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US-12624097-B2 - Nucleic acids, vectors, and cells that encode anti-Sp17 antibodies and other Sp17 binding proteins

US12624097B2US 12624097 B2US12624097 B2US 12624097B2US-12624097-B2

Abstract

This disclosure describes proteins that specifically bind human sperm protein 17 (Sp17) with nanomolar affinity and high specificity. These proteins include a recombinant human anti-Sp17 IgG that is suitable for use as a therapeutic antibody to treat cancers that ectopically express Sp17. Other Sp17-binding proteins are described including antibody fragments, antibody conjugates, and fusion proteins.

Inventors

  • Seah Lim

Assignees

  • Medicovestor, Inc.

Dates

Publication Date
20260512
Application Date
20240109

Claims (20)

  1. 1 . A recombinant nucleic acid, comprising a nucleotide sequence that encodes a protein that comprises an antigen-binding region, wherein: the antigen-binding region comprises a first variable domain and a second variable domain; wherein the first variable domain comprises a VH CDR1 region comprising the amino acid sequence of SEQ ID NO: 5, a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 6, and a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 7; and wherein the second variable domain comprises a VL CDR1 region comprising the amino acid sequence of SEQ ID NO: 8, a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 region comprising the amino acid sequence of SEQ ID NO: 10; and wherein the first variable domain and the second variable domain are paired in the antigen-binding region of the protein such that the antigen-binding region specifically binds human sperm protein 17 (Sp17).
  2. 2 . The recombinant nucleic acid of claim 1 , wherein: the first variable domain comprises an amino acid sequence that is identical to SEQ ID NO: 3; and the second variable domain comprises an amino acid sequence that is identical to SEQ ID NO: 4.
  3. 3 . The recombinant nucleic acid of claim 1 , comprising a nucleotide sequence that is at least 70 percent identical to SEQ ID NO: 1.
  4. 4 . The recombinant nucleic acid of claim 1 , comprising a nucleotide sequence that encodes an amino acid sequence that is at least 90 percent identical to SEQ ID NO: 11.
  5. 5 . The recombinant nucleic acid of claim 1 , comprising a nucleotide sequence that is at least 70 percent identical to SEQ ID NO: 2.
  6. 6 . The recombinant nucleic acid of claim 1 , comprising a nucleotide sequence that encodes an amino acid sequence that is at least 90 percent identical to SEQ ID NO: 12.
  7. 7 . The recombinant nucleic acid of claim 1 , comprising an origin of replication, wherein the recombinant nucleic acid is a plasmid.
  8. 8 . A cell, comprising the recombinant nucleic acid of claim 7 , wherein the cell is a prokaryote.
  9. 9 . A cell, comprising the recombinant nucleic acid of claim 1 , wherein the cell is an immortalized mammalian cell.
  10. 10 . The cell of claim 9 , wherein the cell is a Chinese Hamster Ovary cell or a Human Embryonic Kidney cell; and wherein the cell expresses the protein.
  11. 11 . The cell of claim 9 , wherein the cell expresses a viral vector; and the recombinant nucleic acid comprises a packaging signal for packaging the recombinant nucleic acid in the viral vector.
  12. 12 . A viral vector, comprising the recombinant nucleic acid of claim 1 .
  13. 13 . An isolated human cell, the cell comprising the recombinant nucleic acid as claimed in claim 1 , wherein the human cell expresses the protein.
  14. 14 . The isolated human cell of claim 13 , wherein the human cell is a human peripheral blood mononuclear cell.
  15. 15 . The isolated human cell of claim 13 , wherein the human cell is a T-cell, a natural killer cell, or a monocyte.
  16. 16 . The isolated human cell of claim 13 , wherein the protein is a chimeric antigen receptor.
  17. 17 . A Fab fragment of an antibody encoded by the recombinant nucleic acid of claim 1 .
  18. 18 . An antibody comprising the Fab fragment of claim 17 .
  19. 19 . A bi-specific antibody encoded by the recombinant nucleic acid of claim 1 .
  20. 20 . A bi-specific T-cell engager (BiTE) encoded by the recombinant nucleic acid of claim 1 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63/524,806, filed Jul. 3, 2023, the disclosure of which is incorporated, in its entirety, by this reference. SEQUENCE LISTING This disclosure includes a sequence listing, which has file name “Sequence_Listing_1200590011.xml,” which was created on Jan. 9, 2024, which has a file size of 14,063 bytes, and which is incorporated by reference in its entirety. FIELD OF THE INVENTION The present invention relates to proteins that bind sperm protein 17 (Sp17), which Sp17-binding proteins are derived from an anti-Sp17 Fab and include anti-Sp17 monoclonal antibodies as well as Sp17-binding conjugates and fusion proteins derived from the complementarity determining regions (CDRs) of the Fab. The present invention specifically relates to the use of Sp17-binding proteins in the diagnosis and treatment of various health conditions including cancer. BACKGROUND OF SOME ASPECTS OF THE SPECIFICATION The human Sp17 gene, which is located on chromosome 11q24.2, encodes a highly-conserved, antigenic, 17.4 kilodalton protein Sp17 that is expressed in spermatozoa. The Sp17 protein is involved in acrosome reactions during fertilization. Immunohistochemistry on tissue microarrays and Reverse Transcription-Polymerase Chain Reactions (RT-PCRs) on a panel of RNA from different tissues suggests that Sp17 is expressed in normal testes and absent in other healthy tissues. Sp17 was also identified as an aberrantly expressed tumor antigen in multiple myeloma, lymphoma, ovarian cancer, and non-small cell lung cancer. SUMMARY OF SOME ASPECTS OF THE SPECIFICATION Various aspects of this disclosure relate to the discovery of a novel anti-Sp17 Fab that includes CDRs that have nanomolar affinity for Sp17 and high specificity for Sp17 relative to other human proteins. The CDRs retain their high affinity and fidelity in various antibody formats. A human antibody comprising the CDRs is in pre-clinical development for use as a cancer immunotherapeutic. The CDRs are also compatible with a full range of immunotherapeutic strategies including antibody conjugates, bi-specific proteins (for example, as T-cell engagers), and adoptive cell therapies (for example, as CAR-Ts). The CDRs may also be cloned into antibodies for laboratory research and diagnostics. The following disclosure describes, for example, a chimeric IgG comprising a mouse Fc region that may be used in various assays and diagnostics in combination with an anti-mouse secondary antibody. This disclosure describes its use with flow cytometry, immunohistochemistry, and western blotting. The preceding Background and Summary sections are provided as a brief introduction to the described subject matter as well as a synopsis of some of the technological improvements and advantages that it provides. The Background and Summary shall not be construed as identifying essential aspects of the described subject matter, nor shall they be construed to limit the interpretation of this specification or any patent claim that matures from this specification. BRIEF DESCRIPTION OF THE DRAWINGS A further understanding of this specification may be appreciated with reference to the following drawings. The drawings are exemplary, and neither this specification nor any patent claim that matures from this specification shall be construed as limited by the drawings. FIG. 1 is an image of an SDS-PAGE gel loaded with a molecular weight standard (lane M) and 2 micrograms of recombinant Sp17 protein under reducing conditions (lane 1) and non-reducing conditions (lane 2). FIG. 2 consists of two panels. The left panel is a Coomassie-blue stained SDS-PAGE gel loaded with a molecular weight standard (lane M), the anti-Sp17 antibody chAB2 under reducing conditions (lane 1), and the anti-Sp17 antibody chAB2 under non-reducing conditions (lane 2). The right panel is a western blot of an SDS-PAGE gel loaded with a molecular weight standard (lane M) and the anti-Sp17 antibody chAB2 under reducing conditions (lane E), in which chAB2 was detected using enhanced chemiluminescence (ECL) with anti-mouse secondary antibodies. FIG. 3 is a western blot of an SDS-PAGE gel loaded with a molecular weight standard (lane M) and with Sp17 protein (lanes 1, 2, & 3), in which the Sp17 protein was detected with the anti-Sp17 antibody chAB2. FIG. 4 consists of three panels. The left panel is a graph that depicts flow cytometry results for ID8 cells incubated with an anti-mouse fluorescein isothiocyanate (FITC) antibody, but without a primary antibody. The middle panel is a graph that depicts flow cytometry results for ID8 cells labeled with the anti-Sp17 antibody chAB2 and the anti-mouse FITC antibody. The right panel is a histogram that displays an increased mean fluorescent intensity for the ID8 cells labeled with chAB2 and the anti-mouse FITC antibody of the middle panel relative to ID8 cells incubated with the anti-mouse FITC ant