Therapeutic agents and diagnostic agents and their use

Abstract

The present invention provides binding molecules, and nucleic acid molecules encoding the binding molecules, that have one or more (preferably all) of highly specific binding to the US28 protein of human cytomegalovirus (HCMV), very low levels of non-specific binding to healthy (uninfected) cells, and / or strain-independent binding capabilities. The binding molecules are designed to bind to the extracellular domain 3 (ECD3) of the US28 protein of human cytomegalovirus (HCMV), which is the third of the four extracellular domains presented by US28, corresponding to positions 167-183 of the US28 protein sequence defined by SEQ ID NO:5. The binding molecules of the present invention have been demonstrated to have excellent binding properties, including specific binding specificity for invasive and / or metastatic HCMV-infected cancers, including breast cancer. In certain preferred embodiments, the binding molecules are selected from antibodies (including, for example, BiTE antibodies) and chimeric antigen receptors (CARs), or functional variants, fragments, fusion proteins, and / or conjugates thereof. Cells expressing the binding molecules are also provided, such as CAR-expressing cells, including CAR-T cells, CAR-NK cells, and CAR-M cells.

Claims

1. A binding molecule, wherein the binding molecule is an antibody that has binding specificity to the extracellular domain 3 (ECD3) of the US28 protein of human cytomegalovirus (HCMV), and has binding specificity to an epitope common to and present within both the sequence of TKKNNQCMTDYDYLEVS (SEQ ID NO: 6) and the sequence of TKKDNQCMTDYDYLEVS (SEQ ID NO: 7), or comprises such an antibody. A binding molecule in which the sequence of SEQ ID NO: 6 is the sequence of the 4N variant ECD3 of the HCMV US28 protein, and the sequence of SEQ ID NO: 7 is the sequence of the 4D variant ECD3 of the HCMV US28 protein.

2. The aforementioned antibody (a) V Ｈ -CDR1, V Ｈ -CDR2, V Ｈ -CDR3, V Ｌ -CDR1, V Ｌ - CDR2 and V Ｌ -CDR3 is antibody 1D3 as defined by SEQ ID NOs: 8, 9, 10, 14, 15, and 16, respectively. (b) V Ｈ - CDR1, V Ｈ - CDR2, V Ｈ - CDR3, V Ｌ - CDR1, V Ｌ - CDR2 and V Ｌ - CDR3 are the antibody 1C10 defined by SEQ ID NOs: 112, 113, 114, 117, 83, and 118, respectively (c) V Ｈ -CDR1, V Ｈ -CDR2, V Ｈ -CDR3, V Ｌ -CDR1, V Ｌ - CDR2 and V Ｌ -CDR3 is antibody 1A10, defined by sequence numbers 112, 113, 114, 117, 83, and 118, respectively. (d) V Ｈ -CDR1, V Ｈ -CDR2, V Ｈ -CDR3, V Ｌ -CDR1, V Ｌ - CDR2 and V Ｌ -CDR3 is antibody 1G9 as defined by SEQ ID NOs: 76, 77, 78, 82, 83, and 84, and / or (e) V Ｈ -CDR1, V Ｈ -CDR2, V Ｈ -CDR3, V Ｌ -CDR1, V Ｌ - CDR2 and V Ｌ -CDR3 is antibody 1E8, defined by SEQ ID NOs: 76, 95, 96, 82, 99, and 100, respectively. The binding molecule according to claim 1, comprising six complementarity-determining regions (CDRs) corresponding to all six CDR sequences of the antibody.

3. The aforementioned antibody (a) At least one variable heavy chain (V) comprising or consisting of a sequence selected from the group consisting of sequence numbers 12, 104, 122, 68 and 88 Ｈ ) polypeptides, and (b) A variable light chain (V) comprising or consisting of a sequence selected from the group consisting of sequence numbers 18, 108, 126, 72 and 92. Ｌ ) polypeptides, The binding molecule according to claim 2, comprising:

4. The aforementioned binding molecule, (a) Bivalent antibody, (b) Monovalent antibody, (c)scFv ２ - Fc antibody, (d) bispecific antibody; (e) trispecific antibody, (f) scFv-HSA-scFv antibody, (g) Single-domain antibody, (h) Heavy chain monoglobulin IgG (hcIgG) such as camelid IgG (e.g., VHH antibody) and shark immunoglobulin novel antigen receptor (IgNAR), and single-chain antibodies thereof. (i) Bispecific immune cell engager antibodies, and (j) Monoclonal antibody A binding molecule according to any one of claims 1 to 3, selected from the group consisting of the following.

5. The binding molecule comprises or consists of an antigen-binding fragment or fusion thereof of an antibody as defined in any one of claims 1 to 4, wherein the antigen-binding fragment or fusion thereof has binding specificity to ECD3 of the US28 protein of HCMV, and the binding specificity is to an epitope common to and present in both the sequence of SEQ ID NO: 6 and the sequence of SEQ ID NO: 7, and the antigen-binding fragment is an Fv fragment; a single-stranded Fv fragment (scFv); a disulfide-bonded Fv fragment; a Fab-like fragment; a Fab fragment, a Fab' fragment, or F(ab) ２ A conjugation molecule according to any one of claims 1 to 4, selected from the group consisting of a Fab-like fragment selected from the fragments and a single-domain antibody.

6. A fusion polypeptide comprising the binding molecule described in any one of claims 1 to 5, The sequence of the fusion polypeptide includes a first amino acid sequence fused with a second amino acid sequence. The first amino acid sequence comprises or consists of the binding molecule described in any one of claims 1 to 5. The second amino acid sequence described above is a fusion polypeptide, which is the fusion partner.

7. (i) an extracellular domain comprising or consisting of the binding molecule described in any one of claims 1 to 5, (ii) Transmembrane domain and (iii) A chimeric antigen receptor (CAR) containing an intracellular domain.

8. (a) The transmembrane domain includes the transmembrane domain of a protein; (b) The extracellular domain is connected to the transmembrane domain by a hinge region; (d) The intracellular domain includes an intracellular signaling domain; (e) The intracellular domain includes one or more costimulatory domains; and / or (f) The CAR further includes a leader array, The CAR according to claim 7.

9. A nucleic acid molecule, or a combination of several different nucleic acid molecules, The nucleic acid molecules, individually or in combination, comprise one or more nucleic acid sequences encoding a binding molecule according to any one of claims 1 to 5, a fusion polypeptide according to claim 6, or a CAR according to claim 7 or 8, or the combination of the plurality of different nucleic acid molecules comprises the nucleic acid molecule, or a combination of the plurality of different nucleic acid molecules.

10. A vector comprising the nucleic acid molecule described in claim 9, or a combination of several different nucleic acid molecules.

11. It is a cell, (a) comprising the nucleic acid molecule described in claim 9, or a combination of several different nucleic acid molecules, or the vector described in claim 10; and / or (b) comprising the binding molecule according to any one of claims 1 to 5 and / or the fusion polypeptide according to claim 6, wherein optionally the binding molecule according to any one of claims 1 to 5 is an antibody. cell.

12. It is a cell, (a) comprising a nucleic acid molecule according to claim 9, or a combination of several different nucleic acid molecules, or a vector according to claim 10, wherein the nucleic acid molecule individually or in combination comprises one or more nucleic acid sequences encoding the CAR according to claim 7 or 8, or the combination of several different nucleic acid molecules collectively comprises the same. Optionally, the cells express one or more CARs according to claim 7 or 8, encoded by the nucleic acid molecule according to claim 9, or a combination of several different nucleic acid molecules, or the vector according to claim 10; (b) this A cell as defined in part (a) of claim, wherein the cell is a T cell, a natural killer (NK) cell, or a macrophage; and / or (c) this A cell as defined in part (b) of claim, wherein the cell is a CAR-T cell, a CAR-NK cell, or a CAR-macrophage. If the cells are optionally selected to be CAR-T cells, then the T cells are CD8 ＋ Cells selected from the group consisting of T cells, CD4+ T cells, effector T cells, helper T cells, memory T cells, cytotoxic T lymphocytes (CTLs), EBV-specific T cell receptors (TCRs), or γδ-T cell subtypes.

13. An in vitro method for producing cells, comprising introducing a nucleic acid molecule according to claim 9, or a combination of several different nucleic acid molecules, and / or a vector according to claim 10, into cells.

14. An in vitro method for producing a binding molecule according to any one of claims 1 to 5, a fusion polypeptide according to claim 6, or a CAR according to claim 7 or 8, The method includes expressing the nucleic acid molecule described in claim 9, or a combination of several different nucleic acid molecules, and / or the vector described in claim 10, in a cell. A method comprising the optional step of isolating from the cells a binding molecule according to any one of claims 1 to 5 or a fusion polypeptide according to claim 6, thus produced.

15. A conjugate comprising a conjugating molecule as defined in any one of claims 1 to 5, or a portion conjugated to the fusion polypeptide described in claim 6, The portion is optionally therapeutic, preventive, diagnostic, prognostic, or therapeutic. Conjugate.

16. (i) The portion is a drug; (ii) The portion is radioactive; or (iii) The conjugate according to claim 15, wherein the portion is a radioactive portion of the conjugate suitable for use in radioimmunotherapy (RIT).

17. The conjugate according to claim 15 or 16, wherein the conjugate is an antibody-drug conjugate (ADC).

18. A method for producing the conjugate according to any one of claims 15 to 17, wherein the method is (a) the step of providing a binding molecule according to any one of claims 1 to 5, or a fusion polypeptide according to claim 6, (b) The step of conjugating a portion of the binding molecule according to any one of claims 1 to 5, or the fusion polypeptide according to claim 6, (c) A method comprising the optional step of isolating the conjugate thus produced.

19. A preparation comprising one or more drugs, wherein each of the one or more drugs is independent, i. The binding molecule according to any one of claims 1 to 5, ii. The fusion polypeptide according to claim 6, iii. A nucleic acid molecule according to claim 9, or a combination of several different nucleic acid molecules, vi. The vector according to claim 10, v. The cell according to claim 11 or 12, vi. The conjugate according to any one of claims 15 to 17 A formulation selected from the group consisting of the following.

20. A method for combating HCMV infection in ex vivo or in vitro cell material, wherein the method comprises the step of administering the formulation according to claim 19 to the ex vivo or in vitro cell material.

21. A formulation according to claim 19 for use in combating a disease or condition associated with HCMV infection, (a) The disease or condition is an HCMV infection, and optionally, the HCMV infection includes a multi-strain HCMV infection, and the multi-strain HCMV infection includes infection by one or more different HCMV strains. (b) The disease or condition is a latent HCMV infection, (c) The disease or condition is a soluble HCMV infection, (d) The disease or condition is a congenital single-strain or multi-strain HCMV infection, and / or (e) The disease or condition is an HCMV-infected cancer (multiple strain HCMV-infected cancer, at random) such as latent HCMV-infected cancer (multiple strain latent HCMV-infected cancer, at random). formulation.

22. (a) The person requiring this has been diagnosed with having HCMV-infected cancer cells, and / or (b) The person who needs this is an organ recipient or organ donor, or is expected to become one. The formulation according to claim 21.

23. The subject is one to be administered further substances, such as further therapeutic, prophylactic, diagnostic, prognostic, or therapeutic substances. For example, the formulation for use according to claim 22, wherein the further substance is administered separately, sequentially, or simultaneously with each of the one or more agents.

24. A formulation according to claim 19 for use in pharmaceuticals.

25. A method for evaluating US28 expression in ex vivo biomaterials, wherein the method is: (a) A step of contacting the ex vivo biomaterial with a binding molecule according to any one of claims 1 to 5, a fusion polypeptide according to claim 6, or a conjugate according to any one of claims 15 to 17; and (b) A step of evaluating the ex vivo biomaterial based on direct and / or indirect measurement of the binding of the binding molecule, the fusion polypeptide, or the conjugate to the ex vivo biomaterial. Methods that include...

26. A formulation comprising a conjugate according to any one of claims 1 to 5, a fusion polypeptide according to claim 6, or a conjugate according to any one of claims 15 to 17, for use in a method for diagnosing a disease or condition associated with HCMV by evaluating US28 expression in a subject, wherein the method is (a) A step of bringing the binding molecule, the fusion polypeptide, or the conjugate into contact with the target; and (b) A step of evaluating the object based on direct and / or indirect measurement of the binding of the binding molecule, the fusion polypeptide, or the conjugate to the object. A formulation containing the above.

27. (a) The method includes an ELISA method, and optionally the method is performed on one or more ex vivo biomaterials such as bodily fluids; (b) The method includes flow cytometry, and optionally the method is for evaluating ex vivo blood samples and / or ex vivo bone marrow samples from a subject; (c) The method comprises using a conjugate according to any one of claims 15 to 17, wherein the conjugate comprises a detectable portion such as a radioactive portion, and the method comprises detecting the radioactive portion in the ex vivo biomaterial. For example, the method is an immunopositron emission (PET) imaging method; or (d) The method is an immunohistochemical method performed on a sample of ex vivo biomaterial. The method according to claim 25.

28. The method according to claim 25 or 27, which is performed on an ex vivo biomaterial obtained from a subject for the purpose of diagnosing or evaluating the prognosis of a disease or condition related to HCMV in the ex vivo biomaterial obtained from the subject, wherein, for example, the disease or condition is as defined in claim 21 and / or the subject is as defined in claim 22.

29. A kit for evaluating one or more biological conditions and / or biological properties of a subject and / or an ex vivo biomaterial, wherein the kit comprises a conserved form of a binding molecule according to any one of claims 1 to 5, a fusion polypeptide according to claim 6, or a conjugate according to any one of claims 15 to 17, and the kit is suitable for use in a method for evaluating US28 expression and / or for evaluating HCMV infection and / or diseases or conditions associated with HCMV infection in the subject and / or the ex vivo biomaterial.

30. The kit according to claim 29, wherein the kit is configured for use in an immunoassay selected from ELISA, flow cytometry, immunohistochemistry (IHC), or immuno-based imaging.

31. The kit according to claim 30, wherein the kit is configured for use in immunohistochemistry (IHC) of ex vivo biomaterials.

32. The kit according to claim 31, wherein the ex vivo biomaterial includes a histological specimen.

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