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CN-121975011-A - Monospecific and multispecific antibodies

CN121975011ACN 121975011 ACN121975011 ACN 121975011ACN-121975011-A

Abstract

Disclosed herein are monospecific and multispecific single chain antibodies specific for one or more of CD47, PD-L1, HSA, CD33, LAG3, and CD 16.

Inventors

  • LIANG YANBIN

Assignees

  • 北京烁星生物医药科技有限公司

Dates

Publication Date
20260505
Application Date
20200928
Priority Date
20190927

Claims (20)

  1. 1. A heavy chain Variable (VHH) domain having antigen binding specificity for one of: (a) CD16, wherein the VHH domain has an amino acid sequence as shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) PD-L1 wherein said VHH region has an amino acid sequence as set forth in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) Human Serum Albumin (HSA), wherein the VHH domain has an amino acid sequence shown as one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) CD33, wherein the VHH domain has an amino acid sequence as set forth in one of SEQ ID NO 50 through SEQ ID NO 78; (e) LAG3, wherein the VHH domain has the amino acid sequence shown in one of SEQ ID NO 80 to SEQ ID NO 93, or (F) CD47, wherein said VHH domain has an amino acid sequence as shown in one of SEQ ID NO. 2 to SEQ ID NO. 29 or SEQ ID NO. 223.
  2. 2. A heavy chain-only antibody (HCAb) comprising the VHH domain of claim 1.
  3. 3. A multispecific antibody comprising an antibody binding domain having a first binding specificity and a second antibody binding domain having a second binding specificity different from the first binding specificity, wherein the first binding specificity is specific for CD47, PD-L1, HSA, CD33, LAG3 or CD16, wherein (A) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by an amino acid sequence shown by one of SEQ ID NO. 38, SEQ ID NO. 37, SEQ ID NO. 31 to SEQ ID NO. 36; (c) The HSA binding specificity is represented by the amino acid sequence set forth in one of SEQ ID NOs 40 to 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 50 to SEQ ID NO. 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO 80 to SEQ ID NO 93, and (F) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 2 to SEQ ID NO. 29 or SEQ ID NO. 223.
  4. 4. The multi-specific antibody of claim 3, wherein the second antibody binding domain is specific for CD47, PD-L1, HSA, CD33, LAG3 or CD16, wherein (A) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by an amino acid sequence shown by one of SEQ ID NO. 38, SEQ ID NO. 37, SEQ ID NO. 31 to SEQ ID NO. 36; (c) The HSA binding specificity is represented by the amino acid sequence set forth in one of SEQ ID NOs 40 to 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 50 to SEQ ID NO. 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO 80 to SEQ ID NO 93, and (F) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 2 to SEQ ID NO. 29 or SEQ ID NO. 223.
  5. 5. The multispecific antibody of claim 3, further comprising 1 to 5 additional antibody-binding domains, wherein each additional antibody-binding domain is individually specific for CD47, PD-L1, HSA, CD33, LAG3 or CD16, wherein (A) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by an amino acid sequence shown by one of SEQ ID NO. 38, SEQ ID NO. 37, SEQ ID NO. 31 to SEQ ID NO. 36; (c) The HSA binding specificity is represented by the amino acid sequence set forth in one of SEQ ID NOs 40 to 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 50 to SEQ ID NO. 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO 80 to SEQ ID NO 93, and (F) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 2 to SEQ ID NO. 29 or SEQ ID NO. 223.
  6. 6. The multispecific antibody of claim 4, further comprising 1 to 4 additional antibody-binding domains, wherein each additional antibody-binding domain is specific for CD47, PD-L1, HSA, CD33, LAG3 or CD16, wherein (A) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by an amino acid sequence shown by one of SEQ ID NO. 38, SEQ ID NO. 37, SEQ ID NO. 31 to SEQ ID NO. 36; (c) The HSA binding specificity is represented by the amino acid sequence set forth in one of SEQ ID NOs 40 to 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 50 to SEQ ID NO. 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO 80 to SEQ ID NO 93, and (F) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO. 2 to SEQ ID NO. 29 or SEQ ID NO. 223.
  7. 7. The multispecific antibody of any one of claims 3-6, wherein the antibody is a multispecific single-chain antibody (MVSCA).
  8. 8. The multispecific antibody of any one of claims 3-7, wherein a linker L1 (SEQ ID NO: 100), L2 (SEQ ID NO: 101) or L4 (SEQ ID NO: 103) is interposed between one or more pairs of different antibody binding domains.
  9. 9. The multispecific antibody of any one of claims 3-8, comprising at least one pair of antibody binding domains having the same specificity.
  10. 10. The multispecific antibody of claim 9, wherein the at least one pair of antibody binding domains of identical specificity are adjacent to each other with a linker L3 (SEQ ID NO: 102) interposed therebetween.
  11. 11. The multispecific antibody of any one of claims 3-10, comprising an N-terminally or C-terminally positioned HSA-specific antibody binding domain with a cleavable linker interposed between the antibody binding domain and the antibody binding domain adjacent thereto.
  12. 12. The multispecific antibody of claim 11, wherein the cleavable linker is L11*3（SEQ ID NO:104）、L11*4（SEQ ID NO:105）、L11*5（SEQ ID NO:106）、L11*6（SEQ ID NO:107）、L11*7（SEQ ID NO:108）、L11*8（SEQ ID NO:109）、L11*9（SEQ ID NO:110）、L11*10（SEQ ID NO:111）、L11*11（SEQ ID NO:112）、L11*12（SEQ ID NO:113）、L11*13（SEQ ID NO:114）、L11*14（SEQ ID NO:115）、L11*15（SEQ ID NO:116）、L11*16（SEQ ID NO:117）、L11*17（SEQ ID NO:118） or L11 x 18 (SEQ ID NO: 119).
  13. 13. The multispecific antibody of any one of claims 3-12, wherein all of the antibody binding domains are VHH domains.
  14. 14. The multispecific antibody of any one of claims 3-13, comprising an antibody-binding domain that recognizes HSA, CD47, and PD-L1.
  15. 15. The multispecific antibody of any one of claims 3-13, comprising an antibody-binding domain that recognizes HSA, CD47, and CD 33.
  16. 16. The multispecific antibody of any one of claims 3-13, comprising an antibody-binding domain that recognizes HSA, LAG3, and PD-L1.
  17. 17. The multispecific antibody of any one of claims 3-13, comprising an antibody-binding domain that recognizes HSA, LAG3, and CD 33.
  18. 18. The multispecific antibody of any one of claims 3-13, comprising an antibody-binding domain that recognizes CD16, HSA, and PD-L1.
  19. 19. The multispecific antibody of any one of claims 3-13, comprising an antibody-binding domain that recognizes CD16, HSA, and CD 33.
  20. 20. The multispecific antibody of any one of claims 3-13, CD16, HSA, CD47, and PD-L1.

Description

Monospecific and multispecific antibodies The present invention is a divisional application of 202080082179.3 patent application of the invention of which the application date is 9/28/2020 and the invention name is "monospecific and multispecific antibody". Cross Reference to Related Applications The present application claims the benefit of U.S. provisional patent application 62/907,275 filed on day 27 of 9 and U.S. provisional patent application 62/989,327 filed on day 13 of 2020, both of which are incorporated by reference in their entireties. Disclosure of Invention Monospecific heavy chain-only antibodies (HCAbs) specific for CD47, human Serum Albumin (HSA), PD-L1, CD33, CD16 and LAG3, and multivalent single chain antibodies incorporating two or more HCAb variable domains specific for one or more of these antigens are disclosed herein. Some embodiments are single domain antibodies comprising exclusively or predominantly VHH domains of camelidae antibodies. These embodiments are monospecific and monovalent. Some embodiments are hcabs, or comprise VHH domains fused to one or more constant domains from conventional antibodies (e.g., fc regions of human IgG antibodies). These embodiments are monospecific, but are typically divalent. Other titers are also possible depending on, for example, the choice of constant domain. The Fc regions of IgA and IgM may confer higher titers. Some embodiments comprise two VHH domains (multivalent single chain antibodies) that are specific for the same antigen joined in a single amino acid chain. These embodiments are also monospecific and bivalent. Additional VHH domains can be conjugated to achieve higher titers. Some embodiments comprise two (or more) VHH domains, wherein each VHH domain is specific for a different antigen that is joined in a single amino acid chain (multivalent, multi-specific single chain antibody). These embodiments are multivalent and multispecific. In other embodiments comprising three or more VHH domains, two or more VHH domains can be specific for the same antigen while one or more other VHH domains are specific for different antigens. Such constructs have higher order titers than the specificities. Each of the monospecific embodiments will have specificity for CD47, HSA, PD-L1, CD33, CD16 or LAG 3. Each of the multispecific embodiments is specific for one or more of CD47, HSA, PD-L1, CD33, CD16, and LAG3, but may also be specific for one or more other antigens. Some embodiments are specific for HSA and one or more other antigens. In one aspect of these embodiments, the HSA-specific domain confers extended in vivo half-life, while the other domains provide therapeutic effects. In another aspect of these embodiments, the HSA-specific domain may partially or completely inhibit the binding activity of an adjacent domain. The HSA-specific domains may be joined by a cleavable linker that is cleaved by a protease present at the intended site of action (e.g., in a tumor) such that cleavage releases inhibition of the adjacent domain. Some multispecific embodiments are trispecific. In some embodiments comprising multiple antigen-binding domains, antigen-binding domains derived from conventional VL-VH pairings may be used in place of one or more (but not all) of the VHH domains in the embodiments described above. The antigen binding domains disclosed herein that are specific for a particular antigen may be referred to as means for binding to the antigen. Drawings FIG. 1 depicts flow cytometry analysis of the binding affinity of anti-CD 47 HCAb A09-10 and B6H12 to cell lines overexpressing CD47. FIG. 2 depicts a competitive ELISA binding assay for multispecific antibodies 1511 (SEQ ID NO: 156) and 3321 (SEQ ID NO: 157) having binding specificity for CD 47. FIG. 3 depicts competitive binding assays on Jurkat cell lines using flow cytometry for multispecific molecules 1511 and 3321 that bind CD 47. Fig. 4A depicts a human Red Blood Cell (RBC) hemagglutination assay using multi-specific molecules 1511 and 3321 that bind CD 47. Hu5F9 served as a control. FIG. 4B depicts 1511 and 3321 binding to HL-60 cells and human RBC. FIG. 5 depicts the anti-tumor activity of CD 47-binding multispecific molecule 3321 in Raja-Luc xenograft mice. FIG. 6 depicts flow cytometry binding assays of anti-PD-L1 HCAb, PL14 and PL16 performed on PD-L1 overexpressing CHO cells. Aote Zhu Shankang (Atezolizumab) was used as a control. FIG. 7 cell-based functional assay of multispecific molecule 1511 with binding specificity to PD-L1 and Ab Zhu Shan antibody as control. FIGS. 8A and 8B depict inhibition of MC38-hPD-L1 tumor growth in B-hPD-L1 mice by a PD-L1-binding multispecific molecule 1518 (SEQ ID NO: 135). Fig. 9 depicts the Octet ® binding assay of anti-HSA VHH antibodies. Fig. 10A-10B depict Octet ® binding affinity assays of anti-CD 33 VHH antibodies. Fig. 11A-11B depict Octet ® binding assays of anti-CD 16A VHH molecules CD16F1 (fig. 11A) and CD16E11 (fig. 11B). FIG. 12 depicts a ce