Title:  HIV-vaccines

United States Patent:  6,268,484

Assignee:  Polymun Scientific Immunbiologische Forschung GmbH (Vienna, AT)

Filed:  July 30, 1998

Disclosed are antibodies which can be used for the manufacture of vaccines for active and/or passive immunization of persons in need of such treatment. The invention also provides for human monoclonal antibodies that are functionally equivalent to the above-mentioned antibodies produced by any one of the cell lines CL1 through CL6 (deposited at the European Collection of Animal Cell Cultures (ECACC) at the PHLS in Porton Down, Salisbury, UK). Also provided are hybridoma and/or CHO cell lines producing any one of the antibodies disclosed and claimed herein, Also provided are mixtures of antibodies of the present invention, as well as methods of using individual antibodies or mixtures thereof for the detection, prevention and/or therapeutical treatment of HIV-1 infections in vitro and in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The contents of each of the references cited herein are herein incorporated by reference in their entirety.

When conducting experiments to find novel anti-HIV-1 antibodies the inventors found human monoclonal antibodies which could be shown to efficiently neutralize HIV-1 in vitro including a variety of primary HIV-1 isolates, such as, e.g., primary HIV-1 isolates 92 RW009, 92RW021, 92UG037, 92TH014, 92BR030, N70-2, DJ259 (all obtained from WHO network for HIV-1 isolation and characterization), or WYG, WRF, WRB, WSC, WHM (isolated from Austrian patients).

Surprisingly, it turned out that these antibodies recognize and bind to two different antigenic determinants of the glycoprotein gp160 of HIV-1.

Moreover, it appears that the binding target of these antibodies is extraordinarily unique. In a comparative test involving a mixture of 41 different HIV-1 binding antibodies supplied by laboratories from different companies and research institutes, it was shown that no one of the other antibodies present in the mixture competed with an antibody of the above-identified group, for instance with the human monoclonal antibody from cell line CL2, for binding to the targeted antigenic fragments of gp120/gp160 corresponding to amino acid sequences 79 to 184 and 326 to 400 of processed gp120 of HIV-1 isolate BH10.

Also, investigations of blood serum and blood plasma of HIV-1 infected patients revealed that antibodies of the CL2 type were not present in the samples tested so far. This finding again emphasizes the uniqueness of these HIV-1 neutralizing human monoclonal antibodies and simultaneously indicates that there might exist an extraordinary potential to combat HIV-1 infection, by using these antibodies in a suitable form for the prophylactic and/or therapeutic treatment of human individuals.

Another object of the present invention relates to antibodies of the CL2 type which have been found to bind to the above-mentioned antigenic determinants of gp120/gp160 only if the determinants are present in a glycosylated form; they do not bind to these antigenic glycopeptide fragments when the fragments are deglycosylated, e.g., by the action of Peptide-N-Glycosidase F (EC 3.2.2.18; hereinafter referred to as "PN Gase F").

Still another object of the present invention encompasses human monoclonal antibodies of the CL2 type which are further characterized in that they also specifically bind to a fragment of gp120 produced in the SF9 insect cell/Baculovirus expression system in the absence of tunicamycin, while they do not bind to gp120 fragments expressed in the presence of tunicamycin. Tunicamycin is known for its inhibitory activity toward the glycosylating action of glycosyl transferase in glycoprotein biosynthesis.

Among the antibodies of the CL2 type as disclosed herein, there are also types which inhibit the infection of human lymphocytes by primary HIV-1 isolates such as the ones listed herein, as could successfully be demonstrated by the inventors in in vitro experiments.

The present invention also relates to antibodies of the CL2 type which possess one or more of the above-mentioned properties and which can further be characterized by their special interaction with the anti-idiotypic monoclonal antibodies of hybridoma cell lines CL5 and CL6. While they can be bound by one and/or the other of the two anti-idiotypic antibodies CL5 and CL6, at least part of them is bound by anti-idiotypic huMAb produced by CL6 in a way that results in a specific blockade of the capability of the antibody to inhibit the infection of human lymphocytes by primary HIV-1 isolates.

A further object of the present invention comprises antibodies of the CL2 type which show at least one of the above-mentioned features or properties and which--in addition--have been proved to compete for binding to the antigenic determinants of gp120/160 with the antibody produced by hybridoma call line CL2. The antibodies of this category are therefore--at least functionally--very closely related with the antibody released by CL2, and can be regarded as functional equivalents to it.

Another object of the present invention is directed to the most beneficial human monoclonal antibody produced by hybridoma cell line CL2. This antibody can be used, e.g. for passive immunization of HIV-1 infected individuals, but may even be more useful as a biochemical tool for developing vaccines applicable in the prevention and/or therapy of HIV-1 infections in vivo.

An attractive object of the present invention comprises the use of recombinant CHO cells for the production of the antibodies of the CL2 type. After successful identification of the antigenic determinants recognized and bound by these antibodies, the inventors also succeeded in transforming the respective genetic information into CHO cells, resulting in a stable cell line CL3, which synthesizes the CL2 type antibodies in a more efficient manner than the hybridoma cell line CL2 itself.

In another embodiment, anti-idiotypic antibodies are disclosed which can specifically bind to idiotypic antibodies of the CL2 type and/or which can interact with at least some of them in a fashion that eliminates their anti-HIV protective capability, i.e., bars them from inhibiting the infection of human lymphocytes by primary HIV-1 isolates. Such anti-idiotypic antibodies are therefore expected to be conformationally related to the HIV-1 viruses in that they probably contain similar or even identical antigenic fragments of a viral glycoprotein, e.g., of gp160.

The antibodies of the next embodiment seem to be very interesting because they are of an anti-idiotypic type and combine the features of the anti-idiotypic antibodies of the previous embodiment with their ability to induce--upon administration to a mammal, e.g., a human or animal individual--the production and release of anti-HIV-1 antibodies. Optionally, the induced antibodies are of a nature such that they compete for binding to the above specified antigenic determinants of gp120/160 with at least one antibody of the CL2 type as hereinbefore described in any one of the respective embodiments. A special representative of this group of anti-idiotypic antibodies is the one produced by hybridoma cell line CL6.

While the anti-idiotypic antibodies of the preceding embodiment may be used for active immunization of test animals or HIV-1 endangered and preferably not yet infected persons, the antibodies induced upon such active immunization may serve as components of a vaccine for passive immunization or as subjects of investigation to design and/or synthetically or genetically prepare such antibodies. Optionally, these (idiotypic) antibodies are functional equivalents to the CL2 type antibodies, i.e., they compete with the CL2 type antibodies for binding to the above specified antigenic determinants of gp120/gp160.

In a further--most exciting--embodiment of the invention, the human monoclonal antibodies exhibit strong HIV-1 neutralizing activity and bind to the smaller subunit of gp160, hereinafter referred to as gp41/gp160. Preclinical studies have proved that they are able to significantly reduce--upon intravenous administration to a human HIV-1 infected individual--the level of circulating HIV-1 in the blood serum and/or blood plasma of said individual.

Moreover, at least part of these antibodies may be further characterized in that they also compete with an idiotypic antibody produced by hybridoma cell line CL1 for binding to the gp41/gp160 antigenic determinant. Finally, the antibody produced by said cell line CL1 itself can be regarded as an important member of this group of HIV-1 level reducing antibodies.

Similarty to the situation with the CHO cell line CL3 producing CL2 type antibodies, the inventors also succeeded in cloning a recombinant CHO cell line CL4 producing antibodies which compete with the antibody of CL1 for binding to the gp41/gp160 antigenic determinant and hence may be regarded as more or less close equivalents to the CL1 antibody. Such recombinant CHO cell lines are easier to grow and more efficiently employed in the manufacture of the respective antibodies.

Various in vitro experiments have proved that the CL2 type antibodies as well as tile CL1 type antibodies are able to neutralize a variety of different laboratory and primary HIV-1 isolates including a number of escape mutants, which usually develop upon individual application of any one of these antibodies. It could further be shown that both antibody types are cross-reactive, i.e., they interact synergistically in that each of them is able to capture the escaped HIV-1 mutants of the other antibody. Combined in a mixture, they are therefore a powerful tool to combat HIV-1 infections and AIDS. It is one of the objects of the present invention to provide for a mixture of at least one antibody of the CL1 type and at least one antibody of the CL2 type.

The present invention also relates to a cell line producing any one of the antibodies described above, and in particular, to the cell lines CL1 through CL6 identified by their accession numbers as described below. Viable samples of the hybridoma cell lines CL1 to CL6 producing the monoclonal antibodies herein described were deposited at the European Collection of Animal Cell Cultures (ECACC) at the Public Health Laboratory Service (PHLS), Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 OJG, United Kingdom. They are identified by their accession numbers:

CL1--Accession No. 90091704 (deposited on Sep. 17, 1990);

CL2--Accession No. 93091517 (deposited on Sep. 15, 1993);

CL3--Accession No. 95032235 (deposited on Mar. 22, 1995);

CL4--Accession No. 95032236 (deposited on Mar. 22, 1995);

CL5--Accession No. 95032240 (deposited on Mar. 22, 1995); and

CL6--Accession No. 95032241 (deposited on Mar. 22, 1995).

The corresponding monoclonal antibodies produced by these cell lines are hereinafter termed MAb CL1, MAb CL2 through MAb CL6, when used in the abbreviated form.

In a further embodiment of the present invention, peptide fragments are provided which contain at least one of the antigenic determinants of gp41/gp160 and gp120/gp160 as herein described. It is desired that these peptide fragments are of a nature such that they are able to induce an immune response against HIV-1 infection, optionally the production and/or release of HIV-1 neutralizing antibodies after administration to mammals, e.g., to an animal or a human individual.

In another embodiment, these peptide fragments may be linked to a suitable carrier in order to improve the efficacy of antigen presentation to the immune system. Such carriers may be, for instance, organic polymers including proteins, but any other appropriate and physiologically acceptable carrier may also be used, including tetanus toxoid, cholera toxin, keyhole limpet hemocyan, glutathions S-transferase and all viruses that can be modified by recombinant DNA technology such as, e.g. Rhino-, Polio-, Vaccinia-, or Influenzavirus, etc. It may be advantageous in many cases to have the peptide fragments linked to a modifies, i.e., attenuated and/or recombinant virus such as modified influenza virus or modified hepatitis B virus or to parts f a virus, e.g., to a viral glycoprotein such as, e.g., hemagglutinin of influenza virus or surface antigen of hepatitis B virus, in order to increase the immunological response against HIV-1 viruses and/or infected cells.

It is also an important object of the present invention to provide for the manufacture of a reliable vaccine to protect people from HIV-1 infection and/or to treat patients with already manifest HIV-1 infections in the course of a therapy. Vaccines based on at least one of the idiotypic antibodies of the CL2 and CL1 groups can be employed for active immunization in the prophylaxis and therapy of higher animals including man. Convincing evidence are provided below for the reduction of the HIV-1 level in the plasma and serum of a seropositive patient in the course of a therapeutic treatment in a preclinical study. Also, the preventive potency of the idiotypic antibodies of cell line CL1 was demonstrated in an impressive SCID-mouse trial as well as in a chimpanzee experiment. Neither the antibody-treated mice nor the chimpanzees developed HIV-1 infection upon challenge with live HIV-1 virus, while the animals in the untreated control groups became infected.

The use of at least one anti-idiotypic antibody as hereinbefore described for the manufacture of a vaccine for active immunization can help to successfully combat HIV-1 infection. The anti-idiotypic antibodies--as well as the drugs and vaccines derived therefrom--may primarily be used for the preventive treatment of HIV-1 endangered people and are optimally applied prior to coming into contact with HIV-1 virus. Due to their unique paratope characteristics they may also be administered to already infected patients in order to stimulate the immune system to release the corresponding--and possibly even more powerful--HIV-1 neutralizing antibodies. They may be either directly administered to a person or in combination with at least one suitable carrier and/or additive as usual in the art, and/or along with additional drugs such as, for instance, nucleoside analogues (e.g. AZT, ddl), cytokines (e.g. interleukins), HIV-protease inhibitors, antibiotics, etc. The anti-idiotypic antibodies may, however, also serve as "model templates" for the design and construction of, e.g., fusion proteins carrying their respective antigenic determinant(s) (paratopes).

It might be preferable in many cases to combine an individual antibody or a mixture of at least two different antibodies with an immunoserum and/or an antibiotic, in order to further improve the benefit of an antibody vaccine manufactured accordingly.

In other cases it might be advantageous to use at least one of the herein specified antigenic peptide fragments of gp41/gp160 and gp120/gp160 to substitute the anti-idiotypic antibodies in the corresponding vaccines and drugs. Therefore the present invention also relates to said peptide fragments and to the use thereof for the manufacture of drugs and/or vaccines applicable in the prophylactic and/or therapeutic treatment of HIV-1 endangered or HIV-1 infected people. The fragments are preferably applied as fusion proteins, wherein they are linked to a suitable carrier which might be a recombinant or attenuated virus or a part of a virus such as, e.g., the hemagglutinin of influenza virus or the surface antigen of hepatitis B virus, or another suitable carrier including other viral surface proteins, e.g., surface proteins of Rhinovirus, Poliovirus, Sindbis virus, Coxsackievirus, etc., for efficient presentation of the antigenic site(s) to the immune system. In some cases, the antigenic fragments might, however, also be purely, i.e., without attachment to a carrier, applied in an analytical or therapeutical program. It is of considerable benefit that the fragments can be used for the prevention and/or treatment of HIV-1 infections in human individuals such as persons belonging to one of the high-risk groups of HIV-1 endangered people including medical and scientific staff dealing with HIV-1 viruses and/or infected individuals.

The idiotypic antibodies referred to herein may further be used for the detection and/or determination of HIV-1 infected cells and/or HIV-1 viruses, either as individual antibodies or as an antibody cocktail. Similarly, one or more of the anti-idiotypic antibodies and/or the above-specified peptide fragments can successfully be applied to detect and/or determine anti-HIV-1 antibodies binding to the viruses or to HIV-1 infected cells. Both the idiotypic and anti-idiotypic antibodies of the present invention may therefore be prepared and arranged for an analytical testing procedure and/or for a commercially utilizable test kit.

Finally, it is also an object of the present invention to provide a method of treating HIV-1 infected persons in need of such treatment, and to provide for a method of preventing people from becoming HIV-1 infected. Patients with manifest HIV-1 infections may be therapeutically treated with a vaccine comprising at least one of the idiotypic antibodies of the CL2 and the CL1 type, preferably a mixture thereof. However, in some cases it might be preferable to administer at least one of the anti-idiotypic antibodies and/or antigenic peptide fragments in order to induce additional--possibly even more powerful--antibodies to neutralize the viruses and to reduce the HIV-1 levels in the blood of infected patients.

The vaccine based on antibodies and/or antigenic peptide fragments may further comprise suitable, i.e., physiologically acceptable, carriers--preferably for the preparation of injection solutions--and further additives as usually applied in the art (stabilizers, preservatives, etc.), as well as additional drugs. The patients may be adminstered a dose of approximately 1 to 10 .mu.g/kg body weight, preferably by intravenous injection once a day. For less threatening cases or long-lasting therapies the dose may be lowered to 0.5 to 5 .mu.g/kg body weight per day. The treatment may be repeated in periodic intervals, e.g., two to three times per day, or in daily or weekly intervals, depending on the status of the infection.

Vaccines according to the present invention may comprise any one of the idiotypic or anti-idiotypic antibodies or any one of the peptide fragments disclosed herein, either alone or in combination with suitable carriers and/or linked to carrier molecules. In some cases, e.g., where HIV-1 infection is acute and/or has already considerably progressed, it might be preferable to apply a mixture of idiotypic antibodies, while in other cases it might be more beneficial to apply a mixture of anti-idiotypic antibodies and/or--preferably carrier-linked--gp160 peptide fragments. It is recommended to apply a dose of 0.5 to 10 .mu.g/kg body weight of antibody or carrier-linked gp160 peptide fragments, administered once to three times a day and possibly repeated in periodic intervals, e.g., weekly, monthly or yearly intervals, depending on the status of HIV-1 infection or the estimated threat of an individual of getting HIV infected.

Claim 1 of 8 Claims

We claim:

1. A peptide fragment which consists of one or both amino acid sequences that correspond to amino acid positions 79 to 184 or 326 to 400 (SEQ ID NO:9) of processed gp120 of HIV-1 isolate BH10 (GenBank accession M15654 (SEQ ID NOS:1-10); numbering described in the Swissprot database entry ENV$HIV10).

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