Use of biologically active HIV-1 Tat, fragments or derivatives thereof for preventing or therapeutic vaccination and/or treating other diseases

A biologically active, derivative technology for targeting and/or activating antigen-presenting cells in biologically active HIV-1 T at, fragments or derivatives thereof for prophylactic or therapeutic immunization and/or treatment of other diseases, and/or the area of ​​use of the transported cargo molecules, able to resolve issues such as no claim for protection

Inactive Publication Date: 2004-09-29
INST SUPERIORE DI SANITA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The following were unknown to us at the time of filing of said patent application: a small (picomolar-nanomolar) amount comprising the RGD domain, biologically active Tat or a fragment or derivative thereof, (i) specifically targets APC, and We therefore may not claim its use as a carrier to selectively deliver cargo molecules to them; (ii) cause EC and DC cell maturation and activation and induce Th-1 type immune responses against different antigens, and therefore we may not claim Their use as adjuvants and immunomodulators

Method used

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  • Use of biologically active HIV-1 Tat, fragments or derivatives thereof for preventing or therapeutic vaccination and/or treating other diseases
  • Use of biologically active HIV-1 Tat, fragments or derivatives thereof for preventing or therapeutic vaccination and/or treating other diseases
  • Use of biologically active HIV-1 Tat, fragments or derivatives thereof for preventing or therapeutic vaccination and/or treating other diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0389] Active Tat is efficiently taken up by MDDCs but not TCBs and BLCLs

[0390] Uptake of active Tat by MDDCs, TCBs, and BLCLs was assessed by intracellular immunofluorescence in flow cytometry using specific affinity-purified polyclonal antibodies on permeabilized cells. Figure 1 shows the results for a representative donor whose Tat uptake level represents the median value obtained from the 14 donors tested. Tat uptake by MDDCs is highly efficient and occurs in a dose- and time-dependent manner (attached Figure 1A ). Uptake of Tat was already evident with the lowest dose (0.1 ng / ml). Regardless of the Tat concentration tested, the level of staining always peaked after 5 min of incubation and diminished after 60 min, likely due to protein processing. However, high levels (98%) of Tat uptake remained at the highest Tat dose used (10 μg / ml) up to 60 minutes of incubation. No staining was observed for cells incubated with medium or reconstitution buffer alone (attached F...

Embodiment 2

[0394] The uptake of active Tat by MDDCs increases with cell maturation and is lost due to protein oxidation and inactivation.

[0395] Immature MDDCs take up antigens through phagocytosis and pinocytosis (Bankereau 1998; Bell 1999). Mature DCs lose these activities despite their acquisition of strong antigen-presenting capacity. To verify whether cell maturation affects the uptake of natural Tat, LPS was used to induce MDDC maturation. Compared with immature MDDC, mature MDDC expressed higher levels of surface markers HLA-DR, CD83 and CD86. Either immature or mature cells were subsequently used for uptake assays. Tat uptake was greatly enhanced by MDDC maturation at all protein concentrations tested (attached Figure 2A ). Indeed, culture of mature MDDCs with low Tat concentrations showed similar levels of intracellular staining as observed for immature cells at the highest Tat dose (attached Figure 1A and 2A ).

[0396] Tat contains seven cysteines and is extremely se...

Embodiment 3

[0408] Blockade of active Tat uptake by MDDCs with anti-α5β1 and anti-αvβ3 monoclonal antibodies or their natural ligands FN or VN.

[0409] To verify whether the uptake of Tat by MDDC is subject to the specific receptor-mediated endocytosis pathway, specific monoclonal antibodies or competitor ligands against integrin receptors α5β1 and αvβ3 were tested. These receptors are selected from several receptors that bind Tat on different cell types because they are highly expressed by activated endothelial cells and KS cells that proliferate, migrate and adhere in the presence of Tat and this is regulated by the TatRGD domain Mediated by binding to α5β1 and αvβ3 integrins. as attached Figure 3A , B, both anti-α5β1 and anti-αvβ3 mAbs inhibited the uptake of Tat by MDDCs, and this blockade was complete in the presence of both antibodies (attached Figure 3A , B). The natural ligands for these receptors, FN and VN, similarly block Tat uptake (attached Figure 4A , B). Thus, α5β1...

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Abstract

The present invention concerns a method for prophylactic and / or therapeutic vaccination and / or treatment and / or diagnosis of HIV / AIDS, other infectious diseases, inflammatory and angiogenic diseases and tumours which utilizes a biologically active HIV-1 Tat protein, fragments or derivates thereof, as a module with one or more of the following features: antigen, adjuvant and targeting-delivery system to specific antigen-presenting cells including dendritic cells, endothelial cells and macrophages. In particular, it is claimed that Tat can be used only in its biologically active form as an antigen, alone or combined with one or more other antigens, to prime or to boost protective immune responses against itself as well as other antigens and / or to deliver these antigen(s) as well as active compounds to dendritic cells, endothelial cells and macrophages, due to its capability of targeting these APC and of activating their maturation and functions and of increasing Th-1 type immune responses as an adjuvant. Therefore, due to these characteristics and to the distribution of these cells in the body (during physiological and pathological disorders), biologically active Tat, fragments or derivates thereof containing the RGD region, can be used for preventive, therapeutic and / or diagnostic purposes for HIV / AIDS, other infectious diseases, inflammatory and angiogenic diseases and tumors.

Description

field of invention [0001] The present invention is based on the novel and unexpected discovery of small amounts (picomole-nanomolar) of native, substantially monomeric, biologically active HIV-1 Tat, fragments or derivatives thereof, which (i) are selected for by RGD domain-specific binding α5β1 and integrins expressed on antigen-presenting cells (hereinafter referred to as APCs) such as dendritic cells, macrophages and endothelial cells activated by cytokines, (ii) effectively enter APCs, (iii) promote dendritic cell and endothelial cell maturation and activation, and (iv) entry into class I and class II major histocompatibility complex-restricted antigen presentation pathways. Accordingly, the present invention seeks to exploit these novel discoveries of the aforementioned inherent and interrelated properties of biologically active HIV-1 Tat, fragments or derivatives thereof as antigenic and therapeutic properties for the treatment of certain human diseases (hereinafter coll...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K31/711A61K35/74A61K35/76A61K38/00A61K39/00A61K39/02A61K39/04A61K39/12A61K39/39A61K39/395A61K45/00A61K48/00A61P1/00A61P1/04A61P3/10A61P5/14A61P9/00A61P9/10A61P11/00A61P13/02A61P15/00A61P17/00A61P17/06A61P19/00A61P19/02A61P25/00A61P27/02A61P27/06A61P29/00A61P31/00A61P31/04A61P31/06A61P31/12A61P31/16A61P31/22A61P33/00A61P35/00A61P37/02A61P37/08C07K14/16
CPCA61K2039/53A61K2039/5154C12N2740/16322A61K2039/55516C07K14/005A61K39/39A61P1/00A61P1/04A61P11/00A61P13/02A61P15/00A61P17/00A61P17/06A61P19/00A61P19/02A61P25/00A61P27/02A61P27/06A61P29/00A61P3/10A61P31/00A61P31/04A61P31/06A61P31/12A61P31/16A61P31/18A61P31/22A61P33/00A61P35/00A61P37/02A61P37/04A61P37/08A61P5/14A61P9/00A61P9/10Y02A50/30
Inventor B·恩瑟里
Owner INST SUPERIORE DI SANITA
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