Title:  Methods and compositions for impairing multiplication of HIV-1

United States Patent:  6,399,067

Assignee:  Thymon L.L.C. (Short Hills, NJ)

Filed:  April 28, 2000

A composition which elicits antibodies to multiple known variants of Tat protein of HIV-1 of both the B and non-B clades contains the peptide R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 SEQ ID NO: 23, and preferably an additional at least two variants of a peptide or polypeptide of the formula: R1-Asp-Pro-Y₇ -Leu-Glu-Pro-Trp-Z₁₂ -R2 SEQ ID NO: 8. In this composition, at least one of the two variants contains Arg at Y₇ and Lys at Z₁₂, and in at least a second of the two variants Y₇ is Asn and Z₁₂ is Asn. Vaccinal and pharmaceutical compositions can contain one or more such peptides associated with carrier proteins, associated in multiple antigenic peptides, or as part of recombinant proteins. Diagnostic compositions and uses are described for assessing the immune status of vaccinated patients.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprising at least two variants of a peptide or polypeptide of the Epitope I formula R1-Asp-Pro-Y₇ -Leu-X₉ -Pro-Trp-Z₁₂ -R2 [SEQ ID NO:8], wherein Y₇ is selected from the group consisting of Arg, Lys, Ser and Asn; wherein X₉ is selected from the group consisting of Glu and Asp; wherein Z₁₂ is selected from the group consisting of Lys and Asn; wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl; wherein R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide. In this composition, at least one of the two variants must have the formula wherein Y₇ is Arg and Z₁₂ is Lys, and at least a second of the two variants must have the formula in which Y₇ is Asn and Z₁₂ is Asn. Each peptide of this composition is recognized as an HIV-1 Tat Epitope I by a primate immune system. This formula permits the construction and use of a variety of peptide combinations.

In another aspect, the above-described composition further contains one or more additional peptide or polypeptide(s) which represent other amino acid sequences which correspond to HIV-1 Tat amino acid residues 5 through amino acid residue 12. These optional amino acid sequences are described in detail below. These sequences are preferably from an HIV-1 strain with a Tat protein variant at that location.

In another aspect, this invention provides a composition described above that contains peptides or polypeptides which comprise at least the two required Epitope I peptides, recognized by primates (and preferably additional Epitope I peptides), in combination with one or more HIV-1 Tat Epitopes II, III and/or IV. Epitopes II, III and IV are the HIV-1 Tat peptide formulae described in International Patent Publication No. WO99/02185, incorporated herein by reference. Such compositions can combine appropriate HIV-1 Tat peptides, so as to provide for a composition that induces antibodies reactive with greater than about 95% of all known HIV-1 Tat proteins.

In yet a further aspect, the invention provides an antibody composition comprising at least one antibody, preferably generated in a primate, which specifically binds to a peptide or polypeptide of the formula R1-Asp-Pro-Y₇ -Leu-X₉ -Pro-Trp-Z₁₂ -R2 [SEQ ID NO:8], wherein Y₇ is selected from the group consisting of Arg, Lys, Ser and Asn; wherein X₉ is selected from the group consisting of Glu and Asp; wherein Z₁₂ is selected from the group consisting of Lys and Asn; wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl; wherein R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide. This antibody composition preferably comprises at least two antibodies, i.e., one antibody which binds to the Epitope I variant in which Y₇ is Arg and Z₁₂ is Lys, and at least a second antibody which binds to a second Epitope I variant in which Y₇ is Asn and Z₁₂ is Asn. Other antibodies directed to other variants than the two specified variants may also be included in this composition. These antibodies in the composition bind to Epitope I sequences recognized by the primate immune system, which epitope is present on multiple variants of HIV-1 Tat proteins. These antibodies include a variety of antibody constructs, such as monoclonal antibodies, as described in detail below.

In still another aspect, the invention provides an antibody, particularly a monoclonal antibody, which specifically binds to a primate-recognized epitope of an HIV Tat protein, the epitope comprising the amino acid sequence -Asp-Pro-Y₇ -Leu-X₉ -Pro-Trp-Z₁₂ - [SEQ ID NO:9], wherein Y₇, X₉ and Z₁₂ are defined as above.

In yet another aspect, the invention provides an antibody composition comprising at least one antibody that recognizes Epitope II peptide sequence -Lys-X₄₂ -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 10], where X₄₂ is Gly or Ala, as a distinct epitope from previously described antibodies which recognize the epitope of -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-[SEQ ID NO: 11]. Preferably, the composition comprises one antibody which recognizes both the peptide in which X₄₂ is Gly and the peptide in which X₄₂ is Ala. These antibodies are preferably generated in primates. These antibodies in the composition bind to Epitope II sequences recognized by the primate immune system, which epitope is present on multiple variants of HIV-1 Tat proteins. These antibodies include a variety of antibody constructs, as described in detail below.

In still another aspect, the invention provides an antibody, preferably a monoclonal antibody, that recognizes Epitope II peptide sequence -Lys-X₄₂ -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 10], where X₄₂ is Gly or Ala, as a distinct epitope from the epitope of -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 11], recognized by previously described antibodies.

In yet a further aspect, the invention provides a recombinant or synthetic gene which encodes sequentially a peptide or polypeptide that contains at least two variants of a peptide or polypeptide of the Epitope I formula R1-Asp-Pro-Y₇ -Leu-X₉ -Pro-Trp-Z₁₂ -R2 [SEQ ID NO: 8], as defined above. In this synthetic gene, at least one of the two variants must have the formula wherein Y₇ is Arg and Z₁₂ is Lys, and at least a second of the two variants must have the formula in which Y₇ is Asn and Z₁₂ is Asn. Optionally, this synthetic gene comprises a carboxy terminal Epitope II peptide, as recognized by the primate immune system. Alternatively, the recombinant or synthetic gene contains the seven or eight preferred primate-recognized Epitope I amino acid sequences identified below. The synthetic gene may contain each amino acid sequence separated by a spacer sequence, or may express each peptide/polypeptide in an open reading frame with a carrier protein. The synthetic gene may be separated from the carrier protein by a spacer if the spacer is fused to a primate-recognized Epitope I sequence, leaving an Epitope II sequence at the carboxy terminus of the recombinant protein. Further embodiments include multiple Epitope I peptides of the above formula fused together and to the carrier protein.

In yet a further aspect, the invention provides a synthetic molecule, e.g., a vector, comprising the above-described synthetic gene, operatively linked to regulatory nucleic acid sequences which direct and control expression of the product of the synthetic gene in a host cell.

In another aspect, the invention provides a recombinant microorganism, e.g., a virus or commensal bacterium, which contains the above described synthetic gene or synthetic molecule. This microorganism is capable of expressing multiple copies of the product of the gene or molecule in a host.

Still another aspect of the invention is a pharmaceutical composition useful for inducing antibodies that react with a large number of known HIV-1 Tat proteins, e.g., greater than 95%, and preferably greater than 99%, of the known Tat proteins. These induced antibodies can impair the multiplication of HIV-1. The pharmaceutical composition comprises at least one of the recombinant or synthetic peptide/polypeptide compositions described above, or the synthetic gene/molecule described above; or the recombinant microorganism described above, in a pharmaceutically acceptable carrier.

Still a further aspect of the invention is a pharmaceutical composition useful for impairing the multiplication of HIV-1, this composition containing an above described antibody composition or monoclonal antibody composition.

In yet a further aspect of the invention, a method for reducing the viral levels of HIV-1 involves exposing a human or other primate to antibody-inducing pharmaceutical compositions described above, actively inducing antibodies that react with most HIV-1 Tat proteins, and impairing the multiplication of the virus in vivo. This method is appropriate for an HIV-1 infected subject with a competent immune system, or an uninfected or chronically infected, but asymptomatic, subject. The method induces antibodies which react with HIV-1 Tat proteins, and which reduce viral multiplication during any initial acute infection with HIV-1 and which further minimize chronic viremia which leads to AIDS.

In still another aspect, the invention provides a method for reducing the viral levels of HIV-1 by administering to a human, who is incapable of mounting an effective or rapid immune response to infection with HIV-1, a pharmaceutical composition containing the antibody compositions described above. The method can involve chronically administering the composition.

Yet other aspects of the invention include methods for producing the compositions described above, as well as host cells transfected with such compositions.

Still another aspect of this invention is a kit useful for the measurement and detection of titers and specificities of antibodies induced by immunization with the compositions described above. The kit of the invention includes preferably the two required Epitope I peptides described above, as well as addition peptides of the Epitope I, recognized by primates, and possibly additional peptides of Epitopes II through IV, and coated solid supports, a labeled reagent for detecting the binding of antibodies to these peptides, and miscellaneous substrates and apparatus for evoking or detecting the signals provided by the labels, as well as conventional apparatus for taking blood samples, appropriate vials and other diagnostic assay components.

In yet a further aspect, the invention provides a method for detecting the titers and reactivity patterns of antibodies in subjects immunized with the compositions of this invention. The method includes the steps of incubating dilutions of the subject's biological fluid, e.g. serum, with plates or beads on which are bound one or more peptides of the Epitope I sequences of this invention and optionally, the Epitopes II through IV, washing away unbound biological materials, and measuring any antibody binding to the peptides with labeled reagent, e.g., an anti-human immunoglobulin to which is associated an enzyme. Depending on the type of label employed, the signal produced by the label may be evoked by further adding a substrate which reacts with the enzyme, e.g., producing a color change. Other conventional labels may also be incorporated into this assay design.

Claim 1 of 17 Claims

What is claimed is:

1. A composition comprising a peptide or polypeptide of the formula R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 SEQ ID NO: 8,

wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl; and

wherein R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide.

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