HIV infection blocking combination and method based on CD2 signaling pathway

Abstract

The present invention relates to compositions and methods for blocking viral replication (such as HIV-1 infection) using CD2 receptors. In one embodiment, viral target cells such as blood CD4 T cells can be made resistant to HIV by stimulating CD2 receptors using antibodies, ligands, or small molecules that bind to CD2 receptors. Pre-stimulation of CD2 can be used to enhance anti-HIV immunity and can be used to promote immune responses to HIV infection or anti-HIV vaccines.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Application No. 62 / 570,947, filed on October 11, 2017, the contents of which are hereby incorporated by reference in their entirety into this application. Technical Field

[0003] This statement relates to modulating cellular biological activity, including modulating the susceptibility of cells to bacteria and / or viruses, and delivering compounds and components (such as drugs, nucleic acids and proteins) into cells. Methods, compositions and the like for modulating cellular biological activity are provided herein. Background Art

[0004] CD2 (colony differentiation 2, also known as T11 / Leu-5, LFA-2, LFA-3 receptor, erythrocyte receptor and rosette receptor) is a cell co-stimulatory and surface adhesion molecule expressed on the surface of T cells and NK cells (natural killer cells), but not on B lymphocytes. Therefore, it is used to identify T and NK cells in immunohistochemistry. CD2 is expressed on all peripheral blood T cells and is one of the earliest T cell markers expressed by more than 95% of thymocytes. CD2 monoclonal antibodies inhibit the formation of sheep erythrocyte rosettes, suggesting that it may be an erythrocyte receptor or closely related to it.

[0005] CD2 also interacts with other adhesion molecules such as LFA-3 / CD58 (lymphocyte function-associated antigen-3). The extracellular domain of CD2 contains immunoglobulin-like domains that can mediate the formation of homodimers (Chen et al., 2002). CD2 binds to LEA-3 to mediate T cell adhesion to antigen-presenting cells. Similar CD2 stimulation can also initiate signal transduction pathways that regulate cofilin activity and the actin cytoskeleton and enhance T cell activation. However, CD2 knockout mice show basically normal immune function (Killeen et al., 1992), and it is believed that CD2 has a certain functional redundancy with other T cell co-stimulatory molecules such as CD28 (Hutchcroft et al., 1998). CD2 also interacts with other adhesion molecules such as LFA-3 / CD58 (lymphocyte function-associated antigen-3). The extracellular domain of CD2 contains immunoglobulin-like domains that can mediate the formation of homodimers (Chen et al., 2002). CD2 mediates T cell adhesion to antigen-presenting cells by binding to LEA-3. Similar CD2 stimulation can also initiate signal transduction pathways, regulate cofilin activity and the actin cytoskeleton, and enhance T cell activation. However, CD2 knockout mice have basically normal immune function (Killeen et al., 1992), and it is believed that CD2 has some overlap in function with other T cell co-stimulatory molecules such as CD28 (Hutchcroft et al., 1998). Summary of the invention

[0006] In one aspect, provided herein is a method for blocking HIV infection in a cell comprising pre-stimulating the CD2 receptor with an antibody, ligand or small molecule.

[0007] In one embodiment, the antibody is an anti-CD2 antibody selected from UMCD2, BTI-322, CB.219 and Siplizumab. In another embodiment, the ligand is selected from CD58, Alefacept and a protein that binds to CD2. In another embodiment, the small molecule is a small molecule that interacts with CD2 and acts as an agonist or antagonist. In another embodiment, the cell is a human cell.

[0008] In another aspect, a method for treating or preventing HIV infection is provided herein, comprising administering a combination of pre-stimulated CD2 receptors. In one embodiment, the combination comprises an antibody, a ligand or a small molecule. In one embodiment, the antibody is an anti-CD2 antibody selected from UMCD2, BTI-322, CB.219 and Siplizumab. In another embodiment, the ligand is screened from CD58, Alefacept and a protein that binds to CD2. In another embodiment, the small molecule is a small molecule that interacts with CD2, acting as an agonist or antagonist.

[0009] In another aspect, there is provided a combination for treating or preventing HIV infection, wherein the combination pre-stimulates the CD2 receptor. In one embodiment, the composition comprises an antibody, a ligand or a small molecule.

[0010] On the other hand, the present invention provides a method for blocking viral nuclear migration in immune cells, comprising an antibody, ligand or small molecule that pre-stimulates the CD2 receptor. In one embodiment, the antibody is an anti-CD2 antibody selected from UMCD2, BTI-322, CB.219 and Siplizumab. In another embodiment, it is screened from CD58, Alefacept and a protein that binds to CD2. In another embodiment, the small molecule is a small molecule that interacts with CD2 and acts as an agonist or antagonist. In another embodiment, the cell is a human cell.

[0011] In another aspect, there is provided a combination for degrading viral particles in a cell, wherein the combination pre-stimulates the CD2 receptor.

[0012] On the other hand, the present invention provides a method for enhancing anti-HIV immunity, comprising pre-stimulating the CD2 receptor with an antibody, a ligand or a small molecule. In one embodiment, the antibody is an anti-CD2 antibody selected from UMCD2, BTI-322, CB.219 and Siplizumab. In another embodiment, it is screened from CD58, Alefacept and a protein that binds to CD2. In another embodiment, the small molecule is a small molecule that interacts with CD2 and acts as an agonist or antagonist.

[0013] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, serve to explain the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Pre-stimulation of CD2 inhibits HIV infection of blood CD4 T cells.

[0016] Resting CD4 T cells were negatively selected from the peripheral blood of healthy donors. Cells were pre-stimulated overnight with anti-CD2 antibody-coupled magnetic beads (2 beads per cell). After stimulation, cells were infected with HIV-1 (NL4-3) for 2 h, washed, cultured for 5 days, and then activated with anti-CD3 / CD28 magnetic beads (4 beads per cell).

[0017] Figure 2 : Pre-stimulation of CD2 inhibits HIV infection of blood memory CD4 T cells R5.

[0018] Resting memory CD4 T cells (CD45RO+CD45RA-) were negatively selected from the peripheral blood of healthy blood donors. Cells were pre-stimulated overnight with anti-CD2 antibody-coupled magnetic beads (2 beads per cell). After stimulation, cells were infected with HIV-1 (AD8) for 2 h, washed, cultured for 5 days, and then activated with anti-CD3 / CD28 magnetic beads (4 beads per cell).

[0019] Figure 3 : CD58 pre-stimulation of CD2 inhibits HIV infection of blood CD4 T cells.

[0020] Resting CD4 T cells were negatively selected from the peripheral blood of healthy donors. Cells were pre-stimulated overnight with purified CD58 protein. After stimulation, cells were infected with HIV virus for 2 h, washed, cultured for 5 days, and then activated with anti-CD3 / CD28 microspheres (4 beads per cell).

[0021] Figure 4 : CD2 pre-stimulation does not inhibit T cell activation.

[0022] Resting CD4 T cells were negatively selected from the peripheral blood of healthy donors. Cells were pre-stimulated overnight with anti-CD2 antibody-coupled magnetic beads (2 beads per cell). After stimulation, cells were activated with anti-CD3 / CD28 microspheres (4 microspheres per cell). T cell activation was measured by upregulation of CD25 and CD69.

[0023] Figure 5 : CD2 stimulation does not suppress HIV infection of blood CD4 T cells.

[0024] Resting CD4 T cells were negatively selected from the peripheral blood of healthy donors. Cells were infected with HIV-1 (NL4-3) for 2 h, washed, and stimulated with anti-CD2 antibody-coupled magnetic beads (2 beads per cell). After stimulation, cells were cultured for 5 days and then activated with anti-CD3 / CD28 microspheres (4 beads per cell). DETAILED DESCRIPTION

[0025] Specific implementation cases will be illustrated with drawings and references.

[0026] The present invention relates to combinations and methods for blocking HIV-1 infection using CD2 receptors. In one embodiment, the inventors have demonstrated that by stimulating CD2 receptors using antibodies, ligands or small molecules that bind to CD2 receptors, virus target cells such as blood CD4 T cells can be made resistant to HIV virus.

[0027] The present invention clearly states that pre-stimulation of CD2 receptors can prevent viral replication in immune cells. Therefore, it may be used to treat HIV infection and may also promote the degradation of viral particles in cells and promote the antigen presentation process. Pre-stimulation of CD2 can be used to enhance anti-HIV immunity and can be used to promote immune responses to HIV infection or anti-HIV vaccines.

[0028] All technical terms in the present specification are commonly used in biochemistry, molecular biology and immunology, and can be understood by those skilled in the art in the field of the present invention. These technical terms can be found in: MOLECULAR CLONING:A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; CURRENT PROTOCOLS INMOLECULAR BIOLOGY, ed. Ausubel et al., Greene Publishing Associates andWiley-Interscience, New York, 1988 (with periodic updates); SHORT PROTOCOLSIN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM CURRENT PROTOCOLS INMOLECULAR BIOLOGY, 5.sup.th ed., vol. 1-2, ed. Ausubel et al., John Wiley&Sons, Inc., 2002; GENOME ANALYSIS: A LABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997; CELLULAR AND MOLECULAR IMMUNOLOGY, 4.sup.th ed. Abbas et al., WB Saunders, 1994.

[0029] The combination indicated herein can be introduced into the body by any effective route, including but not limited to oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard route), ocular, nasal, topical, and non-oral routes such as aerosol spray, inhalation, transdermal (epidermal), subcutaneous, intravenous, intramuscular, buccal, sublingual, rectal, vaginal, arterial, mucosal, intrathecal, etc. The combination can be used alone or in combination with any active or inactive ingredients.

[0030] Any individual can receive the combination of the antibody, ligand and / or a small molecule, including those who have been exposed to HIV but have not yet developed HIV infection, as well as those who have developed HIV infection or have progressed to the point of developing one or more clinical symptoms of HIV infection (e.g., AIDS). In addition to treating and / or preventing human infection with HIV, this composition can also be used to treat other organisms (e.g., non-human primates, cats, etc.) infected with HIV or HIV-related viruses (such as SIV, SHIV or FIV). Therefore, the subjects that can be treated include mammals, humans, monkeys, apes, chimpanzees, gorillas, cats, dogs, mice, rats, etc.

[0031] The combinations of antibodies, ligands, and / or small molecules described herein can be used to treat and / or prevent infections caused by HIV virus types, including but not limited to HIV-1 (e.g., A, B, C, D and G subtypes, R5 and R5X4 viruses, etc.), HIV-2 (e.g., R5 and R5X4 viruses, etc.), simian immunodeficiency virus (SIV), simian / human immunodeficiency virus (SHIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) (Wright et al., Vet. Res. Commun., 26:239-50, 2002), HTLV-1 and HTLV-2, etc.

[0032] Priming involves exposing the CD2 receptor to antibodies, ligands, or small molecules prior to exposing the cells to viral infection, such as HIV.

[0033] Post-stimulation refers to exposing the cells to viral infection and then exposing CD2 on the cells to antibodies, ligands or small molecules.

[0034] The antibody refers to an antibody that pre-stimulates the CD2 receptor, such as an anti-CD2 antibody. In some embodiments, the anti-CD2 antibody is selected from UMCD2 (an anti-human CD2 monoclonal antibody), BTI-322 (BTI-322 is a monoclonal anti-CD2 antibody used to treat steroid-resistant acute graft-versus-host disease), CB.219 (monoclonal anti-human CD2 antibody); and Siplizumab (monoclonal anti-human CD2 antibody).

[0035] A ligand is any molecule that binds to the CD2 receptor. One specific ligand is CD58. In some embodiments, the ligand is Alefacept (Alefacept is a fusion protein that binds to the first extracellular domain of LFA3 (CD58, which has constant regions (CH2 and CH3) and the hinge domain of human IgG1). This hybrid molecule can bind to CD2 to treat psoriasis vulgaris).

[0036] Small molecules refer to small molecules that interact with CD2 and act as agonists or antagonists.

[0037] Illustrative examples are listed below. They are typical but not exclusive.

[0038] Case 1: Pre-stimulation of CD2 inhibits HIV infection of blood CD4 T cells

[0039] like Figure 1 As shown, pre-stimulation with CD2 inhibits HIV infection of blood CD4 T cells. Resting CD4 T cells were negatively selected from the peripheral blood of healthy donors. Cells were pre-stimulated overnight with anti-CD2 antibody-coupled magnetic beads (2 beads per cell). After stimulation, cells were infected with HIV-1 (NL4-3) for 2 h, washed, cultured for 5 days, and then activated with anti-CD3 / CD28 magnetic beads (4 beads per cell).

[0040] CD2-mediated inhibition was not limited to CXCR4-specific cytotoxicity (X4). When we tested CCR5 (R5)-specific cytotoxicity HIV-1 (Ad8) infection of memory CD4 T cells, we observed similar inhibition of HIV-1 (Ad8), see Figure 2 In addition, we observed similar inhibitory effects on HIV infection when CD4 T cells were pre-stimulated with the CD2 ligand LFA-3 (CD58), see Figure 3 .

[0041] Case 2: Pre-stimulation of CD2 inhibits R5-phagocytic HIV-infected blood memory CD4 T cells

[0042] like Figure 2 As shown, CD2 pre-stimulation inhibits R5-phagocytic HIV infection of blood memory CD4 T cells. Resting memory CD4 T cells (CD45RO+CD45RA-) were negatively selected from the peripheral blood of healthy donors. Cells were pre-stimulated overnight with anti-CD2 antibody-coupled magnetic beads (2 beads per cell). After stimulation, cells were infected with HIV-1 (AD8) for 2 h, washed, cultured for 5 days, and then activated with anti-CD3 / CD28 magnetic beads (4 beads per cell).

[0043] Case 3: Pre-stimulation of CD2 with CD58 inhibits HIV infection of blood memory CD4 T cells

[0044] like Figure 3As shown, CD2 pre-stimulation with CD58 inhibits HIV infection of blood CD4 T cells. Resting CD4 T cells were negatively selected from the peripheral blood of healthy donors. Cells were pre-stimulated with purified CD58 protein overnight. After stimulation, cells were infected with HIV virus for 2 h, washed, cultured for 5 days, and then activated with anti-CD3 / CD28 magnetic beads (4 beads per cell).

[0045] The inhibition of HIV infection by CD2 stimulation is not due to the possible inhibition of T cell activation or cytotoxicity. Figure 4 As shown, we did not observe a defect in T cell activation when CD2-stimulated resting CD4 T cells were activated using anti-CD3 / CD28 beads. Figure 5 As shown in the figure, resting CD4 T cells infected with HIV-1 for 2 hours and then stimulated with anti-CD2 antibodies had no inhibitory effect on HIV-1 infection. These results suggest that CD2 stimulation can specifically block early viral infection processes, such as viral nuclear migration. It is possible that CD2 signaling alters the cofilin pathway and inhibits viral nuclear migration, as it has been reported that HIV-mediated CXCR4 / cofilin activation is required for viral nuclear migration (Yoder et al., 2008).

[0046] Case 4: cofilin phosphorylation and dephosphorylation levels as a measure of stimulation

[0047] Quantification uses the levels of cofilin phosphorylation and dephosphorylation to measure the extent of stimulation. Cofilin is inactivated by phosphorylation at the Ser-3 site and then reactivated by dephosphorylation. Protein phosphorylation can be measured by any known technique, including but not limited to radioactive 32P radiolabeling, molybdate-based colorimetric assays, immunoblotting or ELISA with phospho-specific antibodies, phospho-staining reagents, and various mass spectrometry and flow cytometry methods.

[0048] References

[0049] Chen, HA, Pfuhl, M., and Driscoll, PC (2002). The pH dependenceof CD2 domain 1 self-association and 15N chemical exchange broadening iscorrelated with the anomalous pKa of Glu41. Biochemistry 41, 14680-14688.

[0050] Hutchcroft, JE, Slavik, JM, Lin, H., Watanabe, T., and Bierer, BE (1998). Uncoupling activation-dependent HS1 phosphorylation from nuclearfactor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 andCD28. J Immunol 161, 4506-4512.

[0051] Killeen, N., Stuart, SG, and Littman, DR (1992). Development and function of T cells in mice with a disrupted CD2 gene. EMBO J 11, 4329-4336.

[0052] Yoder, A., Yu, D., Dong, L., Iyer, SR, Xu,

[0053] Other exemplary embodiments will be apparent to those skilled in the art. The specifications and embodiments of the present invention are to be considered as exemplary only. The true scope and inventive spirit of the present invention are indicated by the following claims.

Claims

1. A non-therapeutic method for blocking HIV infection of healthy cells, comprising pre-stimulating CD2 receptors with CD2 ligands; The CD2 ligand is CD58 and / or Alefacept.

2. The method of claim 1, wherein the cell is a human cell.

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