth. Enzymol . 92:589-601 (1983), which references are entirely incorporated herein by reference.

As used herein, the term "antigen binding region" refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Generally, the antigen binding region will be of murine origin. In other embodiments, the antigen binding region can be derived from other animal species, such as sheep, rabbit, rat or hamster. Preferred sources for the DNA encoding such a non-human antibody include cell lines which produce antibody, preferably hybrid cell lines commonly known as hybridomas . In one embodiment, a preferred hybridoma is the A2 hybridoma cell line.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of selectively binding to an epitope of that antigen. An antigen can have one or more than one epitope.

The term "epitope" is meant to refer to that portion of the antigen capable of being recognized by and bound by an antibody at one or more of the antibody' s antigen binding region. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. By "inhibiting and/or neutralizing epitope" is intended an epitope, which, when bound by an antibody, results in loss of biological activity of the molecule containing the epitope, in vivo or in vi tro, more preferably in vivo, including binding of TNF to a TNF receptor. Epitopes of TNF have been identified within amino acids 1 to about 20, about 56 to about 77, about 108 to about 127 and about 138 to about 149. Preferably, the antibody binds to an epitope comprising at least about 5 amino acids of TNF within TNF residues from about 87 to about 107, about 59 to about 80 or a combination thereof. Generally, epitopes include at least about 5 amino acids and less than about 22 amino acids embracing or overlapping one or more of these regions.

For example, epitopes of TNF which are recognized by, and/or binds with anti-TNF activity, an antibody, and fragments, and variable regions thereof, include:

59-80 : Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly- Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His- Thr-Ile (SEQ ID NO:l); and/or

87-108: Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-

Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro- Glu-Gly (SEQ ID NO: 2) .

The anti-TNF antibodies, and fragments, and variable regions thereof, that are recognized by, and/or binds with anti-TNF activity, these epitopes block the action of TNFα without binding to the putative receptor binding locus as presented by Eck and Sprang (J. Biol . Chem . 264 (29) : 17595-17605 (1989) (amino acids 11-13, 37-42, 49-57 and 155-157 of hTNFα) . Rathjen et al . , International Publication No. W0 91/02078 (published February 21, 1991) , incorporated herein by reference, discloses TNF ligands which can bind additional epitopes of TNF.

Antibody Production Using Hybridomas

The techniques to raise antibodies to small peptide sequences that recognize and bind to those sequences in the free or conjugated form or when presented as a native sequence in the context of a large protein are well known in the art. Such antibodies can be produced by hybridoma or recombinant techniques known in the art .

Murine antibodies which can be used in the preparation of the antibodies useful in the methods and compositions of the present invention have also been described in Rubin et al . , EP 0218868 (published April 22, 1987); Yone et al . , EP 0288088 (published October 26, 1988); Liang, et al . , Biochem . Biophys . Res . Comm . 137:847-854 (1986); Meager, et al . , Hybridoma 5:305-311 (1987); Fendly et al . , Hybridoma 5:359-369 (1987); Bringman, et al . , Hybridoma 6:489-507 (1987); Hirai, et a . , J. Immunol . Meth . 96:57-62 (1987); Mδller, et al . , Cytokine 2:162-169 (1990).

The cell fusions are accomplished by standard procedures well known to those skilled in the field of immunology. Fusion partner cell lines and methods for fusing and selecting hybridomas and screening for mAbs are well known in the art. See, e.g, Ausubel infra , Harlow infra , and Colligan infra , the contents of which references are incorporated entirely herein by reference.

The TNFα- specific murine mAb useful in the methods and compositions of the present invention can be produced in large quantities by injecting hybridoma or transfectoma cells secreting the antibody into the peritoneal cavity of mice and, after appropriate time, harvesting the ascites fluid which contains a high titer of the mAb, and isolating the mAb therefrom. For such in vivo production of the mAb with a hybridoma (e.g., rat or human), hybridoma cells are preferably grown in irradiated or athymic nude mice. Alternatively, the antibodies can be produced by culturing hybridoma or transfectoma cells in vi tro and isolating secreted mAb from the cell culture medium or recombinantly, in eukaryotic or prokaryotic cells.

In one embodiment, the antibody used in the methods and compositions of the present invention is a mAb which binds amino acids of an epitope of TNF recognized by A2 , rA2 or cA2 , produced by a hybridoma or by a recombinant host. In another embodiment, the antibody is a chimeric antibody which recognizes an epitope recognized by A2. In still another embodiment, the antibody is a chimeric antibody designated as chimeric A2 (cA2) .

As examples of antibodies useful in the methods and compositions of the present invention, murine mAb A2 is produced by a cell line designated cl34A. Chimeric antibody cA2 is produced by a cell line designated C168A. "Derivatives" of the antibodies including fragments, regions or proteins encoded by truncated or modified genes to yield molecular species functionally resembling the immunoglobulin fragments are also useful in the methods and compositions of the present invention. The modifications include, but are not limited to, addition of genetic sequences coding for cytotoxic proteins such as plant and bacterial toxins. The fragments and derivatives can be produced from appropriate cells, as is known in the art. Alternatively, anti-TNF antibodies, fragments and regions can be bound to cytotoxic proteins or compounds in vi tro, to provide cytotoxic anti-TNF antibodies which would selectively kill cells having TNF on their surface.

"Fragments" of the antibodies include, for example, Fab, Fab', F(ab')₂ and Fv. These fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody (Wahl et al . , J. Nucl . Med. 24:316-325 (1983)). These fragments are produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments) . Recombinant Expression of Anti-TNF Antibodies

Recombinant and/or chimeric murine-human or human-human antibodies that inhibit TNF can be produced using known techniques based on the teachings provided in U.S. Application No. 08/192,093 (filed February 4, 1994), U.S. Application No. 08/192,102 (filed February 4, 1994), U.S. Application No. 08/192,861 (filed February 4, 1994), U.S. Application No. 08/324,799 (filed on October 18, 1994) and Le, J. et al . , International Publication No. WO 92/16553 (published October 1, 1992) , which references are entirely incorporated herein by reference. See, e.g., Ausubel et al . , eds. Current Protocols in Molecular Biology, Wiley Interscience, New York (1987, 1992, 1993); and Sambrook et al . Molecular Cloning: A Labora tory Manual , Cold Spring Harbor Laboratory Press, New York

(1989) , the contents of which are entirely incorporated herein by reference. See also, e.g., Knight, D.M., et al . , Mol . Immunol 30:1443-1453 (1993); and Siegel, S.A., et al . , Cytokine 7(l):15-25 (1995), the contents of which are entirely incorporated herein by reference.

The DNA encoding an anti-TNF antibody can be genomic DNA or cDNA which encodes at least one of the heavy chain constant region (He) , the heavy chain variable region (He) , the light chain variable region (Lv) and the light chain constant regions (Lc) . A convenient alternative to the use of chromosomal gene fragments as the source of DNA encoding the murine V region antigen-binding segment is the use of cDNA for the construction of chimeric immunoglobulin genes, e.g., as reported by Liu et al . { Proc . Na tl . Acad . Sci . , USA 84:3439 (1987) and J. Immunology 139:3521 (1987)), which references are entirely incorporated herein by reference. The use of cDNA requires that gene expression elements appropriate for the host cell be combined with the gene in order to achieve synthesis of the desired protein. The use of cDNA sequences is advantageous over genomic sequences (which contain introns) , in that cDNA sequences can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems. An example of such a preparation is set forth below. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding the amino acid. The probability that a particular oligonucleotide will, in fact, constitute the actual XXX- encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an anti-TNF antibody or fragment. Such "codon usage rules" are disclosed by Lathe, et al . , J. Mol . Biol . 183 -.1-12 (1985) . Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding anti-TNF variable or constant region sequences is identified.

Although occasionally an amino acid sequence can be encoded by only a single oligonucleotide, frequently the amino acid sequence can be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the protein.

The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding an anti-TNF antibody or fragment including a variable or constant region is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences. /An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the variable or constant region anti-TNF gene (Sambrook et al . , infra) .

A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the variable or constant anti-TNF region (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above-described procedure) , synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing anti-TNF antibodies or variable or constant regions thereof. Single stranded oligonucleotide molecules complementary to the "most probable" variable or constant anti-TNF region peptide coding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art (Belagaje, et al . , J. Biol . Chem . 254:5765-5780 (1979); Maniatis, et al . , In : Molecular Mechanisms in the Control of Gene Expression , Nierlich, et al . , eds., Acad. Press, New York (1976); Wu, et al . , Prog . Nucl . Acid Res . Molec . Biol . 21:101-141 (1978); Khorana, Science 203:614-625 (1979)). Additionally, DNA synthesis can be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York (1989) ; and by Haynes, et al . , in: Nuclei c Acid Hybridiza tion, A Practical Approach, IRL Press, Washington, DC (1985) , which references are entirely incorporated herein by reference. Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (Hsu, et al . , Proc . Natl . Acad . Sci . USA 82:3771-3775 (1985)), fibronectin (Suzuki, et al . , Bur. Mol . Biol . Organ . J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter, et al . , Proc . Natl . Acad. Sci . USA 82:7889-7893 (1985)), tissue-type plasminogen activator (Pennica, et al . , Nature 301:214-221 (1983)) and human placental alkaline phosphatase complementary DΝA (Keun, et al . , Proc . Na tl . Acad . Sci . USA 82:8715-8719 (1985) ) .

In an alternative way of cloning a polynucleotide encoding an anti-TΝF variable or constant region, a library of expression vectors is prepared by cloning DΝA or, more preferably, cDΝA (from a cell capable of expressing an anti-TΝF antibody or variable or constant region) into an expression vector. The library is then screened for members capable of expressing a protein which competitively inhibits the binding of an anti-TΝF antibody, such as A2 or cA2 , and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as anti-TΝF antibodies or fragments thereof. In this embodiment, DΝA, or more preferably cDΝA, is extracted and purified from a cell which is capable of expressing an anti-TΝF antibody or fragment. The purified cDΝA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DΝA or cDΝA fragments . DΝA or cDΝA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DΝA or cDΝA fragment such as in a lambda phage library, expression in prokaryotic cell (e.g., bacteria) or eukaryotic cells, (e.g., mammalian, yeast, insect or, fungus). See, e.g., Ausubel, infra , Harlow, infra , Colligan, infra ; Nyyssonen et al . Bio/Technology 11:591-595 (1993); Marks et al . , Bio/Technology 11:1145-1149 (October 1993). Once nucleic acid encoding such variable or constant anti-TNF regions is isolated, the nucleic acid can be appropriately expressed in a host cell, along with other constant or variable heavy or light chain encoding nucleic acid, in order to provide recombinant monoclonal antibodies that bind TNF with inhibitory activity. Such antibodies preferably include a murine or human anti-TNF variable region which contains a framework residue having complementarity determining residues which are responsible for antigen binding. Human genes which encode the constant (C) regions of the chimeric antibodies, fragments and regions of the present invention can be derived from a human fetal liver library, by known methods. Human C region genes can be derived from any human cell including those which express and produce human immunoglobulins . The human CH region can be derived from any of the known classes or isotypes of human H chains, including gamma, μ, α, δ or e, and subtypes thereof, such as Gl, G2 , G3 and G . Since the H chain isotype is responsible for the various effector functions of an antibody, the choice of CH region will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity (ADCC) . Preferably, the CH region is derived from gamma 1 (IgGl) , gamma 3 (IgG3) , gamma 4 (IgG4) , or μ (IgM) . The human CL region can be derived from either human L chain isotype, kappa or lambda.

Genes encoding human immunoglobulin C regions are obtained from human cells by standard cloning techniques (Sambrook, et al . (Mol ecular Cloning : A Labora tory Manual , 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989) and Ausubel et al . , eds., Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-1993)). Human C region genes are readily available from known clones containing genes representing the two classes of L chains, the five classes of H chains and subclasses thereof. Chimeric antibody fragments, such as F(ab')₂ and Fab, can be prepared by designing a chimeric H chain gene which is appropriately truncated. For example, a chimeric gene encoding an H chain portion of an F(ab')₂ fragment would include DNA sequences encoding the CHI domain and hinge region of the H chain, followed by a tranεlational stop codon to yield the truncated molecule.

Generally, the murine, human and chimeric antibodies, fragments and regions are produced by cloning DNA segments encoding the H and L chain antigen-binding regions of a TNF- specific antibody, and joining these DNA segments to DNA segments encoding CH and CL regions, respectively, to produce murine, human or chimeric immunoglobulin-encoding genes. Thus, in a preferred embodiment, a fused chimeric gene is created which comprises a first DNA segment that encodes at least the antigen-binding region of non-human origin, such as a functionally rearranged V region with joining (J) segment, linked to a second DNA segment encoding at least a part of a human C region. Therefore, cDNA encoding the antibody V and C regions and the method of producing a chimeric antibody can involve several steps, outlined below:

1. isolation of messenger RNA (mRNA) from the cell line producing an anti-TNF antibody and from optional additional antibodies supplying heavy and light constant regions,- cloning and cDNA production therefrom; 2. preparation of a full length cDNA library from purified mRNA from which the appropriate V and/or C region gene segments of the L and H chain genes can be: (i) identified with appropriate probes, (ii) sequenced, and (iii) made compatible with a

C or V gene segment from another antibody for a chimeric antibody;

3. Construction of complete H or L chain coding sequences by linkage of the cloned specific V region gene segments to cloned C region gene, as described above;

4. Expression and production of L and H chains in selected hosts, including prokaryotic and eukaryotic cells to provide murine-murine , human-murine, human-human or human-murine antibodies .

One common feature of all immunoglobulin H and L chain genes and their encoded mRNAs is the J region. H and L chain J regions have different sequences, but a high degree of sequence homology exists (greater than 80%) among each group, especially near the C region. This homology is exploited in this method and consensus sequences of H and L chain J regions can be used to design oligonucleotides for use as primers for introducing useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.

C region cDNA vectors prepared from human cells can be modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence. For example, one can clone the complete human kappa chain C (Ck) region and the complete human gamma-1 C region (C gamma- 1) . In this case, the alternative method based upon genomic C region clones as the source for C region vectors would not allow these genes to be expressed in bacterial systems where enzymes needed to remove intervening sequences are absent. Cloned V region segments are excised and ligated to L or H chain C region vectors. Alternatively, the human C gamma- 1 region can be modified by introducing a termination codon thereby generating a gene sequence which encodes the H chain portion of an Fab molecule. The coding sequences with linked V and C regions are then transferred into appropriate expression vehicles for expression in appropriate hosts, prokaryotic or eukaryotic .

Two coding DNA sequences are said to be "operably linked" if the linkage results in a continuously translatable sequence without alteration or interruption of the triplet reading frame. A DNA coding sequence is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence . Expression vehicles include plasmids or other vectors. Preferred among these are vehicles carrying a functionally complete human CH or CL chain sequence having appropriate restriction sites engineered so that any VH or VL chain sequence with appropriate cohesive ends can be easily inserted therein. Human CH or CL chain sequence-containing vehicles thus serve as intermediates for the expression of any desired complete H or L chain in any appropriate host .

A chimeric antibody, such as a mouse-human or human-human, will typically be synthesized from genes driven by the chromosomal gene promoters native to the mouse H and L chain V regions used in the constructs; splicing usually occurs between the splice donor site in the mouse J region and the splice acceptor site preceding the human C region and also at the splice regions that occur within the human C, region; polyadenylation and transcription termination occur at native chromosomal sites downstream of the human coding regions.

A nucleic acid sequence encoding at least one anti-TNF antibody fragment may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases . Techniques for such manipulations are disclosed, e.g., by Ausubel, supra , Sambrook, supra, entirely incorporated herein by reference, and are well known in the art.

A nucleic acid molecule, such as DNA, is "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as anti-TNF peptides or antibody fragments in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism and is well known in the analogous art. See, e.g., SamJbrooJ: et al . , Molecular Cloning : A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York (1989); and Ausubel, eds. Current Protocols in Molecular Biology, Wiley Interscience, New York (1987, 1993) . Many vector systems are available for the expression of cloned anti-TNF peptide H and L chain genes in mammalian cells (see Glover, ed. , DNA Cloning, Vol . II, pp. 143-238, IRL Press, Washington, DC, 1985) . Different approaches can be followed to obtain complete H2L2 antibodies. It is possible to co-express H and L chains in the same cells to achieve intracellular association and linkage of H and L chains into complete tetrameric H2L2 antibodies. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both H and L chains can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker. Cell lines producing H2L2 molecules via either route could be transfected with plasmids encoding additional copies of peptides, H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines .

IL-2 Native and recombinant forms of native IL-2 are useful in the methods and compositions of the present invention. Recombinant forms of IL-2 include aldeεleukin (Proleukin^® ; Chiron Therapeutics, Emeryville, CA) and teceleukin (Tecin^®; Hoffmann-LaRoche, Nutley, NJ) . "IL-2", as used herein, include native human IL-2 (hIL-2), mammalian homologs, fragments and mutants which possess essentailly the same activity as hIL-2. Mutants of hIL-2 preferably possess essentially the same sequence as hIL-2 (for example, at least 95% sequence identity) .

Administration

Anti-TNF antibodies, IL-2 and the compositions of the present invention can be administered to an individual in a variety of ways. The routes of administration include intradermal, transdermal (e.g., in slow release polymers), intramuscular, intraperitoneal , intravenous, subcutaneous, oral, topical, epidural, buccal, rectal, vaginal and intranasal routes. Any other therapeutically efficacious route of administration can be used, for example, infusion or bolus injection, absorption through epithelial or mucocutaneous linings, or by gene therapy wherein a DNA molecule encoding the therapeutic protein or peptide is administered to the patient, e.g., via a vector, which causes the protein or peptide to be expressed and secreted at therapeutic levels in vivo . In addition, the anti-TNF antibodies, IL-2 and compositions of the present invention can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants (e.g., glycerides) , excipients (e.g., lactose), carriers, diluents and vehicles. If desired, certain sweetening, flavoring and/or coloring agents can also be added.

In a particular embodiment, IL-2 can be administered to an individual by high- or low- dose bolus injection or continuous intravenous infusion. Any other routes of administration can be used, for example, intraperitoneal, subcutaneous, intrapleural , intrathecal, intrahepatic, intralymphatic and intramuscular routes. The anti-TNF antibodies and IL-2 can be administered prophylactically or therapeutically to an individual. Anti-TNF antibodies can be administered prior to, simultaneously with (in the same or different compositions) or sequentially with the administration of IL-2. For parenteral (e.g., intravenous, subcutaneous, intramuscular) administration, anti-TNF antibodies, IL-2 and the compositions of the present invention can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field of art.

For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution. Anti-TNF antibodies and IL-2 are co-administered in therapeutically effective amounts; the compositions of the present invention are administered in a therapeutically effective amount. As used herein, a "therapeutically effective amount" is such that co-administration of anti- TNF antibody and IL-2, or administration of a composition of the present invention, results in inhibition of the biological activity of TNF relative to the biological activity of TNF when therapeutically effective amounts of antibody and IL-2 are not co-administered, or relative to the biological activity of TNF when a therapeutically effective amount of the composition is not administered. A therapeutically effective amount is also that amount of anti-TNF antibody and IL-2 necessary to significantly reduce or eliminate symptoms associated with HIV infection and/or AIDS.

Once a therapeutically effective amount has been administered, a maintenance amount of antibody alone, of IL-2 alone, or of a combination of antibody and IL-2 can be administered to the individual . A maintenance amount is the amount of antibody, IL-2, or combination of antibody and IL-2 necessary to maintain the reduction or elimination of symptoms achieved by the therapeutically effective dose. The maintenance amount can be administered in the form of a single dose, or a series or doses separated by intervals of days or weeks .

The dosage administered to an individual will vary depending upon a variety of factors, including the pharmacodynamic characteristics of the particular antibodies, and its mode and route of administration; size, age, sex, health, body weight and diet of the recipient; nature and extent of symptoms of the disease being treated, kind of concurrent treatment, frequency of treatment, and the effect desired. In vi tro and in vivo methods of determining the inhibition of TNF in an individual are well known to those of skill in the art. Such in vi tro assays can include a TNF cytotoxicity assay (e.g., the WEHI assay or a radioimmunoassay, ELISA) . In vivo methods can include rodent lethality assays and/or primate pathology model systems (Mathison et al . , J. Clin . Invest . , 82:1925-1937 (1988); Beutler et al . , Science 229:869-871 (1985); Tracey et al . , Na ture 330:662-664 (1987); Shimamoto et al . , Immunol . Let t . 27:311-318 (1988); Silva et al . , J. Infect . Dis . 162 : 421-427 (1990); Opal et al . , J. Infect . Diε . 162:1148-1152 (1990); Hinshaw et al . , Circ . Shock 30:279-292 (1990) ) .

Anti-TNF antibody and IL-2 can be co-administered in single or multiple doses depending upon factors such as nature and extent of symptoms, kind of concurrent treatment and the effect desired. Thus, other therapeutic regimens or agents (e.g., multiple drug regimens) can be used in combination with the therapeutic co-administration of anti- TNF antibodies and IL-2. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art. Usually a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1 to 40 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results. Second or subsequent administrations can be administered at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual .

A second or subsequent administration is preferably during or immediately prior to relapse or a flare-up of the disease or symptoms of the disease. For example, second and subsequent administrations can be given between about one day to 30 weeks from the previous administration. Two, three, four or more total administrations can be delivered to the individual, as needed.

Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.

The present invention will now be illustrated by the following example, which is not intended to be limiting in any way.

EXAMPLES

Example 1

A randomized, controlled, open label study was conducted to evaluate the safety and efficacy of combination therapy with IL-2 and a monoclonal anti-TNF antibody (cA2) in patients infected with HIV who had not been previously treated with IL-2. Studv Infusions

The chimeric monoclonal anti-TNF antibody (cA2) was supplied by Centocor, Inc. (Malvern, PA) in one of the following formulations:

1. A sterile solution containing 5 mg cA2 per ml of 0.01 M sodium phosphate in 0.15 M sodium chloride with 0.01% polysorbate 80, pH 7.2.

2. A sterile solution containing 10 mg cA2 per ml of 0.01 M sodium phosphate in 0.15 M sodium chloride with 0.01% polysorbate 80, pH 7.2.

3. Lyophilized solid.

Before use, the appropriate amount of cA2 was diluted to 250 ml in sterile normal saline by the pharmacist (in the NIH Clinical Center pharmacy) , and administered intravenously via a 0.2 μm in-line filter (Pall Biomedical Products, Fast Hills, NY) over 2-3 hours.

Recombinant interleukin-2 (IL-2) was supplied by Chiron Corp. (Emeryville, CA) . Before use, the IL-2 was diluted in a solution of 5% dextrose in water containing 0.1% albumin.

Patients

Forty-five (45) patients who had HIV-1 infection documented by positive ELISA and Western blot, had a CD4 count between 200 and 500 cells/mm³ of blood, had no serious infection other than HIV-1, had no history of prior AIDS-defining opportunistic infection, and had no malignancy other than Kaposi sarcoma which did not require systemic therapy, were enrolled in the trial.

The enrolled patients had no serious abnormalities of the heart, lung, kidney, thyroid, joints, or central nervous system, did not require continuous daily use of anti -hypertensive medication, had no major laboratory test abnormalities, and had received no pentoxifylline, thalidomide, corticosteroid, chemotherapy, or experimental therapy (other than drugs available on an FDA-approved

"parallel track", "expanded access", or "compassionate use" basis, which were permitted) in the 4 weeks prior to study enrollment .

Examples of "expanded-access" anti-retroviral drugs include zidovudine, didanosine, zalcitabine, stavudine and lamivudine .

Treatment Protocol

Patients were randomized to one of three treatment groups (15 patients per group) : IL-2 alone (Group A) ; IL-2 plus anti-TNF monoclonal antibody (cA2) (Group B) ; and IL-2 plus thalidomide (Group C) .

Randomization was stratified by particle-associated plasma HIV RNA levels determined by branched DNA assay (Chiron Corp., Emeryville, CA) at the screening visit to achieve an equal distribution of patients among the three groups whose bDNA measurements are above and below 10,000 RNA copies/ml.

Each of the 15 patients randomized to Group A received a five day course of IL-2 by continuous infusion via peripheral intravenous (IV) access device at a starting dose of 12 million IU/day. Repeat courses of IL-2 were administered approximately every 8 weeks for one year. Immunologic, virologic, and safety variables were examined after completion of each course of IL-2. The dose was reduced by increments of 1-6 million IU/day or temporarily discontinued for serious side effects.

Each of the 15 patients randomized to Group B received an infusion of either 1, 5 or 10 mg/kg per body weight cA2 immediately prior to beginning IL-2 infusion (day 0) . Thirty minutes after complete infusion of cA2 , patients received a five day course of IL-2 by continuous infusion through a peripheral IV line at a starting dose of 12 million IU/day. On day three, a second dose of cA2 (either 1, 5 or 10 mg/kg per body weight) was administered to each patient either through a second IV catheter or piggybacked into the IL-2 infusion and given concurrently with IL-2. Patients were monitored for adverse events during the cA2 infusion and for 1 hour after its completion, with temperature, blood pressure, pulse, and symptoms assessment recorded every 15 minutes. Repeat courses of IL-2 were administered approximately every 8 weeks for one year. Immunologic, virologic, and safety variables were examined after completion of each course of IL-2. The dose of IL-2 was reduced by increments of 1-6 million IU/day or temporarily discontinued for serious side effects.

Each of the 15 patients randomized to Group C received a 300 mg oral loading dose of thalidomide (Calgene Corp., Warren, NJ) the night prior to starting IL-2 infusion and a second dose of 300 mg orally eight hours later (on day 0) . Beginning eight hours after the second 300 mg dose, the thalidomide dosing regimen was 100 mg PO q8h for the first 5 patients enrolled in Group C, and 300 mg q8h for all patients enrolled into Group C thereafter. Thalidomide was dosed in this way around each IL-2 infusion. This regimen continued to complete 72 hours of dosing after completion of IL-2. IL-2 was administered by continuous infusion via peripheral IV access device at a starting dose of 12 million IU/day by a five day course. Repeat courses of IL-2 were administered approximately every 8 weeks for one year. Immunologic, virologic, and safety variables were examined after completion of each course of IL-2. The dose of IL-2 was reduced by increments of 1-6 million IU/day or temporarily discontinued for serious side effects. Antiretroviral agents were administered concomitantly throughout the IL-2 infusion period and during the intervals between infusions.

Patient Monitoring

Patients were monitored according to the plan outlined in Table 1. Toxicities were graded according to the definitions provided in Tables 2 and 3.

Table 1. Monitoring During the Study

SCREEN H t P Q 4 Q 8 DAYS 3 & 5 DAY 4 OF DAY 6 Q 3-6

WEEKS WEEKS OF IL-2 IL-2 MONTHS

NURSING VI SI 1' X X X X

MD VISIT X X

CBC, PLATELETS, DIFF, X X X X RETIC

COAG PROFILE X X X X

ELECTROLYTES X X X X

HEPATIC, RENAL, MINERAL X X X X

THYROID FUNCTION TESTS X X X

HEPATITIS B,C SCREEN X X

TETANUS TOXOID BOOSTER X^*

URINALYSIS X X X

URINE PREGNANCY TEST X x^*4

CHEST X-RAY X

EOG X X x

ECHOCARDIOGRAM X X

IMMUNE STUDIES (FACS X X PROFILES)

HIV ELISA AND WESTERN BLOT X

VIRAL LOAD(BDNA) X X X X^*** X^*"

SERUM P24 ANTIGEN X X X

SERUM TNF X X X X^*** X^*"

SERUM/CELLS FOR BANKING X X J_ X

* If none administered within the past 2 years.

** Within 24 hrs prior to starting IL-2 or thalidomide, and results must be negative. Women of child bearing potential receiving IL-2 plus thalidomide had urine pregnancy testing performed weekly x 2 prior to each course of therapy, every other day during therapy, and l week after completing therapy. All pregnancy testing was performed on first morning voided specimens.

*** The frequency of these tests may be reduced as the study proceeds.

Table 2 : Standard Toxicity Table

GRADE I GRADE II GRADE III GRADE IV MINIMAL MODERATE SEVERE EXTREME

HEMATOLOGIC

Granulocyte 1000-1250/mm3 750 -999/mm3 500-749/mm3 <500/mm3

Platelets 75-150k/mm3 50-74k/mm3 25-49k/mm3 <25k/mm3

Hemoglobin

Male 10.0-12.6g/dl 8.5-9.9g/dl <2U/2wkε c/s >2U/2wks c/s

EPO EPO needed to needed to maintain maintain

HGB>8.4g/dl HGB>8.4g/dl

Female 10.0-11. Og/dl 8.5-9.9 g/dl <2U/2wks c/s >2U/2wks c/s EPO EPO needed to needed to maintain maintain HGB>8.4g/dl HGB>8.4g/dl

HEPATIC

Bilirubin 1.1-2.Omg/dl 2. l-2.5mg/dl 2.6-3.Omg/dl >3. Omg/dl AST(SGOT) 40-150u/l 151-300u/l 301-600u/l >600u/l outside lab 1.2- .8 x nl 4.9-9.6 x nl 9.7-19.3 x nl >19.3 x nl RENAL

Creatmine 1.5-2.0 g/dl 2.1-2.5mg/dl 2.6-3.5mg/dl >3.5mg/dl Proteinuria 1+ 2 + -3 + 4+ and 4+ and <lg/24hr urine >lg/24hr urine

GASTROINTESTINAL

Nausea/vomiting nausea alone emesιs<5x/day ernes is>5x/day Intractable vomiting, IV fluids needed

Diarrhea 3-4 loose 5-7 loose >7 stools/d Bloody stools/d stools/d Diarrhea, IV fluids needed

Anorexia/ anorexia c or ε 5-14% weight 15-19% weight >20% weight Weight Loss <5% weight loss loss loss loss

OTHER

Fatigue/Malaise fatigue without decreased resting in unable to care decrease activity activity in bed bed>50% of day for self <50% of day Table 3 : Standard Toxicity Table

GRADE I GRADE II GRADE III GRADE IV MINIMAL MODERATE SEVERE EXTREME

NEUROLOGIC Parestheεia mild mod discomfort: severe discomfort : OR incapacity: discomfort ^■ non-narcotic narcotic analgesia OR not no rx. analgesia required with responsive to required required symptomatic narcotic im rovement analgesia

Neuro-motor mild weakness mod weakness in marked distal confined to in muscles of feet (unable to weakness (unable to bed or feet but able walk on heels dorsiflex toes or wheelchair to walk and/or and\or toes) , foot drop), and mod. because of mild increase mild weakness in proximal weakness, muscle or decrease in hands, still e g , in hands weakness reflexes able to do most interfering with hand tasks, ADLs and/or and/or loss of requiring assistance previously to walk and/or present reflex unable to rise from or development chair unassisted of hyperreflexia and/or unable to do deep knee bends due to weakness

Neuro- sensory mild mod impairment severe impairment sensory loss impairment (mod. dec. (dec or loss of involves

(dec. sensation, e g , sensation to knees limbs and sensation, vibratory, or wrists) or loss trunk e.g., pinprick, of sensation of at vibratory hot/cold to least mod degree in pinprick, ankles) and/or multiple different hot/cold in joint position body areas (i.e., great toes) in or mild upper and lower focal area or impairment that extremities) symmetrical is not distribution symmetrical

IL-2 toxicities were managed in the following manner:

Grade Dose adiustment, IL-2

1 no change

2 no change

3 decrease IL-2 dose or stop IL-2

4 stop IL-2

If a patient developed grade 3 or grade 4 toxicity while receiving IL-2, and therapy could not be restarted within 2 days, that course of therapy was terminated. For the next course, the dose was reduced in increments of 1 to 6 million IU/day.

Anti-TNF antibody (cA2) toxicities were managed in the following manner:

Grade dose of cA2 1 no change

2 no change

3 decrease until resolved to < grade 3, re-challenge

4 decrease permanently if thought associated

If grade 3 toxicity recurred on rechallenge, cA2 was permanently discontinued. If grade 4 toxicity occurred but was not thought to be associated with cA2 , rechallenge after resolution of toxicity was attempted. If the toxicity recurred, cA2 was permanently discontinued, however, the patient continued to receive IL-2 according to schedule . Thalidomide toxicities were managed in the following manner :

Grade dose of thalidomide

1 no change 2 no change

3 decrease until resolved to ≤ grade 3, re-challenge at next lower dose (300-200-100-50 g)

4 decrease permanently if thought associated

If grade 3 toxicity recurred on rechallenge, thalidomide was permanently discontinued. If grade 4 toxicity occurred but was not thought to be associated with thalidomide, rechallenge after resolution of toxicity was attempted. If the toxicity recurred, thalidomide was permanently discontinued, however, the patient continued to receive IL-2 according to schedule.

Toxicities thought to be related to the specific anti-retroviral agents taken by the patient were handled according to the clinical judgment of the principal investigator or his designee with input from the patient's personal physician. In the setting of a patient taking both IL-2 and indinavir (Crixivan) , IL-2 was continued for hyperbilirubinemia greater than 2.5 mg/dl, since this is a common side effect of indinavir. If the bilirubin is > 5 mg/dl, either the IL-2 or indinavir or both was discontinued and held until the bilirubin was < 2.5 mg/dl. Indinavir or IL-2 was held for a bilirubin > 2.5 mg/dl if a patient had associated symptoms or other laboratory abnormalities such as transaminase elevations, or if the hyperbilirubinemia was not transient or not of unconjugated bilirubin.

A patient was withdrawn from the study when a life-threatening infection or malignancy, or a progressive Kaposi sarcoma requiring systemic chemotherapy, developed, when the patient desired to leave the study, became pregnant or did not comply with the terms of the study, when it was felt by the principal investigator to be in the patient's best interest, when the patient developed an inability to tolerate any licensed anti-retroviral agent in combination with IL-2, and/or when a grade 4 toxicity likely attributable to IL-2 that did not resolve within 4 weeks duration off IL-2.

The primary endpoints of the study were safety and tolerability of the IL-2/TNF inhibitor combination.

Secondary endpoints included changes in CD4+ T cell counts, frequency and severity of IL-2-related side effects, changes in serum TNF levels (< 25% increase in serum TNF levels during the post-infusion period as compared to pre-infusion levels), and plasma viral load changes (≥ 1 log reduction in plasma viral load by bDNA assay as compared to baseline values that is sustained over at least 2 consecutive measurements at 4 week intervals) . The primary and secondary endpoints in patients treated with IL-2 plus TNF inhibitor were compared to those obtained in patients treated with IL-2 alone.

Prospective studies of cardiovascular, pulmonary, and biliary toxicity of intermittent intravenous I -2 administration were performed during the first 12 months on all participants. By comparing these study parameters in the group receiving IL-2 alone with the 2 groups receiving TNF antagonists, the role of TNF as a mediator of cardiopulmonary and biliary side effects of I -2 was evaluated. Enhanced non- invasive cardiac monitoring included electrocardiograms (EKGs) at the beginning and end of each cycle; serum CK and CK/MB determinations on day 0, day 2, and day 4 of each cycle; MUGA scans at day 0 and day 4 of the first and last cycles; and echocardiograms at day 0 and day 4 of the first, third, and last cycles. MUGA scans involved exposure to ionizing radiation. Each of the 4 scans involved administration of 25 mCi of Technetium (Tc99m) . Scans 1 and 2 were separated from scans 3 and 4 by a period of 10 months. Enhanced pulmonary monitoring included pulmonary function tests (spiro etry, lung volume, and diffusion studies) and pulse oximetry at day 0 and day 4 of the second and fifth cycles.

To assess the possible role of TNF in causing edema of the wall of the gallbladder, all participants had biliary sonograms on day 0 and day 4 of the second and fourth cycles of IL-2.

For patients who terminated an IL-2 cycle early, day 4 studies were obtained at the first opportunity following cessation of the IL-2 infusion.

Thalidomide pharmacokinetics

The first 12 patients randomized to Group C (IL-2 plus thalidomide) participated in a pharmacokinetic analysis. At a separate outpatient visit before beginning the first cycle of daily dosing with thalidomide, each patient received a single dose of either 100 mg (the first 5 patients) or 300 mg (all subsequent patients) . Plasma samples were obtained at times 0, 0.5, 1, 1.5, 2, 3, 4, 5, 7, 24, 27 and 31 hours post. Fertile women had a negative urine pregnancy test on a first morning voided sample from one week prior to and on the day of pharmacokinetics before receiving the thalidomide dose. During the first round of IL-2, peak and trough levels of thalidomide were obtained on days 1 and 3; during all subsequent I -2 rounds, random thalidomide levels were obtained twice during each IL-2 round, typically on days 2 and 4. Patients not participating in the formal single dose pharmacokinetic assessment also had random thalidomide levels obtained twice during each IL-2 round, typically on days 2 and 4. Example 2

A randomized, controlled, open label study is conducted to evaluate the safety and efficacy of combination therapy with IL-2 and a monoclonal anti-TNF antibody (cA2) in patients infected with HIV who had been previously treated with IL-2. The study infusions are as described in Example 1.

Patients

Forty-five (45) patients participating on protocols 91-CC-143 (open trial of IL-2 in HIV infection) or 93-CC-113 (randomized trial of IL-2 in HIV infected patients with CD4 counts > 200 cells/mm³) are enrolled in this study. These patients have no history of hypersensitivity or intolerance to thalidomide.

Treatment Protocol

The treatment protocol employed is as set forth in Example 1 except each patient receives IL-2 at their previous dose. If it appears that the TNF inhibitors are associated with an improvement in the adverse effects seen in a patient, then the dose of IL-2 is increased for the next round to see if the higher dose could be tolerated in combination with the inhibitor. The maximum dose of IL-2 administered is 12 million IU/d. In some patients, IL-2 is administered less frequently than every 2 months, based on a previously established algorithm utilizing CD4 count responses :

IL-2 is held if the CD4 count is greater than 2000 cells/mm³, until the CD4 count decreases to less than 1000 cells/mm³. If the CD4 count peaks and plateaus between 1000 and 2000 cells/mm³, IL-2 is held until the CD4 count declines to less than 1000 cells/mm³.

If the CD4 count never increases above 1000 cells/mm³, but appears to have plateaued, it is optional to hold the IL-2 until the counts begin to decrease from the plateau level .

Patient Monitoring

Patients are monitored as described in Example 1. Toxicities are graded and managed as described in Example 1.

Equivalents

Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT Walker, Robert E

McCloskey, Richard V Woody, James N

(li) TITLE OF INVENTION HIV-1 THERAPY WITH IL-2 AND ANTI-TNF ANTIBODY

(ill) NUMBER OF SEQUENCES 2

(iv) CORRESPONDENCE ADDRESS

(A) ADDRESSEE HAMILTON, BROOK, SMITH & REYNOLDS, P C

(B) STREET TWO MILITIA DRIVE

(C) CITY LEXINGTON

(D) STATE MASSACHUSETTS (E) COUNTRY USA

(F) ZIP 02173

(v) COMPUTER READABLE FORM

(A) MEDIUM TYPE Floppy disk

(B) COMPUTER IBM PC compatible (C) OPERATING SYSTEM PC-DOS/MS-DOS

(D) SOFTWARE Patentin Release #1 0, Version #1 30

(vi) CURRENT APPLICATION DATA

(A) APPLICATION NUMBER

(B) FILING DATE (C) CLASSIFICATION

(vin) ATTORNEY/AGENT INFORMATION

(A) NAME Brook, David E

(B) REGISTRATION NUMBER 22,592

(C) REFERENCE/DOCKET NUMBER CTR95-03

(ix) TELECOMMUNICATION INFORMATION

(A) TELEPHONE (617) 861-6240

(B) TELEFAX (617) Θ61-9540 (2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Tyr Ser Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Ser Thr His Val 1 5 10 15

Leu Leu Thr His Thr lie 20

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Tyr Gin Thr Lys Val Asn Leu Leu Ser Ala lie Lys Ser Pro Cys Gin 1 5 10 15

Arg Glu Thr Pro Glu Gly 20

Claims

CLAIMS What is claimed:

1. A method for treating or preventing acquired immunodeficiency disease in an individual in need thereof comprising co-administering interleukin-2 and an anti -tumor necrosis factor chimeric antibody or fragment thereof to the individual, in therapeutically effective amounts.

2. A method of Claim 1 wherein the chimeric antibody and interleukin-2 are administered simultaneously.

3. A method of Claim 1 wherein the chimeric antibody and interleukin-2 are administered sequentially.

4. A method of Claim 1 wherein the chimeric antibody binds to one or more amino acids of hTNFα selected from the group consisting of about 87-108 and about 59-80.

5. A method of Claim 1 wherein the chimeric antibody binds to the epitope of cA2.

6. A method of Claim 5 wherein the chimeric antibody is cA2.

7. A method for treating or preventing human immunodeficiency virus infection in an individual in need thereof comprising co-administering interleukin-2 and an anti-tumor necrosis factor chimeric antibody or a fragment thereof to the individual, in therapeutically effective amounts.

8. A method of Claim 7 wherein the chimeric antibody and interleukin-2 are administered simultaneously.

9. A method of Claim 7 wherein the chimeric antibody and interleukin-2 are administered sequentially.

10. A method of Claim 7 wherein the chimeric antibody binds to one or more amino acids of hTNFoi selected from the group consisting of about 87-108 and about 59-80.

11. A method of Claim 7 wherein the chimeric antibody binds to the epitope of cA2.

12. A method of Claim 11 wherein the chimeric antibody is cA2.

13. A composition comprising interleukin-2 and an anti- tumor necrosis factor chimeric antibody or fragment thereof .

14. A composition of Claim 13 wherein the chimeric antibody binds to one or more amino acids of hTNFα selected from the group consisting of about 87-108 and about 59-80.

15. A composition of Claim 13 wherein the chimeric antibody binds to the epitope of cA2.

16. A composition of Claim 15 wherein the chimeric antibody is cA2.