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WO2024153068A1 - Anticorps à domaine unique anti-pdl1, protéine de fusion et son utilisation - Google Patents

Anticorps à domaine unique anti-pdl1, protéine de fusion et son utilisation Download PDF

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Publication number
WO2024153068A1
WO2024153068A1 PCT/CN2024/072523 CN2024072523W WO2024153068A1 WO 2024153068 A1 WO2024153068 A1 WO 2024153068A1 CN 2024072523 W CN2024072523 W CN 2024072523W WO 2024153068 A1 WO2024153068 A1 WO 2024153068A1
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Prior art keywords
fusion protein
amino acid
pdl1
seq
antibody
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Ceased
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PCT/CN2024/072523
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Inventor
Dawei Sun
Haixia JIANG
Rui Gao
Yanan GENG
Yue Wu
Chunnian WANG
Wenqiang Lu
Xiaoli Guo
Jingfeng Yu
Xiaofeng NIU
Hongtao Lu
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Elpiscience Biopharma Ltd
Elpiscience Suzhou Biopharma Ltd
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Elpiscience Biopharma Ltd
Elpiscience Suzhou Biopharma Ltd
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Priority to KR1020257027445A priority Critical patent/KR20250139314A/ko
Priority to CN202480008177.8A priority patent/CN120603849A/zh
Priority to EP24744243.7A priority patent/EP4652201A1/fr
Publication of WO2024153068A1 publication Critical patent/WO2024153068A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure relates to single domain antibodies to Programmed cell death 1 ligand 1 (PDL1) , fusion proteins and uses thereof.
  • PDL1 Programmed cell death 1 ligand 1
  • PD-L1 Programmed death 1 ligand 1
  • B7-H1 B7 protein family
  • IgV-like domain IgV-like domain
  • IgC-like domain IgC-like domain
  • transmembrane hydrophobic domain IgC-like domain
  • intracellular domain consisting of 30 amino acids.
  • PD-L1 is widely expressed on the surface of various immune cells, epithelial cells, and tumor cells.
  • PD-L1 binds to the programmed death-1 (PD-1) receptor and activates the negative regulation signaling pathway, to inhibit T cell activation or induce T cell apoptosis, thereby suppressing the immune responses (Freeman et al., 2000J Exp Med 192: 1027) .
  • PDL1 has an effect of negatively regulating immune responses, cancer cells induce T cell apoptosis by up-regulating PD-L1 expression, to escape from elimination by immune system, thus leading to the disease progression.
  • Antibody drug development for immunological checkpoints is a potential target for the treatment of various types of cancers.
  • the traditional monoclonal antibodies have a large molecular weight, and thus have difficulty in penetrating the tissues, resulting in low effective concentration in the tumor area and poor therapeutic effect.
  • Single domain antibodies are currently the smallest antibody molecules, and have unique functional characteristics, such as a low molecular weight, high stability, easy expression, simple humanization and low preparation costs, which may overcome the shortcomings of traditional antibodies.
  • the immunogenicity caused by a biological drug often determines its efficacy and safety in clinical use, and thus is a key to its successful development for clinical use.
  • There is still a lack of satisfactory anti-PDL1 single domain antibodies and still remain a need to develop anti-PDL1 single domain antibodies, and fusion proteins involving anti-PDL1 single domain antibodies.
  • the present disclosure relates to anti-PDL1 single domain antibodies as describes herein.
  • the present disclosure relates to single domain antibodies that bind to human PDL1, fusion proteins and pharmaceutical compositions based on the single domain antibody and uses thereof.
  • novel anti-PDL1 single domain antibodies which includes heavy chain complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 is different from an amino acid sequence set forth in SEQ ID NO: 32, 36 or 39 by no more than 3, 2 or 1 amino acid; the CDR2 is different from an amino acid sequence set forth in SEQ ID NO: 33, 35 or 37 by no more than 3, 2 or 1 amino acid; and the CDR3 is different from an amino acid sequence set forth in SEQ ID NO: 34 or 38 by no more than one amino acid.
  • CDRs heavy chain complementarity determining regions
  • the anti-PDL1 single domain antibody includes CDR1, CDR2, and CDR3 having amino acid sequences about 80%or above (e.g. 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 32, 33, and 34, respectively; (2) SEQ ID NOs: 32, 35, and 34, respectively; (3) SEQ ID NOs: 36, 37, and 38, respectively; or (4) SEQ ID NOs: 39, 37, and 38, respectively.
  • the anti-PDL1 single domain antibody includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 1-21. In some embodiments, the anti-PDL1 single domain antibody includes an amino acid sequence 100%identical to one of amino acid sequences set forth in SEQ ID NOs: 1-21.
  • the anti-PDL1 single domain antibody binds to human, cynomolgus and/or mouse PDL1, e.g., human PDL1.
  • the anti-PDL1 single domain antibody blocks the interaction of PD-1/PD-L1, e.g., human PD1 and humanPDL1.
  • the anti-PDL1 single domain antibody enhances IFN- ⁇ production.
  • the anti-PDL1 single domain antibody is a humanized antibody or chimeric antibody.
  • a fusion protein including a first domain and a second domain.
  • the first domain binding to PDL1 includes heavy chain complementarity determining regions CDR1, CDR2, and CDR3 having amino acid sequences about 80%to about 100% (e.g. 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 32, 33, and 34, respectively; (2) SEQ ID NOs: 32, 35, and 34, respectively; (3) SEQ ID NOs: 36, 37, and 38, respectively; or (4) SEQ ID NOs: 39, 37, and 38, respectively.
  • the second domain binds to SIRP ⁇ .
  • the first domain includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 1-21. In some embodiments, the first domain is selected from the group consisting of amino acid sequences set forth in SEQ ID NOs: 1-21.
  • the second domain binds to SIRP ⁇ expressed by effector cells.
  • the second domain binds to human SIRP ⁇ expressing by effector cells (such as macrophages, NK cells, cytotoxic T cells) . In some embodiments, the second domain binds to effector cells expressing SIRP ⁇ , such as human SIRP ⁇ .
  • the second domain includes a heavy chain variable region (VH) paired with a light chain variable region (VL) .
  • VH includes CDR1, CDR2, and CDR3 having amino acid sequences about 80%to about 100% (e.g., 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or more) identical to amino acid sequences set forth in: (1) SEQ ID NOs: 40, 41, and 42, respectively; (2) SEQ ID NOs: 40, 43, and 44, respectively.
  • the VL includes CDR1, CDR2, and CDR3 having amino acid sequences about 80%to about 100% (e.g., 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or more) identical to amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively.
  • the second domain includes a light chain.
  • the light chain is a kappa or lamda light chain.
  • the light chain includes a constant region (CL) having an amino acid sequence set forth in SEQ ID NO: 30 or 31.
  • the second domain includes a heavy chain constant region 1 (CH1) . In some embodiments, the second domain is an anti-SIRP ⁇ Fab fragment.
  • the second domain includes a VH-CH1 fragment paired with the light chain.
  • the VH-CH1 fragment has an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 22, and the light chain has an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 23.
  • the VH-CH1 fragment has an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 24, and the light chain has an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the first domain and the second domain are covalently linked via a Fc fragment.
  • the first domain is fused to N-terminus of Fc fragment and the second domain is fused to C-terminus of Fc fragment.
  • the fusion protein is a symmetric bispecific molecule, wherein two first domains are fused to N-terminus of the Fc fragment and two second domains are fused to C-terminus of the Fc fragment.
  • the Fc fragment is derived from human immunoglobulin, such as IgG4 or IgG1.
  • the Fc fragment is human IgG4 with mutation S228P, or human IgG1 with mutation C220S, numbered by EU numbering scheme.
  • the Fc fragment includes an amino acid sequence set forth in SEQ ID NO: 26 or 27.
  • the Fc fragment and the second domain are covalently linked via a peptide linker, optionally the having the formula (Gly4Ser) n, where n is an integer from 1 to 5. In some embodiments, n is 3 or 4, i.e., (Gly4Ser) 3 or (Gly4Ser) 4.
  • the fusion protein includes a heavy chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 28, and a light chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 48, and a light chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the fusion protein enhances effector cells phagocytosis of tumor cells (e.g., cancer cells) , optionally the tumor cells express target antigen to which the first domain binds, such as human PDL1.
  • the fusion protein binds to human PDL1 and human SIRP ⁇ .
  • the fusion protein blocks the interaction of PD1 and PDL1, optionally, the fusion protein blocks the interaction of PD1 and PDL1 with EC 50 no more than 10, 9, 8, or 7 nM.
  • the EC 50 of binding activity of the fusion protein to the SIRP ⁇ is no more than 10, 5, 4, 3, 2, or 1 nM.
  • the fusion protein barely induces the phagocytosis to human monocytes or human PBMCs. In some embodiments, the fusion protein at least partially reprograms macrophages to M1 or M1-like macrophages.
  • the fusion protein stimulates macrophage to produce inflammatory cytokine, which includes TNF-a, IL-6 and CXCL-10. In some embodiments, the fusion protein barely induces PBMC to produce cytokines, which includes IL-2, IFN- ⁇ , TNF- ⁇ , IL-6 and IL-10.
  • provided herein is an isolated polynucleotide encoding the single domain antibody or the fusion protein described above.
  • a construct including the polynucleotide provided herein.
  • an antibody expressing system including the construct having the isolated polynucleotide described above or having a genome integrated with an exogenous polynucleotide described above, preferably, the expressing system is a cell expression system.
  • provided herein is a method for producing the anti-PDL1 single domain antibody or the fusion protein described above.
  • the method includes, under conditions suitable for expressing the antibody, expressing the antibody or fusion protein using the antibody expressing system described above.
  • composition including the anti-PDL1 single domain antibody or the fusion protein described above, and a pharmaceutically acceptable carrier.
  • kits including the anti-PDL1 single domain antibody, the fusion protein, the isolated polynucleotide, or the construct described above.
  • provided herein is a use of the anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition described above in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease, disorder, or condition.
  • the disease, disorder, or condition is tumor (e.g., cancer) .
  • tumor e.g., cancer
  • at least a tumor cell expresses PDL1, preferably human PDL1.
  • the tumor is a solid tumor.
  • the cancer comprises melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, and colon cancer.
  • provided herein is a method of treating a subject having a tumor, including administrating to the subject a therapeutically effective amount of the anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition provided herein.
  • the subject is mammal animals, such as human and non-human animals (e.g., mouse, and cynomolgus monkey) .
  • mammal animals such as human and non-human animals (e.g., mouse, and cynomolgus monkey) .
  • provided herein is a method of decreasing the rate of tumor growth or the number of tumor cells, including contacting a tumor cell with an effective amount of the anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition described above.
  • a method of killing a tumor cell including contacting a tumor cell with an effective amount of the anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition described above.
  • Figures 1A-1B show ELISA results of serum titer for the 3rd round immunization of alpaca #14 and #15, respectively.
  • Figures 2A-2B show ELISA results of serum titer for the 4th round immunization of alpaca #14 and #15, respectively.
  • Figures 3A-3B show ELISA results of serum titer for the 5th round immunization of alpaca #14 and #15, respectively.
  • Figure 4 shows agarose gel electrophoresis of total RNA from alpaca #14 and #15 (M is DNA marker, 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp and 2000bp from down to up; Lane 1 is RNA from #14 alpaca; Lane 2 is RNA from #15 alpaca) .
  • Figure 5 shows agarose gel electrophoresis of 1st round PCR product amplifying VHH (M is DNA marker, 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp from down to up; Lane 1 is PCR product for #14 alpaca; Lane 2 is PCR product for #15 alpaca)
  • Figure 6 shows agarose gel electrophoresis of 2nd round PCR product amplifying VHH (M is DNA marker, 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp from down to up; Lanes 1 and 2 are PCR products for #14 alpaca; Lanes 3 and 4 are PCR products for #15 alpaca)
  • Figure 7 shows agarose gel electrophoresis of 3rd round PCR product amplifying VHH (M is DNA marker, 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp from down to up; Lanes 1, 2 and 3 are PCR products for #14 alpaca; Lanes 3, 4 and 5 are PCR products for #15 alpaca)
  • Figure 8 shows flow cytometry results of clones 570 (A) and 2719 (B) .
  • Figure 9 shows FACS binding curve of C570 to Raji/hPDL1 cell line and EC 50 values (AEP247 is a benchmark antibody, and human IgG1 is used as negative control) .
  • Figure 10 shows FACS binding curve of C2719 to CHOK1/humanPDL1 cell line and EC 50 values (AEP247 is a benchmark antibody, and human IgG1 is used as negative control) .
  • Figure 11 shows FACS binding curve of C570 to CHOK1/cynoPDL1 cell line and EC 50 values (human IgG1 is used as negative control) .
  • Figure 12A-12C shows Bio-Layer Interferometry binding curves of AEP247, C2719 and C570 to human PDL1 ECD, respectively.
  • Figure 13 shows ELISA results of C570 and C2719 binding to human PDL1 ECD (human IgG1 is used as control) .
  • Figures 14A-14I shows ELISA results of C570 and C2719 binding to human B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, B7-DC, B7-1, B7-2 ECD (human IgG1 is used as control) , respectively.
  • Figure 15 shows blocking activities results in reporter assay of chimeric antibodies C2719 and EC 50 values ( is used as positive control, and hIgG1 is used as isotype control) .
  • Figure 16 shows blocking activities results in reporter assay of chimeric antibodies C570 and EC 50 values ( is used as positive control, and hIgG1 is used as isotype control) .
  • Figure 17 shows MLR assay results of C570, C2719 and AEP247 in enhancing IFN- ⁇ production (PD-1 is used as positive control, CD3/CD28 is used as positive control) .
  • Figure 18 shows concentration-time profiles of C570, C2719 and AEP247 in C57BL/6J-hCD47/hSIRP ⁇ transgenic mouse model.
  • Figure 19 shows concentration-time profiles of C570, C2719 and AEP247 in BALB/c-hPD1/hSIRP ⁇ mice model.
  • Figure 20 shows tumor growth inhibition curves of vehicle, AEP247, C570 and C2719 in MC38-hCD47/PDL1 mouse tumor model.
  • Figure 21 shows tumor growth inhibition curves of saline, AEP247, C570 and C2719 in CT26-hCD47/PDL1 mouse tumor model.
  • Figures 22 shows binding curve of C570 and C570 derived humanized variants to Raji/hPDL1 cell line by FACS detection.
  • Figures 23A-23B show binding curve of C2719 and C2719 derived humanized variants to Raji/hPDL1 cell line by FACS detection (hIgG1 is used as isotype) , respectively.
  • Figure 24 shows EC 50 values and blocking activities results in reporter assay of C570 and C570 derived humanized antibodies (hIgG1 iso is as an isotype control) .
  • Figures 25A-25B show EC 50 values and blocking activities results in reporter assay of C2719 and C2719 derived humanized antibodies (hIgG1 iso is as an isotype control) .
  • Figure 26 is a schematic drawing of an anti-PDL1/SIRP ⁇ bispecific molecule comprising one symmetric antibody with two anti-PDL1 sdAbs are fused to N-terminus of Fc and two anti-SIRP ⁇ Fabs are fused to C-terminus of Fc.
  • Figures 27A-27C show binding curves of representative bispecific antibodies to CHOK1/hPDL1 cells, CHOK1/SIRP ⁇ , Raji/hPDL1 cells by FACS detection, respectively.
  • Figure 28 shows results of macrophage phagocytosis of PDL1+ cancer cells in the presence of an isotype control human IgG1 (hIgG1) , anti-PDL1 antibody C570, anti-SIRP ⁇ antibody hu025.060 and anti-PDL1/SIRP ⁇ bispecific antibody.
  • hIgG1 isotype control human IgG1
  • anti-PDL1 antibody C570 anti-PDL1 antibody C570
  • anti-SIRP ⁇ antibody hu025.060 anti-PDL1/SIRP ⁇ bispecific antibody.
  • Figure 29 shows tumor growth inhibition curves of anti-PDL1 sdAb antibody, anti-SIRP ⁇ monoclonal antibody, anti-PDL1 antibody plus anti-SIRP ⁇ antibody (Combo) , and anti-PDL1/SIRP ⁇ bispecific antibody in BALB/c-hPD1/hSIRPa double knock-in mice.
  • Figure 30 shows the evaluation of ES019-116 in PD1/PDL1 blockade reporter cell line assay.
  • Figure 31A shows the phagocytosis activity of ES019-116 against Raji-PDL1 cells.
  • Figure 31B shows the phagocytosis activity of ES019-116 and PD-L1/SIRP ⁇ -HC2 against Raji-PDL1 cells.
  • Figure 32 shows the macrophage phagocytosis of ES019-116 to human monocytes (32A) and PBMCs (32B) , respectively.
  • Figure 33 shows the phagocytosis of Raji-hPD-L1 cells by macrophages derived from human PBMC in the presence of ES019-116 assessed by FACS-based macrophage phagocytosis assays.
  • hIgG1 represents a human IgG1 isotype control.
  • Figure 34 shows ES019-116 reprogramed macrophages to M1 phenotype by increasing the percentage of CD86 high CD163l ow macrophage population after 24 hours (34A) and 48 hours (34B) treatment, respectively.
  • M1-like macrophage (CD86 high CD163 low ) switch under the indicated treatments was measured by FACS.
  • hIgG1 is a human IgG1 isotype control.
  • Figure 35 shows the macrophage cytokine production TNF-a (A) , IL-6 (B) , CXCL10 (C) and IL-10 (D) stimulated by ES019-116 after 24 hours treatment under the indicated treatments measured by HTRF-based human TNF-a, IL-6, CXCL10 and IL-10 kits.
  • hIgG1 is a human IgG1 isotype control.
  • Figure 36 shows the macrophage cytokine production TNF-a (A) , IL-6 (B) , CXCL10 (C) and IL-10 (D) stimulated by ES019-116 after 48 hours treatment under the indicated treatments measured by HTRF-based human TNF-a, IL-6, CXCL10 and IL-10 kits.
  • hIgG1 is a human IgG1 isotype control.
  • Figure 37A-37E show the cytokine production (IFN- ⁇ (A) , IL-10 (B) , TNF- ⁇ (C) , IL-6 (D) and IL-2 (E) ) by in vitro human PBMC assay induced by ES019.
  • Figure 38 shows the concentration-time curve of ES019-116 in mice model.
  • Figure 39 shows the tumor volume-time curve of anti-PDL1/SIRP ⁇ bispecific antibodies ES019-116 in CT26 syngeneic model in Balb/c-hPD1/hSIRP ⁇ Knock-in mice.
  • Figure 40 shows the tumor volume-time curve of anti-PDL1/SIRP ⁇ bispecific antibodies ES019-116 in in CT26 syngeneic model in Balb/c-hPDL1/hPD1/hCD47/hSIRP ⁇ four knock-in mice.
  • sdAbs novel anti-PDL1 single domain antibodies
  • CDRs heavy chain complementarity determining regions
  • single domain antibody refers to a heavy chain variable region of a cloned antibody, and a single-domain antibody (VHH) constructed to consist of only one heavy chain variable region is the smallest antigen-binding fragment with complete functionality.
  • VHH single-domain antibody
  • an antibody with natural deletion of light chain and heavy chain constant region 1 (CH1) is derived from alpaca immune serum, and then a heavy chain variable region of the antibody is cloned, to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.
  • Single domain antibody includes four “framework regions” that are referred to as “framework region 1” or “FR1” , “framework region 2” or “FR2” , “framework region 3” or “FR3” , and “framework region 4” or “FR4” in the art and hereinafter.
  • the framework regions are spaced apart by three “complementarity determining regions” or “CDRs” that are referred to as “complementarity determining region 1” or “CDR1” , “complementarity determining region 2” or “CDR2” , and “complementarity determining region 3” or “CDR3” in the art and hereinafter.
  • the general structure or sequence of a single-domain antibody can be expressed as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • a single-domain antibody confers an antigen binding specificity to the antibody due to the presence of an antigen-binding site.
  • the CDR1 of anti-PDL1 single domain antibody is different from an amino acid sequence of SEQ ID NO: 32, 36 or 39 by no more than 3, 2 or 1 amino acid. In some embodiments, the CDR1 of anti-PDL1 single domain antibody has the amino acid sequence set forth in SEQ ID NO: 32, 36 or 39.
  • the CDR2 of anti-PDL1 single domain antibody is different from an amino acid sequence of SEQ ID NO: 33, 35 or 37 by no more than 3, 2 or 1 amino acid. In some embodiments, the CDR2 of anti-PDL1 single domain antibody has the amino acid sequence set forth in SEQ ID NO: 33, 35 or 37.
  • the CDR3 of anti-PDL1 single domain antibody is different from an amino acid sequence of SEQ ID NO: 34, or 38 by no more than one amino acid. In some embodiments, the CDR3 of anti-PDL1 single domain antibody has the amino acid sequence set forth in SEQ ID NO: 34 or 38.
  • amino acid refers to an organic compound containing amine (-NH 2 ) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid.
  • amine -NH 2
  • -COOH carboxyl
  • the anti-PDL1 single domain antibody includes CDR1, CDR2, and CDR3 having amino acid sequences about 80%or above (e.g. 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 32, 33, and 34, respectively; (2) SEQ ID NOs: 32, 35, and 34, respectively; (3) SEQ ID NOs: 36, 37, and 38, respectively; or (4) SEQ ID NOs: 39, 37, and 38, respectively.
  • CDR1, CDR2, and CDR3 of the anti-PDL1 single domain antibody have the amino acid sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively; SEQ ID NOs: 32, 35, and 34, respectively; SEQ ID NOs: 36, 37, and 38, respectively; or SEQ ID NOs: 39, 37, and 38, respectively.
  • the CDR is defined by Kabat and/or Chothia, which is well-known to the skilled in the art.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) .
  • Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987) ) .
  • the anti-PDL1 single domain antibody includes an amino acid sequence about 80%or above identical to one of amino acid sequences set forth in SEQ ID NOs: 1-21.
  • the identity is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%.
  • the anti-PDL1 single domain antibody includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%identical to one of amino acid sequences set forth in SEQ ID NOs: 1-21.
  • the anti-PDL1 single domain antibody has an amino acid sequence set forth in one of SEQ ID NOs: 1-21.
  • the percent (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For example, the identity can be determined using the BLAST program of the NCBI database.
  • the anti-PDL1 single domain antibody provided herein binds to human, cynomolgus and/or mouse PDL1, preferably, human PDL1. In certain embodiments, the anti-PDL1 single domain antibody specifically binds to human PDL1 and blocks the interaction of human PD1 and human PDL1.
  • PDL1 is a member of the B7 protein family (referred to as B7-H1) , the other members of B7 protein family, CD86 (B7-2) , B7-DC (PD-L2) , B7-H2, B7-H3 (CD276) , B7-H4, B7-H5, B7-H6, and/or B7-H7, barely bind to anti-PDL1 single domain antibody provided herein.
  • the anti-PDL1 single domain antibody showing good binding specificity to PDL1 (e.g., human PDL1) provided herein may lead to lower adverse reactions and improved clinical safety.
  • the anti-PDL1 single domain antibody provided herein increases the expression of interferon gamma (IFN- ⁇ ) and enhances the response of immune cells, thus, the anti-PDL1 single domain antibody could inhibit the growth of cells expressing PDL1, such as tumor cells.
  • IFN- ⁇ interferon gamma
  • the anti-PDL1 single domain antibody provided herein is humanized antibody or chimeric antibody.
  • the anti-PDL1 single domain antibody provided in the present disclosure can be a humanized antibody retaining at least one of the functional properties of the antibody, for example, the ability to specifically bind to PDL1 or block the interaction between PDL1 and PD1, stimulating killing effect of T cells on target cells, such as tumors.
  • humanized antibody refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, where the rest of the immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin.
  • the antigen binding site may include a complete variable domain fused to a constant domain, or only include a complementarity determining region (CDR) grafted into an appropriate framework region in the variable domain.
  • CDR complementarity determining region
  • the antigen binding site can be wild-type, or modified by one or more amino acid substitutions. For example, modifications are made to allow the antibody to be more similar to human immunoglobulins.
  • Some forms of humanized antibodies retain all CDR sequences (e.g., a humanized single-domain antibody including all three CDRs from alpaca) . Other forms have one or more CDRs that have been altered relative to the original antibody.
  • a “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse or alpaca antibody and the constant regions are derived from a human antibody.
  • humanized antibodies designed according to techniques in the art are all embraced in the present disclosure.
  • the humanized antibody further improves the drug safety, and effectively reduces the immunogenicity of the antibody.
  • the humanized antibody with modified framework region obtained in the present disclosure still maintains a high solubility, making it feasible for use in practical clinical application.
  • the anti-PDL1 single domain antibody shows higher affinity to PDL1 (e.g., human PDL1) ,
  • anti-PDL1 single domain antibody binds to human PDL1 with KD no more than 9.9E-8 M, or no more than 9.9E-9 M detected by Octet rule.
  • the KD value, or affinity constant is the equilibrium dissociation constant, or a ratio of Kd/Ka, between the antibody and its target antigen.
  • Kd refers to dissociation constant
  • Ka refers to association constant.
  • the anti-PDL1 single domain antibody binds to PDL1 (e.g., human PDL1) with EC 50 no more than 3 nM, 2 nM, 1.5 nM, 1 nM 0.65 nM, 0.62 nM, 0.60 nM, 0.58 nM, 0.55 nM, 0.5 nM, 0.45 nM, 0.4 nM, 0.35 nM, 0.3 nM, 0.25 nM, or no more than 0.2 nM detected by FACS (Fluorescence activated Cell Sorting) .
  • PDL1 e.g., human PDL1
  • FACS Fluorescence activated Cell Sorting
  • the anti-PDL1 single domain antibody blocks the interaction between PD-1 and PDL1 with EC 50 no more than 2 mM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 4 nM, 3 nM, 2 nM, 1 nM detected by reporter assay.
  • Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature environment and is an intrinsic property of antibodies that can influence product stability, such as aggregation, during manufacturing and storage.
  • the melting temperature (Tm) value may predict their thermal stability in general. Tm values of the anti-PDL1 single domain antibody provided herein range from 50-75°C, preferably 55-72°C, such as 68°C, 70°C, 71°C, or 72°C, indicating that the antibodies have good thermal stability.
  • the anti-PDL1 single domain antibody provided herein has good stability under pH 3.0-3.5 conditions.
  • the anti-PDL1 single domain antibody provided herein has good freeze-thaw stability. Freeze-thaw stability is often explored to determine the susceptibility of antibodies to temperature cycling which products are frequently exposed. For example, drug substance is often frozen to enable long-term storage. Drug product may be exposed to frozen temperature as part of the lyophilization process.
  • the major degradation pathway of freeze-thaw is aggregates, including precipitates, particles, and soluble particles.
  • the present disclosure relates to a fusion protein including a first domain and a second domain.
  • fusion when used with respect to amino acid sequences (e.g., peptide, polypeptide or protein) refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence.
  • a fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
  • the fusion protein includes bispecific antibody.
  • bispecific antibody or “bispecific molecule” as used herein, refers to an antibody or a molecule that displays a double binding specificity and affinity for two particular epitopes or a composition of antibodies in which all antibodies display a double binding specificity and affinity for two particular epitopes.
  • antibody generally refers to monoclonal antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope.
  • a naturally occurring “antibody” of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL) .
  • the light chain constant region is comprised of one domain.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the single domain antibody, fusion protein, bispecific antibody or monoclonal antibody could include conservatively modified variants, such as FR region.
  • the conservatively modified variants include individual substitutions, deletions or additions to the polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.
  • the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A) , Glycine (G) ; 2) Aspartic acid (D) , Glutamic acid (E) ; 3) Asparagine (N) , Glutamine (Q) ; 4) Arginine (R) , Lysine (K) ; 5) Isoleucine (I) , Leucine (L) , Methionine (M) , Valine (V) ; 6) Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W) ; 7) Serine (S) , Threonine (T) ; and 8) Cysteine (C) , Methionine (M) (see, e.g., Creighton, Proteins (1984) ) .
  • the term "conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • the first domain binds to PDL1 and includes heavy chain complementarity determining regions CDR1, CDR2, and CDR3, the CDR1, CDR2, and CDR3 have the amino acid sequences about 80%to about 100%identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 32, 33, and 34, respectively; (2) SEQ ID NOs: 32, 35, and 34, respectively; (3) SEQ ID NOs: 36, 37, and 38, respectively; or (4) SEQ ID NOs: 39, 37, and 38, respectively.
  • the first domain is an anti-PDL1 single-domain antibody including CDR1, CDR2, and CDR3 having amino acid sequences at least about 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or more, identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 32, 33, and 34, respectively; (2) SEQ ID NOs: 32, 35, and 34, respectively; (3) SEQ ID NOs: 36, 37, and 38, respectively; or (4) SEQ ID NOs: 39, 37, and 38, respectively.
  • CDR1, CDR2, and CDR3 of the first domain have the amino acid sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively; SEQ ID NOs: 32, 35, and 34, respectively; SEQ ID NOs: 36, 37, and 38, respectively; or SEQ ID NOs: 39, 37, and 38, respectively.
  • the first domain includes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% (e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%) identical to one of amino acid sequences set forth in SEQ ID NOs: 1-21.
  • the first domain includes or has an amino acid sequence set forth in one of SEQ ID NOs: 1-21.
  • the second domain of the fusion protein binds to another targeting molecule except PDL1, in some embodiments, the second domain binds to SIRP ⁇ , preferably human SIRP ⁇ . In some embodiments, the second domain binds to SIRP ⁇ expressed by effector cells.
  • the second domain of the fusion protein binds to immune effector cells, e.g., macrophages, NK cells, T cells, B cells, and Dendritic cells, to enhance the immune response directly or indirectly.
  • immune effector cells e.g., macrophages, NK cells, T cells, B cells, and Dendritic cells.
  • the effector cell is macrophage.
  • SIRP ⁇ Signal regulatory protein ⁇
  • SIRP ⁇ is a regulatory membrane glycoprotein from SIRP family. It is mainly expressed by myeloid cells and also by stem cells or neurons. SIRP ⁇ acts as inhibitory receptor and interacts with a broadly expressed transmembrane protein CD47. This interaction negatively controls effector function of innate immune cells such as host cell phagocytosis. SIRP ⁇ diffuses laterally on the macrophage membrane and accumulates at a phagocytic synapse to bind CD47, which inhibits the cytoskeleton-intensive process of phagocytosis by the macrophage.
  • the second domain of the fusion protein binds to SIRP ⁇ (human SIRP ⁇ ) to block the combination of SIRP ⁇ and CD47, eliminating inhibitory effect of SIRP ⁇ .
  • the second domain includes a heavy chain variable region (VH) paired with a light chain variable region (VL) .
  • VH includes CDR1, CDR2, and CDR3 having amino acid sequences about 80%to about 100% (e.g., 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or more) identical to amino acid sequences of SEQ ID NO: 40, 41, and 42, respectively; or, SEQ ID NO: 40, 43, and 44, respectively.
  • the VL includes CDR1, CDR2, and CDR3 having amino acid sequences about 80%to about 100% (e.g., 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or more) identical to amino acid sequences of SEQ ID NO: 45, 46, and 47, respectively.
  • CDR1, CDR2, and CDR3 of the VH of the second domain have amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively; and the CDR1, CDR2, and CDR3 of VL have amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively.
  • CDR1, CDR2, and CDR3 of the VH of the second domain have amino acid sequences set forth in SEQ ID NOs: 40, 43, and 44, respectively; and the CDR1, CDR2, and CDR3 of VL have amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively.
  • the second domain includes a light chain.
  • the light chain is a kappa ( ⁇ ) or lamda ( ⁇ ) light chain.
  • the light chain includes a constant region (CL) having an amino acid sequence about 80%to about 100% (e.g., 80%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or more) identical to the amino acid sequence set forth in SEQ ID NO: 30 or 31.
  • the CL includes an amino acid sequence set forth in SEQ ID NO: 30 or 31.
  • the second domain includes a heavy chain constant region 1 (CH1) . In some embodiments, the second domain is an anti-SIRP ⁇ Fab fragment.
  • Fab fragment refers to the region of the antibody structure that can bind to antigen. It consists of a complete light chain (variable and constant regions) and part of the heavy chain structure (variable and a constant region 1 fragment, VH-CH1) , the light and heavy chains are connected by a disulfide bond. Fab fragments can be obtained by protease digestion of full-length antibodies.
  • the second domain includes a VH-CH1 fragment paired with the light chain.
  • the VH-CH1 fragment has an amino acid sequence about 80%to about 100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequence set forth in SEQ ID NO: 22, and the light chain has an amino acid sequence about 80%to about 100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequence set forth in SEQ ID NO: 23.
  • the VH-CH1 has an amino acid sequence about 80%to about 100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequence set forth in SEQ ID NO: 24, and the light chain has an amino acid sequence about 80%to about 100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the VH-CH1 has an amino acid sequence set forth in SEQ ID NO: 22, and the light chain has an amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the VH-CH1 has an amino acid sequence set forth in SEQ ID NO: 24, and the light chain has an amino acid sequence set forth in SEQ ID NO: 25.
  • the first domain and the second domain are covalently linked via a Fc fragment, optionally, the first domain is fused to N-terminus of the Fc fragment and the second domain is fused to C-terminus of the Fc fragment.
  • the fusion protein is a symmetric bispecific molecule, wherein two first domains are fused to N-terminus of Fc and two second domains are fused to C-terminus of Fc fragment.
  • the C-terminus of the first domain is fused to the N-terminus of the fragment, and/or the C-terminus of the VH-CH1 fragment is fused to the C-terminus of the fragment. See figure 26, showing a schematic drawing of the fusion protein provided.
  • an “Fc region” fragment crystallizable region or “Fc domain” or “Fc fragment” refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system.
  • an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL) .
  • the Fc region comprises two identical protein fragments, derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the Fe region comprises immunoglobulin domains C ⁇ 2 and C ⁇ 3 and the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226 or P230 (or amino acid between these two amino acids) to the carboxy terminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat.
  • the CH2 domain of a human IgG Fc region extends from about amino acid 231 to about amino acid 340, whereas the CH3 domain is positioned on C-terminal side of a CH2 domain in an Fc region, i.e., it extends from about amino acid 341 to about amino acid 447 of an IgG.
  • the Fc region may be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc) .
  • Fc may also refer to this region in isolation or in the context of an Fc-comprising protein polypeptide.
  • the Fc fragment is derived from human immunoglobulin, optionally IgG4 or IgG1. In some embodiments, the Fc fragment is human IgG4 or IgG1 with 0, 1, 2, or more mutations. In some embodiments, the Fc fragment is human IgG4 with mutation S228P, or human IgG1 with mutation C220S, numbered by EU numbering scheme. In some embodiments, the Fc fragment includes an amino acid sequence set forth in SEQ ID NO: 26 or 27.
  • the fusion protein includes a heavy chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 28, and a light chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 48, and a light chain having an amino acid sequence at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the fusion protein includes a heavy chain having an amino acid set forth in SEQ ID NO: 28, and a light chain having an amino acid set forth in SEQ ID NO: 23; or a heavy chain having an amino acid set forth in SEQ ID NO: 48, and a light chain having an amino acid set forth in SEQ ID NO: 25.
  • the Fc fragment and the second domain are covalently linked via a peptide linker
  • the peptide linker has the formula (Gly4Ser) n, where n is 1, 2, 3, 4, or 5. In some embodiments, n is 3 or 4, i.e., the linker is (Gly4Ser) 3, or (Gly4Ser) 4.
  • the fusion protein enhances effector cells phagocytosis of tumor cells (such as cancer cells) . In some embodiments, the fusion protein enhances effector cells phagocytosis of tumor cells expressing target antigen to which the first domain binds, such as human PDL1.
  • target antigen to which the first domain binds such as human PDL1.
  • the fusion protein barely induces the phagocytosis to human monocytes or human PBMCs. This is benefit for improving the safety of the fusion protein and lowering risk to induce systemic cytokine release syndrome.
  • the fusion protein at least partially reprograms macrophages to M1 or M1-like macrophages.
  • the method to estimate the type of macrophages is well-known to those skilled in the art, such as by testing the macrophage population with the indicated biomarker expressing on the specific macrophage, for example, by testing the increased percentage of proinflammatory M1 phenotype (CD86 high CD163 low ) macrophage population, the method to define “high” or “low” is well-known to those skilled in the art, such flow cytometry staining.
  • the fusion protein stimulates macrophage to produce inflammatory cytokine including tumor necrosis factor alpha (TNF- ⁇ ) , interleukin 6 (IL-6) and C-X-C motif chemokine ligand 10 (CXCL-10) .
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-6 interleukin 6
  • CXCL-10 C-X-C motif chemokine ligand 10
  • the fusion protein barely induces PBMC to produce cytokines, which includes interleukin 2 (IL-2) , IFN- ⁇ , TNF- ⁇ , IL-6 and interleukin 10 (IL-10) .
  • cytokines which includes interleukin 2 (IL-2) , IFN- ⁇ , TNF- ⁇ , IL-6 and interleukin 10 (IL-10) .
  • IL-2 interleukin 2
  • IFN- ⁇ IFN- ⁇
  • TNF- ⁇ TNF- ⁇
  • IL-6 interleukin 10
  • the fusion protein blocks the interaction of PD1 and PDL1, optionally, the fusion protein blocks the interaction of PD1 and PDL1 with EC 50 no more than 10, 9, 8, or 7 nM.
  • the fusion protein binds to SIRP ⁇ (e.g., human SIRP ⁇ ) with EC 50 no more than 10, 5, 4, 3, 2, or 1 nM. In some embodiments, the fusion protein binds to SIRP ⁇ (e.g., human SIRP ⁇ ) with Dissociation constant (KD) value no more than 9.9E-07, 1E-07, 9.9E-08, 1E-08, or 9.9E-09.
  • SIRP ⁇ e.g., human SIRP ⁇
  • KD Dissociation constant
  • the fusion protein herein has a good physicalchemistory property with relatively low hydrophobicity, which provides desired productivity and developability.
  • the fusion protein herein also has good thermal stability.
  • the present disclosure provides an isolated polynucleotide encoding an anti-PDL1 single domain antibody described above or a fusion protein described above.
  • the polynucleotide is polymers of DNA, RNA, DNA/RNA hybrids, or modifications thereof. In some embodiments, the polynucleotide is polymers of DNA.
  • the polynucleotide is polymers of RNA.
  • DNA or RNA encoding the anti-PDL1 single domain antibody described above or a fusion protein described above is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
  • the encoding DNA or RNA may also be obtained by synthetic methods.
  • the isolated polynucleotide encoding the anti-PDL1 single domain antibody described above or a fusion protein described above can be inserted into a construct for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • construct components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • a construct includes the isolated polynucleotide provided above.
  • a method of constructing the construct is known to those skilled in the art.
  • the construct can be obtained by in-vitro recombinant DNA technology, DNA synthesis technology, or in-vivo recombinant technology. More specifically, it can be constructed by inserting the isolated polynucleotide into a polyclonal site of an expression vector.
  • the expression vector in the present disclosure generally refers to various commercially available expression vectors well known in the art, for example, bacterial plasm ids, bacteriophages, yeast plasmids, plant cell-infected viruses, mammalian cell-infected viruses such as adenovirus, retrovirus or other vectors.
  • the vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, where the regulatory sequence may include a suitable promoter sequence.
  • the promoter sequence is usually operably linked to a sequence coding the amino acid sequence to be expressed.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutated, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell.
  • the regulatory sequence may further include a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate the transcription. The terminator sequence is linked to the 3′end or terminus of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice may be used in the present disclosure.
  • a suitable vector may contain an origin of replication capable in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.
  • these promoters may include, but not limited to, the lac or trp promoter of Escherichia coli (E.coli) ; the lambda phage PL promoter; and eukaryotic promoters (including CMV immediate-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, methanol oxidase promoter of Pichia pastoris) , and some other known promoters that are capable of controlling gene expression in prokaryotic cells or eukaryotic cells or viruses.
  • E.coli Escherichia coli
  • eukaryotic promoters including CMV immediate-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, methanol oxidase promoter of Pichia pastoris
  • Marker genes can be used to provide phenotypic characters for selection of transformed host cells.
  • marker genes may include, but not limited to, dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance for E. coli.
  • GFP green fluorescent protein
  • the expression vector may further include an enhancer sequence. If an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancer is a cis-acting factor of DNA, typically containing about 10 to 300 base pairs. Enhancer acts on a promoter to enhance gene transcription.
  • the present disclosure provides an antibody expression system, which includes a construct provided above or incorporates an exogenous polynucleotide provided above in the genome.
  • Any cell suitable for the expression of an expression vector can be used as a host cell.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including, but not limited to, Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; or fungal cells such as yeast, and filamentous fungi; plant cells; insect cells derived from Drosophila S2 or Sf9; animal cells such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or a combination thereof.
  • Methods for constructing the expression system should be known to those skilled in the art, for example, including, but not limited to, microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, precipitation with calcium phosphate, or a combination thereof.
  • the present disclosure relates to a pharmaceutical composition including an anti-PDL1 single domain antibody described above or a fusion protein described above, and a pharmaceutically acceptable carrier.
  • the composition is a pharmaceutical composition, which contains the anti-PDL1 single domain antibody or active fragments thereof or the fusion protein described above and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to an ingredient other than the active ingredient in a pharmaceutical composition that is not toxic to the subject.
  • the pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH can be changed according to properties of the formulated substances and disease conditions to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to) intratumoral administration, intraperitoneal administration, intravenous administration, or topical administration.
  • the pharmaceutical composition of the present disclosure contains a safe and effective amount (such as 0.001-99 wt %, preferably 0.01-95 wt %, more preferably 0.1-90 wt %) of the single domain antibody or the fusion protein provided and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient includes (but is not limited to) saline, buffer, glucose, water, glycerol, ethanol and combinations thereof.
  • a pharmaceutical preparation should be matched with the administration mode.
  • the pharmaceutical composition of the present application can be prepared into an injection form, for example, the pharmaceutical composition is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition such as an injection and a solution should be manufactured under sterile conditions.
  • the dosage of active ingredients is a therapeutically effective amount, such as about 10 ⁇ g/kg body weight to about 100 mg/kg body weight per day.
  • the anti-PDL1 single domain antibody described above, or a fusion protein described above can also be used with other therapeutic agents.
  • kits containing the anti-PDL1 single domain antibody, the fusion protein, the isolated polynucleotide, the construct, and/or the pharmaceutical composition provided herein.
  • kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art.
  • Instructions, either as inserts or a labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • the present disclosure provides the use of an anti-PDL1 single domain antibody described above, the fusion protein described above, or the pharmaceutical composition described above in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease, disorder, or condition.
  • the disease, disorder, or condition is a tumor, (such as malignant tumor, cancer) , and at least a tumor cell expresses PDL1, preferably human PDL1.
  • the tumor is a solid tumor.
  • treating/preventing refers to an attempt to alter the natural progress of a disease in a treated individual and can be prevention or clinical intervention implemented during the course of clinical pathology.
  • the desired effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of diseases, alleviating the symptoms, relieving any direct or indirect pathological consequences of the disease, preventing the metastasis, slowing the rate of disease progression, improving or lessening the disease states, and relieving or improving the prognosis.
  • the antibody of the present disclosure is useful for delaying the development of a disease or delaying the progression of a disorder.
  • provided herein is a method for treating a subject having cancer, including administrating to the subject a therapeutically effective amount of anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition provided herein.
  • the “therapeutically effective amount” of the anti-PDL1 single domain antibody, the fusion protein described above, or a pharmaceutical composition provided in the present disclosure preferably causes a reduction in the severity of disease symptoms and increased frequency and duration of asymptomatic period of a disease, disorder or condition, or prevents injury or disability due to illness or suffering.
  • the “therapeutically effective amount” preferably inhibits the cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
  • the term “about” when preceding a numerical value indicates the value plus or minus a range of 20%, 15%, 10%, or 5%.
  • the ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy against human tumors or evaluated by detecting the ability to inhibit cell growth. Such inhibition can be determined in vitro by assays well known to those skilled in the art.
  • the therapeutically effective amount of the anti-PDL1 single domain antibody, the fusion protein, and the pharmaceutical compositions is often able to reduce the tumor size, or otherwise relieve the symptoms of a subject.
  • Those skilled in the art can select an appropriate therapeutically effective dose according to the actual situation, for example, the tumor size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.
  • a prescription for treatment e.g., decision on dosage, etc.
  • a prophylactically effective amount refers to an amount effective for achieving the desired prophylactic effect at a dose and for a period of time required. Usually, but not necessarily, since a prophylactic dose is administered to a subject before the onset of a disease or at an early stage of the disease, the “prophylactically effective amount” is usually lower than the “therapeutically effective amount” .
  • diseases associated with PDL1 expressing cells may include all PDL1 expressing cancers and tumor entities, specifically including, but not limited to, lung cancer, non-small cell lung cancer, melanoma, gastric cancer, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classic Hodgkin lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, which may be early, intermediate, or advanced, such as, metastatic cancers.
  • the subject is mammal animals including human and non-human animals, such as human, mouse, and cynomolgus monkey.
  • the present disclosure provides a method of decreasing the rate of tumor growth or the number of tumor cells, including contacting a tumor cell with an effective amount of anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition described above.
  • the present disclosure provides a method of killing a tumor cell, including contacting a tumor cell with an effective amount of anti-PDL1 single domain antibody, the fusion protein, or the pharmaceutical composition described above.
  • the reference antibodies AEP247 refers to Envafolimab, has been generated according to patent CN107849130B, (SEQ ID NO: 2, Hu56V1) .
  • the variable region sequence of AEP247 is shown in Table 1.
  • Human, cynomolgus and mouse PDL1 stably expressing cell lines Raji/hPDL1, CHOK1/hPDL1, CHOK1/cynoPDL1 and CHOK1/mPDL1, have been generated for chimeric antibody screening and in vitro assays.
  • CHOK1 or Raji cells were transfected with human, cynomolgus and mouse PDL1 expression lentivirus and selectively cultured in medium containing 10 ⁇ g/mL puromycin for 2 weeks. Single cell clones were then isolated by limiting dilution and screened by FACS to obtain the monoclonal cell lines stably expressing PDL1.
  • Human PDL1 extracellular domain (Uniprot Access#Q9NZQ7, AA Met 1-Thr 239) recombinant proteins with human Fc tag or 6xHis-tag were purchased from Sinobiological (Cat: 10084-H08H and Cat: 10084-H02H) for immunization.
  • Biotinylated human PDL1 extracellular domain (Uniprot Access#Q9NZQ7, AA Met 1-Thr 239) and biotinylated cynomolgus PDL1 extracellular domain (GeneBank Access#XP_015292694.1, AA Met 1-Thr 239) were purchased from Sinobiological (Cat: 10084-H08H-B and Cat: 90251-C08H-B) for antibody screening via yeast display.
  • PBMC peripheral blood mononuclear cell
  • PBMC peripheral blood mononuclear cell
  • serum titer of the immunized alpacas was detected by ELISA assay using 6xHis-tagged human PDL1 ECD recombinant protein as antigen and Anti-Alpaca IgG (2+3) HRP as secondary antibody ( Figure 1A-1B, Figure 2A-2B, Figure 3A-3B) .
  • the PBMCs of the 3rd, 4th and 5th round immunization of both alpacas were used for the library construction and antibody screening.
  • the B cells were isolated from the blood of each round of immunization with Ficoll by density gradient centrifugation. And then the total RNA was isolated from these B cells with TRIzol. The RNA isolated from the PBMC was analyzed by electrophoresis on an agarose gel ( Figure 4) . Genomic DNA remained in the total RNA was digested by the DNase I at 37°C for 30 min. After digestion, the RNA was used for 1st strand cDNA synthesis using the Prime Script II 1st Strand cDNA Synthesis Kit (6210B, TAKARA) according to the manufacturer’s user guide.
  • the Prime Script II 1st Strand cDNA Synthesis Kit (6210B, TAKARA
  • the VHH was amplified from the cDNA by two rounds of nest PCR.
  • a set of primers were used to amplify the fragments containing the whole VHH and partial CH2 and with or without CH1 domain for the VH germline.
  • the PCR product was separated by the agarose gel and the DNA with size in 500-750bp was recovered by the NucleoSpin Gel and PCR Clean-up kit ( Figure 5) .
  • a set of nest primers were used to amplify the VHH fragments with the purified DNA as template.
  • the 2nd step PCR product was separated by the agarose gel and the DNA with size in 300-500bp was recovered by the NucleoSpin Gel and PCR Clean-up kit ( Figure 6) .
  • a set of primers with arms homology to the display plasmid were used to amplify the DNA and DNA with 500bp was collected and purified ( Figure 7) .
  • the display plasmid was linearized by restricted enzyme BamHI and NheI.
  • the linearized plasmid was then mixed with the VHH DNA at a weight ratio of 1: 1 and was concentrated by the NaAc to a final concentration higher than 1.5 ⁇ g/ ⁇ l in the deionized water.
  • 20 ⁇ g of the DNA mixture was then electroporated into the yeast competent cell in a 2mm cuvette. 5 independent electroporation was carried out and the yeast was recovered and sampled to count the diversity.
  • the yeasts were mixed and a final library with a total diversity of 1.4 ⁇ E9 was constructed.
  • the antibody screening process contains 3 stages.
  • the PDL1 binders in the library were enriched by 4 rounds of Magnetic Activated Cell Sorting (MACS) , including streptavidin-binder depletion for the 1st round, PDL1 binders enrichment with 50nM biotinylated human-PDL1 for the 2nd round, PDL1 binders enrichment with 50nM biotinylated cyno-PDL1 for the 3rd round, and PDL1 binder enrichment with 10nM biotinylated human-PDL1 for the 4th round. Sorting stage was then carried out to harvest the high binding antibodies with 10nM biotinylated human-PDL1.
  • MCS Magnetic Activated Cell Sorting
  • Colonies could bind to the biotinylated human-PDL1 (only PE positive) but did not form the triplet complex of yeast, biotinylated humanPDL1, and HAC-PD1-Fc (both PE and APC positive) were selected as hits and used for the following confirmation. As shown in Figure 8, clones 570 and 2719 were selected and used for the following confirmation.
  • Example 3 Chimeric antibody generation and characterization
  • variable regions in yeast clones 570 and 2719 were sequenced, and the variable region sequences were shown in Table 2. According to the sequencing results, two human IgG1 chimeric antibody was generated and named as C570 and C2719 respectively, where the prefix “C” stands for chimeric. The benchmark antibody AEP247 was also generated.
  • CDRs were underlined, and were defined by Kabat definition except CDR1, which was defined by a combination of Kabat and Chothia systems.
  • the binding affinity of C570, C2719 and AEP247 to human PDL1 ECD recombinant protein was determined using Bio-Layer Interferometry (Octet) .
  • the association and dissociation curves were fit with 1: 1 binding model, and the Ka/Kd/KD values for each antibody were calculated and summarized in Table 3.
  • the binding curve of antibody AEP247, C2719, C570 were shown in Figure 12A, 12B and 12C, respectively.
  • C570 showed comparable affinity with AEP247, while C2719 showed much higher affinity to hPDL1 (human PDL1) protein.
  • TMB Tetramethylbenzidine
  • Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature environment and is an intrinsic property of antibodies that can influence product stability, such as aggregation, during manufacturing and storage.
  • the melting temperature (Tm) values of hit antibodies in PBS buffer was measured to predict their thermal stability. Briefly, hit antibodies were resolved in 10 mM PBS buffer (pH 7.2-7.4) . The Tm values were then detected by differential scanning fluorimetry (DSF) using QuantStudio 7 Flex Real time PCR system. As shown in Table 4, Tm values of the hit antibodies in PBS buffer range from 55-72°C, indicating their good thermal stability.
  • Retention time in HIC-HPLC assay is a consideration factor for antibody hydrophobicity prediction. 15 ⁇ l of 0.25mg/ml antibody was injected into MacPac-10 HIC column in Agilent 1260 system and detected signal in 280nm/214nm. As shown in Table 4, the retention time of each hit antibodies are less than 13.5min, indicating their good hydrophilicity.
  • the chimeric antibodies C570, C2719 and AEP247 were tested by a Jurkat-NFAT-Luc-PD-1/CHO-K1-aAPC-PD-L1 reporter cell line system (GenScript. Inc) .
  • CHO-K1-aAPC-PD-L1 cells expresses anti-CD3 (OKT3) and PD-L1.
  • Jurkat-NFAT-Luc-PD-1 cell expresses PD-1 and the reporter gene luciferase under the control of NFAT.
  • PD-1/PD-L1 interaction delivers negative signal that inhibit luciferase activity. This inhibition can be relieved by anti-PD-1/PD-L1 blocking mAbs that interrupts PD-1/PD-L1 interaction to restore the luciferase activity.
  • CHO-K1-aAPC-PD-L1 cells were seeded into a 96-well plate, then Jurkat-NFAT-Luc-PD-1 cells and serially diluted antibodies were added into each well.
  • the number ratio of Jurkat-NFAT-Luc-PD-1 cell to CHO-K1-aAPC-PD-L1 cell was 4: 1.
  • the mixed cells were incubated in a 37°C, 5%CO 2 incubator for 6 hours.
  • Equal volume of luciferase detection reagent was added and incubated at R.T. for 5min.
  • the EC 50 values were analyzed by GraphPad Prism 9.0 with Four-Parameter nonlinear fitting, and the EC 50 values were shown under the blocking activity curve of Figures 15-16. As shown in the Figure 15 and 16, C570, C2719 and AEP247 showed strong blocking activity for the PD-1/PD-L1 interaction.
  • CD14+ monocyte was isolated from PBMC and then differentiated into immature dendritic cells (DCs) in the presence of human granulocyte-macrophage colony stimulating factor (GM-CSF) and human IL-4 for 6 days, then interleukin 1 ⁇ (IL-1 ⁇ ) , TNF- ⁇ , IL-6 and prostaglandin E2 (PGE-2) were added to stimulate DC maturation.
  • DCs dendritic cells
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • PGE-2 prostaglandin E2
  • the stimulated DC was validated by FACS analysis for the expression of CD83, CD86 and HLA-DR markers.
  • the mature DCs (mDCs) and CD3+T cells and selected hit antibodies were cocultured for 5 additional days. IFN- ⁇ production in the culture medium were tested. All the 3 tested antibodies were proved to enhance IFN- ⁇ production ( Figure 17) .
  • the epitope binning for C570, C2719 and AEP247 was carried out by octet. Briefly, the HIS1K sensors were hydrolyzed for 10 minutes followed by capture of 6xHis-tagged human PDL1 ECD recombinant protein to 0.3 nm. The PDL1 captured sensors were then exposed to Ab1 and Ab2 in tandem or Ab2 directly. The binding signals of Ab2 to PDL1 under different conditions were detected respectively. Finally, the relative binding signals of Ab2 in tandem were calculated by normalization. High relative binding signals represent low competition between Ab2 and Ab1 for binding antigen. Likewise, low relative association signals represent high competition between Ab2 and Ab1. As shown in Table 5, C570 and C2719 completely competed AEP247 for binding to PDL1.
  • Single dose PK profile of C2719, C570 and AEP247 in mouse were measured using tumor cell MC38-hCD47/hPDL1 (Shanghai Model Organisms Center, Inc) transplanted C57BL/6J/hCD47/hSIRPa transgenic mouse model (Shanghai Model Organisms Center, Inc) .
  • Antibody AEP247 was used as reference control. Mice were administrated with C2719, C570, AEP247 or vehicle saline according to the schedule in Table 6-1.
  • the concentrations of anti-PDL1 antibodies in mouse plasma collected at different time points were determined by a specific quantitative ELISA assay which used Human PD-L1 His Tag protein as capture antibody and goat Anti-human IgG Fc ⁇ -HRP as detection antibody.
  • the assay range of this method is from 10 ng/mL to 1000 ng/mL.
  • PK parameters including T 1/2 , C max , T max , MRT and AUC 0-last were calculated with non-compartment model using WinNonLin 7.0 software.
  • C2719, C570 and AEP247 showed target-mediated drug disposition (TMDD) effect and a relative rapid elimination with T 1/2 ranged from 23.0-53.1 hr.
  • C max of C2719, C570 and AEP247 were 11062 ng/mL, 7592 ng/mL, 4435 ng/mL and T 1/2 were 53.1 hr, 23.0 hr, 34.8 hr, respectively.
  • PK profile ranking was C2719>AEP247>C570, however, as the dose level was only 1 mg/kg in this study, the PK profile might be underestimated due to the TMDD effect.
  • PK profile of C2719, C570 and AEP247 in mouse were measured using BALB/c-hPD1/hSIRP ⁇ mice model bearing CT26-hCD47/hPDL1 tumor cells (hCD47 and hPDL1 overexpression CT26 tumor cells, Gempharmatech Co., Ltd. ) .
  • Antibody AEP247 was used as reference control. Mice were administrated with C2719, C570 or AEP247 according to the schedule in Table 7-1. The concentrations of anti-PDL1 antibodies in mouse plasma collected at different time points were determined by a specific quantitative ELISA assay which used Human PD-L1 His Tag protein as capture antibody and goat Anti-human IgG Fc ⁇ -HRP as detection antibody.
  • the assay range of this method was from 10 ng/mL to 1000ng/mL.
  • PK parameters including T 1/2 , C max , T max , MRT and AUC 0-last were calculated with non-compartment model using WinNonLin 7.0 software.
  • C2719, C570 and AEP247 showed a relative rapid elimination with T 1/2 range from 22.2-42.9 hr due to the TMDD effect from human PDL1 in this model.
  • C max of C2719, C570 and AEP247 were 28519 ng/mL, 33240 ng/mL, 16977 ng/mL and T1/2 were 42.9 hr, 36.1 hr, 22.2 hr, respectively.
  • C2719 showed comparable PK profile compared with C570 and better than reference antibody AEP247.
  • MC38 colorectal cancer cell line was engineered to express human CD47 and PD-L1 instead of mouse proteins (MC38-hCD47/PD-L1, Shanghai Model Organisms Center, Inc) .
  • Cells were subcutaneously inoculated into human CD47 and SIRP ⁇ double knock-in (dKI) C57Bl6 mice (hCD47/SIRP ⁇ dKI, Shanghai Model Organisms Center, Inc) for tumor formation.
  • Mice were divided into 4 groups, each with a mean tumor size of ⁇ 93mm 3 and administered intraperitoneally (i. p. ) with AEP247, C2719 and C570, together with vehicle control.
  • the spare mice were also separated into 3 groups and given a single dose (i. p., 1mg/kg) of vehicle, AEP247, C2719 or C570.
  • Blood was withdrawn at different timepoints to monitor antibody concentration overtime by quantitative ELISA. The design was the same as that in Table 6-1.
  • Tumor growth of treated groups was compared to vehicle group.
  • RTV TVi/TV0, TVi and TV0 are tumor volume of an individual mouse on day i and day 0, respectively) .
  • the first day giving drug was defined as day 0.
  • Statistics of tumor growth differences was carried out by comparing to vehicle group using two-way ANOVA in GraphPad Prism. p value ⁇ 0.05 was considered as significant.
  • Figure 20 showed in vivo tumor growth inhibition curves of C570 and C2719. Combined with Table 8, treatments with AEP247, C570 and C2719 all significantly delayed tumor growth compared to vehicle group. TGI of C570 and C2719 on day11 post treatment initiation (the last day when all groups had all mice alive) was comparable or better than AEP247.
  • CT26 colorectal cancer cell line was engineered to express human CD47 and PDL1 instead of mouse proteins (CT26-hCD47/hPDL1, Gempharmatech Co., Ltd. ) .
  • Cells were subcutaneously inoculated into human PD1 and SIRP ⁇ double KI Balb/c mice (hPD1/hSIRP ⁇ dKI, Gempharmatech Co., Ltd. ) for tumor formation.
  • Mice were divided into 4 groups, each with a mean tumor size of ⁇ 101mm 3 and administered intraperitoneally (i. p. ) with AEP247, C2719 and C570, together with vehicle control.
  • the spare mice were also separated into 3 groups and given a single dose (i. p., 5mg/kg) of AEP247, C2719 or C570.
  • Blood was withdrawn at different timepoints to monitor antibody concentration overtime by quantitative ELISA. The design was shown in Table 9-1.
  • Tumor growth inhibition rate (TGI%) was calculated as the method described in Example 3.2.11, and the results were shown in Table 9-2.
  • Figure 21 showed in vivo tumor growth inhibition curves of C570 and C2719. Combined with Table 9-2, treatment with AEP247, C570 and C2719 all significantly delayed tumor growth compared to vehicle group. Moreover, AEP247, C570 and C2719 administration resulted in 2, 2 and 3 complete remissions out of 6 mice, respectively. TGI of C570 and C2719 on day21 post treatment initiation was comparable or better than AEP247.
  • CDR Complementarity-determining region
  • IGHV3-23*04 was selected as humanization template for C570 based on its homology to the original Alpaca antibody sequence.
  • CDRs were defined using Kabat definition except CDR1, which was defined using a combination of Kabat and Chothia systems.
  • CDR1 was defined using a combination of Kabat and Chothia systems.
  • CDR1 was defined using a combination of Kabat and Chothia systems.
  • CDR2 CDR1
  • CDR1 Chothia systems
  • the resulting variants were produced and the top version of mAbs were designated as H570.001, H570.002, H570.003, and H570.004, where the prefix “H” indicates “humanized” , and the number in the suffix denotes the serial number. All the variants were tested in multiple in vitro assays to select the best ones that retained the property of the parental antibody.
  • VHH sequences of humanized antibody were shown in Table 10. CDRs were underlined and defined by Kabat definition.
  • IGHV3-11*01 was first selected as humanization template for C2719 based on its homology to the original Alpaca antibody sequence.
  • CDRs were defined using Kabat definition except heavy chain CDR1, which was defined using a combination of Kabat and Chothia systems.
  • CDRs and different combinations of canonical residues from C2719 were grafted onto the templates.
  • the resulting variants were produced and designated as H2719.001, H2719.002, H2719.003, H2719.004, H2719.005, H2719.006, H2719.007, H2719.008, H2719.009, H2719.010, H2719.011, H2719.012, H2719.013, H2719.014, and H2719.015, where the prefix “H” indicates “humanized” , and the number in the suffix denotes the serial number. All the variants were tested in multiple in vitro assays to select the best ones that retained the property of the parental antibody.
  • VHH sequences of humanized antibody were shown in Table 11. CDRs were underlined and defined by Kabat definition.
  • the binding affinity of C570 derived humanized variants to human PDL1 ECD protein was determined using Bio-Layer Interferometry (Octet) .
  • the association and dissociation curves were fit with 1: 1 binding model, and the Ka/Kd/KD values for each antibody were calculated and summarized in Table 12-1.
  • Blocking activity of C570 and C2719 derived humanized variants H570.001, H570.002, and H570.003 were tested by reporter assay, which was described in Example 3.2.6. Human IgG1 was used as isotype control.
  • Figure 24 showed that C570 and C570 humanization antibodies block the PD1 and PDL1 interaction, and The EC 50 values were shown under the blocking activity curves. It showed H570.001 and H570.002 had good blocking activity to the PD1/PDL1 binding and was comparable to their parental antibody C570.
  • Blocking activity of C2719, H2719.003, H2719.004, H2719.006, H2719.007, H2719.009, H2719.010, H2719.011, H2719.012, H2719.013, H2719.014, and H2719.015 were tested by reporter assay, which was described in Example 3.2.6. Human IgG1 was used as isotype control.
  • Figures 25A-25B showed that C2719 and C2719 humanization antibodies block the PD1 and PDL1 interaction.
  • the EC 50 values were shown under the blocking activity curves.
  • H2719.004, H2719.006, H2719.007, H2719.009, H2719.010, H2719.013, H2719.014, and H2719.015 had good blocking activity to the PD1/PDL1 binding and was comparable to their parental antibody C2719.
  • Tm melting temperature
  • Tm values were then detected by Differential Scanning Fluorimetry (DSF) using QuantStudio 7 Flex Real time PCR system. As shown in Table 14-1 and Table 14-2, the Tm values of the humanized variants were comparable to their individual parental antibody.
  • humanized variants H570.001, H570.002, H570.003, H570.004, H2719.002, H2719.008, H2719.011, H2719.012, H2719.013, H2719.015, and their parental antibody C570 and C2719 in PBS buffer were titrated with acetic acid to pH 3.0-3.5.
  • ⁇ purity was the purity difference value of samples “Low pH 2h” and samples “T0” .
  • Group T0 in Table 15-1 and Table 15-2 represented initial samples before exposure to the low pH solution for 2 hours.
  • Freeze-thaw stability is often explored to determine the susceptibility of antibodies to temperature cycling which products are frequently exposed.
  • drug substance is often frozen to enable long-term storage.
  • Drug product may be exposed to frozen temperature as part of the lyophilization process.
  • the major degradation pathway of freeze-thaw is aggregates, including precipitates, particles, and soluble particles.
  • Group T0 in Table 16-1 and Table 16-2 represented initial samples without freeze-thaw cycles.
  • Thermal stress at temperatures exceeding normal storage conditions can accelerate degradation, thereby increasing the detectability of potential degradation pathways to provide information about long-term degradation at intended storage conditions.
  • Group T0 in Table 17-1 and Table 17-2 represented initial samples without thermal stress tests.
  • the anti-PDL1/SIRP ⁇ bispecific antibodies were constructed as anti-PDL1 single domain antibody (sdAb) C570 or C570 derived humanized Ab in N-terminus of Fc and anti-SIRP ⁇ Fab fused to C-terminus of Fc ( Figure 26) .
  • sdAb single domain antibody
  • a flexible (Gly4Ser) 3 linker or (Gly4Ser) 4 linker was genetically linked to the N-terminus of the anti-SIRP ⁇ Fab.
  • the amino acid sequences of anti-SIRP ⁇ Fab, anti-PDL1/SIRP ⁇ antibody and Fc fragment were shown in Table 18. CDRs were underlined and defined by Kabat definition.
  • the DNA encoding the light chain and the heavy chain in either the same expression vector or separate expression vectors were used to transfect CHO cell for transfection.
  • the culture media were harvested and the fusion protein was purified by Protein A Sepharose column.
  • ES019-79 or ES019-116 was transiently expressed by CHO-K1 cells (suspension culture) with the transfection of pcDNA3.4 plasmid.
  • the expression titer of ES019-79 was 124mg/L and ES019-116 could reach to 418 mg/L.
  • ES019-79 and ES019-116 were easy to purify because of no mispairing risk.
  • a two-steps purification strategy was used with protein A chromatography followed by cation ion exchange (CEX) by Hi-Trap SP/HP column.
  • Size Exclusion Chromatography SEC was performed using an Agilent 1260 Infinity system with the TSKGel G3000 SWXL size exclusion chromatography column at 25 °C. Samples were diluted to 10 mg/ml with mobile phase before SEC analysis.
  • Capillary Electrophoresis Sodium Dodecyl Sulfate (CE-SDS) was performed for purity analysis on Maurice (Protein Simple) .
  • the SEC-HPLC purity is 94.41% ( ⁇ 1.58%aggregates and ⁇ 4.01%fragments) and the antibody was stored in 10 mM NaAc-HAc, pH5.2 buffer.
  • the final ES019-116 purity analyzed by non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) was 98.5%with 1.5%of fragments, and SEC-HPLC was 99.76%with a 0.24%of aggregates.
  • the percentage of heavy chain and light chain in reduced CE-SDS was 69.4%and 30.3%, respectively.
  • Charge heterogenicity of ES019-116 was analyzed by capillary isoelectric focusing (CIEF) , the pH value (potential of hydrogen) of the main peak was 7.89 which approach to its predicted isoelectric point (PI) of 7.8.
  • the percentage of acidic peak was 25.5%and of the basic peak of 1.1%.
  • ES019-116 was also assessed by hydrophobic interaction chromatography (HIC) -HPLC using MAbPac HIC-10 column, it showed a single peak with the retention time of 7.95 min and no other detected hydrophobic heterogeneity peaks, suggesting ES019-116 has low hydrophobicity.
  • ES019-116 was analyzed by detecting its binding to baculovirus particles (BVP) by ELISA, which was reported as a useful method to evaluate the non-specific binding of therapeutic proteins. Similar with hIgG1 isotype, ES019-116 showed almost no binding to 1%BVP.
  • BVP baculovirus particles
  • CHOK1 cells overexpressing human PDL1 (CHOK1/hPDL1) , CHOK1 cells overexpressing human SIRP ⁇ (CHOK1/SIRP ⁇ ) or CHOK1 cells overexpressing human PDL1 (Raji/hPDL1) were washed with wash buffer and incubated with 100 ⁇ l serial dilution of PDL1/SIRP ⁇ bispecific antibody (ES019-79, ES019-116 or PD-L1/SIRP ⁇ -HC2) for 1 hour at 4°C. Cells were then washed twice with wash buffer and incubated with 100 ⁇ l of anti-human Fc-Alexa Flour 647 for 1 hour at 4°C. Cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences) .
  • PDL1/SIRP ⁇ bispecific antibody ES019-79, ES019-116 or PD-L1/SIRP ⁇ -HC2
  • the anti-PDL1/SIRP ⁇ bispecific antibodies bound to CHO/hPDL1 cells in a dose-dependent manner.
  • the anti-PDL1/SIRP ⁇ bispecific antibodies bound to CHOK1/SIRP ⁇ cell in a dose-dependent manner.
  • MFI maximal mean fluorescence intensity
  • Anti-PDL1/SIRP ⁇ bispecific antibody enhances in vitro macrophage phagocytosis of PDL1+ cancer cells
  • Raji lymphoma tumor cells expressing human PDL1 were labeled with CellTrace Violet staining solution and incubated with mouse bone marrow derived macrophages (BMDMs) labeled with CellTrace Far Red staining solution prepared from C57BL6-hCD47/hSIRP ⁇ double knock-in mice either in the presence of an isotype control human IgG1 (hIgG1) , anti-PDL1 antibody C570, anti-SIRP ⁇ antibody hu025.060 (with human IgG1 isotype) and anti-PDL1/SIRP ⁇ bispecific antibody. After 2 hours, the macrophages were harvested, and analyzed by flow cytometry. CellTrace Far Red and CellTrace Violet double positive events identify macrophages that have phagocytosed CellTrace Violet-labeled tumor cell. Each sample was represented by a different color. Phagocytic index was shown for two separate samples.
  • anti-PDL1 antibody induced ⁇ 50%phagocytosis by antibody dependent cellular phagocytosis (ADCP) and anti-SIRP ⁇ antibody barely induced phagocytosis.
  • Anti-PDL1/SIRP ⁇ bispecific antibody ES019-79 and ES019-116 induced stronger phagocytosis in a dose dependent manner compared to monotherapy.
  • the anti-PDL1/SIRP ⁇ bispecific antibodies stimulated mouse BMDM phagocytosis against Raji/hPDL1 cells better than single treatments.
  • Anti-PDL1/SIRP ⁇ bispecific antibody demonstrates potent in vivo anti-tumor efficacy
  • mice Human BALB/c-hPD1/hSIRP ⁇ double Knock-in mice (Gempharmatech Co., Ltd. ) were inoculated with hCD47/hPDL1 overexpression CT26 tumor cells (CT26-hCD47/hPDL1) . Mice were grouped according to tumor volume into 5 groups when the mean tumor volume reached ⁇ 70-100 mm 3 . Mice were i. p.
  • anti-PDL1 antibody 570 As shown in Figure 29, anti-PDL1 antibody 570, anti-SIRP ⁇ antibody hu025.060 monotherapy and the combination of anti-PDL1 and anti-SIRP ⁇ antibody did not inhibit tumor growth similar to vehicle treatment.
  • Anti-PDL1/SIRP ⁇ bispecific antibody ES019-79 induced strong tumor growth inhibition and almost completed tumor regression in vivo tumor model.
  • ES019-116 and anti-PD-L1 C570 were measured by Biacore surface plasmon resonance (Cytiva) .
  • ES019-116 or C570 was immobilized onto CM5 sensor chips (Cytiva) and then PD-L1 analyte (hPDL1 protein: 2-fold dilution from 60nM) was injected for 180 seconds, followed by a dissociation cycle of 180 seconds.
  • ES019-116 or anti-SIRP ⁇ mAb ES004-hu025.060m1 (hu025.060-IgG1.
  • C220S was immobilized onto CM5 sensor chips (Cytiva) and then SIRP ⁇ analyte (hSIRP ⁇ protein: 2-fold dilution from 60nM) was injected for 180 seconds, followed by a dissociation cycle of 180 seconds.
  • ES019-116 was prepared by 8-folds dilution starting from 200 nM.
  • test samples were loaded in duplicate at room temperature for 1 hour incubation.
  • Peroxidase-conjugated goat anti-hIgG Fc ⁇ fragment (1: 10,000) in ELISA buffer was added and incubated at room temperature for 30 minutes.
  • the phagocytosis activity of ES019-116 was evaluated in human monocyte derived macrophage (hMDM) phagocytosis assay against Raji-PD-L1 tumor cells.
  • hMDM human monocyte derived macrophage
  • Human Raji-PD-L1 tumor cells were labeled with the fluorescent dye CellTrace Far Red and incubated with human monocytes derived macrophages (MDMs) prepared from human PBMC after human M-CSF treatment for 7 days. After incubation of hMDMs and Raji-PD-L1 tumor cells for 2 hours, the macrophages were harvested and stained with fluorescently labeled anti-CD11b, and then analyzed by flow cytometry. CD11b+CellTrace Far Red+ double positive events identify macrophages that have phagocytosed CFSE-labeled tumor cell. Each sample was represented by a different color. Phagocytic index was shown for three separate samples.
  • MDMs monocytes derived macrophages
  • anti-PDL1 C570 induced ⁇ 27%phagocytosis mainly through antibody dependent cellular phagocytosis (ADCP) .
  • ES019-116 showed potent phagocytosis activity of Raji-PD-L1 cells by hMDMs from human PBMC donor. Anti-CD47 antibody were used as control.
  • ES019-116 and PD-L1/SIRP ⁇ -HC2 were also compared by human monocyte derived macrophage (hMDM) against Raji/PD-L1 tumor cells.
  • hMDM human monocyte derived macrophage
  • ES019-116 could induce phagocytosis to PD-L1 expressing tumor cells and but not to human monocytes or human PBMCs, ES019-116 induced phagocytosis is completely dependent on the expression of PD-L1 and it will not cause non-specific phagocytosis in the absence of PD-L1 expression.
  • the macrophage reprogramming assay was performed by in vitro assay.
  • human monocyte derived macrophage hMDM
  • M-CSF human monocyte derived macrophage
  • the macrophages were collected for polarization marker (CD86 and CD163) staining and the culture supernatants were measured for cytokine production (TNF-a, IL-6, CXCL-10 and IL-10) .
  • ES019-116 bispecific antibody could induce strong macrophage phagocytosis of Raji-hPD-L1 tumor cells ( Figure 33) .
  • ES019-116 showed more potent phagocytosis activity than Atezolizumab.
  • ES019-116 could also reprogram M2 like macrophage to M1 like macrophage by increasing the percentage of CD86 high CD163 low macrophage population after 24 hours or 48 hours treatment ( Figure 34A and 34B) .
  • the ES019 induced macrophage polarization effect depended on the presence of tumor cells since there is no such macrophage polarization effect in the absence of tumor cells (macrophage only) .
  • ES019-116 showed more potent macrophage polarization activity than Atezolizumab.
  • ES019-116 treatment could stimulate macrophage to produce inflammatory cytokine in the presence of tumor cells. After 24 hours incubation, ES019-116 could activate macrophage pro-inflammatory cytokine TNF-a and IL-6 production and its cytokine activation efficacy was higher than Atezolizumab. Similar to the result of polarization, there is no pro-inflammatory cytokine production effect in the absence of tumor cells (macrophage only) ( Figure 35) .
  • ES019-116 could further activate macrophage to produce more pro-inflammatory cytokine TNF-a, IL-6 and CXCL-10, but not IL-10 production. Similar to previous result, there is pro-inflammatory cytokine production effect in the absence of tumor cells (macrophage only) ( Figure 36) .
  • ES019 bispecific antibodies ES019-79 and ES019-116 were tested by peripheral blood mononuclear cells (PBMC) of healthy human donors.
  • PBMC peripheral blood mononuclear cells
  • CRS highlighted the capacity of immune stimulatory antibodies to induce significant systemic immune mediated toxicities.
  • the CRS assay using PBMC for the detection of cytokine release is highly recommended for dependable preclinical safety testing of new therapeutic antibodies.
  • Anti-CD3 antibody (Clone: OKT3, a mouse anti-human CD3 antibody) were used as positive control, hIgG1 (human IgG1) and hIgG4 (human IgG4) were isotype control.
  • anti-human PD-L1 C570, Atezolizumab and Avelumab were also tested.
  • IL-2 IFN- ⁇ , TNF- ⁇ , IL-6 and IL-10
  • the in vitro CRS assay result indicated that ES019-79 and ES019-116 didn’t induce any tested cytokines production (IL-2, IFN- ⁇ , TNF- ⁇ , IL-6 and IL-10) from human PBMC donors, so ES019-79 and ES019-116 demonstrated a favorable safety profile due to its low risk to induce systemic cytokine release syndrome.
  • ES019-116 The pharmacokinetics characteristics of ES019-116 was evaluated in a hPD1/hSIRP ⁇ double knock-in BALB/c syngeneic mouse model bearing CT26-hCD47/hPDL1 tumor.
  • the concentrations of ES019-116 was measured by a specific quantitative ELISA method.
  • Human SIRP ⁇ protein was used as the capture protein.
  • Biotinylated Human PD-L1 His Protein and streptavidin-HRP were used as the first and second detection antibody, respectively.
  • the assay detection concentration range was from 20 ng/mL to 2000 ng/mL.
  • Blood samples were collected at 0.25 h, 2 h, 6 h, 24 h, 72 h, 120 h, 144 h, 168 h, 240 h, 336 h and 504 h after single dose of 14 mg/kg ES019-116 by intraperitoneal injection.
  • ES019-116 demonstrated a typical absorption, distribution and elimination phase of IgG antibody.
  • TMDD target-mediated drug disposition
  • ADA anti-drug antibody
  • mice Human Balb/c-hPD1/hSIRP ⁇ double Knock-in mice were inoculated with hCD47/hPDL1 overexpression CT26 tumor cells. Mice were grouped according to tumor volume into 3 groups when the mean tumor volume reached ⁇ 70-100 mm 3 . And then the mice were i. p. dosed with 7 mg/kg or 14 mg/kg ES019-116. Dosing schedule was BIW for 5 doses. Tumor volumes were measured twice per week. 3 days post the 5th dosing, mice were sacrificed and tumors were weighted. Statistics were carried out by 2-way anova comparing the mean tumor volume of different treatment groups to that of isotype control group.
  • TGI Relative tumor inhibition rate
  • mice were inoculated with hCD47/hPDL1 overexpression CT26 tumor cells.
  • Mice were grouped according to tumor volume into 3 groups when the mean tumor volume reached ⁇ 100-120 mm 3 . And then the mice were i. p. dosed with 7 mg/kg or 14 mg/kg ES019-116.
  • Dosing schedule was BIW for 4 doses. Tumor volume were measured twice per week. 3 days post the 4th dosing, mice were sacrificed and tumors were weighted.

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Abstract

L'invention concerne un anticorps à domaine unique anti-PDL1, des protéines de fusion et des compositions pharmaceutiques préparées sur la base de l'anticorps à domaine unique, et l'utilisation de tels anticorps dans la prévention, le diagnostic ou le traitement d'une maladie.
PCT/CN2024/072523 2023-01-18 2024-01-16 Anticorps à domaine unique anti-pdl1, protéine de fusion et son utilisation Ceased WO2024153068A1 (fr)

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CN202480008177.8A CN120603849A (zh) 2023-01-18 2024-01-16 抗pdl1单域抗体、融合蛋白及其用途
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Citations (7)

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Publication number Priority date Publication date Assignee Title
WO2017196867A1 (fr) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anticorps anti-pd-l1
CN108779180A (zh) * 2016-03-23 2018-11-09 迈博斯生物医药(苏州)有限公司 新型抗-pd-l1抗体
CN109970857A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 抗pd-l1抗体及其用途
CN111328286A (zh) * 2017-08-09 2020-06-23 奥里尼斯生物科学有限公司 Pd-1和pd-l1结合剂
CN111433224A (zh) * 2017-11-17 2020-07-17 南京传奇生物科技有限公司 抗pd-l1的单结构域抗体及其变体
CN111848800A (zh) * 2020-07-31 2020-10-30 三优生物医药(上海)有限公司 Pd-l1单结构域抗体及其用途
CN113087796A (zh) * 2021-04-02 2021-07-09 河南省肿瘤医院 一种抗pd-l1抗体及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108779180A (zh) * 2016-03-23 2018-11-09 迈博斯生物医药(苏州)有限公司 新型抗-pd-l1抗体
WO2017196867A1 (fr) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anticorps anti-pd-l1
CN109311985A (zh) * 2016-05-09 2019-02-05 Igm生物科学股份有限公司 抗pd-l1抗体
CN111328286A (zh) * 2017-08-09 2020-06-23 奥里尼斯生物科学有限公司 Pd-1和pd-l1结合剂
CN111433224A (zh) * 2017-11-17 2020-07-17 南京传奇生物科技有限公司 抗pd-l1的单结构域抗体及其变体
CN109970857A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 抗pd-l1抗体及其用途
CN111848800A (zh) * 2020-07-31 2020-10-30 三优生物医药(上海)有限公司 Pd-l1单结构域抗体及其用途
CN113087796A (zh) * 2021-04-02 2021-07-09 河南省肿瘤医院 一种抗pd-l1抗体及其应用

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