Antibodies specific for CD4-binding domain of HIV-1

Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...

Reexamination Certificate

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C435S328000, C435S070200, C435S452000, C530S388350, C530S387300

Reexamination Certificate

active

06309880

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to antibodies which target the CD4-binding region of gp120 HIV-1, and which neutralize HIV-1.
BACKGROUND OF THE INVENTION
Acquired immunodeficiency syndrome (AIDS) is caused by a virus which has at various times been called human T-cell lymphotropic virus type III (HTLV-III), or lymphadenopathy-associated virus (LAV). The virus is currently known as human immunodeficiency virus type 1 (HIV-1).
HIV-1 damages the immune system by infecting and depleting T helper/inducer lymphocytes (hereinafter referred to as “T cells”). T cells are essential because they control the production of antibodies by the B cells, the maturation of cytotoxic T lymphocytes (killer T cells), the maturation and activity of macrophages and natural killer cells, and directly and indirectly, numerous other regulator and effector functions of the immune system.
Infection of a T cell occurs through interaction between an epitope borne by HIV-1 and a receptor site which is located on the T cell surface. This receptor site on the T cell is a protein molecule known as the CD4 antigen. The epitope on HIV-1 is borne by the external envelope glycoprotein gp120 (molecular weight about 120,000 daltons).
The glycoprotein gp120 is produced when a precursor glycoprotein gp 160, made in the HIV-1-infected T cell, is cleaved apart into a transmembrane portion gp41 (molecular weight about 41,000 daltons) and gp120. Glycoprotein gp41 spans through the membrane lipid bilayer of the vizions and of the infected cells and its exterior portion is associated with gp120 through noncovalent binding. Glycoprotein gp120 bears a site which fuses with target cells, whereby the genetic material of the virus enters the cell.
Since the CD4 antigen was identified as the cell-surface receptor for HIV-1, it has been repeatedly shown that soluble forms of CD4 antigen (sCD4) can block the infectivity of the virus. Soluble CD4 inhibits diverse variants of HIV-1, indicating that all these viruses may share a relatively conserved CD4-binding region. Lasky et al. have identified a gp120-specific murine monoclonal antibody (Mab) capable of inhibiting the interaction between gp120 and CD4.
Cell,
50:995-985 (1987).
The epitope recognized by this Mab has been mapped to a conserved region within amino acid residues 413-456 (numbered according to Human Retroviruses and AIDS, Los Alamos National Laboratories, 1990) where HIV-1 and distantly related HIV-2 share significant homology. This domain of gp120 appears to be important in binding to CD4. However, the CD4-binding region of the envelope glycoprotein gp120 does not appear to be immunogenic in HIV-1 infected persons, since there is very little antigenic cross-reactivity against envelope proteins between sera from patients infected with HIV-1 or HIV-2. Clavel, R. et al.,
Science,
233:343-346 (1986).
SUMMARY OF THE INVENTION
This invention pertains to an epitope located in the region of the envelope glycoprotein gp120 of HIV-1 which binds to the CD4 receptor of T cells, and to antibodies which bind to this epitope. The epitope is located at about amino acid residues 423-437 of gp120. Antibody reactive with the epitope can inhibit T cell infection by diverse strains and isolates of HIV-1.
The antibodies of the invention (and related antibodies) can be used as therapeutic agents, or vaccines, against AIDS, AIDS related complex (ARC), or to treat HIV-infected but asymptomatic individuals. The whole antibody, or a fragment, can be used. Alternatively, such antibody can be conjugated to cytotoxic or antiviral agents, or to a microcarrier (for example, a liposome) which contain such an agent, to produce an immunoconjugate which targets the delivery of the agent to HIV-1 infected cells. The targeted delivery of a therapeutic agent can also be achieved with bispecific antibodies derived from the antibody of this invention. Bispecific antibodies have a second specificity for the agent to be delivered to the target.
The antibodies of this invention include those derived wholly from mice or other animals, as well as wholly human antibodies (which are most preferred for in vivo use), or animal/human chimeric antibodies or humanized antibodies, in which only the antigen-binding regions and some of the framework variable regions are animal derived.
Other uses for the monoclonal antibodies of the invention, such as developing anti-idiotypes for use in diagnostic assays or antibody screening, or using the antibodies in screening for HIV-1 virions or infected cells in standard assay formats, such as an ELISA, are also possible.
Peptides corresponding to the gp120 epitopes of this invention could also be used to stimulate a neutralizing immune response against HIV-1. A preferred peptide has the amino acid sequence IINMWQKVGKAMYAP SEQ ID NO:1 or a functional equivalent thereof.
DETAILED DESCRIPTION OF THE INVENTION
The epitopes of this invention are located on gp120 within the region which binds to the CD4 receptor of T cells. The CD4 receptor binding epitope contains a segment having the amino acid sequence IINMWQKVGKAMYAP. This sequence corresponds to residues 423-437 of gp120 (numbered according to Human Retroviruses and AIDS, los Alamos National Laboratories, 1990). Antibodies against the epitope can inhibit HIV-1 infection with high potency. For example, the monoclonal antibodies which were specific for the epitope all inhibited several HIV-1 isolates. In addition, the epitope is highly conserved among HIV-1 strains and isolates; consequently, antibody specific for one or more of these epitopes can inhibit diverse strains and isolates of HIV-1.
Antibodies to this epitope can be made by somatic cell fusion techniques. See generally, G. Kohler and C. Milstein,
Nature,
256, 725 (1975); Fung e al.,
Biotechnology
5:940-946 (1987). In brief, an animal such as a mouse is immunized with gp120 of HIV-1. The gp120 can be purified, or partially purified from viral lysates for this purpose. The purification of gp120 can be accomplished by affinity chromatography with antibody against gp120. After immunization, B cells are taken from the immunized animal and then fused with immortalizing cells such as myeloma cells.
Hybridomas which produce antibody that binds to the epitope can be identified by a screening procedure. In such a screening procedure, the hybrid cells are first tested for production of antibody against gp120, preferably with an enzyme-linked immunosorbent assay (ELISA) or a Western blot assay. The assay is run on each of the hybrid cells and hybrid cells which are positive are selected for further analysis. Those that show highest reactivities are ultimately selected. The antibodies can be tested for crossreactivity among different virus isolates by immunofluorescence staining on the surface of virally-infected cells and by radio-immunoprecipitation assays with metabolically radiolabeled viruses. See e.g., Fung et al., supra.
After identification of antibodies reactive with gp120 by the above-described techniques, the antibodies can be tested for neutralizing activity, which is preferably determined by two assays, the first being a virus neutralization assay, described in Ho, D. D. et al.,
Science,
239:1021-1023 (1988). This assay measures the extent of inhibition of HIV-1 infectivity in H9 cells. The second neutralization test is based on the extent of syncytium inhibition as described by Nara, P. L. et al.,
AIDS Res. Hum. Retroviruses.
3:283-302 (1987). HIV-1 and antibody are added to a well seeded with CD4
+
CEM-SS cells. CEM-SS cells infected by HIV-1 will express gp120 on the cell surface and form syncytium with the neighboring CD4
+
CEM-SS cells. The neutralization of HIV-1 by the Mab is manifested as the inhibition of syncytium formation.
The epitope to which the HIV-1-inhibiting antibodies bind is then identified. This can be accomplished by constructing peptides which correspond to the various regions and which overlap with each other by five amino acids. Reactivity with the peptides can then be examined in a standard immunoassay such as a

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