(12) Patent Application Publication (10) Pub. No.: US 2016/0346380 A1 Stenler Et Al
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US 2016.0346.380A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0346380 A1 Stenler et al. (43) Pub. Date: Dec. 1, 2016 (54) T20 CONSTRUCTS FOR ANTI-HIV (HUMAN (52) U.S. Cl. IMMUNODEFICIENCY VIRUS) THERAPY CPC ............... A61K 39/21 (2013.01); A61K 45/06 AND/OR VACCINES (2013.01); A61K 39/42 (2013.01); A61 K (71) Applicant: Immunomedics, Inc., Morris Plains, NJ 2039/53 (2013.01) (US) (57) ABSTRACT (72) Inventors: Sofia Stenler, Stockholm (SE); Britta Wahren, Stockholm (SE): Chien-Hsing The present invention concerns methods and compositions SS SSA S.sid for treatment of HIV infection using a T20 expression 9, s vector, such as that shown in SEQ ID NO:1 or SEQ ID (21) Appl. No.: 15/164,437 NO:3. The T20 expression vector may be used in a variety of therapeutic applications, such as ex vivo transfection of (22) Filed: May 25, 2016 dendritic cells to induce a host immune response to HIV. Related U.S. Application Data localized transfection in vivo in a gene therapy approach to (60) Provisional application No. 62/167,404, filed on May provide longer term delivery of T20, or in vitro production 28, 2015 s w is of T20 peptide. The T20 may be secreted into the circulation s to act as a fusion inhibitor of HIV infection, or may induce Publication Classification an endogenous immune response to HIV or HIV-infected (51) Int. Cl cells. Alternatively, a DDD peptide may be incorporated in A. iK 39/21 (2006.01) a fusion protein comprising T20 or another antigenic protein A6 IK 39/42 (2006.01) or peptide to enhance the immune response to the protein or A6 IK 45/06 (2006.01) peptide. Patent Application Publication Dec. 1, 2016 Sheet 1 of 5 US 2016/0346380 A1 FIG. I. P W cell lysate MC control Xe control MC Patent Application Publication Dec. 1, 2016 Sheet 2 of 5 US 2016/0346380 A1 FIG 2 Serungsteerina Serr N terra deuxsobiuunues 3§«)*lae, ,"". Patent Application Publication Dec. 1, 2016 Sheet 3 of 5 US 2016/0346380 A1 £(91H. Patent Application Publication Dec. 1, 2016 Sheet 4 of 5 US 2016/0346380 A1 eu?T?seg----- 0 e UO OU Patent Application Publication Dec. 1, 2016 Sheet 5 of 5 US 2016/0346380 A1 e Oc poli paepag$1 uo??estunusuartronesauruuuunuomgæstunuucua o o ||?ununuup e re 93 L JUO OU US 2016/0346380 A1 Dec. 1, 2016 T20 CONSTRUCTS FOR ANTI-HIV (HUMAN expression systems, although performance for in vitro use is IMMUNODEFICIENCY VIRUS) THERAPY also improved (Argyros et al., 2011, J Mol Med (Berlin) AND/OR VACCNES 89:515-29). 0007 Minicircle production typically involves produc RELATED APPLICATIONS tion of a “parental plasmid' and induction of the activity of a site-specific recombinase to excise the prokaryotic vector 0001. This application claims the benefit under 35 U.S.C. sequences (Chen et al., 2003, Mol Ther 8:495-500). The 119(e) of U.S. Provisional Patent Application 62/167,404, resulting minicircles may be recovered by a variety of filed May 28, 2015, the text of which is incorporated herein techniques. Early versions of minicircles lacked an origin of by reference in its entirety. replication and were therefore lost as cell division occurred. More recently, self-replicating minicircles comprising an SEQUENCE LISTING S/MAR (scaffold/matrix attachment region) element have been designed (e.g., Argyros et al., 2011, J Mol Med 0002 The instant application contains a Sequence Listing 89:515-29). Self-replicating minicircles may allow pro which has been submitted in ASCII format via EFS-Web and longed peptide expression from transfected cells. A need is hereby incorporated by reference in its entirety. Said exists for improved minicircle vectors for more effective ASCII copy, created on May 19, 2016, is named therapeutic use. IMM360US1 SL.txt and is 14,349 bytes in size. 0008 T20 (enfuvirtide) is the first HIV fusion inhibitor approved for therapeutic human use (Morris & Kraus, 2005, BACKGROUND OF THE INVENTION J Pediatr Pharmacol Ther 10:215-470. HIV-1 infection is initiated by binding of the gp120 envelope protein to its CD4 0003 Field of the Invention cell surface receptor and a coreceptor (CXCR4 or CCR5) 0004. The present invention concerns methods and com (Martinez-Munoz et al., 2014, Proc. Natl Acad Sci USA positions for treating human immunodeficiency virus (HIV). 111:E1960-69). Binding of gp120 is followed by the release either prophylactically or post-infection. The compositions of and conformation changes in the associated viral trans and methods relate to a T20 minicircle or vaccines com membrane gp41 subunit, which is required for membrane prising a T20 minicircle. A novel T20 minicircle construct is fusion with the host cell (Cai et al., 2011, Curr Top Med provided, comprising the nucleic acid sequence of SEQ ID Chem 11:2959-84). Enfluvirtide works by targeting a con NO:1. The T20 minicircle may be used for anti-HIV therapy formational transition in gp41, preventing creation of an (e.g., enfuVirtide), gene therapy or viral eradication in entry pore for the viral capsid (Cai et al., 2011, Curr Top infected hosts, or for production of anti-HIV vaccines or Med Chem 11:2959-84). Because it is a peptide with poor vaccine enhancement in non-infected hosts. T20 may be oral availability, enfuvirtide is typically administered by used alone or in combination with one or more anti-HIV Subcutaneous injection, after reconstitution by the patient. agents, as discussed below. In some embodiments T20 may Due to the chronic nature of the treatment, the difficulty of be expressed as an unconjugated peptide. Alternatively a administration contributes to poor patient compliance fusion protein or peptide comprising T20 attached to another (Home et al., 2009, AIDS Res Ther 6:2). A need exists for therapeutic peptide may be used. All Such peptide products better T20 vectors that will allow improved means of T20 may be expressed from a minicircle as disclosed herein, or administration. the minicircle itself may be utilized for therapeutic purposes. Enfluvirtide has been used for salvage therapy in patients SUMMARY OF THE INVENTION with multi-drug resistant HIV, alone or in combination with 0009. The present invention fulfills an unresolved need in other anti-viral agents. In certain embodiments, adjuvant the art by providing T20 minicircle (MC) expression vec agents may be utilized to increase the immune response to tors, encoding the production of the T20 HIV fusion inhibi T20 or other antigens. An exemplary antigen is a DDD tor. An exemplary T20 minicircle DNA sequence is shown (dimerization and docking domain) peptide, as disclosed in SEQ ID NO:1. The amino acid sequence of T20 is herein. provided in SEQ ID NO:2. The MC construct exhibits 0005. Description of Related Art several advantages compared to traditional expression vec 0006 Minicircles are episomal DNA vectors that are tors that comprise Substantial amounts of prokaryotic produced as Small (~4 kb) circular expression cassettes, nucleic acid sequences, such as decreased immunogenicity, derived from plasmids but devoid of any prokaryotic DNA a lower potential for inducing host immune response, greater (see, e.g., Wikipedia "Minicircle'). Minicircles have been stability of the construct in host cells, prolonged expression used as transgene carriers for genetic modification of mam and increased production of T20 peptide. malian cells (Id.). Their smaller molecular size enables more (0010. The T20 MC constructs are of use for prevention of efficient transfections and offers Sustained expression over a HIV infection, for example as components of vaccines or period of weeks as compared to standard plasmid vectors vaccine adjuncts, or for treatment of existing HIV infection, that only work for a few days (see, e.g., Kay et al., 2010, for example by administration of expressed T20 peptide or Nature Biotech 28:1287-89). Thus, minicircles may be used by incorporation of a T20 MC expression vector into host for direct transfection of host cells, or may be used for cells for protein expression in vivo. production of cloned peptides which in turn may be used as (0011. The T20 peptide or T20 MC vector may be used therapeutic agents and/or for vaccination. Since minicircles alone or in combination with one or more other known contain no bacterial DNA sequences, they are less likely to anti-HIV agents, selected from the group consisting of be recognized by the host immune system and destroyed fusion inhibitors (e.g., enfuVirtide, maraviroc), integrase (Argyros et al., 2011, J Mol Med (Berlin) 89:515-29). They inhibitors (e.g. raltegravir, elvitegravir, dolutegravir), are therefore more suitable for in vivo use than standard reverse transcriptase inhibitors (e.g., KRV2110, zidovudine, US 2016/0346380 A1 Dec. 1, 2016 abacavir, emitricitabine, tenofovir, etravirine, rilpivirine, DESCRIPTION OF ILLUSTRATIVE didanosine, Zalcitabine, lamivudine, stavudine, nevirapine, EMBODIMENTS efavirenz), protease inhibitors (e.g., saquinavir, indinavir, ritonavir, lopinavir, nelfinavir, amprenavir, darunavir, ataza 0020 All documents, or portions of documents, cited in navir) and anti-HIV antibodies or other binding molecules this application, including but not limited to patents, patent (e.g., P4/D10, 2G12, 2F5, 4E10, Hippeastrum hybrid agglu applications, articles, books, and treatises, are hereby tinin (HHA)). Combination therapy with T20 (enfuvirtide) expressly incorporated by reference in their entirety. has been reported to decrease viral load to below-detectable levels in as many as 90 to 95% of treated patients (see, e.g., DEFINITIONS McGillicket al., 2010, Biochem 49:3575-92). It is generally 0021. As used herein, “a” or “an may mean one or more acknowledged that combination therapy is more effective than one of an item. than single agent therapy in controlling HIV infection and 0022.