AU3831889A

AU3831889A - Combinations of soluble t4 proteins and anti-retroviral agents and methods for treating or preventing aids, arc and hiv infection

Info

Publication number: AU3831889A
Application number: AU38318/89A
Authority: AU
Inventors: Richard A. Fisher; Martin S. Hirsch; Victoria A. Johnson; Robert T. SCHOOLEY; Bruce D. Walker
Original assignee: Biogen Inc
Current assignee: Biogen Inc
Priority date: 1988-06-10
Filing date: 1989-06-08
Publication date: 1990-01-05
Also published as: EP0378643A4; EP0378643A1; JPH02504641A; WO1989011860A1

Description

COMBINATIONS OF SOLUBLE T4 PROTEINS AND ANTI-RETROVIRAL AGENTS AND METHODS FOR TREATING OR PREVENTING AIDS, ARC AND HIV INFECTION

TECHNICAL FIELD OF INVENTION This invention relates to combinations and methods useful for the treatment and prevention of acquired immunodeficiency syndrome, AIDS related complex, and HIV infection. More particularly, this invention relates to pharmaceutically effective combinations of a soluble T4 protein and an anti-retroviral agent for treating or preventing AIDS, ARC and HIV infection. According to this invention, a soluble T4 protein is used in a pharmaceutically effective combination with an anti-retroviral agent, such as AZT or a glucosidase inhibitor, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy.

BACKGROUND OF THE INVENTION

The class of immune regulatory cells known as T cell lymphocytes can be divided into two broad functional classes, the first class comprising T helper or inducer cells - - which mediate T cell proliferation, lymphokine release and helper cell interactions for Ig release; and the second class comprising T cytotoxic or suppressor cells - - which participate in T cell-mediated killing and immune response suppression. In general, these two classes of lymphocytes are distinguished by expression of one of two surface glycoproteins: T4 (m.w. 55,000-62,000 daltons) which is expressed on T helper or inducer cells, probably as a monomeric protein, or T8 (m.w. 32,000 daltons) which is expressed on T cytotoxic or suppressor cells as a dimeric protein.

In immunocompetent individuals, T4 lymphocytes interact with other specialized cell types of the immune system to confer immunity to or defense against infection [E. L. Reinherz and S. F. Schlossman, "The Differentiation Function Of Human T-Cells", Cell, 19, pp. 821-27 (1980)]. More specifically, T4 lymphocytes stimulate production of growth factors which are critical to a functioning immune system. For example, they act to stimulate B cells, the descendants of hemopoietic stem cells, which promote the production of defensive antibodies. They also activate macrophages ("killer cells") to attack infected or otherwise abnormal host cells and they induce monocytes ("scavenger cells") to encompass and destroy invading microbes .

It has been found that the primary target of certain infective agents is the T4 surface protein. These agents include, for example, viruses and retroviruses. When T4 lymphocytes are exposed to such agents, they are rendered nonfunctional. As a result, the host's complex immune defense system is destroyed and the host becomes susceptible to a wide range of opportunistic infections.

Such immunosuppressiσn is seen in patients suffering from acquired immunodeficiency syndrome ("AIDS"). AIDS is a disease characterized by severe or, typically, complete immunosuppression and attendant host susceptibility to a wide range of opportunistic infections and malignancies. In some cases, AIDS infection is accompanied by central nervous system disorders. Complete clinical manifestation of AIDS is usually preceded by AIDS related complex ("ARC"), a syndrome accompanied by symptoms such as persistent generalized lymphadenopathy, fever and weight loss. The human immunodeficiency virus

( "HIV" ) retrovirus is thought to be the etiological agent responsible for AIDS infection and its precursor, ARC [M. G. Sarngadharan et al., "Detection, Isolation And Continuous Production Of Cytopathic Retroviruses (HTLV-III) From Patients With AIDS And Pre-AIDS", Science, 224, pp. 497-508 (1984)].*

Between 85 and 100% of the AIDS/ARCS population test seropositive for HIV [G. N. Shaw et al., "Molecular Characterization Of Human T-Cell Leukemia (Lymphotropic) Virus Type III In The Acquired Immune Deficiency Syndrome", Science, 226, pp. 1165-70 (1984)]. The number of adults in the United States infected with HIV has been estimated to be between 1 and 2.5 million [D. Barnes, "Strategies For An AIDS Vaccine", Science, 233, pp. 1149-53 (1986); M. Rees, "The Sombre View Of AIDS", Nature, 326, pp. 343-45 (1987)]. These estimates include 64,900 individuals who do not belong to a group identified at risk for AIDS [S. L. Sivak and G. P. Wormser, "How Common Is HTLV-III Infection In The United States?", New Eng.

* In this application, human immunodeficiency virus ( "HIV" ) , the generic term adopted by the human retrovirus subcommittee of the International Committee On Taxonomy Of Viruses to refer to independent isolates from AIDS patients, including human T cell lymphotropic virus type III ("HTLV-III"), lymphadenopathy-associated virus ("LAV"), human immunodeficiency virus type 1 ("HIV-1") and AIDS-associated retrovirus ("ARV") will be used. J. Med., 313, p. 1352 (1985)]. The apparent annual rate of diagnosis for those infected with HIV virus is between 1 and 2% - - a rate which may increase significantly in future years. The host range of the HIV virus is associated with cells which bear the T4 surface glycoprotein. Such cells include T4 lymphocytes and brain cells [P. J. Maddon et al., "The T4 Gene Encodes The AIDS Virus Receptor And Is Expressed In The Immune System And The Brain", Cell, 47, pp. 333-48 (1986)]. Upon infection of a host by HIV virus, the T4 lymphocytes are rendered non-functional. The progression of AIDS/ARC syndromes can be correlated with the depletion of T4⁺ lymphocytes, which display the T4 surface glycoprotein. This T cell depletion, with ensuing immunological compromise, may be attributable to both recurrent cycles of infection and lytic growth and from cell-mediated spread of the virus. In addition, clinical observations suggest that the HIV virus is directly responsible for the central nervous system disorders seen in many AIDS patients.

The tropism of the HIV virus for T4⁺ cells is believed to be attributed to the role of the T4 cell surface glycoprotein as the membrane-anchored virus receptor. Because T4 behaves as the HIV virus receptor, its extracellular sequence probably plays a direct role in binding HIV. More specifically, it is believed that HIV envelope selectively binds to the T4 epitope(s), using this interaction to initiate entry into the host cell [A. G. Dalgelish et al., "The CD4 (T4) Antigen Is An Essential Component Of The Receptor For The AIDS Retrovirus", Nature, 312, pp. 763-67 (1984); D. Klatzmann et al., "T-Lymphocyte T4 Molecule Behaves As The Receptor For Human Retrovirus LAV", Nature, 312, pp. 767-68 (1984)]. Accordingly, cellular expression of T4 is believed to be sufficient for HIV binding, with the T4 protein serving as a receptor for the HIV virus.

Therapeutics based upon soluble T4 protein have been proposed for the prevention and treatment of the HIV-related infections AIDS and ARC. The nucleotide sequence and a deduced amino acid sequence for a DNA that purportedly encodes the entire human T4 protein have been reported [P. J. Maddon et al., "The Isolation And Nucleotide Sequence Of A cDNA Encoding The T Cell Surface Protein T4: A New

Member Of The Immunoglobulin Gene Family", Cell, 42, pp. 93-104 (1985)]. Based upon its deduced primary structure, the T4 protein is divided into the following domains: Amino Acid

Structure/Proposed Location Coordinates

Hydrophobic/Secretory Signal -23 to -1

Homology to V-Regions/ +1 to +94 Extracellular Homology to J-Regions/ +95 to +109 Extracellular

Glycosylated Region/ +110 to +374

Extracellular

Hydrophobic/Transmembrane +375 to +395 Sequence

Very Hydrophilic/ +396 to +435 Intracytoplasmic

Soluble T4 proteins have been constructed by truncating the full length T4 protein at amino acid 375, to eliminate the transmembrane and cytoplasmic domains. Such proteins have been produced by recombinant techniques [R. A. Fisher et al., "HIV Infection Is Blocked In Vitro By Recombinant Soluble CD4", Nature, 331, pp. 76-78 (1988)]. Soluble T4 proteins advantageously interfere with the T4/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 surface protein. And soluble T4 proteins inhibit interaction between T4 lymphocytes and antigen presenting cells and targets of T4 lymphocyte mediated killing. By acting as soluble virus receptors, soluble T4 proteins are useful as anti-viral therapeutics to inhibit HIV binding to T4⁺ cells and virally induced syncytium formation.

Other potential anti-retroviral agents target the reverse transcriptase enzyme of HIV as a unique step in the life cycle of the virus. Such agents utilize HIV reverse transcriptase inhibition as the mechanism of action. These agents include, for example, suramin, azidothymidine ("AZT") and dideoxycytidine [H. Mitsuya et al., "3'-Azido-3' -Deoxythymidine (BW A509U): An Antiviral Agent That Inhibits The Infectivity And Cytopathic Effect Of Human T-Lymphotropic Virus Type III/Lymphadenopathy-Associated Virus In Vitro", Proc. Natl. Acad. Sci. USA, 82, pp. 7096-7100 (1985); H. Mitsuya and S. Broder, "Inhibition Of The In Vitro Infectivity And Cytopathic Effect Of Human T-Lymphotropic Virus Type III/Lymphodenopathy-Associated Virus (HTLV-III/LAV) By 2',3'-Dideoxynucleosides", Proc. Natl. Acad. Sci. USA, 83, pp. 1911-15 (1986); R. Yarchoan et al., "Administration Of 3'-Azido-3'- Deoxythymidine, An Inhibitor Of HTLV-III/LAV Replication, To Patients With AIDS or AIDS-Related Complex", Lancet, pp. 575-80 (March 15, 1986)].

Although each of these agents has exhibited activity against HIV in vitro, only AZT has demonstrated clinical benefits in properly designed placebo controlled clinical trials. An increasing number of patients receiving AZT, however, tolerate only low doses of the drug. Certain dosage regimens of AZT have been reported to be lymphotoxic [Yarchoan et al., supra]. And evidence utilizing an ELISA assay for serum HIV p24 antigen suggests that doses of AZT lower than 200 mg/4 hr may be less effective in controlling viral replication [R. E. Chaisson et al., "Significant Changes In HIV Antigen Level In The Serum Of Patients Treated With Azidothymidine", N. Eng. J. Med., 315, pp. 1610-11 (1986)]. AZT administration in effective amounts has been accompanied by undesirable and debilitating side effects, such as granulocytopenia and anemia. Over the long term, therefore, hematologic toxicity appears to be a significant limiting factor in the use of AZT in the treatment of AIDS and ARC [D. D. Richman et al., "The Toxicity Of Azidothymidine (AZT) In The Treatment Of Patients With AIDS And AIDS-Related Complex: A Double-Blind, Placebo-Controlled Trial", N. Eng. J. Med., 317, pp. 192-97 (1987)].

Proposed methods for treating AIDS and ARC have also focused on the development of agents exhibiting anti-retroviral activity against steps in the viral replicative cycle other than reverse transcription [PCT patent application WO 87/03903]. Such methods include the administration of glucosidase inhibitors, such as the plant alkaloid castanospermine, which modify glycosylation of envelope glycoproteins of HIV infected cells by interfering with the normal processing of N-linked oligosaccharide chains on those glycoproteins, leading to reduced expression of a functional envelope protein at the cell surface and inhibition of production of infectious virus particles. Such anti-retroviral agents, however, may exert toxic effects on cellular metabolism at higher doses when given as monotherapy.

To date, therefore, the need exists for the development of immunotherapeutic agents, methods and strategies for the treatment or prevention of AIDS, ARC and HIV infection which avoid the disad vantages of conventional agents while providing effective therapy for those diseases.

DISCLOSURE OF THE INVENTION

The present invention solves the problems referred to above by providing pharmaceutically effective combinations and methods for the treatment and prevention of acquired immunodeficiency syndrome, AIDS related complex and HIV infection. According to this invention, a soluble T4 protein is used in a pharmaceutically effective combination with an anti-retroviral agent, such as azidothymidine ("AZT") or a glucosidase inhibitor, for treating or preventing AIDS, ARC and HIV infection, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent each being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy.

Advantageously, the use of a soluble T4 protein in the combinations and methods of this invention increases the effectiveness of conventional anti-retroviral agents in the treatment of AIDS, ARC and HIV infection. Combination therapies according to this invention exert a synergistic effect in inhibiting HIV replication, because each component agent of the combination acts at a different site of HIV virus replication. These combinations demonstrate greater anti-retroviral activity than the sum of their separate effects.

The use of the combinations and methods of this invention reduces the dosages which would be required by therapies based on the use of conventional anti-retroviral agents alone. Accordingly, the combinations and methods of this invention reduce or eliminate the side effects of conventional single anti-retroviral agent therapies, while not interfering with the anti-retroviral activity of those agents. And the combinations and methods of this invention reduce potential resistance to single agent therapies, while minimizing any associated toxicity.

Combinations of soluble T4 protein and anti-retroviral agents that attack the HIV-1 replicative cycle at multiple sites according to this invention permit the interception of several events early in that cycle, prior to integration. Accordingly, the likelihood of subsequent productive and latent HIV-1 infections of T4 cells is diminished or avoided. Furthermore, in view of the conserved nature of T4 binding by diverse isolates of HIV-1 and HIV-2 and the activity of an anti-retroviral agent such as AZT, against both viruses [Q. J. Sattentau and R. A. Weiss, "The CD4 Antigen: Physiologic Ligand and HIV Receptor", Cell, 52, pp. 631-33 (1988); H. Mitsuya and S. Broder "Inhibition Of Infectivity And Replication Of HIV-2 And SIV In Helper T-Cells By 2',3'-Dideoxynucleosides In Vitro", AIDS Res, and Hum. Retroviruses, 4, pp. 107-13 (1988)] the combination regimens of this invention advantageously exhibit broad anti-retroviral activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the nucleotide sequence and the derived amino acid sequence of T4 cDNA of plasmid p170-2.

Figure 2 depicts the nucleotide sequence and the derived amino acid sequence of T4 cDNA of plasmid pBG381. In Figures 1 and 2, the amino acids are represented by single letter codes as follows:

Phe : F Leu: L Ile: I Met: M

Val : V Ser : S Pro : P Thr : T

Ala : A Tyr : Y His : H Gln: Q

Asn : N Lys : K Asp : D Glu : E

Cys : C Trp : w Arg: R Gly: G

* = position at which a stop codon is present. In Figure 1, the T4 protein translation start (AA_-23) is located at the methionine at nucleotides 1199-1201 and the mature N-terminus is located at the asparagine (AA₃) at nucleotides 1274-1276. In Figure 2, the T4 protein translation start (AA_-23) is located at the methionine at nucleoides 1207-1209 and the mature N-terminus is located at the asparagine (AA₃) at nucleotides 1282-1285.

Figure 3 depicts, in tabular form, the parameters of various assays demonstrating the effects of the combinations of this invention in inhibiting HIV replication in vitro.

Figure 4 depicts, in tabular form, the combination index values for various combinations of this invention.

Figure 5 depicts, in graphic form, the results of various assays demonstrating the effects of the combinations of this invention in inhibiting HIV replication in vitro. Figure 6 depicts, in graphic form, the effects of the combinations of this invention in inhibiting HIV replication in vitro over a 14 day period.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to therapeutic or prophylactic combinations and methods for treating AIDS, ARC and HIV infection. More particularly, this invention relates to pharmaceutically effective combinations comprising a soluble T4 protein and an anti-retroviral agent, wherein the dosage of each compound is less than that required for a desired therapeutic or prophylactic effect when either agent is administered as a monotherapy. Such combinations advantageously avoid the side effects of high level dosages of anti-retroviral agents. According to one embodiment, the method of this invention comprises the step of treating a patient in a pharmaceutically acceptable manner with a dosage of a soluble T4 protein together with a dosage of an anti-retroviral agent, such as AZT or a glucosidase inhibitor, said dosages each being less than that required for a desired therapeutic or prophylactic effect when either compound is used alone, for a. period of time sufficient to lessen the immunocompromising effects of HIV infection or to prevent intracellular spread of HIV infection.

The use of a soluble T4 protein in the compositions and methods of this invention advantageously enhances the antiviral activity of the anti-retroviral agent, AZT or the glucosidase inhibitor, in the treatment of AIDS, ARC and HIV infection. Additionally, the use of a soluble T4 protein in combination with such anti-retroviral agents may reduce the dosage of treatment which would be required by therapies based upon those anti-retroviral agents alone. Finally, combination therapies according to this invention advantageously permit administration of anti-retroviral agents in dosages formerly considered too low to result in anti-retroviral effects if given alone.

The combinations and methods of this invention may be used to treat humans having AIDS, ARC, HIV infection or antibodies to HIV. These cominations and methods may also be used for treating AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, in mammals including humans.

According to this invention, patients are treated by the pharmaceutically acceptable administration of a pharmaceutically effective combination of a soluble T4 protein and an anti-retroviral agent, such as AZT or castanospermine, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent each being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy, for a period of time sufficient to reduce the effects of retroviral infection or to prevent intracellular spread of a retrovirus.

Glucosidase inhibitors useful in the combinations and methods of this invention include, but are not limited to, castanospermine and deoxynojirimycin and deoxynojirimycin derivatives.

As used in this application, "soluble T4 protein" includes all proteins, polypeptides and peptides which are natural or recombinant soluble T4 proteins, or soluble derivatives thereof, and which are characterized by the immunotherapeutic (anti-retroviral) activity of soluble T4 protein. They include soluble T4-like compounds from a variety of sources, such as soluble T4 protein derived from natural sources, recombinant soluble T4 protein and synthetic or semi-synthetic soluble T4 protein. Such soluble T4-like compounds advantageously interfere with the T4/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 surface protein.

Soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA_-23-AA₃₆₂ of Figure 1, a polypeptide of the formula AA₁-AA₃₆₂ of Figure 1, a polypeptide of the formula Met-AA₁-AA₃₆₂ of Figure 1, a polypeptide of the formula AA₁-AA₃₇₄ of Figure 1, a polypeptide of the formula Met-AA_1-374 of Figure 1, a polypeptide of the formula AA₁-AA₃₇₇ of Figure 1, a polypeptide of the formula Met-AA_1-377 of Figure 1, a polypeptide of the formula AA_-23-AA₃₇₄ of Figure 1, a polypeptide of the formula AA_-23-AA₃₇₇ of Figure 1, or portions thereof. Other soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA_-23-AA₁₈₂ of Figure 1, a polypeptide of the formula Met-AA₁-AA₁₈₂ of Figure 1, a polypeptide of the formula AA₁-AA₁₈₂ of Figure 1, a polypeptide of the formula AA_-23-AA₁₈₂ of Figure 1, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA₁-AA₁₈₂ of Figure 1, followed by the amino acids asparagine-leucine-glutamine-histidine- serine-leucine, a polypeptide of the formula Met-AA_1-182 of Figure 1, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA_-23-AA₁₁₃ of Figure 1, a polypeptide of the formula AA₁-AA.₁₁₃ of Figure 1, a polypeptide of the formula Met-AA_1-113 of Figure 1, a polypeptide of the formula AA_-23-AA₁₃₁ of Figure 1, a polypeptide of the formula AA₁-AA₁₃₁ of Figure 1, a polypeptide of the formula Met-AA_1-113 of Figure 1, a polypeptide of formula

AA_-23-AA₁₄₅ of Figure 1, a polypeptide of the formula AA₁-AA₁₄₅ of Figure 1, a polypeptide of the formula Met-AA_1-145 of Figure 1, a polypeptide of the formula AA_-23-AA₁₆₆ of Figure 1, a polypeptide of the formula AA₁-AA₁₆₆ of Figure 1, a polypeptide of the formula Met-AA_{1- 1 66} of Figure 1 , or portions thereof.

Additionally, soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA_-23-AA₃₆₂ of mature T4 protein, a polypeptide of the formula AA_1-362 of mature T4 protein, a polypeptide of the formula Met-AA_1-362 of mature T4 protein, a polypeptide of the formula AA_1-374 of mature T4 protein, a polypeptide of the formula Met-AA_1-374 of mature T4 protein, a polypeptide of the formula AA_1-377 of mature T4 protein, a polypeptide of the formula Met-AA_1-377 of mature T4 protein, a polypeptide of the formula AA_-23-AA₃₇₄ of mature T4 protein, a polypeptide of the formula AA_-23-AA₃₇₇ of mature T4 protein, or portions thereof.

And soluble T4 proteins include a polypeptide selected from the group consisting of a polypeptide of the formula AA_-23-AA₁₈₂ of mature T4 protein, a polypeptide of the formula AA₁-AA₁₈₂ of mature T4 protein, a polypeptide of the formula Met-AA_1-182 of mature T4 protein, a polypeptide of the formula AA_-23-AA₁₈₂ of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA₁-AA₁₈₂ of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula Met-AA_1-182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA_-23-AA₁₁₃ of mature T4 protein, a polypeptide of the formula AA₁-AA₁₁₃ of mature T4 protein, a polypeptide of the formula Met-AA_1-113 of mature T4 protein, a polypeptide of the formula AA_-23-AA₁₃₁ of mature T4 protein, a polypeptide of the formula AA₁-AA₁₃₁ of mature T4 protein, a polypeptide of the formula Met-AA₁-AA₁₃₁ of mature T4 protein, a polypeptide of the formula AA_-23-AA₁₄₅ of mature T4 protein, a polypeptide of the formula AA₁-AA₁₄₅ of mature T4 protein, a polypeptide of the formula Met-AA_1-145 of mature T4 protein, a polypeptide of the formula AA_-23-AA₁₆₆ of mature T4 protein, a polypeptide of the formula AA₁-AA₁₆₆ of mature T4 protein, a polypeptide of the formula Met-AA_1-166 of mature T4 protein, or portions thereof.

The amino terminal amino acid of mature T4 protein isolated from T cells begins at lysine, the third amino acid of the sequence depicted in Figure 1. Accordingly, soluble T4 proteins also include polypeptides of the formula AA₃-AA₃₇₇ of Figure 1, or portions thereof. And such polypeptides include polypeptides selected from the group consistng of a polypeptide of the formula AA₃-AA₃₆₂ of Figure 1, a polypeptide of the formula AA₃-AA₃₇₄ of Figure 1, a polypeptide of the formula AA₃-AA₁₈₂ of Figure 1, a polypeptide of the formula AA₃-AA₁₁₃ of Figure 1, a polypeptide of the formula AA₃-AA-₁₃₁ of Figure 1, a polypeptide of the formula AA₃-AA₁₄₅ of Figure 1, a polypeptide of the formula AA₃-AA₁₆₆ of Figure 1. Soluble T4 proteins also include the above-recited polypeptides preceded by an N-terminal methionine group.

Soluble T4 proteins useful in the combination and methods of this invention may be produced in a variety of ways. We have depicted in Figure 1 the nucleotide sequence of full-length T4 cDNA obtained from deposited clone p170-2 and the amino acid sequence deduced therefrom. The T4 cDNA of p170-2 is almost identical to the approximately 1,700 bp sequence reported by Maddon et al., supra. The T4 cDNA of p170-2, however, contains three nucleotide substitutions that, in the translation product of this cDNA, produce a protein containing three amino acid substitutions compared to the sequence reported by Maddon et al. These differences are at amino acid position 3, where the asparagine of Maddon et al. is replaced with lysine; position 64, where the tryptophan of Maddon et al. is replaced with arginine and at position 231, where the phenylalanine of Maddon et al. is replaced with serine. The asparagine reported at position 3 of Maddon et al. instead of lysine was the result of a DNA sequencing error [D. R. Littman et al., "Corrected CD4 Sequence", Cell, 55, p. 541 (1988)]. Soluble T4 protein constructs may be produced by truncating the full length T4 sequence at various positions to remove the coding regions for the transmembrane and intracytoplasmic domains, while retaining the extracellular region believed to be responsible for HIV binding. More particularly, soluble T4 proteins may be produced by conventional techniques of oligonucleotide directed mutagenesis, restriction digestion, followed by insertion of linkers, or chewing back full-length T4 protein with enzymes.

Prior to such constructions, the cDNA coding sequence of a full length T4 clone, such as p170-2, may be modified in sequential steps of site-directed mutagenesis and restriction fragment substitution to modify the amino acids at positions 64 and 231. For example, one may employ oligonucleotide-directed mutagenesis to modify amino acid 64. Subsequently, restriction fragment substitution with a fragment including the serine 231 codon of a partial T4 cDNA isolated from a T4 positive lymphocyte cell line [O. Acuto et al., Cell, 34, pp. 717-26 (1983)] library in λgt 11 may be used to modify the amino acid at position 231 [Fisher et al., supra].

DNA sequences coding for soluble T4 proteins may be used to transform eukaryotic and prokaryotic host cells by conventional recombinant techniques to produce recombinant soluble T4 proteins in clinically and commercially useful amounts. Such soluble T4 proteins include those produced according to the processes set forth in United States patent application 094,322, filed September 4, 1987, United States patent application 141,649, filed January 7, 1988, and PCT patent application WO 89/01940, filed September 1, 1988, the disclosures of which are hereby incorporated by reference.

Microorganisms and recombinant DNA molecules characterized by DNA sequences coding for soluble T4 proteins are exemplified by cultures deposited in the In Vitro International, Inc. cul ture collection in Linthicum, Maryland, and identified as:

Deposit Accession No Deposit Date

EC100: E.coli JM83/pEC100 IVI 10146 September 2, 1987

BG377: E.coli MC1061/pBG377 IVI 10147 September 2, 1987

BG380: E.coli MC1061/pBG380 IVI 10148 September 2, 1987

BG381: E.coli MC1061/pBG381 IVI 10149 September 2, 1987

BG-391: E.coli MC1061/pBG391 IVI 10151 January 6, 1988

BG-392: E.coli MC1061/pBG392 IVI 10152 January 6, 1988

BG-393: E.coli MC1061/pBG393 IVI 10153 January 6, 1988

BG-394: E.coli MC1061/pBG394 IVI 10154 January 6, 1988

BG-396: E.coli MC1061/pBG396 IVI 10155 January 6, 1988

203-5: E.coli SG936/p203-5 IVI 10156 January 6, 1988

211-11: E.coli A89/pBG211-11 IVI 10183 August 24, 1988

214-10: E.coli A89/pBG214-10 IVI 10184 August 24, 1988

215-7: E.coli A89/pBG215-7 IVI 10185 August 24, 1988

Alternatively, soluble T4 proteins may be chemically synthesized by conventional peptide synthesis techniques, such as solid phase synthesis. [R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc, 83, pp. 2149-54 (1963)].

The compositions used in therapies according to this invention may be in a variety of conventional depot forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusable solutions. The preferred form depends upon the intended mode of administration and therapeutic application. The compositions of this invention also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosebased substances and polyethylene glycol. Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropyleneblock polymers, polyethylene glycol and wood wax alcohols. Preferably, the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.

According to this invention, the antiretroviral agent, such as AZT or castanospermine, may be administered to the patient in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramuscularly - - including paravertebrally and periarticularly - - subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical routes.

Generally, the soluble T4 protein may be formulated and administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides (e.g., α-IFN). Any pharmaceutically acceptable dosage route, including, parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the soluble T4 protein. An effective dose may be in the range of from less than about 0.1 to 1.0 mg/kg body weight, it being recognized that lower and higher doses may also be useful.

In accordance with this invention, the anti-retroviral agent, such as AZT or a glucosidase inhibitor and the soluble T4 protein are administered sequentially or concurrently to the patient. The most effective mode of administration and dosage regimen of anti-retroviral agent and soluble T4 protein will depend upon the severity and course of infection, previous therapy, the patient's health status and response to treatment and the judgment of the treating physician.

The anti-retroviral agent and the soluble T4 protein may be administered to the patient at one time or over a series of treatments. The soluble T4 protein and the anti-retroviral agent may be administered sequentially to the patient, with the anti-retroviral agent being administered before, after, or both before and after treatment with the soluble T4 protein. Concurrent administration involves treatment with soluble T4 protein at least on the same day (within 24 hours) of treatment with the anti-retroviral agent and may involve continued treatment with the anti-retroviral agent on days that the soluble T4 protein is not administered. Other dosage regimens of AZT or glucosidase inhibitor and soluble T4 protein are also useful.

According to one embodiment of this invention, conventional modes of administration of AZT or castanospermine may be used. For example, AZT may be administered orally at a dosage of less than about 200-250 mg, six times a day, i.e., less than about 1.2 g to 1.5 g per day. Alternatively, castanospermine may be administered at a dosage of less than about 100 mg per day. Dosages of the particular anti-retroviral agent may be titrated to the individual patient.

Advantageously, in the methods and combinations of this invention AZT, castanospermine and soluble T4 protein may be administered in amounts less than the conventional dosage, for example, less than about 75% of the conventional dosage, when each is administered as a monotherapy.

Once improvement of the patient's condition has occurred, a maintenance dose of the combination is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

EXAMPLES

These examples demonstrate the in vitro effects of anti-retroviral agents, such as AZT or a glucosidase inhibitor, and soluble T4 protein alone, or in combination, in inhibiting HIV replication in vitro. These examples demonstrate that combinations of AZT or glucosidase inhibitors with soluble T4 protein were effective to a greater degree than treatments with any one of AZT, glucosidase inhibitor or soluble T4 protein alone.

In these examples, the soluble T4 protein used was recombinant soluble T4 protein ("rsT4") supplied by Biogen Research Corp. (Cambridge,

Massachusetts). That recombinant soluble T4 protein was derived from a Chinese hamster ovary cell transfected with animal cell expression vector pBG381 [R. A. Fisher et al., "HIV Infection Is Blocked ln Vitro By Recombinant Soluble CD4" , Nature, 331, pp. 76-78 (1988)]. pBG381 is characterized by DNA coding for AA_-23 to AA₃₇₇ of T4 protein, as depicted in Figure 2. The concentration of the stock rsT4 was assigned by an optical density measurement with an extinction coefficient determination such that 2.04 A₂₈₀ optical density units/ml = 1 mg/ml. The rsT4 was stored at a concentration of 125 μg/ml in PBS in aliquots of 0.5 ml/vial at -20°C prior to use. The AZT was obtained in powder form from

Dr. P. A. Furman, Burroughs-Wellcome Co., Research Triangle Park, North Carolina. It was dissolved in sterile phosphate buffered saline and stored at a concentration of 1 mM in aliquots of 0.5 ml/vial at -20°C prior to use. And the glucosidase inhibitor, castanospermine, was a gift of Dr. L. Rohrschneider, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Assays Of Anti-Viral Activity In these examples, we evaluated the anti-viral activities of various combinations of this invention using modifications of various in vitro assays used to study anti-viral agents [D. D. Ho et al., "Recombinant Human Interferon Alpha/Suppresses HTLV-III Replication In Vitro", Lancet, 1, pp. 602-04 (1985); K. L. Hartshorn et al., "Syner gistic Inhibition Of Human T-Cell Lymphotropic Virus Type III Replication In Vitro By Phosphonoformate And Recombinant Interferon Alpha-A Interferon", Antimicrob. Ag. Chemother., 30, pp. 189-91 (1986)]. For each of these assays, we cultured uninfected H9 cells, a gift from Dr. Robert C. Gallo, National Cancer Institute, Bethesda, Maryland, [M. Popovic et al., "Detection, Isolation And Continuous Production Of Cytopathic Retrovirus (HTLV-III) From Patients With AIDS And Pre-AIDS",

Science, 224, pp. 497-500 (1984)] at an initial concentration of 0.4 x 10⁶ cells/ml in 5 ml final volume of R-20 culture medium comprising RPMI medium, 20% heat-inactivated fetal calf serum, HEPES buffer, L-glutamine, and antibiotics (penicillin, 250 U/ml and streptomycin, 250 μg/ml) in a T-25 flask (Falcon, Becton Dickinson Laboratory, Lincoln Park, New Jersey).

In assays 1-4, uninfected H9 cells (2 x 10⁶ cells) were suspended in 5 ml of R-20 medium in T-25 flasks. The multiplicities of infection varied, ranging from 500-3500 tissue culture infectious doses TCID₅₀ of cell-free HIV-1 (HTLV-IIIB) (obtained from Dr. Robert C. Gallo) per 1 x 10⁶ cells. The culture medium was changed on days 3, 5, 7 and 10, with 2 ml of cell suspension being resuspended in 5 ml of replacement medium containing the original concentrations of antiretroviral agent(s). Various graded concentrations of recombinant soluble T4 protein or AZT, either alone or in combination, were added to the cell cultures simultaneously with the HIV-1 isolate. The culture medium was exchanged on days 3, 5, 7 and 10 and again recombinant soluble T4 protein, AZT, or both, were added with the medium changes to maintain the concentration of each of those agents originally present. In assays 5 and 6, peripheral blood mononuclear cells (PBMCs) from HIV-1-seronegative donors were obtained by Ficoll-Hypague density gradient centrifugation. The cells were then treated with phytohemagglutinin (PHA-P) (Difco Labs, Detroit, Michigan) (10 μg/ml) and cultured in R-20 medium supplemented with 10% interleukin-2 (IL-2) (Electro-Nucleonics, Inc., Silver Spring, Maryland). Four-day PHA-stimulated PBMCs (5 x 10 cells in 5 ml of R-20 medium with 10% IL-2 ) from a single HIV-1 seronegative donor were exposed to 3000 TCID₅₀ of HIV-1 per 10⁶ cells. Various graded concentrations of recombinant soluble T4 protein or AZT, either alone or in combination, were added to the cell cultures simulataneously with the HIV-1 isolate. The culture medium was changed on days 4, 7 and 10, and a 2 ml aliquot of cell suspension was resuspended in 5 ml of replacement medium containing the original concentrations of antiretroviral agent(s). In assays 7 and 8, BT4 cells (provided by

E. Amento, Massachusetts General Hospital) were maintained in Iscove's modified Dulbecco medium with 10% fetal calf serum and antibiotics as above. Uninfected BT4 cells (2 x 10⁶ cells) were suspended in 5 ml of R-20 medium and exposed to 10⁶ TCID₅₀ of

HIV-1 per 10⁶ cells. Various graded concentrations of recombinant soluble T4 protein or AZT, either alone or in combination, were added to the cell cultures simulataneously with the HIV-1 isolate. The culture medium was changed on days 3, 5, 7 and 10, with 2 ml of cell suspension being resuspended in 5 ml replacement medium containing the original concentrations of antiretroviral agent(s).

In these assays, cell-free supernatants of the cultures were harvested on one or more of days 5, 7, 10, 12, and 14 for determination of HIV-1 p24 antigen production, viral reverse transcriptase activity (RT) or yield of infectious virus to evaluate the effects of the agents alone, or in combination, on HIV replication in vitro. In addition, in assay 4, cell pellets from H9 cultures were prepared for determination of HIV antigen expression by indirect immunofluorescence assay and by virus yield assay essentially according to the protocol set forth in K. L. Hartshorn, "Synergistic Inhibition Of Human Immunodeficiency Virus In Vitro By Azidothymidine And Recombinant Alpha Interferon", Antimicrob. Ag. Chemother., 31, pp. 168-72 (1987).

For each virus replication assay, uninfected cells were exposed to HIV-1 inoculum without a subsequent wash. Simultaneously, multiply-diluted fixed-ratio combinations of agents, or single agents, were added to each flask. Cell counts were performed on all harvest days to exclude additive toxicity of combinations of agents. In all assays, uninfected and infected cultures were maintained in parallel, as well as drug-treated toxicity controls. Uninfected drug-treated toxicity controls were maintained at the highest concentration of each agent tested (either alone or in combination). We assessed cell proliferation and viability by the trypan blue dye exclusion method.

Reverse Transcriptase Assay

We measured reverse transcriptase activity as an indicator of the effects of combinations of AZT and soluble T4 protein according to this invention on HIV viral replication [as described by

M. Popovic et al., Science, 224, pp. 497-500 (1984); R. C. Gallo et al., Science, 224, pp. 500-03 (1984); or D. D. Ho et al., Proc. Natl. Acad. Sci. USA, 81, pp. 7588-90 (1984); D. D. Ho et al., Science, 226, pp. 451-53 (1984)] More specifically, in the reverse transcriptase assay, we employed the reagents listed below. The DTT was obtained from Boehringer Mannheim Bio-Chemicals, Indianapolis, Indiana. The PEG 8000 was obtained from Fisher Scientific, Medford,

Massachusetts. The tRNA used was type X-S, obtained from Sigma Chemical, St. Louis, Missouri. The 3H-TTP was obtained from DuPont, New England Nuclear Research Products, Boston, Massachusetts.

Buffer A

*1.0 M Tris (pH 7.8) 12.5 ml

*0.1 M EDTA 1.25 ml

*10% Triton X-100 1.25 ml

*Glycerol 250.00 ml

Water 235.10 ml

DTT (crystal) 0.77 g

KC1 (crystal) 3.72 g

500.00 ml

Solution 2

*10% Triton X-100 45.00 ml double distilled water 455.00 ml

KC1 (crystal) 1.63 g

500.00 ml

PEG (30%), 0.4 M NaCl

PEG 8000 150.00 g

NaCl 11.70 g

Water to 500.00 ml

Trichloroacetic Acid (10%)

TCA 50.00 g Sodium pyrophosphate 4.46 g

Water to 500.00 ml

* Solutions made in distilled water. Trichloroacetic Acid (5%)

TCA 1000.00 g

Sodium Pyrophosphate 178.00 g

Water to 20.00 1 Universal Buffer

0.01 M Tris (pH 8.0 ) and 0.015 M NaCl solution

0.2 M Dithiothreitol

0.309 g DTT

10.0 ml universal buffer This solution was stored frozen at -20°C and thawed only once before use.

10 mg/ml tRNA tRNA was made 10 mg/ml in universal buffer and stored at -20°C.

10 units/ml dA or rA template

Oligo dT template primers (# 27-7878 and # 27-7868, Pharmacia/P-L Biochemicals, Piscataway, New Jersey) were dissolved in 2.5 ml universal buffer to give a 10 unit/ml solution. The solution was stored in 0.5 ml aliquots at -20°C and thawed before use.

Optionally, the assay samples may be prepared by first disrupting the virus particles by adding 0.9% Triton-X-199 in 1.5 M KCl (50-100 μl) and clarifying by low-speed centrifugation. Subsequently, the samples may be pelleted by ultracentrifugation at 100,000 Xg for 2 hours and the virus pellets may then be resuspended for PEG precipitation.

We carried out PEG precipitation of virus particles from cell-free supernatants by adding to 2 or 3 ml of clarified culture supernatant, one-half as much of the PEG precipitation solution, vortexed well and placed the mixture on ice overnight at 4°C. We then centrifuged at 800 Xg (~2100 rpm) for 45 minutes to pellet precipitate. Subsequently, we aspirated off all the supernatant, let the precipitate stand, then aspirated again to provide a dry pellet. We then resuspended the precipitate in a buffer comprising 150 μl buffer A and 75 μl solution 2, where the original supernatant used was 3 ml, with proportionate volumes of resuspension buffer being used for different volumes of supernatant. We resuspended the pellet by placing a pasteur pipette in the tube and vortexing. We froze the sample at -20°C if not assayed immediately. All the steps of the PEG precipitation were carried out using capped centrifuge tubes. Any step requiring exposure to air was carried out in a biohazard hood until after the addition of buffer A and solution 2, which inactivated the virus.

We assayed samples using the following reverse transcriptase (RT) cocktail: rA cocktail X dA cocktail X # samples # samples

RT cocktail (μl/tube) (μl/tube)

1 M Tris 4 4 (pH 7.8)

0.2 M DTT 4 4

0.2 M MgCl₂ 5 5 double-distilled 47 47 water

Universal buffer 22.5 22.5

3H-TTP 2.5 2.5

Oligo dT, poly rA 5 5 (or dA)

We prepared the RT cocktail by multiplying the volume of reagents by the number of samples plus 2 for controls (the dA cocktail was an internal negative control) plus 2 for pipetting loss. Subsequently, we added 90 μl of rA or dA cocktail to eppendorf tubes kept in an ice-water bath (1 rA and 1 dA tube for each sample). We then added 10 μl of each sample to an rA and a dA tube, vortexed and then incubated in a 37°C water bath for 1 hour.

In order to terminate reverse transcriptase activity, we removed samples from the water bath and placed them in an ice water bath. We then added 10 μl of cold tRNA solution to each tube, followed by 1 ml of cold 10% TCA solution. We let the tubes stand in the ice water bath for 20-30 minutes.

We then soaked 2.4 cm glass fiber filters (Millipore Corporation, Bedford, Massachusetts) in 5% TCA solution. We placed the filters on a sampling manifold (Millipore, Model 1225) attached to a vacuum source. Subsequently, we applied a sample to the filter and rinsed each sample tube four times into the manifold with the 5% TCA solution using a Cornwall syringe set for 1 ml. We then washed the filters twice with 5% TCA by filling the manifold wells rapidly while the vacuum was on. We dried the filters by turning off the vacuum and filling each well of the manifold with 70% ethanol, allowing the filters to stand for about 15 seconds before reapplying the vacuum.

Subsequently, we placed the filters under a heat lamp for 10-20 minutes to dry. The dried filters were placed in 18 ml scintillation vials (Beckman Instruments, Wakefield, Massachusetts). We then added 10-12 ml of Betaflour scintillation fluid for counting (National Diagnostics, Manville, New Jersey) to each vial and capped the vials. We also tested each sample using a non-specific poly (dA)-oligo(dT)_12-18 template. We calculated assay results by subtracting dA counts/min from rA counts/min and then multiplying the result by 7.5 to convert to net counts/min/ml of original culture supernatant sample. Samples having values of 10 3 counts/mm/ml or greater were considered positive for HIV-1.

p24 Radioimmunoassay

We tested soluble T4 protein and castanospermine as inhibitors of viral replication in a p24 competition radioimmunoassay, an HIV virus replication assay according to Hartshorn et al., Antimicrob. Aq. Chemother., 31, supra, and J. Sodroski et al., Nature, 322, pp. 470-74 (1986). We carried out the assay essentially as described. In this assay, we evaluated the ability of soluble T4 protein and castanospermine, alone or in combination, to block HIV replication, as measured by HIV p24 antigen production.

We sampled cell-free culture supernatant fluid for HIV p24 antigen as follows. We obtained an assay kit [HTLV-III p24 Radioimmunoassay System, Catalogue No. NEK-040, NEK-040a, Biotechnology Systems, DuPont-NEN Research Products, Billerica, Massachusetts] which contains affinity purified 1²⁵I labelled HIV p24 antigen, a rabbit anti-p24 antibody and a second goat anti-rabbit antibody which is used to precipitate antigen-antibody complexes. We carried out the assay according to the protocol included with the kit. More specifically, we mixed a sample to be assayed or one of a series of amounts of unlabelled p24 antigen with a fixed amount of ¹²⁵I labelled p24 and a fixed limited amount of rabbit anti-p24 antibody. We incubated the samples overnight at room temperature and then added a goat anti-rabbit immunoglobulin preparation for 15 minutes at 4°C. We centrifuged the samples and aspirated the supernatant fluid. Pelleted ¹²⁵I labelled p24 was quantitated for each sample by gamma counting and a standard curve for the ¹²⁵I p24 displaced by the known amounts of antigen added to standard tubes was constructed. We then calculated the ¹²⁵I labelled p24 displaced by the antigen present in the unknown samples by interpolation using the standard curve constructed from the known amounts of p24 antigen contained in the standard samples.

p24 ELISA

We tested soluble T4 protein and AZT as inhibitors of viral replication in a p24 ELISA.

Specifically, we sampled cell-free culture supernatant fluid for p24 antigen as follows. We obtained an assay kit [HIV p24 Core Antigen ELISA, Catalogue Nos. NEK-045, NEK-046 and NEK-047, DuPont-NEN Research Products, Billerica, Massachusetts], according to the protocol included with the kit, which contains rabbit polyclonal antibodies to p24 antigen immobilized to microplate wells to capture any p24 antigen lysed from a test sample. The captured p24 antigen was complexed with biotinylated polyclonal antibodies to p24 antigen and probed with a stxeptavidin-horseradish peroxidase conjugate. We then incubated the complex with orthophenyldiamineHCl, which produces yellow color proportional to the amount of p24 antigen captured. We then measured the absorbance of each well using a microplate reader and calibrated against the absorbance of a p24 antigen standard curve. Mathematical Analysis Of Drug Interactions

We evaluated the agent interactions by the median effect principle and. the isobologram technique with computer software using an IBM-PC (Armonk, New York) [J. Chou and T.-C. Chou, "Dose-Effect Analysis With Microcomputers: Quantitation Of ED₅₀, LDC₅₀ Synergism And Antagonism, Low-Dose Risk, Receptor Binding And Enzyme Kinetics. A Computer Software For Apple II Or IBM PC (Elsevier Biosoft, Cambridge, England, 1986)]. A multiple drug effect analysis [T. C. Chou and P. Talalay, "Quantitative Analysis Of Dose-Effect Relationships: The Combined Effects Of Multiple Drugs Or Enzyme Inhibitors", Adv. Enzyme Regul., 22, pp. 27-55 (1984)] was used to calculate combined agent effects. This method involves the plotting of dose-effect curves for each agent and for multiply-diluted fixed-ratio combinations of the agents using the median effect equation. Based on this method, we determined the combination index (CI) for various combinations according to this invention. CI values were determined from the median-effect plot parameters m( slope) and D (ED₅₀) of each agent and their combination based on the isobologram equation. A combination index value of less than 1 indicates synergy, while a value equal to 1 indicates additive effects and a value greater than 1 indicates antagonism. We also analyzed the data by the isobologram technique, which evaluates drug interactions by a dose-oriented geometric method.

Infection kinetics were dependent on the multiplicity of infection and the cell type utilized. In order to examine the activity of the two antiviral agents assayed under conditions during which appreciable viral replication was ongoing, combination indices are presented for assays which met the following criteria; (1) an infected/uninfected cell number ratio ≥ 30%, (2) an infected control RT activity value ≥ 5 x 10⁴ counts/mm/ml and (3) ongoing viral replication, such that p24 antigen and reverse transcription activity values on day x were ≥ x-n, where day x-n was the previous harvest day. The CI values indicated for the PBMC assays represent calculations based on data corrected for viable cell numbers. In the BT4 assays, only days 10 and 12 in culture were analyzed, due to slower replication kinetics in that cell type.

In order to facilitate the calculation of CI values, some combination data with 100% inhibition were assumed to be 99% inhibited and some single agent data with 0% inhibition were assumed to be 1% inhibited. These assumptions would be expected to result in a slight underestimation of the degrees of synergism. Data from some assays was not subject to quantative computer analysis because of (1) a flat dose effect curve (m < 0.4) or (2) a low linear correlation coefficient in the median-effect plot (r < 0.8).

Results: Soluble T4 Protein and Castanospermine

In the p24 radioimmunoassay, we measured inhibition of HIV replication by various graded concentrations of soluble T4 protein and castanospermine, either alone or in combination, in a culture of 0.4 x 10⁶ cells/ml uninfected H9 cells in 5 ml of R-20 culture medium which was exposed to 500 TCID₅₀ of HIV-1 per 1 x 10 ⁶ cells. The results are shown in the tables below. p24 ASSAY OF HIV-1 REPLICATION INHIBITION (Day 5)

HIV-1 p24 Level (ng of protein/ml) At The Following Concentration Of rsT4 (μg/ml)

Castanospermine

(μg/ml) 0 0.03125 0.125 0.5 2.0

0 11.3 6.2 5.6 5.5 5.9

0.5 10.0 5.8

2.0 8.3 5.9

8.0 7.0 6.3

32.0 6.5 6.3 p24 ASSAY OF HIV-1 REPLICATION INHIBITION (Day 7)

Castanospermine

(μg/ml) 0 0.03125 0.125 0.5 2.0

0 9.5 3.35 1.8 1.5 1.45

0.5 10.0 2.30

2.0 5.2 1.70

8.0 2.4 1.65

32.0 2.1 1.50 p24 ASSAY OF HIV-1 REPLICATION INHIBITION (Day 10) HIV-1 p24 Level (ng of protein/ml) At

The Following Concentration Of rsT4 (μg/ml)

Castanospermine (μg/ml) 0 0.03125 0.125 0.5 2.0

0 25.0 11.0 1.6 0 0

0.5 20.0 3.7

2.0 10.0

8.0 2.15

32.0 1.1

- - for assay days 5, 7 and 10, uninfected control = 0 Mathematical analysis of these results demonstrated that combinations of castanospermine and soluble T4 protein were synergistically inhibitory for HIV-1 replication over a wide range of concentrations. More specifically, the combination index values for the combinations of soluble T4 protein and castanospermine ranged from 0.58 to 0.39 for 70-95% inhibition of viral replication on day 10, indicating that the combinations were synergistically inhibitory for HIV-1 replication. Advantageously, no additional toxic side effects were associated with those combinations.

Results: Soluble T4 Protein And AZT

In Figure 3, we have summarized the parameters of additional assays demonstrating the effects of combinations of soluble T4 protein and AZT according to this invention in inhibiting HIV replication in vitro. Figure 4 depicts, in tabular form, the combination index values for rsT4 and AZT combinations in assays 1-8. In these assays, the concentrations of soluble T4 protein and AZT required to fully inhibit HIV-1 replication, as single agents and in combination, varied depending on the input virus inoculum, the cell type tested, and the sensitivity of the HIV-1 replicative assay utilized, as expected in biologic assays. These assays, however, clearly demonstrate the synergistic effects of soluble T4 protein and AZT in combinations according to this invention. In the reverse transcriptase assay, we measured inhibition of HIV replication in a culture of 0.4 x 10⁶ cells/ml of uninfected H9 cells in 5 ml of R-20 culture medium which was exposed to 500 TCID₅₀ of HIV-1 per 1 x 10⁶ cells. Various graded concentrations of recombinant soluble T4 protein or AZT, either alone or in combination, were added to the cell cultures simultaneously with the HIV-1 isolate . The results are shown in the table below .

REVERSE TRANSCRIPTASE ASSAY OF HIV- 1 REPLICATION INHIBITION (Day 7)

Mean RT Values (x 10⁴ CPM/ml)

At The Following Concentration Of rsT4 (μg/ml)

AZT

(μM) 0 0 .0078 0.03125 0.125 0.5

0 3.2 3.4 2.3 0.18

0.01 2.1 5.7

0.04 1.2 1.6

0.16 2.2

0.64 2.4 0.24

REVERSE TRANSCRIPTASE ASSAY OF HIV-1 REPLICATION INHIBITION (Day 10) Mean RT Values (x 10⁴ CPM/ml)

At The Following Concentration Of rsT4 (μg/ml)

AZT

(μM) 0 0.0078 0.03125 0.125 0.5

0 100 94 76 2.8 0.45

0.01 170 260

0.04 130 81

0.16 88 3.4

0.64 19 0 REVERSE TRANSCRIPTASE ASSAY OF HIV-1 REPLICATION INHIBITION (Day 12)

Mean RT Values (x 10⁴ CPM/ml)

At The Following Concentration Of rsT4 (μg/ml)

AZT

(μM) 0 0.0078 0.03125 0.125 0.5

0 130 110 290 22 3.1

0.01 200 180

0.04 210 120

0.16 120 13

0.64 110 0

- - for assay days 7, 10 and 12, uninfected control = 0

Mathematical analysis of these results demonstrated that combinations of soluble T4 protein and AZT were synergistically inhibitory at higher doses against HIV-1 viral replication when compared to the effect of each agent alone. More specifically, the combination index values for the combinations of soluble T4 and AZT ranged from 1.38 - 0.495 for days 10 and 12, the days analyzed, for 70-95% inhibition of viral replication. Synergy was observed in high dose combinations. There was no additional toxicity of the combinations over the single agents alone.

We also carried out additional assays, as set forth in Figure 3. For example, we carried out assays 1 and 2 using virus inocula of 3500 TCID₅₀ of HIV-1 per 1 x 10⁶ H9 cells and concentrations of 0.01 - 0.50 μg/ml soluble T4 protein and 0.01 - 0.64 μM AZT. Synergistic interactions between soluble T4 protein and AZT occurred at the highest dosages tested in combination - - rsT4 at 0.5 μg/ml and AZT at 0.64 μM. CI values were <1, indicating synergism, when 95% inhibition of infection was achieved in all except one instance.

Assay 3 was carried out using a virus inoculum of 1500 TCID₅₀ of HIV-1 per 1 x 10⁶ cells and concentrations of 0.002 - 0.125 μg/ml soluble T4 protein and 0.01 - 0.64 μM AZT. We found that concentrations of soluble T4 protein from 0.031 - 0.125 μg/ml in combination with 0.16 - 0.64 μM AZT inhibited HIV-1 synergistically, as measured by p24 antigen production and RT activity, with one exception, in which a Cl value of slightly greater than 1 was seen for p24 antigen production on day 10 (Figure 4).

We carried out assay 4 using a virus inoculum of 500 TCID₅₀ of HIV-1 per 1 x 10⁶ cells and concentrations of 0.001 - 0.320 μg/ml soluble T4 protein and 0.01 - 2.56 μM AZT. As demonstrated in Figure 5, combinations of ≥ 0.02 μg/ml soluble T4 protein and ≥ 0.16 μM AZT inhibited HIV-1 synergistically on day 10, as measured by p24 antigen production, RT activity, yield of infectious virus and

HIV-1 antigen expression by immunofluorescence. As shown in Figure 4, the Cl values for each of the p24 antigen production, reverse transcriptase, yield of infectious virus and HIV-1 antigen expression by immunofluorescence assays were less than 1, indicating that these agents act synergistically to inhibit virus replication.

We carried out assays 5 and 6 using a virus inocula of 3000 TCID₅₀ of HIV-1 per 1 x 10⁶ PBMC cells and concentrations of 0.02 - 0.32 μg/ml soluble T4 protein and 0.003 - 0.040 μm AZT. In each assay, we observed a dose-dependent inhibition of HIV-1 replication throughout 10 days of culture with soluble T4 protein and AZT when each was present as a single agent. In assay 5, combinations of ≥ 0.08 μg/ml rsT4 and ≥ 0.01 μM AZT inhibited HIV-1 synergistically on day 10. The results are shown in the table below. p24 ASSAY OF HIV-1 REPLICATION INHIBITION (Day 10)

HIV-1 p24 Level (ng of protein/10⁶ cells)

At The Following Concentration Of rsT4 (μg/ml)

AZT

(μM) 0 0.02 0.04 0.08 0.16 0.32

0 50.8 45.8 47.1 37.3 36.0 20.7

0.003 41.7 57.4

0.005 26.8 29.2

00..001100 2233..44 6.8

0.020 4.8 0.1

0.040 0.6 0.1 - - for assay day 10, uninfected control = 0

Figure 6 depicts the results of assay 5 over a 14 day period. Even though soluble T4 protein (0.02 - 0.32 μg/ml) and AZT (0.003 - 0.040 μM) as single agents were less effective against HIV-1 replication by day 14 of the assay, combinations of ≥ 0.16 μg/ml soluble T4 protein and ≥ 0.02 μM AZT demonstrated synergistic interactions which increased over time and was evident on day 14 (Figures 4 and 6). In assay 6, which was carried out in duplicate, similar synergism was observed throughout the 10 day assay period (Figure 4). We carried out assays 7 and 8 using virus inocula of 1 x 10⁶ TCID₅₀ of HIV-1 per 1 x 10⁶ BT4 cells and concentrations of 0.001 - 0.320 μg/ml soluble T4 protein and 0.01 - 2.56 μM AZT.

Both soluble T4 protein and AZT as single agents resulted in a dose dependent inhibition of HIV-1 replication over these concentration ranges, with rsT4 at 0.32 μg/ml and AZT at 2.56 μM exhibiting 90-99% inhibition by day 12. In assay 7 , we observed strong synergistic inhibition, which increased over time , at every concentration of soluble T4 protein and AZT tested in combination over the 12 day course of the assay . Combinations containing as little as 0. 001 μg/ml soluble T4 protein and 0 .01 μM AZT were also synergistic . These results are shown in the table below .

p24 ASSAY OF HIV-1 REPLICATION INHIBITION (Day 12) HIV- 1 p24 Level (ng of protein/ 10 cells )

At The Following Concentration Of rsT4 (μg/ml)

AZT

(μM) 0 0.001 0.005 0.020 0.080

0 71.1 89.0 39.2 10.4 0.6

0.01 47.8 19.7

0.04 20.7 8.7

0.16 11.9 2.9

0.64 7.6 0.3 - - for assay day 12, uninfected control = 0

In all of the assays described above, these agents, either alone or in combination, did not display toxicity in parallel control cultures over the concentration ranges tested. Microorganisms and recombinant DNA molecules which may be employed in the processes of this invention are exemplified by cultures deposited in the In Vitro International, Inc. Culture Collection in Linthicum, Maryland, on June 9, 1988, and identified as 170-2: E.coli JA221/p170-2. This culture was assigned accession number IVI 10172.

While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic constructions can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

Claims

CLAIMS We Claim:

1. A pharmaceutically effective combination against AIDS, ARC or HIV infection comprising a soluble T4 protein and an anti-retroviral agent, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent each being less than that required for a desired therapeutic or prophylactic effect when either compound is administered a monotherapy.

2. The combination according to claim 1, wherein the anti-retroviral agent is azidothymidine.

3. The combination according to claim 1, wherein the soluble T4 protein is recombinant soluble T4 protein.

4. The combination according to claim 1, wherein the amount of soluble T4 protein in the combination is less than about 0.1 mg/kg body weight/ day to 1.0 mg/kg body weight/day.

5. The combination according to claim 1, wherein the amount of azidothymidine in the combination is less than about 1.2 g/day to 1.5 g/day.

6. The combination according to claim 1, wherein the anti-retroviral agent is a glucosidase inhibitor.

7. The combination according to claim 6, wherein the glucosidase inhibitor is selected from the group consisting of castanospermine and deoxynojirimycin.

8. The combination according to claim 7, wherein the glucosidase inhibitor is castanospermine.

9. The combination according to claim 8, wherein the amount of castanospermine in the combination is less than about 100 mg/day.

10. The combination according to claim 8, wherein the amount of soluble T4 protein in the comination is less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.

11. The combination according to claim 1, wherein the soluble T4 protein is recombinant soluble T4 protein.

12. A method for treating or preventing AIDS, ARC or HIV infection in humans comprising the step of administering to a human a combination according to claim 1.

13. A method for treating or preventing immunodeficiency-causing retroviruses in mammals comprising the step of administering to a mammal a combination according to claim 1.

14. The use of a soluble T4 protein and an anti-retroviral agent for the production of a combination which is pharmaceutically effective for the treatment or prevention of AIDS, ARC OR HIV infection, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent each being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy.

15. The use according to claim 14, wherein the combination is effective to reduce the effects of HIV infection or to prevent intracellular spread of HIV infection.

16. The use according to claim 15, wherein the soluble T4 protein is recombinant soluble T4 protein.

17. The use according to claim 15, wherein the anti-retroviral agent is azidothymidine.

18. The use according to claim 17, wherein the azidothymidine is administered at a dosage of less than about 1.2 g/day to 1.5 g/day.

19. The use according to claim 15, wherein the soluble T4 protein is administered at a dosage of less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.

20. The use acording to claim 15, wherein the anti-retroviral agent is a glucosidase inhibitor.

21. The use according to claim 20, wherein the glucosidase inhibitor is selected from the group consisting of castanospermine and deoxynojirimycin.

22. The use according to claim 21, wherein the glucosidase inhibitor is castanospermine.

23. The use according to claim 22, wherein the castanospermine is administered at a dosage of less than about 100 mg/day.

24. The use according to claim 23, wherein the soluble T4 protein is administered at a dosage of less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.