Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-11
2001-02-20
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S003000, C435S004000, C435S005000, C435S007240, C435S325000, C424S130100, C424S134100, C424S141100, C424S147100, C424S148100
Reexamination Certificate
active
06190871
ABSTRACT:
1. TECHNICAL FIELD
This invention is in the field of immunotherapy. Specifically, the invention relates to the development of antibodies and derivatives thereof, cell lines that secrete the antibodies, and their method of use in the prevention, detection, amelioration or treatment of HIV disease, primarily AIDS (Acquired Immunodeficiency Syndrome) and ARC (AIDS Related Complex).
2. BACKGROUND ART
Recent scientific inquiry has focused upon an extracellular glycoprotein of Human Immunodeficiency Virus Type 1 (HIV-1), namely gp120. This glycoprotein is believed to play a vital role in attachment of HIV-1 to a host cell, by binding specifically to the host cell's cellular receptor CD4 and therefore important for the development of an AIDS vaccine. However, the gene encoding gp120 is highly variable among different HIV-1 strains, including sequential isolates from the same patient (1-4). The greatest variability has been demonstrated in several hypervariable regions which are flanked by more conserved regions (3, 4).
In response to the AIDS virus, the host immune system will produce antibodies targeted against various antigenic sites, or determinants, of gp120. Some of those antibodies will have a neutralizing effect and will inhibit HIV infectivity. It is believed that this neutralizing effect is due to the antibodies' ability to interfere with HIV's cellular attachment. It is also believed that this effect may explain in part, the rather long latency period between the initial seroconversion and the onset of clinical symptoms.
Much of our present knowledge about which domains of gp120 are immunogenic has come from studies of antibodies raised in laboratory animals. It was first observed that a variety of animals immunized with purified or recombinant gp120 or gp160 developed strain specific neutralizing antibodies (5-7). Subsequently, polyclonal animal antisera raised to recombinant or synthetic peptides representing the third hypervariable domain (V3) of gp120 (covering amino acid residues 307-330) were found to manifest similar strain specific neutralizing activity. This observation identified the V3 domain as a major target of neutralization, at least in laboratory animals (8,9). The V3 hypervariable region is flanked by conserved cysteine residues which may form a disulfide bond and define a “loop” region containing the largely conserved sequence Gly-Pro-Gly in its center. Synthetic loop region peptides have been found to elicit the production of antibodies that neutralize only virus obtained from isolates from which the synthetic peptide was derived. Hence, the V3 loop induces type-specific neutralizing antibodies; but these antibodies do not account for the broad virus-neutralizing activity detected in the sera of most infected persons.
Several type-specific neutralizing murine monoclonal antibodies have been produced that bind to epitopes within the V3 domain (10-12), allowing precise mapping of these epitopes. Murine monoclonal antibodies have also been generated to epitopes associated with the CD4 binding region near the COOH terminal of gp120 (14-15); but the extent that this region is also involved in neutralization has not yet been conclusively established. Although the peptide sequence in this region is relatively well conserved, Lasky et al (14) detected minor sequence differences in several virus strains. This finding has raised the possibility that some antigenic variation may also occur within the CD4 binding domain.
Our present knowledge about which epitopes of gp120 stimulate humoral responses in chronically infected humans is much more limited. In contrast to the type-specific neutralizing activity of animal antisera, sera of HIV infected patients generally contain more broadly reactive, group specific neutralizing antibodies (15-17). Such broadly neutralizing antibodies may be directed against a conserved site on gp41, against antibodies specific for gag proteins (31-35), or possibly against conformational epitopes on gp120 (42). Nevertheless, several groups of investigators have also observed that some patient sera manifest distinct patterns of strain-restricted neutralizing activity when tested against a variety of strains (5,6,18,19). Moreover, patient antibodies that have undergone affinity purification to gp120 from one virus strain have been shown to possess type-specific neutralizing activity against that strain (6). These observations suggest that the multiplicity of antibodies commonly detected early in HIV-1 positive patient sera to variable epitopes may mask important later antibody responses to broad neutralization epitopes, which are believed to develop approximately six-twelve months after initial HIV seroconversion. Consequently, the need exists for the development of such agents with broad binding and neutralizing capabilities for the prevention, diagnosis and treatment of HIV and AIDS related infections. The present invention satisfies this need and provides related advantages as well.
The papers cited throughout this application are incorporated herein by reference.
3. DISCLOSURE OF THE INVENTION
We have discovered four (4) previously undescribed and unrecognized human monoclonal antibodies which exhibit binding affinity for a specific conserved antigenic determinant of gp120. Tests indicate this binding to be dependent upon the tertiary or non-linear structure of the glycoprotein, and is important for both HIV-1 neutralization and CD4 binding. In addition, and in part because of the unexpected and surprising potency with which HIV infectivity is neutralized by these monoclonal antibodies, we believe the HIV determinant to be located in the CD4 binding region of gp120.
a. Definitions
As used herein, the terms “immunoreagent,” “immunoreagent equivalent” and “immunoreagent derivative” refer to antibodies, antibody fragments, antisera, anti-idiotypes, polypeptides or derivatives thereof, whether human, hybrid, chimeric or recombinant, purified or produced by any method, whether conjugated to toxins to enhance immunotoxicity or other molecules to enhance immunogenicity, and the cell lines which produce the antibodies, which bind in an equivalent manner to gp120, have similar neutralization activity, or otherwise mimic this epitope, and their use for the prevention, diagnosis, treatment or amelioration of HIV related disease.
As used herein, references to “gp120 antigen,” “gp120 antigenic determinant” and “epitope of HIV-1 gp120,” refer to that portion of the HIV-1 virus which is capable of combining with antibodies and derivatives thereof, T cell receptors and immunoreagents which mimic T cell receptors, and which is capable of inducing an immune response.
As used herein, the terms “reactive” and “reactivity” refer to an ability of the immunoreagent to recognize, bind to and/or mimic an antigen, antigenic determinant and/or epitope.
b. Method of Production
Because epitopes recognized by immunized animals may differ from those that stimulate antibody responses in naturally infected humans, we have produced human monoclonal antibodies (HMabs) that represent immunogenic responses of chronically infected patients to gp120 antigens. This production incidentally has allowed more precise identification and characterization of both conserved and variant gp120 determinants recognized by the human immune system. These IgG HMabs were produced by B cell lines derived by EBV transformation of peripheral blood B lymphocytes obtained from three asymptomatic HIV-1 infected patients. However, these antibodies and derivatives thereof, can also be produced in animals or with the help of hybridoma methods (monoclonal antibodies in mice or monoclonal antibodies in humans) or in yeast, bacteria or mammalian cells by recormbinant methods.
In particular, N70-1.5e, N70-1.7B, N70-2.1H and Y76-4.8D bind to a previously undescribed and unrecognized conformation dependent epitope on gp120, and exhibit surprisingly great potency for HIV neutralization. This epitope also appears to be responsible for inducing the broad HIV neutralizing and cellular binding inh
Ho David D.
Robinson James E.
Cedars-Sinai Medical Center
Lyon & Lyon LLP
Nelson Brett
Stucker Jeffrey
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