WO1999033477A1

WO1999033477A1 - Drug-directed mutagenesis/drug-driven selection for lethal mutants

Info

Publication number: WO1999033477A1
Application number: PCT/US1998/027766
Authority: WO
Inventors: Robert Lloyd, Jr.
Original assignee: Visible Genetics Inc.
Priority date: 1997-12-24
Filing date: 1998-12-24
Publication date: 1999-07-08
Also published as: EP1047432A1

Abstract

A series of mutations in the nucleic acids of a therapeutic target (e.g., virus such as HIV, bacteria, parasites and cancer cells) are selected for or induced by administration of a series or combination of drugs which select for drug resistant forms of the pathogen with specific mutations. In the case of patients infected with the HIV virus, a series of anti-viral drugs are administered which eventually select for a virus which is unable to replicate or is non-infectious. It is believed that the drugs cause (or select for) a series of mutations in the viral nucleic acids which code for amino acids near the active site of the HIV polymerase enzyme, thereby rendering the virus unable to replicate. Further, in the case of HIV, this can be accomplished using clinically available drugs already used to treat patients suffering from HIV infection.

Description

Drug-Directed Mutagenesis/Drug-Driven Selection for Lethal Mutants

DESCRIPTION

Background of the Invention

The present invention relates to a method of administering therapeutic agents in combination with monitoring of nucleic acid sequences to treat a patient infected with a pathogen, including viruses and bacteria, and to treat cancer. In particular, the invention relates to the treatment of a patient by systematic administration of therapeutic agents, including drugs, which result in changes to the nucleic acid sequence of the pathogen or cancer cells (either by selection or by introduction of specific mutations), thereby causing the selection of a desired form of the pathogen (non-replicative or non-harmful). The method of the invention has particularly applicability in the treat of human immunodeficiency virus (HTV) infection.

Several known antiviral drugs are presently used to treat persons infected with the HTV virus. Those antiviral drugs which are used to reduce the activity of the Reverse Transcriptase ("RT") enzyme are divided into two classes, the Nucleoside Reverse Transcriptase Inhibitor drugs (NRTI class drugs) and the Non-Nucleoside Reverse Transcriptase Inhibitor class drugs (NNRTI class drugs). The NRTI class antiviral drugs include Zidovudine (such as AZT and Retrovir) as well as Lamivudine (such as 3TC and Lamivudine/Epivir). The NNRTI class antiviral drugs include Nevirapine (such as Viramune) .

It is known that the use of antiviral drugs in the treatment of HTV, either used individually or in combination, frequently leads to resistence in the HTV virus. It is known that the use of antiviral drugs selects for resistant strains of the HIV virus which are then able to proliferate. As a result of this resistance, the drug becomes ineffective in preventing further proliferation of the virus, and a new drug must be used to which the virus is not resistant. Similar selection or creation of drug-resistance mutants has been observed with pathogenic bacteria, such as Mycobacterium tuberculosis and in cancer cells. In some cases, strains may be selected which are effectively resistant to all known therapeutic agents. Therefore, in the treatment of cancer cells and various pathogens, including HTV, the selection of resistant strains is to be avoided. Chow et al. ( 1993 Nature 361(6413):650-654) disclosed that HTV-1 infected cell cultures were treated with antiviral drugs AZT and dideoxyinosine (ddl) resulted in the sterilization of the cell culture (i.e. the virus was no longer detectable) by the induction of specific identified mutations in the RT gene. These experiments were termed "convergent combination therapy ' In effect, these experiments disclose variations of already-known forms of administering anti-HTV drugs to HIV infected patients, generally known as drug cocktails. However, when an HTV-1 virus was mutated with these mutations (codons 74, 215, and 181) by site-directed mutagenesis, the HTV-1 virus survived and was able to replicate (see Brendan Larder et al. "Convergent Combination Therapy can select viable multi-drug— resistant HIN-1 in vitro" 1993 Nature

365 (6445): 451-453 ). Therefore, the prior art has not been able to provide a method of using antiviral drugs, either singly or in combination, which will sterilize HIV-1 infected cells.

Summary of the Invention

A series of mutations in the nucleic acids of a therapeutic target (e.g., virus such as HTV, bacteria, parasites and cancer cells) are selected for or induced by administration of a series or combination of drugs which select for drug resistant forms of the pathogen with specific mutations. In the case of patients infected with the HTV virus, a series of anti-viral drugs are administered which eventually select for a virus which is unable to replicate or is non-infectious. It is believed that the drugs cause (or select for) a series of mutations in the viral nucleic acids which code for amino acids near the active site of the HTV polymerase enzyme, thereby rendering the virus unable to replicate. Further, in the case of HIV, this can be accomplished using clinically available drugs already used to treat patients suffering from HTV infection.

Brief Description of the Figures

Fig. 1 shows the genotype, viral load and CD4 levels for a human patient infected with HIV-1 at various times during treatment. Detailed Description of The Invention

The present invention can be used generally to develop and maintain an appropriate therapeutic regimen for conditions where mutation rates of the therapeutic target cells or organisms are potentially significant and where induction of resistance to the therapeutic drugs is of concern. Such conditions include cancer, where resistance to chemotherapy agents may develop; viral infection, including hepatitis B, cytomegalovirus (CMV) and HIV; and bacterial infections such as tuberculosis. The application of the present invention is illustrated in the context of HIV infections.

The present invention is applicable to the treatment of pathogenic infections and other conditions such as cancers where a set of drug-induced mutations has been identified for the target cell or pathogen which, when present in combination, lead to a non-virulent or reproduction-incompetent cell or pathogen. Such infections and conditions are referred to in the specification and claims of this application as a "target condition ' and the pathogen or cells associated with the target condition are referred to as the "therapeutic targets." It should be remembered that it is the effect of the combination of mutations that is of importance, and that in some cases, including the HIN mutations discussed below, the individual mutations may actually cause undesirable drug resistance.

In accordance with the invention, the target condition is treated by administering to an individual a series or combination of therapeutic agents. The therapeutic agents used are effective to induce the set of mutations which combine to produce a non-virulent or reproduction-incompetent therapeutic target. As used here, the term "induce" refers to the formation of the desired mutation, regardless of the mechanism. Thus, the therapeutic agent may provide selective pressure favoring a mutation within the set of desired mutations, or they may promote specific mutagenesis at the desired site.

During the treatment, representative therapeutic target are periodically evaluated for the presence of the desired mutations. The treatment protocol is adjusted based on these evaluations to increase the proportion of therapeutic targets with the desired set of mutations. It will be appreciated that even where a starting population is genetically homogeneous, the induction of mutations produces a heterogeneous population. Thus, the term "representative therapeutic targets" refers to a population of therapeutic target cells or organisms which is representative of the varies species of therapeutic targets found in the individual being treated.

Evaluation for the presence of the desired mutation can be performed by any appropriate technique and can be performed on genomic DNA, genomic RNA, proviral DNA or other accessible nucleic acids reflecting the induced mutations, as appropriate to the specific therapeutic target. For example, mutations may be detected and quantified by restriction fragment length polymorphism (RFLP), where applicable, or through the use of sequence-specific hybridization probes. A preferred technique for performing the evaluation, however, is sequencing of the nucleic acids of the therapeutic target, at least in the region of the mutation site of interest.

Sequencing can be performed by any of the numerous methodologies known in the art. Sequencing methodologies based on the S anger sequencing method are preferred. Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977). Examples of such methods are found in US Patents Nos. 5,834,189, 5,830,657, 5,789,168, 5,427,911,

5,171,534, and 4,729,947, which are incorporated herein by reference.

The interval at which the periodic evaluations are suitably performed will vary depending on factors including the nature of the target condition (e.g., period and consequences of disease progression) and the mutation rate of the therapeutic targets. For HIV, a suitable frequency is once every three months.

The invention can be exemplified with particular reference to managing treatment of individuals suffering from HTV infection. In accordance with this embodiment of the invention, standard clinically available anti-HTV drugs are used to confer resistance and force the selection for specific mutant strains of the virus, which are confirmed by DNA sequencing of the HTV virus. In particular, a triple-drug cocktail containing AZT, 3TC and Nevirapine is used. Such cocktails have been used for treatment with some success, but almost invariably after some period of time the observed viral load increases. In the past, this increase in viral load has been seen as an indication that the therapy had become ineffective, and the therapy was stopped, to be replaced with some other drug or drug combination. Applying the present invention, however, the actual mutational composition of the virus present in the patient is analyzed and used as the basis for determining future drug treatment.

The goal of the present invention when managing HIV therapy is to induce a specific triple mutation in a large proportion of the HTV population in a patient. The three mutations of interest are all found in the HTV reverse transcriptase gene, at codons

181, 184 and 215. Table 1 illustrates that virus with one or two of these mutations grow, but virus with all three mutations do not. Periodic sequencing is used to monitor the formation of mutant strains in response to therapy, permitting modification of the therapeutic regime to maintain selected pressure for desirable mutations while avoiding induction on virulent and drug-resistant strains. It will be appreciated that many patients may have already been exposed to therapy with one or another drug or drug combination, and thus may already have a set of induced mutations present. Thus, a somewhat different protocol is appropriate depending on whether the patient is naive or previously treated.

An HTV-1 infected person treated according to the method of the present invention will result in a sustained homeostasis of viral output (see enclosed patient in

Table 1), which can be sustained over a long period of time. It appears that viral levels of 5,000 to 10,000 viral copies represent homeostasis. It should further be noted that patients with viral loads of less than 10,000 have a prognosis of surviving for 12 years or longer, while those with viral loads of 50,000 have a prognosis of surviving only 5 to 7 years. Thus, the reduction of viral load using the combination therapy of the invention is associated with a therapeutic benefit.

Table 1 shows the location of known mutations in the RT gene of the HTV-1 genome which are caused by the use of known anti-HTV drugs. As shown, the use of the drug 3TC (or FTC) to treat HTV-1 results in the selection of HIV-1 virus having a mutation at codon 184 in the RT gene corresponding to a nucleotide base change from

ATG to GTG or ATA (amino acid change from Methionine [Met] to Valine [Val] or Isoleucine [He] ). Shinazi et al. 1993 875-881 Anti. Agents and Chemo.; See also Smith et al. 1997 2357-2362 J. Virol, in which Feline Imunodeficiency Virus (FIV) was tested. As an apparent result of this mutation, the use of the anti-viral drug 3TC or FTC results in the selection of mutant strains of the HTV-1 virus in which the activity of the HTV RT enzyme is greatly reduced (i.e. from a transcription rate of 2000 bases per min. to 200 bases per min.) .

The use of the drug Nevirapine (or DMP266) to treat HIV-1 results in the selection of HTV virus having mutations at codon 181 and codon 188 in the RT gene. These codon changes correspond to a base change at codon 181 from TAT to TGT or ATT

(amino acid change from Tyrosine [Tyr] to Cysteine [Cys] or Isoleucine [He]) and a base change at codon 188 from TAT to TGT (amino acid change from Tyrosine [Tyr] to Cysteine [Cys]). The use of the drug AZT to treat HIV-1 results in the selection of HTV virus having a mutation at codon 215 in the RT gene. This codon change corresponds to a base change at codon 215 from ACC to TAC or TTC (amino acid change from

Threonine [Thr] to Tyrosine [Tyr] or Phenylalanine [Phe]).

These drugs force or select for virus having mutations within or near the known active site of the RT enzyme located at codons 183 to 186 having normal amino acid motif of Y M D D (i.e. Tyr Met Asp Asp). The YMDD amino acid motif is highly conserved in most retroviruses. It is believed each of these mutations, considered individually, alters the amino acid structure of the RT gene and results in a reduced bond potential between the RT enzyme and the enzyme's substrate. Thus, it is believed that when each of these anti-viral drugs, 3TC (or FTC), Nevirapine (or DMP266), and AZT, are taken individually, they each select for virus having specific mutations which reduce the activity of the Reverse Transcriptase enzyme and thereby reduce its rate of replication.

If only one or two these drugs are taken, the activity of the reverse transcriptase is only partially diminished and thus the virus is still able to replicate and the patient's viral load eventually rises and returns to high levels.

According to the present invention, if all of these drugs are administered to HTV-1 infected cells to produce virus having all of these mutations at codons 181, 184, and

215 , as confirmed by DNA sequencing, then the virus population will be unable to replicate or will be non-infectious (i.e. a lethal mutant). Therefore, the present invention teaches a method of selecting for resistant forms of the virus by treatment of available antiviral drugs in order to also select for specific mutations in RT gene which effectively prevents the virus from replicating at high levels. According to the present invention, a patient would be maintained on regime of these drugs to maintain selective pressure for virus having these specific mutations (codons 181, 184, and 215). Nevirapine also causes the 188 mutation and it is believed that this mutation further impairs the virus due to its proximity to the RT active site and thus represents a desirable additional mutation.

In the case of a naive patient, the patient is preferably first treated with a combination of drugs (for example AZT and 3TC) to induce mutations at codons 184 and

215 of the RT gene of HTV-1. A protease inhibitor, e.g., Nelfinavir, Saquinavir, Ritonavir or Indinavir, may also be included in this therapeutic mixture. This treatment will be effective for a period of time, but eventually the viral load will increase. At this stage, the conventional wisdom would be to stop treatment with AZT and 3TC. In accordance with the present invention, however, the cocktail is modified to a mixture of AZT, 3TC and

Nevirapine after confirming the presence of mutations in codons 184 and 215. The addition of Nevirapine induces mutations at codon 181, to produce the desired triple mutant. The occurrence of these mutations are confirmed by suitable forms of DNA analysis such as DNA sequencing. It is important to expose the virus to Nevirapine after 3TC (or FTC) and AZT. If Nevirapine is not supplied last, Nevirapine will also eventually select for other mutations, including a mutation at codon 103, which results in viable virus.

When a patient has been previously treated with antiviral drugs, particularly nucleosides, the first important step is to check the viral population for the presence of mutations. If high levels of mutations are present, a period of cessation of therapy may be appropriate prior to the adoption of a therapeutic regimen in accordance with the invention, to reduce the frequency of drug-induced mutations.

It is important to ensure that the virus is exposed to the drugs for a sufficient period to select for the appropriate mutations. Sequencing of the viral nucleic acids is performed in order to confirm that the proper mutations have occurred. The rates of proliferation of the particular virus as well as the rate of occurrence of these mutations will vary in each infected person. For example, in the case of a non-mutated Reverse Transcriptase enzyme, it is known that the rate of misincorporation of nucleotides is 1 misincorporation in 10,000 incorporations. Thus, in the case of HTV-l infected patients, the actual rate of mutation will vary between different patients with different viral loads and different rates of proliferation. As a result of these variations, the actual sequence of the viral nucleic acids at these codons must be monitored in each patient to obtain/select for these specific mutations in the virus.

Further, according to the present invention, DNA sequencing is essential to monitor mutations that arise or are selected for in the virus as a result of exposure to these drugs, and also to avoid undesired mutations. For example, it is known that prolonged exposure to nucleosides can lead to a mutation at codon 151 in the RT gene corresponding to a codon change from CAG to ATG (also known as a multi-drug resistance mutation). An HTV- 1 virus having the 151 mutation is resistant to all known NRTI class drugs and permits the virus to proliferate which results in the rapid demise of the infected person Therefore, according to this invention, it is important to use a DNA sequencing step to confirm that the mutations at codons 184, 215 and 181 have occurred and to avoid selecting for resistance mutations, such as mutations at codon 151 (caused by long term use of AZT) or codon 103 (caused by long term use of Nevirapine).

The operation of the present invention in managing HIV therapy will now be further described with reference to the following non-limiting examples.

EXAMPLE 1 An experiment was conducted where the genome of viable HTV-1 virus was mutated directly by known methods of site directed mutagenesis in order to produce virus with several mutations in the RT gene. These mutations corresponded to the same mutations which occur on the RT gene of HTV-1 when the drugs 3TC, AZT and Nevirapine are used to treat an HTV-1 infected cell. These mutated HTV strains were then electroporated into CEMss cells. The ability of the virus to replicate was then assessed. One group of mutated HTV-1 virus had a mutation at codon 181 corresponding to a base change from TAT to TGT or ATT (amino acid change from Tyrosine [Tyr] to Cysteine

[Cys] or Isoleucine [He]). A second group of HTV-1 virus had a mutation at codon 188 corresponding to a base change from TAT to TGT (amino acid change from Tyrosine [Tyr] to Cysteine [Cys]). A third group of mutated HIV-1 virus had a mutation at codon 184 corresponding to a nucleotide base change from ATG to GTG or ATA (amino acid change from Methionine [Met] to Valine [Val] or Isoleucine [He] ). A fourth group of mutated HTV-1 virus had a mutation at codon 215 which corresponds to a base change from ACC to TAC or TTC (amino acid change from Threonine [Thr] to Tyrosine [Tyr] or Phenylalanine [Phe] ). Finally, HTV-1 virus having combinations of these specific mutation were also tested and the viability of the virus was assessed. As shown, HIV-1 virus having only one mutation or a combination of two mutations still retained the ability to grow (replicate) . More significantly, when virus having all three mutations at codons

181,184 and 215 are electroporated into the CEMss cells, the virus does not show growth (inability to replicate or non-infectious).

EXAMPLE 2 A human patient infected with HIV-1 was evaluated at intervals during prior treatment and treatment in accordance with the invention. Fig. 1 shows the relationship of viral load and CD4 levels over time in response to changes in drug treatment of the patient. The HTV-1 nucleic acids were at various times sequenced in order to determine the specific genotype (DNA sequence) in both the RT gene and the Protease gene of the HTV-l virus, and these genotypes are indicated in Fig. 1 as well.

The patient had been previously treated with ddl to which a combination of the drugs AZT and 3TC were added. The drug ddl was then removed from the treatment and the protease inhibitor Saquinavir was added to the treatment regime. Then Saquinavir was removed and the protease inhibitor Crivixan (Indinavir) was substituted. As can be seen, the patient's viral load began to rise sharply from 12/96.

At 5/97, Crixivan and Combovir (3TC, and AZT) were removed and treatment with D4T and 3TC began. Treatment with Nevirapine began at 4/97. As shown, prior to treatment with Nevirapine, the patient had HTV-1 virus with mutations at codon 184 and 215 resulting from treatment with 3TC and AZT. At 6/97 the increase in viral load was observedm showing that the virus is now resistant Nevirapine and has a mutation at codon 181. DNA sequencing and clonal analysis (n=20) was conducted to confirm this. From the period between 8/97 to 8/98, the viral load remained relatively constant (See actual viral load -Table 2), and the level of CD4⁺ increased. DNA sequencing (and clonal analysis) was conducted and confirmed the existence of all three mutations (181, 184, and 215) until 2/98, at which time the 181 mutation disappeared and a new Nevirapine related mutation at codon 190 appeared. The 190 mutation is consistent with long-term use of Nevirapine and is believed to give more fitness to the virus, while still providing resistance to Nevirapine. (Boyer et al., Antimicorbial Agents and Chemotherapy 42: 447-452 (1998). According to the invention, once the the 190 mutation appears, the patient should no longer be treated with Nevirapine. DNA sequencing should be conducted regularly every 1 or 2 months until the 190 mutation is no longer detected, at which time Nevirapine is readministered. In the present example, Nevirapine was removed at 8/98 and replaced with Delavirdine As shown, a drop in viral load resulted by 10/98. The drop resulted because the new drug selected out the predominant HIV-1 virus strain which was still sensitive to this new drug. The patient's viral load will rise as resistance to Delavirdine is acquired. Once the 190 mutation is no longer detected, via DNA sequencing, the patient will again be treated with Nevirapine so as to select virus with the 181 mutation and provide the combination of mutations which are associated with maintenance of a stable, relatively low viral load. The HTV-l virus DNA will be regularly sequenced and Nevirapine will be removed on recurrence of the 190 mutation and loss of the 181 mutation.

It is contemplated that treating HTV-1 infected cells with the combination of drugs to produce these mutations can be used to treat HTV-1 infected patients and can maintain their viral loads at low levels. It is also contemplated that method of the present invention may be used intermittently in order to avoid the selection of HIV-1 mutants having undesired mutations, such as the mutation at codon 151 (the multi-drug resistance mutation) upon long term use of these NRTI class drugs.

It is also contemplated that selecting for HTV-1 virus having these mutations according to the present invention, will have a secondary effect on the patient's immune system. The mutated virus will be able to infect cells (T cells) in vivo, but will not be able to integrate into the cell's DNA due to the mutated RT enzyme product (due to the mutations at codon 184, 181 and 215 in the RT gene). As a result, it is believed that the host cell (T cell) will not shed its CD4 surface proteins before an immune response can be mounted by the host organism against the HIV-1 infected cell. All the required antigens necessary for epitope recognition by antibodies will be present. Therefore, it is believed that the present invention provides a means of selecting for a weakened/attenuated non-replicating form of virus which permits the body to build up an immune response to attack infected cells. In essence, the present invention discloses a means of creating a vaccine by selection of a weakened form of HIV in vivo, by use of drugs, thereby vaccinating the patient.

The patient will also be monitored for the continued existence of the mutations art codon 215. Once this mutation is no longer detected, e.g. via DNA sequencing, the patient will again be treated with AZT so as to select virus with the 215 mutation and provide the combination of mutations which are associated with maintenance of a stable, relatively low viral load.

TABLE 1

Table 1. cont'd

Table 2

Claims

CLAMS

1. A method for managing treatment of an individual suffering from a target condition for which a set of mutations has been identified for associated therapeutic targets which, when present in combination, lead to a non-virulent or reproduction- incompetent mutation targets, comprising the steps of: (a) treating the target condition using a treatment protocol comprising administering to the individual a series or combination of therapeutic agents effective to induce the set of mutations which in combination result in conversion of the associated therapeutic target into a non-virulent or reproduction-incompetent form; and (b) periodically during the treatment evaluating representative therapeutic targets derived from the individual for the presence of each of the plurality of mutations within the set of mutations and adjusting the treatment protocol to increase the proportion of the therapeutic targets having the combination of mutations which result in conversion of the therapeutic target into a non-virulent or reproduction-incompetent form.

2. The method of claim 1, wherein the evaluation is performed by sequencing relevant portions of the therapeutic target's genetic material.

3. The method of claim 1, wherein the therapeutic target is human immunodeficiency virus (HIN).

4. The method of claim 3, wherein the treatment protocol induces mutations in codons 181, 184 and 215 of the HIV reverse transcriptase gene.

5. The method of claim 4, wherein the treatment protocol comprises administering the drugs 3TC or FTC, AZT and Nevirapine in series or in combination.

6. The method of claim 4, wherein the individual is a naive individual who has not previously undergone treatment for HTV infection, and wherein the individual is first treated with a first combination of drugs to induce mutations at codons 184 and 215 of the HIN reverse transcriptase gene.

7. The method of claim 6, wherein the first combination of drugs further comprises a protease inhibitor.

8. The method of claim 6, wherein the first combination of drugs comprises AZT and 3TC or FTC.

9. The method of claim 6, wherein the individual is treated with the first combination of drugs until a decrease in effectiveness is detected, at which time the treatment protocol is changed to administration of a second combination of drugs comprising the members of the first drug combination plus a drug effective to induce mutations in codon 181 of the HTV reverse transcriptase gene.

10. The method of claim 9, wherein the first combination of drugs further comprises a protease inhibitor.

11. The method of claim 10, wherein the first combination of drugs comprises AZT and 3TC or FTC.

12. The method of claim 9, wherein the first combination of drugs comprises AZT and 3TC or FTC.

13. The method of claim 9, wherein the second combination of drugs comprises AZT, 3TC or FTC, and Nevirapine.