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US-12624101-B2 - Monoclonal antibodies against neonatal Nav1.5

US12624101B2US 12624101 B2US12624101 B2US 12624101B2US-12624101-B2

Abstract

Describe herein are binding molecules which specifically bind to neonatal 5′-exon splice variants of the a subunit of Na v 1.5 (nNa v 1.5), including monoclonal antibodies and antigen-binding fragments, bispecific antibodies and antibody-drug conjugates derived from such monoclonal antibodies, and NK and T cells comprising chimeric antigen receptors derived from such monoclonal antibodies, and their use in diagnostic and therapeutic methods.

Inventors

  • Mustafa Bilgin Ali Djamgoz
  • Carsten Faltum

Assignees

  • Celex Oncology Innovations Limited

Dates

Publication Date
20260512
Application Date
20210121
Priority Date
20200121

Claims (18)

  1. 1 . A monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds to neonatal 5′-exon splice variants of the α subunit of Nav1.5 (nNav1.5), comprising a VH region comprising complementarity determining region (CDR) 1, CDR2, and CDR3 amino acid sequences comprising SEQ ID NOs: 14, 15, and 16, respectively, and a VL region comprising CDR1, CDR2, and CDR3 amino acid sequences comprising SEQ ID NOs: 17, 18, and 19, respectively.
  2. 2 . The monoclonal antibody according to claim 1 , which binds to the peptide segment corresponding to residues 206 to 219 of SEQ ID NO:2.
  3. 3 . The monoclonal antibody according to claim 2 , which binds more readily to a nNav1.5 comprising the amino acid sequence of residues 206 to 219 of SEQ ID NO: 2 than to an adult 3′-exon splice variant of the α subunit of Nav1.5 (aNav1.5) comprising the amino acid sequence of residues 206 to 219 of SEQ ID NO:1.
  4. 4 . The monoclonal antibody according to claim 3 , which reduces the VGSC current of a VGSC comprising a nNav1.5.
  5. 5 . The monoclonal antibody according to claim 4 , wherein the nNav1.5 is a human nNav1.5.
  6. 6 . The monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 , comprising a VH region at least 80%, such as at least 90%, 95%, 97%, 98% or 99% identical, to the VH region encoded by SEQ ID NO: 10, and a VL region at least 80%, such as at least 90%, 95%, 97%, 98% or 99% identical, to the VL region encoded by SEQ ID NO: 11.
  7. 7 . The monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 , comprising a VH region comprising the amino acid sequence of SEQ ID NO: 12 and a VL region comprising the amino acid sequence of SEQ ID NO:13.
  8. 8 . The monoclonal antibody according to claim 1 , wherein all constant domains, framework regions, or constant domains and framework regions, are human.
  9. 9 . The monoclonal antibody according to claim 8 , which is chimeric, or humanized.
  10. 10 . A chimeric antigen receptor (CAR), bispecific antibody, or antibody-drug conjugate (ADC) comprising a monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 .
  11. 11 . A nucleic acid sequence encoding the monoclonal antibody, or antigen-binding fragment thereof, of claim 1 .
  12. 12 . An expression vector comprising the nucleic acid sequence of claim 11 .
  13. 13 . A cell comprising the nucleic acid sequence of claim 11 .
  14. 14 . A method of producing a monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 , the method comprising culturing a cell comprising a nucleic acid sequence encoding the monoclonal antibody or the antigen-binding fragment, and recovering the monoclonal antibody or the antigen-binding fragment.
  15. 15 . A method for treatment or amelioration of metastatic cancer in a subject, comprising administering to the subject an effective amount of a monoclonal antibody, or an antigen-binding fragment thereof, according to claim 1 .
  16. 16 . A method for reducing the risk for metastatic cancer in a subject, comprising administering to the subject an effective amount of a monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 .
  17. 17 . A method for detecting the presence of metastatic cancer in a subject, the method comprising administering a labelled antibody or antigen-binding fragment thereof, which specifically recognizes nNav1.5, to the subject and subsequently measuring signal distribution derived from the labelled antibody or antigen-binding fragment in said subject, where a localised dense signal in a part of the body of the subject is indicative of the presence of metastatic disease in said part of the body, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 .
  18. 18 . A method for detecting the presence of neonatal 5′-exon splice variants of the α subunit of Nav1.5 (nNav1.5) in a biological sample from a subject, the method comprising contacting the biological sample with an antibody or antigen-binding fragment thereof which specifically recognizes nNav1.5, and subsequently detecting binding of the antibody or antigen-binding fragment to the biological sample, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, or antigen-binding fragment thereof, according to claim 1 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage filing under 35 U.S.C. 371 of PCT/EP2021/051317, filed Jan. 21, 2021, which was published by the International Bureau in English on Jul. 29, 2021, and which claims priority from European Application No. 20152789.2, filed Jan. 21, 2020, each of which is hereby incorporated in its entirety by reference in this application. FIELD OF THE INVENTION The present invention relates to the field of cancer therapy. In particular, the present invention relates to the field of treatment of metastasizing cancers and to therapeutic means and methods. BACKGROUND OF THE INVENTION Several major human carcinomas express functional voltage-gated Na+ channels (VGSCs) which promote their cellular invasiveness in vitro and metastasis in vivo (Djamgoz et al., 2019). In humans, there are nine different VGSC alpha subunits or “Nav” proteins (Nav1.1 to Nav1.9), encoded by the genes SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A and SCN11A, respectively. The alpha subunit of VGSCs is a transmembrane protein with 4 repetitive transmembrane domains (termed DI, DII, DIII and DIV), which each contains 6 transmembrane spanning sections that are termed S1-S6. Na+ ions flow through a pore formed by S5 and S6 combined from all 4 domains. The S4 segment plays a central role in voltage sensing and channel activation. The smaller beta subunits contain an N-terminal extracellular immunoglobulin (Ig) loop, a transmembrane domain and an intracellular domain, and function as regulatory and adhesion molecules. Human Nav1.5 is encoded by the gene SCN5A, a highly conserved gene located on human chromosome 3, where it spans more than 100 kb. The gene consists of 28 exons, of which exon 1 and in part exon 2 form the 5′ untranslated region (5′UTR), and exon 28 forms the 3′ untranslated region (3′UTR) of the RNA. More than 10 different splice isoforms have been described for SCN5A, of which several harbour different functional properties. Importantly, Nav1.5 is developmentally regulated via alternative splicing of exon 6, giving rise to ‘adult’ and ‘neonatal’ or ‘foetal’ variants of the Nav1.5 protein that differ in the S3-S4 region of DI by several amino acids. The foetal and adult form of the protein can have 7 amino acid differences in the DI:S3-S4 region of the channel protein. It has previously been found that the foetal/neonatal isoform of Nav1.5 (nNav1.5) is associated with metastatic cancers and in vitro data have demonstrated that it is possible to interfere with the metastatic ability of malignant cells by blocking nNav1.5 (Djamgoz et al., 2019). Chioni et al. (2005) describes the generation of an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised ‘neonatal’ but not ‘adult’ Nav1.5 when tested on cells specifically over-expressing one or other of these Nav1.5 spliced forms. However, there is still a need for therapeutic and diagnostic means and methods for cancers, particularly cancers associated with nNav1.5 expression. It is an object of embodiments of the invention to provide such therapeutic and diagnostic means and methods. SUMMARY OF THE INVENTION The present inventor has identified monoclonal antibodies which specifically bind to nNav1.5. So, in one aspect, the present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds to neonatal 5′-exon splice variants of the a subunit of Nav1.5 (nNav1.5), optionally comprising human constant heavy (CH) and constant light (CL) domains and/or human framework (FR) regions. In one aspect, the present invention provides a chimeric antigen receptor (CAR) which specifically binds to neonatal 5′-exon splice variants of the a subunit of Nav1.5 (nNav1.5), optionally comprising an antigen-binding fragment derived from the monoclonal antibody of the preceding aspect. These and other aspects and embodiments of the invention are disclosed in more detail below. LEGENDS TO THE FIGURES FIG. 1 shows Nav1.5 splice variant sequences encoded by exon 6 of the SCN5A gene and a consensus sequence (SEQ ID NO:4). FIG. 2 shows the results of immunocytochemical tests, using mAbs obtained from hybridomas and EBNA cell lines stably expressing nNav1.5 or aNav1.5. See Example 1 for details. FIG. 3 shows the results of patch-clamp recordings performed on EBNA cells stably expressing neonatal Nav1.5 as previously described by Chioni et al. (2005). The blocking ability of mAb candidates (supernatants) was tested by short-term application using a ‘puff’ pipette whilst clamping the cells at different holding potentials. The figure shows the dose dependent blockage of nNav1.5 currents by the A3 and C2 mAbs. Each data point denotes means+/− standard error (n=4-6). The dotted horizontal line denotes ‘null effect’ for ease of comparison. FIG. 4 shows the VH (A) and VL (B) amino acid sequences of the A3 antibody (SEQ ID NO: 12 and 13, respectively), with the amino acid positions