Antibodies that bind to LILRB4 and CD3, and their applications

Abstract

The present invention provides an antibody capable of binding to LILRB4, and a bispecific antibody capable of binding to both LILRB4 and CD3. Further, the invention provides a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, host cells, and a method. The present invention further relates to a method for treating solid tumors, multiple myeloma, or leukemia such as acute myeloid leukemia (AML) using the anti-LILRB4 × CD3 biantibody of the present invention.

Claims

1. A VHH antibody that specifically binds to LILRB4, It includes three complementarity determination regions (CDRs) contained in the VHH, which is represented by any one of sequence numbers 11, 16, 21, 25, 30, 32, 36, 39, and 42. Preferably, the CDR sequence is defined according to IMGT. VHH antibody.

2. It includes the complementarity determination regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, of which, (i) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 12, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 13, VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 14, or (ii) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 17, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 18, and VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:

19. (iii) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 75, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 22, and VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:

23. (iv) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 26, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 27, and VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:

28. (v) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 17, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 33 or 37, and VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 34 or 40. (vi) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 17, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 33 or 37, VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 34, or (vii) VHH CDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 17, VHH CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 33 or 37, and VHH CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:

40. The VHH antibody according to claim 1.

3. A heavy chain variable region is included in or consists of, the heavy chain variable region is (i) an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one selected from SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39, and 42, or consists of such an amino acid sequence. (ii) Consists of or consists of an amino acid sequence represented by any one selected from SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42, or (iii) Compared to the amino acid sequence represented by any one selected from SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39, and 42, the amino acid sequence includes one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably amino acid-conservative substitutions), and preferably, the amino acid changes do not occur in the CDR region. The VHH antibody according to claim 1.

4. A heavy chain antibody that specifically binds to LILRB4, comprising the VHH antibody described in any one of claims 1 to 3. Heavy chain antibodies.

5. The present invention comprises a VHH antibody according to any one of claims 1 to 3, which is linked to an antibody constant region or Fc region, preferably the antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3, or human IgG4, and optionally the VHH antibody and the Fc region are linked via a hinge region or a part thereof, preferably the amino acid sequence of the hinge region portion is EPKSS (SEQ ID NO: 59) or EPKSC (SEQ ID NO: 67). The heavy chain antibody according to claim 4.

6. The VHH antibody according to any one of claims 1 to 3, which is linked to the antibody Fc region, wherein the Fc region is an Fc region derived from human IgG1, IgG2, IgG3 or IgG4, and preferably the Fc region is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 55 or 68, or consisting of such an amino acid sequence. (ii) Consists of or consists of the amino acid sequence shown in SEQ ID NO: 55 or 68, or (iii) Compared to the amino acid sequence shown in Sequence ID No. 55 or 68, the amino acid sequence includes one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably amino acid-conservative substitutions), The heavy chain antibody according to claim 4.

7. (i) an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one selected from SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41, or 43, or consists of such an amino acid sequence, (ii) Consists of or consists of an amino acid sequence represented by any one selected from sequence numbers 15, 20, 24, 29, 31, 35, 38, 41, or 43, or (iii) Compared to the amino acid sequence represented by any one selected from SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41, or 43, the amino acid sequence includes one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably amino acid-conservative substitutions), and preferably, the amino acid changes do not occur in the CDR region. The heavy chain antibody according to claim 4.

8. The aforementioned antibody is a chimeric antibody or a humanized antibody. A VHH antibody according to any one of claims 1 to 3, or a heavy chain antibody according to any one of claims 4 to 7.

9. The material comprises a first antigen-binding region and a second antigen-binding region, wherein the second antigen-binding region specifically binds to LILRB4 and comprises a VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8. Bispecific antibodies.

10. The first antigen-binding region specifically binds to CD3. The bispecific antibody according to claim 9.

11. The first antigen-binding region comprises VH and VL, wherein VH comprises three complementarity-determining regions (HCDRs) derived from the heavy chain variable region, namely HCDR1, HCDR2, and HCDR3, and VL comprises three complementarity-determining regions (LCDRs) derived from the light chain variable region, namely LCDR1, LCDR2, and LCDR3, of which, (i) HCDR1, HCDR2, and HCDR3 are the three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH indicated by Sequence ID 3, and LCDR1, LCDR2, and LCDR3 are the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL indicated by any one of Sequence ID 4, or (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 5, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 6, HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 7, LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 8, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 9, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

10. The bispecific antibody according to claim 10.

12. The first antigen-binding region includes VH and VL, of which, The aforementioned VH includes, or comprises, the amino acid sequence shown in Sequence ID No. 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. Furthermore / or, the VL includes or consists of the amino acid sequence shown in Sequence ID No. 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith. The bispecific antibody according to claim 11.

13. The first antigen-binding region is a Fab that specifically binds to CD3. A bispecific antibody according to any one of claims 10 to 12.

14. The aforementioned Fab encompasses CH1, of which the CH1 is derived from IgG1, IgG2, IgG3, or IgG4, and preferably from IgG1. The bispecific antibody according to claim 13.

15. The aforementioned CH1 is, (i) an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from Sequence ID No. 51, or consists of such an amino acid sequence, (ii) an amino acid sequence selected from Sequence ID No. 51, or consisting thereof The bispecific antibody according to claim 14.

16. The first antigen-binding region is an scFv that specifically binds to CD3, the scFv encompassing VH and VL as defined in claim 11 or 12, and optionally the VH and VL being linked via a linker, the linker encompassing, for example, an amino acid sequence (G4S)n, where n = 1, 2, 3, 4, or 5, preferably n = 3 or 4, more preferably n = 3. A bispecific antibody according to any one of claims 10 to 12.

17. The second antigen-binding region is the VHH described in any one of claims 1 to 3 and 8. A bispecific antibody according to any one of claims 9 to 16.

18. The aforementioned bispecific antibody is an IgG-like bispecific antibody containing an Fc dimer, wherein the two Fc regions containing the Fc dimer are homologous or distinct. A bispecific antibody according to any one of claims 9 to 17.

19. The two Fc regions are different, and preferably, corresponding knob mutations and Hole mutations are introduced into the two Fc regions, respectively. The bispecific antibody according to claim 18.

20. One Fc-region polypeptide contains the knob mutation T366W, and the other Fc-region polypeptide contains the hole mutations T366S, L368A, and Y407V, or One Fc-region polypeptide contains knob mutations T366W and Y349C, and the other Fc-region polypeptide contains hole mutations T366S, L368A, Y407V and S354C, or One Fc-region polypeptide contains knob mutations T366W and S354C, while the other Fc-region polypeptide contains hole mutations T366S, L368A, Y407V and Y349C. The Fc region optionally further includes one or more mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation, the D265A mutation, or the P329A mutation, for example, the L234A / L235A mutation, the D265A mutation, and the P329A mutation. The bispecific antibody according to claim 19.

21. One or both of the Fc regions include a hinge region, for example, EPKSS or EPKSC. A bispecific antibody according to any one of claims 18 to 20.

22. (i) The Fc region containing the knob mutation is a) Consists of or consists of the amino acid sequence of SEQ ID NO: 49, 50, or 73, or b) Consists of or comprises amino acid sequences that have at least 90% identity with SEQ ID NOs: 49, 50, or 73, e.g., 95%, 96%, 97%, 99%, or higher, and include knob mutations (e.g., S354C and T366W), and / or The Fc region containing the (ii) hole mutation is a) Containing or consisting of the amino acid sequence of SEQ ID NO: 47, 48, or 74, or b) Having at least 90% identity with SEQ ID NOs. 47, 48, or 74, for example, 95%, 96%, 97%, 99%, or more, and containing or consisting of an amino acid sequence that includes hole mutations (e.g., Y349C, T366S, L368A, and Y407V), The bispecific antibody according to claim 21.

23. The bispecific antibody comprises a first antigen-binding region, a second antigen-binding region, and an Fc dimer, wherein the first antigen-binding region is a Fab fragment that specifically binds to CD3, and the second antigen-binding region is a VHH that specifically binds to LILRB4, for example, the VHH described in any one of claims 1 to 3 and 8, for example, the bispecific antibody comprises one first antigen-binding region and one or two second antigen-binding regions. A bispecific antibody according to any one of claims 9 to 22.

24. It contains one anti-LILRB4 VHH, one anti-CD3 antibody Fab fragment, and an Fc heterodimer, of which, The Fab fragment comprises VH-CH1 and VL-CL, and the VHH comprises the heavy chain variable region. Of these, the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region (for example, the Fc region containing the Knob mutation) to form the first heavy chain. The VHH is fused to the CH2 or hinge region of the second Fc region (for example, the Fc region containing the Hole mutation) to form a second heavy chain. and The VL-CL of the aforementioned Fab fragment constitutes a light chain. The bispecific antibody according to claim 23.

25. The aforementioned The first heavy chain comprises or consists of the amino acid sequence shown in Sequence ID No. 44, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence. The second heavy chain comprises or consists of the amino acid sequence shown in Sequence ID No. 45, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned amino acid sequence, and / or The light chain includes, or consists of, the amino acid sequence shown in Sequence ID No. 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence. The bispecific antibody according to claim 24.

26. It contains two anti-LILRB4 VHHs, one anti-CD3 antibody Fab fragment, and an Fc heterodimer, of which, The Fab fragment comprises VH-CH1 and VL-CL, and the VHH comprises the heavy chain variable region. Of these, the C-terminus of the first VHH fragment is fused to the N-terminus of the VH of the Fab fragment, and the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the Fc region (e.g., including a knob mutation) to form the first heavy chain. The second VHH is fused to the CH2 or hinge region of the Fc region (e.g., including a hole mutation) to form a second heavy chain. and The VL-CL of the aforementioned Fab fragment constitutes a light chain. The bispecific antibody according to claim 23.

27. The first VHH and the second VHH are homologous or different. The bispecific antibody according to claim 26.

28. The fusion of the first VHH and the Fab is carried out via a linked peptide, for example, (GGGGS)n, where n = 1, 2, 3, or 4. The bispecific antibody according to claim 26 or 27.

29. The aforementioned The first heavy chain comprises or consists of the amino acid sequence shown in Sequence ID No. 46, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence. The second heavy chain comprises or consists of the amino acid sequence shown in Sequence ID No. 45, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned amino acid sequence, and / or The light chain includes, or consists of, the amino acid sequence shown in Sequence ID No. 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence. The bispecific antibody according to claim 26.

30. It contains two anti-LILRB4 VHHs, one anti-CD3 antibody scFv and Fc heterodimers, of which, The scFv sequentially includes VH and VL from the N-terminus to the C-terminus, and the VHH includes the heavy chain variable region. Of these, the C-terminus of the first VHH is fused to the N-terminus of the VH of the scFv, and the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the Fc region (for example, including a knob mutation), thereby forming the first heavy chain, The second VHH is fused with an Fc region (e.g., containing a hole mutation) to form a second heavy chain (for example, the C-terminus of the VHH is fused with the CH2 or hinge region of the second Fc region). The bispecific antibody according to claim 23.

31. The VH and VL of the scFv are linked via a linker or linking peptide, for example, the linker or linking peptide contains or consists of the amino acid sequence shown in Sequence ID No.

70. The bispecific antibody according to claim 30.

32. The first VHH and the second VHH are homologous or different. The bispecific antibody according to claim 30 or 31.

33. The fusion of the first VHH and the scFv is carried out via a linked peptide, for example, (GGGGS)n, where n = 1, 2, 3, or 4. A bispecific antibody according to any one of claims 30 to 32.

34. The aforementioned The first heavy chain comprises or consists of the amino acid sequence shown in Sequence ID No. 72, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, and / or The second heavy chain includes, or consists of, the amino acid sequence shown in Sequence ID No. 45, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned amino acid sequence. The bispecific antibody according to claim 30.

35. A multispecific antibody comprising a VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, wherein the multispecific antibody is a bispecific antibody. Multispecific antibodies.

36. A nucleic acid sequence comprising: a VHH antibody according to any one of claims 1 to 3 and 8, a heavy chain antibody according to any one of claims 4 to 8, a chain of any one of the bispecific antibodies according to any one of claims 9 to 34, or a chain of any one of the multispecific antibodies according to claim 35; Nucleic acid molecule.

37. The nucleic acid molecule described in claim 36, Expression vector.

38. A host cell comprising the nucleic acid molecule described in claim 36 or the expression vector described in claim 37, preferably the host cell being prokaryotic or eukaryotic, for example, a 293 cell or a CHO cell, for example, a 293F cell or a 293T cell or a CHO-S cell. host cell.

39. A method for preparing a VHH antibody according to any one of claims 1 to 3 and 8, a heavy chain antibody according to any one of claims 4 to 8, a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35, comprising: culturing host cells containing a nucleic acid molecule according to claim 36 or an expression vector according to claim 37 under conditions suitable for the chain expression of the antibody; and optionally recovering the antibody from the host cells (or host cell culture medium). method.

40. The present invention comprises a VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, or a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35. Immune complex.

41. A VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, or a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35, or an immune complex according to claim 40, and optionally a medicinal auxiliary material. Pharmaceutical composition, drug, or preparation.

42. A VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, or a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35, or an immune complex according to claim 40, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators), comprising Drug combination products.

43. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, or a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35, or an immune complex according to claim 40, or a pharmaceutical composition, drug or preparation according to claim 41, or a drug combination product according to claim 42. method.

44. The tumor cells of the aforementioned cancer have LILRB4 protein and / or nucleic acid levels, or have elevated LILRB4 protein and / or nucleic acid levels (e.g., elevated expression). The method according to claim 43.

45. The cancer is a solid tumor or a hematological malignancy, for example, a solid tumor, multiple myeloma, or leukemia, for example, acute myeloid leukemia (AML), for example, monocytic AML and non-monocytic AML, for example, human myelomonocytic leukemia cells, human promyelocytic acute leukemia, or human acute myeloid leukemia. The method according to claim 43 or 44.

46. This further includes administering the drug in combination with other therapies, such as treatment methods (e.g., radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators). The method according to any one of claims 43 to 45.

47. A method for detecting the presence of LILRB4 in a biological sample, (i) Contacting a biological sample with a VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, or a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35, under conditions that enable its binding to LILRB4, (ii) To detect whether a complex has been formed between the antibody or bispecific antibody and LILRB4, Includes, Among these, the formation of the composite indicates the presence of LILRB4. method.

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