US-12622964-B2 - Modular phage vector platform for sonodynamic therapy

US12622964B2US 12622964 B2US12622964 B2US 12622964B2US-12622964-B2

Abstract

The present invention relates to a phage or a composition that comprises it, wherein said phage expresses at least one specific recognition element and is furthermore conjugated with at least 300 molecules of a sonosensitiser. The invention further relates to the use of said phage in a sonodynamic therapy.

Inventors

Alberto DANIELLI
Matteo Calvaresi
Matteo Di Giosia
Andrea Cantelli
Francesco Starinieri
Michela Nigro
Edoardo Sarti

Assignees

Alma Mater Studiorum—Universita' di Bologna

Dates

Publication Date: 20260512
Application Date: 20200626
Priority Date: 20190626

Claims (16)

1 . A method for treating a tumour or an infection, said method comprising administering to a subject a phage expressing at least one specific recognition element, said specific recognition element being conjugated with at least 300 molecules of a sonosensitiser or a composition comprising the phage and excipients and/or additional substances and/or pharmaceutically accepted carriers wherein the infection is selected from a bacterial, viral or fungal infection, and wherein the phage is administered in association or in combination with ultrasound.
2 . The method according to claim 1 , wherein said tumor is a malignant tumor and said infection is a bacterial infection.
3 . The method according to claim 2 , wherein the tumor is a tumor with overexpression of at least one structure selected from: EGFR, HER2 + , EGFRvIII, fibronectin, CD99, GD2 and combinations thereof.
4 . The method according to claim 1 , wherein the phage is administered in association or in combination with at least one antitumoral, or in association with at least one antibiotic.
5 . The method according to claim 1 , wherein the phage is administered in association or in combination with a treatment of a malignant tumor.
6 . The method according to claim 1 , wherein the phage is administered in before or after an operation to remove the malignant tumor.
7 . The method according to claim 1 , wherein the sonosensitiser is selected from: Bacteriochlorin, Phthalocyanines, Pheophorbide A and B, Bacteriopheophorbide, Naphthalocyanine, BODIPY stains, Anthracycline, Tetracycline, Fluoroquinolones, Phenothiazine, Perylene, Perylenequinone, Curcumin, Quinones, Anthraquinones, Fullerenes, Endohedral Metallofullerenes, Acridines, Isoquinole alkaloids, Xanthenes, Porphyrins, Chlorins and Cyanine.
8 . The method according to claim 1 , wherein the sonosensitiser is selected from: a xanthen, a porfirin, a chlorin, a cyanine, and combinations thereof.
9 . The method according to claim 1 , wherein the xanthen is selected from rose bengal, eosin, erythrosin B, fluorescein and rhodamine; the porphyirin is selected from protoporphyirin IX, hematoporphyirin, porfimer sodium and ATX-70; the chlorin is selected from chlorin e6, temoporfin and verteporfin; the cyanine is selected from cyanine IR-806, indocyanine green and cyanine IR-780, and combinations thereof.
10 . The method according to claim 1 , wherein said phage is conjugated with at least 400 sonosensitiser molecules.
11 . The method according to claim 1 , wherein the phage is selected from the M13 phage, fd phage, Ff phage, T4 and MS2 phage.
12 . The method according to claim 1 , wherein said specific recognition element is selected from: an antibody, a receptor, a protein, a peptide and combinations thereof.
13 . The method according to claim 1 , wherein said specific recognition element is selected from: EGFR, HER2 + , EGFRvIII, CD99, GD2, fibronectin, bacterial fimbriae and flagella and a peptide binding to the outer membrane of Gram-negative bacteria.
14 . The method according to claim 1 , wherein said phage is conjugated with at least 500 sonosensitizer molecules.
15 . The method according to claim 1 , wherein said phage is conjugated with at least 700 sonosensitizer molecules.
16 . The method according to claim 1 , wherein the phage is M13 phage.

Description

This application is a U.S. national stage of PCT/IB2020/056058 filed on 26 Jun. 20202 which claims priority to and the benefit of Italian patent application No. 102019000010131 filed on 26 Jun. 2019, and claims priority to and the benefit of Italian patent application No. 102019000022200 filed on 26 Nov. 2019, the content of which are all incorporated herein by reference in their entireties. FIELD OF THE INVENTION The present invention relates to a phage or a composition comprising it, wherein said phage expresses at least one specific recognition element and is furthermore conjugated with at least 300 molecules of a sonosensitiser. In addition, the invention relates to the use of said phage in a sonodynamic therapy. PRIOR ART Bacteriophages (phages) are ubiquitous viruses which infect bacteria while being inactive against eukaryotic cells. Phages are biocompatible, uniform in size and in morphology, and relatively stable at high temperatures, in a broad pH range. For this reason, phages, such as, for example, the M13 phage, have been receiving growing attention as an ordered protein platform for the assembly of functional molecules and nanostructured materials. Directing molecules and therapeutic effectors can be easily exposed or conjugated on their surface. The M13 phage can be directed at virtually any type of cell through the exposure of specific peptides or antibodies. Furthermore, its excellent safety profile makes it highly suitable for in vivo applications. US2007/0020241 describes bacteriophages conjugated to photosensitisers and said conjugate has shown to be effective in killing bacteria when irradiated with light of suitable wavelength. Li, K., et al., in Chemical Modification of M13 Bacteriophage and Its Application in Cancer Cell Imaging. Bioconjug. Chem. (2010) 21, 1369-1377, describe the conjugation of photoreactive molecules to the M13 phage, wherein a quenching effect is noted when more than 500 molecules are conjugated to the phage. Progress beyond phototherapy has been achieved with sonodynamic therapy (SDT), an approach that involves a combination of low-intensity ultrasound and specialised chemical agents known as sonosensitisers. Activation of the sensitiser by ultrasound generates reactive oxygen species (ROS) responsible for cytotoxicity. Being able to penetrate more deeply into tissues than light irradiation, ultrasound also enables treatment in deep regions. By way of example, ultrasound can be focused in a region of a tumour in order to activate a sonosensitiser, thus offering the possibility of striking tumours in a targeted manner. The available sonosensitisers include sonosensitisers based on porphyrins or on xanthens. Use has also been made of small molecules, including curcumin, indocyanine green (ICG), acridine orange, and hypocrellin B. One of the limits most strongly perceived with SDT is tied to the high ultrasonic powers that need to be deployed in order to obtain an effective activation of sonosensitisers. It is an object of the present invention to provide a system for an effective SDT. SUMMARY OF THE INVENTION A first aspect of the present invention relates to a phage conjugated with at least one sonosensitiser. The phage preferably expresses at least one specific recognition element adapted to favour binding of the phage with target cells, such as, for example, bacterial cells or tumour cells. A second aspect of the present invention relates to a composition comprising the phage and excipients and/or additional substances and/or pharmaceutically accepted carriers. A third aspect of the present invention relates to the phage or to the composition that comprises it for use as a medicament, preferably in a sonodynamic therapy (SDT). A fourth aspect of the present invention relates to the phage or to the composition that comprises it for use in the treatment or follow-up of a tumour, preferably a malignant tumour. A further aspect of the present invention relates to the phage or to the composition that comprises it for use in the treatment or prevention of an infection selected from a bacterial, viral or fungal infection, preferably a bacterial infection. A further aspect of the present invention relates to a method for the treatment or follow-up of a tumour, preferably a malignant tumour, or for the treatment or prevention of an infection. Said method comprises at least a step of administering an effective amount of a phage, or of a composition that comprises it as described in detail above, to an individual affected by a tumour or an infection. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the absorbance spectra of the a) wild-type M13 phage and conjugates b) phage-Rose Bengal, c) phage-Chlorin e6, d) phage Protoporphyrin IX, and e) phage-Cyanine IR-806. FIG. 2 shows the cytotoxic effect of phages conjugated with chlorin e6 (Ce6) on an epidermoid carcinoma A431 cell line in the presence of ultrasound radiation. FIG. 3 shows the cytotoxic effect of phages conjugated w