ANTIVIRAL IMMUNOTHERAPY BY MEMBRANE RECEPTOR LIGATION

Abstract

The present invention relates to a cytotoxic agent for the prophylaxis and/or treatment of a viral infection which is configured for the selective binding to a membrane receptor of virus-infected T lymphocytes, a pharmaceutical composition containing said cytotoxic agent, the use of the cytotoxic agent for the prophylaxis and/or treatment of viral infections, a method of finding cytotoxic agents, the use of a membrane receptor of virus-infected T lymphocytes which is overexpressed in comparison to non-infected T lymphocytes for the diagnosis of a viral infection.

Claims

1. A cytotoxic agent for the prophylaxis or treatment of a viral infection, which is configured for the selective binding to a membrane receptor of virus infected T lymphocytes, wherein the membrane receptor is selected from the group consisting of: CD134, CD132, CD71, CD70, CD54, CD39, BTLA, CD97, CD2, CD63, CD50, CD161, CD218, CD226, CD7, CD49d, and CD29.

2. The cytotoxic agent of claim 1, which is coupled to a binding molecule which is selective for the membrane receptor.

3. The cytotoxic agent of claim 2, wherein the binding molecule is selected from the group consisting of: immunoglobulin, immunoglobulin fragment, aptamer, lower molecular compound, receptor, receptor fragment, cytokine.

4. The cytotoxic agent of claim 3, wherein the immunoglobulin is an agonistic antibody.

5. The cytotoxic agent of claim 3, wherein the immunoglobulin is selected from the group consisting of: MEDI6469 (9612), MEDI6383, MEDI0562, Hu106-222/Hu119-122 (UTMDACC).

6. The cytotoxic agent of claim 1, wherein the virus-infected T lymphocytes are virus-infected CD4.sup.+ T lymphocytes.

7. The cytotoxic agent of claim 1, wherein the virus infection is an HIV infection.

8. The cytotoxic agent of claim 1 comprising a cytostatic agent.

9. The cytotoxic agent of claim 8, wherein the cytostatic agent is selected from the group consisting of: alkylating agents, platinum analogues, intercalants, antibiotics, mitosis inhibitors, taxanes.

10. The cytotoxic agent of claim 1 comprising a virostatic agent.

11. A pharmaceutical composition comprising the cytotoxic agent of claim 1 and a pharmaceutically acceptable carrier.

12. A method for the prophylaxis or treatment of a viral infection, comprising the administration to a living being in need in a therapeutically or prophylactically effective amount a cytotoxic agent, said cytotoxic agent is configured for the selective binding to a membrane receptor of virus infected T lymphocytes, wherein the membrane receptor is selected from the group consisting of: CD134, CD132, CD71, CD70, CD54, CD39, BTLA, CD97, CD2, CD63, CD50, CD161, CD218, CD226, CD7, CD49d, and CD29.

13. A method for the finding of cytotoxic agents, which comprises the selection of such compounds which can bind selectively to a membrane receptor of virus-infected T lymphocytes, wherein the membrane receptor is selected from the group consisting of: CD134, CD132, CD71, CD70, CD54, CD39, BTLA, CD97, CD2, CD63, CD50, CD161, CD218, CD226, CD7, CD49d, and CD29.

14. A method for the diagnosis of a viral infection in a living being, comprising the determination of an overexpression of a membrane receptor of virus-infected T lymphocytes over non-infected T lymphocytes in said living being, wherein the membrane receptor is selected from the group consisting of: CD134, CD132, CD71, CD70, CD54, CD39, BTLA, CD97, CD2, CD63, CD50, CD161, CD218, CD226, CD7, CD49d, and CD29.

15. A method for the determination of membrane receptors of virus-infected T lymphocytes, which are suitable as a target of cytostatic agents, comprising the infection of T lymphocytes with a virus and the determination of any overexpression of membrane receptors on the virus-infected T lymphocytes over non-infected T lymphocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG. 1A shows schematically the establishment of a screening method for the determination of surface structures in HIV-1 infected T lymphocytes, which is the basis for the finding of the cytotoxic agents according to the invention, and FIG. 1B shows FACS analysis of stained CD4+ T cells;

[0078] FIG. 2A shows the increase in expression of various membrane receptors on HIV-1 infected versus non-infected primary human CD4.sup.+ T lymphocytes, and FIG. 2B shows the increased expression of CD134 on CD4.sup.+ T lymphocytes isolated from HIV patients.

EXAMPLES

1. Screening of HIV-1 Infected T Lymphocytes

[0079] CD4+ T lymphocytes were isolated from the peripheral blood of healthy donors. The isolated CD4+ T lymphocytes were stimulated with phytohaemagglutinin (PHA) for three days. Subsequently, the cells so stimulated were infected with HIV-1 expressing CFP (‘cyan fluorescent protein’) via an internal ribosomal entry site (IRES) together with the nef open reading frame. The surface of the infected CD4+ T lymphocytes was stained for 48 to 72 hours after the infection with 332 PE conjugated antibodies presented in four 96 well micro titer plates. To switch off the autofluorescence, infected and non-infected cells were filtered against allophycocyanin (APC). For the analysis, the PE average values of infected CFP-positive cells and non-infected CFP-negative cells were used. The establishment of the receptor surface screening in HIV-1 infected CD4+ T lymphocytes is shown schematically in FIGS. 1A and 1B.

2. Overexpression of Membrane Receptors in HIV-1 Infected T Lymphocytes

[0080] 48 up to 72 hours after the infection of CD4+ T lymphocytes, the overexpression of membrane receptors that have already been described in the state of the art in connection with an HIV-infection, such as CD45RO, CD25, CD150 and CD279 is shown as expected. These results confirm the robustness and reliability of the developed screening method.

[0081] Surprisingly, however, the inventors were able to find a large number of other overexpressed membrane receptors on the infected cells, which are so far not described. The highest increase in cell surface expression was for CD134 (OX40), which is a T cell co-stimulatory molecule and cytokine receptor; see FIG. 2A.

[0082] The values indicate the X-fold increase in receptor expression on infected versus non-infected T lymphocytes (n=5, p value for all receptor molecules shown <0.01). Receptors for which an increase in the expression of CD4+ T lymphocytes in HIV infection has already been described are marked in black. For all other white marked receptors the overexpression in HIV infected CD4+ T lymphocytes was unknown and therefore surprising.

[0083] CD4+ T lymphocytes were isolated from the blood of HIV-1 infected individuals. The surface of the cells was analyzed for the expression of CD134 and the interior of the cell for p24 of HIV-1. The measurement was carried out by flow cytometry. The result is shown in FIG. 2B.

[0084] The graph shows the mean fluorescence intensity (MFI) of CD134 in p24− (not infected) versus p24+ (infected) T cells from ten patients. A statistical calculation was done with the paired student T test. It was thus confirmed that the CD134 expression also strongly increases on infected CD4+ T lymphocytes from the blood of HIV-1 patients.

[0085] Thus, based on the CD134 expression, an HIV-1 infected CD4+ T lymphocyte cell can be specifically distinguished from a non-infected cell. CD134 is therefore an ideal target for an anti-HIV-1 immunotherapy.

3. Cytotoxic Agent for the Treatment of an HIV Infection

[0086] At present, some antibodies against human CD134 are already available in the state of the art. An example of this is the antibody developed by the company MedImmune with the designation MEDI6469 or 9B12, respectively. This agonistic antibody is currently only used for the treatment of solid tumors. It has already been tested for safety and anti-tumor activity in humans (Curti et al., 2013, loc. cit.). The 9B12 antibody is currently being tested in a clinical trial to determine its suitability for tumor therapy. According to the inventors' findings, this antibody is also potentially suitable for eliminating virus-infected CD4+ T lymphocytes and thus removing the pathogenic viruses from the infected host.

[0087] The detection of the cytotoxic property is as follows:

[0088] PBMCs are isolated from the blood of healthy donors. The isolated cells are infected with HIV-1 which expresses GFP or CFP. This allows the identification of the infected cell population due to the expression of the chromophore. Various amounts of the anti CD134 antibody, for example MEDI6469 (9B12), are added. An anti-mouse PE is used as the secondary antibody to detect by flow cytometry cells that bind the antibody. This experiment allows to evaluate that the antibody specifically binds to HIV-1 infected cells due to the cellular CD134 expression and not to non-infected cells. It also allows to determine whether different doses have an influence on the level of specific or non-specific binding to HIV-1-infected and non-infected cells.

[0089] Next, for a period of 12 days, the viral replication and the amount of infected cells in the HIV-1 infected PBMC-population are observed. Different amounts of the anti-CD134 antibody are added to the infected cultures. At 2 days intervals, the absolute number of infected CD4+ T cells, non-infected CD4+ T cells, the absolute levels of CD3+ cells and of CD8+ T cells which represent cytotoxic T lymphocytes are quantified. The T cell activation and proliferation is analyzed by staining on CD69 and Ki67. The levels of monocytes are measured by the expression of the CD14 receptor.

[0090] In addition, the virus production is determined on a reporter cell line by quantifying the release of p24 into the supernatant and the amount of infectious, released virions. Controls include non-infected PBMC cultures with or without treatment with an anti-CD134 antibody and HIV-1 infected PBMC without any treatment. At least three different donors will be analyzed. These experiments allow the conclusion that the CD134 ligation on ex vivo infected primary cells has the potential to eliminate infected cells or suppress viral replication and that non-infected cells are not affected or eliminated by the treatment.

[0091] The efficiency of an anti-CD134 treatment can be verified in an established humanized mouse model for the HIV-1 infection. Such a mouse model is provided by the company “Transcure”. These mice are infected with HIV-1 and left untreated (n=5) or three different doses of the anti-CD134 antibody were administered (n=5, each dose) to ligate CD134 and specifically eliminate HIV-1 infected cells which express high levels of CD134. The viral load, CD4+ T lymphocyte counts and the percentage of infected cells are observed. Overall, these experiments show that the ligation of CD134 by an antibody directed against this, such as MEDI6469 (9612), has the potential to eliminate HIV-1 infected cells from an organism and thus represents a new anti-HIV-1 immunotherapy.