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BR-122026002529-A2 - Bonding molecule, isolated nucleic acid molecule, and composition.

BR122026002529A2BR 122026002529 A2BR122026002529 A2BR 122026002529A2BR-122026002529-A2

Abstract

This document provides compositions and methods for the repurposing of recombinant viral capsid proteins/capsids/vectors, for example, in vivo, with a multispecific binding molecule, such as a bispecific antibody, that binds specifically to a heterologous epitope displayed by the capsid protein and a protein expressed in the cell of interest for the targeted delivery of a nucleotide of interest.

Inventors

  • Christos Kyratsous
  • ANDREW J. MURPHY
  • Cheng Wang
  • Leah Sabin

Assignees

  • REGENERON PHARMACEUTICALS, INC.

Dates

Publication Date
20260310
Application Date
20180627
Priority Date
20170627

Claims (17)

  1. 1. Binding molecule, characterized in that it comprises a paratope that binds to SEQ ID NO:6, wherein the antibody paratope comprises HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3 sequences of an scFv comprising the amino acid sequence as set out in SEQ ID NO: 37.
  2. 2. Isolated nucleic acid molecule encoding the linker molecule as defined in claim 1, characterized by comprising the nucleic acid sequence as set forth as SEQ ID NO:28.
  3. 3. Binding molecule according to claim 1, characterized in that the antibody paratope is an Fv domain.
  4. 4. Linking molecule according to claim 3, characterized in that the Fv domain is directly fused to a first constant domain of the heavy chain.
  5. 5. Binding molecule according to claim 4, characterized in that the binding molecule comprises a bispecific antibody, and the bispecific antibody further comprises a redirecting linker comprising a distinct Fv domain fused to a second constant domain of the heavy chain.
  6. 6. A binding molecule according to claim 5, characterized in that the first and second constant domains of the heavy chain bind to protein A with differential binding affinities.
  7. 7. Binding molecule according to claim 1, characterized in that the binding molecule is a bispecific antibody comprising the antibody paratope and a redirecting ligand, wherein the redirecting ligand comprises a tetrameric antibody structure comprising two identical immunoglobulin (Ig) heavy chains and two identical Ig light chains, and the antibody paratope is fused to the C-terminal or N-terminal of one or both Ig heavy chains and/or to the C-terminal or N-terminal of one or both Ig light chains.
  8. 8. Binding molecule according to claim 7, characterized in that the antibody paratope is an Fv domain.
  9. 9. Binding molecule according to claim 8, characterized in that the paratope of the antibody is a scFv.
  10. 10. Linking molecule according to claim 9, characterized in that the scFv comprises the amino acid sequence as set forth in SEQ ID NO: 37.
  11. 11. A linking molecule according to any one of claims 1 to 10, characterized in that the linking molecule further comprises a redirecting ligand.
  12. 12. Binding molecule according to claim 11, characterized in that the redirecting ligand binds specifically to a protein or cell surface marker expressed by a target cell.
  13. 13. A binding molecule according to claim 11 or 12, characterized in that the redirecting ligand binds specifically to asialoglycoprotein 1 (ASGR1), CD3, GCGR, ENTPD3, or CD71 (transferrin receptor).
  14. 14. A linking molecule according to claim 11 or 12, characterized in that the redirecting ligand specifically binds to a protein or cell surface marker expressed by a liver cell, a brain cell, a neuronal cell, a T cell, a kidney cell, an intestinal cell, a pancreatic cell, a cancer cell, a muscle cell, or a cell infected with a heterologous pathogen.
  15. 15. A linking molecule according to any one of claims 11 to 14, characterized in that the redirecting ligand specifically binds to a protein or cell surface marker expressed by a human cell.
  16. 16. A linking molecule according to claim 11 or claim 12, characterized in that: (i) the target cell is a human liver cell, wherein the redirecting ligand binds to the human asialoglycoprotein receptor 1 (hASGR1) expressed by the human liver cell; (ii) the redirecting ligand binds to the GABA receptor expressed by a human neuronal cell; (iii) the redirecting ligand binds to the transferrin receptor expressed by a human neuronal cell; (iv) the target cell is a human T cell, wherein the redirecting ligand binds to CD3 expressed by the human T cell; (v) the target cell is a human T cell, wherein the redirecting ligand binds to CD3ε expressed by the human T cell; (vi) the target cell is a human hematopoietic cell, wherein the redirecting ligand binds to CD34 expressed by the hematopoietic cell; (vii) the target cell is a human cancer cell, wherein the redirection ligand binds to a tumor-associated antigen expressed by the human cancer cell; (viii) the target cell is a human cancer cell, wherein the redirection ligand binds to a tumor-associated antigen selected from the group consisting of E6, E7, and Her2; (ix) the redirection ligand binds to the human glucagon receptor (hGCGR); or (x) the target cell is a human intestinal cell or a human pancreatic cell, wherein the redirection ligand binds to human ENTPD3 expressed by the human intestinal cell or the human pancreatic cell.
  17. 17. Composition, characterized by comprising: (a) a recombinant adeno-associated virus (AAV) vector comprising an AAV capsid encapsulating a nucleotide of interest, wherein the AAV capsid comprises a recombinant AAV capsid protein modified to comprise a heterologous epitope, wherein the heterologous epitope comprises an amino acid sequence EQKLISEEDL (established as SEQ ID NO:6), and wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope; and (b) the linking molecule as defined in any one of claims 1-16, wherein the AAV capsid exhibits: (i) reduced or abolished tropism in the absence of the linking molecule; (e) and (j) modified tropism, after combination with the binding molecule, compared to a reference AAV capsid, wherein the reference AAV capsid comprises a reference AAV capsid protein that is identical to the recombinant AAV capsid protein except for the absence of the heterologous epitope.

Description

Reference to a list of sequences sent as a text file via the EFS network. [001] The sequence listing, written in the file 10335WO01_ST25.txt with 88 kilobytes, was created on June 27, 2018 and is incorporated herein by reference. TECHNICAL FIELD [002] The disclosure in this document refers generally to recombinant viral vectors modified for tropism and compositions comprising the same, useful for the targeted introduction of genetic material into cells and/or tissues. FUNDAMENTALS OF THE INVENTION [003] Gene delivery to specific target cells has become one of the most important technologies in modern medicine for the diagnosis and gene therapy of a variety of chronic and genetic diseases. To date, progress in the clinical application of gene therapy has been limited by the lack of ideal gene delivery vehicles. To achieve therapeutic success, gene delivery vehicles must be able to transduce target cells while avoiding transduction of non-target cells. Specifically, when the native tropism of the virus does not meet immediate therapeutic needs, there is a need for recombinant viral vectors in which the natural tropism is removed or diminished and the desired tropism is successfully manipulated. (Buchholz et al.,) [004] In recent years, most progress in vector development has been achieved using naked viruses (i.e., viruses comprising a capsid formed by viral capsid proteins without an envelope (e.g., lipid bilayer)), such as adeno-associated viruses (AAVs) and adenoviruses (Ads), as well as enveloped viruses (i.e., viruses for which the capsid is surrounded by a lipid bilayer) such as retroviruses, lentiviruses, and herpes simplex viruses. AAV-based vectors have been the focus of much research, as AAVs are non-enveloped viruses that are only mildly immunogenic, yet capable of transducing a wide range of species and tissues in vivo without evidence of toxicity. [005] AAVs are small, non-enveloped, single-stranded DNA viruses. The AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames that encode the Rep and Cap proteins, respectively. The two ITRs are the only cis elements essential for AAV replication, packaging, and integration. The Rep reading frame encodes four proteins with molecular weights of 78 kDa, 68 kDa, 52 kDa, and 40 kDa. These proteins function primarily in regulating AAV replication and integration into the chromosomes of a host cell. The Cap reading frame encodes three structural viral proteins (VPs) (capsids) with molecular weights of 83–85 kDa (VP1), 72–73 kDa (VP2), and 61–62 kDa (VP3). Over 80% of the total proteins in AAV virions comprise VP3; In mature virions, VP1, VP2, and VP3 are found in relative abundances of approximately 1:1:10. In vitro, the three proteins spontaneously assemble into virion-like structures, for example, viral capsids. It therefore appears that the formation of viral capsids in infected cells proceeds independently of viral DNA synthesis (reviewed by Kotin et al. (1994) Hum. Gene Ther. 5:793). [006] Among all known AAV serotypes, AAV2 is perhaps the best characterized serotype, because its infectious clone was the first to be made. (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA 79:2077-2081). Subsequently, complete sequences for AAV3A, AAV3B, AAV4, and AAV6 were also determined. (Rutledge et al. (1998) J. Virol. 72:309-319; Chiorini et al. (1997) J. Virol. 71:6823-6833; S. Muramatsu et al. (1996) Virol. 221:208-217). Generally, all AAVs share more than 80% nucleotide sequence identity. [007] AAV is a promising vector for human gene therapy, since, unlike other viral vectors, AAVs have not been shown to be associated with any known human disease and are generally not considered pathogenic. (Muzyczka et al. (1992) Current Topics in Microbiology and Immunology 158:97-129). Furthermore, AAV safely transports post-mitotic tissues with relatively low immunogenicity and is able to integrate into host chromosomes in a site-specific manner and into tissue-cultured cells on chromosome 19 if Rep proteins are provided trans. (Kotin et al. (1990) Proc. Natl. Acad. Sci. USA 87:2211-2215; Samulski et al. (1991) EMBO J. 10(12):3941-3950; Balague et al. (1997) J. Virol. 71:3299-3306; Surosky et al. (1997) J. Virol. 71:7951-7959). Integrated AAV genomes have been shown to allow long-term gene expression in various tissues, including muscle, liver, and brain (Fisher (1997) Nature Med. 3(3):306-312; Snyder et al. (1997) Nature Genetics 16:270-276; Xiao et al. (1997) Experimental Neurology 144:113-124; Xiao et al. (1996) J. Virol. 70(11):8098-8108). [008] Several viruses, including AAVs, infect cells through a virus/ligand:cell/receptor interaction, ultimately resulting in endocytosis of the virus by the infected cell. This ligand:receptor interaction is the focus of most research on viral vectors; for example, it can be manipulated to redirect a natural tropism of viruses from a cell naturally permissive to infection by wild-t