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Immunoconjugates comprising cd4 and immunoglobin molecules for the treatment of HIV infection

a technology of immunoglobin and conjugate, which is applied in the field of cd4 polypeptides, can solve the problem of inability to efficiently neutralize primary isolates of hiv

Inactive Publication Date: 2009-11-19
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The CD4 fusion proteins demonstrate improved neutralization of HIV by increasing the avidity for gp120, effectively inhibiting viral entry and replication at lower concentrations compared to sCD4.

Problems solved by technology

However, toxicities associated with long term HAART have put a high priority on the design and development of less toxic therapies.
Among the problems inherent to sCD4 was its inability to efficiently neutralize primary isolates of HIV.

Method used

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  • Immunoconjugates comprising cd4 and immunoglobin molecules for the treatment of HIV infection
  • Immunoconjugates comprising cd4 and immunoglobin molecules for the treatment of HIV infection
  • Immunoconjugates comprising cd4 and immunoglobin molecules for the treatment of HIV infection

Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Methods

[0150]Virus entry. Virion entry into primary lymphocytes was measured using a quantitative real-time PCR assay based upon the generation of early LTR transcripts, adapted from a method previously described (Chun et al., Nature 387(6629):183, 1997). Briefly, freshly isolated peripheral blood mononuclear cells (PBMCs) were activated (OKT3 (1 μg / ml) / IL2 (50 u / ml) for three days and then depleted of CD8+ T-cells by magnetic bead selection (Dynal, Lake Success N.Y.). 3×106 cells were incubated in a volume of 100 μl with the addition of titered viral stocks (Advanced Biotechnologies Columbia, Md.) at a multiplicity of infection (MOI) of 0.1 for two hours at 37° C. Where specified, monomeric sCD4 and D1D2-Ig□tp (see below) were preincubated with virus stocks for 10 minutes at 37° C. prior to cell inoculation. Cells were washed with PBS, pelleted through a 100% fetal bovine serum (FBS) cushion (heat inactivated), and then resuspended in DMEM / FBS (heat inactivated) and i...

example 2

Expression of a CD4 Fusion Protein

[0160]D1D2-Igαtp is expressed as a highly oligomerized protein. It was first asked whether D1D2-Igαtp was expressed in a highly oligomerized form. To this end, was purified from culture supernatants by protein-A affinity chromatography, and analyzed by standard size exclusion chromatography. When passed over an analytic superdex-200 gel-filtration column a major peak appeared in that fraction corresponding to a molecular weight greater than 650 kDa. A minor fraction, comprising less than 5% of total protein eluted in the 50-100 kDa range. Because the major fraction appeared close to the void volume of the column, it was not possible to accurately estimate its molecular weight from these data. These fractions were then reduced and electrophoresed under denaturing conditions. Western blot analysis with a polyclonal antisera specific for CD4 indicated that D1D2-Igαtp resided primarily in the peak fraction (FIG. 1). When this blot was re-probed with a g...

example 3

Comparison of D1D2-Igαtp and Monomeric sCD4 in a Quantitative HIV Entry Assay

[0161]To determine the efficiency with which D1D2-Igαtp inhibited HIV entry a real-time PCR based quantitative viral entry assay was established. Virion entry was detected by measuring the level of the initial reverse transcription products in the R and U5 regions of the HIV-1 LTR. The target cells utilized in this assay were three day activated, CD8+ T-cell depleted PBMCs. After optimization, the linear range of this assay typically fell between 25 and 200,000 copies of reverse transcribed product. Two viruses, JR-FL and Bal, both of which utilize the CCR5 coreceptor and were derived after minimal passage of primary isolates, were then employed. To establish the conditions under which sCD4 would enhance viral entry, viral inoculi were briefly pre-incubated with various concentrations of monomeric sCD4 and then carried out the entry assay.

[0162]Under these conditions sCD4 at a concentration of either 6.25 o...

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Abstract

Nucleic acids encoding recombinant CD4-fusion proteins are disclosed herein that include a CD4 polypeptide ligated at its C-terminus with a portion of an immunoglobulin comprising a hinge region and a constant domain of a mammalian immunoglobulin heavy chain. The portion of the IgG is fused at its C-terminus with a polypeptide comprising a tailpiece from the C terminus of the heavy chain of an IgA antibody or a tailpiece from a C terminus of the heavy chain of an IgM antibody. Also disclosed herein are methods for using these CD4-fusion proteins.

Description

PRIORITY CLAIM[0001]This application is a divisional of U.S. patent application Ser. No. 10 / 493,676, filed on Jul. 27, 2004, which is the § 371 U.S. National Stage of PCT Application No. PCT / US02 / 34393, filed Oct. 24, 2002, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Patent Application No. 60 / 346,231, filed Oct. 25, 2001. The prior applications are incorporated by reference herein in their entirety.FIELD OF THE INVENTION[0002]This invention relates to the field of CD4 polypeptides, specifically to CD4 fusion proteins of use in the treatment of an immunodeficiency virus infection such as a human immunodeficiency virus (HIV).BACKGROUND OF THE INVENTION[0003]The primary immunologic abnormality resulting from infection by HIV is the progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein (Lane et al., Ann. Rev. Immunol. 3:477, 1985). CD4 is a non-polymorphic glycoprotei...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07H21/00C12N15/63C12N5/00C12N5/08C12N7/06A61P37/04A61K38/00C07K14/73C12P21/08
CPCA61K38/00C07K2319/30C07K2319/00C07K14/70514A61P37/04
Inventor ARTHOS, JAMESCICALA, CLAUDIAFAUCI, ANTHONY S.
Owner UNITED STATES OF AMERICA
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