Nucleoside Reverse Transcriptase Inhibitor and Anti- HIV-1 Antibodies

Field of the Invention The invention relates to treatment of HIV-1 infection and

HIV-1 disease. Background of the Invention

It is well known that AIDS is one of the most serious health

problems facing the world. Monoclonal antibodies which bind to a neutralizing epitope of the human immunodeficiency virus type

1 ("HIV-1") — the virus which causes AIDS — have been suggested for both treating and preventing HIV-1 infection. Most neutralizing anti-HIV-1 antibodies target specific regions of the envelope glycoproteins gpl20 and gp41. Some of these regions are the principal neutralizing determinant in the V3 region, the C4 region, the V2 loop, and the CD4-binding domain. Monoclonal antibodies which target the principal neutralizing deterrninant ("PND") have been shown to protect from infection in animal models. The anti-HTV-1 antibodies referred to prevent infection of

T cells with the virus. The T cells are critical to the proper functioning of the immune system. When HIV-1 infects human T cells, it ultimately destroys these cells by lysing them. As the

number of T cells declines, the victim's immune system is increasingly compromised, and opportunistic infections and tumors which are characteristic of AIDS result. The invariable outcome of the infection is the death of the victim, usually as a direct result of one or more of these complications.

A number of nucleoside reverse transcriptase inhibitors including Zidovudine™ (also known as "AZT"), dideoxyinosine ("ddl"), and dideoxycytidine ("ddC") are approved for use in treatment of HIV-disease. There are also indications, particularly for AZT, that these inhibitors can prevent infection of newborn whose mothers are HIV-1 seropositive. This mode of infection is known as vertical transmission.

Anti-HIV monoclonal antibodies are also intended for use in preventing vertical transmission, or otherwise protecting persons who are exposed to the virus. Such exposure can occur, for example, when a hospital worker is accidentally punctured with an HTV-1 contaminated needle or other sharp object, or through unprotected sexual contact with an infected person. Adrninisterrng anti-HTV monoclonal antibodies to inhibit progression of disease or to prevent infection is known as passive immunization.

To date, no one has shown a synergistic effect in inhibiting HIV-1 infection through a combination of anti-HIV-1 monoclonal

antibodies and a nucleoside reverse transcriptase inhibitor. Summary of the Invention

The invention includes the use of nucleoside reverse transcriptase inhibitors, preferably AZT, in combination with one or more neutralizing anti-HIV-1 antibodies, to treat or prevent

HIV-1 infection. Polyclonal or monoclonal antibodies can be used, with the preferred antibodies being those which target a neutralizing domain of gpl20 or gp41, and the more preferred being those targeting the PND or gpl20. The most preferred anti- gpl20 monoclonal antibody is AIDS-439, which is a genetically engineered chimeric mouse/human monoclonal antibody with human IgGl constant regions and variable regions corresponding to the murine monoclonal antibody BAT123. A transfectoma cell line producing AIDS-439 is on deposit at the American Type Culture Collection ("ATCC"), 12301 Parklawn Drive, Rockville

Maryland 20853 under Accession Number CRL 10499. A hybridoma cell line producing BAT123 is on deposit at the ATCC under Accession Number HB 10438.

It has been discovered in in vitro tests that when certain nucleoside reverse transcriptase inhibitors are used in combination with neutralizing anti-HTV-1 monoclonal antibodies, there is a synergistic effect, resulting in greater viral neutralization than

would be expected merely by adding the dosages of the products together. The same effect is likely to occur if neutralizing polyclonal antibodies are used, particularly if such polyclonals bind to the PND. In addition to use in therapy or prevention of HTV-1 infection, neutralizing anti-HIV-1 antibodies, polyclonal and monoclonal, can also be used to assay a biological fluid sample for HrV-1 virions or for HTV-1 -infected cells, or to quantify the concentration of HIV-1 or of infected cells, present in a biological fluid. This is useful for diagnosis of HIV-1 infection and detection of HIV-1 contamination in a culture or another sample. These antibodies can be used in standard assay formats, such as the ELJSA format or the immunofluorescence format described below. Description of Making and Using the Invention As noted above, the invention includes the use of nucleoside reverse transcriptase inhibitors, preferably AZT, in combination with neutralizing anti-HTV-1 antibodies, to treat or prevent HTV-1 infection. Preferred nucleoside reverse transcriptase inhibitors are AZT, ddl, and ddC. The dosage ranges for these inhibitors are all readily available from the pharmacopeias, as these products are approved for sale. The recommended dosages should be used when practicing the

invention, although other dosages which do not result in toxicity and which result in synergistic neutralization are also acceptable. The neutralizing anti-HIV-1 antibodies for use in the invention are preferably those targeting the PND, the C4 region, the V2 loop, or the CD4-binding domain. A number of such antibodies are known and available. AIDS-439 targets the PND. G3-519, G3-508 and G45-60 (the cells lines producing them are on deposit at the ATCC under Accession Numbers HB 10747, HB 10748, and HB 10749, respectively) target the C4 region. BAT 085, G3-136 and G3-4 (the cell lines producing them are on deposit at the ATCC under Accession Numbers HB 11118, HB 10932, and HB 10733, respectively) target the V2 loop. There are also a number of other monoclonal antibodies which target these same regions, or otherwise neutralize HIV-1, and which can be used in the invention.

The recommended dosage for the monoclonal antibodies can be estimated based on in vitro studies and animal model studies. To perform this extrapolation, one determines the dosage in animal models (such as the hu-PBL-SCID mouse) which is effective in protecting the animals from HTV-l infection, when they are administered the same dose of HIV-1. Then one multiplies this dosage by the ratio of the mass of the human over

that of the animal. This provides the initial estimate of the dosage

to be administered to d e human. From mere, a more precise determination is made by administering a range of doses to a number of different test subjects. An example of how an animal model experiment in hu-PBL-SCID mice can be carried out is set forth below.

Example 1: Protection of hu-PBL-SCID Mice from Infection A study was made of me ability of two monoclonal antibodies of the invention, BAT123 and AIDS-439, to protect hu- PBL-SCID mice from infection by HIV-1. The protocol for this study was as follows.

SCID mice were reconstituted by intraperitoneal (i.p.) injection of 2 x 10 ⁷ human peripheral blood lymphocytes (PBL).

After 14 days, the reconstituted mice were checked for human immunoglobulin. To be used in me study, me reconstituted hu-

PBL-SCLD mice were required to show a human immunoglobulin level of at least 10 μg/ml in their sera.

The hu-PBL-SCID mice for use in the study were divided into three groups with six mice in each group. One group was a control group, and received tl.. irrelevant immunoglobulin PNTU.

Anomer group received BAT123 and the remaining group received

AIDS-439. All antibodies were injected i.p. at a dose of 40 mg/kg

of animal weight. One hour after antibody administration, me animals were inoculated i.p. with HIV-1 _IIIB , at a dose adequate to infect at least 80% of the animals, as calculated based on a prior virus titration in otiier hu-PBL-SCID mice. This dosage is 10 times die dosage needed to infect 50% of me animals.

Serum samples were taken from me animals at selected intervals in order to study me pharmacokinetics of me injected antibodies. The animals were sacrificed three weeks after inoculation and their spleen cells and peritoneal lavage were cultured for HIV-l for four weeks. Cells from me peritoneal lavage and spleen cells were analyzed for HTV-l infection by co- cultivation, and only spleen cells were analyzed by PCR.

As determined using a co-cultivation p24 antigen assay of both me peritoneal lavage and me spleen cells, and by PCR of spleen cells, none of me 12 hu-PBL-SCID mice which received

BAT123 and AIDS-439 showed any HTV-l infection. Five out of the six control mice showed infection of their spleen cells as determined by co-cultivation, and two of the five showed infection by PCR. Only one control animal showed infection of me cells from me peritoneal lavage.

It was not unexpected that only one control animal showed infection of cells from me peritoneal lavage, as far fewer of me

peritoneal lavage cells were cultured. Further, it was not

unexpected mat fewer control animals showed infection using PCR as compared with co-cultivation, because fewer cells were analyzed using PCR. The results from me co-cultivation, showing five of six control animals infected, are believed to be more sensitive man the PCR results.

A range of the same dosages of AIDS-439 (40 mg/kg of animal weight) could be used in humans, in beginning me determination of the appropriate dosage to use. The same procedure could be used to determine me appropriate dosage of other anti-HTV-l antibodies. Example II: Safety and Tolerability of ATDS-439

A clinical trial was conducted in 12 male CDC stage TV AIDS patients to determine the safety and tolerability of me AIDS- 439, and whemer it had biological activity in vivo.

Patients were screened from the closely monitored AIDS patients at the University Hospital of Zurich. Twelve male AIDS patients selected were above age 18. Each had CD4 ⁺ lymphocyte counts of 10-230/mm ³ with proven viremia at CDC stage IV clinical status, i^., up to 3 opportunistic Cl or C2 type infections wim a life expectancy of at least 6 months. All patients were wimdrawn from AZT treatment four to six weeks before the trial

began.

These patients were HIV-l antigenemic (positive by HIV-l antigen assays and tissue culture infectious dose (TCID) assays). These patients were divided into d ree groups and entered into a dose schedule as described in Table I. The gpl20 of patient HIV-

1 isolates from eight patients in Groups 2 and 3 were tested reactive wim AIDS-439 in a specially designed capture ELISA.

After appropriate data evaluation whnin 5 days following the infusion of the highest dose of 200 mg to Group 3 patients, a decision was made to continue the trial with up to eight doses of

AIDS-439 identical to me highest dose for each of me three groups, to be given three weeks apart (see Table I).

The 12 selected patients were carefully evaluated, beginning 6-8 weeks before the trial. Monitoring and recording activities for each patient included a medical history, clinical examination, and various laboratory tests, including hematology, clinical chemistry, general immunology, HIV antigen test, HIV-l viremia, special immunology, and urinalysis.

Follow-up clinical and laboratory evaluation was scheduled at regular intervals during treatment. HIV-l antigenemia was monitored by me Abbott HTV Ag test and HTV viremia was monitored by tissue culture for TCID, PCR, and branched DNA

amplification techniques. The pharmacokinetics of AIDS-439 were

determined using a double-antibody capture ELISA employing an anti-idiotypic antibody to AIDS-439. Emergence of human anti- antibodies to AIDS-439 in patients also was examined. CD4 ⁺ , CD3 ⁺ ,and CD8 ⁺ cells from peripheral blood were enumerated with use of specific monoclonal antibody reagents and flow cytometry. All laboratory work complied with good clinical practices.

Clinical and laboratory analysis data in combination wim the weekly physical examinations were the basis for review of the clinical status of each patient throughout the trial. Good tolerability was defined as lack of subjective or objective symptoms following administration of AIDS-439.

TABLE I AIDS-439 DOSE SCHEDULE

Group 1 Grou]p2 Group 3

Infusion n = 4 mg n = 4 mg n = 4 mg

1° 4 1 4 10 4 25

2° 4 50 4 100 4 200

3° 3* 50 4 100 4 200

4° 3 50 3* 100 4 200

5° 3 50 3 100 4 200

6° 2* 50 3 100 4 200

7° 2 50 3 100 4 200

8° 2 50 3 100 4 200

* indicates a patient dropping out

AIDS-439 was well tolerated, even up to a cumulative dose of 1,425 mg over 170 days. However, one patient reported tiredness at an interim stage in the trial. Among the patients receiving the highest dose of AIDS-439 (Group 3), all of mem showed stabilization of body weight and all survived for me entire 170 day trial period. This indicates mat AIDS-439 can be safely administered at a dose up to at least 200 mg per administration, cumulatively over several weeks. Such dosages can be used in me present invention. The same protocol detailed above can be used to determine the safe and effective dosage of otiier anti-HIV antibodies. In summary, one uses several different dosages in

various patients and monitors their reaction and their clinical response over a period of time.

Example ELI: Synergy in Inhibition of HTV-l Infection by a Combination of AZT and AIDS-439 The following is an analysis of the inhibition of HIV-l TTTR infection of CEM-SS cells (a human T cell line) by a combination of AZT and AIDS-439. Progressively increasing dosages of

AIDS-439 and AZT were added to a culture of CEM-SS cells, a human T cell line known to be subject to HTV-l infection. An infectivity assay was men perfo ^~ ' ;d on me CEM-SS cells, using the method of Nara, P. et al., AIDS Res. Human Retroviruses

3:283-302 (1987). The results are set forth in Table π below.

TABLE II

Percent Inhibition of HIV-HϋB Infection of CEM-SS Cells by a combination of AZT and ATDS-439

AIDS-439 ( g/ml)

AZT *M)

0 0.039 0.078 0.156

0 0 1 19 51

0.0031 0.1 15

0.0063 1 47

0.0125 26 75

0.0250 41

0.050 58

0.10 69

0.20 77

0.40 90

AIDS-439 tøg/ml)

AZT M)

0.313 0.625 1.250 2.50 5.0

0 68 89 94 97 98

0.0031

0.0063

0.0125

0.0250 91

0.050 98

0.10 99

0.20 99

0.40 99

A synergy analysis was men carried out on me results of the AZT and AIDS-439 combination. This analysis was conducted by first determining CI, combination indices, which were

calculated based on the median-effect equation for mutually non¬

exclusive interaction (for drugs wim distinct mechanisms of action) as described by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984). For CI < 1, this indicates synergism; for CI = 1, the effect is additive; and for CI > 1, this indicates antagonism. IC ₅₀ is me median inhibitory concentration (μg/ml), r is the correlation coefficient as determined from me median-effect plot, and m is the slope of the plot. The results of this analysis are set forth in Table III below.

TABLE III

Synergy analysis of AZT and AJLDS-439 combinations against HTV-lUULB infection of CEM-SS cells by syncytium- f o r m i n g a s s a y

Parameter

Inhibitor m ICso r

AIDS-439 1.61 0.257 0.96

AZT 1.69 0.0174 0.92

AIDS-439/AZT (46.8:l) ⁺ 1.71 0.089 0.99

concentration ratio, /tg/ml

CI at % Inhibition

Inhibitor

50 70 90

AIDS-439

AZT

AIDS-439/AZT (46.8: 1) ⁺ 0.48 0.47 0.45

⁺ concentration ratio, μg/ml

This synergy analysis in Table HI shows tiiat there is a synergistic effect from a combination of AZT and AIDS-439. The same synergistic effect is expected from omer combinations of neutralizing anti-HTV-1 antibodies and nucleoside reverse transcriptase inhibitors.

Example TV: Use of Antibodies for Detection or Quantitation of HTV-l Virions or Infected Cells

As noted above, anti-HIV-1 antibodies, monoclonal and

polyclonal, can be used for detection or quantitation of HIV-l virions or infected cells. A protocol for carrying this out is

described below.

(i) Detecting HTV-l virions and HIV-l gp!20 in specimens

The antibodies of me invention, either alone or in combination, can be immobilized on inert solid matrices or magnetic beads, either directly or indirectly through a cross-linking agent or a specific binding agent (e.g. protein A, goat anti-mouse IgG, or goat anti-human IgG). The biological fluid test samples are then incubated wim the antibody-coated matrices. HIV-l virions or gpl20 reactive with the antibodies will bind to me matrices. The bound virions or gpl20 can men be detected wim either monoclonal or polyclonal anti-HTV-1 antibodies, which can men be reacted wim enzyme-linked secondary detecting antibodies for quantitation based on color reaction. Alternatively, the captured virions can be detected by otiier means, ejg. fluorescence, chemiluminescence, or PCR.

(ii) Detecting HIV-l -Infected Cells in a Specimen The monoclonal antibodies of the invention can be used to detect and to quantitate the HTV-l -infected cells in patient blood samples by direct or indirect immunofluorescence procedures. The

protocol is well-known by tiiose skilled in me art, and is described specifically in U.S. Application Serial No. 07/950,571.

The terms, expressions and examples herein are exemplary only and not limiting, and tiiose skilled in me art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. All such equivalents are intended to be encompassed by the following claims.