AU4964290A - Compositions and methods for treating or preventing aids, arc and hiv infection - Google Patents
Compositions and methods for treating or preventing aids, arc and hiv infectionInfo
- Publication number
- AU4964290A AU4964290A AU49642/90A AU4964290A AU4964290A AU 4964290 A AU4964290 A AU 4964290A AU 49642/90 A AU49642/90 A AU 49642/90A AU 4964290 A AU4964290 A AU 4964290A AU 4964290 A AU4964290 A AU 4964290A
- Authority
- AU
- Australia
- Prior art keywords
- protein
- soluble
- patient
- administered
- rst4
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- AIDS & HIV (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING AIDS, ARC AND HIV INFECTION
TECHNICAL FIELD OF INVENTION
This invention relates to vaccines, compositions, 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 compositions for treating or preventing AIDS, ARC, and HIV infection that are characterized by an immunologically effective amount of a soluble T4 protein which elicits in a treated patient the formation of antibodies to soluble T4 protein or, alternatively, the release of other proteins which, in turn, are effective to protect against or to lessen the spread, severity or immunocompromising effects of AIDS, ARC or HIV infection.
BACKGROUND OF THE INVENTION
The class of immune regulatory cells known as T 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 or CD4 (m.w. 55,000-62,000 daltons) which is expressed on T helper or inducer cells, probably as a monomeric protein, or T8 or CD8 (m.w. 32,000 daltons) which is expressed on T cytotoxic or suppressor cells as a dimeriσ protein.
In immύnocompetent 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 hematopoietic 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, some viruses and retroviruses. When T4 lymphocytes are exposed to such agents, they are rendered non-functional. 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 immunosuppression 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 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
* In this application, human immunodeficiency virus ("HIV"), the generic term adopted by the human retrovirus subcommittee of the International Committee On Taxonomy Of Viruses refers to independent isolates from AIDS patients, including human T cell lymphotropic virus type III ("HTLV-III"), lymphadenopathy-associated virus ("LAV") , human immunodeficiency virus type l ("HIV-1") and AIDS-associated retrovirus ("ARV") .
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 protein 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," Natur . 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 treatment and prevention 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:
A ino Acid
Structure/Proposed Location Coordinates
Hydrophobia/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.
Proposed methods for treating or preventing AIDS and ARC have also focused on the development of anti-retroviral agents which 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, azidothy idine ("AZT") and dideoxycytidine [H. Mitsuya et al., "38-Azido-3' -Deoxythymidine (BW A509U) : An Antiviral Agent That Inhibits The Infectivity And Cytopathic Effect Of Human T-Lymphotropic Virus Type I I/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-31-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. f supra]. 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. Enq. J. Med.. 317, pp. 192-97 (1987)].
Other prophylactic and therapeutic regimens are based on 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 effective im unotherapeutic agents, methods, and strategies for the treatment or prevention of AIDS, ARC, and HIV infection which avoid the disadvantages of conventional agents.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred to above by providing pharmaceutically effective compositions and methods for the treatment and prevention of acquired immunodeficiency syndrome, AIDS related complex and HIV infection. The compositions and methods of this invention are characterized by a soluble T4 protein which elicits in a treated patient the formation of antibodies to soluble T4 protein or, alternatively, the release of other proteins which, in turn, are effective to protect against or to lessen the spread, severity or immunocompromising effects of AIDS, ARC, and HIV infection.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the nucleotide sequence and the derived amino acid sequence of T4 cDNA of plasmid pl70-2. 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 lie: I Met: M
Val: V Ser: S Pro: P Thr: T
Ala: A Tyr: Y His: H Gin: Q
Asns 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 (AA ) at nucleotides 1274-1276. In Figure 2, the T4 protein translation start
(AA_2_) is located at the methionine at nucleotides 1207-1209 and the mature N-terminus is located at the asparagine (AA3) at nucleotides 1282-1285.
Figure 3A depicts the reverse transcriptase activities of bone marrow cells of normal monkeys subjected in vitro to exogenous SIV prior to, during and after rsT4 treatment.
Figure 3B depicts in tabular form the reverse transcriptase activities generated in PBL from a normal monkey exposed m vitro to exogenous SIV in the presence of plasma of an rsT4-treated monkey.
Figure 4 depicts, in tabular form, the reverse transcriptase activities of bone marrow cell cultures from SIV-infected monkeys and of peripheral
blood lymphocyte (SIV-infected monkeys) /H9 co-cultures before, during and after those monkeys were treated with rsT4.
Figure 5 depicts, in graphic form, the effects of rsT4 treatment on functional abnormalities caused by - SIVmac-induced disease activity - in rsT4- treated SIVmac-infected monkeys,
Figure 6 depicts, in graphic form, the effects of rsT4 retreatment on functional abnormalities caused by SIVmac-induced disease activity in a monkey - previously treated with rsT4.
Figure 7 depicts, in tabular form, the increase in CFU-GM and BFU-E colony counts in rsT4- treated SIVmac-infected monkey***s. Figure 8 depicts, in tabular form, the in vitro effect of either plasma from rsT4-treated monkeys or rsT4 on colony forming cells from bone marrow of
SIVmac-infected monkey***s.
Figure 9 depicts, in tabular form, the in vitro effect of plasma from rsT4-treated monkeys on colony-** forming3 cells from bone marrow of SIVmac- infected monkeys harvested during rsT4 treatment.
Figure 10 depicts, in tabular form, the in vitro effect of plasma from rsT4-treated monkeys on colony *** forming3 cells from bone marrow of SIVmac infected monkeys after completion of rsT4 treatment.
Figure 11 depicts, in tabular form, the in vitro augmentation of CFU-GM in bone marrow of SIVmac infected monkeys following addition of rsT4 or plasma from an rsT4-treated monkey.
Figure 12 depicts, in tabular form, the augmentation of CFU-GM by IgG fractions and non-IgG fractions of plasma of an rsT4-treated monkey.
Figure 13 depicts the surface bound immunoglobulin on circulating lymphocytes in an rsT4- treated monkey.
Figure 14 depicts the binding of immunoglobulin in plasma of an rsT4-treated monkey to CD4+ but not CD8+ human lymphocytes.
Figure 15 depicts the binding of immunoglobulin in plasma of an rsT4-treated monkey to CD4+ but not CD8+ rhesus monkey lymphocytes. Figure 16 depicts the binding of plasma immunoglobulin from an rsT4 immunized monkey to monkey PBLs.
Figure 17 depicts, in graphic form, inhibition of reverse transcriptase activity in SIV infected monkey PBLs, by plasma of an rsT4-immunized monkey.
Figure 18 depicts, in tabular form, inhibition of reverse transcriptase activity in SIV infected bone marrow macrophages, by plasma of an rsT4- immunized monkey.
Figure 19 depicts, in tabular form, CFU-GM from bone marrow cells of three SIVmac-infected monkey•*■*s in the presence of plasma from an rsT4-immunized monkey. Figure 20 depicts, in tabular form, that bone marrow macrophages from normal rhesus monkeys become resistant to SIV infection following rsT4/CFA immunization.
Figure 21 depicts in tabular form, that PBLs from normal rhesus monkeys become resistant to SIVmac infection following rsT4 immunization.
Figure 22 depicts, in tabular form, that virus isolations from PBLs and bone marrow cells become negative following immunization of SIVmac infected monkeys with rsT4 in adjuvant.
Figure 23 depicts, in graphic form, the increase of bone marrow CFU-GM and BFU-E in SIVmac infected monkeys following immunization with rsT4 in adjuvant.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to pharmaceutically acceptable compositions and methods for treating or preventing AIDS, ARC, and HIV infection. More particularly, this invention relates to pharmaceuti- cally effective compositions comprising an immunologically effective amount of a soluble T4 protein which elicits in a treated patient the formation of antibodies to soluble T4 protein or, alternatively, the release of other proteins which, in turn, are effective to protect against or to lessen the spread, severity or immunocompromising effects of AIDS, ARC or HIV infection. According to one embodiment, the method of this invention comprises the step of treating or immunizing a patient in a pharmaceutically acceptable manner with an immunologically effective amount of a soluble T4 protein, for a period of time sufficient to lessen the immunocompromising effects of HIV infection, to prevent HIV infection, or to prevent intracellular spread of HIV infection. The vaccines, compositions and methods of this invention may be used to treat or prevent AIDS, ARC, or HIV infection in mammals, including humans. These vaccines, compositions and methods may also be used for treating or preventing AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, in mammals including humans.
As used in this application, the term "immunologically effective" denotes the ability to elicit in a treated patient the formation of antibodies
to soluble T4 protein which are effective to protect the patient for some period of time against AIDS, ARC or HIV infection or to lessen the spread, severity or immunocompromising effects of AIDS, ARC or HIV infection. The term "immunologically effective" also denotes the ability to elicit in a treated patient the production or release of factors such as lymphokines, colony stimulating factors or other proteins which enhance the immune response in a patient to a degree which is effective to protect against AIDS, ARC or HIV infection or to lessen the severity, spread or immunocompromising effects of AIDS, ARC or HIV infection.
The compositions and methods of this invention are characterized by an indirect mechanism of activity of the soluble T4 protein which is prophylactic or therapeutic. Without being bound by theory, we believe that the indirect mechanism of action of soluble T4 protein which characterizes the methods and compositions of this invention may be due to the generation of antibodies to soluble T4 protein which themselves protect against or are effective in treating HIV infection. Alternatively, this indirect mechanism of soluble T4 protein activity may be mediated by the generation of lymphokines, such as interleukins, colony stimulating factors, or other proteins which enhance immune responses in a patient which, in turn, are effective in protecting against or treating HIV infection. 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 i munotherapeutic (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 1-A 362 of Figure 1, a polypeptide of the formula Met-AA -AA of Figure 1, a polypeptide of the formula AA1-AA3?4 of Figure 1, a polypeptide of the formula Met-AA1_374 of Figure 1, a polypeptide of the formula AA1~AA37? of Figure 1, a polypeptide of the formula Met-AA of Figure 1, a polypeptide of the formula AA_23-AA of Figure 1, a polypeptide of the formula AA_23-AA3?7 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,-3,6-.2_ of mature
T4 protein, a polypeptide of the formula Met-AA 1.—3_6.2. of mature T4 protein, a polypeptide of the formula AA of mature T4 protein, a polypeptide of the formula Met- AA of mature T4 protein, a polypeptide of the formula AA _ of mature T4 protein, a polypeptide of the formula Met-AA of mature T4 protein, a polypeptide of the formula AA_23-AA3?4 ' of mature T4 protein, a polypeptide of the formula AA -AA3 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 AA -AA of Figure 1, or portions thereof. Such polypeptides include polypeptides selected from the group consistng of a polypeptide of the formula A 3 to AA3g2 of Figure 1, a polypeptide of the formula A to A 3 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 vaccines, compositions 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 pl70-2 and the amino acid sequence deduced therefrom. The T4 cDNA of pl70-2 is almost identical to the approximately 1,700 bp sequence reported by Maddon et al. , supra. The T4 cDNA of pl70- 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 pl70-2, 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 [R. A. Fisher et al., Nature, 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 PCT/US88/02940, 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. culture collection, in Linthicum, Maryland, on September 2, 1987 and identified as:
EC100: E.coli JM83/pEC100 - IVI 10146 BG377S E:coli MC1061/pBG377 - IVI 10147 BG380: E.COli MC1061/pBG380 - IVI 10148 BG381: E.COli MC1061/pBG381 - IVI 10149.
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)].
According to one embodiment of the present invention, the soluble T4 protein is preferably modified or subjected to treatments to modify the protein to enhance its immunogenic character in the intended recipient. For example, various amino acid substitutions, modifications or deletions may be carried out during preparation of the soluble T4 protein. Alternatively, a soluble T4 protein may be modified by the addition of various pharmaceutically acceptable adjuvants protein prior to administration. Such an adjuvant may contain, for example, a muramyl dipeptide derivative and a carrier which includes a detergent and a combination of free fatty acids. Either type of such modification may be one that increases the immunogenicity of the soluble T4 protein beyond that of a soluble T4 protein endogenous to the patient or its species.
The vaccines and compositions of 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 or prophylactic application,. Such dosage forms may include pharmaceuti¬ cally 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, cellulose-based substances, and polyethylene glycol. Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
The vaccines and compositions of this invention may also include other components or be subject to other treatments during preparation to enhance their immunogenic character or to improve their tolerance in patients.
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.
For example, soluble T4 protein 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, intra- cutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical routes. Preferably, the compositions of the invention are in the form of a unit dose- and will usually be administered to the patient intramuscularly.
The soluble T4 protein may be administered to the patient at one time or over a series of treatments. The most effective mode of administration and dosage regimen of soluble T4 protein will depend upon the particular composition and/or adjuvant used for treatment, the severity and course of infection, previous therapy, the patient's health status and response to treatment, and the judgment of the treating physician.
According to one embodiment of this invention, a daily dose equal to or less than about 1 mg/kg body weight of a soluble T4 protein may be administered to the patient, via one or several administrations, or released from a depot form per day of treatment over a treatment period of between about 1 to 30 days. Subsequent boosters may be administered as needed to maintain the initial therapeutic or prophylactic effect.
For example, a typical dosage regimen for treatment or prevention of HIV infection using a soluble T4 protein which has been modified to enhance its immunogenic character in the intended patient would involve the administration of about 0.5 mg/kg body
weight to the patient once a day for about 30 days. Patients may require intermittent boosters of about 1.0 mg/kg body weight daily, once a week, on a weekly basis. In a preferred embodiment of this invention, the soluble T4 protein or modified soluble T4 protein is administered with an adjuvant, in order to increase its immunogenicity. Useful adjuvants include simple metal salts such as aluminum hydroxide, as well as oil based adjuvants such as complete and incomplete
Freund's adjuvant. When an oil based adjuvant is used, the soluble T4 protein usually is administered in an emulsion with the adjuvant. Most preferably, the soluble T4 protein is administered in an emulsion with incomplete Freund's adjuvant.
When the soluble T4 protein is mixed with an adjuvant, the mixture usually would be administered at several sites intramuscularly, intradermally or subcutaneously. Intramuscular administration is most preferred.
Where an adjuvant is added to the soluble T4 protein prior to administration about 1 mg/kg body weight could be administered on one day, followed by boosters of 1 mg/kg body weight once a week until the desired response is achieved. Thereafter, a booster of about 1 mg/kg body weight may be administered every one month.
Where the soluble T4 protein is administered in an emulsion with incomplete Freund's adjuvant, about 1 mg per individual could be administered on one day, followed by boosters of 1 mg per individual about once a month for two months, and thereafter about once every three months.
It shall, of course, be understood that the dosage and length of treatment will vary depending on
such factors as the level of immunogenicity of the soluble T4 protein used, whether an adjuvant is administered with the soluble T4 protein, the nature of any adjuvant used, and the immune status of the individual being treated. For example, the more highly immunogenic the soluble T4 protein, the lower the dosage and necessary treatment time. Similarly, the dosage and necessary treatment time will be lowered if the soluble T4 protein is administered with an adjuvant.
It should also be understood that dosage regimens according to this invention may include the administration of more than 1.0 mg/kg body weight/day over a given treatment period. 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 effects of soluble T4 protein in inhibiting viral replication and in improving functional abnormalities associated with SIVmac-induced disease activity.
In these examples, the soluble T4 protein used was recombinant human soluble T4 protein ("rsT4") or ("rsCD4") 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 In Vitro By Recombinant Soluble CD4," Nature, 331, pp. 76-78 (1988)]. pBG381 is characterized by DNA
coding for AA_23 to AA37? of T4 protein, as depicted in Figure 2.
We obtained two rhesus monkeys (Macaca ulatta) (colony born and raised at the New England Regional Primate Research Center, *Southborough,
Massachusetts) each weighing 5-7 kg, who were free of infectious disease and in good health. We also obtained four SIV -infected rhesus monkeys (colony born and raised at the New England Regional Primate Research Center) of similar weight. SIVmac-infection had been induced in these four animals by intravenous injection of tissue culture supernatant containing cell-free SIVmac virus. We also induced SIVmac infection in three additional rhesus monkeys (colony born and raised at the New England Regional Primate
Research Center) of similar weight who were not part of the rsT4-treatment group.
Three of the virus-infected monkeys had relatively normal immune parameters at the start of treatment; the fourth had a circulating absolute T4+ lymphocyte count of less than 100 per mm3 (normal rhesus monkeys have >1,000 T4+ PBLs per mm3). Each monkey had been housed in a captive primate colony for more than 6 months prior to administration of the soluble T4 protein. Throughout the administration period, each monkey was maintained on a conventional diet of monkey chow supplemented with fresh fruit.
The monkeys described in the treatment and non-treatment groups were designated as follows: Normal/rsT4-treated
Mm 156-84 Mm 158-84
SIVmac-infected'/rsT4-treated
Mm 156-85
Mm 159-86
Mm 167-84 Mm 202-84
SIVmac-infected/'no rsT4 treatment
Mm 129-86 Mm 244-86 Mm 335-78 Over a period of 50 days, each normal animal and four of the infected animals received recombinant human soluble T4 protein once daily by intramuscular injection to the large muscles of the thighs or buttocks, for a total dose of 2 mg/day/monkey of rsT4.3 (from the pBG381-transformed cell line BG 381) . Serum samples for clearance determination and to assess liver and renal functions were collected on days 8, 15, and 44 of treatment, as well as on days 8 and 18 following treatment. One of the infected monkeys, Mm 156-85, was re-treated for an additional 50 day period commencing on the 96th day after the end of the first course of treatment. We found that none of the animals experienced any significant adverse reactions to the rsT4. The distribution of lymphocyte subsets in the peripheral blood of all the animals (T2+, T4+, T8+, IL-2 receptor"1" and Bl+) and the function of those cells (proliferation following stimulation with xenogenic cells and pokeweed mitogen) did not change during the course of treatment. The serum chemistry studies to assess liver and renal function were unchanged when compared to baseline values. Although the animals experienced a transient drop in the number of bone marrow granulocyte-macrophage (CFU-GMs) progenitor colonies with an associated transient neutropenia, this
transient bone marrow suppression never became clinically significant. During treatment, none of the normal or infected monkeys showed changes in the distribution of lymphocyte subsets in their peripheral blood, in PBL function or in serum chemistry assessment of liver and renal function.*
PrepBaration of SIVmac Culture
We prepared an SIV -containing culture by exp *■anding SIVma„c isolate 251 (available from the New England Regional Primate Research Center, Southborough, Massachusetts) in a culture of peripheral blood lymphocytes (PBLs) obtained from a healthy human donor. Specifically, we isolated PBLs from a blood sample on a Ficoll-diatrizoate density gradient (sp. gr. 1.077) and incubated them for 3 days in culture medium (RPMI
1640 + 10% fetal calf serum) supplemented with 1 μg/ml Concanavalin A. The stimulated PBLs were then washed in Hanks' buffered saline solution and 3 x lθ7 cells were brought up to a 30 ml volume in culture medium
* The infected monkey (Mm 167-84) that showed evidence of severe immunological impairment at the start of treatment died, on the third day after the completion of the 50 day treatment period, of severe Coombs positive he olytic anemia. In the last 10 days before death, the animal's hemoglobin dropped from 7.7 to 3.7 grams per deciliter. Lesions seen at necropsy included a large amount of clotted blood in the small intestine, severe myocardial fibrosis and histiocytic infiltration of the splenic lymphoid follicles. These last two findings are not uncommon in rhesus monkeys chronically infected with SIV. The presence of blood in the proximal small bowel indicated that the animal may have died from an acute gastrointestinal hemorrhage, possibly secondary to stress induced by the acute hemolytic process. Such significant hemolytic anemias have been reported in AIDS patients [Z. A.
Schreiber et al.. Blood. 52, p. 117a (1983)].
Accordingly, death may have been a natural consequence of the SIVmac infection,
supplemented with 2 units/ml IL-2 (IL-2 obtained from Du Pont) . Following a 2 hour incubation at 37°C, we washed the cells, added 3 ml SIVmac isolate 251 containing tissue culture medium and incubated at 37°C. We harvested the supernatant at the time of peak reverse transcriptase (RT) activity (1.8 x 105 cpm/ml of RT activity) as described in M. Kannagi et al., "In Vitro Growth Characteristics of Simian T-Lymphotropic Virus Type III," Proc. Natl. Acad. Sci. USA. 82, pp. 7053-57 (1985) and M. D. Daniel et al., Science. 228, pp« 1201-04 (1985) . The supernatant was passed through 0.45 μm filters, divided into 1 ml aliquots and stored at -70°C until use.
Preparation of Bone Marrow Cell Cultures Heparinized bone marrow samples were obtained from ketamine anesthetized monkeys by posterior iliac crest aspiration. Mononuclear cells were isolated from the bone marrow aspirates by density gradient centrifugation, washed in Hanks' balanced salt solution and cultured in Iscove's modified Dulbecco's MEM (IMDM) supplemented with 12.5% FBS and 12.5% horse serum in 4 or 8 chamber tissue culture slides (Lab-Tek, Miles Scientific, Naperville, Illinois) at a cell concentration of 1 x 106/ml. After 7 days of culture at 37°C, nonadherent cells were removed.
Preparation Of PBL/H9 Co-Cultures
Peripheral blood lymphocytes were isolated from blood samples of monkeys by Ficoll-diatrizoate density gradient centrifugation (sp. gr. 1.077) and stimulated with 1 μg/ml Concanavalin A for 3 days as described supra. The stimulated PBLs were washed in Hanks' buffered saline solution and 1 x lθ6 cells were added to 1 x 106 uninfected H9 cells (a gift from
Dr. Robert C. Gallo, National Cancer Institute, Bethesda, Maryland) and the co-culture was maintained at 37°C for a minimum of 3 weeks. We monitored reverse transcriptase activity in culture supernatants as an indication of virus replication.
Assays Of Anti-Viral Activity
In these examples, we evaluated the antiviral activities of the compositions of this invention using modifications of various in vitro assays used to study anti-viral agents and neutralizing antibodies.
Reverse Transcriptase Assay
Bone marrow cell samples, prepared as described supra. were incubated with a 1:20 dilution of SIV culture supernatant (1.8 x 105 cpm/ml of RT activity) at a cell concentration of 1 x 106 per ml for 2 hours at 37°C. The cells were then washed twice in Hanks' balanced salt solution and placed in culture in complete medium. Alternatively, bone marrow cell samples obtained from infected monkeys, or PBL/H9 co- cultures prepared as described supra. were placed in culture in complete medium. The culture medium was changed every 3-4 days. Culture supernatant was harvested every 2-3 days and replaced with an equal volume of culture medium (RPMI 1640 + 10% fetal calf serum) .
We then measured RT activities of harvested culture supernatants as described below. -Supernatants with RT activity 5x background or greater, i.e., greater than about 1000 cpm/ml, were scored as positive for infection with SIVmac
We measured reverse transcriptase activity as an indicator of the effects of the compositions of this invention on HIV viral replication as described in
M. Kannagi et al., "In Vitro Growth Characteristics Of Simian T-Lymphotrophic Virus Type III," Proc. Natl. Acad. Sci. USA, 82, pp. 7053-57 (1985). Specifically, assay samples were prepared by placing 1.4 ml of test sample in a 1.5 ml Eppendorf tube and centrifuging at 12,000 xg for 90 minutes. We then removed the supernatant and incubated the pelleted virus on ice for 10 minutes with 20 μl of dissociation buffer [0.01 M Tris-HCl (pH 7.3)/0.2% Triton X-100/0.001 M EDTA/0.05 M dithiothreitol/0.06 M KC1] . We then mixed 15 μl of dissociated virus solution with 60 μl of RT cocktail [0.05 M Tris-HCl (pH 8=2)/0.007 M MgCl2/0.06 M KCl/0„08 mg poly(rC) oθligo(dG) primer per ml/0c007 M dithiothreitol/3.3 μCi [α32P]dGTP (3000Ci/mmol; lCi=37GBq; Amersham) ] and incubated at 37°C for 60 minutes. We then applied 60 μl of each sample onto a Whatman 3 disk and washed each disk in a beaker with 5% tricholoroacetic acid/2% sodium pyrophosphate. The disks were then dried, and the radioactivity of each disk was measured.
Colony Formation Assays .-. -
Bone marrow granulocyte-monocyte (CFU-GM) and erythrocyte (BFU-E) progenitor cell growth of bone marrow cell samples, prepared as described supra, were quantitated as follows.
We established a two layer culture for the quantitation of CFU-GM colonies. The underlayer of 1 ml 0.5% Nobel agar contained 60 jig ml eσombinant human granulocyte-macrophage colony stimulating factor (a gift of Genetics Institute, Inc. , Cambridge, Massachusetts) . The overlayer contained 105 bone marrow cells in 1 ml IMDM supplemented with 12.5% FBS and 12.5% horse serum in 0.3% Nobel agar. We layered the overlayer onto the underlayer in 35 X 10 mm Lux
tissue culture dishes (Nunc, Inc. , Naperville, Illinois) and the two layer cultures were maintained at 37°C in a 5% CO -humidified atmosphere.
BFU-E colonies were assessed in cultures of 105 bone marrow cells maintained in 0.9% w/v methylcellulose (Dow Chemical, Midland, Michigan) in IMDM supplemented with 30% FBS, 0.9% deionized bovine serum albumin (Fraction V, Sigma, St. Louis, Missouri) , 5 x 10 M 2-mercaptoethanol, containing 60 ng/ml recombinant human granulocyte-macrophage colony stimulating factor, 1 unit/ml sheep erythropoietin (Step III, Connaught Laboratory, Willowdale, Ontario) and 5% phytohemaglutinin-stimulated rhesus monkey conditioned medium. The phytohemaglutinin-stimulated rhesus monkey conditioned medium was prepared as follows. PBLs were isolated from heparinized blood of normal rhesus monkeys by Ficoll-diatrizoate density gradient centrifugation, as described supra. The PBLs were then incubated 5 days in culture medium (RPMI 1640 + 10% fetal calf serum) supplemented with lOμg/ml phytohemaglutinin. After incubation, the cells were pelleted, and the supernatant was used as the conditioned medium
CFU-GM and BFU-E colonies of greater than 50 cells were then counted under an inverted microscope 12-14 days after the cultures were established.
Total neutrophil counts in the peripheral blood were determined from complete blood counts and differentials done on EDTA—anticoagulated blood samples.
Inhibition of SIV Infection of Normal Bone Marrow Macrophages .
We subjected bone marrow cell cultures to
SIVmac and measured RT activities of culture
supernatants as described supra. We found that more than 90% of bone marrow cultures established from non- infected normal monkeys could be reproducibly infected with exogenously introduced SIV (data not shown) . Bone marrow from both of the rsT4 treated normal monkeys was susceptible to infection in vitro with exogenously introduced SIV prior to and 4 weeks following completion of treatment. However, three attempts at infection of bone marrow cell cultures from the rsT4 treated normal monkeys during the 50 day treatment period and a single attempt at 18 days after treatment was completed did not yield positive cultures.
Specifically, we sampled bone marrow from the rsT4-treated normal monkeys on the days noted below and introduced SIVmac into cultures of these cells 19 days prior to treatment, on days 10, 30 and 40 during treatment, and on days 18, 32, 87, 108, 128, 156, 163, and 183 after ("post") treatment. RT activity was measured in supernatants of these cultures. The results are shown in the table below and in Figure 3A.
REVERSE TRANSCRIPTION ASSAY " OF ST mac IMPLICATION INHIBITION Normal monke Mm 156-84
REVERSE TRANSCRIPTION ASSAY OF SIV REPLICATION INHIBITION (Normal monkev Mm 158-84)
We also found that SIVmac infection of normal monkey bone marrow macrophages was inhibited to a greater degree by plasma of rsT4-treated monkeys than by rsT4 itself. In this assay, we prepared bone marrow cell cultures as described supra and added either rsT4
or plasma from an rsT4-treated monkey. We then subjected the cell cultures to exogenous SIV as described supra.
As shown in the table below, ' SIVmac infection of normal bone marrow cell cultures was inhibited to a greater degree by plasma of the rsT4-treated monkey than by rsT4 itself, even at rsT4 concentrations exceeding the peak plasma levels of rsT4 achieved in the plasma of treated animals during the treatment period.*
RT (cpm/ml) Treatment Assay Day 19
Control 7489 rsT4 (200 μg/ml) 6333 (20 μg/ml) 5727
Plasma (αrsT4 Ab+) 1:5 331
(day 47 of treatment (Mm 156-85) )
1:25 1284 As shown in Figure 3B, plasma from day 42 post-treatment of the rsT4-treated monkey Mm 156-84 also efficiently inhibited SIV -replication in PBLs of a normal rhesus monkey.
We also measured the reverse transcriptase activities of bone marrow cultures of rsT4-treated monkeys or from co-cultures of peripheral blood lymphocytes of the treated monkeys and H9 cells before, during and after the rsT4-treatment period.
* We observed that peak plasma levels of rsT4 in the treated animals were reached two hours after the first administration of rsT4 on day 1 of the treatment period. In normal monkey 158-84, a peak plasma level of 1457 ng/ml of rsT4 was present 2 hours after administration of rsT4 on treatment day l. in contrast, on day 47 of treatment, the peak plasma reached 2 hours after administration was 243 ng/ml.
As illustrated in Figure 4, co-cultivation of PBLs of Mml56-85, Mm 159-86 and Mm202-84 with H9 cells yielded SIV prior to the start of treatment and up until approximately the end of the second week of the rsT4 treatment period. Virus isolation attempts thereafter were negative until, at the earliest, one month after completion of treatment.
SIVmac rep clication in bone marrow cultures,' a reflection of virus replication in macrophages rather than T lymphocytes, showed parallel changes. While virus was readily isolated from bone marrow prior to the initiation of treatment of Mm 156-85, Mm 159-86 and Mm 202-84, virus isolation attempts during the course of treatment were negative. Virus isolations from bone marrow reverted to positive on day 18 after completion of treatment in Mm 156-85 and remained negative as late as 45 days following treatment in Mm 159-86 and Mm 202-84.
In Mm 167-84, the monkey that was immunologically compromised at the outset of treatment, virus was isolated only episodically from PBLs following co-cultivation with H9 cells both prior to and during rsT4 treatment. While isolation of virus from the bone marrow of Mm 167-84 was positive before initiating treatment, three attempts at isolating virus during treatment were negative.
Thus, in the rsT4-treated SIVmac-infected monkeys, virus was more difficult to isolate during periods of rsT4 treatment than before d a ter treatment. We believe that these observations reflect a quantitative decrease in replication of virus in the infected monkeys due to the rsT4 treatment.
Improvement in Functional Abnormalities Caused
—bv- SIVmac-Induced Disease Activity—:
We also found that functional abnormalities reflecting SIV -induced disease activity in the rsT4- treated SIV -infected monkeys improved as a result of treatment with soluble T4 protein. Prior to rsT4 treatment of the SIVmac-infected monkey-*s,' the in vitro growth of both CFU-GM and BFU-E colonies from the bone marrow of these monkeys was depressed, as commonly found in SIVmac-infected monkeys and HIV-1-infected humans [C. C. Stella et al. , J. Clin. Invest.. 80, pp. 286-93 (1987)]. CFU-GM colonies in a series of assays of 10 normal animals were 222 ± 52/5 x 104 cultured bone marrow cells and BFU-E counts were 76 ± 12/2 x 105 cultured bone marrow cells. As a result of treatment, the depressed CFU-GM and BFU-E * colony counts improved in the infected animals. As shown in Figures 5-7, the number of colony forming and erythrocyte progenitor cells from the bone marrow of the SIV-infected animals increased during the rsT4 treatment period.
After treatment, a decline in bone marrow CFU-GM and BFU-E colony counts to baseline values occurred in two of the three surviving SIV-infected animals. The colony count of Mm 202-84 remained elevated. A transient neutropenia occurred in two of the four monkeys during the first two weeks of treatment. As shown in Figure 5, however, we observed an eventual rise in total peripheral blood neutrophil counts in all four animals which accompanied the increased colony-forming cell counts from their bone marrows.
Mm 156-85 was retreated in exactly the manner described supra after a period of 85 days following the first treatment. As shown in Figure 6, the same
increases in CFU-GM and BFU-E were seen associated with this treatment.
We also studied the .in vitro effect of rsT4 or plasma from rsT4-treated monkeys on the colony forming ^ cells from bone marrow of SIVmac-infected monkeys. The results are depicted in Figure 8, in which the "rsT4 Added" control was no added rsT4 and the "Plasma Source" control was plasma from a normal monkey who had not been treated with rsT4. The "pre Mm" plasma source was plasma obtained from an infected monkey prior to rsT4 treatment. The plasma was maintained in the cultures at a 1:40 final dilution. As shown in Figure 8, the plasma of rsT4- treated monkeys was more effective in increasing colony forming cells than rsT4, even when high concentrations of rsT4 were added.
Figures 9 and 10 depict the in vitro effect of plasma from rsT4-treated monkeys on colony forming cells from bone marrow of SIVmac-infected monkey■*s harvested during rsT4 treatment (Figure 9) and harvested after rsT4 treatment (Figure 10) . In those figures, the "Plasma Source" controls and the "pre-Mm" designations are the same as those referred to for Figure 8. As shown in Figure 9, colony forming cells in the infected animals increased over the course of treatment. Furthermore, upon re-treatment of an infected animal, CFU-GM cell counts again increased. As shown in Figure 10, the increase in CFU-GM colonies of infected monkeys caused -by plasma from rsT treated monkeys was still evident for some time after the end of the treatment period.
Figure 11 depicts the augmentation of CFU-GM in vitro in bone marrow from SIVmac-infected monkey■*s following addition of either rsT4 or plasma from rsT4- treated monkey. In Figure 11, the control used was
bone marrow without added rsT4 or added plasma. As shown in Figure 11, the plasma from an rsT4-treated monkey caused a greater increase in CFU-GM colony counts of SIVmae-infected monkeys when comp rared with concentrations of rsT4 far exceeding those achieved in the plasma of the rsT -treated monkey.
Figure 12 depicts the results of an assay carried out to determine what portion of the plasma was responsible for the colony formation augmenting activity observed. Specifically, we passed the plasma over a protein A Sepharose column. The flowthrough of the column was collected and retained as the "non-IgG" fraction. The bound material was then eluted as follows. The column was incubated for 30 minutes at room temperature in approximately 1 column volume of elution buffer (0.58 M acetic acid, pH 2.2/0.15 M NaCl) . The column was then eluted with NaHCO - neutralized elution buffer, and the eluate was dialyzed overnight at room temperature against phosphate buffered saline. This dialyzed eluate was retained as the " gG fraction". We then tested those fractions for their CFU-GM augmenting activity. In Figure 12, the control used was a bone marrow culture alone. As shown in Figure 12, the CFU-GM augmenting activity resides in the IgG fraction of the plasma of the rsT4-treated monkeys.
Characterization of the Antibody Response Induced Bv rsT4 Treatment
We believe that the antibody response to soluble T4 protein developed by the rsT4-treated monkeys was characterized by functional anti-viral properties responsible for the therapeutic effects observed. Accordingly, we next characterized the antibody response observed.
First, we determined whether peripheral blood lymphocytes circulating in the rsT4-treated rhesus monkeys had antibodies bound to their surface membranes. More sp cecifically***, we evaluated two SIVmac infected rsT4-treated animals (Mm 202-84 and Mm 156-85) as follows. PBLs from heparinized blood were isolated 5 days after completion of a 50 day course of rsT4 treatment as described supra. We incubated the PBLs in vitro with FITC-conjugated goat anti-human IgG (Tago) at a dilution of 1:40 in phosphate buffered saline for 30 minutes. The cells were then washed in Hanks' buffered salt solution (HBSS) and analyzed by flow cytometry (Epics CS, Coulter Electronics, Hialiah, Florida) . [See generally H. M. Shapiro, Practical Flow Cytometry, Alan R. Liss, Inc., New York, New York (1985).]
The PBLs of Mm 202-84 and Mm 156-85 exhibited surface fluorescence of 31% and 40%, respectively. PBLs of Mm 202-84 continued to demonstrate surface staining as late as 71 days after completion of rsT4 treatment. PBLs of Mm 156-85 exhibited less than 7% cell surface fluorescence by 20 days after treatment was completed. However, upon retreatment of this animal with single intramuscular inoculations of 4 mg rsT4 in a 0.8 ml volume on day 20, and 5 mg in a 1.0 ml volume on day 34 following completion of the first treatment course, an increase in the number of cells with bound surface Ig was observed. As shown in Figure 13, this level reached 50% of the cells by 42 days after completion of the 50 day treatment course.
Thus, the SIVmac-infected rsT4-treated rhesus monkey -s clearly developed rsT4-treatment related immunoglobulin (Ig) coating of some of their circulating PBLs. This suggested that the treated monkeys developed antibody
responses with specificity for molecules expressed by some but not all circulating lymphocytes.
We then defined the specificity of the antibody response observed. Specifically, we tested plasma from Mia 156-84 for its ability to bind to purified CD8+ and CD4+ human PBLs. We had previously generated populations by antibody-mediated complement lysis utilizing monoclonal anti-CD4 (l9Thy5D7) and anti-CD8 (7PT3F9) antibodies [Miskell and Shiigi, Selected Methods in Cellular Immunology, Freeman & Co. 1980, p. 211]. The anti-CD4 and anti-CD8 monoclonal antibodies used were gifts from Dr. S. F. Schlossman, Dana Farber Institute, Boston, Massachusetts. These antibodies are characterized in Leukocyte Typing II. Springer-Verlag, New York, New York, Reinherz et al., eds. (1986) . First, we incubated the CD4+ and CD8+ cell populations with the plasma of the rsT4-treated animal Mm 156-84 at a 1:40 dilution for 30 minutes at room temperature. Then, we washed the cells in HBSS and incubated for 30 minutes at room temperature in a 1:40 dilution of FITC-conjugated goat anti-human Ig. The cells were then washed in HBSS 3 times and analyzed by flow cytometry (Epics CS) . As shown in Figure 14, approximately 94% of the CD4+ cells but less than 6% of the CD8+ cells exhibited fluorescence. Thus, Ig in the plasma of the rsT4-treated animal bound human CD4+ but not CD8+ cells.
We next assessed the plasma of an rsT4- treated monkey for binding to CD4+ and CD8+ rhesus monkey PBLs. PBLs from a normal rhesus monkey were isolated as described supra and reacted with 10 μl of either phycoerythrin conjugated anti-CD4 (0KT4, Ortho Diagnostics) or phycoerythrin conjugated anti-CD8 (OKT8, Ortho Diagnostics) for 30 minutes. We then washed the cells in HBSS and incubated them for 30
minutes in a 1:40 dilution of plasma from monkey Mm 156-84. The cells were washed in HBSS and incubated in phosphate buffered saline for 30 minutes at room temperature with a 1:40 dilution of FITC-conjugated goat anti-human Ig (Tago) and washed again with HBSS. We fixed the cells in 0.1% paraformaldehyde-PBS, and analyzed them by two color flow cytometry (Epics CS) . As shown in Figure 15, the plasma stained CD4+ but did not stain CD8+ rhesus monkey PBLs. Thirty percent of the PBLs stained with both plasma and 0KT4, 19% of the PBLs stained with 0KT4 and not plasma.
Immunization Of Normal Rhesus Monkeys With rsT4 In Adjuvant
As demonstrated above, the plasma of the rsT4-treated"monkeys is highly efficient at blocking in vitro SIVmac rep clication and at enhancing** bone marrow hematopoietic function. In addition, the rsT4-treated monkeys developed an anti-CD4 antibody response. Based on these observations, we believed that very low doses of rsT4 delivered in adjuvant would provide a highly efficient way to generate an antibody response containing anti-SIV activity. To assess this belief, we obtained two normal rhesus monkeys, Mm 152-81 and Mm 346-80. Both monkeys were colony born and raised at the New England Regional Primate Research Center,
Southborough, Massachusetts. Each weighed 5-7 kg and was free of infectious disease and in good health. We immunized these animals with l mg rsT4 in an emulsion with complete Freund's adjuvant (Sigma) on day 0 and another 1 mg in an emulsion with incomplete Freund's adjuvant (Sigma) on day 30. These immunizations were each delivered subcutaneously with a total volume of 0.4 ml in 6 separate sites on the back. The animals were then bled twice monthly.
We tested plasma from Mm 346-80 for Ig binding to normal rhesus monkey PBLs which had been Con A activated and cultivated in IL-2 containing medium as described supra. As shown in Figure 16, binding to activated rhesus PBLs was detectable by day 28 following initial immunization and was maximal on day 90, with binding detected on 34% of the cells. Binding continued to be detected as late as 167 days following initial immunization. The plasma of the rsT4-immunized monkey Mm
346-80 was then assessed for anti-SIVmac activity■* in in vitro cultures of PBLs and bone marrow. The techniques utilized for infection and determination of reverse transcriptase activity are described supra. As shown in Figure 17, addition of Mm 346-80 plasma at a 1:40 final dilution in RPMI 1640 plus 10% fetal calf serum resulted in an almost ten-fold inhibition of the RT activity generated by the PBLs from the SIV -infected rhesus monkey Mm 351-85. As a positive control, Figure 17 also depicts the affect on reverse transcriptase activity of the anti-CD4 monoclonal antibody 19Thy5D7 (see supra) . As shown in Figure 18, the plasma from Mm 346-80 also caused a ten-fold inhibition of SIVmac replication in bone marrow cells of a normal rhesus monkey.
We then tested the plasma of an rsT4- immunized rhesus monkey for its affect on hematopoietic function in vitro using methods described supra. As shown in Figure 19, a 1:25 and a 1:125 final dilution of plasma of Mm 346-80 caused a significant increase in the CFU-GM from bone marrow of three SIVmac-infected rhesus monkeys (Mm 244-86, Mm 108-84 and Mm 169-79) .
We also tested the rsT4-immunized animals to determine whether any anti-viral activity was detectable in their bone marrow cells or PBLs. These
determinations were carried out as described supra for assessment of in vitro susceptibility of bone marrow cells to infection with SIVmac. Following •" immunization, both bone marrow cells and PBLs from the rsT4-immunized rhesus monkeys Mm 152-81 and Mm 346-80 were periodically placed in culture with various dilutions of cell-free SIVmac. The RT activity in these culture supernatants was determined as described supra. As shown in Figures 20 and 21, significant resistance to SIV replication was detectable by day 62 after the initial immunization and remained until approximately day 145. Thereafter, the PBLs and bone marrow cells of these animals appeared to be as susceptible to SIV infection as they were prior to immunization.
Immunization of SIV -Infected Monkeys With rsT4 in Adiuvanc
We next determined the therapeutic efficacy of rsT4 in adjuvant. The three SIVmac-infected monkeys utilized (Mm 179-86, Mm 104-86 and Mm 388-87) were colony born and raised at the New England Regional Primate Research Center, Southborough, Massachusetts. Each weighed 5-7 kg and was infected experimentally with SIV isolate 251 4-12 months prior to initiating this study. Two of the monkeys (Mm 179-86 and Mm 104- 86) were immunized with rsT4 in complete Freund's adjuvant on day 0 and in incomplete Freund's adjuvant on day 30 as described supra. The third monkey (Mm 388-87) was used as a control and immunized with adjuvant as described supra. but with 1 mg human serum albumin (HSA) in place of rsT4. The presence of virus in the bone marrow and PBL of these animals was then determined periodically, as described supra.
As shown in Figure 23, virus isolations from bone marrow cells and PBLs were positive prior to the
initial immunizations in all three monkeys. Virus isolations remained positive in the HSA-treated animal throughout the course of the assay. However following the second rsT4 immunization on day 30, virus isolations from bone marrow and PBL from Mm 179-86 became negative until day 100. Moreover, except for one positive virus isolation from PBLs and bone marrow of monkey Mm- 104-86, virus isolations from Mm 104-86 also became negative from the time of the second immunization on day 30 until approximately day 100.
As shown in Figure 24, during this treatment, CFU-GM and BFU-E from bone marrow cells of the two rsT4-immunized monkeys but not from bone marrow cells of the HSA-immunized monkey rose to the normal range. This increase in hematopoietic potential indicates an improvement in an SIV -induced functional abnormality. Advantangeously, therefore, the improved clinical and virologic parameters observed following daily treatment with soluble T4 protein may alternatively be achieved by immunization with low doses of soluble T4 protein in adjuvant.
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 composi¬ tions 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 (33)
1. A method" for treating or preventing AIDS, ARC or HIV infection in a patient comprising the step of administering to the patient a pharmaceutically acceptable composition which comprises an immunologically effective amount of a soluble T4 protein.
2. The method according to claim 1, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.
3. The method according to claim 2, wherein the soluble T4 protein is. administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.
4. The method according to claim 2, wherein the soluble T4 protein is modified by .he addition of a pharmaceutically acceptable adjuvant.
5. The method according to claim 4, wherein the soluble T4 protein is administered to the patient at a dosage of about 1 mg/kg body weight/day.
6. The method according to claim 3 or 5, wherein the composition is administered to the patient for a period of time between about 1 and 30 days.
7. A pharmaceutically acceptable composition for treating or preventing AIDS, ARC or HIV infection in a patient which comprises an immunologically effective amount of a soluble T4 protein.
8. The composition according to claim 7, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.
9. The composition according to claim 7, further comprising a pharmaceutically acceptable carrier.
10. The use of a pharmaceutically acceptable composition which comprises an immunologically effective amount of a soluble T4 protein for the treatment or prevention of AIDS, ARC or HIV infection.
11. The use according to claim 10, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.
12. The use according to claim 11, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.
13. The use according to claim 11, wherein the soluble T4 protein is modified by the addition of a pharmaceutically acceptable adjuvant.
14. The use according to claim 13, wherein the soluble T4 protein is administered to the patient at a dosage of about l mg/kg body weight/day.
15. The use according to claim 12 or 14, wherein the soluble T4 protein is administered to the patient for a period of time between about 1 and 30 days.
16. The method according to claim 1 or 2, wherein the pharmaceutically acceptable composition further comprises an adjuvant.
17. The method according to claim 16, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight to 1.0 mg/kg body weight per administration.
18. The method according to claim 17, wherein the soluble T4 protein is administered once a week until the desired immunological effect is achieved.
19. The method according to claim 18, wherein after the desired immunological effect is achieved, the soluble T4 protein is administered once a month.
20. The method according to claim 1 or 2, wherein the pharmaceutically acceptable composition further comprises incomplete Freund's adjuvant.
21. The method according to claim 20, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg to to 1.0 mg per patient per administration.
22. The method according to claim 21, wherein the soluble T4 protein is administered about once a month for two months and is thereafter administered about once every three months.
23. A pharmaceutically acceptable composition for treating or preventing AIDS, ARC or HIV infection which comprises an immunologically effective amount of a soluble T4 protein and an adjuvant.
24. A pharmaceutically acceptable composition for treating or preventing AIDS, ARC or HIV infection in a patient which comprises an immunologically effective amount of an adjuvant and a soluble T4 protein that is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.
25. The composition according to claim 23 or 24, further comprising a pharmaceutically acceptable carrier.
26. The use of a pharmaceutically acceptable composition which comprises an immunologically effective amount of a soluble T4 protein and an adjuvant for the treatment or prevention of AIDS, ARC or HIV infection in a patient.
27. The use according to claim 26, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.
28. The use according to claim 27, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight to 1.0 mg/kg body weight per administration.
29. The use according to claim 28, wherein the soluble T4 protein is administered once a week until the desired immunological effect is achieved.
30. The use according to claim 29, wherein after the desired immunological effect is achieved, the soluble T4 protein is administered once a month.
31. The use according to claim 24 or 25, wherein the pharmaceutically acceptable composition further comprises incomplete Freund's adjuvant.
32. The use according to claim 31, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg to to 1.0 mg per patient per administration.
33. The use according to claim 32, wherein the soluble T4 protein is administered about once a month for two months and is thereafter administered about once every three months.
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US30009689A | 1989-01-18 | 1989-01-18 | |
US300096 | 1989-01-18 |
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EP (1) | EP0454767A1 (en) |
JP (1) | JPH04504842A (en) |
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US5756096A (en) * | 1991-07-25 | 1998-05-26 | Idec Pharmaceuticals Corporation | Recombinant antibodies for human therapy |
US6136310A (en) * | 1991-07-25 | 2000-10-24 | Idec Pharmaceuticals Corporation | Recombinant anti-CD4 antibodies for human therapy |
IE922437A1 (en) * | 1991-07-25 | 1993-01-27 | Idec Pharma Corp | Recombinant antibodies for human therapy |
JP4646157B2 (en) * | 2007-12-25 | 2011-03-09 | 財団法人山形県産業技術振興機構 | Organic electroluminescent material and device using the same |
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1990
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- 1990-01-18 EP EP90902495A patent/EP0454767A1/en not_active Withdrawn
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