AGONISTS IN THE COSTIMULATION OF TcR/CD3-INDUCED T-LYMPHOCYTES

Abstract: 2-Chloro-5-nitrobenzoic acid and various other chloro and nitro substitute benzoic acid derivatives are disclosed for administration in tablet or injectable solution form for treatment of HIV infection. The active compounds have surprising efficacy in various treatment regimes and have particular application to effect regression of lesions in HIV-related Kaposi's sarcoma. The compounds act as costimulators of CD3-triggered T-lymphocyte proliferation to increase the immunological challenge to the retrovirus presented by the natural immune system.

Description: Agonists in the Costi ulation of TcR/CD3-induced T-Lvmphocytes The invention relates to agents active as agonists of intracellular proliferative signalling in anti-CD28 costimulated anti-TcR/CD3 complex-induced T-lymphocyte cells. The invention is concerned generally with the application of the agonists to enhance T-cell proliferation per se but is especially concerned with the enhancement of T-cell proliferative response to reinforce an immune system challenged by a viral infection such as HIV infection. Administration of the agonists is effective to combat HIV in human patients, improvements in CD4 count, body weight gain and improved general patient condition being achievable whilst, in animal (eg murine) models, various blood parameters increase in level, including haemoglobin concentration, red blood cell count, lymphocytes count and haematocrit. HIV is an infection which attacks the iitutiune system itself. Serum virus enters the cytoplasm of T-cells, following which dsDNA is reverse transcripted from viral ssRNA and integrates into the genome of the cell. Cell activation leads to proviral transcription, formation of a series of viral mRNAs and subsequently production of structual viral proteins and assembly of virions which are free to bud from the cell. Continued viral replication leads to T-cell death. The membrane envelope of HIV-l contains two linked glycoproteins, gpl20 and gp41, cleaved from a common precursor, gpl60. The gpl20 protein binds to CD4 and the virus enters cells carrying this marker. These include CD4 + T cells, and cells of the monocyte/macrophage lineage, such as the dendritic cells of lymphoid tissue and skin (Langerhans ■ cells) , and the microglia of the central nervous system. However, T-lymphocytes (mostly CD4+ cells) form the major HIV reservoir and the highest concentration of HIV-infected T- lymphocytes is in lymph nodes. The nucleocapsid of HIV contains four proteins, p24, pl7, p9 and p7, which are cleaved from the 53 kDa molecule (p53) encoded by the gag gene of the virus. Individuals infected with HIV make antibodies to gpl20, gp41 and, most prominently of the gag proteins, to p24. Because of difficulties in detecting the virus itself, infection is defined by the appearance in the serum of antibodies to any or all of these proteins. Seroconversion can take up to 3 months from the initial infection. Having entered a CD4 cell, HIV loses its coat, and a single- stranded DNA copy of the viral RNA is made. This is mediated by HIV reverse transcriptase. Ultimately, a complementary strand of DNA is made to give a double-stranded DNA replica of the viral genome. This is incorporated into the host genome. A DNA copy of the viral RNA may remain dormant within the cell for months or years. Infectious viral particles are subsequently made, particularly when an infected T cell is activated. Shortly after the primary infection, as many as 1 in 100 T cells may contain HIV. Host defence mechanisms decrease the viral burden at first, but ultimately the virus overcomes them and progressively infects more and more T cells. HIV-2 is fundamentally similar in lifecycle and structure to HIV-l although there is little if any serological cross- reaction between the envelope antigens. HIV-2 is less pathogenic in the sense that patients stay healthy and alive for longer than individuals infected with HIV-l. When an individual is first infected with HIV he or she may develop a transient illness characterized by fever, lymphodenopathy, a rash and inflammation of the meninges. This occurs in about 15% of infected individuals. The remainder are asymptomatic at the time of initial infection. As time passes, an individual with asymptomatic HIV infection may, however, develop lymphadenopathy progressing to fever, night sweats, weight loss and perhaps minor infections such as thrush and other yeast organisms in the mouth (Candida albicans) . The asymptomatic phase of HIV infection typically has a duration of about 10 years. During that time, it is evident that the turnover in T-cells and virus is very large indeed without the immune system failing. Apart from lymphodenopathy and its developments, infected individuals remain as free of other diseases during the asymptomatic phase of the infection as would ordinarily be the case, and in addition maintain relatively stable CD4+ cell counts, relatively small numbers of HIV-infected cells and strong T c -cell responses to pathogens recognized by the immune system. Eventually, however, a threshold is reached at which the infection progresses to the symptomatic phase known as AIDS. T-cell death during the symptomatic phase of the infection exceeds replenishment through proliferation with the result that the normal CD4+ count of a healthy human subject is reduced from CD4+ = 1 to 1.3 X IO 3 cells/mm 3 to CD4+ = 0.8 X IO 3 cells/mm 3 by WHO HIV Clinical Stage 2, and by WHO Clinical Stage 3 this has decreased to CD4+ = 0.7 X 10 3/mm3. Enumeration of CD4 cells and viral load in the patient's blood is the best indicators of the progress of the infection. When the absolute number of CD4 + T cells falls below 350/ml, the patient begins to lose cell-mediated immunity and opportunistic infections ensue. Among the many organisms that can affect the immunoco promised host, especially patients with AIDS, are Pneumocystiε carinii , Candida albicans , Mycobacterium avium-intracellulare, Toxoplasma gondii , Cryptosporidium spp. and genital and anal herpes simplex. During the early phases of the infection there is polyclonal expansion of B cells and the serum contains large amounts of IgG, IgM and IgA. In the late stages of AIDS, the amount of immunoglobulin in the serum falls dramatically and the antibody titres to gpl20 and p24 decrease concomitantly. The symptomatic phase of the infection is accordingly characterized by failures in both the recognition and effector faculties of the immune system with the result that pathogenic infections are inadequately challenged and the host eventually suffers death. Large numbers of substances have been proposed in recent years having anti-viral properties addressable to various viral infections ranging from herpes to HIV. Because of its virulence, the number of anti-viral agents proposed for use in HIV therapy has been the largest. The majority of these agents act viricidally directly against the viral organism itself or cytotoxically to kill viral host cells and thus the virus itself. The only therapy for HIV infection thus far which has had some measure of success, has been the use of dideoxynucleosides, principally azidothymidine (AZT or zidovudine) and now the proteinase inhibitors (saquinavir is now approved) . These compounds inhibit the reverse transcriptase of HIV, so impairing the production of DNA replicas of the virus. Resistance to AZT develops and renders AZT of limited usefulness. However, AZT given to infected pregnant women effectively reduces transplacental virus transmission by 75%. CDNB (l-chloro-2,4-dinitrobenzene) acts as an immunomodulator of dendritic cells (Stricken et al, Immunology Letters, 368, 1-6, 1993) when applied topically. However, studies have shown that when addressed to CD3-stimulated peripheral blood T-cells, CDNB is an immunosuppressant. CDNB has been found to deplete lymphocytes of intracellular glutathione. Glutathione is a key substance in maintaining the T-cell receptor (TcR) /CD3-dependent transmembrane signal transduction which is crucial to lymphocyte proliferative response (Kavanagh et al, Toxicology and Applied Pharmacology, 119, 99-99, 1993) . Tucaresol has been found to act as a T-cell costimulator in peripheral blood mononuclear cells (PBMC) . Tucaresol is 4-(2- formyl-3-hydroxyphenoxymethyl) - benzoic acid and has been found to form a Schiff base with CD4+ cell surface amines in an extracellular reaction which does not involve macromolecular interactions between T-cell markers and APC counter-receptors (Rhodes et al, Nature, 377, 71-75, 1995). UK Patent Application No 2 284 153 A discloses various benzoic acid derivatives for use in HIV therapy. Increase in CD4 count is disclosed as a result of such therapy and immunomodulation is indicated for at least some of the active substances disclosed without suggesting that the results achieved are due to enhancement of T-lymphocyte proliferative response through costimulation of TcR/CD3-stimulated T-cells. Anti-viral assays disclosed show IC 5 Q data of interest coupled with low toxicity. UK Patent Application No 2 288 333 A discloses a broader range of similar compounds, including certain sulfonic acids, for anti-viral use. In addition to TcR engagement of an antigenic peptide bound to major histocompatibility complex (MHC) receptors, other costimulatory signals are necessary for T cell activation. The most important of the costimulatory signals identified to date is provided by the interaction of CD28 on T cells with its ligands CD80 and CD86 on antigen-presenting cells. CD28 signal transduction can prevent apoptosis in cultures of HIV- infected cells and can induce expression of the Bcl-X L cell survival gene. Anti-CD28 generated signals are essential in TcR/CD3-induced lymphocytes but are not required in cell lines, such as lymphoblastoid cells, produced by oncogenesis using inserted foreign viral oncogenes. According to the invention, there is provided use of a compound as defined below, as a signalling agonist operative in anti-CD28 costimulated anti-TcR/CD3 complex-induced T- ly phocyte cells, for the preparation of a medicament for use in the enhancement of T-lymphocyte proliferative response, said proliferative response enhancement being effective in T- lymphocytes which are virally infected or uninfected, and being unaccompanied by substantial stimulation of viral replication in T-lymphocytes which are virally infected, and said agonist being an aromatic monocyclic compound whose molecules consist of a phenyl nucleus substituted with at least one electrophilic group and with at least one labile leaving group. In selecting the substituent groupings for a compound according to the invention an essential feature is the provision within any particular aromatic ring context of at least one labile group substituent and at least one electrophilic group substituent. Moreover, a group which may be classified as labile within one particular ring context may be classifiable as electrophilic within another alternative ring context. Furthermore, where there are at least two nitro substituents the labile group substituent may be a ring hydrogen. Generally speaking the compound of the invention may be of the general formula:- wherein X is COQ where Q is hydroxy, amino or substituted 2 amino, or the group OR 3 in which R 3 is a metal or alkyl; X is hydrogen, halogen, nitro, amino or hydroxy; X , X and X are, each independently, hydrogen, halogen or nitro; and X is hydrogen, nitro, optionally substituted amino or halogen. More preferred are compounds of formula (I) wherein X is COQ where Q is hydroxy, amino or substituted amino, or the group OR 3 in which R 3 is a metal or alkyl; X is hydrogen or halogen; X and X are, each independently, hydrogen, halogen or nitro; X is hydrogen; and X is hydrogen, nitro, optionally substituted amino or halogen. Amongst the compounds of formula (I) , those most preferred are:- 2,4-dichloro-3, 5-dinitrobenzoic acid 4-chloro-3 , 5-dinitrobenzoic acid 2-chloro-3 , 5-dinitrobenzoic acid 2-chloro-5-nitrobenzoic acid 2,5-dichlorobenzoic acid 2 ,4-dinitrobenzoic acid 3, 5-dinitrobenzoic acid 2, 5-dichloro-4-nitrobenzoic acid 2,4-dichloro-5-nitrobenzoic acid 2, 6-dichloro-4-nitrobenzoic acid 3,5-dichloro-4-nitrobenzoic acid 4-chloro-3-nitrobenzoic acid 2-chloro-4-nitrobenzoic acid 3,4-dichlorobenzoic acid the alkali metal salt of the foregoing named acid compounds. The compounds for use as agonists in the invention may be prepared by known process techniques for preparing benzene substituted compounds. Such techniques are described in various standard texts, for example, "Organic Syntheses" 1963 Collective Volume 4, pages 364 to 366, by Harry P. Schultz and published by John Wiley and Sons Inc. The agonists used in the invention may be formulated for use as pharmaceutical compositions (eg for iv, ip, oral or sc administration) comprising at least one active compound and a diluent or carrier. Such a composition may be in bulk form or, more preferably, unit dosage form. For example, the composition may be formulated as a tablet, capsule, powder, solution or suspension. Soft gel capsules may be especially convenient. The composition may be a liposo al formulation or administered in a slow sustained release delivery system. The compositions may be prepared in accordance with conventional pharmaceutical practice. The diluents, excipients or carriers n f p ablv used are well known in the formulation art and the form chosen for any particular treatment regimen will depend on the given context and the physician's choice. The agonists may be administered in solution in sterile deionised water. Solution may be facilitated using dimethyl sulphoxide (DMSO) when required or alternatively an alcohol, a glycol or a vegetable oil may be used as a vehicle for the costimulation compounds. The agonist compounds are most favourably administered in corn oil or as a solution in DMSO/sterile water. When used as sodium salts the active compounds are preferably administered as an aqueous solution. In using the agonists (eg a chloronitrobenzoic acid such as 2- chloro-5-nitrobenzoic acid) , dosage may be from about 50mg/kg typically up to about lOOOmg/kg (eg up to about 400mg/kg, conveniently about 200mg/kg or less) . Thus, it is to be expected that a typical dosage for humans will be from about 5mg/kg upwards (eg up to about 80mg/kg - such as up to 20mg/kg) . When administered in injectable liquid form, concentration will typically be 250mg/ml or less. Patient tolerance was found to be of a higher order for concentrations below that threshold although concentrations of more than 250mg/ml may be used. Typically, a preferred concentration is less than lOO g/ml, with concentrations below 50mg/ml (eg 40mg/ml) being preferred. Intravenous treatment will generally take place by administration very slowly over a period, typically a period of approximately 20 minutes or more. Administration will typically be by catheter, for example a catheter left in situ after flashing with 2ml of saline. Of course, the catheter will in practice then be used for taking blood samples for pharmacokinetic measurements and removed prior to the patient's departure from the clinic. For oral administration, enteric coated tablets will often be preferred. Maximum strength will generally be less than 500mg and preferably 400mg or less. Thus, for example, a range of strengths for oral administration (whether in tablet or other solid form) will be lOOmg, 200mg or 400mg. Patients who are subject to oral administration will generally fast for 8 hours prior to first dose, with the fast continuing for 4 hours after first dose. Free access to fluid will generally be allowed during this fasting period. Activity 1. Introduction The agonists of the invention act immunologically to enhance T- lymphocyte proliferation. H9 lymphoblastoid and donor PMBC assays demonstrate the enhanced production of intracellular DNA associated with proliferation by the cells when exposed to the agonist in an anti-TcR/CD3 stimulated state. This enhancement is evident in both chronically-infected and uninfected cells but may be more pronounced in uninfected cells. In acutely infected cells, increasing the virus input reduced anti-CD3-induced incorporation but without any significant effect on constimulation by the agonist as measured by the mean costimulatory indices with increasing TCID50 (see Section 5.1.5 hereafter) . There is some evidence that the CD28-stimulation associated with the agonists renders the cells intracellularly anti-viral so that HIV will not internalise at the cell membrane. Anti-viral assays in relation to the various agonists demonstrate some anti-viral activity for the agonists per se. 2. Primary Assay 2.1 Acute Infection Assay High titre virus stocks of the human immunodeficiency virus HIV- 1 RF were grown in H9 cells with RPMI 1640 (Flow laboratories) supplemented with 10% fetal calf serum, penicillin (lOOIU/ml) . Cell debris was removed by low speed centrifugation, and the supernatant stored at -70°C until required. In a typical assay C8166 T-lymphoblastoid CD4+ cells were incubated with 10XTCID50 HTV-I-,- , at 37°C for 90 minutes and then washed three times with phosphate buffered saline (PBS) . Cell aliquots (2 x 10 ) were resuspended in 1.5 ml growth medium in 6 ml tubes, and compounds in log dilutions [200mM to 0.2mM] were added immediately. 20 mM stock solutions of each compound were made up in 70% alcohol. The compounds were stored as a powder and made up freshly in distilled water before each experiment or were stored as a 20 mM stock solution in 70% alcohol. The final concentration of alcohol in the tissue culture medium was 1%. The cells were then incubated at 37°C in 5% C0 2 . At 72 hours post-infection 200 ml of supernatant was taken from each culture and assayed for HIV (Kingchington et al, 1989, Robert et al 1990) using an antigen capture ELISA which recognizes all the core proteins equally (Coulter Electronics, Luton, UK) . The following controls were used: (i) supematants taken from uninfected and infected cells, (ii) infected cells treated with AZT (Roche Products UK, Ltd) , (iii) infected cells treated with ddC (Roche) and (iv) infected cells treated with R031-8959 (Roche) an inhibitor of HIV proteinase. The IC 5 Q activities of AZT and dde in infected cells were an average 20 nM and 200 nM respectively (Figure 1) . The ELISA plates were read with a spectrophotometer. Compounds were tested in duplicate at each concentration, and the data shown is the average of at least two assays. This assay assesses the activity of compounds by measuring their inhibition of HIV core antigen levels. 2.2 Chronically Infected Cell Assay Chronically infected cells (H9RF) were washed three times to remove extracellular virus and incubated with the active compounds (eg 200-0.2 mM) for four days. HIV-l antigen in the supernatant was then measured using an ELISA. 2.3 Toxicity Assay To test for compound toxicity, aliquots of 2 x 10 of uninfected cells were cultured with the compounds in the same dilutions for 72 hours. The cells were then washed with PBSA and resuspended in 200ml of growth medium containing 14C protein hydrolysate. After 12 hours the cells were harvested and the 14C incorporation measured. Uninfected, untreated cells were used as controls. Toxicity is expressed as inhibition of uptake of 14C protein hydrolysate. 2 .4 Assay results 2 .4 .1 4-Chloro-3 , 5-nitrobenzoic acid The results of these assays for 4-chloro-3,5-dinitrobenzoic acid are summarised in Table 1 below. The IC 50 is the drug concentration that causes a 50% reduction in HIV core antigen levels as detected by the Coulter P24 antigen assay and is determined by doubling dilutions of supernatant taken from tubes containing untreated acutely infected cells. The CD 50 is the concentration of drug that causes a 50% inhibition of cells as measured by 14C protein hydrolysate uptake. The therapeutic index (TI) is determined by dividing the CD 50 by the IC 50 . TABLE 1 Code Compound IC 50 CD 50 Tl 4-chloro-3,5-dinitro- benzoic acid 30μm 70μm 2.33 2.4 .2 Other compounds Following the methodology set forth earlier for performance assay against HIV, more extensive assays were performed as reported in Tables 2 below for various compounds in free acid, sodium salt and methyl estar form; the compound signified by the code 15-Na(C17) is referred to hereinafter either as such or as the abbreviated code C17:- TABLE 2.1 STRUCTURE-ACTIVITY RELATIONSHIP AGAINST HIV VIRUS SUBSTTTUTE SHEET (RULE 26) CODE COMPOUNDS Ag IC50 Tox CC50 16 2, 4-dichloro-3 , 5-dinitrobenzoic acid 3-Me 2,4-dichloro-3,5-dinitrobenzoic acid methyl ester 17 4-chloro-3,5-dinitrobenzoic acid 17-Me 4-chloro-3,5-dinitrobenzoic acid methyl ester 18 2-chloro-3,5-dinitrobenzoic acid 18-Me 2-chloro-3, 5-dinitrobenzoic acid methyl ester 4 4-chloro-3-nitrobenzoic acid 7 2-chloro-4-nitrobenzoic acid 19 3,4-dichlorobenzoic acid 20 2,5-dichlorobenzoic acid 21 4-chlorobenzoic acid 15-Na(C17) 2-chloro-5-nitrobenzoic acid sodium salt TABLE 2.2 C-Compounds IC50 CC50 SI (Antiviral) (Toxicity) (Selectivity Index) Aαainst HIV-IIIB 16 5 70 14 36 70 2 33 70 2 35 60 2 Average 27 70 16 10 30 3 2.5 20 8 Aαainst HIV-1RF 16 7 60 8.5 - - 56 16 56 3.5 Average 11.5 57 5 Against chronically infected cells 16 16 30 2 16 95 6 Average 16 63 4 Against HIV-1IIIB 3-Me 0.3 7 23 17 40 100 2.5 30 70 2.3 Average 35 85 2.4 17-Me 5 60 12 18 23 150 6 5 >200 >10 Average 22 >175 8 18 >1 35 >35 10 30 3 18-Me 10 60 >200 >200 7 >200 >200 19 >200 >200 20 >200 >200 Against HIV-I IIIB 15-Na(C17) >1 >1000 >1000 5 >1000 >200 3. Patient Tests A pilot study was conducted for safety, pharmacokinetics and preliminary activity of C17 in patients with HIV-l infection and HIV-related Kaposi's sarcoma. In the pilot study, 10 patients were enrolled but only 6 were eligible for evaluation. These patients were positive for serum antibody to HIV-l as determined by both enzyme-linked immunosorbent assay (ELISA) and Western Blot. These patients had WHO Clinical Stage 2 to 3 for HIV infection and disease and fulfilled the inclusion and exclusion criteria as per the Clinical Trial Protocol. Their CD4 ranged from 0.072 to 0.812 x 10 3 cells/mm3 (normal range 1.0 x 1.3 x 103 cells/mm ) . Patient 6 had AIDS-related Kaposi's sarcoma confirmed on histology. C17 was prepared for intravenous administration under good manufacturing practice in strength of 40mg/ml following the method set forth below for producing a 250mg/ml solution, additional water being added at Step 7:- 1. To 275ml water for injection add 266.4ml 4N sodium hydroxide solution and mix. 2. Add with stirring 182.5g of 2-chloro-5-nitrobenzoic acid 3. Add additional 4N sodium hydroxide if needed to obtain solution. 4. Filter solution through a Sterivex GV 0.22μm filter. 5. Dilute to approximately 680ml with water for injection. 6. Adjust to pH 7.2-7 with hydrochloric acid. 7. Make-up to 730ml water for injection. Adjust pH if necessary. 8. Transfer solution to aseptic room and filter through Sterivex GV 0.22μm filter into injection vials. Seal with rubber stoppers and aluminium closures. Effect terminal sterilization by autoclave. SUBSTTTUTE SHEET (RULE 26) The patients received lOmg/kg body weight/day of C17 by deep intramuscular injection daily for 5 days weekly. The CD4 counts were estimate before and at about 2 weeks after the beginning of the therapy. The CD4 counts in all 6 patients showed an increase which was associated with clinical improvements in patient general condition including weight gain and a marked decrease in opportunistic infections and diarrhoea. Patient observations are shown in Tables 3 below. In the case of Patient 6, the sarcoma lesions disappeared. SUBSTTTUTESHEET(RULE26) TABLE 3.0 (Summarizing Tables 12.1 to 12.6) PATIENTS 1 TO 6 IMMUNOLOGY:- PATIENT TABLE 3.1 PATIENT 1 IMMUNOLOGY:- DAY 20 Lymphocytes x 10 mm 2.1 2.0 CD4 % 19 38 CD4 ABS/mm ' 0.399 0.760 CD8 ABS/mm ' 0.567 0.840 CD4/CD8 0.70 0.90 TABLE 3.2 PATIENT 2 IMMUNOLOGY:- DAY 1 14 Lymphocytes X 10 3mm- -3 2.0 1.9 CD4 % 18 27 CD4 ABS/mm 3 0.360 0.513 CD8 ABS/mm 3 0.940 0.874 CD4/CD8 0.38 0.59 SUBSTTTUTE SHEET (RULE 26) TABLE 3.3 PATIENT 3 IMMUNOLOGY:- DAY 20 ) -3 Lymphocytes X io'mm 0.9 1.1 CD4 % 8 17 CD4 ABS/mm 3 0.072 0.187 CD8 ABS/mm 3 0.270 0.682 CD4/CD8 0.27 0.27 TABLE 3.4 PATIENT 4 IMMUNOLOGY:- DAY 14 -3 Lymphocytes X io 3 mm 1.8 2.0 CD4 % 6 22 CD4 ABS/mm 3 0.108 0.440 CD8 ABS/mm 3 1.350 1.260 CD4/CD8 0.08 0.35 SUBSTTTUTE SHEET (RULE 26) TABLE 3.5 PATIENT 5 IMMUNOLOGY:- DAY 21 i -3 Lymphocytes X 10 " mm 2.8 3.1 CD4 % 29 33 CD4 ABS/mm 3 0.812 1.023 CD8 ABS/mm 3 1.456 1.550 CD4/CD8 0.56 0.66 TABLE 3.6 PATIENT 6 IMMUNOLOGY:- DAY 34 Lymphocytes X 10'mm " -3 2.1 1.9 CD4 % 18 23 CD4 ABS/mm 3 0.378 0.437 CD8 ABS/mm 3 1.218 0.931 CD4/CD8 0.31 0.47 4. Murine Hae atology Preliminary data on the administration of C17 in mice and rats showed increases in red blood cell count, haemoglobin concentration, macrophage count, white blood cell count (WBC), lymphocyte count and haematocrit (HTC) on day 7 when maximum SUBSTTTUTESHEET(RULE26) tolerated dose (MTD) was given by 5 consecutive daily intravenous injections. These values returned to normal on day 14. The MTD value were 950mg and 900mg/kg body weight/day respectively in mouse and rat. The data are reported in Figures 2 to 9. 5. Immunology 5 .1 H9 lymphoblastoid cell assays . 5.1 .1 Substituted Benzoic Acid . H9 cells were maintained in pyrogen-free RPMI 1640 containing 10% bovine calf serum. Stocks of antibiotics and glutamine were dissolved in pyrogen-free water. Flat-bottomed microtitre plates were precoated with goat anti-mouse IgG Fc (Fab )2 fragments (Sigma, Poole, England) (lOμg/ml; 200μg/well in sodium bicarbonate buffer, (pH 9.6) ovemight at 4°C. Each treated well was washed twice with phosphate buffered saline (PBS) . An azide free anti-CD3 mAb (5μg/ml (UCHTl) , Autogen Bioclear, Potterne, England) was immobilised in the wells for 4h at 4°C, and the plates were washed twice with PBS. 2xl0 5 H9 cells (in 200 μl) were then added to each well. Compounds 1-15 set forth in Table 4 below were dissolved in dimethyl sulphoxide (DMSO) and added to cultures in the concentration range of 5-1000 μM. DMSO had a final concentration of 1% in the culture medium. All samples were (at least) in triplicates. Controls included cultures containing H9 cells alone and H9 cells stimulated with anti-CD3 mAb with and without DMSO. Plates were warmed to 37°C before adding the compounds to the cells. This overcame problems imposed by the relative insolubility of the acid derivatives and high melting point of DMSO (18°C) . DMSO iε routinely used as a solvent in antiviral assays with T lymphoblastoid cells (Roberts, et al 1990) . SUBSTTTUTESHEET(RULE26) Plates were incubated in a humidified 5% C0 2 atmosphere for 3 days. Proliferative responses were measured on day 3 by pulsing the cultures with 0.5 uCi/well of [methyl- 3 H]thymidine 6h before harvesting of cells for the measurement of radiolabel incorporated into newly synthesised DNA. Cells were harvested using a Skatron cell harvester and filterdisc transfer system (Skatron Instruments Ltd, Newmarket, Suffolk, England). TABLE 4 COMPOUND CHEMICAL STRUCTURE 1 5-chloro-2-nitrobenzoic acid 2 2-amino-5-nitrobenzoic acid 3 2,4-dichloro-3 ,5-dinitrobenzoic acid 4 4-chloro-3-nitrobenzoic acid 5 2-nitrobenzoic acid 6 2-hydroxy-5-nitrobenzoic acid 7 2-chloro-4-nitrobenzoic acid 8 2-chlorobenzoic acid 9 2-chloro-3,5-dinitrobenzoic acid 10 benzoic acid 11 5-methyl-2-nitrobenzoic acid 12 3,5-dinitrobenzoic acid 13 2,4-dinitrobenzoic acid 14 2-bromo-5-nitrobenzoic acid 15 2-chloro-5-nitrobenzoic acid The results are shown in Figures 10.1, 10.2 and 10.3 5.1 .2 Sodium Benzoate Analogs The assay methodology of Paragraph 5.1.1 above was repeated with, as active agent, the sodium benzoates listed in Table 5 below but using the higher concentrations of active agent shown in Figure 11:- SUBSTTTUTESHEET(RULE26) TABLE 5 1-Na Sodium 5-chloro-2-nitrobenzoate 5-Na Sodium 2-nitrobenzoate 7-Na Sodium 2-chloro-4-nitrobenzoate 10-Na Sodium benzoic acid 11-Na Sodium 5-methyl-2-nitrobenzoate 13-Na Sodium 2,4-dinitrobenzoate 14-Na Sodium 2-bromo-5-nitrobenzoate 15-Na(C17) Sodium 2-chloro-5-nitrobenzoate The results are shown in Figure 11. 5.1 .3 H9 lymphoblastoid cell assays with cells uninfected /chronically infected Uninfected H9 lymphoblastoid cells and H9 cells chronically infected with HIV-IIIB were maintained in pyrogen-free RPMI 1640 medium containing 10% fetal calf serum. Cells were transferred to fresh medium 18 hours before each experiment. Costimulation of the two cell lines was carried out in parallel, using Compound 15-Na(C17) and the methodology set forth in Paragraph 5.1.2, the concentration of C17 being 3000μM. Plates were incubated for three days and pulsed with [methyl- 3H]thymidine as described above. The results are shown in Table 6 below and in Figure 12. TABLE 6 Uninfected H9 cells 0 μM 3000 μM cpm SEM (Number cpm SEM (Number of samples) of samples) 6017 +/- 442 (n=3) 20889 576 (n=4) 4875 +/- 586 (n=6) 26124 847 (n=7) 4905 +/- 165 (n=6) 29647 1506 (n=5) SUBSTTTUTE SHEET (RULE 26) Chronically Infected H9 IIIB cells 0 μM 3000 μM 26148 +/- 2505 (n=4) 43306 +/- 1905 (n=4) 28941 +/- 1539 (n=6) 46330 +/- 1564 (n=7) 32278 +/- 920 (n=5) 38872 +/- 1431 (n=8) 5.1 .4 H9 lymphoblastoid cell assays with mixed uninfected /chronically infected cells The assay set forth in Paragraph 5.1.3 was repeated with mixtures of uninfected and chronically-infected cells in ratios 10:1 to 10000:1. The results are shown in Figure 13. 5.1 .5 Assays with C8166 cells acutely infected with HIV-IIIB Uninfected C8166 T lymphoblastoid cells were maintained in pyrogen free RPMI 1640 medium containing 10% fetal calf serum. Cells were transferred to fresh medium 18 hours before each experiment. 96 well plates were treated with F(ab)2 and anti-CD3 as described above. In a typical assay C8166 cells were incubated with 10- 100TCID50 HIV-l at 37°C for 90 minutes and then washed three times with culture medium. Cell aliquots (5 x IO 4 ) were resuspended in 200μl of growth medium either with or without 1500μM of C17. All samples were in triplicate. Uninfected cells were used as controls. Duplicate sets of plates were then incubated at 37°C in 5% C0 2 . At 72 hours post-infection one plate was pulsed with [methyl- 3Hjthymi•di■ne as described above and the other assayed for p24. For p24 analysis 200μl of supernatant was taken from each well and assayed (Kinchington, et al 1989) using an antigen capture ELISA which recognises all the core proteins equally (Coulter Electronics, Luton, UK). The ELISA plates were read with a SUBSTTTUTESHEET(RULE26) spectrophotometer. The results are shown in Figure 14. The data shown represents a typical assay. 5 .1 .6 Antigen release assay - mixed uninfected/ chronically infected cellε The H9 cells referred to in Paragraph 5.1.4 were tested for HIV p24 antigen release in the cultures. The results are shown in Figure 15. 5 .1 . 7 Antigen release assay - acutely infected cells The C8166 cells referred to in Paragraph 5.1.5 were tested for HIV p24 antigen release in the cultures. The results are shown in Figure 16. 5.2 PBMC Assays 5.2 .1 Standard PBMC assay PBMC were isolated using Ficoll-Paque gradient from Buffy coat preparations obtained from blood donors (North East London Transfusion Service) and cultured in pyrogen-free RPMI-complete medium as described (Kinchington and Ng, 1996) . The sodium salt of 2-chloro-5-nitrobenzoic acid (ie Compound C17) was dissolved in water and added to cultures at final concentrations of 1500 and 3000 μM. All samples were (at least) in triplicates. Controls included cultures containing PBMC alone and PBMC stimulated with anti-CD3 mAb only. Plates were incubated for three days and pulsed with [methyl- H]thymidine as described above. Table 7 shows the results for PBMC from a number of donors (n=ll) :- TABLE 7 Donor Anti-CD3/AgonistStandard Anti-CD3 Standard PBMC (1500 μM) deviation alone deviation Average cpm Average cpm 101787 +/ - 981 82626 +/- 2929 92981 +/- 11873 57720 +/- 1744 3 132343 +/- 1551 90549 +/- 13247 99686 +/ - 4688 86829 +/- 3677 101895 +/- 4436 85518 +/- 7417 6 134635 +/- 2712 117128 +/- 10584 112476 +/ - 2098 88441 +/- 7013 (3000 μM) 8 126112 +/- 5308 88441 +/- 7013 9 158199 +/- 9795 102089 +/- 6081 10 126804 +/ - 16529 57720 +/- 1744 11 144037 +/- 18714 117128 +/- 10584 The average results are shown in Figure 17. 5.2 .2 Assay after B7-1 and B7-2 ligation blocking The effects of B7-1 (CD80) and B7-2 (CD86) molecules on blocking anti-CD3/Cl7 costimulation were investigated by preincubation of PBMC with azide-free anti-B7-l or anti-B7-2 mAb (20μg/ml) (Autogen Bioclear, Potterne England) at 4°C for 30 minutes prior to transfer to anti-CD3 antibody coated wells. Plates were incubated for three days and pulsed with [methyl- H]thymidine as described above. The results are shown in Figures 18.1 and 18.2 5.2 .3 Further assays Further PBMC assays were carried out using the protocol presented below. Costimulation was determined by [methyl- H] thymidine uptake assay, the results being shown in Figure 19.1. SUBSTTTUTESHEET(RULE26) p24 HIV-antigen assay was conducted, the results being shown in Figure 19.2 wherein "d(number)" means Day number (eg Day 3) post-infection at the fifth day. Rantes chemokine assay was conducted, and the results are shown in Figure 19.3. Figure 19.4 is a control for uninfected PBMC. Chemokine MlPI-α and MlPI-β assays were conducted, and the results are shown in Figure 19.5 (infected) and 19.6 (control- uninfected) . Protocol Day 1 Prepare PBMC from buffy coat (see Paragraph 5.2.1 above). Coat 96 well plates with F(ab)2 (lOμg/ml) fragments and leave overnight at 4°C Day 2 Wash plates with PBS x 3 and add anti-CD3 (5μg/ml) to appropriate wells and leave for 2 hours at 37°C. Wash off excess anti-CD3 and add PBMC (2 x 10 /200μl) to wells containing:- + anti-CD3/+ C17 (1500μM) abbreviated +/+ + anti-CD3/- C17 +/- - anti-CD3/+ C17 -/+ + anti-CD3/+ IL-2 (10 units/ml) +/+IL-2 To separate culture of PBMC in flask, add IL-2 (10 units/ml) + PHA(2μg/ml) and 10TCID50 of HIV-1,^ strain. Days 3, A , 5, 6 and 7 Add 3H-Thymidine and measure incorporation after 6 hours. SUBSTTTUTE SHEET (RULE 26) Day 5 (day of infection) Remove infected PBMC from infected PBMC supply in flask using special cell scraper and wash the cells x 3 with PBS to remove excess virus. Remove lOOμl of supernatant from each sample well in the 96 well plates and add 2 x 10 4 of the chronically infected PBMC to each sample well in fresh medium containing C17 or IL-2 where required. Day 8 Remove lOOμl supernatant from each sample well and freeze (-20) in labelled 96 well plate. Day 12 Remove lOOμl supernatant from each sample well and freeze (-20) in labelled 96 well plate. Day 15 Remove lOOμl supernatant from each sample well and freeze (-20) in labelled 96 well plate. Day 16 (or when convenient) Evaluate p24 levels in each sample in parallel using a commercial ELISA. Include p24 standard for quantification. Evaluate chemokine levels in each sample in parallel using a commercial ELISA. Include standard for quantification. The costimulation is maximum at 5 days. After this it plateaus. The graph represented in Figure 19. l shows changes in 3H- Thymidine rather than absolute incorporation from time 0. 5.2 .4 Costimulation in HIV-infected PBMC:p24 and viral RNA measurements SUBSTTTUTESHEET(RULE26) A timed study of the events of anti-CD3/C17 costimulation showed that the maximum effect occurred after 5 days (Figure 20.1). T- cell growth declined after day 3 with anti-CD3 alone and with anti-CD3/IL-2 T-cell growth peaked at 4 days and then declined (Figure 20.1). The dose response in all cases levelled at day 6 and further increases in T-cell growth, as measured by 3H- Thymidine uptake, was small. PBMC infected from the same batch on day 1 were added to uninfected costimulated cells on day 5 in the ratios of 1:10 respectively. Supernatant was removed from each sample at days 3, 7 and 10 post-infection for p24 analysis and subsequently fresh growth medium containing C17 or IL-2 where necessary were added to each sample on days 3 and 7 post-infection. The data indicates that at days 7 and 10 post-infection anti-CD3/C17 costimulation reduces HIV p24 release into the culture supematants when compared to cultures either lacking C17 or containing IL-2 (Figure 20.2). The standard deviations are large due to the small number of samples in this assay. Preliminary studies measuring HIV-RNA in the supematants have shown that at 24 hours post-infection anti-CD3/C17 costimulation reduces viral RNA when compared to anti-CD3 alone or anti-CD3 + IL-2 (Figure 20.3) . These measurements are made using PCR technology which accurately measure HIV-RNA copy. Further work will extend the time points to 72 hours and measure integrated HIV-DNA with reasonable expectation that they will show a reduction in integrated viral DNA attributable to C17. For PCR analysis 5 x 10 7 PBMC were incubated in 25ml flasks for 5 days. Flasks contained either anti-CD3 alone, anti-CD3 + CNBA- Na, CNBA-Na or anti-CD3 + IL-2. Cells were removed from each flask using a cell scraper. PBMC were resuspended in lml of the original supernatant and high titre was added to each sample and incubated for 1.5 hours at 37°C. Each sample was washed three times with PBS and aliquots of 2 x 10 cells were resuspended in 96 well plates containing anti-CD3 , CNBA-Na or SUBSTTTUTESHEET(RULE26) IL-2 as appropriate. Samples were then incubated at 37 C in 5% CO 2 . At 2 and 24 hours post-infection lOOμl supernatant was removed from each sample, spun in a Eppendorf centrifuge to remove any cell debris and the supernatant denatured in guanadinimu thiocyanate buffer. RNA was extracted using silica gel beads and then added to the PCR mix which first transcribed the RNA and then amplified the HIV-DNA using gag-primers. The DNA was biotinylated and added to streptavidin coated wells where it was denatured and the chemiluminescent substrate was added. Plates were read after 1 hour incubation at 450nm. Samples were compared with controls containing known copy numbers. 6. Summary of Activity The lymphoblastoid assays reported in Figures 10.1, 10.2 and 10.3 demonstrate that the benzoic acid derivatives assayed enhance anti-TcR/CD3-stimulated T-lymphoblastoid cell proliferation in a dose-dependent manner. Compounds 1, 2, 7, 11, 14 and 15 demonstrated increased proliferative response as concentration of the compound in question increased up to lμM. Compounds 3, 4 and 9, however, gradually blocked thymidine up¬ take with increasing compound concentration. Compounds 5, 10 and 13 had substantially no effect at concentrations of up to lμM. Compound 6 consistently enhanced T-lymphoblastoid proliferation at 500μM concentration but its operation proved concentration-sensitive in the concentration range used as evidenced by the fact that proliferative effect rapidly fell away with both increasing and decreasing concentrations. Assays carried out with these compounds in the concentration range lOOOμM to 1500μM showed some small further increase in costimulating activity. At higher concentrations the acids concerned reach maximum solubility. Acids neutralized with sodium hydroxide to form the corresponding benzoate sodium salts demonstrated continued SUBSTTTUTE SHEET (RULE 26) improvement in costimulatory effect at concentrations of lOOOμM to l500μM and above (Figure 11) . These benzoates are, of course, relatively readily soluble at concentrations up to 3000μM. The sodium salts of Compounds 1-Na, 7-Na, 14-Na and 15-Na(C27) showed especially high capacity to enhance T- ly phoblastoid proliferation at such concentrations. The dose-dependent enhancement in proliferative response achieved by the C7 sodium benzoate salt of Compound 15 was 10-fold at 3000μM concentration and 9-fold at 1500μM concentration. In PBMC assay (Figure 17), Compound 15-Na, namely the benzoate sodium salt C17, enhanced [methyl- 3H-]thymidine uptake by 25% and 56%, respectively, for anti-TcR/CD3-induced PBMC at compound concentrations of 1500μM and 3000μM as compared to anti-TcR/CD3 stimulation alone. The benzoate did not significantly increase proliferative response of the CD4+ cells in the PBMC donation in the absence of anti-TcR/CD3 complex stimulation. C17 was also shown (Figure 12) to effect costimulation in chronically HIV IIIB-infected H9 lymphoblastoid cells and to do so in a dose-dependent manner (and also, as shown in Figure 13, to effect costimulation in a range of mixtures of uninfected and chronically infected cells) . Baseline DNA/RNA synthesis rate in chronically infected cultures (both anti- TcR/CD3-stimulated and unstimulated) was significantly higher than for uninfected cells. However, the costimulatory effect of C17 on TcR/CD3-induced proliferation was greatest in uninfected cell cultures. Average increases in anti-CD3- induced [methyl- H-]thymidine uptake costimulated with C17 were 400% in uninfected cells and 50% in chronically-infected cells. The greatest concentration of infected T-lymphocytes in vivo is in the lymph nodes with only a relatively small percentage of T-cells in serum being infected by HIV. B7-1 and B7-2 ( CD80 and CD86 ) are APC-presented ligands for the CD28 marker on T- lymphocytes . C17 in a concentration of 1500μM significantly enhances PBMC proliferation which is CD3- induced. Preincubation of the PBMC cultures with CD80 and CD86 presented by APC in the donation cancelled the effect of C17 so that the proliferative responses to lOOOμM C17, 3000μM C17 and zero C17 are indistinguishable (Figures 18.1 and 18.2) . In acutely infected cells with increasing viral load the anti- CD3-induced proliferation (ie thymidine uptake) is reduced but the costimulatory effect remains unchanged. Antigen (p24) release with treatment of these cultures (Figure 15) never exceeded the untreated cultures. This demonstrates that the agonist compound does not enhance viral replication despite the fact that HIV replication is mediated by the cell nucleus. Figure 15 shows that in the case of the mixed cultures of Paragraph 5.1.4 (uninfected and chronically infected cells) HIV p24 antigen levels are generally less for costimulated cultures than for cultures which are only TcR/CD3-induced. 7. Mechanism Activation of a T-cell by antigen engagement to the TCR/CD3 complex causes the cell to produce and excrete cytokines, and in particular to introduce IL-2 to serum. Serum IL-2 engages the cell's IL-2 receptor (IL-2R) to stimulate cell proliferation. However, IL-2R in resting T-cells is composed of beta- and gamma-polypeptide chains only and has the capacity to bind IL-2 only weakly. Proliferative response in practice requires that the beta- and gamma peptides be associated with an α-chain polypeptide (p55) . IL-2R[p55] is expressed by the IL-2Rα gene. The kB element of the IL-2Rα gene promoter region is regulated by the binding thereto of the transcription factor NF-kB. However, NF-kB is a Rel protein which exists cytoplas ically as an inactive complex SUBSTTTUTESHEET(RULE26) with an inhibitory I-kB protein substrate which must be dissociated by phosphorylation to release the Rel (eg tyrosyl phosphorylation or phosphorylation of Ser or Thr) protein for binding to the IL-2Rα gene promoter. C17 has been found to induce tyrosine kinase activity in such manner as may enhance tyrosyl phophorylation of the Rel protein : I-kB substrate to release the NF-kB IL-2Rα gene promoter transcription factor. C17 clearly does have a modifying effect on one or more signal pathways downstream of the TcR/CD3 complex and may enhance IL- 2 production in addition to its possible role in enhancing expression of IL-2R[p55]. It is also possible that C17 may in some way secure the extracellular domain of IL-2Rα against the surface enzymatic cleaving which may otherwise cause receptor shedding of the binding fragment of the chain. Rel proteins such as NF-kB are, of course, only one of many families of intracellular protein substrates in T-lymphocytes. Protein substrates containing sarc homology (sh) domains bind to activated p56lck sarc ki.nase i.mmobi.lized on the i.nteri.or surface of the cell membrane and associated with the cytoplasmic domain of the CD3/CD4 receptor. Kinase p56lck is activated on antigen binding to CD3/CD4 by tyrosyl phosphorylation to attach a phosphate grouping to the kinase substrate, and it is to the phosphate grouping that the sh domain protein binds. C17 is thought to encourage tyrosyl phosphorylation of kinase p56lck. sh Domai.n protei.n bi.ndi.ng i.s part of a chain reaction producing a signal pathway to the nucleus to induce the cell to proliferate. The belief is that, in addition to the other attributes referred to herein, the active compounds of the invention, eg C17, stimulate T-cells to secrete Rantes chemokineε and/or MlPl-α (or β) chemokines for binding to T-lymphocyte and/or macrophage CCR5 cell surface coreceptors to block HIV binding and thus HIV will not enter into and integrate into the cell) . The contents of PCT Application No PCT/GB96/00650 are hereby repeated herein.

Claims: Claims : 1. Use of a compound as defined below, as a signalling agonist operative in the anti TcR/CD3-CD28-initiated intracellular proliferative signal pathway in anti-CD28 costimulated anti-TcR/CD3 complex-induced T-lymphocyte cells, for the preparation of a medicament for use in the enhancement of T-lymphocyte proliferative response, said proliferative response enhancement being effective in T-lymphocytes which are virally uninfected, acutely virally infected or chronically virally infected and being unaccompanied by substantial stimulation of viral replication in T-lymphocytes which are virally acutely infected or virally chronically infected, and said agonist being an aromatic monocyclic compound whose molecules consist of a phenyl nucleus substituted with at least one electrophilic group and with at least one labile leaving group. 2. Use as claimed in Claim 1 wherein the agonist demonstrates optimally in an H9 lymphoblastoid cell costimulation assay conducted as set forth in sub-paragraphs (a) to (f) below, an increase in CD3-triggered T-cell DNA synthesis as measured by the uptake expressed in cpm of [methyl- 3 H] thymidine by TcR/CD3-stimulated uninfected H9 lymphoblastoid cells of about 50% or more cpm as compared to the same TcR/CD3-stimulated uninfected H9 lymphoblastoid cells assayed in the absence of the agonist:- (a) the agonist is dissolved in DMSO; (b) uninfected H9 lymphoblastoid cells are cultured by maintaining them in either pyrogen-free or normal RPMI 1640 culture medium containing 10% bovine fetal calf serum in the presence of glutamine and antibiotic; (c) cell aliquots containing 5 x IO 4 H9 lymphoblastoid cells resuspended in 200μl culture medium are added to SUBSTTTUTE SHEET (RULE 26) prepared wells containing 200μg of lOμg/ml goat anti- mouse IgG Fc (Fab) 2 in sodium bicarbonate buffer at pH9.6 immobilized azide-free anti-CD3 mAb at a concentration of 5μg/ml; (d) DMSO agonist solution is added to the wells to form cultures having concentrations of agonist over the range 5 to 1000 μM compound per litre and 1% DMSO; (e) the cultures are incubated in humidified 5% C0 2 atmosphere for 3 days at a temperature of 37°C; and (f) the incubated cultures are pulsed on the third day with 0.5 uCi/well of [methyl- H] thymidine 6 hours before harvesting the cells for measurement of radiolabel incorporation. 3. Use as claimed in Claim 1 or Claim 2 wherein the agonist demonstrates in a repetition of the H9 lymphoblastoid cell costimulation assay defined in Claim 2 conducted with the modifications set forth in sub-paragraphs (a) and (b) below, an increase in CD3-triggered T-cell DNA synthesis as measured 3 by the uptake expressed in cpm of [methyl- H] thymidine by TcR/CD3-stimulated uninfected/HIV-IIIB-chronically infected H9 lymphoblastoid cell mixtures of about 50% or more cpm as compared to the same TcR/CD3-stimulated cell mixtures assayed in the absence of the agonist: - (a) the H9 lymphoblastoid cells are a mixture of uninfected H9 lymphoblastoid cells with H9 lymphoblastoid cells chronically infected with HIV-IIIB; and (b) the assay is conducted at uninfected: infected cell ratios of from 10 to 10 3 . 4. Use as claimed in any one of Claims 1 to 3 wherein the agonist demonstrates in a repetition of the H9 lymphoblastoid cell costimulation assay defined in Claim 2 conducted with the SUBSTTTUTE SHEET (RULE 26) modifications set forth in sub-paragraphs (a) to (e) below, increases in CD3-triggered T-cell DNA synthesis as measured by the uptake expressed in cpm of [methyl- H] thymidine by TcR/CD3-stimulated HIV-IIIB acutely infected C8166 lymphoblastoid cells of about 50% or more cpm at 10TCID50 to 50TCID50 and about 25% or more cpm at 500TCID50 as compared to the same TcR/CD3-stimulated cell mixtures assayed in the absence of the agonist:- (a) the C8166 lymphoblastoid cells are H9 lymphoblastoid cells acutely infected with HIV-IIIB; (b) the C8166 lymphoblastoid cells are cultured by maintaining them in pyrogen-free RPMI 1640 culture medium containing 10% bovine fetal calf serum in the presence of penicillin; (c) cell debris is removed by low speed centrifugation and the cell culture then incubated with HIV-IIIB at a temperature of 37°C for 90 minutes and at viral loads in the range 10TCID50 to 500TCID50; (d) cell aliquots containing 5 x 10 C8166 lymphoblastoid cells resuspended in 200μl culture medium after quadruple washing of the culture are added to prepared wells containing 200μg of lOμg/ml goat anti-mouse IgG Fc (Fab') 2 in sodium bicarbonate buffer at pH9.6 and 5μg/l immobilized azide-free anti-CD3 mAb; and (e) agonist is added to the wells in amounts of I500μ</well 5. Use as claimed in any preceding claim wherein the agonist demonstrates in donor peripheral blood mononuclear cell (PBMC) costimulation assay (n=9) conducted as set forth below, an increase in CD3-triggered T-cell DNA synthesis as measured by the uptake expressed in cpm of [methyl- 3 H] thymidine by TcR/CD3-stimulated uninfected PBMC of 25% or more SUBSTTTUTESHEET(RULE26) cpm as compared to the same TcR/CD3-stimulated uninfected PBMC assayed in the absence of the agonist: - (a) donor PBMC is isolated using Ficall-Paque gradient from Buffy coat preparations obtained from donors; (b) the PBMC isolate is cultured in either pyrogen-free or normal RPMI 1640 culture medium containing 10% bovine fetal calf serum in the presence of glutamine and antibiotic; (c) the agonist is dissolved in water and added to the cultures at final concentrations of 1500 and 3000μM; (d) the resulting cultures are incubated in humidified 5% C0 2 atmosphere for three days at a temperature of 37°C; (e) the incubated cultures are pulsed on the third day with 0.5 uCi/well of [methyl- H] thymidine 6 hours before harvesting the cells for measurement of radiolabel incorporation. 6. Use as claimed in any preceding claim wherein the agonist demonstrates in a repetition of the H9 lymphoblastoid cell costimulation assay defined by Claim 2, Claim 3 or Claim 4 or the PBMC costimulation assay defined by Claim 5 with the modification that the H9 lymphoblastoid cells or PBMC are not TcR/CD3-stimulated, no increase in T-cell DNA synthesis as 3 measured by the uptake expressed in cpm of [methyl- H] thymidine by H9 lymphoblastoid cells as compared to a DMSO control assay. 7. Use as claimed in any preceding claim wherein the agonist demonstrates, in a repetition of the PBMC costimulation assay defined in Claim 5 conducted with the modification that the PBMC culture resulting from step (b) recited in Claim 5 is pre-incubated with B7-1(CD80) and B7-2(CD86) mAb's, no increase in T-cell DNA synthesis as measured by the uptake expressed in cpm of [methyl- H] thymidine by PBMC as compared to the same TcR/CD3-stimulated PBMC assayed in the absence of agonist. 8. Use as claimed in any preceding claim wherein the agonist acts as an agonist of tyrosyl phosphorylation of intracellular tyrosine kinase substrate in anti-TcR/CD3-stimulated CD28 costimulated T-lymphocytes in PBMC in the absence of butylated hydroxytoluene. 9. Use as claimed in any preceding claim wherein the agonist promotes genetic expression of the α-chain interleukin-2 receptor component Il-2R[p55] in anti-TcR/CD3-stimulated CD28- costi ulated T-lymphocytes. 10. Use as claimed in any preceding claim wherein the agonist is a compound whose molecules consist of a phenyl nucleus substituted with two electrophilic groups and a labile leaving group. 11. Use as claimed in any preceding claim wherein the agonist is one in which the substituents are in 1, 2, 4- or 1, 2, 5- configuration. 12. Use as claimed in Claim 11 wherein the phenyl nucleus in the agonist has 1-substitution with a first electrophilic group, 2-substitution with a second electrophilic group or labile group and 4- or 5-substitution with a labile leaving group or electrophilic group. 13. Use as claimed in any preceding claim wherein the electrophilic group in the agonist is (i) a group of formula - SO 3 X in which X is hydrogen or a metal or (ii) a group of formula -C00Y in which Y is hydroxy, alkyl, a metal or optionally substituted amino. SUBSTTTUTE SHEET (RULE 26) 14. Use as claimed in any preceding claim wherein the labile leaving group in the agonist is halogen, amino, hydroxy or alkyl. 15. Use as claimed in any preceding claim wherein the agonist is a benzoic acid derivative having the formula:- wherein R_, R 2 , R 3 , R 4 and R 5 are, each independently hydrogen, Cx to C 4 alkyl, nitro or halo, and wherein Rς, is hydrogen, an ester-forming moiety or a salt-forming moiety; subject to the following two provisos:- (a) at least one of R 1( R 2 , R 3 , R 4 and R 5 is other than hydrogen, and (b) the halo atom(s) when present is (are) chlorine or bromine. 16. Use as claimed in Claim 15 wherein the benzoic acid derivative has the general formula:- SUBSTTTUTE SHEET (RULE 26) wherein X_ and X 2 are, each independently, hydrogen or chloro and wherein X 3 and X are, each independently, hydrogen or nitro, provided that at least two of X lf X 2 , X 3 and X 4 are other than hydrogen, or a salt or ester thereof. 17. Use as claimed in Claim 15 or Claim 16 wherein the benzoic acid derivative is a compound of the formula presented in Claim 15 or that presented in Claim 16 wherein in either case X_ is chloro, X 2 is chloro or hydrogen, X 3 is nitro and X 4 is nitro or hydrogen. 18. Use as claimed in Claim 17 wherein the benzoic acid derivative is a compound of the formula presented in Claim 15 or that presented in Claim 16 wherein in either case Xj and X are other than 4-chloro. 19. Use as claimed in any one of Claims 15 to 18 wherein the benzoic acid derivative is a compound of the formula presented in Claim 15 or that presented in Claim 16 wherein X_ and X 2 together represent 2-chloro-, 4-chloro-, 2,5-dichloro-, 2, 4- dichloro-, 2, 6-dichloro-, 3, 5-dichloro- or 3, 4-dichloro- and wherein X 3 and X 4 together represent two hydrogen atoms, 3- nitro-, 4-nitro-, 5-nitro-, 2, 4-dinitro- or 3, 5-dinitro-, provided that:- (i) when X 3 and X 4 together represent 3, 5-dinitro-, X_ and X 2 together represent 2-chloro-, (ii) when X 3 and X 4 together represent 5-nitro-, λ and X 2 together represent 2- chloro- or 2, 4-dichloro-, (iii)when X 3 and X together represent two hydrogen atoms, and X 2 together represent 3, 4-dichloro-, (iv) when X 3 and X together represent 4-nitro-, X λ and X 2 together represent 2, 5- dichloro-, 3, 5-dichloro-2- chloro- or 2, 6-dichloro-, (v) when X 3 and X 4 together represent 3-nitro-, X 2 and X 2 together represent 4- chloro-. SUBSTTTUTESHEET(RULE26) 20. Use as claimed in Claim 19 wherein the benzoic acid derivative is a compound of the formula presented in Claim 15 or that presented in Claim 16 wherein in either case X lf X 2 , X 3 and X 4 are as there defined subject to provisos (I), (ϋ), (iv) and (v) and subject to the further proviso that when X 3 and X 4 together represent 5-nitro-, Xi and X 2 together represent 2, 4-dichloro-. 21. Use as claimed in Claim 15 wherein the benzoic acid derivative is a compound as set forth by name below or an ester or salt thereof: - 21.1 2, 4-dichloro-3 , 5-dinitrobenzoic acid 21.2 4-chloro-3, 5-dinitrobenzoic acid 21.3 2-chloro-3, 5-dinitrobenzoic acid 21.4 2, 5-dichlorobenzoic acid 21.5 2, 4-dinitrobenzoic acid 21.6 2, 5-dichloro-4-nitrobenzoic acid 21.7 2, 4-dichloro-5-nitrobenzoic acid 21.8 2, 6-dichloro-4-nitrobenzoic acid 21.9 3, 5-dichloro-4-nitrobenzoic acid 21.10 4-chloro-3-nitrobenzoic acid 21.11 3, 4-dichlorobenzoic acid 21.12 2-chloro-5-nitrobenzoic acid 22. Use as claimed in any one of Claims 1 to 14 wherein the agonist is a compound of formula:- wherein R_ is hydrogen, C_ - C 4 alkyl or a metal, one of X 2 , X 4 and X 5 is nitro or hydrogen, one is halogen, amino, C} - C 4 SUBSTTTUTESHEET(RULE26) alkyl or hydroxy and the other is hydrogen provided that X 4 is hydrogen when X 5 is other than hydrogen. 23. Use as claimed in any one of Claims 1 to 14 wherein the agonist is a compound of the following general formula:- wherein R 10 is hydrogen, C ! -C alkyl or a metal, one of X 2 , X 4 and X 5 is C ! -C alkyl or amino, another of X 2 , X 4 and X 5 is nitro and the other of X 2 , X 4 and X 5 is hydrogen provided that X 4 and X 5 are not simultaneously a substituent. 24. Use as claimed in Claim 23 wherein the agonist is a compound of the general formula IIB set forth and defined in Claim 23 wherein the C 1 -C 4 alkyl group is methyl or ethyl. 25. Use as claimed in Claim 24 wherein the agonist is 5- methyl-2-nitrobenzoic acid, 5-nitro-2-methylbenzoic acid or a c l ~ 4 alkyl ester or metal salt of either. 26. Use as claimed in any one of Claims 1 to 14 wherein the agonist is a compound of the following general formula:- SUBSTTTUTE SHEET (RULE 26) wherein R_oo is hydrogen, Cι~C 4 alkyl or a metal, one of X 2 , X 4 and X 5 is halogen, another of X 2 , X 4 and X 5 is nitro, hydrogen or halogen and the remaining one of X 2 , X 4 and X 5 is hydrogen provided that X 4 and X 5 are not simultaneously a substituent. 27. Use as claimed in Claim 26 wherein the agonist is one of the compounds set forth by name below or an alkyl ester or metal salt thereof:- 27.1 5-chloro-2-nitrobenzoic acid 27.2 2-chloro-4-nitrobenzoic acid 27.3 2-chlorobenzoic acid 27.4 2-bromo-5-nitrobenzoic acid 27.5 2-chloro-5-nitrobenzoic acid 28. Use as claimed in any one of Claims 1 to 14 wherein the agonist is one of the compounds set forth by name below or an alkyl ester or metal salt thereof:- 28.1 2-amino-5-nitrobenzoic acid 28.2 2-hydroxy-5-nitrobenzoic acid 28.3 3, 5-dinitrobenzoic acid 29. Use as claimed in any preceding claim wherein the aromatic monocyclic compound is a sodium or other alkali metal benzoate salt. 30. Use of a halo-, nitro- or halonitro- benzoic acid compound in acid, alkyl ester or metal salt form as a signalling agonist operative in the anti TcR/CD3-CD28- initiated intracellular proliferative signal pathway in anti- CD28 costimulated anti-TcR/CD3 complex-induced T-lymphocyte cells, for the preparation of a medicament for use in the enhancement of T-lymphocyte proliferative response, said proliferative response enhancement being effective in T- ly phocytes which are virally uninfected, acutely virally infected or chronically virally infected and being unaccompanied by substantial stimulation of viral replication in T-lymphocytes which are virally acutely infected or virally chronically infected. 31. Use of a compound as defined below for the manufacture of a medicament for use in the treatment of HIV-infected patients, said compound having the general formula:- wherein X is a group of for ula-cOQ in which Q is hydroxy, optionally substituted amino or has the formula -OR 3 in which R 3 is alkyl or metal; X 2 i•s hydrogen or halogen; 3 4 X and X are, each independently, hydrogen, halogen or nitro; X ,6 is hydrogen; and X is hydrogen, nitro, optionally substituted amino or halogen; provided that said compound has at least one electrophilic substituent and at least one labile leaving substituent present in the molecule; said compound acting as an agonist for the proliferation of T-cells, preferably as a signalling agonist operative in the anti TcR/CD3-CD28-initiated intracellular proliferative signal pathway in anti-CD28 costimulated anti-TcR/CD3 complex-induced T-lymphocyte cells, for the enhancement of T-lymphocyte proliferative response, said proliferative response enhancement being effective in T- lymphocytes which are virally uninfected, acutely virally infected or chronically virally infected and being unaccompanied by substantial stimulation of viral replication in T-lymphocytes which are virally acutely infected or virally chronically infected. 32. Use as claimed in Claim 31 wherein said agonist is a compound having the general formula:- COOR 6 X. wherein X is hydrogen or halogen, X 3 is hydrogen, nitro or halogen, X 4 is hydrogen, nitro, amino or halogen, X 5 is hydrogen, nitro or halogen and R 6 is hydrogen, a metal or an alkyl group, subject to the proviso that the substituents on the phenyl nucleus include at least one halogen substituent and at least one nitro substituent. 33. Use as claimed in any one of Claims 30 to 32 wherein the agonist demonstrates optimally in an H9 lymphoblastoid cell costimulation assay conducted as set forth in sub-paragraphs SUBSTTTUTESHEET(RULE26) (a) to (f) below, an increase in CD3-triggered T-cell DNA synthesis as measured by the uptake expressed in cpm of [methyl- 3 H] thymidine by TcR/CD3-stimulated uninfected H9 lymphoblastoid cells of about 50% or more cpm as compared to the same TcR/CD3-stimulated uninfected H9 lymphoblastoid cells assayed in the absence of the agonist:- (a) the agonist is dissolved in DMSO; (b) uninfected H9 lymphoblastoid cells are cultured by maintaining them in either pyrogen-free or normal RPMI 1640 culture medium containing 10% bovine fetal calf serum in the presence of glutamine and antibiotic; (c) cell aliquots containing 5 x io 4 H9 lymphoblastoid cells resuspended in 200μl culture medium are added to prepared wells containing 200μg of lOμg/ml goat anti- mouse IgG Fc (Fab) 2 in sodium bicarbonate buffer at pH9.6 immobilized azide-free anti-CD3 mAb at a concentration of 5μg/ml; (d) DMSO agonist solution is added to the wells to form cultures having concentrations of agonist over the range 5 to 1000 μM compound per litre and 1% DMSO; (e) the cultures are incubated in humidified 5% C0 2 atmosphere for 3 days at a temperature of 37°C; and (f) the incubated cultures are pulsed on the third day with 0.5 uCi/well of [methyl- 3 H] thymidine 6 hours before harvesting the cells for measurement of radiolabel incorporation. 34. A compound of the general formula IIB set forth and defined in Claim 23 for use as a T-lymphocyte proliferation agonist. SUBSTTTUTESHEET(RULE26) 35. A compound as claimed in Claim 34 wherein X is other than alkyl. 36. 5-Methyl-2-nitrobenzoic acid, or an alkyl ester or metal salt thereof, for use as a T-lymphocyte proliferation agonist. 37. A compound of the general formula III set forth and defined in Claim 26, for use as a T-lymphocyte proliferation agonist, wherein one of X 2 , X 4 and X 5 is bromine, another of X 2 , X 4 and X 5 is bromine, hydrogen or nitro and the other of X 2 , X and X 5 is hydrogen, the compound being 2-, 4-, 5-, 2,4- or 2,5- substituted. 38. 5-Chloro-2-nitrobenzoic acid, or a Ci-C 4 alkyl ester or metal salt thereof, for use as a T-lymphocyte proliferation agonist. 39. 2-Chlorobenzoic acid, or a C ! -C alkyl ester or metal salt thereof, for use as a T-lymphocyte proliferation agonist. 40. 2-Bromo-5-nitrobenzoic acid, or a C_-C 4 alkyl ester or metal salt thereof, for use as a T-lymphocyte proliferation agonist. 41. A compound as claimed in Claim 39 or Claim 40 in the form of the sodium benzoate salt. 42. A method for the enhancement of T-lymphocyte proliferative response in a mammalian subject, which method comprises administering to anti-TcR/CD3-CD28-costimulated T- lymphocytes a signalling agonist operative in the anti- TcR/CD3-CD28 intracellular proliferation signal pathway in so- costimulated T-lymphocyte cells, the agonist being a compound as defined in any preceding claim and the T-lymphocyte proliferation enhancement being unaccompanied by substantial stimulation of viral replication in T-lymphocytes which are acutely or chronically virally infected and the subject having SUBSTTTUTESHEET(RULE26) T-lymphocytes which are virally uninfected, acutely virally infected and/or chronically virally infected. 43. A compound as specifically disclosed herein by formula or chemical name for use as a T-lymphocyte proliferation agonist, said compound being de facto known per se but not known for a medical use. 44. A product containing a signalling agonist as defined in any preceding claim and a second component, said second component being an anti-viral agent and the two components being presented as a combined preparation for simultaneous, separate or sequential use in anti-viral immunotherapy. 45. Use of an agonist as defined in any preceding claim for the manufacture of a medicament for use in the challenge of a viral infection in which MHC-bound retroviral antigen is presented by an APC to a T-lymphocyte as an anti-TcR/CD3- co plex stimulus of the T-lymphocyte and in which said anti- TCR/CD3 stimulus is accompanied by anti-CD28 stimulation of the CD28 marker of the T-cell by ligation with APC-presented B7-1 and B7-2 immunoglobulin. 46. Use of an aromatic monocyclic compound whose molecules consist of a phenyl nucleus substituted with an electrophilic group and with a labile leaving group for the manufacture of a medicament for use in the treatment of HIV-infection by action upon T-lymphocytes which are anti-TcR/CD3 stimulated to impede the proliferation of HIV within the cell. 47. Use of an aromatic monocyclic compound whose molecules consist of a phenyl nucleus substituted with at least one electrophilic group and with at least one labile leaving group for the manufacture of a medicament for us in the treatment of HIV infection by the stimulation of T-cells to secrete Rantes chemokine and/or a chemokine MICl such as chemokine MICl-α and/or chemokine MICl-β. SUBSTTTUTESHEET(RULE26) 48. A method of ex-vivo treatment of an HIV patient suffering from severe (eg terminal) AIDS comprising removal of T-cells from the patient, costimulating the removed T-cells ex-vivo by interaction with CD3 antibody and CD28 antibody in the presence of an aromatic monocyclic compound whose molecules consist of a phenyl nucleus substituted with at least one electrophilic group and with at least one labile leaving group, purifying the so-treated T-cells of any free said antibody and returning the T-cells to the patient so purified and in the absence of a fatal or near fatal dose of the aromatic monocyclic compound. 49. Use of a T-cell proliferation agonist as defined in any preceding claim for the manufacture of a medicament for the treatment of HIV-infection in combination therapy. SUBSTTTUTE SHEET (RULE 26)

Inventors: AYUKO WASHINGTON ODUR, KINCHINGTON DEREK, NG TONY TSZ-CHEONG, AYUKO, WASHINGTON, ODUR, KINCHINGTON, DEREK, NG, TONY,TSZ-CHEONG
Applicants: AYUKO WASHINGTON ODUR, KINCHINGTON DEREK, NG TONY TSZ CHEONG, RADOPATH LTD, AYUKO, WASHINGTON, ODUR, KINCHINGTON, DEREK, NG, TONY,TSZ-CHEONG, RADOPATH LIMITED
Application year: 1997
Main classification: A61K31
IPC codes: A61K31