WO2023221936A1 - 双功能蛋白质及其制剂和用途 - Google Patents

双功能蛋白质及其制剂和用途 Download PDF

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WO2023221936A1
WO2023221936A1 PCT/CN2023/094267 CN2023094267W WO2023221936A1 WO 2023221936 A1 WO2023221936 A1 WO 2023221936A1 CN 2023094267 W CN2023094267 W CN 2023094267W WO 2023221936 A1 WO2023221936 A1 WO 2023221936A1
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seq
sequence
cancer
binding
antigen
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PCT/CN2023/094267
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French (fr)
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WO2023221936A9 (zh
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张凡
蒙盼
钱雪明
李红俊
滕菲
郭欢欢
顾怡
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苏州创胜医药集团有限公司
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure relates to bifunctional protein molecules that target immune checkpoint molecules (eg, PD-L1) and block the activity of anti-tumor immunosuppressive (ATIS) cytokines (eg, IL-1 or TGF ⁇ ) or stimulate immunity, and drugs thereof Preparations, methods of preparation and uses thereof.
  • target immune checkpoint molecules eg, PD-L1
  • AVS anti-tumor immunosuppressive
  • cytokines eg, IL-1 or TGF ⁇
  • PD-L1 is frequently overexpressed in different tumors and its interaction with PD-1 on T cells enables cancer cells to evade T cell-mediated immune responses (Okazaki T et al., Nature Immunology, 2013, 14(12):1212-1218). Therefore, blocking the PD-1/PD-L1 interaction can restore T cell activation and anti-tumor responses (Callahan M K et al., Immunity, 2016, 44(5):1069-1078).
  • Antibody-based PD-1/PD-L1 blocking therapies such as atezolizumab (Rittmeyer A et al., “The Lancet”, 2017, 389(10066):255-265), avelumab (Hamilton G et al., “Expert Opinion on Biological Therapy", 2017, 17(4):515-523) and durvalumab (Brower V, "The Lancet Oncology", 2016, 17(7):e275)
  • the success means a breakthrough in the fight against human cancers, especially solid tumors.
  • an association has been shown between PD-L1 expression by tumor cells and/or infiltrating immune cells and clinical response to PD-1/PD-L1 targeted therapies, this association is not perfect (Herbst, R. et al.
  • TGF- ⁇ transforming growth factor beta
  • TGF- ⁇ transforming growth factor beta
  • EMT epithelial-mesenchymal transition
  • TGF- ⁇ blocking antibodies and anti-PD-L1 reduced TGF- ⁇ signaling in stromal cells, promoted T cell infiltration into the tumor center, and stimulated strong anti-tumor immunity and tumor regression ( Mariathasan S et al., “Nature”, 2018, 554(7693):544-548).
  • the present disclosure provides proteins including a PD-L1 binding moiety linked to a TGF ⁇ binding moiety, an IL-1 binding moiety, an immunostimulatory polypeptide (eg, soluble LAG3 or soluble CD4) or a CD47 binding moiety, pharmaceutical formulations, preparation methods and use.
  • proteins including a PD-L1 binding moiety linked to a TGF ⁇ binding moiety, an IL-1 binding moiety, an immunostimulatory polypeptide (eg, soluble LAG3 or soluble CD4) or a CD47 binding moiety, pharmaceutical formulations, preparation methods and use.
  • the present disclosure provides protein formulations including:
  • Bifunctional molecules including:
  • first portion that binds to an immune checkpoint molecule, said first portion preferably being an antibody directed against PD-L1 or an antigen-binding fragment thereof, and
  • TGF ⁇ binding part is preferably a soluble TGF ⁇ receptor (TGF ⁇ R) or a TGF ⁇ binding fragment or variant thereof, or an antibody against TGF ⁇ and an antigen-binding fragment thereof;
  • protein formulations comprising:
  • a bifunctional molecule comprising a first part that binds to PD-L1 and a second part that i) blocks the activity of immunosuppressive cytokines or ii) stimulates immunity; wherein the first part includes a molecule directed against PD-L1 Antibodies or antigen-binding fragments thereof;
  • Embodiment 1 A protein formulation comprising:
  • a bifunctional molecule which includes a first part that binds to an immune checkpoint molecule and a second part that blocks interleukin-1 (IL-1) activity;
  • Embodiment 2 The protein formulation of embodiment 1, wherein the first portion comprises an agonist of an immunostimulatory checkpoint molecule, optionally, the immunostimulatory checkpoint molecule is selected from the group consisting of: CD27 , CD70, CD28, CD80(B7-1), CD86(B7-2), CD40, CD40L(CD154), CD122, CD137, CD137L, OX40(CD134), OX40L(CD252), GITR, ICOS(CD278) and ICOSLG (CD275), CD2, ICAM-1, LFA-1(CD11a/CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160 and CD83.
  • the immunostimulatory checkpoint molecule is selected from the group consisting of: CD27 , CD70, CD28, CD80(B7-1), CD86(B7-2), CD40, CD40L(CD154), CD122, CD137, CD137L, OX40
  • Embodiment 3 The protein formulation of embodiment 1, wherein the first portion comprises an antagonist of an immunosuppressive checkpoint molecule, optionally, the immunosuppressive checkpoint molecule is selected from the group consisting of: A2AR , B7-H3(CD276), B7-H4(VTCN1), BTLA(CD272), CTLA-4(CD152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L1, PD-L2 , TIM-3, VISTA, SIGLEC7(CD328), TIGIT, PVR(CD155), SIGLEC9(CD329), CD160, LAIR1, 2B4(CD244), CD47, B7-H5.
  • the immunosuppressive checkpoint molecule is selected from the group consisting of: A2AR , B7-H3(CD276), B7-H4(VTCN1), BTLA(CD272), CTLA-4(CD152), IDO1, IDO2, TDO, KIR,
  • Embodiment 4 The protein formulation of embodiment 3, wherein the immune checkpoint molecule is PD-L1.
  • Embodiment 5 The protein formulation according to any one of the preceding embodiments, wherein the first portion comprises an antibody to PD-L1 or an antigen-binding fragment thereof, and the second portion comprises an IL-1 binding portion or IL -1 receptor (IL-1R) binding portion.
  • the first portion comprises an antibody to PD-L1 or an antigen-binding fragment thereof
  • the second portion comprises an IL-1 binding portion or IL -1 receptor (IL-1R) binding portion.
  • IL-1R IL -1 receptor
  • Embodiment 6 The protein formulation of Embodiment 5, wherein the IL-1 binding moiety comprises IL-1R, or a fragment or variant thereof, or an antibody directed against IL-1, or an antigen-binding fragment thereof.
  • Embodiment 7 The protein formulation of embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region from an anti-IL-1 ⁇ antibody or an anti-IL-1 ⁇ antibody and/or A light chain variable region, the anti-IL-1 ⁇ antibody selected from the group consisting of: XB2001, lutikizumab, LY2189102, and bermekimab, or the anti-IL-1 ⁇ antibody Selected from the group consisting of: SSGJ-613, CDP484, canakinumab, and gevokizumab.
  • Embodiment 8 The protein preparation of embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region HCDR1 including the sequence including SEQ ID NO:104 or SEQ ID NO:112, HCDR2 including the sequence including SEQ ID NO:105 or SEQ ID NO:113, and the sequence including SEQ ID NO:106 or SEQ ID NO:114 HCDR3, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and comprising SEQ ID NO :109 or LCDR3 of the sequence SEQ ID NO:117.
  • the heavy chain variable region HCDR1 including the sequence including SEQ ID NO:104 or SEQ ID NO:112
  • HCDR2 including the sequence including SEQ ID NO:105 or SEQ ID NO:113
  • Embodiment 9 The protein preparation according to embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 104, HCDR2 comprising the sequence of SEQ ID NO: 105, and HCDR3 comprising the sequence of SEQ ID NO: 106, the light chain variable region comprising SEQ ID NO: 107 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:108, and LCDR3 of the sequence including SEQ ID NO:109.
  • the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 104
  • HCDR2 comprising the sequence of SEQ ID NO: 105
  • HCDR3 comprising the sequence of SEQ ID NO: 106
  • the light chain variable region comprising SEQ ID NO: 107 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:108, and LCDR3 of
  • Embodiment 10 The protein preparation according to embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 112, HCDR2 comprising the sequence of SEQ ID NO: 113, and HCDR3 comprising the sequence of SEQ ID NO: 114, the light chain variable region comprising SEQ ID NO: 115 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:116, and LCDR3 of the sequence including SEQ ID NO:117.
  • the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 112
  • HCDR2 comprising the sequence of SEQ ID NO: 113
  • HCDR3 comprising the sequence of SEQ ID NO: 114
  • the light chain variable region comprising SEQ ID NO: 115 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:116, and LCDR3 of the
  • Embodiment 11 The protein preparation according to embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region Comprising a sequence selected from the group consisting of: SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region includes a sequence selected from the group consisting of Sequences: SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences having at least 80% sequence identity therewith.
  • Embodiment 12 The protein preparation of embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region Comprising a sequence selected from the group consisting of: SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 103 and a homologous sequence having at least 80% sequence identity thereto.
  • Embodiment 13 The protein preparation of embodiment 6, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region Contains a sequence selected from the group consisting of Column: SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 111, and having at least 80% sequence identity thereto. % sequence identity of homologous sequences.
  • Embodiment 14 The protein formulation of embodiment 5, wherein the IL-1R binding moiety includes an interleukin-1 receptor antagonist, or a fragment or variant thereof, or an antibody directed against IL-1R, or an antigen binding thereof. fragment.
  • Embodiment 15 The protein preparation according to embodiment 14, wherein the antibody against IL-1R or an antigen-binding fragment thereof comprises an antibody selected from the group consisting of spesolimab, astegolimab , imsidolimab, AMG 108, melrilimab, nidanilimab (nidanilimab), MEDI8968, REGN6490, HB0034 and CSC012 antibodies heavy chain variable region and/or light chain variable region.
  • Embodiment 16 A protein formulation comprising:
  • a bifunctional molecule comprising a first part that binds to PD-L1 and a) a second part that blocks the activity of immunosuppressive cytokines or b) stimulates immunity, wherein the first part includes a protein that targets PD-L1
  • HCDR1 comprising DYYMN (SEQ ID NO: 1) or a homologous sequence with at least 80% sequence identity to DYYMN,
  • HCDR3 comprising WGDGPFAY (SEQ ID NO:3) or a homologous sequence with at least 80% sequence identity to WGDGPFAY, and/or
  • the light chain variable region includes:
  • LCDR1 includes a sequence selected from the group consisting of KASQNVX 4 X 5 X 6 VA (SEQ ID NO: 20) or homologous sequences having at least 80% sequence identity with KASQNVX 4 X 5 X 6 VA,
  • LCDR2 includes a sequence selected from the group consisting of SX 7 SX 8 RYT (SEQ ID NO: 21) or a homologous sequence having at least 80% sequence identity to SX 7 SX 8 RYT, and
  • LCDR3 includes a sequence selected from the group consisting of QQYSNYPT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity with QQYSNYPT;
  • X 1 is G or A
  • X 2 is G or D or Q or E or L
  • X 3 is S or M or Q or L or V
  • X 4 is G or P or K
  • X 5 is A or G
  • X 6 is A or I
  • X 7 is A or N or R or V
  • X 8 is N or H or V or D;
  • Embodiment 17 The protein formulation of embodiment 16, wherein the heavy chain variable region comprises:
  • HCDR1 includes the sequence of SEQ ID NO:1,
  • HCDR2 includes a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18, and
  • HCDR3 includes the sequence of SEQ ID NO:3,
  • Light chain variable region the light chain variable region includes:
  • LCDR1 includes a sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9,
  • LCDR2 includes a sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and
  • f)LCDR3 includes the sequence of SEQ ID NO:6.
  • Embodiment 18 The protein formulation of embodiment 17, wherein the heavy chain variable region is selected from the group consisting of:
  • a heavy chain variable region including the following: HCDR1 including the sequence of SEQ ID NO:1, HCDR2 including the sequence of SEQ ID NO:2 and HCDR3 including the sequence of SEQ ID NO:3;
  • a heavy chain variable region including: HCDR1 including the sequence of SEQ ID NO:1, HCDR2 including the sequence of SEQ ID NO:13 and HCDR3 including the sequence of SEQ ID NO:3;
  • a heavy chain variable region including: HCDR1 including the sequence of SEQ ID NO:1, HCDR2 including the sequence of SEQ ID NO:14 and HCDR3 including the sequence of SEQ ID NO:3;
  • HCDR1 comprising the sequence of SEQ ID NO: 1
  • HCDR2 comprising the sequence of SEQ ID NO: 15
  • HCDR3 comprising the sequence of SEQ ID NO: 3;
  • a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 17 and HCDR3 comprising the sequence of SEQ ID NO: 3.
  • Embodiment 19 The protein formulation of embodiment 17 or 18, wherein the light chain variable region is selected from the group consisting of:
  • a light chain variable region including the following: LCDR1 including the sequence of SEQ ID NO:4, LCDR2 including the sequence of SEQ ID NO:5 and LCDR3 including the sequence of SEQ ID NO:6;
  • a light chain variable region comprising the following: LCDR1 comprising the sequence of SEQ ID NO: 9, LCDR2 comprising the sequence of SEQ ID NO: 5 and LCDR3 comprising the sequence of SEQ ID NO: 6;
  • a light chain variable region including: LCDR1 including the sequence of SEQ ID NO:8, LCDR2 including the sequence of SEQ ID NO:5 and LCDR3 including the sequence of SEQ ID NO:6;
  • a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO: 4, LCDR2 comprising the sequence of SEQ ID NO: 12 and LCDR3 comprising the sequence of SEQ ID NO: 6; and
  • a light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO:4, LCDR2 comprising the sequence of SEQ ID NO:11 and LCDR3 comprising the sequence of SEQ ID NO:6.
  • Embodiment 20 The protein formulation of any one of embodiments 16 to 19, wherein the antibody to PD-L1 or antigen-binding fragment thereof further comprises one or more of heavy chain HFR1, HFR2, HFR3 and HFR4. and/or one or more of the light chains LFR1, LFR2, LFR3 and LFR4, where:
  • the HFR1 includes QVQLVQSGAEVKKPGASVKVSCKASGYX 9 FT (SEQ ID NO: 40) or a homologous sequence with at least 80% sequence identity to QVQLVQSGAEVKKPGASVKVSCKASGYX 9 FT,
  • said HFR2 includes WVRQAPGQX 10 LEWMG (SEQ ID NO: 41) or a homologous sequence having at least 80% sequence identity with WVRQAPGQX 10 LEWMG,
  • the HFR3 sequence includes RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 (SEQ ID NO: 42) or a homologous sequence with at least 80% sequence identity to RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 ,
  • the HFR4 includes WGQGTLVTVSS (SEQ ID NO:25) or a homologous sequence having at least 80% sequence identity with WGQGTLVTVSS,
  • the LFR1 includes DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:26) or a homologous sequence with at least 80% sequence identity to DIQMTQSPSSLSSASVGDRVTITC,
  • said LFR2 includes WYQQKPGKX 14 PKLLIY (SEQ ID NO: 43) or a homologous sequence with at least 80% sequence identity to WYQQKPGKX 14 PKLLIY,
  • said LFR3 includes GVPX 15 RFSGSGSGTDFTX 17 TISSLQPEDIATYYC (SEQ ID NO: 44) or a homologous sequence with at least 80% sequence identity to GVPX 15 RFSGSGSGTDFTX 17 TISSLQPEDIATYYC, and
  • the LFR4 includes FGQGTKLEIK (SEQ ID NO: 29) or a homologous sequence with at least 80% sequence identity to FGQGTKLEIK,
  • X 9 is T or V
  • X 10 is G or S
  • X 11 is T or K
  • X 12 is A or V
  • X 13 is R or K
  • X 14 is A or S
  • X 15 is S or D
  • X 16 is M or V
  • X 17 is F or L.
  • Embodiment 21 The protein formulation of embodiment 20, wherein:
  • the HFR1 includes a sequence selected from the group consisting of SEQ ID NO: 22 and 30,
  • the HFR2 includes a sequence selected from the group consisting of SEQ ID NO: 23 and 31,
  • the HFR3 includes a sequence selected from the group consisting of SEQ ID NO: 24 and 32-35,
  • the HFR4 includes the sequence of SEQ ID NO: 25,
  • the LFR1 includes a sequence from the group consisting of SEQ ID NO: 26,
  • the LFR2 includes a sequence selected from the group consisting of SEQ ID NO: 27 and 36,
  • the LFR3 includes a sequence selected from the group consisting of SEQ ID NO: 28 and 37-38, 39, 45, and
  • the LFR4 includes the sequence of SEQ ID NO:29.
  • Embodiment 22 The protein formulation of any one of embodiments 16 to 21, wherein the heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and combinations thereof Homologous sequences having at least 80% sequence identity.
  • Embodiment 23 The protein formulation of any one of embodiments 16 to 22, wherein the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and homologous sequences thereof having at least 80% sequence identity therewith.
  • Embodiment 24 The protein formulation of embodiment 16, wherein the antibody directed against PD-L1 or an antigen-binding fragment thereof comprises a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of :SEQ ID NO:49/54, 50/54, 51/54, 52/54, 49/55, 50/55, 51/55, 52/55, 58/62, 58/63, 58/64, 58 /65, 59/62, 59/63, 59/64, 59/65, 60/62, 60/63, 60/64 and 60/65 and homologous sequences thereof having at least 80% sequence identity thereto.
  • Embodiment 25 The protein formulation of any one of embodiments 16 to 24, wherein the antibody against PD-L1 or antigen-binding fragment thereof further comprises one or more amino acid residue substitutions or modifications, but remains Specific binding specificity and/or affinity for PD-L1.
  • Embodiment 26 The protein formulation of embodiment 25, wherein at least one of said substitutions or modifications is in one or more of said CDR sequences and/or in said VH or VL In one or more non-CDR regions of the sequence.
  • Embodiment 27 The protein formulation according to any one of embodiments 16 to 26, wherein the antibody directed against PD-L1 or an antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a constant region of a human Ig , or optionally the constant region of a human IgG.
  • Embodiment 28 The protein formulation of embodiment 27, wherein the constant region comprises the Fc region of human IgGl, IgG2, IgG3 or IgG4.
  • Embodiment 29 The protein formulation of embodiment 28, wherein the constant region comprises an Fc variant having reduced effector function relative to a corresponding wild-type Fc region.
  • Embodiment 30 The protein formulation of embodiment 29, wherein the Fc variant comprises one or more amino acid residue substitutions selected from the group consisting of: 220S, 226S, 228P, 229S, 233P, 234V, 234G , 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S and any combination thereof, wherein the numbering of the residues in the Fc region is the numbering of the EU index in Kabat.
  • Embodiment 31 The protein formulation of any one of embodiments 29 to 30, wherein the Fc variant comprises a combination of mutations selected from the group consisting of: a) K322A, L234A and L235A; b) P331S, L234F and L235E; c) L234A and L235A; c) N297A; d) N297Q; e) N297G; f) L235E; g) L234A and L235A (IgG1); A330S and P331S (IgG2); j) V234A, G237A, P238S, H268A, V309L, A330S and P331S (IgG2), wherein the numbering of the residues in the Fc region is the numbering of the EU index in Kabat.
  • the Fc variant comprises a combination of mutations selected from the group consisting of: a) K322A, L234A and L235A; b
  • Embodiment 32 The protein formulation of any one of embodiments 29 to 31, wherein the Fc variant comprises the amino acid sequence of SEQ ID NO: 81.
  • Embodiment 33 The protein formulation of any one of embodiments 16 to 32, wherein the antibody to PD-L1 or antigen-binding fragment thereof is humanized.
  • Embodiment 34 The protein formulation of any one of embodiments 16 to 33, wherein the antigen-binding fragment is a diabody, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide bond Stable Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer ( Bivalent diabodies), multispecific antibodies, camelized single domain antibodies, Nanobodies, domain antibodies and bivalent domain antibodies.
  • the antigen-binding fragment is a diabody, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide bond Stable Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulf
  • Embodiment 35 The protein formulation of any one of embodiments 16 to 34, wherein the antibody or antigen-binding fragment thereof is capable of binding to both human PD-L1 and cynomolgus monkey PD-L1.
  • Embodiment 36 The protein formulation of any one of embodiments 1 to 15, wherein the first portion comprises competing for binding to PD- Antibodies to L1 or antigen-binding fragments thereof.
  • Embodiment 37 The protein formulation of any one of embodiments 16 to 36, wherein the immunosuppressive cytokine comprises a cytokine in the transforming growth factor beta (TGF- ⁇ ) superfamily, IL-1 or vascular Endothelial growth factor (VEGF).
  • TGF- ⁇ transforming growth factor beta
  • VEGF vascular Endothelial growth factor
  • Embodiment 38 The protein formulation of embodiment 37, wherein the immunosuppressive cytokines in the TGF- ⁇ superfamily include TGF- ⁇ , bone morphogenetic protein (BMP), activin, NODAL, and growth and differentiation. factor(GDF).
  • TGF- ⁇ TGF- ⁇
  • BMP bone morphogenetic protein
  • activin activin
  • NODAL NODAL
  • GDF growth and differentiation. factor
  • Embodiment 39 The protein formulation of any one of embodiments 16 to 38, wherein the immunosuppressive cytokine is TGF- ⁇ .
  • Embodiment 40 The protein formulation of embodiments 16 to 39, wherein the second portion comprises a TGF ⁇ binding moiety.
  • Embodiment 41 The protein formulation of embodiment 40, wherein the TGF[beta] binding moiety comprises a soluble TGF[beta] receptor (TGF[beta]R) or a TGF[beta] binding fragment or variant thereof, or an antibody directed against TGF[beta] and an antigen-binding fragment thereof.
  • TGF[beta]R soluble TGF[beta] receptor
  • TGF[beta]R soluble TGF[beta] receptor
  • TGF[beta]R TGF[beta] binding fragment or variant thereof
  • an antibody directed against TGF[beta] and an antigen-binding fragment thereof an antibody directed against TGF[beta] and an antigen-binding fragment thereof.
  • Embodiment 42 The protein formulation of embodiment 41, wherein the soluble TGF ⁇ R comprises the extracellular domain (ECD) of the TGF ⁇ R or a TGF ⁇ binding fragment or variant thereof.
  • ECD extracellular domain
  • Embodiment 43 The protein formulation of embodiment 42, wherein the TGF ⁇ R is selected from the group consisting of: TGF ⁇ receptor I (TGF ⁇ RI), TGF ⁇ receptor II (TGF ⁇ RII), TGF ⁇ receptor III (TGF ⁇ RIII), and Any combination.
  • TGF ⁇ R TGF ⁇ receptor I
  • TGF ⁇ RII TGF ⁇ receptor II
  • TGF ⁇ RIII TGF ⁇ receptor III
  • Embodiment 44 The protein formulation of embodiment 42, wherein the TGF ⁇ R is TGF ⁇ RII.
  • Embodiment 45 The protein formulation of embodiment 44, wherein the TGF ⁇ RII selectively binds to TGF ⁇ 1 relative to TGF ⁇ 2 and TGF ⁇ 3.
  • Embodiment 46 The protein formulation of embodiment 45, wherein the TGF ⁇ 1 is human TGF ⁇ 1 or mouse TGF ⁇ 1.
  • Embodiment 47 The protein preparation according to any one of embodiments 42 to 46, wherein the ECD of TGF ⁇ R comprises the amino acid sequence of SEQ ID NO: 66, 79, 78, 77 or is at least 80% identical to the amino acid sequence. % sequence identity but still retain a specific binding specificity and/or affinity for TGF- ⁇ .
  • Embodiment 48 The protein formulation of embodiment 36, wherein the second portion comprises an IL-1 binding moiety or an IL-1 receptor (IL-1R) binding moiety.
  • IL-1R IL-1 receptor
  • Embodiment 49 The protein formulation of embodiment 48, wherein the IL-1 binding portion comprises soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody directed against IL-1, or thereof. Antigen-binding fragments.
  • Embodiment 50 The protein formulation of embodiment 49, wherein the antibody to IL-1 or antigen-binding fragment thereof comprises a heavy chain variable region from an anti-IL-1 ⁇ antibody or an anti-IL-1 ⁇ antibody and/or A light chain variable region, the anti-IL-1 ⁇ antibody selected from the group consisting of: XB2001, lutikizumab, LY2189102, and bermekimab, or the anti-IL-1 ⁇ antibody Selected from the group consisting of: SSGJ-613, CDP484, canakinumab, and gevokizumab.
  • the antibody to IL-1 or antigen-binding fragment thereof comprises a heavy chain variable region from an anti-IL-1 ⁇ antibody or an anti-IL-1 ⁇ antibody and/or A light chain variable region, the anti-IL-1 ⁇ antibody selected from the group consisting of: XB2001, lutikizumab, LY2189102, and bermekimab, or the anti-IL-1 ⁇ antibody Selected from the group consisting of: SSGJ-613,
  • Embodiment 51 The protein formulation of embodiment 49, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region HCDR1 including the sequence including SEQ ID NO:104 or SEQ ID NO:112, HCDR2 including the sequence including SEQ ID NO:105 or SEQ ID NO:113, and the sequence including SEQ ID NO:106 or SEQ ID NO:114 HCDR3, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and comprising SEQ ID NO :109 or LCDR3 of the sequence SEQ ID NO:117.
  • the heavy chain variable region HCDR1 including the sequence including SEQ ID NO:104 or SEQ ID NO:112
  • HCDR2 including the sequence including SEQ ID NO:105 or SEQ ID NO:113
  • Embodiment 52 The protein formulation of embodiment 49, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 104, HCDR2 comprising the sequence of SEQ ID NO: 105, and HCDR3 comprising the sequence of SEQ ID NO: 106, the light chain variable region comprising SEQ ID NO: 107 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:108, and LCDR3 of the sequence including SEQ ID NO:109.
  • the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 104
  • HCDR2 comprising the sequence of SEQ ID NO: 105
  • HCDR3 comprising the sequence of SEQ ID NO: 106
  • the light chain variable region comprising SEQ ID NO: 107 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:108, and LCDR3 of
  • Embodiment 53 The protein formulation of embodiment 49, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 112, HCDR2 comprising the sequence of SEQ ID NO: 113, and HCDR3 comprising the sequence of SEQ ID NO: 114, the light chain variable region comprising SEQ ID NO: 115 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:116, and LCDR3 of the sequence including SEQ ID NO:117.
  • the heavy chain variable region HCDR1 comprising the sequence of SEQ ID NO: 112
  • HCDR2 comprising the sequence of SEQ ID NO: 113
  • HCDR3 comprising the sequence of SEQ ID NO: 114
  • the light chain variable region comprising SEQ ID NO: 115 LCDR1 of the sequence, LCDR2 of the sequence including SEQ ID NO:116, and LCDR3 of the
  • Embodiment 54 The protein formulation of embodiment 49, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region Comprising a sequence selected from the group consisting of: SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region includes a sequence selected from the group consisting of Sequences: SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences having at least 80% sequence identity therewith.
  • Embodiment 55 The protein formulation of embodiment 49, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region Comprising a sequence selected from the group consisting of: SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 103 and a homologous sequence having at least 80% sequence identity thereto.
  • Embodiment 56 The protein formulation of embodiment 49, wherein the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region Comprising a sequence selected from the group consisting of: SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO. :111, and a homologous sequence having at least 80% sequence identity therewith.
  • Embodiment 57 The protein formulation of embodiment 49, wherein the bifunctional molecule comprises a heavy chain of the amino acid sequence of SEQ ID NO: 118 or SEQ ID NO: 120, and/or comprises SEQ ID NO: 119 or SEQ The light chain of the amino acid sequence of ID NO:121.
  • Embodiment 58 The protein formulation of embodiment 49, wherein the IL-1 binding moiety comprises: the extracellular domain (ECD) of IL-1RI; IL-1RI, the ECD of IL-1RI, IL-1RII, or The ECD of IL-1RII, or an IL-1 binding fragment or variant of any of the ECD of IL-1RAP or IL-1RAP, IL-1sRI, or IL-1sRII.
  • ECD extracellular domain
  • Embodiment 59 The protein formulation of embodiment 48, wherein the IL-1R binding portion comprises IL-IRa, or an IL-1 binding fragment or variant thereof, or an antibody directed against IL-1R, or an antigen-binding fragment thereof.
  • Embodiment 60 The protein preparation of embodiment 59, wherein the antibody against IL-1R or an antigen-binding fragment thereof comprises an antibody selected from the group consisting of spesolimab, astegolimab , imsidolimab, AMG 108, melrilimab, nidanilimab (nidanilimab), MEDI8968, REGN6490, HB0034 and CSC012 antibodies heavy chain variable region and/or light chain variable region.
  • Embodiment 61 The protein formulation of embodiment 60, wherein the IL-1R binding portion comprises the amino acid sequence of SEQ ID NO: 67 or 76, or has at least 80% sequence identity with SEQ ID NO: 67 or 76 The amino acid sequence, or IL-1 binding fragment or variant thereof.
  • Embodiment 62 The protein formulation of any one of embodiments 48 to 49, wherein the IL-1 is IL-1 ⁇ or IL-1 ⁇ .
  • Embodiment 63 The protein formulation of embodiment 62, wherein the IL-1 ⁇ is human IL-1 ⁇ .
  • Embodiment 64 The protein formulation of any one of embodiments 16 to 36, wherein the second portion stimulates anti-tumor immunity and includes an immunostimulatory polypeptide.
  • Embodiment 65 The protein formulation of embodiment 64, wherein the immunostimulatory polypeptide comprises interleukin (IL)-2 (IL-2), IL-15, IL-21, IL-10, IL- 12. IL-23, IL-27, IL-35, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble CD4, soluble LAG-3, IFN- ⁇ or their functional equivalents.
  • IL interleukin
  • IL-15 interleukin-21
  • IL-10 IL- 12.
  • IL-23, IL-27, IL-35 granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble CD4, soluble LAG-3, IFN- ⁇ or their functional equivalents.
  • Embodiment 66 The protein formulation of embodiment 65, wherein said soluble LAG-3 comprises the extracellular domain (ECD) of said LAG-3 or a MHCII binding variant thereof.
  • ECD extracellular domain
  • Embodiment 67 The protein formulation of any one of embodiments 16 to 36, wherein the second portion stimulates anti-tumor immunity and includes an antagonist of immunosuppressive receptor signaling.
  • Embodiment 68 The protein formulation of embodiment 67, wherein the immunosuppressive receptor is signal regulatory protein alpha (SIRP ⁇ ).
  • SIRP ⁇ signal regulatory protein alpha
  • Embodiment 69 The protein formulation of embodiment 68, wherein the second moiety blocks the interaction between CD47 and SIRP ⁇ .
  • Embodiment 70 The protein formulation of embodiment 69, wherein the second portion comprises a CD47 binding domain or a SIRP ⁇ binding domain.
  • Embodiment 71 The protein formulation of embodiment 70, wherein the CD47 binding domain comprises soluble SIRP ⁇ , or a CD47 binding fragment or variant thereof, or an anti-CD47 antibody, or an antigen binding fragment thereof.
  • Embodiment 72 The protein formulation of embodiment 71, wherein said soluble SIRP ⁇ comprises said The extracellular domain (ECD) of SIRP ⁇ or its CD47-binding variant.
  • ECD extracellular domain
  • Embodiment 73 The protein formulation of embodiment 70, wherein the CD47 binding domain comprises the amino acid sequence of SEQ ID NO: 84 or has at least 80% sequence identity with the amino acid sequence but retains binding to CD47 specific amino acid sequence.
  • Embodiment 74 The protein formulation of embodiment 70, wherein the SIRP ⁇ binding domain comprises soluble CD47, or a SIRP ⁇ binding fragment or variant thereof, or an anti-SIRP ⁇ antibody, or an antigen-binding fragment thereof.
  • Embodiment 75 The protein formulation of embodiment 74, wherein the soluble CD47 comprises the extracellular domain (ECD) of CD47, or a SIRP ⁇ binding fragment or variant thereof, an anti-SIRP ⁇ antibody, or an antigen-binding fragment thereof.
  • ECD extracellular domain
  • Embodiment 76 The protein formulation of any one of the preceding embodiments, further comprising a linker connecting the first moiety and the second moiety.
  • Embodiment 77 The protein formulation of embodiment 76, wherein the linker is selected from the group consisting of cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers.
  • Embodiment 78 The protein formulation of embodiment 77, wherein the linker comprises the amino acid sequence of ((G)nS)m, wherein m and n are independently an integer selected from 0 to 30.
  • Embodiment 79 The protein formulation of any one of embodiments 16 to 78, wherein the bifunctional molecule includes one or more of the second moieties.
  • Embodiment 80 The protein formulation of embodiment 79, wherein at least one second moiety of the plurality of second moieties is linked to the N-terminus or C-terminus of the polypeptide chain of the first moiety.
  • Embodiment 81 The protein formulation of embodiment 79, wherein at least one second portion of said plurality of second portions is linked to: a) the N-terminus or C-terminus of the heavy chain of said first portion, or b ) the N-terminus or C-terminus of the light chain of the first part.
  • Embodiment 82 The protein formulation of embodiment 79, wherein at least one second moiety of the plurality of second moieties is linked to the C-terminus of the heavy chain constant region of the first moiety.
  • Embodiment 83 The protein formulation of embodiment 79, wherein each second portion of the plurality of second portions is individually linked to the C-terminus of each heavy chain constant region of the first portion.
  • Embodiment 84 The protein formulation of embodiment 79, wherein the bifunctional molecule includes more than one of the second moieties, each of the more than one second moiety being linked to: the first The N-terminus of the heavy chain of a portion, the C-terminus of the heavy chain of the first portion, the N-terminus of the light chain of the first portion, the C-terminus of the light chain of the first portion, or any combination thereof.
  • Embodiment 85 The protein formulation of any one of embodiments 79 to 84, wherein the bifunctional molecule comprises a homodimeric heavy chain or a heterodimeric heavy chain.
  • Embodiment 86 The protein formulation of any one of embodiments 79 to 84, wherein the heavy chain is heterodimeric with respect to the presence or position of the second moiety.
  • Embodiment 87 The protein formulation of embodiment 86, wherein the heterodimeric heavy chains comprise one heavy chain having the second portion and the other heavy chain not having the second portion.
  • Embodiment 88 The protein formulation of embodiment 86, wherein the heterodimer heavy chain further comprises a heterodimer associated in a manner that hinders homodimerization and/or facilitates heterodimerization. Fc area.
  • Embodiment 89 The protein formulation of embodiment 86, wherein the heterodimeric Fc region is capable of associating into a heterodimer through a pestle structure, hydrophobic interaction, electrostatic interaction, hydrophilic interaction or increased flexibility. .
  • Embodiment 90 The protein formulation of any one of embodiments 86 to 89, wherein the heterodimeric Fc region includes Y349C, T366S, L368A or Y407V or any combination thereof in one Fc polypeptide chain and in another An Fc polypeptide chain includes S354C or T366W or a combination thereof, wherein the numbering of the residues in the Fc polypeptide chain is the numbering of the EU index in Kabat.
  • Embodiment 91 The protein formulation of any one of the preceding embodiments, further linked to one or more conjugate moieties.
  • Embodiment 92 The protein formulation of Embodiment 91, wherein the conjugate moiety includes a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label , DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents or other anti-cancer drugs, such as androgen receptor inhibitors.
  • the conjugate moiety includes a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label , DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents or other anti-cancer drugs, such as androgen receptor inhibitors.
  • Embodiment 93 The protein formulation according to any one of embodiments 16 to 35, 37 to 47 and 76-92, wherein the first portion of the bifunctional molecule comprises an antibody to PD-L1 or an antigen-binding fragment thereof, and the second Parts include the TGF ⁇ binding moiety,
  • the antibody against PD-L1 or an antigen-binding fragment thereof includes a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NO: 49/54, 50/54, 51 /54, 52/54, 49/55, 50/55, 51/55, 52/55, 58/62, 58/63, 58/64, 58/65, 59/62, 59/63, 59/64 , 59/65, 60/62, 60/63, 60/64 and 60/65 and their homologous sequences having at least 80% sequence identity therewith; and/or
  • the TGF ⁇ binding portion includes a soluble TGF ⁇ receptor (TGF ⁇ R) or a TGF ⁇ binding fragment or variant thereof, wherein the soluble TGF ⁇ R includes an extracellular domain (ECD) of the TGF ⁇ R or a TGF ⁇ binding fragment or variant thereof, and
  • ECD of TGF ⁇ R includes the amino acid sequence of SEQ ID NO: 66, 79, 78, 77 or has at least 80% sequence identity with the amino acid sequence but still retains specific binding specificity and/or affinity for TGF- ⁇ sequence.
  • Embodiment 94 The protein formulation according to Embodiment 93, wherein the first portion of the bifunctional molecule comprises an antibody to PD-L1 or an antigen-binding fragment thereof, and the second portion comprises a TGF ⁇ binding moiety,
  • the antibody against PD-L1 or an antigen-binding fragment thereof includes a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NO: 59/64 and having at least 80% sequence identity of its homologous sequence; and/or
  • the TGF ⁇ binding portion includes a soluble TGF ⁇ receptor (TGF ⁇ R) or a TGF ⁇ binding fragment or variant thereof, wherein the soluble TGF ⁇ R includes an extracellular domain (ECD) of the TGF ⁇ R or a TGF ⁇ binding fragment or variant thereof, and
  • ECD of TGF ⁇ R includes the amino acid sequence of SEQ ID NO: 66 or a sequence that has at least 80% sequence identity with the amino acid sequence but still retains specific binding specificity and/or affinity for TGF- ⁇ .
  • Embodiment 95 The protein formulation according to any one of the preceding embodiments, wherein the concentration of the bifunctional molecule is about 1-200 mg/mL, such as about 1-150 mg/mL, about 100-200 mg/mL, about 1- 100 mg/mL, about 10-100 mg/mL, about 10-50 mg/mL, about 20-30 mg/mL, about 20 mg/mL, or about 30 mg/mL.
  • Embodiment 96 The protein formulation according to any one of the preceding embodiments, wherein the buffer is acetate, histidine, citrate, succinate, malate, glutamate, phosphate or Lactate buffer, such as acetate buffer or histidine buffer; the acetate buffer is, for example, acetic acid-sodium acetate buffer; the histidine buffer, for example, is histidine-hydrochloride buffer agent.
  • the buffer is acetate, histidine, citrate, succinate, malate, glutamate, phosphate or Lactate buffer, such as acetate buffer or histidine buffer
  • the acetate buffer is, for example, acetic acid-sodium acetate buffer
  • the histidine buffer for example, is histidine-hydrochloride buffer agent.
  • Embodiment 97 The protein formulation according to any one of the preceding embodiments, wherein the concentration of the buffer is about 5-100mM, such as about 10-50mM, about 15-30mM, about 10-20mM, about 25mM or about 20mM. .
  • Embodiment 98 The protein formulation according to any one of the preceding embodiments, wherein the buffer is an acetate buffer, such as an acetic acid-sodium acetate buffer, of about 10-50mM, about 15-30mM, or about 20mM.
  • an acetate buffer such as an acetic acid-sodium acetate buffer
  • Embodiment 99 The protein formulation according to any one of the preceding embodiments, wherein the buffer is about 10-50mM, about 15-30mM or about 20mM histidine buffer, for example a histidine-hydrochloride buffer .
  • Embodiment 100 The protein formulation according to any one of the preceding embodiments, wherein the pH of the formulation is in the range of about 4.5-6.0, such as about 5.0-5.6, about 5.0-5.5, about 5.3, or 5.3 ⁇ 5%.
  • Embodiment 101 The protein formulation according to any one of the preceding embodiments, wherein the surfactant is a nonionic surfactant, such as polysorbate, poloxamer, Brij or Triton X, such as polysorbate 80 or polysorbate Sorbate 20.
  • a nonionic surfactant such as polysorbate, poloxamer, Brij or Triton X, such as polysorbate 80 or polysorbate Sorbate 20.
  • Embodiment 102 The protein formulation according to any one of the preceding embodiments, wherein the surfactant concentration is about 0.01-0.5% (w/v), such as about 0.01-0.1% (w/v), 0.02-0.1 % (w/v), about 0.025%-0.1% (w/v) or about 0.05% (w/v).
  • Embodiment 103 The protein formulation according to any one of the preceding embodiments, wherein the surfactant is about 0.01-0.5% (w/v), about 0.01-0.1% (w/v), 0.02-0.1% ( w/v), about 0.025% to 0.1% (w/v), or about 0.05% (w/v) of a polysorbate, such as polysorbate 80 or polysorbate 20.
  • a polysorbate such as polysorbate 80 or polysorbate 20.
  • Embodiment 104 The protein formulation according to any one of the preceding embodiments, wherein the surfactant is about 0.01-0.5% (w/v), about 0.01-0.1% (w/v), 0.02-0.1% ( w/v), about 0.025%-0.1% (w/v), or about 0.05% (w/v) polysorbate 80.
  • Embodiment 105 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises a stabilizer.
  • Embodiment 106 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises a stabilizer selected from one or more of amino acids, inorganic salts, sugars, polyols and chelating agents, preferably Arginine hydrochloride, arginine or NaCl.
  • a stabilizer selected from one or more of amino acids, inorganic salts, sugars, polyols and chelating agents, preferably Arginine hydrochloride, arginine or NaCl.
  • Embodiment 107 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises a stabilizer at a concentration of about 10-300mM, such as about 100-300mM, about 120-300mM, about 100- 200mM, about 130-170mM, about 140-160mM or about 150mM.
  • Embodiment 108 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises arginine hydrochloride or arginine.
  • Embodiment 109 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 100-200mM, about 130-170mM, about 140-160mM, or about 150mM arginine hydrochloride or arginine.
  • Embodiment 110 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 100- 200mM, about 130-170mM, about 140-160mM or about 150mM arginine hydrochloride.
  • Embodiment 111 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 130-170mM, about 140-160mM, or about 150mM arginine hydrochloride.
  • Embodiment 112. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises NaCl.
  • Embodiment 113 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 100-200mM, about 130-170mM, or about 135mM NaCl.
  • Embodiment 114 The protein formulation according to any one of the preceding embodiments, wherein the formulation optionally comprises a chelating agent, such as EDTA ⁇ 2Na, at a concentration of, for example, about 30-350 ⁇ M, such as 75-300 ⁇ M.
  • a chelating agent such as EDTA ⁇ 2Na
  • Embodiment 115 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 0.01-0.5% (w/v), about 0.01-0.1% (w/v), 0.02-0.1% (w/ v), about 0.025%-0.1% (w/v), or about 0.05% (w/v) polysorbate, such as polysorbate 80 or polysorbate 20, and about 30-350 ⁇ M, such as 75-300 ⁇ M EDTA ⁇ 2Na.
  • Embodiment 116 The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises from about 0.025% to 0.1% (w/v) (eg, about 0.05% (w/v)) of a polysorbate (eg, poly Sorbitate 80) and approximately 30-350 ⁇ M EDTA ⁇ 2Na.
  • w/v 0.1%
  • w/v polysorbate
  • 30-350 ⁇ M EDTA ⁇ 2Na approximately 30-350 ⁇ M EDTA ⁇ 2Na.
  • Embodiment 117 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.0-5.6.
  • Embodiment 118 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.0-5.6.
  • Embodiment 119 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.0-5.6.
  • Embodiment 120 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.3 ⁇ 5%.
  • Embodiment 121 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.3 ⁇ 5%.
  • Embodiment 122 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.3 ⁇ 5%.
  • Embodiment 123 The protein formulation according to any one of the preceding embodiments, comprising:
  • the pH is 5.3.
  • Embodiment 124 The protein formulation according to any one of the preceding embodiments, wherein the bifunctional molecule is formulated with a chelating agent, such as EDTA ⁇ 2Na (e.g., about 0.3-10mM, about 0.1-20mM, about 0.3-15mM , about 0.3-10mM or about 0.5-5mM EDTA ⁇ 2Na) for treatment, and then the chelating agent is removed; for example, the treatment is performed in a buffer at room temperature; for example, the chelating agent is processed by dialysis, ultrasonic Removed by filtration or diafiltration (for example, dialysis with a buffer); optionally adding other auxiliary materials after removing the chelating agent to prepare the preparation.
  • a chelating agent such as EDTA ⁇ 2Na (e.g., about 0.3-10mM, about 0.1-20mM, about 0.3-15mM , about 0.3-10mM or about 0.5-5mM EDTA ⁇ 2Na) for treatment, and then
  • Embodiment 125 The protein formulation according to any one of the preceding embodiments, wherein the bifunctional molecule is formulated with a chelating agent, such as EDTA ⁇ 2Na (e.g., about 0.3-10mM, about 0.1-20mM, about 0.3-15mM , about 0.3-10mM or about 0.5-5mM EDTA ⁇ 2Na) in buffer at room temperature (for example, overnight), and then the chelating agent is removed by dialysis, ultrafiltration or diafiltration with buffer.
  • a chelating agent such as EDTA ⁇ 2Na (e.g., about 0.3-10mM, about 0.1-20mM, about 0.3-15mM , about 0.3-10mM or about 0.5-5mM EDTA ⁇ 2Na) in buffer at room temperature (for example, overnight), and then the chelating agent is removed by dialysis, ultrafiltration or diafiltration with buffer.
  • a chelating agent such as EDTA ⁇ 2Na (e.g., about
  • Embodiment 126 The protein formulation according to any one of the preceding embodiments, wherein the bifunctional molecule is formulated with a chelating agent, such as EDTA ⁇ 2Na (e.g., about 0.3-10mM, about 0.1-20mM, about 0.3-15mM , about 0.3-10mM or about 0.5-5mM EDTA ⁇ 2Na) in a buffer at room temperature (for example, overnight), then remove the chelating agent by dialysis, ultrafiltration or diafiltration with the buffer, and then add other Pharmaceutically acceptable excipients are used to prepare the preparation.
  • a chelating agent such as EDTA ⁇ 2Na (e.g., about 0.3-10mM, about 0.1-20mM, about 0.3-15mM , about 0.3-10mM or about 0.5-5mM EDTA ⁇ 2Na) in a buffer at room temperature (for example, overnight), then remove the chelating agent by dialysis, ultrafiltration or diafiltration with the buffer, and then add other
  • Embodiment 127 The protein formulation according to any of the preceding embodiments in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form.
  • Embodiment 128 The protein formulation according to any one of the preceding embodiments, which is an injection, such as intravenous, intramuscular or subcutaneous injection.
  • an injection such as intravenous, intramuscular or subcutaneous injection.
  • Embodiment 129 The protein formulation according to any one of the preceding embodiments, which is a liquid injection.
  • Embodiment 130 The protein formulation according to any one of embodiments 1-129 for treating, preventing or alleviating a PD-L1 related disease in a subject.
  • Embodiment 131 The protein formulation for use according to embodiment 130, wherein the disease is an immune-related disease or disorder, cancer, or an infectious disease.
  • Embodiment 132 The protein formulation for use according to Embodiment 131, wherein the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and Bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, prostate cancer, gastroesophageal cancer, rectum Cancer, anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glue tumors and adenocarcinomas.
  • lung cancer e.g., non-small cell lung cancer
  • liver cancer pancreatic cancer
  • breast cancer bronchi
  • Embodiment 133 The protein formulation for use according to any one of embodiments 130-132, wherein the subject has been identified as having cancer cells expressing PD-L1.
  • Embodiment 134 The protein formulation for use according to any one of embodiments 130-133, wherein the PD-L1 related disease is resistant to PD-L1/PD-1 monotherapy.
  • Embodiment 135. The protein formulation for use according to any one of embodiments 130-134, wherein the subject is a human.
  • Embodiment 136 The protein formulation according to any one of embodiments 1-129 for the treatment, prevention or alleviation of a disease that would benefit from the suppression of immunosuppressive cytokines, the induction of sustained immune responses or the stimulation of anti-tumor immunity. or disease;
  • the immunosuppressive cytokine is TGF ⁇ ;
  • the disease or condition is a TGF ⁇ -related disease or condition;
  • the TGF ⁇ -related disease is cancer, fibrotic disease or kidney disease;
  • the immunosuppressive cytokine is IL-1;
  • the disease or condition is an IL-1 related disease or condition;
  • the disease or condition would benefit from induction of MHCII signaling by stimulating MHCII signaling with an immunostimulatory polypeptide inducing a sustained immune response; for example, the immunostimulatory polypeptide is soluble LAG-3; or
  • the disease or condition would benefit from stimulating anti-tumor immunity by inhibiting immunosuppressive receptor signaling; for example, the immunosuppressive receptor is SIRP ⁇ .
  • Embodiment 137 The protein formulation for use according to any one of embodiments 130-136, wherein the protein formulation is administered in combination with a second therapeutic agent.
  • Embodiment 138 The protein formulation for use according to embodiment 137, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer agents, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents , gene therapy agents, hormone therapy agents or cytokines.
  • the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer agents, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents , gene therapy agents, hormone therapy agents or cytokines.
  • Embodiment 139 A pharmaceutical combination comprising a protein formulation according to any one of embodiments 1-129, and an agent selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents , cell therapy agent, gene therapy agent, hormone therapy agent or second therapeutic agent of cytokines.
  • Embodiment 140 Use of the protein formulation according to any one of embodiments 1-129 in the preparation of a medicament for treating, preventing or alleviating PD-L1 related diseases in a subject.
  • Embodiment 141 Use according to embodiment 140, wherein the disease is an immune-related disease or disorder, cancer, or an infectious disease.
  • Embodiment 142 Use according to embodiment 141, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and cholangiocarcinoma , ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, prostate cancer, gastroesophageal cancer, rectal cancer, Anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma and adenocarcinoma.
  • lung cancer e.g., non-small cell lung cancer
  • liver cancer pancreatic cancer
  • breast cancer bronchial cancer
  • Embodiment 143 The use according to any one of embodiments 140-142, wherein the subject has been identified as having cancer cells expressing PD-L1.
  • Embodiment 144 Use according to any one of embodiments 140-143, wherein the PD-L1 related disease is resistant to PD-L1/PD-1 monotherapy.
  • Embodiment 145 The use according to any one of embodiments 140-144, wherein the subject is a human.
  • Embodiment 146 Use of a protein formulation according to any one of embodiments 1 to 129 for the manufacture of a medicament for treatment, prevention or relief that would benefit from the suppression of immunosuppressive cytokines, the induction of a sustained immune response or diseases or conditions that stimulate anti-tumor immunity;
  • the immunosuppressive cytokine is TGF ⁇ ;
  • the disease or condition is a TGF ⁇ -related disease or condition;
  • the TGF ⁇ -related disease is cancer, fibrotic disease or kidney disease;
  • the immunosuppressive cytokine is IL-1;
  • the disease or condition is an IL-1 related disease or condition;
  • the disease or condition would benefit from the induction of a sustained immune response by stimulating MHCII signaling with an immunostimulatory polypeptide; for example, the immunostimulatory polypeptide is soluble LAG-3; or
  • the disease or condition would benefit from stimulating anti-tumor immunity through inhibition of immunosuppressive receptor signaling immune; for example, the immunosuppressive receptor is SIRP ⁇ .
  • Embodiment 147 The use according to any one of embodiments 140-146, wherein the agent is administered in combination with a second therapeutic agent.
  • Embodiment 148 Use according to embodiment 147, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapeutic agents, hormonal treatments, or cytokines.
  • the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapeutic agents, hormonal treatments, or cytokines.
  • Embodiment 149 A method of treating, preventing, or alleviating a PD-L1-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein formulation according to any one of embodiments 1-129.
  • Embodiment 150 The method according to embodiment 149, wherein the disease is an immune-related disease or disorder, cancer, or an infectious disease.
  • Embodiment 151 The method of embodiment 150, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and cholangiocarcinoma , ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, prostate cancer, gastroesophageal cancer, rectal cancer, Anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma and adenocarcinoma.
  • lung cancer e.g., non-small cell lung cancer
  • liver cancer pancreatic cancer
  • breast cancer bronchial cancer
  • Embodiment 152 The method according to any one of embodiments 149-151, wherein the subject has been identified as having cancer cells expressing PD-L1.
  • Embodiment 153 The method according to any one of embodiments 149-152, wherein said PD-L1 related disease is resistant to PD-L1/PD-1 monotherapy
  • Embodiment 154 The method according to any one of embodiments 149-153, wherein the subject is a human.
  • Embodiment 155 A method of treating, preventing, or alleviating a disease or condition in a subject that would benefit from inhibition of immunosuppressive cytokines, induction of sustained immune response, or stimulation of anti-tumor immunity, said method comprising administering an effective amount of a protein formulation according to any one of embodiments 1-129;
  • the immunosuppressive cytokine is TGF ⁇ ;
  • the disease or condition is a TGF ⁇ -related disease or condition;
  • the TGF ⁇ -related disease is cancer, fibrotic disease or kidney disease;
  • the immunosuppressive cytokine is IL-1;
  • the disease or condition is an IL-1 related disease or condition;
  • the disease or condition would benefit from the induction of a sustained immune response by stimulating MHCII signaling with an immunostimulatory polypeptide; for example, the immunostimulatory polypeptide is soluble LAG-3; or
  • the disease or condition would benefit from stimulating anti-tumor immunity by inhibiting immunosuppressive receptor signaling; for example, the immunosuppressive receptor is SIRP ⁇ .
  • Embodiment 156 The method according to any one of embodiments 149-155, further comprising administering a therapeutically effective amount of a second therapeutic agent.
  • Embodiment 157 The method according to embodiment 156, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapeutic agents, hormonal treatments, or cytokines.
  • the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapeutic agents, hormonal treatments, or cytokines.
  • Embodiment 158 A method for preparing a protein formulation according to any one of embodiments 1-129, comprising the following steps:
  • Embodiment 159 The preparation method according to embodiment 158, wherein the bifunctional molecule of step a is treated with a chelating agent, such as EDTA ⁇ 2Na (for example, about 0.3-10mM, about 0.1-20mM, about 0.3-15mM, about 0.3-10mM or About 0.5-5mM EDTA ⁇ 2Na) is treated, and then the chelating agent is removed; for example, the treatment is carried out in a buffer at room temperature; for example, the chelating agent is removed by dialysis, ultrafiltration or diafiltration. (e.g. dialysis with buffer, ultrafiltration or diafiltration).
  • a chelating agent such as EDTA ⁇ 2Na (for example, about 0.3-10mM, about 0.1-20mM, about 0.3-15mM, about 0.3-10mM or About 0.5-5mM EDTA ⁇ 2Na) is treated, and then the chelating agent is removed; for example, the treatment is carried out in a buffer at room temperature; for example
  • Embodiment 160 The preparation method according to embodiment 158, wherein the bifunctional molecule of step a is treated with a chelating agent, such as EDTA ⁇ 2Na (for example, about 0.3-10mM, about 0.1-20mM, about 0.3-15mM, about 0.3-10mM or EDTA ⁇ 2Na) at about 0.5-5 mM in buffer at room temperature (eg overnight) and the chelating agent is removed by dialysis, ultrafiltration or diafiltration against buffer.
  • a chelating agent such as EDTA ⁇ 2Na (for example, about 0.3-10mM, about 0.1-20mM, about 0.3-15mM, about 0.3-10mM or EDTA ⁇ 2Na) at about 0.5-5 mM in buffer at room temperature (eg overnight) and the chelating agent is removed by dialysis, ultrafiltration or diafiltration against buffer.
  • a chelating agent such as EDTA ⁇ 2Na (for example, about 0.3-10mM, about 0.1-20m
  • the present disclosure also provides the following embodiments:
  • the present disclosure provides a bifunctional molecule comprising a first portion that binds to an immune checkpoint molecule and a second portion that blocks the activity of interleukin-1 (IL-1).
  • IL-1 interleukin-1
  • the first portion includes an agonist of an immunostimulatory checkpoint molecule
  • the immunostimulatory checkpoint molecule is selected from the group consisting of: CD27, CD70, CD28, CD80 (B7- 1), CD86(B7-2), CD40, CD40L(CD154), CD122, CD137, CD137L, OX40(CD134), OX40L(CD252), GITR, ICOS(CD278) and ICOSLG(CD275), CD2, ICAM-1 , LFA-1(CD11a/CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160 and CD83.
  • the first portion includes an antagonist of an immunosuppressive checkpoint molecule
  • the immunosuppressive checkpoint molecule is selected from the group consisting of: A2AR, B7-H3 (CD276), B7 -H4(VTCN1), BTLA(CD272), CTLA-4(CD152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC7( CD328), TIGIT, PVR (CD155), SIGLEC9 (CD329), CD160, LAIR1, 2B4 (CD244), CD47 and B7-H5.
  • the immune checkpoint molecule is PD-L1.
  • the first portion includes an antibody to PD-L1 or an antigen-binding fragment thereof
  • the second portion includes an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion.
  • IL-1R IL-1 receptor
  • the IL-1 binding portion includes IL-1R, or an IL-1 binding fragment or variant thereof, or an antibody directed against IL-1, or an antigen-binding fragment thereof.
  • the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 104 or SEQ HCDR1 of the sequence of ID NO:112, HCDR2 of the sequence of SEQ ID NO:105 or SEQ ID NO:113, and HCDR2 of the sequence of SEQ ID NO:106 or SEQ ID NO:113 HCDR3 of the sequence of ID NO:114, the light chain variable region comprising LCDR1 of the sequence of SEQ ID NO:107 or SEQ ID NO:115, LCDR2 of the sequence of SEQ ID NO:108 or SEQ ID NO:116 , and LCDR3 comprising the sequence of SEQ ID NO:109 or SEQ ID NO:117.
  • the heavy chain variable region comprising SEQ ID NO: 104 or SEQ HCDR1 of the sequence of ID NO:112, HCDR2 of the sequence of SEQ ID NO:105 or SEQ ID NO:113, and HCDR2 of the sequence
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 104 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 105, and HCDR3 comprising the sequence of SEQ ID NO: 106, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 107, comprising SEQ ID NO: LCDR2 of the sequence 108, and LCDR3 including the sequence of SEQ ID NO:109.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 112 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 113, and HCDR3 comprising the sequence of SEQ ID NO: 114, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 115, comprising SEQ ID NO: LCDR2 of the sequence 116, and LCDR3 including the sequence of SEQ ID NO:117.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103, SEQ ID NO: 111, and a homologous sequence having at least 80% sequence identity thereto.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103 and having at least 80% sequence identity thereto homologous sequences.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 111, and having at least 80% sequence identity thereto Sequence identity of homologous sequences.
  • the IL-1R binding moiety includes an interleukin-1 receptor antagonist, or fragment or variant thereof, or an antibody directed against IL-1R, or an antigen-binding fragment thereof.
  • a bifunctional molecule in another aspect, includes a first portion that binds to PD-L1 and a second portion that a) blocks the activity of immunosuppressive cytokines or b) stimulates immunity, wherein said first portion includes a protein that targets PD-L1
  • An antibody of L1 or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof includes a heavy chain variable (VH) region and/or a light chain variable (VL) region, wherein the heavy chain variable region includes:
  • HCDR1 comprising DYYMN (SEQ ID NO: 1) or a homologous sequence with at least 80% sequence identity to DYYMN,
  • HCDR3 comprising WGDGPFAY (SEQ ID NO:3) or a homologous sequence having at least 80% sequence identity to WGDGPFAY, and/or
  • the light chain variable region includes:
  • LCDR1 includes a sequence selected from the group consisting of KASQNVX 4 X 5 X 6 VA (SEQ ID NO: 20) or homologous sequences having at least 80% sequence identity with KASQNVX 4 X 5 X 6 VA,
  • LCDR2 includes a sequence selected from the group consisting of SX 7 SX 8 RYT (SEQ ID NO: 21) or a homologous sequence having at least 80% sequence identity to SX 7 SX 8 RYT, and
  • LCDR3 includes a sequence selected from the group consisting of QQYSNYPT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity with QQYSNYPT;
  • X 1 is G or A
  • X 2 is G or D or Q or E or L
  • X 3 is S or M or Q or L or V
  • X 4 is G or P or K
  • X 5 is A or G
  • X is A or I
  • X is A or N or R or V
  • X is N or H or V or D.
  • the heavy chain variable region includes:
  • HCDR1 includes the sequence of SEQ ID NO:1,
  • HCDR2 includes a sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:18, and
  • HCDR3 includes the sequence of SEQ ID NO:3,
  • Light chain variable region the light chain variable region includes:
  • LCDR1 includes a sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9,
  • LCDR2 includes a sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and
  • f)LCDR3 includes the sequence of SEQ ID NO:6.
  • the heavy chain variable region is selected from the group consisting of:
  • a heavy chain variable region including the following: HCDR1 including the sequence of SEQ ID NO:1, HCDR2 including the sequence of SEQ ID NO:2 and HCDR3 including the sequence of SEQ ID NO:3;
  • a heavy chain variable region including: HCDR1 including the sequence of SEQ ID NO:1, HCDR2 including the sequence of SEQ ID NO:13 and HCDR3 including the sequence of SEQ ID NO:3;
  • a heavy chain variable region including: HCDR1 including the sequence of SEQ ID NO:1, HCDR2 including the sequence of SEQ ID NO:14 and HCDR3 including the sequence of SEQ ID NO:3;
  • a heavy chain variable region comprising the following: HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 15 and HCDR3 comprising the sequence of SEQ ID NO: 3;
  • HCDR1 comprising the sequence of SEQ ID NO: 1
  • HCDR2 comprising the sequence of SEQ ID NO: 17
  • HCDR3 comprising the sequence of SEQ ID NO: 3;
  • HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:18 and HCDR3 comprising the sequence of SEQ ID NO:3.
  • the light chain variable region is selected from the group consisting of:
  • a light chain variable region including the following: LCDR1 including the sequence of SEQ ID NO:4, LCDR2 including the sequence of SEQ ID NO:5 and LCDR3 including the sequence of SEQ ID NO:6;
  • a light chain variable region comprising the following: LCDR1 comprising the sequence of SEQ ID NO: 9, LCDR2 comprising the sequence of SEQ ID NO: 5 and LCDR3 comprising the sequence of SEQ ID NO: 6;
  • a light chain variable region including: LCDR1 including the sequence of SEQ ID NO:8, LCDR2 including the sequence of SEQ ID NO:5 and LCDR3 including the sequence of SEQ ID NO:6;
  • a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO: 4, LCDR2 comprising the sequence of SEQ ID NO: 12 and LCDR3 comprising the sequence of SEQ ID NO: 6; and
  • a light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO:4, LCDR2 comprising the sequence of SEQ ID NO:11 and LCDR3 comprising the sequence of SEQ ID NO:6.
  • the antibody against PD-L1 or antigen-binding fragment thereof further includes one or more of heavy chain HFR1, HFR2, HFR3, and HFR4, and/or light chain LFR1, LFR2, LFR3, and LFR4. one or more of, where:
  • the HFR1 includes the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYX 9 FT (SEQ ID NO: 40) or a homologous sequence having at least 80% sequence identity with the amino acid sequence,
  • said HFR2 comprises the amino acid sequence WVRQAPGQX 10 LEWMG (SEQ ID NO: 41) or a homologous sequence having at least 80% sequence identity with said amino acid sequence,
  • the HFR3 sequence includes the amino acid sequence RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 (SEQ ID NO: 42) or a homologous sequence having at least 80% sequence identity with the amino acid sequence,
  • the HFR4 includes the amino acid sequence WGQGTLVTVSS (SEQ ID NO:25) or a homologous sequence having at least 80% sequence identity with the amino acid sequence,
  • the LFR1 includes the amino acid sequence DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO:26) or a homologous sequence having at least 80% sequence identity with the amino acid sequence,
  • said LFR2 comprises the amino acid sequence WYQQKPGKX 14 PKLLIY (SEQ ID NO: 43) or a homologous sequence having at least 80% sequence identity with said amino acid sequence,
  • said LFR3 comprises the amino acid sequence GVPX 15 RFSGSGSGTDFTX 17 TISSLQPEDIATYYC (SEQ ID NO: 44) or a homologous sequence having at least 80% sequence identity with said amino acid sequence, and
  • the LFR4 includes the amino acid sequence FGQGTKLEIK (SEQ ID NO: 29) or a homologous sequence having at least 80% sequence identity with the amino acid sequence,
  • X 9 is T or V
  • X 10 is G or S
  • X 11 is T or K
  • X 12 is A or V
  • X 13 is R or K
  • X 14 is A or S
  • X 15 is S or D
  • X 16 is M or V
  • X 17 is F or L.
  • the HFR1 includes a sequence selected from the group consisting of SEQ ID NO: 22 and 30,
  • the HFR2 includes a sequence selected from the group consisting of SEQ ID NO: 23 and 31,
  • the HFR3 includes a sequence selected from the group consisting of SEQ ID NO: 24 and 32-35,
  • the HFR4 includes the sequence of SEQ ID NO: 25,
  • the LFR1 includes a sequence from the group consisting of SEQ ID NO: 26,
  • the LFR2 includes a sequence selected from the group consisting of SEQ ID NO: 27 and 36,
  • the LFR3 includes a sequence selected from the group consisting of SEQ ID NO: 28 and 37-38, 39, 45, and
  • the LFR4 includes the sequence of SEQ ID NO:29.
  • the heavy chain variable region includes a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO :52, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and homologous sequences having at least 80% sequence identity therewith.
  • the light chain variable region includes a sequence selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO :62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and homologous sequences having at least 80% sequence identity therewith.
  • the antibody directed against PD-L1 or an antigen-binding fragment thereof includes a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NO: 49/54, 50/54, 51/54, 52/54, 49/55, 50/55, 51/55, 52/55, 58/62, 58/63, 58/64, 58/65, 59/62, 59/ 63, 59/64, 59/65, 60/62, 60/63, 60/64 and 60/65.
  • the antibody directed against PD-L1 or antigen-binding fragment thereof further includes one or more amino acid residue substitutions or modifications but still retains specific binding specificity and/or affinity for PD-L1.
  • At least one of the substitutions or modifications is in one or more of the CDR sequences and/or in one of the non-CDR regions of the VH or VL sequence or in multiple non-CDR regions.
  • the antibody directed against PD-L1 or an antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a constant region of a human Ig, or optionally a constant region of a human IgG.
  • the constant region includes the Fc region of human IgG1, IgG2, IgG3, or IgG4.
  • the Fc region of human IgG1 includes or is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%) SEQ ID NO:80 % or 99%) sequence identity.
  • the constant region includes an Fc variant that has reduced effector function relative to a corresponding wild-type Fc region.
  • the Fc region includes one or more amino acid residue modifications or substitutions such that effector function is reduced relative to SEQ ID NO:80.
  • the Fc region includes one or more amino acid residue substitutions selected from the group consisting of: 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G , 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S and any combination thereof, wherein the Fc region
  • the numbering of the residues in is the numbering of the EU index in Kabat.
  • the Fc region includes a combination of mutations selected from the group consisting of: a) K322A, L234A, and L235A; b) P331S, L234F, and L235E; c) L234A and L235A; c) N297A; d) N297Q; e) N297G; f) L235E; g) L234A and L235A (IgG1); h) F234A and L235A (IgG4); i) H268Q, V309L, A330S and P331S (IgG2); j) V234A, G237A, P238S, H268A , V309L, A330S and P331S (IgG2), wherein the numbering of the residues in the Fc region is the numbering of the EU index in Kabat.
  • the Fc variant includes the amino acid sequence of SEQ ID NO: 81.
  • the antibody to PD-L1 or antigen-binding fragment thereof is humanized.
  • the antigen-binding fragment is a diabody, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific Sexual dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized ) single domain antibodies, Nanobodies, domain antibodies and bivalent domain antibodies.
  • the antibody or antigen-binding fragment thereof is capable of binding to both human PD-L1 and cynomolgus PD-L1.
  • the first portion includes an antibody or antigen-binding fragment thereof that competes for binding to PD-L1 with an antibody or antigen-binding fragment thereof provided herein.
  • the immunosuppressive cytokines include cytokines of the transforming growth factor beta (TGF-beta) superfamily, IL-1, or vascular endothelial growth factor (VEGF).
  • TGF-beta transforming growth factor beta
  • IL-1 IL-1
  • VEGF vascular endothelial growth factor
  • the immunosuppressive cytokines in the TGF- ⁇ superfamily include TGF- ⁇ , bone morphogenetic protein (BMP), activin, NODAL, and growth and differentiation factors (GDF).
  • BMP bone morphogenetic protein
  • GDF growth and differentiation factors
  • the immunosuppressive cytokine is TGF- ⁇ .
  • the second portion includes a TGF ⁇ binding moiety.
  • the TGF ⁇ binding moiety includes a soluble TGF ⁇ receptor (TGF ⁇ R) or a TGF ⁇ binding fragment or variant thereof, or an antibody directed against TGF ⁇ and an antigen-binding fragment thereof.
  • TGF ⁇ R soluble TGF ⁇ receptor
  • the soluble TGF ⁇ R includes the extracellular domain (ECD) of the TGF ⁇ R or a TGF ⁇ binding fragment or variant thereof.
  • the TGF ⁇ R is selected from the group consisting of TGF ⁇ receptor I (TGF ⁇ RI), TGF ⁇ receptor II (TGF ⁇ RII), TGF ⁇ receptor III (TGF ⁇ RIII), and any combination thereof.
  • the TGF ⁇ R is TGF ⁇ RII.
  • the TGF ⁇ RII selectively binds to TGF ⁇ 1 relative to TGF ⁇ 2 and TGF ⁇ 3.
  • the TGF ⁇ 1 is human TGF ⁇ 1 or mouse TGF ⁇ 1.
  • the ECD of TGF ⁇ R includes or has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 66, 79, 78, 77 but still retains a specific binding specificity for TGF- ⁇ properties and/or affinity of the sequence.
  • the second portion includes an IL-1 binding moiety or an IL-1 receptor (IL-1R) binding moiety.
  • IL-1R IL-1 receptor
  • the IL-1 binding portion includes soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody directed against IL-1 or an antigen-binding fragment thereof.
  • the IL-1 binding moiety includes: the extracellular domain (ECD) of IL-1RI; IL-1RI, the ECD of IL-1RI, IL-1RII, or the ECD of IL-1RII, or IL-1RII.
  • ECD extracellular domain
  • the IL-1R binding portion includes IL-1Ra, or an IL-1 binding fragment or variant thereof, or an antibody directed against IL-1R, or an antigen-binding fragment thereof.
  • the IL-1R binding portion includes the amino acid sequence of SEQ ID NO: 67 or 76, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 67 or 76, or IL-1 thereof. Combine fragments or variants.
  • the IL-1 is IL-1 ⁇ or IL-1 ⁇ .
  • the IL-1 ⁇ is human IL-1 ⁇ .
  • the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 104 or SEQ HCDR1 of the sequence of SEQ ID NO:112, HCDR2 of the sequence of SEQ ID NO:105 or SEQ ID NO:113, and HCDR3 of the sequence of SEQ ID NO:106 or SEQ ID NO:114, the light chain variable The region includes LCDR1 including the sequence of SEQ ID NO:107 or SEQ ID NO:115, LCDR2 including the sequence of SEQ ID NO:108 or SEQ ID NO:116, and LCDR2 including the sequence of SEQ ID NO:109 or SEQ ID NO:117 Sequence of LCDR3.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 104 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 105, and HCDR3 comprising the sequence of SEQ ID NO: 106, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 107, comprising SEQ ID NO: LCDR2 of the sequence 108, and LCDR3 including the sequence of SEQ ID NO:109.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 112 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 113, and HCDR3 comprising the sequence of SEQ ID NO: 114, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 115, comprising SEQ ID NO: LCDR2 of the sequence 116, and LCDR3 including the sequence of SEQ ID NO:117.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103, SEQ ID NO: 111, and a homologous sequence having at least 80% sequence identity thereto.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103 and having at least 80% sequence identity thereto homologous sequences.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of :SEQ ID NO:110, and has to A homologous sequence having at least 80% sequence identity, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 111, and a homologous sequence having at least 80% sequence identity thereto.
  • the bifunctional molecule comprises a heavy chain of the amino acid sequence of SEQ ID NO: 118 or SEQ ID NO: 120, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 121. chain.
  • the second portion stimulates anti-tumor immunity and includes an immunostimulatory polypeptide.
  • the immunostimulatory polypeptides include interleukin (IL)-2 (IL-2), IL-15, IL-21, IL-10, IL-12, IL-23, IL-27 , IL-35, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble CD4, soluble LAG-3 or IFN- ⁇ or their functional equivalents.
  • IL-2 interleukin-2
  • IL-15 interleukin-21
  • IL-10 IL-12
  • IL-23 IL-27
  • IL-35 granulocyte-macrophage colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • CD4 soluble LAG-3 or IFN- ⁇ or their functional equivalents.
  • the soluble LAG-3 includes the extracellular domain (ECD) of the LAG-3 or a MHCII-binding fragment or variant thereof.
  • the second portion stimulates anti-tumor immunity and includes an antagonist of immunosuppressive receptor signaling.
  • the immunosuppressive receptor is signal regulatory protein alpha (SIRP ⁇ ).
  • the second moiety blocks the interaction between CD47 and SIRP ⁇ .
  • the second portion includes a CD47 binding domain or a SIRP ⁇ binding domain.
  • the CD47 binding domain includes soluble SIRP ⁇ , or a CD47 binding fragment or variant thereof, or an anti-CD47 antibody, or an antigen-binding fragment thereof.
  • the soluble SIRP ⁇ includes the extracellular domain (ECD) of the SIRP ⁇ or a CD47-binding fragment or variant thereof.
  • the soluble SIRP ⁇ includes the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence but retains binding specificity for CD47.
  • the SIRP ⁇ binding domain includes soluble CD47, or a SIRP ⁇ binding fragment or variant thereof, or an anti-SIRP ⁇ antibody, or an antigen-binding fragment thereof.
  • the soluble CD47 includes the extracellular domain (ECD) of CD47, or a SIRP ⁇ binding fragment or variant thereof, an anti-SIRP ⁇ antibody, or an antigen-binding fragment thereof.
  • ECD extracellular domain
  • the bifunctional molecule further includes a linker connecting the first moiety and the second moiety.
  • the linker is selected from the group consisting of cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers.
  • the linker includes the amino acid sequence of ((G)nS)m, wherein m and n are independently an integer selected from 0 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, n is 2, 3, 4, or 5, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 68.
  • the bifunctional molecule includes one or more of the second moieties.
  • At least one second moiety of the plurality of second moieties is linked to the N-terminus or C-terminus of the polypeptide chain of the first moiety.
  • At least one second portion of the plurality of second portions is connected to: a) the N-terminus or C-terminus of the heavy chain of the first portion, or b) the light chain of the first portion. N-terminal or C-terminal.
  • At least one second portion of the plurality of second portions is linked to the C-terminus of the heavy chain constant region of the first portion.
  • each second portion of the plurality of second portions is individually linked to the C-terminus of each heavy chain constant region of the first portion.
  • the bifunctional molecule includes more than one second part in the second part, and the more than one second part is respectively connected to: the N-terminus of the heavy chain of the first part, the The C-terminus of the heavy chain of the first part, the N-terminus of the light chain of the first part, the C-terminus of the light chain of the first part, or any combination thereof.
  • the bifunctional molecule includes a homodimeric heavy chain or a heterodimeric heavy chain.
  • the heavy chain is heterodimeric with respect to the presence or location of the second moiety.
  • the heterodimeric heavy chains include one heavy chain having the second portion and the other heavy chain not having the second portion.
  • the heterodimeric heavy chain further includes a heterodimeric Fc region associated in a manner that hinders homodimerization and/or favors heterodimerization.
  • the first and said heterodimeric Fc regions are capable of associating into heterodimers through knobs-into-holes, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility. body.
  • the heterodimeric Fc region includes Y349C, T366S, L368A or Y407V or any combination thereof in one Fc region and S354C or T366W or a combination thereof in another Fc region, wherein said Fc
  • the numbering of residues in a region is that of the EU index as in Kabat.
  • the bifunctional molecule is further linked to one or more conjugate moieties.
  • the conjugate moiety includes a clearance modifier, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA alkylating agent, a topoisomer Enzyme inhibitors, tubulin binders, or other anticancer drugs such as androgen receptor inhibitors.
  • the present disclosure further provides a pharmaceutical composition or kit comprising a bifunctional molecule provided herein and a pharmaceutically acceptable carrier.
  • the present disclosure further provides an isolated polynucleotide encoding a bifunctional molecule provided herein.
  • the present disclosure further provides a vector comprising an isolated polynucleotide provided herein.
  • the present disclosure further provides a host cell comprising a vector provided herein.
  • the present disclosure further provides a method of expressing a bifunctional molecule provided herein, the method comprising culturing a host cell provided herein under conditions of an expression vector.
  • the present disclosure further provides a method of treating, preventing, or alleviating a PD-L1-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a bifunctional molecule provided herein and/or the pharmaceutical compositions or kits provided herein.
  • the disease is an immune-related disease or disorder, cancer, an autoimmune disease, or an infectious disease.
  • the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and bile duct cancer, ovarian cancer, testicular cancer , kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, prostate cancer, gastroesophageal cancer, rectal cancer, anal cancer, gastrointestinal cancer , skin cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma and adenocarcinoma.
  • lung cancer e.g., non-small cell lung cancer
  • liver cancer pancreatic cancer
  • breast cancer bronchial cancer
  • bone cancer e.g., bronchial cancer
  • the subject has been identified as having cancer cells that express PD-L1.
  • the subject is human.
  • the method further comprises administering a therapeutically effective amount of a second therapeutic agent.
  • the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapy agents, hormone therapeutic agents or cytokines.
  • the present disclosure provides use of a bifunctional molecule provided herein in the manufacture of a medicament for treating a PD-L1 related disease or condition in a subject.
  • the present disclosure provides a method of treating, preventing, or alleviating a disease or condition in a subject that would benefit from the suppression of immunosuppressive cytokines, the induction of a sustained immune response, or the anti-tumor Stimulation of immunity, the method comprising administering an effective amount of a bifunctional molecule provided herein.
  • the immunosuppressive cytokine is TGF ⁇ .
  • the disease or condition is a TGF ⁇ -related disease or condition.
  • the TGF ⁇ -related disease is cancer, fibrotic disease, or renal disease.
  • the immunosuppressive cytokine is IL-1.
  • the disease or condition is an IL-1 related disease or condition.
  • the disease or condition would benefit from stimulating anti-tumor immunity by inhibiting immunosuppressive receptor signaling (eg, SIRPa signaling).
  • immunosuppressive receptor signaling eg, SIRPa signaling
  • Figure 1 shows binding of humanized 4B6 antibody to human PD-L1 measured by ELISA.
  • Figure 2 shows the binding of Hu4B6_HgLa to human PD-L1 measured by ELISA.
  • Figures 3A-3C show binding of AM-4B6-IgG1-TGF ⁇ RII variants to PD-L1 measured by ELISA.
  • Figure 4 shows affinity fractionation of AM-4B6-IgG1-TGF ⁇ RII variants using flow cytometry.
  • Figures 5A and 5B show blockade of PD-L1/PD-1 or PD-L1/B7-1 by AM-4B6-IgG1-TGF ⁇ RII variants.
  • Figure 6 shows blockade of PD-L1/PD-1 by AM-4B6-IgG1-TGF ⁇ RII variants using cell-based assays.
  • Figure 7 shows SDS-PAGE of AM4B6_hlgG1_TBRII(20-136) expressed in a stable cell line.
  • Figures 8A and 8B show binding to human PD-L1 or cynomolgus monkey PD-L1 measured by ELISA analysis.
  • Figures 9A-9C show binding to human PD-L1 and other members of the B7 family and other members of the TGF ⁇ superfamily as measured by ELISA analysis.
  • Figures 10A-10F show binding to PD-L1 expressing cells measured by FACS analysis.
  • Figure 11 shows binding to human PD-L1 on activated human T cells measured by FACS analysis.
  • Figures 12A-12B show blockade of binding of human PD-L1 to human PD-1 or cynomolgus PD-L1 to cynomolgus PD-1 as measured by ELISA analysis.
  • Figure 13 shows simultaneous binding to hPD-L1 and TGFb1 measured by ELISA analysis.
  • Figure 14 shows blocking of hPD-L1/hPD-1 using a reporter gene assay.
  • Figure 15 shows blocking of TGF ⁇ 1 signaling using the TGF- ⁇ reporter HEK-293 cell line.
  • Figure 16 shows the effect of AM4B6-hlgG1-TGF ⁇ RII' on IFN ⁇ release from PBMC stimulated by tuberculin (TB).
  • Figures 17A-17B show anti-tumor activity in the MC38-hPD-L1 tumor model.
  • Figures 18A-18B show anti-tumor activity in the H460 tumor model.
  • Figures 19A-19B show anti-tumor activity in the EMT6-hPD-L1 tumor model.
  • Figures 20A-20C show in vivo pharmacokinetic and pharmacodynamic studies of AM4B6-hlgG1-TGF ⁇ RII.
  • Figure 21 shows the binding activity of AM4B6-hlgG1-IL-1RA to human PD-L1 measured by ELISA.
  • Figure 22 shows the binding activity of AM4B6-hlgG1-IL-1RA to human PD-L1 measured by FACS analysis.
  • Figure 23 shows blockade of PD-L1/PD-1 by AM4B6-hlgG1-IL-1RA using a cell-based assay.
  • Figure 24 shows the blocking activity of AM4B6-IgG1-IL-1RA on human IL-1 ⁇ measured by ELISA.
  • Figure 25 shows the blocking activity of AM4B6-hlgG1-IL-1RA on human IL-1 ⁇ on reporter cells.
  • Figure 26 shows SEC-HPLC purity of asymmetric bifunctional antibodies.
  • Figure 27 shows binding of bifunctional molecules to human PD-L1 measured by ELISA.
  • Figure 28 shows binding of bifunctional molecules to human CD47 measured by ELISA.
  • Figure 29 shows the ELISA binding activity of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6 bispecific antibodies to hIL-1 ⁇ protein.
  • Figure 30 shows the ELISA binding activity of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6 bispecific antibodies to hPD-L1 protein.
  • Figure 31 shows measurement of binding of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6 bispecific antibodies to PD-L1 expressing 293T cells by FACS.
  • Figure 32 shows the cell-based PD1/PD-L1 blocking activity of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6.
  • Figure 33 shows the blocking activity of IgG-scFv-XOMA052-AM4B6 on any IL-1 ⁇ on HDF cells.
  • Figure 34 shows the blocking activity of IgG-scFv-ACZ885-AM4B6 on hIL-1 ⁇ on reporter cells.
  • Figure 35 shows the JMP software analysis results of ⁇ HMW% and ⁇ LMW% detected by SEC and NR CE-SDS after the samples of prescriptions F1-F12 were placed at 40°C for 4 weeks, showing The effects of pH, PS80 and other excipients on protein stability were studied.
  • Figure 36 shows the JMP software analysis results of ⁇ HMW% and ⁇ LMW% detected by SEC and NR CE-SDS after the samples of prescriptions F1-F12 were stirred at 25°C for 3 hours, showing Effect of pH, PS80 and other excipients on protein stability.
  • Figure 37 and Figure 38 show samples of prescriptions F11 and F13, which were placed at 40°C for 4 weeks and subjected to SEC and NR
  • the changes in HMW% and LMW% detected by CE-SDS show the impact of buffers on protein stability.
  • Figure 39 and Figure 40 show samples of prescriptions F14 and F15, which were placed at 40°C for 6 weeks and subjected to SEC and NR
  • the changes in HMW% and LMW% detected by CE-SDS show the impact of buffers on protein stability.
  • Figures 41 to 43 (Figure 41: Polymer changes under light conditions; Figure 42: Polymer changes under 40°C conditions; Figure 43: Polymer changes under 25°C conditions) respectively show the samples of prescriptions F16 and F17. After being exposed to light for 7 days and placed at 40°C for 6 weeks or at 25°C for 6 weeks, the change in HMW% detected by SEC shows the impact of excipients on protein stability.
  • Figure 44- Figure 45 (Figure 44: Change in PS80 concentration under 40°C; Figure 45: Change in PS80 concentration under 25°C) respectively show samples of prescriptions F18-F21, placed at 40°C for one month or at 25°C.
  • the degradation results of PS80 under 6-month conditions show the protective effect of disodium edetate on PS80.
  • Figure 46- Figure 49 (Figure 46: Changes in polymers under 25°C; Figure 47: Changes in small molecule fragments under 25°C; Figure 48: Changes in polymers under 5°C; Figure 49: Small molecule fragments under 5°C Changes) respectively show the changes in HMW% and LMW% detected by SEC and NR CE-SDS when samples of prescriptions F18-F21 were placed at 25°C for 4 weeks or 5°C for 6 months, showing the impact of excipients on protein stability. Impact.
  • Figure 50 shows the PS80 of the preparation obtained by adding different concentrations of disodium edetate or not adding disodium edetate during the preparation process and leaving it at 40°C for 4 weeks. Degradation comparison results.
  • an antibody means one antibody or more than one antibody. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not limiting.
  • the terms “comprises,” “includes,” or “containing” mean the inclusion of stated elements, values, or steps, but not the exclusion of any other elements, values, or steps.
  • the terms “comprises,” “includes,” or “contains” are used, combinations of the stated elements, values, or steps are also encompassed unless otherwise specified.
  • reference is made to an antibody variable region that "comprises” a particular sequence it is also intended to encompass antibody variable regions that consist of that particular sequence.
  • formulation and “pharmaceutical formulation” are used interchangeably and refer to a composition suitable for administration to an animal, preferably a mammal, including a human, comprising at least one active ingredient and at least one inactive ingredient or Preparations.
  • the formulations of the present disclosure are, for example, in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form.
  • Liquid formulation refers to a formulation in liquid form.
  • the formulations of the present disclosure are preferably liquid formulations, such as injections, intravenous injections, intramuscular injections or subcutaneous injections.
  • buffer refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation.
  • acetate histidine, glutamate, citrate, succinate, malate, phosphate or lactate, and/or their respective free acids or bases, and various salts and/or or mixtures of acids and bases thereof.
  • Preferred pharmaceutically acceptable buffers include, but are not limited to, acetate buffers and histidine buffers.
  • Acetate buffer or “acetate buffer” includes acetic acid-sodium acetate, histidine-acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • “Histidine buffer” or “histidine buffer” includes histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, and the like.
  • the above buffering agents are usually used at a concentration of about 1-100mM, such as about 5-100mM, about 10-50mM, about 15-30mM, or about 20mM.
  • the buffer is capable of maintaining the pH of the liquid formulation of the present disclosure in the range of about 4.5-7.0, such as about 4.5-6.0, about 5.0-5.6, or about 5.0-5.5, such as about 5.3 or 5.3 ⁇ 5%.
  • surfactant refers to a pharmaceutically acceptable excipient that has surface activity.
  • nonionic surfactants are used.
  • Pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid ester (Tween), polyoxyethylene alkyl ether (Brij), alkylphenyl polyoxyethylene ether (Triton X), polyoxyethylene Ethylene-polyoxypropylene copolymers (poloxamer, Pluronic), and sodium dodecyl sulfate (SDS), such as polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer Mu 188, Brij series, Triton X.
  • the concentration is typically about 0.001-1%, such as about 0.01-0.1%, about 0.02%-0.1%, about 0.025%-0.1%, or about 0.05%.
  • surfactant concentrations are described as percentages expressed in weight/volume (w/v, g/100 mL).
  • stabilizer means a stabilizer other than the surfactants described above that is a pharmaceutically acceptable agent that protects the active pharmaceutical ingredient and/or formulation in a stable or unchanged state during manufacture, storage, and use. Excipients, such as to help prevent aggregation, oxidation, color changes, etc.
  • Stabilizers include, but are not limited to, sugars (including monosaccharides, such as glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, neuraminic acid; and oligosaccharides, such as sucrose, trehalose, lactose, maltose, and Raffinose), amino acids (including but not limited to arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine , phenylalanine, tyrosine, tryptophan, methionine, serine, proline.
  • sugars including monosaccharides, such as glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, neuraminic acid
  • oligosaccharides such as suc
  • amino acids used are preferably in the L-form.
  • amino acids used are preferably in the L-form.
  • inorganic salts for example, in the form of a hydrochloride salt, such as arginine hydrochloride, histidine hydrochloride or lysine hydrochloride.
  • the concentration is, for example, about 20 to about 250mM, about 100 to about 200mM, such as about 150mM), a salt (for example, an inorganic salt , such as sodium chloride, magnesium chloride, calcium chloride), chelating agents (such as EDTA (edetic acid), EDTA salts, such as EDTA ⁇ 2Na), polyols (such as sorbitol, mannitol, xylitol, dextran , glycerol, arabitol, propylene glycol, polyethylene glycol), cyclodextrin (such as hydroxypropyl- ⁇ -cyclodextrin, sulfobutylethyl- ⁇ -cyclodextrin, ⁇ -cyclodextrin), Polyethylene glycol (eg PEG 3000, PEG 3350, PEG 4000, PEG 6000), albumin (eg human serum albumin (HSA), bovine serum albumin (BSA)).
  • a salt
  • the stabilizer is, for example, arginine hydrochloride, NaCl, sucrose or sorbitol, preferably arginine hydrochloride or NaCl, for example about 150mM acid arginine or approximately 135mM NaCl.
  • the stabilizer can be present at about 1-500mM, such as about 10-300mM, about 100-300mM, about 120-300mM, about 100-200mM, about 135-280mM, about 135-200mM, about 130-170mM, about 140-160mM, Concentrations of 135, 150, 260 or 280mM are present in the formulations.
  • the concentration of amino acid or inorganic salt stabilizer is about 120-300mM, about 100-200mM, about 135-200mM, about 130-170mM, about 140-160mM, about 135mM or about 150mM.
  • concentration of arginine hydrochloride is about 120-300mM, about 100-200mM, about 135-200mM, about 130-170mM, about 140-160mM, or about 150mM.
  • the concentration of the chelating agent, such as EDTA ⁇ 2Na is about 30-350 ⁇ M, such as about 75-350 ⁇ M, such as about 75, about 150 or about 300 ⁇ M. More than one stabilizer selected from the same or different groups may be present in the formulations of the present disclosure. Stabilizers in the formulations of the present disclosure may also function as osmotic pressure regulators at the same time.
  • osmotic pressure regulator refers to a pharmaceutically acceptable excipient used to adjust the osmotic pressure of a solution.
  • osmotic pressure regulators include, but are not limited to, sugars (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars such as glucose, sucrose, and trehalose), amino acids (including alcohols, etc.) amino acid, glycine, cysteine, histidine), salts (e.g., inorganic salts, such as sodium chloride, magnesium chloride, calcium chloride), and polyols (e.g., sorbitol, mannitol, xylitol, right Glycoside, glycerin, arabitol, propylene glycol, polyethylene glycol).
  • sugars including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars,
  • lyoprotectants include, but are not limited to, sugars, polyols (such as, for example, sugar alcohols), and amino acids.
  • Preferred lyoprotectants may be selected from sugars such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine, Galactosamine, meglumine, polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine or mixtures thereof. Lyoprotectants are generally used in an amount of about 10 to 500mM, preferably in an amount of about 10 to about 300mM and more preferably in an amount of about 100 to about 300mM.
  • a subgroup of stabilizers are antioxidants, which include, but are not limited to, ascorbic acid, glutathione, cysteine, methionine, citric acid, EDTA.
  • the antioxidant may be used in an amount of about 0.01 to about 100mM, preferably in an amount of about 5 to about 50mM and more preferably in an amount of about 5 to about 25mM.
  • the term "about" when used in connection with a numerical value is intended to encompass a range of numerical values having a lower limit of 10% less than the specified numerical value and an upper limit of 10% greater than the specified numerical value, i.e., a range of ⁇ 10%, e.g. ⁇ 5%, for example ⁇ 3%.
  • w/v refers to "weight/volume” in g/100mL.
  • antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen.
  • Natural intact antibodies include two heavy (H) chains and two light (L) chains.
  • Mammalian heavy chains are divided into ⁇ , ⁇ , ⁇ , ⁇ and ⁇ . Each heavy chain consists of a variable region (VH) and a first, second, third and optional fourth constant region (CH1, CH2, CH3, CH4);
  • mammalian light chains are divided into lambda or kappa, and each light chain consists of a variable region (VL) and a constant region.
  • Antibodies have a "Y" shape, where the stem of the Y-shaped structure consists of the second and third constant regions of two heavy chains held together by disulfide bonds.
  • Each arm of Y includes the variable and first constant regions of a single heavy chain combined with the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable region of each chain usually contains three hypervariable regions, called complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3).
  • CDRs complementarity determining regions
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be determined by Kabat, IMGT, Chothia or Al-Lazikani rules to define or identify (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997) ); Chothia, C. et al., "J Mol Biol.” December 5; 186(3):651-63 (1985); Chothia, C. and Lesk, AM, "Molecular Biology Journal of Science, 196, 901 (1987); Chothia, C. et al., Nature.
  • FRs framework regions
  • light chain FR includes LFR1, LFR2, LFR3, and LFR4
  • heavy chain FR includes HFR1, HFR2, HFR3, and HFR4
  • effector function refers to cell-mediated or complement-mediated cellular functions resulting from interactions between the Fc region of an antibody and the C1q complement protein or Fc receptor (FcR) on immune cells. Toxic effects.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • the antibodies provided herein encompass any antigen-binding fragment thereof.
  • antigen-binding fragment refers to an antibody fragment formed from a portion of an antibody including one or more CDRs, or any other antibody fragment that binds to an antigen but does not include the intact native antibody structure.
  • antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv- dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single structures Domain antibodies, nanobodies, domain antibodies and bivalent domain antibodies.
  • Antigen-binding fragments are capable of binding to the same antigen to which the parent antibody binds.
  • the "Fab” of an antibody refers to a part of an antibody composed of a single light chain (including variable region and constant region) and a single heavy chain variable region and the first constant region bound by a disulfide bond.
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F(ab') 2 refers to the dimer of Fab'.
  • Fc of an antibody refers to the second and third constant domains of the first heavy chain connected via disulfide bonds to the second and third constant structures of the second heavy chain.
  • the binding domain makes up part of the antibody.
  • the Fc of IgM and IgE isotype antibodies further includes a fourth constant domain.
  • the Fc portion of an antibody is responsible for a variety of different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Fv refers to the smallest fragment of the antibody that contains an intact antigen-binding site. Fv fragments are composed of the variable region of a single light chain combined with the variable region of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked directly to each other or through a peptide linker sequence (Huston JS et al. National Academy of Sciences Proceedings of the Academy (Proc Natl Acad Sci USA), 85:5879(1988)).
  • Single chain Fv-Fc antibody or “scFv-Fc” refers to an engineered antibody consisting of an scFv linked to the Fc portion of an antibody.
  • “Camelized single domain antibody”, “heavy chain antibody” or “HCAb” refers to an antibody containing two V H domains but no light chain (Riechmann L. and Muyldermans S., Journal of Immunological Methods (J Immunol Methods) ⁇ . December 10; 231(1-2):25-38(1999); Muyldermans S., “J Biotechnol.”June; 74(4):277-302( 2001); WO94/04678; WO94/25591; U.S. Patent No. 6,005,079).
  • Heavy chain antibodies are originally derived from the family Camelidae (camels, dromedaries, and llamas).
  • variable region of the heavy chain antibody represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. 11 Month; 21(13):3490-8. Epub 2007 June 15 (2007)).
  • Nanobody refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
  • Diabodies or “dAbs” include small antibody fragments having two antigen-binding sites, wherein the fragments include a VH domain and a VL domain linked on the same polypeptide chain (VH-VL or VL-VH) (See, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8(1993); EP404097; WO93/11161).
  • VH-VL or VL-VH VH-VL or VL-VH
  • a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain, creating two antigen-binding sites.
  • the two antigen binding sites can target the same or different antigens (or epitopes).
  • a "bispecific ds diabody” is a diabody that targets two different antigens (or epitopes).
  • Domain antibody refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH domains are covalently linked by a peptide linker to form a bivalent or multivalent domain antibody.
  • the two VH domains of a bivalent domain antibody can target the same or different antigens.
  • the term “valency” refers to the presence of a specified number of antigen-binding sites in a given molecule.
  • the term “monovalent” refers to an antibody or antigen-binding fragment that has only one antigen-binding site; and the term “multivalent” refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites.
  • the terms “bivalent,” “tetravalent,” and “hexavalent” refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in the antigen-binding molecule.
  • the antibody or antigen-binding fragment thereof is bivalent.
  • a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes.
  • the two epitopes may be present on the same antigen, or they may be present on two different antigens.
  • a "scFv dimer” is a bivalent diabody or bispecific scFv (BsFv) that includes two VH-VL moieties that dimerize (joined by a peptide linker) such that one moiety of VH Collaborate with another part's VL Two binding sites are formed, which can target the same antigen (or epitope) or different antigens (or epitopes).
  • a "scFv dimer” is a bispecific diabody comprising mutually associated VH1-VL2 (linked by a peptide linker) and VL1-VH2 (also linked by a peptide linker). ), so that VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperative pair has different antigen specificity.
  • dsFv refers to a disulfide-stabilized Fv fragment in which the connection between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • “(dsFv)2" or “(dsFv-dsFv')” includes three peptide chains: two VH moieties connected by a peptide linker (e.g., a long flexible linker) and each connected by a disulfide bond. The two VL parts are combined.
  • dsFv-dsFv' is bispecific, wherein each pair of heavy and light chains paired by a disulfide bond has a different antigen specificity.
  • chimeric refers to an antibody or antigen binding having a portion of a heavy chain and/or light chain derived from one species and the remainder of the heavy chain and/or light chain derived from a different species. fragment.
  • a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (eg, derived from a mouse).
  • the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • humanized as used herein is meant to include antibodies or antigen-binding fragments that include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans, when applicable.
  • affinity refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, an antibody) or a fragment thereof and an antigen.
  • Specific binding refers to a non-random binding reaction between two molecules, for example, the reaction between an antibody and an antigen. Specific binding may be characterized by binding affinity, expressed, for example, by a K value, ie, the ratio of off-rate to association rate (k off / kon ) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. KD can be determined by using any conventional method known in the art, including, but not limited to, surface plasmon resonance, Octet methods, microthermophoresis, HPLC-MS methods, and FACS assays.
  • K D value can indicate the binding of the antibody or its antigen Specific binding between the fragment and PD-L1 (e.g., human PD-L1 or cynomolgus PD-L1).
  • the ability to "compete for binding to PD-L1" refers to the ability of a first antibody or an antigen-binding fragment thereof to inhibit the binding interaction between PD-L1 and a second anti-PD-L1 antibody to any detectable extent.
  • the antibody or antigen-binding fragment that competes for binding to PD-L1 inhibits the binding interaction between PD-L1 and the second anti-PD-L1 antibody by at least 85% or at least 90%. In some embodiments, this inhibition may be greater than 95% or greater than 99%.
  • amino acid refers to organic compounds containing amino ( -NH2 ) and carboxyl (-COOH) functional groups as well as side chains unique to each amino acid.
  • Amino acid names are also represented in this disclosure by standard one-letter or three-letter codes, summarized below:
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid residues with hydrophobic side chains such as Met, Ala, Val, Leu and Ile
  • amino acid residues with neutral hydrophilic side chains such as Cys, Ser, Thr, Asn and Gln
  • amino acid residues with acidic side chains e.g. Asp, Glu
  • amino acid residues with basic side chains e.g. His, Lys and Arg
  • amino acid residues with aromatic side chains e.g. Conservative substitutions are made between Trp, Tyr and Phe. It is known in the art that conservative substitutions generally do not cause significant changes in the conformational structure of the protein and therefore can retain the biological activity of the protein.
  • Percent (%) sequence identity when applied to an amino acid sequence (or a nucleic acid sequence) is defined as the difference between a candidate sequence and a reference sequence after sequence alignment and the introduction of gaps where necessary to achieve maximum correspondence. The percentage of amino acid (or nucleic acid) residues that are identical to the amino acid (or nucleic acid) residues in the protein.
  • BLASTN BLASTN
  • BLASTp available on the website of the US National Center for Biotechnology Information (NCBI)
  • NCBI National Center for Biotechnology Information
  • different residue positions may differ by conservative amino acid substitutions.
  • a "conservative amino acid substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity).
  • R group side chain
  • conservative amino acid substitutions will not materially alter the functional properties of the protein.
  • the percentage or degree of similarity may be adjusted upward to correct Conservative nature of positive substitutions. Methods for making this adjustment are well known to those skilled in the art. See, eg, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.
  • homologous sequence refers to a polynucleotide sequence (or complement thereof) or amino acid sequence that, when optionally aligned with another sequence, has at least 80% (eg, at least 85% , 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • an “isolated” substance has been artificially altered from its natural state. If an “isolated” composition or substance occurs in nature, it has been altered or removed from its original environment, or both.
  • a polynucleotide or polypeptide naturally occurring in a living animal is not “isolated” if the polynucleotide or polypeptide is sufficiently separated from and exists in a substantially pure state from the materials with which it naturally coexists. Can be thought of as “separated”.
  • Isolated “nucleic acid” or “polynucleotide” are used interchangeably and refer to the sequence of an isolated nucleic acid molecule.
  • an "isolated antibody or antigen-binding fragment thereof” refers to a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of antibodies or antigen-binding fragments, the purity of which is determined by electrophoresis methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reversed-phase HPLC).
  • electrophoresis methods such as SDS-PAGE, isoelectric focusing, capillary electrophoresis
  • chromatographic methods such as ion exchange chromatography or reversed-phase HPLC.
  • subject includes humans and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians and reptiles.
  • patient or “subject” are used interchangeably herein.
  • Treating or treating a condition includes preventing or alleviating the condition, slowing the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, alleviating or ending the relationship with the condition. Symptoms associated with a condition, production of complete or partial resolution of the condition, cure of the condition, or some combination thereof.
  • vector refers to a vehicle into which a genetic element is operably inserted and the genetic element is expressed so as to produce the protein, RNA or DNA encoded by the genetic element or to replicate the genetic element. tool.
  • Vectors can be used to transform, transduce or transfect host cells so that the genetic elements they carry can be expressed in the host cells. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses. .
  • Vectors can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also include materials that facilitate their entry into cells, including, but not limited to, viral particles, liposomes, or protein coatings.
  • the vector may be an expression vector or a cloning vector.
  • the disclosure provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence. and at least one selection marker.
  • a "host cell” refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.
  • soluble refers to the ability of a molecule (eg, a protein) to dissolve in a solvent (eg, liquids and aqueous environments).
  • a solvent eg, liquids and aqueous environments
  • transforming growth factor beta and "TGF ⁇ ” refer to any TGF ⁇ family protein, including precursors, having the full-length, native amino acid sequence of any TGF- ⁇ from a subject (e.g., human) and latent forms of mature TGF ⁇ and associated or non-associated complexes ("latent TGF ⁇ ").
  • latent TGF ⁇ latent forms of mature TGF ⁇ and associated or non-associated complexes
  • References in this article to such TGF ⁇ will be understood Reference to any of the currently identified forms, including TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3 isoforms and potential forms thereof, as well as future identified human TGF ⁇ species, includes sequences derived from any known TGF ⁇ and at least about 75% identical to that sequence.
  • TGF ⁇ 1 TGF ⁇ 2
  • TGF ⁇ 3 TGF- ⁇ as defined in the literature, such as Derynck et al., Nature, Cancer Res., 47:707 (1987); Seyedin et al., Journal of Biochemistry, 261:5693-5695 (1986); deMartin et al., EMBO J., 6:3673 (1987); Kuppner et al., International Cancer Journal (Int. J. Cancer)", 42: 562 (1988).
  • the terms "transforming growth factor beta”, “TGF ⁇ ”, “TGFbeta”, “TGF- ⁇ ” and “TGF-beta” are used interchangeably in this disclosure.
  • human TGF ⁇ 1 refers to the TGF ⁇ 1 protein encoded by the human TGFBl gene (eg, the wild-type human TGFBl gene).
  • An exemplary wild-type human TGF ⁇ 1 protein is provided by GenBank accession number NP_000651.3.
  • human TGF[beta]2 refers to the TGF[beta]2 protein encoded by the human TGFB2 gene (eg, wild-type human TGFB2 gene).
  • Exemplary wild-type human TGF ⁇ 2 proteins are provided by GenBank accession numbers NP_001129071.1 and NP_003229.1.
  • human TGF[beta]3 refers to the TGF[beta]3 protein encoded by the human TGFB3 gene (eg, wild-type human TGFB3 gene).
  • Exemplary wild-type human TGF ⁇ 3 proteins are provided by GenBank accession numbers NP_003230.1, NP_001316868.1, and NP_001316867.1.
  • mouse TGF ⁇ 1 refers to the mouse TGF ⁇ 1 gene (e.g., wild-type mouse TGFBl gene), the mouse TGFB2 gene (e.g., The TGF ⁇ 1 protein, TGF ⁇ 2 protein and TGF ⁇ 3 protein encoded by the wild-type mouse TGFB2 gene) and the mouse TGFB3 gene (e.g., the wild-type mouse TGFB3 gene).
  • Exemplary wild-type mouse (Mus musculus) TGF ⁇ 1 proteins are provided by GenBank accession numbers NP_035707.1 and CAA08900.1.
  • An exemplary wild-type mouse TGF ⁇ 2 protein is provided by GenBank accession number NP_033393.2.
  • An exemplary wild-type mouse TGF ⁇ 3 protein is provided by GenBank accession number AAA40422.1.
  • TGF ⁇ receptor refers to any receptor that binds at least one TGF ⁇ isoform.
  • TGF ⁇ receptors include TGF ⁇ receptor I (TGF ⁇ RI), TGF ⁇ receptor II (TGF ⁇ RII) or TGF ⁇ receptor III (TGF ⁇ RIII).
  • TGF ⁇ receptor I or "TGF ⁇ RI” refers to the human TGF ⁇ receptor type 1 sequence, including wild-type TGF ⁇ RI and all isoforms and variants thereof known to be capable of binding to at least one TGF ⁇ isoform.
  • An exemplary amino acid sequence for wild-type TGF ⁇ RI is available under GenBank accession number ABD46753.1 or UniProtKB-P36897 and is also included herein as SEQ ID NO:69.
  • the variant TGF ⁇ RI may have an amino acid sequence identical to SEQ ID NO: 69 by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity and retain at least 25%, 35%, 50%, 75%, 90%, 95% or 99% of the TGF ⁇ binding activity of the wild-type sequence (e.g., SEQ ID NO: 69).
  • TGF ⁇ receptor II or “TGF ⁇ RII” refers to the human TGF ⁇ receptor type 2 isoform A sequence, including wild-type TGF ⁇ RII and all isoforms thereof known to be capable of binding to at least one TGF ⁇ isoform. Types and variants.
  • wild-type TGF ⁇ RII isoform A or isoform 1 is available under GenBank accession number NP_001020018.1 or UniProtKB-P37173-1 and is also included herein as SEQ ID NO:70
  • wild-type TGF ⁇ RII Isoform B is available under GenBank accession number NP_003233.4 or UniProtKB-P37173-2 and is also included herein as SEQ ID NO:71.
  • Variant TGF ⁇ RII may have the same effect as SEQ ID NO: 70 or 71 Sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identical and retain the wild-type sequence (e.g. SEQ ID At least 25%, 35%, 50%, 75%, 90%, 95% or 99% of the TGF ⁇ binding activity of NO: 70 or 71).
  • TGF ⁇ receptor III refers to the human TGF ⁇ receptor type 3 sequence, including wild-type TGF ⁇ RII and all isoforms and variants.
  • An exemplary amino acid sequence for wild-type TGF ⁇ RIII is available under GenBank accession number NP_003234.2 or UniProtKB-Q03167 and is also included herein as SEQ ID NO:72.
  • the term "variant" with respect to a reference protein or peptide refers to a modified form of the reference protein or peptide, such as a functional equivalent, fragment, fusion, derivative, mimetic, or any combination thereof , the modified form has at least 70% similarity to the reference sequence (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %) sequence identity and retain at least 25% (e.g., 35%, 50%, 75%, 90%, 95%, or 99%) of the biological activity or binding activity of the reference sequence (e.g., the wild-type sequence).
  • the variant may be a fragment, mutant, fusion, truncation or any combination thereof of the reference protein or peptide.
  • interleukin-1 includes IL-1 ⁇ and IL-1 ⁇ , their precursors (e.g., pro-IL-1 ⁇ and pro-IL-1 ⁇ ), homologs Types and variants.
  • human IL-1 ⁇ refers to the IL-1 ⁇ protein, as well as isoforms and variants, encoded by the human IL1A gene (eg, wild-type human IL1A gene).
  • UniProtKB-P01583 provides an exemplary wild-type human IL1 ⁇ protein.
  • human IL-1 ⁇ refers to the IL-1 ⁇ protein encoded by the human IL1B gene (eg, wild-type human IL1B gene).
  • exemplary wild-type human IL1 ⁇ protein is provided by GenBank accession number NP_000567.1 or UniProtKB-C9JVK0.
  • IL-1 receptor refers to a receptor that can bind to IL-1, including all wild-type receptors, isoforms of which it is capable of binding to IL-1 and variations.
  • IL-1RI IL-1 receptor I
  • IL-1RII IL-1 receptor II
  • IL-1RII acts as a decoy receptor that binds ligand without transducing a signal. Proteolytic cleavage of IL-1RII results in the formation of soluble receptors, such as IL-1sRI and IL-1sRII, which bind ligands without transducing signals (for details see Thomas G.
  • IL-1sRI and IL-1sRII are proteolytic cleavage products of IL-1RII and may be a group of extracellular domain fragments of IL-1RII.
  • the term IL-1R is also intended to encompass the coreceptor IL-1RAP, which can associate with IL-1RI bound to IL-1 ⁇ to form a high-affinity interleukin 1 receptor complex, which The complex mediates interleukin 1-dependent activation of NF- ⁇ -B and other pathways.
  • IL-1RI includes wild-type IL-1RI as well as all isoforms and variants thereof capable of binding to IL-1 ⁇ and/or IL-1 ⁇ .
  • An exemplary amino acid sequence for wild-type IL-1RI is available under UniProtKB-P14778 and is also included herein as SEQ ID NO:73.
  • IL-1RII includes wild-type IL-1RII as well as all isoforms and variants thereof capable of binding to IL-1 ⁇ and/or IL-1 ⁇ .
  • An exemplary amino acid sequence for wild-type IL-1RII is available under UniProtKB-P27930 and is also included herein as SEQ ID NO:75.
  • IL-1RAP includes wild-type IL-1RAP as well as all isoforms and variants thereof capable of binding to the IL-1R that binds to IL-1 ⁇ .
  • the exemplary amino acid sequence of wild-type IL-1RAP is available under UniProtKB-Q9NPH3 and is also included herein as SEQ ID NO:74.
  • IL-1sRI includes all soluble forms of IL-1RI that can be produced by proteolytic cleavage involving metalloproteases.
  • the molecular weight of naturally occurring IL-1sRI can range from about 45 kDa to 60 kDa. This term also encompasses all isoforms and variants of IL-1sRI capable of binding to IL-1 ⁇ and/or IL-1 ⁇ .
  • IL-1sRII includes all soluble forms of IL-1RII that can be produced by proteolytic cleavage involving metalloproteases.
  • the molecular weight of naturally occurring IL-1sRII can range from about 45 kDa to 60 kDa. This term also encompasses all isoforms and variants of IL-1sRII capable of binding to IL-1 ⁇ and/or IL-1 ⁇ .
  • IL-1 receptor antagonist generally includes any protein that can compete with IL-1 ⁇ or IL-1 ⁇ for binding to the IL-1 receptor and inhibit IL-1 ⁇ or IL-1 ⁇ activity.
  • IL-1 receptor antagonists may include naturally occurring antagonists such as IL-1Ra, IL-1sRI, and IL-1sRII, as well as others that block the binding of IL-1 ⁇ or IL-1 ⁇ to the IL-1 receptor. , specifically artificial antagonists of IL-1RI.
  • IL-1Ra includes wild-type IL-1Ra as well as all isoforms and variants thereof capable of binding to IL-1 ⁇ and/or IL-1 ⁇ .
  • An exemplary amino acid sequence for wild-type IL-1Ra is available under UniProtKB-P18510 and is also included herein as SEQ ID NO:76.
  • cancer refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, invasion, or metastasis, and may be benign or malignant, and includes solid tumors and non-solid cancers (e.g., Hematological malignancies) such as leukemia.
  • solid tumor refers to a solid mass of neoplastic and/or malignant cells.
  • pharmaceutically acceptable means that the specified carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with the other ingredients making up the formulation, and is physiologically compatible with its recipient. compatible.
  • fusion refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur naturally, such as by chemical bonding or recombinant means.
  • Amino acid sequence e.g., a peptide, polypeptide, or protein
  • the fusion amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.
  • the present disclosure provides a bifunctional molecule that includes a first portion that binds to an immune checkpoint molecule and a second portion that blocks the activity of interleukin-1 (IL-1).
  • the bifunctional molecules provided herein work by blocking the interaction between IL-1 and the IL-1 receptor with an IL-1 binding moiety or an IL-1 receptor (IL-1R) binding moiety (i.e., the second portion of the bifunctional molecule). The interaction allows blocking and/or reducing IL-1 activity in the tumor microenvironment.
  • the IL-1 binding moiety and/or the IL-1R binding moiety can be linked to a portion that targets immune checkpoint molecules (i.e., the first portion of the bifunctional molecule) that can be found on the surface of certain tumor cells or immune cells.
  • IL-1 is an inflammatory cytokine. Inflammation is an important component of the tumor microenvironment, and IL-1 plays a key role in carcinogenesis and tumor progression (A. Mantovani et al., Immunol Rev. 2018 Jan; 281(1) ):57–61.). IL-1 plays a role at different levels in tumor initiation and progression, including driving chronic uncontrolled inflammation, tumor angiogenesis, activation of the IL-17 pathway, induction of myeloid-derived suppressor cells (MDSCs), and macrophages. Cell recruitment, invasion and metastasis (ibid.).
  • MDSCs myeloid-derived suppressor cells
  • Immune checkpoint molecules are expressed on certain immune cells such as T cells, natural killer cells, etc. Some cancer cells can also express certain immune checkpoint molecules, which may block activation of immune checkpoints, allowing cancer cells to avoid immune system surveillance.
  • the present disclosure provides a novel bifunctional molecule that can be used to treat immune checkpoint-related diseases, such as cancer and autoimmune diseases. , infectious diseases, etc.
  • the first moiety includes an agonist of a checkpoint molecule having immunostimulatory or costimulatory activity.
  • immunostimulatory checkpoint molecules may include, but are not limited to, CD27, CD70, CD28, CD80(B7-1), CD86(B7-2), CD40, CD40L(CD154), CD122, CD137, CD137L, OX40(CD134) , OX40L(CD252), GITR, ICOS(CD278) and ICOSLG(CD275), CD2, ICAM-1, LFA-1(CD11a/CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160 and CD83.
  • the first portion includes an inhibitor of a checkpoint molecule that has immunosuppressive or co-suppressive activity.
  • immunosuppressive checkpoint molecules may include, but are not limited to, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, LAG3 , NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC7(CD328), TIGIT, PVR(CD155), SIGLEC9(CD329), CD160, LAIR1, 2B4(CD244), CD47 and B7 -H5.
  • the immune checkpoint molecule is PD-L1.
  • the first portion includes an antibody portion directed against PD-L1 or an antigen-binding fragment thereof.
  • the first portion includes an antagonist antibody portion directed against PD-L1 or an antigen-binding fragment thereof.
  • the second portion includes an IL-1 binding moiety or an IL-1 receptor (IL-1R) binding moiety.
  • IL-1R IL-1 receptor
  • Both IL-1 ⁇ and IL-1 ⁇ are pro-inflammatory and bind to the IL-1R.
  • IL-1R Upon binding to IL-1 ⁇ or IL-1 ⁇ , IL-1R can recruit the IL-1R accessory protein and adapter protein MyD88 to the receptor complex, causing the activation of downstream signaling cascades and ultimately activating a host of immunity and inflammation Gene.
  • the inventors found that blocking the activity of IL-1 or its binding to the IL-1R would be useful in combination with modulation of immune checkpoint molecules.
  • IL-1 is IL-1 ⁇ or IL-1 ⁇ . In some embodiments, IL-1 ⁇ is human IL-1 ⁇ .
  • the second portion includes an IL-1 binding moiety.
  • the IL-1 binding moiety specifically binds to IL-1 ⁇ or IL-1 ⁇ .
  • the IL-1 binding portion includes soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody directed against IL-1 or an antigen-binding fragment thereof.
  • Soluble IL-1R can be a domain or fragment of IL-1R, such as the extracellular domain (ECD) of IL-1R.
  • the soluble IL-1R may be IL-1sRI or IL-1sRII, which are isoforms that are naturally soluble and capable of binding to IL-1.
  • IL-1RI or the ECD of IL-1RI, or IL-1RII or the ECD of IL-1RII, or IL-1RAP or the ECD of IL-1RAP, or shortened fragments of IL-1sRI or IL-1sRII may be sufficient. Binds to IL-1 (eg, IL-1 ⁇ or IL-1 ⁇ ) so long as such fragments contain an IL-1 binding domain. Accordingly, the disclosure also encompasses any of IL-1RI, the ECD of IL-1RI, or IL-1RII, or the ECD of IL-1RII, or IL-1RAP, or the ECD of IL-1RAP, IL-1sRI, and IL-1sRII.
  • the IL-1 binding portion includes the amino acid sequence of SEQ ID NO: 73, 74, or 75, or an IL-1 binding fragment or variant thereof. In some embodiments, the IL-1 binding portion includes an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 73, 74, and 75, or an IL-1 binding fragment or variant thereof.
  • the IL-1 binding moiety includes an antibody directed against IL-1 or an antigen-binding fragment thereof.
  • Antibodies directed against IL-1 or antigen-binding fragments thereof may also be used, as long as such antibodies or antigen-binding fragments can block the binding of IL-1 (eg, IL-1 ⁇ or IL-1 ⁇ ) to the IL-1R.
  • the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 104 or SEQ HCDR1 of the sequence of SEQ ID NO:112, HCDR2 of the sequence of SEQ ID NO:105 or SEQ ID NO:113, and HCDR3 of the sequence of SEQ ID NO:106 or SEQ ID NO:114, the light chain variable The region includes LCDR1 including the sequence of SEQ ID NO:107 or SEQ ID NO:115, LCDR2 including the sequence of SEQ ID NO:108 or SEQ ID NO:116, and LCDR2 including the sequence of SEQ ID NO:109 or SEQ ID NO:117 Sequence of LCDR3.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 104 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 105, and HCDR3 comprising the sequence of SEQ ID NO: 106, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 107, comprising SEQ ID NO: LCDR2 of the sequence 108, and LCDR3 including the sequence of SEQ ID NO:109.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 112 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 113, and HCDR3 comprising the sequence of SEQ ID NO: 114, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 115, comprising SEQ ID NO: LCDR2 of the sequence 116, and LCDR3 including the sequence of SEQ ID NO:117.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103, SEQ ID NO: 111, and a homologous sequence having at least 80% sequence identity thereto.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103 and having at least 80% sequence identity thereto homologous sequences.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 111, and having at least 80% sequence identity thereto Sequence identity of homologous sequences.
  • the second portion includes an IL-1R binding portion.
  • the IL-1R binding portion includes IL-IRa or an IL-1R binding fragment or variant thereof.
  • IL-1Ra is an antagonist of IL-1R and can compete with IL-1 ⁇ or IL-1 ⁇ for binding to IL-1R.
  • the skilled artisan will understand that shortened fragments of IL-1Ra may be sufficient for binding to IL-1R and/or competing with IL-1 ⁇ or IL-1 ⁇ .
  • the IL-1R binding moiety includes a truncated form of IL-IRa.
  • the IL-1R binding portion includes the amino acid sequence of SEQ ID NO: 67 or 76, or any IL-1 binding fragment or variant thereof.
  • the IL-1R binding portion includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 67 or 76, or any IL-1 binding fragment or variant thereof.
  • variants of wild-type IL-1Ra may also be used in the present disclosure, so long as such variants are able to compete with IL-1 ⁇ or IL-1 ⁇ for binding to the IL-1R.
  • the IL-1R binding portion includes an antibody to IL-1R or an antigen-binding fragment thereof.
  • Antibodies directed against IL-1R or antigen-binding fragments thereof may also be used, as long as such antibodies or antigen-binding fragments can compete with IL-1 ⁇ or IL-1 ⁇ for binding to IL-1R.
  • PD-1/PD-L1 axis checkpoint inhibitors e.g., PD-L1 antibodies
  • TME tumor microenvironment
  • immunosuppressive cytokines Signaling by such immunosuppressive cytokines in the local microenvironment can reduce tumor-infiltrating T cells and bias them towards Tregs and attenuate the activation of immune effector cells.
  • the present disclosure provides a novel bifunctional molecule that includes a first portion that binds to PD-L1 and a second portion that a) blocks the activity of immunosuppressive cytokines or b) stimulates anti-tumor immunity.
  • the molecule may be a compound, peptide, polypeptide, protein, or any combination thereof.
  • the second part can restore the immune response in the tumor microenvironment by blocking immunosuppressive activity or cytokines or increasing or stimulating immunity.
  • bifunctional molecules provided herein include a first portion that binds to PD-L1 (i.e., a PD-L1 binding portion) and a second portion that blocks the activity of an immunosuppressive cytokine.
  • immunosuppressive cytokines include cytokines of the transforming growth factor beta (TGF-beta) superfamily, IL-1, or vascular endothelial growth factor (VEGF).
  • TGF-beta transforming growth factor beta
  • VEGF vascular endothelial growth factor
  • the immunosuppressive cytokines in the TGF- ⁇ superfamily include bone morphogenetic proteins (BMP), activin, NODAL, and growth and differentiation factors (GDF).
  • the immunosuppressive cytokine is TGF- ⁇ . In some embodiments, the immunosuppressive cytokine is IL-1.
  • the second portion includes a TGF ⁇ binding moiety. In some embodiments, the second portion includes an IL-1 binding moiety.
  • binding moiety and “binding fragment” refer to a portion or fragment that has the ability to specifically bind to a target molecule or complex.
  • TGF ⁇ binding moiety refers to a substance that has the ability to specifically bind to one or more family members or isoforms of the TGF ⁇ family (e.g., TGF ⁇ 1, TGF ⁇ 2, or TGF ⁇ 3). part of the ability.
  • IL-1 binding moiety refers to a moiety that has the ability to specifically bind to one or more family members of the IL-1 family (eg, IL-1 ⁇ , IL-1 ⁇ ).
  • bifunctional molecules provided herein include a first portion that binds to PD-L1 (i.e., a PD-L1 binding portion) and a second portion that stimulates anti-tumor immunity.
  • the second portion includes an immunostimulatory polypeptide or functional equivalent or variant thereof.
  • the immunostimulatory polypeptide is interleukin (IL)-2 (IL-2), IL-15, IL-21, IL-10, IL-12, IL-23, IL-27 , IL-35, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble CD4, soluble LAG-3 or IFN- ⁇ or their functional equivalents.
  • the second moiety includes an antagonist of immunosuppressive receptor signaling.
  • the immunosuppressive receptor is SIRP ⁇ .
  • the bifunctional molecule includes one or more of the second moieties.
  • the one or more of the second portions can be of the same type, e.g., each of the one or more second portions can block an immune The activity of inhibitory cytokines, or each of the one or more second moieties, may stimulate anti-tumor immunity.
  • the one or more of the second portions may be of different types.
  • each second portion of the plurality of second portions may have the same sequence or may have a different amino acid sequence.
  • the TGF ⁇ binding moiety includes a soluble TGF ⁇ receptor (TGF ⁇ R) or a TGF ⁇ binding fragment or variant thereof, or an antibody directed against TGF ⁇ and an antigen-binding fragment thereof.
  • TGF ⁇ R soluble TGF ⁇ receptor
  • TGF ⁇ binding moiety may also be referred to as “TGF ⁇ Trap” in this disclosure. Therefore, proteins that target both PD-L1 and TGF ⁇ may also be referred to as "anti-PD-L1/TGF ⁇ traps" in this disclosure.
  • the TGF ⁇ binding moiety binds human and/or mouse TGF ⁇ . In some embodiments, the TGF ⁇ binding moiety is capable of antagonizing and/or inhibiting the TGF ⁇ signaling pathway. In some embodiments, the TGF ⁇ binding moiety is capable of antagonizing and/or inhibiting TGF ⁇ .
  • a TGF ⁇ binding moiety may include any moiety that specifically binds to one or more family members or isoforms of the TGF ⁇ family.
  • TGF ⁇ binding moieties include moieties that bind to TGF ⁇ 1 (eg, human TGF ⁇ 1), TGF ⁇ 2 (eg, human TGF ⁇ 2), and/or TGF ⁇ 3 (eg, human TGF ⁇ 3) or have similar or improved TGF ⁇ binding affinity.
  • the TGF ⁇ binding moiety includes a moiety that binds TGF ⁇ 1 (eg, human TGF ⁇ 1).
  • the TGF ⁇ binding moiety includes a moiety that binds TGF ⁇ 2 (eg, human TGF ⁇ 2).
  • the TGF ⁇ binding moiety includes a moiety that binds TGF ⁇ 3 (eg, human TGF ⁇ 3). In some embodiments, a TGF ⁇ binding moiety includes a moiety that specifically binds to both TGF ⁇ 1 (eg, human TGF ⁇ 1) and TGF ⁇ 2 (eg, human TGF ⁇ 2). In some embodiments, a TGF ⁇ binding moiety includes a moiety that specifically binds to both TGF ⁇ 1 (eg, human TGF ⁇ 1) and TGF ⁇ 3 (eg, human TGF ⁇ 3).
  • a TGF ⁇ binding moiety includes a moiety that specifically binds to both TGF ⁇ 2 (eg, human TGF ⁇ 2) and TGF ⁇ 3 (eg, human TGF ⁇ 3).
  • a TGF ⁇ binding moiety includes a moiety that specifically binds to each of TGF ⁇ 1 (eg, human TGF ⁇ 1), TGF ⁇ 2 (eg, human TGF ⁇ 2), and TGF ⁇ 3 (eg, human TGF ⁇ 3).
  • TGF ⁇ 1 eg, human TGF ⁇ 1
  • TGF ⁇ 2 eg, human TGF ⁇ 2
  • TGF ⁇ 3 eg, human TGF ⁇ 3
  • the TGF ⁇ binding moiety includes a moiety that selectively binds to TGF ⁇ 1 relative to TGF ⁇ 2 and/or TGF ⁇ 3.
  • the TGF ⁇ binding moiety includes a moiety that specifically binds human TGF ⁇ 1 and mouse TGF ⁇ 1 with similar affinity.
  • the TGF ⁇ binding portion of the present disclosure includes a soluble TGF ⁇ receptor (TGF ⁇ R) or a TGF ⁇ binding fragment or variant thereof.
  • TGF ⁇ R soluble TGF ⁇ receptor
  • TGF ⁇ receptors include TGF ⁇ RI, TGF ⁇ RII, and TGF ⁇ RIII.
  • the TGF ⁇ receptor is selected from the group consisting of TGF ⁇ receptor I (TGF ⁇ RI), TGF ⁇ receptor II (TGF ⁇ RII), TGF ⁇ receptor III (TGF ⁇ RIII), and any combination thereof.
  • the TGF ⁇ receptor is TGF ⁇ RI (eg, human TGF ⁇ RI).
  • the TGF ⁇ receptor is TGF ⁇ RII (eg, human TGF ⁇ RII).
  • TGF ⁇ RIII eg, human TGF ⁇ RIII.
  • the TGF ⁇ binding portion includes the extracellular domain (ECD) of a TGF ⁇ receptor (eg, human TGF ⁇ receptor) or a TGF ⁇ binding fragment or variant thereof.
  • ECD of a TGF ⁇ receptor includes an ECD of TGF ⁇ RI (e.g., human TGF ⁇ RI), an ECD of TGF ⁇ RII (e.g., human TGF ⁇ RII), an ECD of TGF ⁇ RIII (e.g., human TGF ⁇ RIII), or any combination thereof.
  • the ECD of TGF ⁇ RII includes the amino acid sequence of SEQ ID NO: 66, 79 or is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90% identical to the amino acid sequence). %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity but still retains binding specificity for TGF ⁇ .
  • the ECD of TGF ⁇ RI includes the amino acid sequence of SEQ ID NO: 77 or is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity but still retains binding specificity for TGF ⁇ .
  • the ECD of TGF ⁇ RIII includes the amino acid sequence of SEQ ID NO: 78 or is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity but still retains binding specificity for TGF ⁇ .
  • TGF ⁇ binding moieties include antibodies to TGF ⁇ and antigen-binding fragments thereof.
  • Exemplary anti-TGF ⁇ antibodies include fresolimumab and metelimumab as well as anti-TGF ⁇ antibodies or antigen-binding fragments thereof as described in: e.g., US7494651B2, US8383780B2, US8012482B2, WO 2017141208A1, Each document in the literature is incorporated by reference in its entirety.
  • the TGF ⁇ binding portion includes a combination of one or more ECDs of one or more TGF ⁇ receptors and/or one or more anti-TGF ⁇ antibodies or antigen-binding fragments thereof.
  • the one or more ECDs may be the same or different.
  • the TGF ⁇ binding moiety may comprise identical repeats of the ECD of the TGF ⁇ receptor, or alternatively may comprise a combination of different ECD sequences from the same TGF ⁇ receptor, or alternatively may comprise a combination of different ECDs from different TGF ⁇ receptors.
  • the one or more anti-TGF ⁇ antibodies may be the same or different.
  • the TGF ⁇ binding moiety includes a combination (or fusion) of an ECD selected from the group consisting of: an ECD of TGF ⁇ RI (e.g., human TGF ⁇ RI), an ECD of TGF ⁇ RII (e.g., human TGF ⁇ RII), TGF ⁇ RIII (e.g., ECD of human TGF ⁇ RIII) or any combination thereof.
  • an ECD of TGF ⁇ RI e.g., human TGF ⁇ RI
  • an ECD of TGF ⁇ RII e.g., human TGF ⁇ RII
  • TGF ⁇ RIII e.g., ECD of human TGF ⁇ RIII
  • the TGF ⁇ binding portion includes a combination (or fusion) of one or more anti-TGF ⁇ antibodies or antigen-binding fragments thereof.
  • the TGF ⁇ binding moiety includes a combination (or fusion) of an ECD selected from the group consisting of an ECD of TGF ⁇ RI (e.g., human TGF ⁇ RI), an ECD of TGF ⁇ RII (e.g., human TGF ⁇ RII) , an ECD of TGF ⁇ RIII (eg, human TGF ⁇ RIII); one or more anti-TGF ⁇ antibodies or antigen-binding fragments thereof; or any combination thereof.
  • an ECD of TGF ⁇ RI e.g., human TGF ⁇ RI
  • an ECD of TGF ⁇ RII e.g., human TGF ⁇ RII
  • an ECD of TGF ⁇ RIII eg, human TGF ⁇ RIII
  • one or more anti-TGF ⁇ antibodies or antigen-binding fragments thereof or any combination thereof.
  • the second portion includes an IL-1 binding moiety.
  • IL-1 is IL-1 ⁇ or IL-1 ⁇ .
  • IL-1 ⁇ is human IL-1 ⁇ .
  • the IL-1 binding moiety specifically binds to IL-1 ⁇ or IL-1 ⁇ . In some embodiments, the IL-1 binding moiety includes a moiety that selectively binds to IL-1 ⁇ relative to IL-1 ⁇ or a moiety that selectively binds to IL-1 ⁇ relative to IL-1 ⁇ .
  • the IL-1 binding portion includes soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody directed against IL-1 or an antigen-binding fragment thereof.
  • Soluble IL-1R may include domains or fragments or variants of IL-1R, for example, the extracellular domain (ECD) of IL-1R.
  • soluble IL-1R may also include IL-1sRI or IL-1sRII, which are isoforms that are naturally soluble and capable of binding to IL-1.
  • IL-1R or the ECD of IL-1R, or shortened fragments of IL-1sRI or IL-1sRII may be sufficient to bind to IL-1 (e.g., IL-1 ⁇ or IL-1 ⁇ ), so long as such fragments contain IL-1 binding domain.
  • the IL-1 binding portions provided herein may also include IL-1 binding fragments of any of IL-1R, the ECD of IL-1R, IL-1sRI, and IL-1sRII.
  • the IL-1 binding portion includes the amino acid sequence of SEQ ID NO: 73, 74, or 75, or an IL-1 binding fragment or variant thereof.
  • the IL-1 binding portion includes an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 73, 74, and 75, or an IL-1 binding fragment or variant thereof.
  • the IL-1 binding moiety includes an antibody directed against IL-1 or an antigen-binding fragment thereof.
  • Antibodies directed against IL-1 or antigen-binding fragments thereof may also be used, as long as such antibodies or antigen-binding fragments can block the binding of IL-1 (eg, IL-1 ⁇ or IL-1 ⁇ ) to the IL-1R.
  • the antibody against IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 104 or SEQ HCDR1 of the sequence of SEQ ID NO:112, HCDR2 of the sequence of SEQ ID NO:105 or SEQ ID NO:113, and HCDR3 of the sequence of SEQ ID NO:106 or SEQ ID NO:114, the light chain variable The region includes LCDR1 including the sequence of SEQ ID NO:107 or SEQ ID NO:115, LCDR2 including the sequence of SEQ ID NO:108 or SEQ ID NO:116, and LCDR2 including the sequence of SEQ ID NO:109 or SEQ ID NO:117 Sequence of LCDR3.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 104 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 105, and HCDR3 comprising the sequence of SEQ ID NO: 106, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 107, comprising SEQ ID NO: LCDR2 of the sequence 108, and LCDR3 including the sequence of SEQ ID NO:109.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 112 HCDR1, HCDR2 comprising the sequence of SEQ ID NO: 113, and HCDR3 comprising the sequence of SEQ ID NO: 114, the light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO: 115, comprising SEQ ID NO: LCDR2 of the sequence 116, and LCDR3 including the sequence of SEQ ID NO:117.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103, SEQ ID NO: 111, and a homologous sequence having at least 80% sequence identity thereto.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 103 and having at least 80% sequence identity thereto homologous sequences.
  • the antibody to IL-1 or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a sequence selected from the group consisting of : SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, the light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 111, and having at least 80% sequence identity thereto Sequence identity of homologous sequences.
  • the IL-1 binding moiety includes a combination of one or more moieties selected from the group consisting of: IL-1R, ECD of IL-1R, IL-1sRI, IL-1sRII, IL-1 Antibodies, any IL-1 binding fragments thereof, and any combinations thereof.
  • Such one or more parts may be connected by direct keys or may be connected by suitable joints.
  • the IL-1R binding portion includes IL-IRa or an IL-1R binding fragment or variant thereof.
  • IL-1Ra is an antagonist of IL-1R and can compete with IL-1 ⁇ or IL-1 ⁇ for binding to IL-1R.
  • the skilled artisan will understand that shortened fragments of IL-1Ra may be sufficient for binding to IL-1R and/or competing with IL-1 ⁇ or IL-1 ⁇ .
  • the IL-1R binding moiety includes a truncated form of IL-IRa.
  • the IL-1R binding portion includes the amino acid sequence of SEQ ID NO: 67 or any IL-1 binding fragment or variant thereof.
  • the IL-1R binding portion includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 67, or any IL-1 binding fragment or variant thereof.
  • variants of wild-type IL-1Ra may also be used in the present disclosure, so long as such variants are able to compete with IL-1 ⁇ or IL-1 ⁇ for binding to the IL-1R.
  • the IL-1R binding portion includes an antibody to IL-1R or an antigen-binding fragment thereof.
  • Antibodies directed against IL-1R or antigen-binding fragments thereof may also be used, as long as such antibodies or antigen-binding fragments can compete with IL-1 ⁇ or IL-1 ⁇ for binding to IL-1R.
  • the IL-1R binding moiety includes a combination of one or more moieties selected from the group consisting of IL-1Ra, an antibody directed against IL-1R, any IL-1R binding fragment or variant thereof, and Any combination.
  • Such one or more parts may be connected by direct keys or may be connected by suitable joints.
  • the second portion includes an immunostimulatory polypeptide or functional equivalent or variant thereof.
  • the immunostimulatory polypeptide is soluble CD4, soluble LAG-3, or functional equivalents thereof.
  • soluble LAG-3 includes the extracellular domain (ECD) of LAG-3 or an MHC class II (MHCII) binding fragment or variant thereof.
  • ECD extracellular domain
  • MHCII MHC class II
  • LAG-3 (Uniprot number: Q61790) belongs to the immunoglobulin (Ig) superfamily and is a type I transmembrane protein including 503 amino acids.
  • Lag-3 includes an intracellular domain (ICD), a transmembrane domain (TMD), and an extracellular domain (ECD).
  • ECD includes four Ig-like domains, namely D1 to D4, where D1 includes 9 ⁇ -chains: A, B, C, C', C", D, E, F and G chains. Between C chain and C' Between the strands, there are additional sequences with about 30 amino acids that form "extra loops.” Such "extra loops" have been reported to be involved in the interaction between LAG-3 and MHCII.
  • soluble LAG-3 includes the amino acid sequence of additional loops, D1 domains, D1 plus D2 domains, or any MHC II binding fragment or variant thereof. In some embodiments, soluble LAG-3 includes SEQ ID NO: 99 , SEQ ID NO:100, the amino acid sequence of SEQ ID NO:101, or any MHC II binding fragment or variant thereof.
  • LAG-3 is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells.
  • the primary ligand of LAG-3 is MHC class II, and LAG-3 binds to MHC class II with higher affinity than CD4.
  • a linker peptide (CP) is present between D4 and the TMD of LAG-3, where cleavage occurs in the presence of metalloproteases ADAM10 and/or ADAM17 to produce cleaved soluble LAG-3. See, for example, Huard et al., Proc Natl Acad Sci U S A 1997;94:5744-9; Workman et al., J Immunol 2002;169:5392– 5. doi:10.4049/jimmunol.169.10.5392; and Lawrence et al., J Immunother Cancer. 2015;3(Suppl 2):P216, which are incorporated herein by reference.
  • LAG-3 also encodes alternative splice variants of LAG-3 that can be translated into soluble forms.
  • Soluble LAG-3 activates antigen-presenting cells (APCs) through MHCII signaling, thereby increasing antigen-specific T cell responses in vivo.
  • APCs antigen-presenting cells
  • soluble LAG-3 activates dendritic cells (DCs) and has been reported to be involved in the pro-inflammatory activity of bystander T cells through cytokine activation (such as TNF- ⁇ and/or IL-12 activation), and it can directly activate DC. See, eg, Triebel, Trends Immunol., 2003, 24:619-622, which is incorporated herein by reference.
  • DCs dendritic cells
  • soluble LAG-3 includes Eftilagimod alpha (IMP321) or an MHC II binding fragment or variant thereof.
  • IMP321 is a soluble dimeric recombinant form of LAG-3.
  • IMP321 induces a sustained immune response by stimulating dendritic cells via MHCII molecules.
  • Combination therapy of MP321 and anti-PD-1 antibodies or anti-PD-L1 antibodies has been shown to synergistically activate T cells (specifically CD8+ T cells). See e.g. Luc et al., Future Oncol Actions Search in PubMed Search in NLM Catalog Add to Search.
  • the second moiety includes an antagonist of immunosuppressive receptor signaling.
  • the immunosuppressive receptor is SIRP ⁇ .
  • SIRPa which is interchangeable with the term “signal regulatory protein alpha,” refers to an inhibitory receptor expressed primarily on myeloid cells and dendritic cells.
  • SIRP ⁇ belongs to the SIRPs family, which also includes several other transmembrane glycoproteins, including SIRP ⁇ and SIRP ⁇ . Each member of the SIRPs family contains three similar extracellular Ig-like domains, but different transmembrane and cytoplasmic domains.
  • SIRPa can bind to CD47, which transmits the "don't eat me” signal to inhibit phagocytosis, and blocking CD47-mediated engagement of SIRPa on phagocytes can cause the removal of viable cells carrying the "eat me” signal.
  • CD47 is a ubiquitously expressed transmembrane glycoprotein with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. CD47 functions as a cellular ligand for SIRP ⁇ . Tumor cells often overexpress CD47 to avoid macrophage-mediated destruction. The interaction between CD47 and SIRP ⁇ has been shown to be involved in the regulation of macrophage-mediated phagocytosis (Takenaka et al., Nature Immunol., 8(12):1313-1323, 2007).
  • the second moiety blocks the interaction between CD47 and SIRP ⁇ .
  • therapies that block the interaction of CD47 with SIRP ⁇ stimulate phagocytosis of cancer cells in vitro and anti-tumor immune responses in vivo.
  • the second portion may include a CD47 binding domain or a SIRP ⁇ binding domain.
  • the immunosuppressive receptor is signal regulatory protein alpha (SIRP ⁇ ).
  • SIRP ⁇ signal regulatory protein alpha
  • the second moiety blocks the interaction between CD47 and SIRP ⁇ .
  • the second portion includes a CD47 binding domain or a SIRP ⁇ binding domain.
  • the CD47 binding domain includes soluble SIRP ⁇ , or a CD47-binding fragment thereof, or an anti-CD47 antibody, or an antigen-binding fragment thereof.
  • the soluble SIRP ⁇ includes the extracellular domain (ECD) of the SIRP ⁇ or a CD47-binding fragment or variant thereof.
  • the soluble SIRP ⁇ includes the amino acid sequence of SEQ ID NO: 84 or is at least 80% identical to the amino acid sequence (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity but still retains binding specificity for CD47.
  • the soluble SIRP ⁇ is an engineered high-affinity SIRP ⁇ variant that effectively antagonizes CD47 on cancer cells but does not itself induce macrophage phagocytosis.
  • SIRP ⁇ variants include one or more mutations relative to SEQ ID NO: 98 selected from the group consisting of: L4V, L4I, V6I, V6L, A21V, V27I, V27L, I31T, I31S, I31F , E47V, E47L, K53R, E54Q, H56P, H56R, V63I, S66T, S66G, K68R, V92I, F94L, F94V and F103V.
  • SIRPa variants include combinations of mutations selected from the group consisting of: 1) V27I, K53R, S66T, K68R, F103V; 2) L4V, V27L, E47V, K53R, E54Q, S66G, K68R, V92I ;3)L4V, V6I, A21V, V27I, I31T, E47L, K53R, H56P, S66T, K68R, F94L; 4) V6I, V27I, I31S, E47V, K53R, E54Q, H56P, S66G, V92I, F94L; 5) L4I , A21V, V27I, I31F, E47V, K53R, E54Q, H56R, S66G, F94V, F103V; 6) L4V, V6I, V27I, I31F, E47V, K53R, H56R, S66G, K68R, V92I, F103V;
  • the SIRP ⁇ binding domain includes soluble CD47, or a SIRP ⁇ binding fragment thereof, or an anti-SIRP ⁇ antibody, or an antigen-binding fragment thereof.
  • the soluble CD47 includes the extracellular domain (ECD) of CD47 or a SIRP ⁇ binding fragment thereof, an anti-SIRP ⁇ antibody or an antigen-binding fragment thereof.
  • the CD47 binding domain includes anti-CD47 antibodies and antigen-binding fragments thereof.
  • Exemplary anti-CD47 antibodies include, but are not limited to, humanized 5F9 antibody, B6H12 antibody, and ZF1 antibody. See Lu et al., Tumor Index "OncoTargets and Therapy", Volume 13, DOI https://doi.org/10.2147/OTT.S249822, which is incorporated herein by reference.
  • the SIRP ⁇ binding domain includes an anti-SIRP ⁇ antibody or antigen-binding fragment thereof.
  • Exemplary anti-SIRPa antibodies include, but are not limited to, BI765064 and AL008. See, for example, WO 2019073080A1, WO 2019175218A1 and WO 2018107058A1, which are incorporated herein by reference.
  • the CD47 binding domain includes a combination of one or more ECDs of one or more SIRP ⁇ , SIRP ⁇ , or SIRP ⁇ and/or one or more anti-CD47 antibodies or antigen-binding fragments thereof.
  • the one or more ECDs may be the same or different.
  • a CD47 binding domain may comprise identical repeats of the ECD of SIRP ⁇ , SIRP ⁇ or SIRP ⁇ , or alternatively may comprise a combination of different ECD sequences from the same SIRP ⁇ , SIRP ⁇ or SIRP ⁇ , or alternatively may comprise sequences from different SIRP ⁇ , SIRP ⁇ or a combination of different ECDs of SIRP ⁇ .
  • the one or more anti-CD47 antibodies can be the same or different.
  • the CD47 binding domain includes a combination (or fusion) of ECDs selected from the group consisting of: ECD of SIRP ⁇ , ECD of SIRP ⁇ , ECD of SIRP ⁇ , or any combination thereof.
  • the CD47 binding domain includes a combination (or fusion) of one or more anti-CD47 antibodies or antigen-binding fragments thereof.
  • the CD47 binding domain includes a combination (or fusion) of: an ECD selected from the group consisting of: ECD of SIRP ⁇ , ECD of SIRP ⁇ , ECD of SIRP ⁇ ; one or more anti- CD47 antibody or antigen-binding fragment thereof; or any combination thereof.
  • bifunctional molecules provided herein include a first moiety that is a PD-L1 binding moiety.
  • the PD-L1 binding moiety of the present disclosure binds to PD-L1 (eg, human PD-L1 or cynomolgus PD-L1). In some embodiments, the PD-L1 binding portions of the present disclosure bind to human PD-L1. In some embodiments, the PD-L1 binding moiety of the present disclosure binds to cynomolgus monkey PD-L1.
  • the PD-L1 binding portions of the present disclosure include anti-PD-L1 antibody portions.
  • exemplary anti-PD-L1 antibodies are disclosed in the Anti-PD-L1 Antibodies section and the Illustrative Anti-PD-L1 Antibodies section of this disclosure.
  • an anti-PD-L1 antibody portion includes one or more CDRs. In some embodiments, an anti-PD-L1 antibody portion includes one or more CDRs described in the Illustrative Anti-PD-L1 Antibodies section of this disclosure. In some embodiments, an anti-PD-L1 antibody portion includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the anti-PD-L1 antibody portion includes the VH and VL of an anti-PD-L1 antibody as disclosed in the Illustrative Anti-PD-L1 Antibody section of this disclosure.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-L1 antibody portion further includes a heavy chain constant domain appended to the carboxy terminus of the heavy chain variable region.
  • the heavy chain constant region is derived from the group consisting of IgA, IgG, and IgM.
  • the heavy chain constant region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM.
  • the anti-PD-L1 antibody portion further includes a light chain constant domain appended to the carboxy terminus of the light chain variable region.
  • the light chain constant region is derived from a kappa light chain or a lambda light chain.
  • the heavy chain constant region includes Including the amino acid sequence of SEQ ID NO: 80 or 81.
  • the light chain constant region includes the amino acid sequence of SEQ ID NO:82.
  • the second part may be connected to any part of the first part.
  • a second portion such as a TGF ⁇ binding portion or an IL-1 binding portion
  • a first portion such as a PD-L1 binding portion (eg, an anti-PD-L1 antibody portion).
  • the PD-L1 binding moiety includes one or more polypeptide chains, such as antibody heavy and light chains.
  • the bifunctional molecule includes one or more of the second moieties.
  • at least one second moiety of the plurality of second moieties is linked to the amino terminus (N-terminus) or carboxyl group (C-terminus) of the polypeptide chain of the first moiety.
  • the at least one second portion of the plurality of second portions is connected to the N-terminus or C-terminus of the heavy chain of the first portion or to the N-terminus or C-terminus of the light chain of the first portion. end.
  • the at least one second portion of the plurality of second portions is linked to the C-terminus of the heavy chain constant region of the first portion. In some embodiments, each second portion of the plurality of second portions is individually linked to the C-terminus of each heavy chain constant region of the first portion.
  • the bifunctional molecule includes at least two of the second portions, each of the at least two second portions being respectively connected to each of the first portions.
  • the C-terminus of each heavy chain, or each of the at least two second portions is respectively connected to the C-terminus of each light chain of the first portion.
  • the bifunctional molecule includes at least two of the second portions, each of the at least two second portions being respectively connected to each of the first portions.
  • the N-terminus of each heavy chain, or each second portion of the at least two second portions is respectively connected to the N-terminus of each light chain of the first portion.
  • the bifunctional molecule includes more than one second part in the second part, and the more than one second part is respectively connected to: the N-terminus of the heavy chain of the first part, the The C-terminus of the heavy chain of the first part, the N-terminus of the light chain of the first part, the C-terminus of the light chain of the first part, or any combination thereof.
  • the bifunctional molecule may comprise at least two of the second moieties, one of the at least two second moieties linked to the C-terminus of a heavy chain of the first moiety, And another second part is attached to the C-terminus of the light chain of said first part.
  • the bifunctional molecule may comprise at least two of the second moieties, one of the at least two second moieties linked to the N-terminus of a heavy chain of the first moiety, And another second moiety is attached to the N-terminus of the light chain of said first moiety.
  • one or more TGF ⁇ binding moieties, one or more IL-1 binding moieties, one or more immunostimulatory polypeptides (e.g., soluble LAG3 or soluble CD4), or one or more CD47 binding moieties Attached to the anti-PD-L1 antibody portion at one or more positions selected from the group consisting of: 1) the N-terminus of the heavy chain variable region, 2) the N-terminus of the light chain variable region of the anti-PD-L1 antibody portion end, 3) the C-terminus of the heavy chain variable region; 4) the C-terminus of the light chain variable region; 5) the C-terminus of the heavy chain constant region; 6) the C-terminus of the light chain constant region; and 7) any combination thereof .
  • immunostimulatory polypeptides e.g., soluble LAG3 or soluble CD4
  • CD47 binding moieties Attached to the anti-PD-L1 antibody portion at one or more positions selected from the group consisting of: 1) the N-terminus of
  • the bifunctional molecule includes a homodimeric heavy chain. In some embodiments, the bifunctional molecule includes a heterodimeric heavy chain. The heavy chain is heterodimeric with respect to the presence or location of the second portion. In some embodiments, the heterodimeric heavy chains include one heavy chain having the second portion and the other heavy chain not having the second portion.
  • the second part may be connected to the first part directly or via a joint.
  • the direct link may be a chemical link (such as a covalent bond).
  • the bifunctional molecule further includes a linker connecting the first moiety and the second moiety.
  • linker may be any suitable bifunctional moiety capable of reacting with at least two entities to be joined, thereby bonding the entities to form a molecule or holding the entities closely enough association.
  • the linker may be integrated into the resulting linker molecule or structure with or without its reactive functional groups.
  • the linker is selected from the group consisting of cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers.
  • the linker includes a peptide linker.
  • Peptide linkers can be composed of amino acid residues linked together by peptide bonds.
  • the peptide linker may further include one or more unnatural amino acids.
  • a peptide linker includes at least 1, 2, 3, 4, 5, 8, 10, 15, 20, 30, 50 or more amino acid residues linked by peptide bonds and is capable of linking two or Amino acid sequences of more polypeptides.
  • Peptide linkers may or may not have secondary structure.
  • any suitable peptide linker can be used.
  • Many peptide linker sequences are known in the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure (Structure) ⁇ 2:1121-1123(1994).
  • the peptide linker can include or consist of amino acid residues selected from the group consisting of glycine, serine, alanine, methionine, asparagine, and glutamine.
  • the peptide linker can be composed of mostly non-sterically hindered amino acids such as glycine and alanine.
  • the linker is polyglycine, polyalanine, a combination of glycine and alanine (such as poly(Gly-Ala)) or a combination of glycine and serine (such as poly(Gly-Ser)).
  • the linker includes the amino acid sequence of ((G)nS)m, wherein m and n are independently an integer selected from: 0 to 30, 1 to 29, 2 to 28, 3 to 27, 4 to 26, 5 to 25, 6 to 24, 7 to 23, 8 to 22, 9 to 21, 10 to 20, 11 to 19, 12 to 18, 13 to 17, 14 to 16 or 5.
  • m is 4 and n is 4.
  • the linker includes the amino acid sequence of SEQ ID NO: 68. In some embodiments, the linker comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least Amino acid sequences with 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the PD-L1 binding portion of the bifunctional molecules provided herein includes a portion comprising an anti-PD-L1 antibody or antigen-binding fragment thereof.
  • anti-PD-L1 antibodies and antigen-binding fragments thereof are capable of specifically binding to PD-L1.
  • the anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein provide no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, or no more than Binds specifically to human PD-L1 with a K D value of 0.4 nM.
  • the Biacore assay is based on surface plasmon resonance technology, see for example Murphy, M. et al., Current protocols in protein science, Chapter 19, Single Yuan 19.14, 2006.
  • the KD value is measured by a method as described in Example 6 of this disclosure.
  • Binding of antibodies or antigen-binding fragments thereof to human PD-L1 provided herein may also be expressed as a "half-maximal effective concentration" (EC50 ) value, which refers to the concentration of the antibody at which 50% of its maximum binding is observed.
  • EC50 values can be measured by binding assays known in the art, such as direct or indirect binding assays, such as enzyme-linked immunosorbent assays (ELISA), fluorescence-activated cell sorting (FACS) assays, and other binding assays.
  • the antibodies and antigen-binding fragments thereof provided herein have an EC 50 (i.e., 50% Binding concentration) specifically binds to PD-L1.
  • the antibodies and antigen-binding fragments thereof provided herein provide no more than 1.4 nM, no more than 1.3 nM, no more than 1.2 nM, no more than 1.1 nM, no more than 1.0 nM, no more than 1.0 nM, as measured by FACS assay. Specifically binds to PD-L1 with an EC50 (i.e., 50% binding concentration) of more than 0.3 nM, no more than 0.25 nM, or no more than 0.21 nM.
  • an EC50 i.e., 50% binding concentration
  • anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein specifically bind to PD-L1. In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein do not bind to other members of the B7 family.
  • anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein are capable of blocking the interaction between PD-L1 and its binding partners (e.g., PD-1 and B7-1), as determined by ELISA
  • the measured IC50 of the interaction does not exceed 2.2, 2.1, 2.0, 1.9, 1.8 or 1.2ug/ml.
  • anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein are capable of blocking the interaction between PD-L1 and its binding partner (e.g., PD-1), as measured by cell-based assays , the EC50 of the interaction does not exceed 1.3, 1.2, 1.1, 1.0, 0.9 or 0.8nM.
  • the anti-PD-L1 antibodies i.e., antibodies directed against PD-L1 and antigen-binding fragments thereof of the present disclosure include one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR, the one or more CDRs comprising a sequence selected from the group consisting of: DYYMN (SEQ ID NO: 1), DINPNNX 1 ) , KASQNVX 4 _ _ _ G or D or Q or E or L, X 3 is S or M or Q or L or V, X 4 is G or P or K, X 5 is A or G, X 6 is A or I, X 7 is A or N or R or V, X 8 is N or H or V or D.
  • one or more CDRs comprising a sequence selected from the group consisting of: DYYMN (SEQ ID NO: 1), DINPNNX 1 ) , KASQNVX 4 _ _ _ G or D or Q or E or L, X 3 is S
  • the heavy chain variable region includes:
  • HCDR1 includes the sequence of SEQ ID NO:1,
  • HCDR2 includes a sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:18, and
  • HCDR3 includes the sequence of SEQ ID NO:3,
  • Light chain variable region the light chain variable region includes:
  • LCDR1 includes a sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9,
  • LCDR2 includes a sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and
  • f)LCDR3 includes the sequence of SEQ ID NO:6.
  • the heavy chain variable region is selected from the group consisting of:
  • a heavy chain variable region comprising the following: HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 2 and HCDR3 comprising the sequence of SEQ ID NO: 3;
  • a heavy chain variable region comprising the following: HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 13 and HCDR3 comprising the sequence of SEQ ID NO: 3;
  • a heavy chain variable region comprising the following: HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 14 and HCDR3 comprising the sequence of SEQ ID NO: 3;
  • a heavy chain variable region comprising: HCDR1 including the sequence of SEQ ID NO: 1, HCDR2 including the sequence of SEQ ID NO: 15, and HCDR3 including the sequence of SEQ ID NO: 3; and
  • HCDR1 comprising the sequence of SEQ ID NO: 1
  • HCDR2 comprising the sequence of SEQ ID NO: 17
  • HCDR3 comprising the sequence of SEQ ID NO: 3;
  • a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO: 1, HCDR2 comprising the sequence of SEQ ID NO: 18 and HCDR3 comprising the sequence of SEQ ID NO: 3.
  • the light chain variable region is selected from the group consisting of:
  • a light chain variable region including the following: LCDR1 including the sequence of SEQ ID NO:4, LCDR2 including the sequence of SEQ ID NO:5 and LCDR3 including the sequence of SEQ ID NO:6;
  • a light chain variable region comprising the following: LCDR1 comprising the sequence of SEQ ID NO: 9, LCDR2 comprising the sequence of SEQ ID NO: 5 and LCDR3 comprising the sequence of SEQ ID NO: 6;
  • a light chain variable region including: LCDR1 including the sequence of SEQ ID NO:8, LCDR2 including the sequence of SEQ ID NO:5 and LCDR3 including the sequence of SEQ ID NO:6;
  • a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO: 4, LCDR2 comprising the sequence of SEQ ID NO: 12 and LCDR3 comprising the sequence of SEQ ID NO: 6; and
  • a light chain variable region comprising LCDR1 comprising the sequence of SEQ ID NO:4, LCDR2 comprising the sequence of SEQ ID NO:11 and LCDR3 comprising the sequence of SEQ ID NO:6.
  • antibody “4B6” refers to a monoclonal antibody that includes a heavy chain variable region having the sequence of SEQ ID NO: 46 and a light chain variable region having the sequence of SEQ ID NO: 47.
  • the present disclosure provides anti-PD-L1 antibodies comprising one or more (eg, 1, 2, 3, 4, 5, or 6) CDR sequences of antibody 4B6 or a variant of antibody 4B6 and their Antigen-binding fragments.
  • CDR boundaries are defined or qualified by Kabat rules.
  • the present disclosure provides anti-PD-L1 antibodies and antigen-binding fragments thereof, the antibodies and antigen-binding fragments thereof comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; comprising a compound selected from the group consisting of SEQ ID NO: HCDR2 having the amino acid sequence of the group consisting of 2, 13, 14, 15, 17 and 18; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and/or comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 7, 8-9 LCDR1 consisting of the amino acid sequence of the group; LCDR2 including the amino acid sequence selected from the group consisting of SEQ ID NO: 5, 10, 11-12; and LCDR3 including the amino acid sequence of SEQ ID NO:6.
  • HCDR1 comprising the amino acid sequence of SEQ ID NO: 1
  • HCDR2 having the amino acid sequence of the group consisting of 2, 13, 14, 15, 17 and 18
  • HCDR3 comprising the amino acid sequence of SEQ ID NO:
  • CDRs are known to be responsible for antigen binding. However, it has been discovered that not all 6 CDRs are indispensable or unchangeable. In other words, one or more CDRs in anti-PD-L1 antibody 4B6 can be replaced or altered or modified while still substantially retaining specific binding affinity for PD-L1.
  • the antibodies and antigen-binding fragments thereof provided herein include suitable framework region (FR) sequences so long as the antibodies and antigen-binding fragments thereof can specifically bind to PD-L1.
  • suitable framework region FR
  • the CDR sequences provided in Table 1 above were obtained from mouse antibodies, but the CDR sequences can be transplanted into any suitable species such as mouse, human, rat, rabbit, etc. using suitable methods known in the art such as recombinant technology. Any suitable FR sequence.
  • the antibodies and antigen-binding fragments thereof provided herein are humanized.
  • Humanized antibodies or antigen-binding fragments thereof are desirable in terms of their reduced immunogenicity in humans.
  • Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are grafted to human or substantially human FR sequences.
  • Humanization of an antibody or antigen-binding fragment can essentially be accomplished by substituting a non-human (e.g., murine) CDR gene for the corresponding human CDR gene in the human immunoglobulin gene (see, e.g., Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).
  • Suitable human heavy and light chain variable domains can be selected for this purpose using methods known in the art.
  • a "best fit" approach may be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences and the closest non-human Human sequences of the query sequence were identified and used as human scaffolds for transplantation of non-human CDR sequences (see, e.g., Sims et al. (1993) J Immunol 151:2296; Chothia et al. (1987) J Mol Biol .Mot.Biol.) ⁇ 196:901).
  • Frameworks derived from consensus sequences of human antibodies can be used to graft non-human CDRs (see, e.g., Carter et al. (1992) Proc Natl Acad Sci USA 89:4285; Presta et al. (1993) J Immunol. 151:2623).
  • the present disclosure provides 12 humanized antibodies to 4B6, named Hu4B6_Hg.2La.1, Hu4B6_Hg.2La.2, Hu4B6_Hg.2La.4, Hu4B6_Hg.2La.6, Hu4B6_Hg. 3La.1, Hu4B6_Hg.3La.2, Hu4B6_Hg.3La.4, Hu4B6_Hg.3La.6, Hu4B6_Hg.5La.1, Hu4B6_Hg.5La.2, Hu4B6_Hg.5La.4 and Hu4B6_Hg.5La.6.
  • SEQ ID NOs for the heavy and light chain variable regions of the humanized antibodies are shown in Table 5.
  • the CDRs for each of the 12 humanized antibodies of 4B6 are shown in Table 5 (underlined sequences). CDR boundaries are defined or qualified by Kabat rules.
  • Table 3a shows the amino acid sequences of the variant CDRs of humanized 4B6, and Table 3b below shows the FRs of the heavy and light chain variable regions of humanized 4B6.
  • Table 4 shows the FR amino acid sequence of each heavy chain and light chain of the 12 humanized antibodies of chimeric antibody 4B6, which are named Hu4B6_Hg.2La.1 and Hu4B6_Hg.2La respectively. 2.
  • Table 5 The heavy chain variable regions and light chain variable regions of these 12 humanized antibodies are shown in Table 5.
  • Table 3a Amino acid sequences of CDR variants of humanized antibodies of 4B6.
  • Table 4 FR amino acid sequence of each humanized heavy chain and light chain variable region of humanized antibody 4B6.
  • Table 5 below shows 3 variants of the humanized 4B6 heavy chain variable region (i.e., Hu4B6_Hg.2, Hu4B6_Hg.3, and Hu4B6_Hg.5) and 4 variants of the humanized 4B6 light chain variable region. (i.e., AM4B6_La.1, AM4B6_La.2, AM4B6_La.4, AM4B6_La.6).
  • the humanized anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein consist of substantially fully human sequences, except for non-human CDR sequences.
  • the variable FR region and the constant region are entirely or essentially derived from human immunoglobulin sequences.
  • the human FR sequence and the human constant region sequence can be derived from different human immunoglobulin genes, for example, the FR sequence is derived from one human antibody and the constant region is derived from another human antibody.
  • the humanized antibody or antigen-binding fragment thereof includes human heavy chain HFR1-4 and/or light chain LFR1-4.
  • a human-derived FR region can include the same amino acid sequence as the human immunoglobulin from which it is derived.
  • one or more amino acid residues of the human FR are substituted with corresponding residues from the parent non-human antibody. In some embodiments, this may be desirable to bring the humanized antibody or fragment thereof into close proximity to the non-human parent antibody structure, thereby optimizing binding properties (eg, increasing binding affinity).
  • the humanized antibodies or antigen-binding fragments thereof provided herein comprise no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 in each of the plurality of human FR sequences.
  • one or more amino acids of the human FR sequence are randomly mutated to increase binding affinity. In some embodiments, one or more amino acids of the human FR sequence are backmutated to the corresponding amino acids of the parent non-human antibody to increase binding affinity.
  • the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include: heavy chain HFR1 comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYX 9 FT (SEQ ID NO: 40) or with the sequence of A homologous sequence with at least 80% sequence identity; heavy chain HFR2 comprising the sequence of WVRQAPGQX 10 LEWMG (SEQ ID NO: 41) or a homology with at least 80% sequence identity with said sequence Sequence; heavy chain HFR3 comprising the sequence of RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 (SEQ ID NO: 42) or a homologous sequence having at least 80% sequence identity to said sequence; and heavy chain HFR4 , the heavy chain HFR4 includes the sequence of WGQGTLVTVSS (SEQ ID NO: 25) or a homologous sequence with at least 80% sequence identity to the sequence, wherein X 9 is T or V
  • the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include: light chain LFR1 that includes or has the sequence of DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO:26) Homologous sequences with at least 80% sequence identity; light chain LFR2 comprising or having at least 80% sequence identity with the sequence of WYQQKPGKX 14 PKLLIY (SEQ ID NO: 43) The homologous sequence of; light chain LFR3, the light chain LFR3 comprising the sequence of GVPX 15 RFSGSGTDFTX 17 TISSLQPEDIATYYC (SEQ ID NO: 44) or a homologous sequence having at least 80% sequence identity with said sequence; and light chain LFR4 , the light chain LFR4 includes the sequence of FGQGTKLEIK (SEQ ID NO: 29) or a homologous sequence with at least 80% sequence identity to the sequence, wherein X 14 is A or S, X
  • HFR1 includes a sequence selected from the group consisting of SEQ ID NO:22 and 30, HFR2 includes a sequence selected from the group consisting of SEQ ID NO:23 and 31, and HFR3 includes a sequence selected from the group consisting of SEQ ID NO:24 and Sequences from the group consisting of 32-35, HFR4 includes the sequence of SEQ ID NO: 25, LFR1 includes a sequence selected from the group consisting of SEQ ID NO: 26, and LFR2 includes a sequence selected from the group consisting of SEQ ID NO: 27 and 36 , LFR3 includes a sequence selected from the group consisting of SEQ ID NO: 28 and 37-38, 39, 45, and LFR4 includes the sequence of SEQ ID NO: 29.
  • humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include HFR1, HFR2, HFR3, and/or HFR4 sequences comprised in a heavy chain variable region, the heavy chain variable region Select from the group consisting of: Hu4B6_Hg.2(SEQ ID NO:58), AM4B6_Hg.3(SEQ ID NO:59), AM4B6_Hg.5(SEQ ID NO:60).
  • humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include LFR1, LFR2, LFR3, and/or LFR4 sequences comprised in a light chain variable region. Select from the group consisting of: AM4B6_La.1(SEQ ID NO:62), AM4B6_La.2(SEQ ID NO:63), AM4B6_La.4(SEQ ID NO:64), and AM4B6_La.6(SEQ ID NO:65) .
  • the heavy chain variable region includes a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO :52, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and homologous sequences having at least 80% sequence identity therewith.
  • the light chain variable region includes a sequence selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO :62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and homologous sequences having at least 80% sequence identity therewith.
  • the antibody directed against PD-L1 or an antigen-binding fragment thereof includes a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NO: 49/54, 50/54, 51/54, 52/54, 49/55, 50/55, 51/55, 52/55, 58/62, 58/63, 58/64, 58/65, 59/62, 59/ 63, 59/64, 59/65, 60/62, 60/63, 60/64 and 60/65.
  • exemplary humanized anti-PD-L1 antibodies retain specific binding ability or affinity for PD-L1 and are superior in this respect to the parent mouse antibody 4B6.
  • the anti-PD-L1 antibodies and antigen-binding fragments provided herein include all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain.
  • an anti-PD-L1 antibody or antigen-binding fragment thereof provided herein is a single domain antibody consisting of all or a portion of a heavy chain variable domain provided herein. More information on such single domain antibodies is available in the art (see, eg, U.S. Patent No. 6,248,516).
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, optionally further comprising a heavy chain and/or light chain constant region.
  • the heavy chain constant region includes the CH1, hinge, and/or CH2-CH3 region (or optional CH2-CH3-CH4 region).
  • anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein include human IgG1, IgG2, IgG3, IgG4, Heavy chain constant region of IgA1, IgA2 or IgM.
  • the light chain constant region includes C ⁇ or C ⁇ .
  • the constant regions of the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein may be identical to the wild-type constant region sequence or differ in one or more mutations.
  • the anti-PD-L1 antibodies, or antigen-binding fragments thereof, provided herein have specific binding affinity for human PD-L1 sufficient to provide diagnostic and/or therapeutic use.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, labeled antibodies, bivalent Antibodies, anti-idiotypic antibodies or fusion proteins.
  • Recombinant antibodies are antibodies produced using recombinant methods in vitro rather than in animals.
  • the PD-L1 binding moiety includes an anti-PD-L1 antibody, or antigen-binding fragment thereof, with a pair of heavy chain variable regions and a light chain selected from the group consisting of: Antibodies or antigen-binding fragments of variable region sequences compete for binding to PD-L1: SEQ ID NO: 49/54, 50/54, 51/54, 52/54, 49/55, 50/55, 51/55, 52 /55, 58/62, 58/63, 58/64, 58/65, 59/62, 59/63, 59/64, 59/65, 60/62, 60/63, 60/64 and 60/65 .
  • anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.
  • the antibody variant is in one or more of the plurality of CDR sequences provided in Table 1 above, the heavy chain variable region or the light chain provided in Tables 3a, 3b and 5 above.
  • One or more modifications or substitutions are included in one or more of the plurality of non-CDR sequences of the variable region and/or in the constant region (eg, Fc region).
  • Such variants retain the binding specificity for PD-L1 of their parent antibody, but possess one or more desired properties conferred by modifications or substitutions.
  • an antibody variant may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, increased Pharmacokinetic half-life, pH sensitivity, and/or compatibility with conjugation (eg, one or more introduced cysteine residues).
  • Parent antibody sequences can be screened to identify suitable or preferred residues to be modified or substituted using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine), and yield Modified antibodies and screen them for properties of interest. If a substitution at a specific amino acid position exhibits a functional change of interest, then that position can be identified as a potential residue for modification or substitution. The potential residues can be further evaluated by substitution with another residue (eg, a cysteine residue, a positively charged residue, etc.).
  • target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • neutral or negatively charged amino acids e.g.,
  • Affinity variants of the antibody may be characterized by one or more CDR sequences provided in Table 1 above, one or more FR sequences provided in Tables 3b and 4 above, or heavy or light chain variables provided in Table 5 above.
  • the region sequence contains modifications or substitutions.
  • One skilled in the art can easily identify the FR sequences based on the CDR sequences in Table 1 above and the variable region sequences in Table 5 above, since it is well known in the art that in a variable region, a CDR region is flanked by two FR regions.
  • Affinity variants retain the specific binding affinity for PD-L1 of the parent antibody or even have a higher specific binding affinity for PD-L1 than the parent antibody.
  • at least one (or all) substitutions in a CDR sequence, FR sequence, or variable region sequence include conservative substitutions.
  • the humanized anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein have one or more CDR sequences of a plurality of CDR sequences and/or one or more FR sequences of a plurality of FR sequences. Includes one or more amino acid residue substitutions.
  • affinity variants include a total of no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences.
  • an anti-PD-L1 antibody or antigen-binding fragment thereof includes 1, 2, or 3 CDR sequences that are at least identical to one (or more) of the sequences listed in Tables 1 and 3a above. 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity, but still in a relative Specific binding affinity for PD-L1 is retained at similar or even higher levels than its parent antibody.
  • an anti-PD-L1 antibody or antigen-binding fragment thereof includes one or more variable region sequences that are consistent with one (or more) of the variable region sequences listed in Table 5 above. ) has a sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity, However, specific binding affinity for PD-L1 is retained at a similar or even higher level relative to its parent antibody.
  • a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Table 5 above.
  • substitutions, insertions, or deletions occur in regions outside of the CDRs (eg, in the FRs).
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibodies or antigen-binding fragments thereof.
  • Anti-PD-L1 antibodies or antigen-binding fragments thereof may include one or more modifications that introduce or remove glycosylation sites.
  • Glycosylation sites are amino acid residues with side chains to which carbohydrate moieties (eg, oligosaccharide structures) can be attached.
  • Glycosylation of antibodies is usually N-linked or O-linked.
  • N-linked refers to the side chain of a carbohydrate moiety attached to an asparagine residue (e.g., the asparagine residue in tripeptide sequences such as asparagine-X-serine and asparagine-X-threonine) , where X is any amino acid except proline.
  • O-linked glycosylation refers to linking one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine.
  • the native glycosylation site can be conveniently removed, for example by altering the amino acid sequence such that the above mentioned tripeptide sequence (for N-linked glycosylation sites) or the serine or threonine residue ( For O-linked glycosylation sites) one of them is substituted.
  • New glycosylation sites can be generated in a similar manner by introducing such tripeptide sequences or serine or threonine residues.
  • the anti-PD-L1 antibodies and antigen-binding fragments provided herein include one or more mutations to remove one or more deamidation sites.
  • the anti-PD-L1 antibodies and antigen-binding sheets provided herein The segment includes a mutation at G55 (eg, G55A) in the heavy chain. These mutations were tested and are not believed to negatively affect the binding affinity of the antibodies provided herein.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein also encompass cysteine-engineered variants that include one or more introduced free cysteine amino acid residues.
  • a free cysteine residue is a cysteine residue that is not part of a disulfide bond.
  • Engineered variants of cysteine can be used to combine, for example, cytotoxic and/or imaging compounds, labels or radioactive isotopes at the site of the engineered cysteine by, for example, a maleimide or haloacetyl group. etc. conjugation.
  • Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
  • Anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein also encompass Fc variants that include one or more amino acid residue modifications or substitutions at the Fc region and/or hinge region, e.g., to provide altered Effector functions such as ADCC and CDC.
  • Methods of altering ADCC activity through antibody engineering have been described in the art, see, for example, Shields RL. et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE. et al., Journal of Immunology (J Immunol.)" 2000.164(8):4178-84; Steurer W. et al., "Journal of Immunology” 1995,155(3):1165-74; Idusogie EE.
  • the CDC activity of the antibodies or antigen-binding fragments provided herein can also be altered, for example, by improving or reducing C1q binding and/or CDC (see, e.g., WO99/51642; Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821); and WO94/29351 for other examples of Fc region variants.
  • One or more amino acids selected from amino acid residues 329, 331 and 322 of the Fc region can be replaced with different amino acid residues to alter C1q binding and/or to reduce or eliminate complement-dependent cytotoxicity (CDC) (see Idusogie et al., U.S. Patent No. 6,194,551).
  • One or more amino acid substitutions may also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94/29351 by Bodmer et al.).
  • Fc variants provided herein have reduced effector function relative to a wild-type Fc (e.g., an Fc of IgG1) and include one or more amino acid substitutions at a position selected from the group consisting of: 220, 226, 228, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, 331 in Fc area and 332 (see WO2016/196228; Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al.
  • substitutions that reduce effector function include, but are not limited to, 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R , 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S or any combination thereof (see WO2016/196228; and Strohl (2009) "Current Reviews of Biotechnology" 20:685 -691).
  • an anti-PD-L1 antibody or antigen-binding fragment thereof provided herein has reduced effector function and includes one or more amino acid substitutions in IgG1 at a position selected from the group consisting of: 234, 235, 237, 238, 268, 297, 309, 330 and 331.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and include one or more amino acid substitutions selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S and any combination thereof.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and include the L234A and L235A mutations. In some embodiments, anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and include L234F, L235E, and P331S.
  • a set of substituted L234F, L235E, and P331S localized in the CH2 region of the Fc domain can eliminate FC ⁇ R and C1q binding, rendering the antibody unable to elicit ADCC or CDC (Oganesyan et al., Acta Crystallographica D (Acta Crystallogr.D) ⁇ 64:700-704(2008)).
  • PCT/US2013/36872 has shown that combining these mutations in a variant Fc domain (e.g., a variant Fc domain in an antibody) results in an Fc domain having Reduced thermal stability.
  • the Fc variant includes the amino acid sequence of SEQ ID NO: 81.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG2 isotype and include one or more amino acid substitutions selected from the group consisting of: H268Q, V309L, A330S, P331S, V234A , G237A, P238S, H268A and any combination thereof (for example, H268Q/V309L/A330S/P331S, V234A/G237A/P238S/H268A/V309L/A330S/P331S).
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG4 isotype and include one or more amino acid substitutions selected from the group consisting of: S228P, N297A, N297Q, N297G, L235E , F234A, L235A and any combination thereof.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are IgG2/IgG4 cross-isotypes. Examples of IgG2/IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256–1264 (2007).
  • an anti-PD-L1 antibody or antigen-binding fragment thereof includes one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • Such a variant may have an extended pharmacokinetic half-life because it binds to FcRn at acidic pH, protecting it from degradation in lysosomes, and is subsequently translocated and released outside the cell.
  • Methods of engineering antibodies or antigen-binding fragments thereof to increase binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R .
  • an anti-PD-L1 antibody or antigen-binding fragment thereof includes one or more amino acid substitutions in the interface of the Fc region to facilitate and/or promote heterodimerization.
  • modifications include introducing protrusions into the first Fc polypeptide and cavities into the second Fc polypeptide, where the protrusions can be positioned in the cavities so as to facilitate the interaction of the first Fc polypeptide with the second Fc polypeptide to form heterogeneous dimers or complexes.
  • the PD-L1 binding portion of the bifunctional molecules provided herein also encompasses anti-PD-L1 antigen-binding fragments.
  • antigen-binding fragments are known in the art and can be based on the anti-PD-L1 antibodies provided herein and different variants thereof (e.g. Affinity variants, glycosylation variants, Fc variants, cysteine engineered variants, etc.) are developed including, for example, their CDRs as set forth in Tables 1 and 3a above and their variable sequences Exemplary antibodies are shown in Tables 2 and 5.
  • anti-PD-L1 antigen-binding fragments provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv ) 2.
  • Bispecific dsFv dsFv-dsFv'
  • disulfide bond-stabilized diabody ds diabody
  • single-chain antibody molecule scFv
  • scFv dimer bivalent diabody
  • multispecific antibody camelized single domain antibodies, nanobodies, domain antibodies and bivalent domain antibodies.
  • a variety of techniques can be used to generate such antigen-binding fragments.
  • Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science , 229:81 (1985)), recombinant expression by host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., For ScFv) and chemical coupling of two Fab'-SH fragments to form an F(ab') 2 fragment (Carter et al., Bio/Technology 10:163-167 (1992)).
  • Other techniques for producing antibody fragments will be apparent to those skilled in the art.
  • the antigen-binding fragment is a scFv.
  • the generation of scFv is described in: eg WO93/16185; US Patent No. 5,571,894; and No. 5,587,458.
  • ScFv can be fused to an effector protein at the amino or carboxyl terminus to provide a fusion protein (see, for example, "Antibody Engineering", edited by Borrebaeck).
  • anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule with more than two valences is considered multivalent, covering for example trivalent, tetravalent, hexavalent, etc.
  • a bivalent molecule can be monospecific if both binding sites specifically bind to the same antigen or the same epitope. In some embodiments, this provides stronger binding to the antigen or epitope than the monovalent counterpart.
  • multivalent molecules can also be monospecific.
  • the first valence of the binding site and the second valence of the binding site are structurally the same (i.e., have the same sequence) or are structurally different ( i.e., have different sequences but the same specificity).
  • a bivalent can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and a third binding site is specific for a second antigen (or epitope) sexual.
  • an anti-PD-L1 antibody or antigen-binding fragment thereof is bispecific.
  • the PD-L1 binding antibody or antigen-binding fragment thereof is further linked to additional functionality that has a different binding specificity than the anti-PD-L1 antibody or antigen-binding fragment thereof. domain.
  • the bispecific antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second antigen or PD-L1 in addition to PD-L1 (and in addition to the target bound by the second moiety). a second epitope (or a second epitope on the target that the second moiety binds).
  • bifunctional molecules provided herein are capable of binding to PD-L1 and a target bound by the second moiety. In some embodiments, bifunctional molecules provided herein are capable of binding to both PD-L1 and TGF ⁇ , or to PD- L1 and IL-1 combine, or combine with both PD-L1 and IL-1R, or combine with both PD-L1 and MHCII, or combine with both PD-L1 and CD47, or combine with PD-L1 and SIRP ⁇ combines both.
  • a bifunctional molecule targeting PD-L1 and TGF ⁇ of the present disclosure reacts at no more than 2.0 nM (e.g., no more than 2.0 nM, no more than 1.2 nM, no more than 1.1 nM) as measured by an ELISA assay. , no more than 1.0 nM, no more than 0.9 nM, no more than 0.8 nM) EC50 specifically binds to human TGF ⁇ 1.
  • proteins targeting PD-L1 and TGF ⁇ of the present disclosure are capable of binding to both PD-L1 and TGF ⁇ simultaneously as measured by ELISA assays.
  • a bifunctional molecule targeting PD-L1 and TGF ⁇ of the present disclosure is capable of operating at no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, or Binds specifically to human PD-L1 with a KD value of no more than 0.4 nM.
  • a bifunctional molecule targeting PD-L1 and TGF ⁇ of the present disclosure can be administered at no more than 2.0 nM (e.g., no more than 2.0 nM, no more than 1.2 nM, no more than 1.1) as measured by an ELISA assay.
  • the bifunctional molecules targeting PD-L1 and TGF ⁇ of the present disclosure can exhibit synergistic effects on tumor growth inhibition in a dose-dependent manner.
  • bifunctional molecules targeting PD-L1 and TGF ⁇ of the present disclosure can exhibit enhanced infiltration of anti-tumor immune cells into the tumor microenvironment compared to molecules including only immune checkpoint molecules.
  • bifunctional molecules targeting PD-L1 and TGF ⁇ of the present disclosure are capable of selectively reducing plasma levels of at least 90% (e.g., at least 80%, 70%, 60%, 50%, 40%, 30 % or 20%) of TGF ⁇ 1 and such reduction can be maintained for at least 10, 14 or 21 days.
  • the bifunctional molecule includes a heterodimeric heavy chain.
  • the heavy chain is heterodimeric with respect to the presence or location of the second portion.
  • the heterodimeric heavy chains include one heavy chain having the second portion and the other heavy chain not having the second portion, wherein the second portion includes a CD47 binding domain ( For example, soluble SIRP ⁇ ) or SIRP ⁇ binding domain.
  • the heterodimeric heavy chains include one heavy chain having the second portion and the other heavy chain not having the second portion.
  • the heterodimeric heavy chain may further comprise a heterodimeric Fc region associated in a manner that hinders homodimerization and/or facilitates heterodimerization.
  • heterodimeric Fc regions can be selected so that they are not identical and that they preferentially form heterodimers between each other rather than forming homodimers within themselves.
  • the heterodimeric Fc region is capable of associating into a heterodimer by forming a pestle structure, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.
  • the heterodimeric Fc region includes CH2 and/or CH3 domains, respectively, mutated to form a pestle structure.
  • the pestle structure can be obtained by replacing small amino acid residues in the first CH2/CH3 polypeptide with larger amino acid residues, and the hammer structure can be obtained by replacing larger residues with smaller residues.
  • the heterodimeric Fc region includes the first CH3 domain of the IgG1 isotype containing the S354C and T366W substitutions (SEQ ID NO: 96, pestle structure) and the first CH3 domain of the IgG1 isotype containing the Y349C, T366S, L368A, and Y407V substitutions ( The second CH3 domain of the IgG1 isotype of SEQ ID NO:97, structure).
  • the bifunctional molecule comprises a heavy chain of the amino acid sequence of SEQ ID NO: 118 or SEQ ID NO: 120, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 121. chain.
  • the bifunctional molecule further includes one or more conjugate moieties.
  • the conjugate moiety can be attached to a bifunctional molecule.
  • a conjugate moiety is a moiety that can be attached to a bifunctional molecule. It is contemplated that a variety of conjugate moieties can be attached to the bifunctional molecules provided herein (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (editor), Carger Press, New York, (1989)).
  • conjugate moieties can be connected to bifunctional molecules by covalent binding, affinity binding, intercalation, coordination binding, complexation, association, blending or addition.
  • bifunctional molecules can be linked to one or more conjugates via a linker.
  • bifunctional molecules provided herein can be engineered to contain specific sites outside of the epitope binding moiety that can be used to bind to one or more conjugate moieties.
  • sites may include one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent attachment to the conjugate moiety.
  • the bifunctional molecule can be linked to the conjugate moiety indirectly or through another conjugate moiety.
  • bifunctional molecules provided herein can be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin.
  • the conjugate moiety includes a clearance modifying agent (e.g., a half-life extending polymer such as PEG), a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label) labels, enzyme substrate labels), DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, purified fractions or other anticancer drugs.
  • a clearance modifying agent e.g., a half-life extending polymer such as PEG
  • chemotherapeutic agent e.g., a toxin, a radioactive isotope, a lanthanide
  • a detectable label e.
  • a "toxin” can be any agent that is harmful to cells or that can damage or kill cells.
  • toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecahine, mitomycin, etoposide, tenoposide, vinblastine Vincristine, MMAE, MMAF, DM1, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione ), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine ( tetracaine), lidocaine, propranolol, puromycin and its analogs, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, Cytarabine, 5-fluorouracil, dacarbazine),
  • detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidation enzyme, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase or ⁇ -D-galactosidase), radioactive isotopes (e.g., 123I, 124I, 125I, 131I, 35S, 3H , 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi and 32P, other lanthanide elements), luminescent markers, chromophore parts, digoxigenin ), biotin/avidin, DNA molecules or gold for detection.
  • fluorescent labels e.g.,
  • the conjugate moiety can be a clearance modifier that helps increase the half-life of the bifunctional molecule.
  • Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like.
  • the polymer can be of any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules.
  • the conjugate moiety can be a purification moiety, such as magnetic beads.
  • the bifunctional molecules provided herein are used as the basis for conjugates.
  • the formulations of the present disclosure may also contain other pharmaceutically acceptable excipients, such as vehicles (aqueous or non-aqueous), osmotic pressure regulators, viscosity reducing excipients, preservatives, wetting agents, emulsifiers and dispersing agents.
  • vehicles aqueous or non-aqueous
  • osmotic pressure regulators e.g parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Preservatives are typically used at a concentration of about 0.001% (w/v) to about 2% (w/v).
  • Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propylparaben, and benzalkonium chloride.
  • bifunctional molecules especially PD-L1/TGF- ⁇ protein
  • PD-L1/TGF- ⁇ protein can maintain long-term stability and meet clinical use requirements.
  • the solution provided by the present disclosure also solves the problem of polysorbate 80 degradation and avoids the possible impact of polysorbate 80 degradation products on protein quality.
  • formulations of the present disclosure are liquid formulations.
  • the liquid preparation is an injection.
  • the liquid preparation includes water for injection.
  • formulations of the present disclosure are maintained at about -80°C to about 45°C, such as -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C.
  • the purity of the bifunctional protein molecules in the preparation decreases by no more than 10%, such as no more than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1, as detected by SEC method. %.
  • the formulation of the present disclosure is at about -80°C to about 45°C, such as -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C.
  • the purity of the bifunctional protein molecule will not decrease by more than 10%, such as not more than 5%, 4%, 3%, 2%, 1%, 0.5, using the non-reducing CE-SDS method. % or 0.1%.
  • the present disclosure provides methods of treating, preventing, or alleviating PD-L1-related disease in a subject, comprising administering to the subject a therapeutically effective amount of a formulation provided herein.
  • the subject is human.
  • PD-1 related conditions and disorders may be immune-related diseases or disorders, cancer, autoimmune diseases, or infectious diseases.
  • PD-1 associated conditions and disorders include cancer, e.g., non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer Carcinoma, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycoses fungoids , Merkel cell cancer and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma tumors, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal cancer, CHL
  • PD-1 related conditions and disorders include autoimmune diseases.
  • Autoimmune diseases include, but are not limited to, acquired immune deficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease disease (autoimmune Addison's disease), autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenia Purpura (ATP), Behcet's disease, cardiomyopathy, sprue-dermatitis herpetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, juvenile dermatomyositis, Discoid l
  • PD-1 associated conditions and disorders include infectious diseases.
  • Infectious diseases include, for example, chronic viral infections, such as fungal infections, parasitic/protozoal infections, or chronic viral infections, such as malaria, coccidioidomycosis immitis, histoplasmosis, onychomycosis, aspergillus Mycosis, blastomycosis, candidiasis albicans, paracoccidioidomycosis (paracoccidiiomycosis), microsporidiosis, Acanthamoeba keratitis (Acanthamoeba keratitis), amebiasis, ascariasis, babesiosis, Bagworm disease, raccoon shellascariasis (Baylisascariasis), Chagas disease (Chagas disease), clonorchiasis, Cochliomyia, cryptosporidiosis, schizophrenia, dracunculi
  • the PD-L1-related disease is a cancer that expresses PD-L1 or a cancer that overexpresses PD-L1.
  • PD-L1-expressing cancer is a type of cancer involving cancer cells or tumor cells that have the PD-L1 protein on their cell surface.
  • Cancers that overexpress PD-L1 are cancers that have significantly higher levels of PD-L1 on the cell surface of cancer or tumor cells compared to non-cancer cells of the same tissue type.
  • PD-L1 expression or overexpression can be determined in a diagnostic or prognostic assay by assessing increased levels of PD-L1 present on the cell surface (eg, by immunohistochemical assay; IHC). Alternatively or additionally, detection can be performed, for example, by fluorescence in situ hybridization (FISH; see WO98/45479, published in October 1998), southern blotting or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). to measure the levels of nucleic acid encoding PD-L1 in cells.
  • PD-L1 overexpression can also be studied by measuring shed antigens (eg, PD-L1 extracellular domain or soluble PD-L1) in biological fluids such as serum.
  • cells in a patient can be exposed to an anti-PD-L1 antibody, which is optionally labeled with a detectable label (e.g., a radioisotope), and can be obtained, for example, by external scanning of the radioactivity or by analysis of cells previously exposed to A patient's biopsy evaluates the binding of antibodies to the patient's cells.
  • a detectable label e.g., a radioisotope
  • the subject has been identified as likely to respond to a PD-1 antagonist.
  • the presence or level of PD-L1 on a biological sample of interest can indicate whether the subject from which the biological sample is derived is likely to respond to a PD-1 antagonist.
  • the test sample is derived from cancer cells or tissue, or tumor-infiltrating immune cells.
  • the presence or upregulated level of PD-L1 in a test biological sample is indicative of the likelihood of a response.
  • the term "upregulation" refers to an overall increase in the protein level of PD-L1 in a test sample of not less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or greater.
  • the reference sample may be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained.
  • a reference sample may be a non-diseased sample adjacent to or in the vicinity of a test sample (eg, a tumor).
  • the PD-L1-related disease is resistant to PD-L1/PD-1 monotherapy.
  • PD-L1/PD-1 monotherapy refers to a monotherapy that acts by inhibiting or reducing PD-L1 and PD-1 interaction or signaling.
  • exemplary PD-L1/PD-1 monotherapy may include anti-PD-L1 antibody therapy, anti-PD-1 antibody therapy, or monotherapy involving small molecule inhibitors directed against PD-1 or PD-L1.
  • "Resistant" means that the disease has no response or sensitivity or has a reduced response or sensitivity to PD-L1/PD-1 monotherapy. Decreased response may be indicated by, for example, the need for increased dosage to achieve a given efficacy.
  • the disease may not respond to PD-L1/PD-1 monotherapy.
  • cancer cells or tumor size may increase despite treatment with PD-L1/PD-1 monotherapy, or the disease may show regression to its previous state, e.g., resumption of previous symptoms after partial recovery.
  • Resistance to PD-L1/PD-1 monotherapy can be de novo or acquired.
  • the present disclosure provides methods of treating, preventing, or alleviating a disease or condition in a subject that would benefit from the suppression of immunosuppressive cytokines, the induction of sustained immune responses, or the induction of anti-tumor immunity.
  • Stimulation the method includes administering an effective amount of a formulation provided herein.
  • the immunosuppressive cytokine is TGF ⁇ or IL-1. In some embodiments, the immunosuppressive cytokine is TGF ⁇ 1 or IL-1 ⁇ .
  • the disease or condition is a TGF ⁇ -related disease or condition.
  • the TGF ⁇ -related disease is cancer, fibrotic disease, or renal disease.
  • the TGF ⁇ -associated disease is cancer.
  • the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer , endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma, skin cancer, squamous cell carcinoma Skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.
  • the TGF ⁇ -related disease is a fibrotic disease.
  • Fibrotic diseases are diseases or conditions involving fibrosis. Fibrosis is a scarring process that is a common feature of chronic damage to organs such as the lungs, liver, kidneys, skin, heart, intestines, or muscles. Fibrosis is characterized by elevated transforming growth factor- ⁇ (TGF- ⁇ ) activity, leading to increased deposition and changes in the extracellular matrix and other fibrosis-related proteins.
  • TGF- ⁇ transforming growth factor- ⁇
  • Fibrotic diseases can include fibrotic diseases of the lungs, liver, kidneys, eyes, skin, heart, intestines, or muscles.
  • fibrotic diseases of the lung include pulmonary fibrosis, cystic fibrosis, pulmonary hypertension, progressive massive fibrosis, bronchiolitis obliterans, airway remodeling associated with chronic asthma or idiopathic lung.
  • fibrotic diseases of the liver include cirrhosis or non-alcoholic steatohepatitis.
  • fibrotic diseases of the kidney include renal fibrosis, ischemic kidney injury, tubulointerstitial fibrosis, diabetic nephropathy, nephrosclerosis or nephrotoxicity.
  • fibrotic diseases of the eye include corneal fibrosis and subretinal fibrosis.
  • fibrotic diseases of the skin include nephrogenic systemic fibrosis, keloids or scleroderma.
  • fibrotic diseases of the heart include endocardial fibrosis or old myocardial infarction.
  • the disease or condition is an IL-1 related disease or condition.
  • the IL-1 associated disease is an autoinflammatory disease, metabolic syndrome, acute inflammation, chronic inflammation, or malignancy.
  • the disease or condition will benefit from the induction of a sustained immune response by stimulating MHCII signaling with an immunostimulatory polypeptide (eg, soluble LAG-3).
  • an immunostimulatory polypeptide eg, soluble LAG-3
  • the disease or condition is cancer, viral infection, parasitic infection, or a combination thereof.
  • the disease or condition would benefit from stimulation of anti-tumor immunity by inhibiting immunosuppressive receptor signaling.
  • the immunosuppressive receptor is SIRP ⁇ .
  • the disease, disorder or condition is associated with SIRPa, such as cancer, solid tumors, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia disease, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type 2 diabetes, transplant dysfunction, or arthritis.
  • the cancer is anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and cholangiocarcinoma, pancreatic cancer, breast cancer, liver cancer , ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small bowel cancer, urethra cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, Colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary gland Cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CLL), chronic
  • the therapeutically effective amount of the bifunctional molecules provided herein will depend on various factors known in the art, such as weight, age, past medical history, current drug therapy, subject's health and potential for cross-reactivity, allergies, sensitivity and adverse side effects, as well as route of administration and extent of disease progression.
  • bifunctional molecules provided herein can be administered at a therapeutically effective dose of about 0.01 mg/kg to about 100 mg/kg.
  • compositions provided herein may be administered by routes known in the art, for example, parenterally (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or parenterally (e.g., oral , intranasal, intraocular, sublingual, rectal or topical) route.
  • parenterally e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection
  • parenterally e.g., oral , intranasal, intraocular, sublingual, rectal or topical route.
  • the bifunctional molecules provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent.
  • the bifunctional molecules disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent, an anti-cancer drug, radiotherapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy, a cell therapy , gene therapy, hormone therapy, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators or cytokines.
  • immunotherapy refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or generally strengthens the immune system.
  • immunotherapies include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.
  • Targeted therapy is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or that help Target molecules in the cancer microenvironment for cancer growth and survival.
  • Targeted therapy targets therapeutic agents to tumors, thereby sparing normal tissue from the effects of the therapeutic agent.
  • bifunctional molecules provided herein administered in combination with one or more additional therapeutic agents can be administered simultaneously with one or more additional therapeutic agents, and in some of these embodiments , the bifunctional molecule and the additional therapeutic agent can be administered as part of the same pharmaceutical composition.
  • a bifunctional molecule administered "in combination" with another therapeutic agent need not be administered at the same time as the agent or in the same composition as the agent.
  • a bifunctional molecule that is administered before or after another agent is considered to be administered "in combination with" that agent, as that phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered by different routes.
  • the present disclosure also provides the bifunctional molecules provided herein and/or the pharmaceutical compositions and pharmaceutical preparations provided herein for use in the manufacture of treating PD-L1 related diseases and/or TGF- ⁇ related diseases in a subject. and/or use in medicines for IL-1-related diseases and/or CD47-related diseases.
  • Example 1 Generation, expression and purification of humanized 4B6 antibody
  • Anti-PD-L1 mAb 4B6 derived from patent WO 2017161976A1 and including the VH sequence of SEQ ID NO:46 and the VL sequence of SEQ ID NO:47 shown below, is a potent PD-1/PD-L1 blocker. This antibody is produced from a mouse hybridoma antibody and therefore requires appropriate humanization. The sequence of the variable domain of mouse antibody 4B6 was used to identify the germline sequence with the highest homology to its corresponding murine framework. Computer modeling is used to design humanized variants with complementarity determining region (CDR) grafting and backmutation.
  • CDR complementarity determining region
  • the italicized part indicates the frame (FR), and the underlined part indicates the CDR sequence.
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the human germline framework sequences VK/1-33 of the light chain and VH/1-2 of the heavy chain were used for CDR transplantation, respectively.
  • Heavy chain variants 1, 2, 3, and 4 were modified by directly grafting the three CDRs to the VH germline sequence (SEQ ID NO: 48) and additionally modifying the VH variants M69V, R71V, VH variant of 1 (SEQ ID NO:49) M69V, R71V, R94K of VH variant 2 (SEQ ID NO:50) M69V, R71V, T73K of VH variant 3 (SEQ ID NO:51) , T28V and VH variant 4 (SEQ ID NO:52) M69V, R71V, A93V, R94K, T73K, T28V, G44S were obtained by reverse mutation respectively.
  • VH/1-2 (4B6-VH germline, SEQ ID NO: 48):
  • VH/1-2 variant 1 (4B6_Ha, SEQ ID NO:49):
  • VH/1-2 variant 2 (4B6_Hb, SEQ ID NO:50):
  • VH/1-2 variant 3 (Hu4B6_Hc, SEQ ID NO:51):
  • VH/1-2 variant 4 (Hu4B6_Hd, SEQ ID NO:52):
  • VL variants 1 and 2 Light chain variants 1 and 2 (VL variants 1 and 2) were modified by grafting three CDRs directly to the germline sequence (SEQ ID NO:53) and the additional F73L mutation of VL variant 1 (SEQ ID NO:54) And F73L, A43S, S60D of VL variant 2 (SEQ ID NO:55) were obtained by reverse mutation respectively.
  • VK/1-33 variant 1 (Hu4B6_La, SEQ ID NO:54):
  • VK/1-33 variant 2 (Hu4B6_Lb, SEQ ID NO:55):
  • the cDNAs for the heavy and light chain variable regions described above were synthesized and then fused to the constant region sequences of human IgGl and human kappa.
  • the resulting antibody gene sequence was cloned into an expression vector.
  • Large-scale DNA was prepared using the Plasmid Maxiprep System from Qiagen for humanized 4B6 variant expression as shown in Table 6 and using the ExpiFectamine TM CHO reagent from Invitrogen according to the manufacturer protocol for cell transfection. The supernatant was harvested when cell viability exceeded 60% and filtered through a 0.22um filter capsule to remove cell debris. The filtered supernatant was then loaded onto a pre-equilibrated Protein-A affinity column.
  • the Protein A resin was washed with equilibration buffer (PBS) and the antibody was then eluted using 25mM citrate (pH 3.5).
  • the purified antibody solution was adjusted to pH 6.0-7.0 by using 1M Tris-base (pH 9.0). Endotoxin is controlled below 1EU/mg. Finally, purified antibodies were characterized by SDS-PAGE.
  • Hu4B6_HaLa indicates that there are two mutations (heavy chain Hu4B6_Ha and light chain Hu4B6_La) on the humanized murine antibody Hu4B6_HaLa, and so on.
  • Hu4B6_L0 and Hu4B6_H0 were obtained by CDR grafting that lacked key reverse mutations and therefore were not used for expression.
  • Binding of humanized antibodies was assessed by ELISA method. Briefly, human PD-L1-His was immobilized on the plate. The humanized 4B6 antibodies listed in Table 6 were serially diluted and added in PBS and incubated for 1 hour. Next, goat pAb to human IgG-HRP and TMB was added to detect binding at OD450nm.
  • Hu4B6-HgLa retains the activity of chimeric 4B6, as an antagonist drug, higher affinity is preferred.
  • Several panning cycles based on the Hu4B6-HgLa sequence, site-directed mutagenesis in the CDRs and in vitro off-rate dependent selection were further used for affinity maturation.
  • the VL and VH domains of 4B6-HgLa were amplified by overlapping PCR and connected through peptide linker (G 4 S) 3 to form scFv, and then subcloned into the phagemid vector pComb3x (Wuhan MiaoLingBio), P0862) as a wild-type sequence that undergoes affinity maturation through the SfiI cleavage site.
  • CDR1 and CDR2 of the heavy and light chains In order to study the individual contributions of CDR1 and CDR2 of the heavy and light chains to 4B6 affinity maturation, a SPM (small perturbation mutagenesis) phage library of each of the above CDRs will be constructed, since antibody CDR3 of both chains is usually involved in antigen binding. It plays an important role.
  • the CDR1 and CDR2 sequences of both chains were aligned with the germline sequences, and the germlines of the variable regions of the heavy and light chains were IGHV1-2 and IGKV1-33, respectively. The results of bioinformatics analysis of germline CDR sequences were used to guide library design.
  • degenerate primers are designed to increase the diversity of the mutation library.
  • the scFv genes were ligated with the pComb3XSS phage display vector to generate scFv libraries.
  • Codon-based primers for each CDR (including HCDR1, HCDR2, LCDR1, and LCDR2 listed in Table 2) were established as independent libraries, and the 4B6 affinity maturation library was divided into 4 libraries library.
  • HCDR1 The capacity of HCDR1 is 1.76 ⁇ 10 8 CFM
  • the capacity of HCDR2 is 1.81 ⁇ 10 8 CFM
  • the capacity of LCDR1 is 2.34 ⁇ 10 8 CFM
  • the capacity of LCDR2 is 2.00 ⁇ 10 8 CFM.
  • hPD-L1 (Acro Biosystems, PD1-H5229) antigen was coated onto an ELISA plate and reacted with 200 ⁇ L phage (1 ⁇ 10 10 pfu/ml phage display library) at 37°C for 1 hour. After washing, TG1 (Lucigen, 60502-2) with an OD600 of approximately 0.5 was added directly to the wells for infection and incubated with the phage for 15 minutes. A sufficient volume of M13KO7 helper phage (NEB, N0315S) was cultured to mid-log phase for library phagemid rescue, and the phage were generated and purified for the next round of screening. The screening process was repeated for 3 rounds, with the antigen concentration reduced to 2.5 ⁇ g/ml for round 2 and to 1 ⁇ g/ml for round 3.
  • 96 clones from each library were picked and subjected to phage ELISA to detect their binding activities. Briefly, 1 ⁇ g/ml hPD-L1 (Acro Biosystems, PD1-H5229) antigen was coated onto an ELISA plate and left at 4°C overnight. Then add 300 ⁇ L of 3% (w/v) skim milk to block for 1 hour at room temperature. After 1 hour, 100 ⁇ l of the supernatant containing the monoclonal antibody fragment phage was added together with PBS as a negative control and incubated at 37°C for 1 hour.
  • hPD-L1 Acro Biosystems, PD1-H5229
  • Biolayer interferometry was used to test the binding affinity of 4B6scFv variants to human PD-L1-Fc (Sino Biological, 70110-D02H) antigen. Materials and procedures are shown in Table 8 and Table 9, respectively. The results are shown in Table 10-12. Based on the binding affinity results, 4 light chain variants (L-CDR1-2, L-CDR1-3, L-CDR2-2, L-CDR2-3) and 3 heavy chain variants (H-CDR2-2, H-CDR2-3, H-CDR2-5) were selected for future construction.
  • TGF ⁇ RII(1-136) has the amino acid sequence shown in SEQ ID NO:79:
  • amino acid sequence of hIgG1 is as follows (SEQ ID NO:80):
  • TGF ⁇ RII (1-136) is linked to the carboxyl terminus of hlgG1 via a peptide linker (G4S) 4 G (SEQ ID NO: 68).
  • amino acid sequence of h ⁇ is as follows (SEQ ID NO:82):
  • Co-transfection of heavy and light chains was performed using ExpiFectamine TM CHO reagent from Invitrogen (Thermo, A29129) according to the manufacturer's protocol. The supernatant was harvested on day 10 and purified by affinity chromatography.
  • hPD-L1 (Acro Biosystems, PD1-H5229) antigen was coated onto the ELISA plate and left at 4°C overnight. Then 300 ⁇ l of 3% (w/v) skim milk was added to block for 1 hour at room temperature. After 1 hour, 100 ⁇ l of AM-4B6-hlgG1-TGF ⁇ RII variant or original 4B6-hlgG1-TGF ⁇ RII at concentrations ranging from 100 nM to 0.006 nM (four-fold serial dilution) was added together with PBS as a negative control and incubated at room temperature. Incubate for 1 hour.
  • AM-4B6-hlgG1-TGF ⁇ RII variant or original 4B6-hlgG1-TGF ⁇ RII was immobilized on the surface of the S-series Protein A chip.
  • Human PD-L1 was diluted to the appropriate concentration gradient (0 nM, 1.875 nM, 3.75 nM, 7.5 nM, 15 nM, 30 nM, 60 nM) and injected into the sample channel of Biacore 2000.
  • the results are shown in Table 15.
  • AM-4B6-hlgG1-TGF ⁇ RII variant 7 has improved binding affinity to human PD-L1 than the original 4B6-hlgG1-TGF ⁇ RII About 15 times.
  • 293T-PD-L1-CD3L cells were generated by MabSpace Bioscience for the characterization of PD-L1 antibodies.
  • Cells were transfected with human PD-L1 and anti-CD3scFv.
  • AM-4B6-hlgG1-TGF ⁇ RII variant or original 4B6-hlgG1-TGF ⁇ RII was serially diluted (5-fold dilution) to obtain 8 concentrations in dilution buffer (PBS with 2% BSA).
  • diluted antibodies were added to the plate and incubated at 4°C for 30 minutes. After washing twice with dilution buffer, PE-conjugated donkey anti-human IgG (H+L) (Jacksonimmuno, 709-116-149) was added to the plate and incubated at 4°C for 30 minutes. After washing, cells were resuspended in 200 ⁇ l PBS and analyzed by flow cytometry.
  • H+L PE-conjugated donkey anti-human IgG
  • these five variants bind to PD-L1 expressed on the surface of 293T-PD-L1-CD3L cells with similar EC50.
  • the EC50 of variant 7 is slightly lower than the other variants, which is consistent with the binding affinity results measured by Biacore.
  • Example 7 PD-1/PD-L1 blocking activity of AM-4B6-hlgG1-TGFbRII variant
  • PD-1/PD-L1 and B7-1/PD-L1 blockade measured by ELISA assay
  • hPD-L1-Fc antigen 0.5 ⁇ g/ml hPD-L1-Fc antigen was coated onto ELISA plates and left at 4°C overnight.
  • 300 ⁇ L of blocking buffer to block for 1 hour at room temperature.
  • 50 ⁇ l of AM-4B6-hlgG1-TGF ⁇ RII variant 7 or original 4B6-hlgG1-TGF ⁇ RII at a concentration ranging from 100 nM to 0.024 nM (four-fold serial dilution) and 50 ⁇ l of PD- at a concentration of 1 ⁇ g/ml. L1-his and incubated for 1 hour at room temperature.
  • 293T-PD-L1-CD3L cells express PD-L1 and anti-CD3 scFv
  • Jurkat-NFAT-Luc-PD1 cells express PD-1 and carry an NFAT signal that can be activated with CD3 stimulation.
  • NFAT activation leads to downstream luciferase gene transcription and expression, which can be detected by its substrate.
  • the two cells were produced by Mabos Biosciences.
  • 293T-PD-L1-CD3L cells were harvested and resuspended at a density of 2 ⁇ 10 cells/ml.
  • AM-4B6-hlgG1-TGF ⁇ RII variant or original 4B6-hlgG1-TGF ⁇ RII was serially diluted (3-fold dilution) to obtain 8 concentrations in RPMI medium with 2% FBS.
  • Jurkat-NFAT-Luc-PD1 cells were harvested and resuspended in RPMI medium containing 2% FBS at a density of 4 ⁇ 10 cells/ml. Finally, 20 ⁇ l of cells per well and 5 ng/ml TGF- ⁇ (R&D, 240-B-010) were added to the half-well plate and incubated at 37 °C, 5% CO for 5 h. 60 ⁇ l of OneGlo detection reagent (Promega, E6120) was added to each well and incubated at room temperature for 5 minutes. The luminescence signal is read by a microplate reader. Data were analyzed via GraphPad Prism.
  • variant 7 had the most potent blocking activity in this cell-based assay compared to other variants. Therefore, the 4B6 Fab portion of AM-4B6-hlgG1-TGF ⁇ RII variant 7 was referred to as AM4B6, and the AM4B6-hlgG1-TGF ⁇ RII fusion protein was further evaluated in the following experiments.
  • Example 8 In vitro generation and characterization of AM4B6-hlgG1-TGF ⁇ RII'
  • TGF ⁇ RII ECD_20-136 is soluble and retains the ability to bind TGF ⁇ 1 (Kim-Ming Lo et al., US9676863 B2, 2017; Christian C. et al., "Protein Expression and Purification” ⁇ ,2000,20:98–104).
  • the full length of the extracellular domain of TGF ⁇ RII_1-136 was replaced with a truncated extracellular domain and developability and stability were assessed.
  • TGF- ⁇ RII truncated TGF- ⁇ RII
  • SEQ ID NO: 66 SEQ ID NO: 66
  • TGF ⁇ RII' to distinguish it from TGF- ⁇ RII(1-136).
  • AM4B6-hlgG1-TGF ⁇ RII' represents a molecule with TGF- ⁇ RII (20-136).
  • AM4B6-hlgG1-TGF ⁇ RII' competed with cynomolgus monkeys at an EC50 similar to that of human PD-L1.
  • PD-L1 cross-reactivity.
  • TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 from four common species published on the Uniport website (https://www.uniprot.org/): human, cynomolgus monkey, mouse and rat, TGF ⁇ members are quite conserved.
  • the sequences of human TGF ⁇ 1 and cynomolgus TGF ⁇ 1 are identical; mouse TGF ⁇ 1 and rat TGF ⁇ 1 are identical; human TGF ⁇ 2 and cynomolgus TGF ⁇ 2 are identical; mouse TGF ⁇ 2 and rat TGF ⁇ 2 are identical; human TGF ⁇ 3, cynomolgus TGF ⁇ 3, and mouse TGF ⁇ 3 are identical.
  • TGF ⁇ 1 and TGF ⁇ 3 the procedure was as follows: add 0.5 ⁇ g/ml human TGF ⁇ 1 (Sino Biological, 10804-HNAC) or mouse TGF ⁇ 1 (Novoprotein, CK33) or human TGF ⁇ 3 (Genscript, Z03430) or rat TGF ⁇ 3 ( Novoprotein, CJ44) antigen was coated onto an ELISA plate and left at 4°C overnight. Then 300 ⁇ l of 3% (w/v) skim milk was added to block for 1 hour at room temperature.
  • TGF ⁇ 2 For TGF ⁇ 2, the testing procedure was different: 2 ⁇ g/ml of AM4B6-hlgG1-TGF ⁇ RII' or control hIgG1-TGF ⁇ RII' was coated onto an ELISA plate and left at 4°C overnight. Then 300 ⁇ l of 3% (w/v) skim milk was added to block for 1 hour at room temperature. After 1 hour, 100 ⁇ l of human TGF ⁇ 2 or mouse TGF ⁇ 2 at concentrations ranging from 39.4 nM to 0.3 nM (two-fold serial dilution) was added and incubated for 1 hour at room temperature.
  • TGF ⁇ 2 biotinylated antibody (1:10000, R&D, BAF302) was added. After 1 hour of incubation at room temperature and washing, 100 ⁇ l of HRP-Streptavidin (1:5000, Abcam, ab7403) was added and the plate was incubated for 1 hour at room temperature. After washing, mixed TMB substrate reagents (InnoReagents, TMB-S-003) were added and incubated at room temperature for 5 minutes, and terminated by the addition of 0.1 M H2SO4 . OD450nm was recorded by a microplate reader.
  • hPD-L1 (Acro Biosystems, PD1-H5229) or hPD-L2 or B7-2 or B7-1 or B7-H2 or B7-H3 or B7-H4 or VISTA was coated into the ELISA Plate and place at 4 °C overnight.
  • hPD-L1 Acro Biosystems, PD1-H5229
  • hPD-L2 or B7-2 or B7-1 or B7-H2 or B7-H3 or B7-H4 or VISTA was coated into the ELISA Plate and place at 4 °C overnight.
  • TGF ⁇ superfamily 0.5 ⁇ g/ml human activin A, BMP-2, LAP, or TGF ⁇ 1 was coated overnight at 4°C. Then 300 ⁇ l of 3% (w/v) skim milk was added to block for 1 hour at room temperature.
  • AM4B6-hlgG1-TGF ⁇ RII' specifically binds to PD-L1 but not other antigens that also belong to the B7 family.
  • AM4B6-hlgG1-TGF ⁇ RII' specifically bound to TGF- ⁇ 1 but not other antigens also belonging to the TGF ⁇ superfamily.
  • MC38/hPD-L1 was generated by deleting mPD-L1 via the CRISPR-Cas9 system and then using lentivirus to transduce hPD-L1. This cell line was provided by the laboratory of Professor Qin Xiaofeng at the Center of Systems Medicine, Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences (Huang, Anfei et al. "Scientific Reports” ⁇ 7(2017):42687.). MC-38/hPD-L1 cells were cultured in RPMI1640+10% FBS. EMT-6/hPD-L1 is a mouse breast cancer cell line that stably expresses the transfected human PD-L1 gene.
  • EMT-6/hPD-L1 cells were cultured in Waymouth's (1 ⁇ ) MB752/1 + 15% FBS.
  • NCI-H460 cells were purchased from COBIOER Ltd. (COBIOER Ltd). It is a human lung epithelial tumor cell line with PD-L1 expression.
  • NCI-H460 cells were cultured in RPMI1640+10% FBS.
  • NCI-H292 cells were purchased from COBIOER Co., Ltd. It is a human lung epithelial tumor cell line with PD-L1 expression.
  • NCI-H292 cells were cultured in RPMI1640+10% FBS+1 nM sodium pyruvate solution.
  • the FACS analysis protocol was the same as that in Section 3 of Example 6.
  • PBMC Human or cynomolgus monkey PBMC (TPCS, catalog number PB025C) were recovered from liquid nitrogen and resuspended in RPMI1640 with 10% FBS. 5 ⁇ g/ml of PHA (Sigma, Cat. No. L8902) was added to stimulate PBMC activation and the cells were cultured for 3 days. Activated PBMC were collected and centrifuged and resuspended in PBS at a density of 2 ⁇ 10 cells/ml and added to the plate at 100ul per well.
  • PHA Sigma, Cat. No. L8902
  • AM4B6-hlgG1-TGF ⁇ RII' or AM4B6 or control hlgG1-TGF ⁇ RII' were serially diluted (5-fold dilution) to obtain 10 concentrations in dilution buffer (PBS with 2% BSA). After centrifugation and removal of the supernatant from the plate, the diluted antibodies were added to the plate with activated PBMC and incubated at 4 °C for 1 h. After washing twice with dilution buffer, Alexa488-labeled mouse anti-human CD3 (Biolegend, Cat. No. 300320) and APC-labeled anti-human IgG secondary antibodies (BD, Cat. No. 550931) were added and incubated at 4°C for 30 minute. After washing, cells were resuspended in 150 ⁇ l PBS and analyzed by flow cytometry.
  • dilution buffer PBS with 2% BSA
  • AM4B6-hlgG1-TGF ⁇ RII' can bind to PD-L1 expressed on these cancer cell lines and activated human or cynomolgus monkey T cells with similar affinity to AM4B6 mAb alone.
  • Human PBMC (TPCS, catalog number PB025C) were recovered from liquid nitrogen and resuspended in RPMI1640 with 10% FBS.
  • PHA Sigma, Cat. No. L8902
  • Activated PBMC were collected and centrifuged and resuspended in PBS at a density of 2 ⁇ 10 cells/ml and added to the plate at 100ul per well.
  • AM4B6-hlgG1-TGF ⁇ RII' or AM4B6 or control hlgG1-TGF ⁇ RII' were serially diluted (5-fold dilution) to obtain 10 concentrations in dilution buffer (PBS with 2% BSA). After centrifugation and removal of the supernatant from the plate, the diluted antibodies were added to the plate with activated PBMC and incubated at 4 °C for 1 h. After washing twice with dilution buffer, Alexa488-labeled mouse anti-human CD3 (Biolegend, catalog number 300320) was added and APC-labeled anti-human IgG secondary antibody (BD, Cat. No. 550931) and incubated for 30 minutes at 4°C. After washing, cells were resuspended in 150 ⁇ l PBS and analyzed by flow cytometry.
  • dilution buffer PBS with 2% BSA
  • AM4B6-hlgG1-TGF ⁇ RII' can bind to PD-L1 expressed on activated human T cells.
  • AM4B6-hlgG1-TGF ⁇ RII' can also completely block cynomolgus PD-L1/cynomolgus PD- at an IC50 similar to that of blocking human PD-L1/human PD-1. 1.
  • hTGF ⁇ -1 0.5 ⁇ g/ml hTGF ⁇ -1 was coated onto the ELISA plate and left at 4°C overnight. Then add 300 ⁇ l of blocking buffer to block for 1 h at room temperature. After 1 hour, 100 ⁇ l of AM4B6-hlgG1-TGF ⁇ RII' or AM4B6 or control hlgG1-TGF ⁇ RII' at concentrations ranging from 100 nM to 0.02 nM (four-fold serial dilution) was added and incubated for 1 hour at room temperature. 0.5% PBS+Tween-20 was used for 3 washes, and then 0.5 ⁇ g/ml of hPD-L1-biotin was added per well.
  • AM4B6-hlgG1-TGF ⁇ RII' composed of anti-PD-L1 antibodies AM4B6 and TGF ⁇ RII' can bind to two targets simultaneously, indicating its bispecific or bifunctional properties.
  • AM4B6-hlgG1-TGF ⁇ RII can block PD-L1/PD-1 inhibition and subsequently reverse signaling activation, identical to AM4B6 mAb alone.
  • the TGF ⁇ reporter HEK-293 cell line was purchased from Genomeditech (catalog: GM-C05346) and incubated in a 37°C incubator with 5% carbon dioxide containing 10% FBS, 4 ⁇ g/ml blasticidin, 400 ⁇ g/ml neomycin, Culture in DMEM medium containing 125 ⁇ g/ml hygromycin, 0.75 ⁇ g/ml puromycin and 1% Pen/Strep.
  • AM4B6-hlgG1-TGF ⁇ RII' showed potent blocking activity on TGF ⁇ 1 signaling.
  • the IC50 was 0.35 nM, while AM4B6 mAb alone had no blocking activity, indicating that the blocking activity is TGF ⁇ 1 specific.
  • Human PBMC were recovered from liquid nitrogen and resuspended at a density of 2 ⁇ 10 ⁇ 6/mL. Add TB to a final concentration of 1.33 ⁇ g/mL; incubate at 37°C for 5 days. On the sixth day, induced PBMC were collected and centrifuged, washed once with PBS, resuspended in fresh medium, adjusted to a density of 1 ⁇ 10 ⁇ 6/ml, and seeded into a 96-well cell plate at 180 ⁇ L/well. Add diluted antibodies at 20 ⁇ L/well to 96-well cell culture plates. The control group and blank group were added with 20 ⁇ L PBS.
  • AM4B6-hlgG1-TGF ⁇ RII' induced significantly higher levels of IFN- ⁇ release than AM4B6 mAb alone, indicating that its activating activity is more potent due to its bispecific binding and blocking activities.
  • effector cell Jurkat-NFAT Luc-Fc ⁇ RIIIa-158V cell line was constructed by Mabspace Biosciences (Suzhou) Co., Limited.
  • Target cells HEK-293T-hPD-L1 cells (purchased from Crown Biosciences Inc., catalog: 2005).
  • HEK-293T-hPD-L1 cells to the cell culture plate at 10,000 cells/12.5 ⁇ l/well.
  • AM4B6-hlgG1-TGF ⁇ RII' dilutions with final concentrations ranging from 200 nM to 0.003 nM were then added at 12.5 ⁇ l/well.
  • the plate was then placed in a 37°C incubator to incubate the antibodies and cells for 30 minutes.
  • Jurkat-NFAT Luc-Fc ⁇ RIIIa-158V cells were then added to the wells at 60,000 cells/25 ⁇ l/well.
  • the plates were then placed in a 37°C incubator for 6 hours.
  • ONE-GloTM Luciferase Assay System was added at 50 ⁇ l/well, and after incubation for 10 min at room temperature, the plate was read with a microplate reader.
  • the target cells were also HEK-293T-hPD-L1 cells.
  • HEK-293T-hPD-L1 cells were added to the cell culture plate at 10,000 cells/25 wells.
  • AM4B6-hlgG1-TGF ⁇ RII' dilutions with final concentrations ranging from 200 nM to 0.3 nM were then added at 12.5 ⁇ l/well.
  • the plate was then placed in a 37°C incubator to incubate the antibodies and cells for 30 minutes.
  • HEK-293T-hPD-L1 cells were treated with 40% complement at 50 ⁇ l/well (final concentration 20%) and then incubated at 37°C for 80 minutes.
  • ONE-GloTM Luciferase Assay System was added at 100 ⁇ l/well, and after incubation for 10 min at room temperature, the plate was read with a microplate reader.
  • Knockout of endogenous mouse PD-L1 in the mouse tumor cell line MC38 (ATCC) using the recently developed efficient CRISPR/Cas9 system was designed, and cells were transfected with a hit-and-run CRISPR/Cas9+sgRNA construct. and select knockout cells.
  • Cells with complete knockout of endogenous PD-L1 in mice were characterized by FACS analysis to identify cell surface expression of PD-L1 at steady state or under interferon gamma stimulation, which was subsequently verified by TA cloning and targeted genomic region sequencing.
  • human PD-L1 replacement cell line To generate a human PD-L1 replacement cell line, the coding sequence of human PD-L1 cDNA was cloned into an FG12-derived lentiviral vector. Then use the expression human PD- Lentivirus of L1 infected mouse PD-L1 knockout cells, and FACS analysis confirmed high-level and stable expression of human PD-L1 in the established cell line.
  • This engineered cell of MC38 was named MC38-hPD-L1.
  • TGI Tumor growth inhibition

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Abstract

提供了包括与TGFβ结合部分、IL-1结合部分、免疫刺激性多肽(例如、可溶性LAG3或可溶性CD4)或CD47结合部分连接的PD-L1结合部分的蛋白质，其药物制剂、制备方法和用途。

Description

双功能蛋白质及其制剂和用途
相关申请的交叉引用
本申请要求2022年5月17日提交的专利申请CN202210541662.4的优先权权益，其公开内容通过引用并入本文。
技术领域
本公开涉及靶向免疫检查点分子(例如，PD-L1)并且阻断抗肿瘤免疫抑制(ATIS)细胞因子(例如，IL-1或TGFβ)的活性或刺激免疫的双功能蛋白质分子，其药物制剂，其制备方法和用途。
背景技术
PD-L1在不同的肿瘤中经常过度表达并且其与T细胞上的PD-1相互作用使癌细胞能够避开T细胞介导的免疫应答(Okazaki T等人,《自然免疫学》,2013,14(12):1212-1218)。因此，阻断PD-1/PD-L1相互作用可以恢复T细胞激活和抗肿瘤应答(Callahan M K等人,《免疫力》,2016,44(5):1069-1078)。基于抗体的PD-1/PD-L1阻断疗法如阿特珠单抗(atezolizumab)(Rittmeyer A等人,《柳叶刀(The Lancet)》,2017,389(10066):255-265)、阿维鲁单抗(avelumab)(Hamilton G等人,《生物治疗专家观点(Expert Opinion on Biological Therapy)》,2017,17(4):515-523)和德瓦鲁单抗(durvalumab)(Brower V,《柳叶刀肿瘤学(The Lancet Oncology)》,2016,17(7):e275)的成功意味着在对抗人类癌症，尤其是实体瘤方面取得了突破性进展。尽管已显示肿瘤细胞和/或浸润性免疫细胞表达PD-L1与对PD-1/PD-L1靶向疗法的临床应答之间存在关联，但这种关联并非完美无缺(Herbst,R.等人,《自然》515,563–567(2014)；Taube J M等人,《临床癌症研究(Clinical cancer research)》,2014,20(19):5064-5074)。只有少数PD-L1阳性肿瘤对这些治疗有应答，然而某些PD-L1阴性肿瘤仍对治疗有应答。这增加了另外的因素控制患者对PD-1/PD-L1靶向疗法的应答的可能性，并且必须鉴定另外的预测性生物标志物以改善这些药剂的临床使用。
Mariathasan S等人发现缺乏应答与成纤维细胞中转化生长因子β(TGF-β，TGFβ)信号传导的特征相关(Mariathasan S等人,《自然》,2018,554(7693):544-548)。David JM等人还发现，作为一种已知诱导上皮间充质转化(EMT)并抑制抗肿瘤免疫的多效性细胞因子，TGF-β可以上调多种上皮NSCLC细胞系中的肿瘤PD-L1表达，并且上调与作为TGF-β信号传导的关键下游效应子的Smad2的磷酸化有关(David J M等人,《肿瘤免疫学(Oncoimmunology)》,2017,6(10):e1349589)。在小鼠中，治疗性施用TGF-β阻断抗体和抗PD-L1减少了基质细胞中的TGF-β信号传导，促进T细胞渗透到肿瘤中心，并激发强烈的抗肿瘤免疫和肿瘤消退(Mariathasan S等人,《自然》,2018,554(7693):544-548)。
然而，抗PD-L1抗体的低亲和力仍然对实现高治疗功效和低毒副作用提出了挑战。
因此，需要具有对PD-L1具有高结合亲和力、改善的治疗功效和减少的毒副作用的治疗分子及其药物制剂。
本公开内容
本公开提供了包括与TGFβ结合部分、IL-1结合部分、免疫刺激性多肽(例如，可溶性LAG3或可溶性CD4)或CD47结合部分连接的PD-L1结合部分的蛋白质，其药物制剂、制备方法和用途。
本公开提供了蛋白质制剂，其包括：
(1)双功能分子，其包括：
与免疫检查点分子结合的第一部分，所述第一部分优选是针对PD-L1的抗体或其抗原结合片段，和
i)阻断免疫抑制性细胞因子的活性或ii)刺激免疫的第二部分，所述第二部分优选是TGFβ结合部分、IL-1结合部分、LAG-3结合部分或Sirpa结合部分，其中所述TGFβ结合部分优选是可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体、或针对TGFβ的抗体和其抗原结合片段；
(2)缓冲剂；和
(3)表面活性剂。
更具体而言，本公开还提供了蛋白质制剂，其包括：
(1)双功能分子，其包括与PD-L1结合的第一部分和i)阻断免疫抑制性细胞因子的活性或ii)刺激免疫的第二部分；其中所述第一部分包括针对PD-L1的抗体或其抗原结合片段；
(2)缓冲剂；和
(3)表面活性剂。对此，本公开还提供了以下更具体实施方案：
实施方案1.蛋白质制剂，其包括：
(1)双功能分子，其包括与免疫检查点分子结合的第一部分和阻断白细胞介素-1(IL-1)活性的第二部分；
(2)缓冲剂；和
(3)表面活性剂。
实施方案2.根据实施方案1所述的蛋白质制剂，其中所述第一部分包括免疫刺激性检查点分子的激动剂，任选地，所述免疫刺激性检查点分子选自由以下组成的组：CD27、CD70、CD28、CD80(B7-1)、CD86(B7-2)、CD40、CD40L(CD154)、CD122、CD137、CD137L、OX40(CD134)、OX40L(CD252)、GITR、ICOS(CD278)和ICOSLG(CD275)、CD2、ICAM-1、LFA-1(CD11a/CD18)、CD30、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160和CD83。
实施方案3.根据实施方案1所述的蛋白质制剂，其中所述第一部分包括免疫抑制性检查点分子的拮抗剂，任选地，所述免疫抑制性检查点分子选自由以下组成的组：A2AR、B7-H3(CD276)、B7-H4(VTCN1)、BTLA(CD272)、CTLA-4(CD152)、IDO1、IDO2、TDO、KIR、LAG3、NOX2、PD-1、PD-L1、PD-L2、TIM-3、VISTA、SIGLEC7(CD328)、TIGIT、PVR(CD155)、SIGLEC9(CD329)、CD160、LAIR1、2B4(CD244)、CD47、B7-H5。
实施方案4.根据实施方案3所述的蛋白质制剂，其中所述免疫检查点分子是PD-L1。
实施方案5.根据前述实施方案中任一项所述的蛋白质制剂，其中所述第一部分包括针对PD-L1的抗体或其抗原结合片段，并且所述第二部分包括IL-1结合部分或IL-1受体(IL-1R)结合部分。
实施方案6.根据实施方案5所述的蛋白质制剂，其中所述IL-1结合部分包括IL-1R或其片段或变体、或针对IL-1的抗体或其抗原结合片段。
实施方案7.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含来自抗IL-1α抗体或抗IL-1β抗体的重链可变区和/或轻链可变区，所述抗IL-1α抗体选自由以下组成的组：XB2001、鲁吉珠单抗(lutikizumab)、LY2189102和贝迈奇单抗(bermekimab)，或所述抗IL-1β抗体选自由以下组成的组：SSGJ-613、CDP484、卡那单抗(canakinumab)和吉伏组单抗(gevokizumab)。
实施方案8.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104或SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:105或SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:106或SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107或SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:108或SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:109或SEQ ID NO:117的序列的LCDR3.
实施方案9.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104的序列的HCDR1，包括SEQ ID NO:105的序列的HCDR2，和包括SEQ ID NO:106的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107的序列的LCDR1，包括SEQ ID NO:108的序列的LCDR2，和包括SEQ ID NO:109的序列的LCDR3.
实施方案10.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:117的序列的LCDR3.
实施方案11.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102,SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103,SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
实施方案12.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103和与其具有至少80％序列同一性的同源序列。
实施方案13.根据实施方案6所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序 列：SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
实施方案14.根据实施方案5所述的蛋白质制剂，其中所述IL-1R结合部分包括白细胞介素-1受体拮抗剂或其片段或变体、或针对IL-1R的抗体或其抗原结合片段。
实施方案15.根据实施方案14所述的蛋白质制剂，其中所述针对IL-1R的抗体或其抗原结合片段包含来自选自司柏索利单抗(spesolimab)、艾特利单抗(astegolimab)、imsidolimab、AMG 108、melrilimab、尼达尼单抗(nidanilimab)、MEDI8968、REGN6490、HB0034和CSC012的抗体的重链可变区和/或轻链可变区。
实施方案16.蛋白质制剂，其包括：
(1)双功能分子，其包括与PD-L1结合的第一部分和a)阻断免疫抑制性细胞因子的活性或b)刺激免疫的第二部分，其中所述第一部分包括针对PD-L1的抗体或其抗原结合片段，所述抗体或其抗原结合片段包括重链可变(VH)区和/或轻链可变(VL)区，其中所述重链可变区包括：
a)HCDR1，所述HCDR1包括DYYMN(SEQ ID NO:1)或与DYYMN具有至少80％序列同一性的同源序列，
b)HCDR2，所述HCDR2包括DINPNNX1X2TX3YNHKFKG(SEQ ID NO:19)或与DINPNNX1X2TX3YNHKFKG具有至少80％序列同一性的同源序列，以及
c)HCDR3，所述HCDR3包括WGDGPFAY(SEQ ID NO:3)或与WGDGPFAY具有至少80％序列同一性的同源序列，和/或
其中所述轻链可变区包括：
d)LCDR1包括选自由KASQNVX4X5X6VA(SEQ ID NO:20)或与KASQNVX4X5X6VA具有至少80％序列同一性的同源序列组成的组的序列，
e)LCDR2包括选自由SX7SX8RYT(SEQ ID NO:21)或与SX7SX8RYT具有至少80％序列同一性的同源序列组成的组的序列，以及
f)LCDR3包括选自由QQYSNYPT(SEQ ID NO:6)或与QQYSNYPT具有至少80％序列同一性的同源序列组成的组的序列；
其中X1是G或A，X2是G或D或Q或E或L，X3是S或M或Q或L或V，X4是G或P或K，X5是A或G，X6是A或I，X7是A或N或R或V，并且X8是N或H或V或D；
(2)缓冲剂；和
(3)表面活性剂。
实施方案17.根据实施方案16所述的蛋白质制剂，其中所述重链可变区包括：
a)HCDR1包括SEQ ID NO:1的序列，
b)HCDR2包括选自由以下组成的组的序列：SEQ ID NO:2、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17和SEQ ID NO:18，以及
c)HCDR3包括SEQ ID NO:3的序列，
和/或
轻链可变区，所述轻链可变区包括：
d)LCDR1包括选自由以下组成的组的序列：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9，
e)LCDR2包括选自由以下组成的组的序列：SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12，以及
f)LCDR3包括SEQ ID NO:6的序列。
实施方案18.根据实施方案17所述的蛋白质制剂，其中所述重链可变区选自由以下组成的组：
a)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:2的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
b)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:13的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
c)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:14的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
d)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:15的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；以及
e)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:17的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3。
实施方案19.根据实施方案17或18所述的蛋白质制剂，其中所述轻链可变区选自由以下组成的组：
a)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
b)包括以下的轻链可变区：包括SEQ ID NO:9的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
c)包括以下的轻链可变区：包括SEQ ID NO:8的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
d)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:12的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；以及
e)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:11的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3。
实施方案20.根据实施方案16到19中任一项所述的蛋白质制剂，其中所述针对PD-L1的抗体或其抗原结合片段进一步包括重链HFR1、HFR2、HFR3和HFR4中的一个或多个，和/或轻链LFR1、LFR2、LFR3和LFR4中的一个或多个，其中：
a)所述HFR1包括QVQLVQSGAEVKKPGASVKVSCKASGYX9FT(SEQ ID NO:40)或与QVQLVQSGAEVKKPGASVKVSCKASGYX9FT具有至少80％序列同一性的同源序列，
b)所述HFR2包括WVRQAPGQX10LEWMG(SEQ ID NO:41)或与WVRQAPGQX10LEWMG具有至少80％序列同一性的同源序列，
c)所述HFR3序列包括RVTX16TVDX11SISTAYMELSRLRSDDTAVYYCX12X13(SEQ ID NO:42)或与RVTX16TVDX11SISTAYMELSRLRSDDTAVYYCX12X13具有至少80％序列同一性的同源序列，
d)所述HFR4包括WGQGTLVTVSS(SEQ ID NO:25)或与WGQGTLVTVSS具有至少80％序列同一性的同源序列，
e)所述LFR1包括DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:26)或与DIQMTQSPSSLSASVGDRVTITC具有至少80％序列同一性的同源序列，
f)所述LFR2包括WYQQKPGKX14PKLLIY(SEQ ID NO:43)或与WYQQKPGKX14PKLLIY具有至少80％序列同一性的同源序列，
g)所述LFR3包括GVPX15RFSGSGSGTDFTX17TISSLQPEDIATYYC(SEQ ID NO:44)或与GVPX15RFSGSGSGTDFTX17TISSLQPEDIATYYC具有至少80％序列同一性的同源序列，并且
h)所述LFR4包括FGQGTKLEIK(SEQ ID NO:29)或与FGQGTKLEIK具有至少80％序列同一性的同源序列，
其中X9是T或V，X10是G或S，X11是T或K，X12是A或V，X13是R或K，X14是A或S，X15是S或D，X16是M或V，并且X17是F或L。
实施方案21.根据实施方案20所述的蛋白质制剂，其中：
所述HFR1包括选自由SEQ ID NO:22和30组成的组的序列，
所述HFR2包括选自由SEQ ID NO:23和31组成的组的序列，
所述HFR3包括选自由SEQ ID NO:24和32-35组成的组的序列，
所述HFR4包括SEQ ID NO:25的序列，
所述LFR1包括来自由SEQ ID NO:26组成的组的序列，
所述LFR2包括选自由SEQ ID NO:27和36组成的组的序列，
所述LFR3包括选自由SEQ ID NO:28和37-38、39、45组成的组的序列，并且
所述LFR4包括SEQ ID NO:29的序列。
实施方案22.根据实施方案16到21中任一项所述的蛋白质制剂，其中所述重链可变区包括选自由以下组成的组的序列：SEQ ID NO:46、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60和与其具有至少80％序列同一性的其同源序列。
实施方案23.根据实施方案16到22中任一项所述的蛋白质制剂，其中所述轻链可变区包括选自由以下组成的组的序列：SEQ ID NO:47、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65和与其具有至少80％序列同一性的其同源序列。
实施方案24.根据实施方案16所述的蛋白质制剂，其中所述针对PD-L1的抗体或其抗原结合片段包括选自由以下组成的组的一对重链可变区和轻链可变区序列：SEQ ID NO:49/54、50/54、51/54、52/54、49/55、50/55、51/55、52/55、58/62、58/63、58/64、58/65、59/62、59/63、59/64、59/65、60/62、60/63、60/64和60/65和与其具有至少80％序列同一性的其同源序列。
实施方案25.根据实施方案16到24中任一项所述的蛋白质制剂，其中所述针对PD-L1的抗体或其抗原结合片段进一步包括一个或多个氨基酸残基取代或修饰，但仍保留对PD-L1的特定结合特异性和/或亲和力。
实施方案26.根据实施方案25所述的蛋白质制剂，其中所述取代或修饰中的至少一个取代或修饰在所述CDR序列中的一个或多个CDR序列中和/或在所述VH或VL序列的非CDR区中的一个或多个非CDR区中。
实施方案27.根据实施方案16到26中任一项所述的蛋白质制剂，其中所述针对PD-L1的抗体或其抗原结合片段进一步包括免疫球蛋白恒定区，任选地人Ig的恒定区，或任选地人IgG的恒定区。
实施方案28.根据实施方案27所述的蛋白质制剂，其中所述恒定区包括人IgG1、IgG2、IgG3或IgG4的Fc区。
实施方案29.根据实施方案28所述的蛋白质制剂，其中所述恒定区包括Fc变体，所述Fc变体相对于对应野生型Fc区具有降低的效应子功能。
实施方案30.根据实施方案29所述的蛋白质制剂，其中所述Fc变体包括选自由以下组成的组的一个或多个氨基酸残基取代：220S、226S、228P、229S、233P、234V、234G、234A、234F、234A、235A、235G、235E、236E、236R、237A、237K、238S、267R、268A、268Q、269R、297A、297Q、297G、309L、318A、322A、325L、328R、330S、331S和其任何组合，其中所述Fc区中的残基的编号为如Kabat中的EU索引的编号。
实施方案31.根据实施方案29到30中任一项所述的蛋白质制剂，其中所述Fc变体包括选自由以下组成的组的突变的组合：a)K322A、L234A和L235A；b)P331S、L234F和L235E；c)L234A和L235A；c)N297A；d)N297Q；e)N297G；f)L235E；g)L234A和L235A(IgG1)；h)F234A和L235A(IgG4)；i)H268Q、V309L、A330S和P331S(IgG2)；j)V234A、G237A、P238S、H268A、V309L、A330S和P331S(IgG2)，其中所述Fc区中的残基的编号为如Kabat中的EU索引的编号。
实施方案32.根据实施方案29到31中任一项所述的蛋白质制剂，其中所述Fc变体包括SEQ ID NO:81的氨基酸序列。
实施方案33.根据实施方案16到32中任一项所述的蛋白质制剂，其中所述针对PD-L1的抗体或其抗原结合片段是人源化的。
实施方案34.根据实施方案16到33中任一项所述的蛋白质制剂，其中所述抗原结合片段是双抗体、Fab、Fab'、F(ab')2、Fd、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双抗体(ds双抗体)、单链抗体分子(scFv)、scFv二聚体(二价双抗体)、多特异性抗体、骆驼化单结构域抗体、纳米抗体、结构域抗体和二价结构域抗体。
实施方案35.根据实施方案16到34中任一项所述的蛋白质制剂，其中所述抗体或其抗原结合片段能够与人PD-L1和食蟹猴PD-L1两者结合。
实施方案36.根据实施方案1到15中任一项所述的蛋白质制剂，其中所述第一部分包括与根据实施方案16到35中任一项所述的抗体或其抗原结合片段竞争结合PD-L1的抗体或其抗原结合片段。
实施方案37.根据实施方案16到36中任一项所述的蛋白质制剂，其中所述免疫抑制性细胞因子包括转化生长因子β(TGF-β)超家族中的细胞因子、IL-1或血管内皮生长因子(VEGF)。
实施方案38.根据实施方案37所述的蛋白质制剂，其中所述TGF-β超家族中的免疫抑制性细胞因子包括TGF-β、骨形态发生蛋白(BMP)、激活素、NODAL和生长和分化因子(GDF)。
实施方案39.根据实施方案16到38中任一项所述的蛋白质制剂，其中所述免疫抑制性细胞因子是TGF-β。
实施方案40.根据实施方案16到39所述的蛋白质制剂，其中所述第二部分包括TGFβ结合部分。
实施方案41.根据实施方案40所述的蛋白质制剂，其中所述TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体、或针对TGFβ的抗体和其抗原结合片段。
实施方案42.根据实施方案41所述的蛋白质制剂，其中所述可溶性TGFβR包括所述TGFβR的胞外结构域(ECD)或其TGFβ结合片段或变体。
实施方案43.根据实施方案42所述的蛋白质制剂，其中所述TGFβR选自由以下组成的组：TGFβ受体I(TGFβRI)、TGFβ受体II(TGFβRII)、TGFβ受体III(TGFβRIII)和其任何组合。
实施方案44.根据实施方案42所述的蛋白质制剂，其中所述TGFβR是TGFβRII。
实施方案45.根据实施方案44所述的蛋白质制剂，其中所述TGFβRII相对于TGFβ2和TGFβ3而选择性地结合到TGFβ1。
实施方案46.根据实施方案45所述的蛋白质制剂，其中所述TGFβ1是人TGFβ1或小鼠TGFβ1。
实施方案47.根据实施方案42到46中任一项所述的蛋白质制剂，其中TGFβR的所述ECD包括SEQ ID NO:66、79、78、77的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对TGF-β的特定结合特异性和/或亲和力的序列。
实施方案48.根据实施方案36所述的蛋白质制剂，其中所述第二部分包括IL-1结合部分或IL-1受体(IL-1R)结合部分。
实施方案49.根据实施方案48所述的蛋白质制剂，其中所述IL-1结合部分包括可溶性IL-1R、IL-1R的IL-1结合片段或变体、或针对IL-1的抗体或其抗原结合片段。
实施方案50.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含来自抗IL-1α抗体或抗IL-1β抗体的重链可变区和/或轻链可变区，所述抗IL-1α抗体选自由以下组成的组：XB2001、鲁吉珠单抗(lutikizumab)、LY2189102和贝迈奇单抗(bermekimab)，或所述抗IL-1β抗体选自由以下组成的组：SSGJ-613、CDP484、卡那单抗(canakinumab)和吉伏组单抗(gevokizumab)。
实施方案51.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104或SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:105或SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:106或SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107或SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:108或SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:109或SEQ ID NO:117的序列的LCDR3.
实施方案52.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104的序列的HCDR1，包括SEQ ID NO:105的序列的HCDR2，和包括SEQ ID NO:106的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107的序列的LCDR1，包括SEQ ID NO:108的序列的LCDR2，和包括SEQ ID NO:109的序列的LCDR3.
实施方案53.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:117的序列的LCDR3.
实施方案54.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102,SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103,SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
实施方案55.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103和与其具有至少80％序列同一性的同源序列。
实施方案56.根据实施方案49所述的蛋白质制剂，其中所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
实施方案57.根据实施方案49所述的蛋白质制剂，其中所述双功能分子包含SEQ ID NO:118或SEQ ID NO:120的氨基酸序列的重链，和/或包含SEQ ID NO:119或SEQ ID NO:121的氨基酸序列的轻链。
实施方案58.根据实施方案49所述的蛋白质制剂，其中所述IL-1结合部分包括：IL-1RI的胞外结构域(ECD)；IL-1RI、IL-1RI的ECD、IL-1RII或IL-1RII的ECD、或IL-1RAP或IL-1RAP的ECD、IL-1sRI或IL-1sRII中的任一者的IL-1结合片段或变体。
实施方案59.根据实施方案48所述的蛋白质制剂，其中所述IL-1R结合部分包括IL-1Ra或其IL-1结合片段或变体、或针对IL-1R的抗体或其抗原结合片段。
实施方案60.根据实施方案59所述的蛋白质制剂，其中所述针对IL-1R的抗体或其抗原结合片段包含来自选自司柏索利单抗(spesolimab)、艾特利单抗(astegolimab)、imsidolimab、AMG 108、melrilimab、尼达尼单抗(nidanilimab)、MEDI8968、REGN6490、HB0034和CSC012的抗体的重链可变区和/或轻链可变区。
实施方案61.根据实施方案60所述的蛋白质制剂，其中所述IL-1R结合部分包括SEQ ID NO:67或76的氨基酸序列、或与SEQ ID NO:67或76具有至少80％序列同一性的氨基酸序列、或其IL-1结合片段或变体。
实施方案62.根据实施方案48到49中任一项所述的蛋白质制剂，其中所述IL-1是IL-1α或IL-1β。
实施方案63.根据实施方案62所述的蛋白质制剂，其中所述IL-1β是人IL-1β。
实施方案64.根据实施方案16到36中任一项所述的蛋白质制剂，其中所述第二部分刺激抗肿瘤免疫并且包括免疫刺激性多肽。
实施方案65.根据实施方案64所述的蛋白质制剂，其中所述免疫刺激性多肽包括白细胞介素(IL)-2(IL-2)、IL-15、IL-21、IL-10、IL-12、IL-23、IL-27、IL-35、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、可溶性CD4、可溶性LAG-3、IFN-α或其功能等效物。
实施方案66.根据实施方案65所述的蛋白质制剂，其中所述可溶性LAG-3包括所述LAG-3的胞外结构域(ECD)或其MHCII结合变体。
实施方案67.根据实施方案16到36中任一项所述的蛋白质制剂，其中所述第二部分刺激抗肿瘤免疫并且包括免疫抑制性受体信号传导的拮抗剂。
实施方案68.根据实施方案67所述的蛋白质制剂，其中所述免疫抑制性受体是信号调节蛋白α(SIRPα)。
实施方案69.根据实施方案68所述的蛋白质制剂，其中所述第二部分阻断CD47与SIRPα之间的相互作用。
实施方案70.根据实施方案69所述的蛋白质制剂，其中所述第二部分包括CD47结合结构域或SIRPα结合结构域。
实施方案71.根据实施方案70所述的蛋白质制剂，其中所述CD47结合结构域包括可溶性SIRPα或其CD47结合片段或变体、或抗CD47抗体或其抗原结合片段。
实施方案72.根据实施方案71所述的蛋白质制剂，其中所述可溶性SIRPα包括所述 SIRPα的胞外结构域(ECD)或其CD47结合变体。
实施方案73.根据实施方案70所述的蛋白质制剂，其中所述CD47结合结构域包括SEQ ID NO:84的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对CD47的结合特异性的氨基酸序列。
实施方案74.根据实施方案70所述的蛋白质制剂，其中所述SIRPα结合结构域包括可溶性CD47或其SIRPα结合片段或变体、或抗SIRPα抗体或其抗原结合片段。
实施方案75.根据实施方案74所述的蛋白质制剂，其中所述可溶性CD47包括所述CD47的胞外结构域(ECD)或其SIRPα结合片段或变体、抗SIRPα抗体或其抗原结合片段。
实施方案76.根据前述实施方案中任一项所述的蛋白质制剂，其进一步包括连接所述第一部分和所述第二部分的接头。
实施方案77.根据实施方案76所述的蛋白质制剂，其中所述接头选自由以下组成的组：可切割接头、不可切割接头、肽接头、柔性接头、刚性接头、螺旋接头和非螺旋接头。
实施方案78.根据实施方案77所述的蛋白质制剂，其中所述接头包括((G)nS)m的氨基酸序列，其中m和n独立地是选自0到30的整数。
实施方案79.根据实施方案16到78中任一项所述的蛋白质制剂，其中所述双功能分子包括所述第二部分中的一个或多个第二部分。
实施方案80.根据实施方案79所述的蛋白质制剂，其中所述多个第二部分中的至少一个第二部分连接到所述第一部分的多肽链的N端或C端。
实施方案81.根据实施方案79所述的蛋白质制剂，其中所述多个第二部分中的至少一个第二部分连接到：a)所述第一部分的重链的N端或C端，或b)所述第一部分的轻链的N端或C端。
实施方案82.根据实施方案79所述的蛋白质制剂，其中所述多个第二部分中的至少一个第二部分连接到所述第一部分的重链恒定区的C端。
实施方案83.根据实施方案79所述的蛋白质制剂，其中所述多个第二部分中的每个第二部分分别连接到所述第一部分的每个重链恒定区的C端。
实施方案84.根据实施方案79所述的蛋白质制剂，其中所述双功能分子包括所述第二部分中的多于一个第二部分，所述多于一个第二部分分别连接到：所述第一部分的重链的N端、所述第一部分的重链的C端、所述第一部分的轻链的N端、所述第一部分的轻链的C端或其任何组合。
实施方案85.根据实施方案79到84中任一项所述的蛋白质制剂，其中所述双功能分子包括同二聚体重链或异二聚体重链。
实施方案86.根据实施方案79到84中任一项所述的蛋白质制剂，其中所述重链就所述第二部分的存在或位置而言是异二聚体的。
实施方案87.根据实施方案86所述的蛋白质制剂，其中所述异二聚体重链包括具有所述第二部分的一条重链，而另一条重链不具有所述第二部分。
实施方案88.根据实施方案86所述的蛋白质制剂，其中所述异二聚体重链进一步包括以阻碍同二聚体化和/或有利于异二聚体化的方式缔合的异二聚体Fc区。
实施方案89.根据实施方案86所述的蛋白质制剂，其中所述异二聚体Fc区能够通过杵臼结构、疏水相互作用、静电相互作用、亲水相互作用或增加的柔性缔合成异二聚体。
实施方案90.根据实施方案86到89中任一项所述的蛋白质制剂，其中所述异二聚体Fc区在一条Fc多肽链中包括Y349C、T366S、L368A或Y407V或其任何组合并且在另一条Fc多肽链中包括S354C或T366W或其组合，其中所述Fc多肽链中的残基的编号为如Kabat中的EU索引的编号。
实施方案91.根据前述实施方案中任一项所述的蛋白质制剂，其进一步连接到一个或多个缀合物部分。
实施方案92.根据实施方案91所述的蛋白质制剂，其中所述缀合物部分包括清除修饰剂、化学治疗剂、毒素、放射性同位素、镧系元素、发光标记、荧光标记、酶-底物标记、DNA烷化剂、拓扑异构酶抑制剂、微管蛋白结合剂或其它抗癌药物，如雄激素受体抑制剂。
实施方案93.根据实施方案16至35、37至47和76-92中任一项的蛋白质制剂，其中所述双功能分子第一部分包括针对PD-L1的抗体或其抗原结合片段，且第二部分包括TGFβ结合部分，
其中所述针对PD-L1的抗体或其抗原结合片段包括选自由以下组成的组的一对重链可变区和轻链可变区序列：SEQ ID NO:49/54、50/54、51/54、52/54、49/55、50/55、51/55、52/55、58/62、58/63、58/64、58/65、59/62、59/63、59/64、59/65、60/62、60/63、60/64和60/65及与其具有至少80％序列同一性的其同源序列；和/或
其中所述TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体，其中所述可溶性TGFβR包括所述TGFβR的胞外结构域(ECD)或其TGFβ结合片段或变体，且TGFβR的所述ECD包括SEQ ID NO:66、79、78、77的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对TGF-β的特定结合特异性和/或亲和力的序列。
实施方案94.根据实施方案93的蛋白质制剂，其中所述双功能分子第一部分包括针对PD-L1的抗体或其抗原结合片段，且第二部分包括TGFβ结合部分，
其中所述针对PD-L1的抗体或其抗原结合片段包括选自由以下组成的组的一对重链可变区和轻链可变区序列：SEQ ID NO:59/64及与其具有至少80％序列同一性的其同源序列；和/或
其中所述TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体，其中所述可溶性TGFβR包括所述TGFβR的胞外结构域(ECD)或其TGFβ结合片段或变体，且TGFβR的所述ECD包括SEQ ID NO:66的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对TGF-β的特定结合特异性和/或亲和力的序列。
实施方案95.根据前述实施方案中任一项的蛋白质制剂，其中所述双功能分子的浓度是约1-200mg/mL，例如约1-150mg/mL、约100-200mg/mL、约1-100mg/mL、约10-100mg/mL、约10-50mg/mL、约20-30mg/mL、约20mg/mL或约30mg/mL。
实施方案96.根据前述实施方案中任一项的蛋白质制剂，其中所述缓冲剂是醋酸盐、组氨酸、柠檬酸盐、琥珀酸盐、苹果酸盐、谷氨酸盐、磷酸盐或乳酸盐缓冲剂，例如醋酸盐缓冲剂或组氨酸缓冲剂；所述醋酸盐缓冲剂例如是醋酸-醋酸钠缓冲剂；所述组氨酸缓冲剂例如是组氨酸-盐酸缓冲剂。
实施方案97.根据前述实施方案中任一项的蛋白质制剂，其中所述缓冲剂的浓度是约5-100mM，例如约10-50mM、约15-30mM、约10-20mM、约25mM或约20mM。
实施方案98.根据前述实施方案中任一项的蛋白质制剂，其中所述缓冲剂是约10-50mM、约15-30mM或约20mM的醋酸盐缓冲剂，例如醋酸-醋酸钠缓冲剂。
实施方案99.根据前述实施方案中任一项的蛋白质制剂，其中所述缓冲剂是约10-50mM、约15-30mM或约20mM的组氨酸缓冲剂，例如是组氨酸-盐酸缓冲剂。
实施方案100.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂的pH在约4.5-6.0的范围，例如约5.0-5.6、约5.0-5.5、约5.3、或5.3±5％。
实施方案101.根据前述实施方案中任一项的蛋白质制剂，其中所述表面活性剂是非离子表面活性剂，例如聚山梨酯、泊洛沙姆、Brij或Triton X，例如聚山梨酯80或聚山梨酯20。
实施方案102.根据前述实施方案中任一项的蛋白质制剂，其中所述表面活性剂浓度是约0.01-0.5％(w/v)，例如约0.01-0.1％(w/v)、0.02-0.1％(w/v)、约0.025％-0.1％(w/v)或约0.05％(w/v)。
实施方案103.根据前述实施方案中任一项的蛋白质制剂，其中所述表面活性剂是约0.01-0.5％(w/v)、约0.01-0.1％(w/v)、0.02-0.1％(w/v)、约0.025％-0.1％(w/v)、或约0.05％(w/v)的聚山梨酯、例如聚山梨酯80或聚山梨酯20。
实施方案104.根据前述实施方案中任一项的蛋白质制剂，其中所述表面活性剂是约0.01-0.5％(w/v)、约0.01-0.1％(w/v)、0.02-0.1％(w/v)、约0.025％-0.1％(w/v)、或约0.05％(w/v)的聚山梨酯80。
实施方案105.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含稳定剂。
实施方案106.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含稳定剂，所述稳定剂选自氨基酸、无机盐、糖、多元醇和螯合剂中的一种或多种，优选盐酸精氨酸、精氨酸或NaCl。
实施方案107.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含稳定剂，所述稳定剂的浓度是约10-300mM，例如约100-300mM、约120-300mM、约100-200mM、约130-170mM、约140-160mM或约150mM。
实施方案108.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含盐酸精氨酸或精氨酸。
实施方案109.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含约100-200mM、约130-170mM、约140-160mM或约150mM的盐酸精氨酸或精氨酸。
实施方案110.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含约100- 200mM、约130-170mM、约140-160mM或约150mM的盐酸精氨酸。
实施方案111.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含约130-170mM、约140-160mM或约150mM的盐酸精氨酸。
实施方案112.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含NaCl。
实施方案113.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含约100-200mM、约130-170mM或约135mM的NaCl。
实施方案114.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂任选地包含螯合剂，例如EDTA·2Na，其浓度例如为约30-350μM、例如75-300μM。
实施方案115.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含约0.01-0.5％(w/v)、约0.01-0.1％(w/v)、0.02-0.1％(w/v)、约0.025％-0.1％(w/v)、或约0.05％(w/v)的聚山梨酯、例如聚山梨酯80或聚山梨酯20，以及约30-350μM、例如75-300μM的EDTA·2Na。
实施方案116.根据前述实施方案中任一项的蛋白质制剂，其中所述制剂包含约0.025％-0.1％(w/v)(例如约0.05％(w/v))的聚山梨酯(例如聚山梨酯80)和约30-350μM的EDTA·2Na。
实施方案117.根据前述实施方案中任一项的蛋白质制剂，其包括：
10-50mg/mL的所述双功能分子；
10-50mM的缓冲剂；
0.025％-0.1％(w/v)的聚山梨酯80或聚山梨酯20；
100-200mM的盐酸精氨酸；以及
任选地30-350μM的EDTA·2Na；
pH是5.0-5.6。
实施方案118.根据前述实施方案中任一项的蛋白质制剂，其包括：
10-50mg/mL的所述双功能分子；
10-50mM的醋酸盐缓冲剂或组氨酸缓冲剂；
0.025％-0.1％(w/v)的聚山梨酯80或聚山梨酯20；
100-200mM的盐酸精氨酸；以及
任选地30-350μM的EDTA·2Na；
pH是5.0-5.6。
实施方案119.根据前述实施方案中任一项的蛋白质制剂，其包括：
10-50mg/mL的所述双功能分子；
10-50mM的醋酸盐缓冲剂或组氨酸缓冲剂；
0.025％-0.1％(w/v)的聚山梨酯80或聚山梨酯20；
130-170mM的盐酸精氨酸；以及
任选地30-350μM的EDTA·2Na；
pH是5.0-5.6。
实施方案120.根据前述实施方案中任一项的蛋白质制剂，其包括：
10-50mg/mL的所述双功能分子；
10-50mM的醋酸盐缓冲剂或组氨酸缓冲剂；
0.025％-0.1％(w/v)的聚山梨酯80或聚山梨酯20；
140-160mM的盐酸精氨酸；以及
任选地30-350μM的EDTA·2Na；
pH是5.3±5％。
实施方案121.根据前述实施方案中任一项的蛋白质制剂，其包括：
10-50mg/mL的所述双功能分子；
10-50mM的醋酸盐缓冲剂或组氨酸缓冲剂；
0.025％-0.1％(w/v)的聚山梨酯80或聚山梨酯20；以及
140-160mM的盐酸精氨酸；
pH是5.3±5％。
实施方案122.根据前述实施方案中任一项的蛋白质制剂，其包括：
20-30mg/mL的所述双功能分子；
20-30mM的醋酸盐缓冲剂或组氨酸缓冲剂；
0.025％-0.1％(w/v)的聚山梨酯80；以及
140-160mM的盐酸精氨酸；
pH是5.3±5％。
实施方案123.根据前述实施方案中任一项的蛋白质制剂，其包括：
30mg/mL的所述双功能分子；
20mM的醋酸盐缓冲剂或组氨酸缓冲剂；
0.05％(w/v)的聚山梨酯80；以及
150mM的盐酸精氨酸；
pH是5.3。
实施方案124.根据前述实施方案中任一项的蛋白质制剂，其中所述双功能分子在配制过程中用螯合剂、例如EDTA·2Na(例如约0.3-10mM、约0.1-20mM、约0.3-15mM、约0.3-10mM或约0.5-5mM的EDTA·2Na)进行处理，然后将螯合剂除去；例如所述处理是在缓冲剂中、在室温下进行的；例如所述螯合剂是通过透析、超滤或渗滤除去的(例如用缓冲液透析)；任选地在除去螯合剂后加入其他辅料，从而配制得到所述制剂。
实施方案125.根据前述实施方案中任一项的蛋白质制剂，其中所述双功能分子在配制过程中用螯合剂、例如EDTA·2Na(例如约0.3-10mM、约0.1-20mM、约0.3-15mM、约0.3-10mM或约0.5-5mM的EDTA·2Na)在缓冲剂中、在室温下进行处理(例如过夜)，然后通过用缓冲液透析、超滤或渗滤将螯合剂除去。
实施方案126.根据前述实施方案中任一项的蛋白质制剂，其中所述双功能分子在配制过程中用螯合剂、例如EDTA·2Na(例如约0.3-10mM、约0.1-20mM、约0.3-15mM、约0.3-10mM或约0.5-5mM的EDTA·2Na)在缓冲剂中、在室温下进行处理(例如过夜)，然后通过用缓冲液透析、超滤或渗滤将螯合剂除去，然后加入其他药学上可接受的辅料，从而配制得到所述制剂。
实施方案127.根据前述实施方案中任一项的蛋白质制剂，其为液体形式、冻干形式或由冻干形式重构的液体形式。
实施方案128.根据前述实施方案中任一项的蛋白质制剂，其为注射剂，例如静脉注射剂、肌内注射剂或皮下注射剂。
实施方案129.根据前述实施方案中任一项的蛋白质制剂，其为液体注射剂。
实施方案130.根据实施方案1-129中任一项的蛋白质制剂，其用于治疗、预防或减轻受试者的PD-L1相关疾病。
实施方案131.根据实施方案130使用的蛋白质制剂，其中所述疾病是免疫相关疾病或病症、癌症或传染病。
实施方案132.根据实施方案131使用的蛋白质制剂，其中所述癌症选自由以下组成的组：肺癌(例如，非小细胞肺癌)、肝癌、胰腺癌、乳腺癌、支气管癌、骨癌、肝和胆管癌、卵巢癌、睾丸癌、肾癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、前列腺癌、胃食管癌、直肠癌、肛门癌、胃肠癌、皮肤癌、垂体癌、胃癌、阴道癌、甲状腺癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、胶质瘤和腺癌。
实施方案133.根据实施方案130-132中任一项使用的蛋白质制剂，其中所述受试者已被鉴定为具有表达PD-L1的癌细胞。
实施方案134.根据实施方案130-133中任一项使用的蛋白质制剂，其中所述PD-L1相关疾病对PD-L1/PD-1单一疗法具有抗性。
实施方案135.根据实施方案130-134中任一项使用的蛋白质制剂，其中所述受试者是人。
实施方案136.根据实施方案1-129中任一项的蛋白质制剂，其用于治疗、预防或减轻将受益于免疫抑制性细胞因子的抑制、持续免疫应答的诱导或抗肿瘤免疫的刺激的疾病或病状；
其中，例如所述免疫抑制性细胞因子是TGFβ；例如所述疾病或病状是TGFβ相关疾病或病状；例如所述TGFβ相关疾病是癌症、纤维化疾病或肾病；
其中，例如所述免疫抑制性细胞因子是IL-1；例如所述疾病或病状是IL-1相关疾病或病状；
其中，例如所述疾病或病状将受益于通过用免疫刺激性多肽刺激MHCII信号传导而诱 导持续免疫应答；例如所述免疫刺激性多肽是可溶性LAG-3；或
其中，例如所述疾病或病状将受益于通过抑制免疫抑制性受体信号传导而刺激抗肿瘤免疫；例如所述免疫抑制性受体是SIRPα。
实施方案137.根据实施方案130-136中任一项使用的蛋白质制剂，其中蛋白质制剂与第二治疗剂联合施用。
实施方案138.根据实施方案137使用的蛋白质制剂，其中所述第二治疗剂选自化学治疗剂、抗癌药物、放射疗法、免疫治疗剂、抗血管生成剂、靶向治疗剂、细胞治疗剂、基因治疗剂、激素治疗剂或细胞因子。
实施方案139.药物组合，其包含根据实施方案1-129中任一项的蛋白质制剂，以及选自化学治疗剂、抗癌药物、放射疗法、免疫治疗剂、抗血管生成剂、靶向治疗剂、细胞治疗剂、基因治疗剂、激素治疗剂或细胞因子的第二治疗剂。
实施方案140.根据实施方案1-129中任一项的蛋白质制剂在制备药物中的用途，所述药物用于治疗、预防或减轻受试者的PD-L1相关疾病。
实施方案141.根据实施方案140的用途，其中所述疾病是免疫相关疾病或病症、癌症或传染病。
实施方案142.根据实施方案141的用途，其中所述癌症选自由以下组成的组：肺癌(例如，非小细胞肺癌)、肝癌、胰腺癌、乳腺癌、支气管癌、骨癌、肝和胆管癌、卵巢癌、睾丸癌、肾癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、前列腺癌、胃食管癌、直肠癌、肛门癌、胃肠癌、皮肤癌、垂体癌、胃癌、阴道癌、甲状腺癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、胶质瘤和腺癌。
实施方案143.根据实施方案140-142中任一项的用途，其中所述受试者已被鉴定为具有表达PD-L1的癌细胞。
实施方案144.根据实施方案140-143中任一项的用途，其中所述PD-L1相关疾病对PD-L1/PD-1单一疗法具有抗性。
实施方案145.根据实施方案140-144中任一项的用途，其中所述受试者是人。
实施方案146.根据实施方案1-129中任一项的蛋白质制剂在制备药物中的用途，所述药物用于治疗、预防或减轻将受益于免疫抑制性细胞因子的抑制、持续免疫应答的诱导或抗肿瘤免疫的刺激的疾病或病状；
其中，例如所述免疫抑制性细胞因子是TGFβ；例如所述疾病或病状是TGFβ相关疾病或病状；例如所述TGFβ相关疾病是癌症、纤维化疾病或肾病；
其中，例如所述免疫抑制性细胞因子是IL-1；例如所述疾病或病状是IL-1相关疾病或病状；
其中，例如所述疾病或病状将受益于通过用免疫刺激性多肽刺激MHCII信号传导而诱导持续免疫应答；例如所述免疫刺激性多肽是可溶性LAG-3；或
其中，例如所述疾病或病状将受益于通过抑制免疫抑制性受体信号传导而刺激抗肿瘤免 疫；例如所述免疫抑制性受体是SIRPα。
实施方案147.根据实施方案140-146中任一项的用途，所述药物与第二治疗剂联合施用。
实施方案148.根据实施方案147的用途，其中所述第二治疗剂选自化学治疗剂、抗癌药物、放射疗法、免疫治疗剂、抗血管生成剂、靶向治疗剂、细胞治疗剂、基因治疗剂、激素治疗剂或细胞因子。
实施方案149.治疗、预防或减轻受试者的PD-L1相关疾病的方法，所述方法包括向所述受试者施用治疗有效量的根据实施方案1-129中任一项的蛋白质制剂。
实施方案150.根据实施方案149的方法，其中所述疾病是免疫相关疾病或病症、癌症或传染病。
实施方案151.根据实施方案150的方法，其中所述癌症选自由以下组成的组：肺癌(例如，非小细胞肺癌)、肝癌、胰腺癌、乳腺癌、支气管癌、骨癌、肝和胆管癌、卵巢癌、睾丸癌、肾癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、前列腺癌、胃食管癌、直肠癌、肛门癌、胃肠癌、皮肤癌、垂体癌、胃癌、阴道癌、甲状腺癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、胶质瘤和腺癌。
实施方案152.根据实施方案149-151中任一项的方法，其中所述受试者已被鉴定为具有表达PD-L1的癌细胞。
实施方案153.根据实施方案149-152中任一项的方法，其中述PD-L1相关疾病对PD-L1/PD-1单一疗法具有抗性
实施方案154.根据实施方案149-153中任一项的方法，其中所述受试者是人。
实施方案155.治疗、预防或减轻受试者的疾病或病状的方法，所述疾病或病状将受益于免疫抑制性细胞因子的抑制、持续免疫应答的诱导或抗肿瘤免疫的刺激，所述方法包括施用有效量的根据实施方案1-129中任一项的蛋白质制剂；
其中，例如所述免疫抑制性细胞因子是TGFβ；例如所述疾病或病状是TGFβ相关疾病或病状；例如所述TGFβ相关疾病是癌症、纤维化疾病或肾病；
其中，例如所述免疫抑制性细胞因子是IL-1；例如所述疾病或病状是IL-1相关疾病或病状；
其中，例如所述疾病或病状将受益于通过用免疫刺激性多肽刺激MHCII信号传导而诱导持续免疫应答；例如所述免疫刺激性多肽是可溶性LAG-3；或
其中，例如所述疾病或病状将受益于通过抑制免疫抑制性受体信号传导而刺激抗肿瘤免疫；例如所述免疫抑制性受体是SIRPα。
实施方案156.根据实施方案149-155中任一项的方法，其进一步包括施用治疗有效量的第二治疗剂。
实施方案157.根据实施方案156的方法，其中所述第二治疗剂选自化学治疗剂、抗癌药物、放射疗法、免疫治疗剂、抗血管生成剂、靶向治疗剂、细胞治疗剂、基因治疗剂、激素治疗剂或细胞因子。
实施方案158.根据实施方案1-129中任一项的蛋白质制剂的制备方法，其包括以下步骤：
a.提供在缓冲剂中的根据实施方案1-129中任一项所述的双功能分子；
b.加入实施方案1-129中任一项所述的表面活性剂，任选的稳定剂，以及任选的其他药学上可接受的辅料；
c.任选地无菌过滤。
实施方案159.根据实施方案158的制备方法，其中将步骤a的双功能分子用螯合剂、例如EDTA·2Na(例如约0.3-10mM、约0.1-20mM、约0.3-15mM、约0.3-10mM或约0.5-5mM的EDTA·2Na)进行处理，然后将螯合剂除去；例如所述处理是在缓冲剂中、在室温下进行的；例如所述螯合剂是通过透析、超滤或渗滤除去的(例如用缓冲液透析、超滤或渗滤)。
实施方案160.根据实施方案158的制备方法，其中将步骤a的双功能分子用螯合剂、例如EDTA·2Na(例如约0.3-10mM、约0.1-20mM、约0.3-15mM、约0.3-10mM或约0.5-5mM的EDTA·2Na)在缓冲剂中、在室温下进行处理(例如过夜)，然后通过用缓冲液透析、超滤或渗滤将螯合剂除去。
关于上文、尤其是上文的实施方案中所述的双功能分子，本公开还提供了以下实施方案：
在一个方面，本公开提供了一种双功能分子，其包括与免疫检查点分子结合的第一部分和阻断白细胞介素-1(IL-1)的活性的第二部分。
在一些实施方案中，所述第一部分包括免疫刺激性检查点分子的激动剂，任选地，所述免疫刺激性检查点分子选自由以下组成的组：CD27、CD70、CD28、CD80(B7-1)、CD86(B7-2)、CD40、CD40L(CD154)、CD122、CD137、CD137L、OX40(CD134)、OX40L(CD252)、GITR、ICOS(CD278)和ICOSLG(CD275)、CD2、ICAM-1、LFA-1(CD11a/CD18)、CD30、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160和CD83。
在一些实施方案中，所述第一部分包括免疫抑制性检查点分子的拮抗剂，任选地，所述免疫抑制性检查点分子选自由以下组成的组：A2AR、B7-H3(CD276)、B7-H4(VTCN1)、BTLA(CD272)、CTLA-4(CD152)、IDO1、IDO2、TDO、KIR、LAG3、NOX2、PD-1、PD-L1、PD-L2、TIM-3、VISTA、SIGLEC7(CD328)、TIGIT、PVR(CD155)、SIGLEC9(CD329)、CD160、LAIR1、2B4(CD244)、CD47和B7-H5。
在一些实施方案中，所述免疫检查点分子是PD-L1。
在一些实施方案中，所述第一部分包括针对PD-L1的抗体或其抗原结合片段，并且所述第二部分包括IL-1结合部分或IL-1受体(IL-1R)结合部分。
在一些实施方案中，所述IL-1结合部分包括IL-1R或其IL-1结合片段或变体、或针对IL-1的抗体或其抗原结合片段。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104或SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:105或SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:106或SEQ  ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107或SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:108或SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:109或SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104的序列的HCDR1，包括SEQ ID NO:105的序列的HCDR2，和包括SEQ ID NO:106的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107的序列的LCDR1，包括SEQ ID NO:108的序列的LCDR2，和包括SEQ ID NO:109的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102,SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103,SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述IL-1R结合部分包括白细胞介素-1受体拮抗剂或其片段或变体、或针对IL-1R的抗体或其抗原结合片段。
在另一方面，一种双功能分子包括与PD-L1结合的第一部分和a)阻断免疫抑制性细胞因子的活性或b)刺激免疫的第二部分，其中所述第一部分包括针对PD-L1的抗体或其抗原结合片段，所述抗体或其抗原结合片段包括重链可变(VH)区和/或轻链可变(VL)区，其中所述重链可变区包括：
a)HCDR1，所述HCDR1包括DYYMN(SEQ ID NO:1)或与DYYMN具有至少80％序列同一性的同源序列，
b)HCDR2，所述HCDR2包括DINPNNX1X2TX3YNHKFKG(SEQ ID NO:19)或与DINPNNX1X2TX3YNHKFKG具有至少80％序列同一性的同源序列，以及
c)HCDR3，所述HCDR3包括WGDGPFAY(SEQ ID NO:3)或与WGDGPFAY具有至少80％序列同一性的同源序列，和/或
其中所述轻链可变区包括：
d)LCDR1包括选自由KASQNVX4X5X6VA(SEQ ID NO:20)或与KASQNVX4X5X6VA具有至少80％序列同一性的同源序列组成的组的序列，
e)LCDR2包括选自由SX7SX8RYT(SEQ ID NO:21)或与SX7SX8RYT具有至少80％序列同一性的同源序列组成的组的序列，以及
f)LCDR3包括选自由QQYSNYPT(SEQ ID NO:6)或与QQYSNYPT具有至少80％序列同一性的同源序列组成的组的序列；
其中X1是G或A，X2是G或D或Q或E或L，X3是S或M或Q或L或V，X4是G或P或K，X5是A或G，X6是A或I，X7是A或N或R或V，并且X8是N或H或V或D。
在一些实施方案中，所述重链可变区包括：
a)HCDR1包括SEQ ID NO:1的序列，
b)HCDR2包括选自由以下组成的组的序列：SEQ ID NO:2、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17和SEQ ID NO:18，以及
c)HCDR3包括SEQ ID NO:3的序列，
和/或
轻链可变区，所述轻链可变区包括：
d)LCDR1包括选自由以下组成的组的序列：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9，
e)LCDR2包括选自由以下组成的组的序列：SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12，以及
f)LCDR3包括SEQ ID NO:6的序列。
在一些实施方案中，所述重链可变区选自由以下组成的组：
a)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:2的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
b)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:13的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
c)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:14的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
d)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:15的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
e)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:17的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；以及
f)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:18的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3。
在一些实施方案中，所述轻链可变区选自由以下组成的组：
a)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
b)包括以下的轻链可变区：包括SEQ ID NO:9的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
c)包括以下的轻链可变区：包括SEQ ID NO:8的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
d)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:12的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；以及
e)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:11的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3。
在一些实施方案中，所述针对PD-L1的抗体或其抗原结合片段进一步包括重链HFR1、HFR2、HFR3和HFR4中的一个或多个，和/或轻链LFR1、LFR2、LFR3和LFR4中的一个或多个，其中：
a)所述HFR1包括氨基酸序列QVQLVQSGAEVKKPGASVKVSCKASGYX9FT(SEQ ID NO:40)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
b)所述HFR2包括氨基酸序列WVRQAPGQX10LEWMG(SEQ ID NO:41)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
c)所述HFR3序列包括氨基酸序列RVTX16TVDX11SISTAYMELSRLRSDDTAVYYCX12X13(SEQ ID NO:42)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
d)所述HFR4包括氨基酸序列WGQGTLVTVSS(SEQ ID NO:25)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
e)所述LFR1包括氨基酸序列DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:26)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
f)所述LFR2包括氨基酸序列WYQQKPGKX14PKLLIY(SEQ ID NO:43)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
g)所述LFR3包括氨基酸序列GVPX15RFSGSGSGTDFTX17TISSLQPEDIATYYC(SEQ ID NO:44)或与所述氨基酸序列具有至少80％序列同一性的同源序列，并且
h)所述LFR4包括氨基酸序列FGQGTKLEIK(SEQ ID NO:29)或与所述氨基酸序列具有至少80％序列同一性的同源序列，
其中X9是T或V，X10是G或S，X11是T或K，X12是A或V，X13是R或K，X14是A或S，X15是S或D，X16是M或V，并且X17是F或L。
在一些实施方案中，
所述HFR1包括选自由SEQ ID NO:22和30组成的组的序列，
所述HFR2包括选自由SEQ ID NO:23和31组成的组的序列，
所述HFR3包括选自由SEQ ID NO:24和32-35组成的组的序列，
所述HFR4包括SEQ ID NO:25的序列，
所述LFR1包括来自由SEQ ID NO:26组成的组的序列，
所述LFR2包括选自由SEQ ID NO:27和36组成的组的序列，
所述LFR3包括选自由SEQ ID NO:28和37-38、39、45组成的组的序列，并且
所述LFR4包括SEQ ID NO:29的序列。
在一些实施方案中，所述重链可变区包括选自由以下组成的组的序列：SEQ ID NO:46、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60和与其具有至少80％序列同一性的其同源序列。
在一些实施方案中，所述轻链可变区包括选自由以下组成的组的序列：SEQ ID NO:47、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65和与其具有至少80％序列同一性的其同源序列。
在一些实施方案中，所述针对PD-L1的抗体或其抗原结合片段包括选自由以下组成的组的一对重链可变区和轻链可变区序列：SEQ ID NO:49/54、50/54、51/54、52/54、49/55、50/55、51/55、52/55、58/62、58/63、58/64、58/65、59/62、59/63、59/64、59/65、60/62、60/63、60/64和60/65。
在一些实施方案中，所述针对PD-L1的抗体或其抗原结合片段进一步包括一个或多个氨基酸残基取代或修饰，但仍保留对PD-L1的特定结合特异性和/或亲和力。
在一些实施方案中，所述取代或修饰中的至少一个取代或修饰在所述CDR序列中的一个或多个CDR序列中和/或在所述VH或VL序列的非CDR区中的一个或多个非CDR区中。
在一些实施方案中，所述针对PD-L1的抗体或其抗原结合片段进一步包括免疫球蛋白恒定区，任选地人Ig的恒定区，或任选地人IgG的恒定区。
在一些实施方案中，所述恒定区包括人IgG1、IgG2、IgG3或IgG4的Fc区。
在一些实施方案中，所述人IgG1的Fc区包括SEQ ID NO:80或与SEQ ID NO:80具有至少80％(例如，至少85％、90％、95％、96％、97％、98％或99％)序列同一性的其变体。
在一些实施方案中，所述恒定区包括Fc变体，所述Fc变体相对于对应野生型Fc区具有降低的效应子功能。在一些实施方案中，所述Fc区包括一个或多个氨基酸残基修饰或取代，从而使得相对于SEQ ID NO:80降低效应子功能。
在一些实施方案中，所述Fc区包括选自由以下组成的组的一个或多个氨基酸残基取代：220S、226S、228P、229S、233P、234V、234G、234A、234F、234A、235A、235G、235E、236E、236R、237A、237K、238S、267R、268A、268Q、269R、297A、297Q、297G、309L、318A、322A、325L、328R、330S、331S和其任何组合，其中所述Fc区中的残基的编号为如Kabat中的EU索引的编号。
在一些实施方案中，所述Fc区包括选自由以下组成的组的突变的组合：a)K322A、L234A和L235A；b)P331S、L234F和L235E；c)L234A和L235A；c)N297A；d)N297Q；e)N297G；f)L235E；g)L234A和L235A(IgG1)；h)F234A和L235A(IgG4)；i)H268Q、V309L、A330S和P331S(IgG2)；j)V234A、G237A、P238S、H268A、V309L、A330S和P331S(IgG2)，其中所述Fc区中的残基的编号为如Kabat中的EU索引的编号。
在一些实施方案中，所述Fc变体包括SEQ ID NO:81的氨基酸序列。
在一些实施方案中，所述针对PD-L1的抗体或其抗原结合片段是人源化的。
在一些实施方案中，所述抗原结合片段是双抗体、Fab、Fab'、F(ab')2、Fd、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双抗体(ds双抗体)、单链抗体分子(scFv)、scFv二聚体(二价双抗体)、多特异性抗体、骆驼化(camelized)单结构域抗体、纳米抗体、结构域抗体和二价结构域抗体。
在一些实施方案中，所述抗体或其抗原结合片段能够与人PD-L1和食蟹猴PD-L1两者结合。
在一些实施方案中，所述第一部分包括与本文提供的抗体或其抗原结合片段竞争结合PD-L1的抗体或其抗原结合片段。
在一些实施方案中，所述免疫抑制性细胞因子包括转化生长因子β(TGF-β)超家族中的细胞因子、IL-1或血管内皮生长因子(VEGF)。
在一些实施方案中，所述TGF-β超家族中的免疫抑制性细胞因子包括TGF-β、骨形态发生蛋白(BMP)、激活素、NODAL和生长和分化因子(GDF)。
在一些实施方案中，所述免疫抑制性细胞因子是TGF-β。
在一些实施方案中，所述第二部分包括TGFβ结合部分。
在一些实施方案中，所述TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体、或针对TGFβ的抗体和其抗原结合片段。
在一些实施方案中，所述可溶性TGFβR包括所述TGFβR的胞外结构域(ECD)或其TGFβ结合片段或变体。
在一些实施方案中，所述TGFβR选自由以下组成的组：TGFβ受体I(TGFβRI)、TGFβ受体II(TGFβRII)、TGFβ受体III(TGFβRIII)和其任何组合。
在一些实施方案中，所述TGFβR是TGFβRII。
在一些实施方案中，所述TGFβRII相对于TGFβ2和TGFβ3而选择性地结合到TGFβ1。
在一些实施方案中，所述TGFβ1是人TGFβ1或小鼠TGFβ1。
在一些实施方案中，TGFβR的所述ECD包括SEQ ID NO:66、79、78、77的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对TGF-β的特定结合特异性和/或亲和力的序列。
在一些实施方案中，所述第二部分包括IL-1结合部分或IL-1受体(IL-1R)结合部分。
在一些实施方案中，所述IL-1结合部分包括可溶性IL-1R、IL-1R的IL-1结合片段或变体、或针对IL-1的抗体或其抗原结合片段。
在一些实施方案中，所述IL-1结合部分包括：IL-1RI的胞外结构域(ECD)；IL-1RI、IL-1RI的ECD、IL-1RII或IL-1RII的ECD、或IL-1RAP或IL-1RAP的ECD、IL-1sRI或IL-1sRII中的任一者的IL-1结合片段或变体。
在一些实施方案中，所述IL-1R结合部分包括IL-1Ra或其IL-1结合片段或变体、或针对IL-1R的抗体或其抗原结合片段。
在一些实施方案中，所述IL-1R结合部分包括SEQ ID NO:67或76的氨基酸序列、或与SEQ ID NO:67或76具有至少80％序列同一性的氨基酸序列、或其IL-1结合片段或变体。
在一些实施方案中，所述IL-1是IL-1α或IL-1β。
在一些实施方案中，所述IL-1β是人IL-1β。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104或SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:105或SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:106或SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107或SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:108或SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:109或SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104的序列的HCDR1，包括SEQ ID NO:105的序列的HCDR2，和包括SEQ ID NO:106的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107的序列的LCDR1，包括SEQ ID NO:108的序列的LCDR2，和包括SEQ ID NO:109的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102,SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103,SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:110,和与其具有至 少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述双功能分子包含SEQ ID NO:118或SEQ ID NO:120的氨基酸序列的重链，和/或包含SEQ ID NO:119或SEQ ID NO:121的氨基酸序列的轻链。
在一些实施方案中，所述第二部分刺激抗肿瘤免疫并且包括免疫刺激性多肽。
在一些实施方案中，所述免疫刺激性多肽包括白细胞介素(IL)-2(IL-2)、IL-15、IL-21、IL-10、IL-12、IL-23、IL-27、IL-35、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、可溶性CD4、可溶性LAG-3或IFN-α或其功能等效物。
在一些实施方案中，所述可溶性LAG-3包括所述LAG-3的胞外结构域(ECD)或其MHCII结合片段或变体。
在一些实施方案中，所述第二部分刺激抗肿瘤免疫并且包括免疫抑制性受体信号传导的拮抗剂。
在一些实施方案中，所述免疫抑制性受体是信号调节蛋白α(SIRPα)。
在一些实施方案中，所述第二部分阻断CD47与SIRPα之间的相互作用。
在一些实施方案中，所述第二部分包括CD47结合结构域或SIRPα结合结构域。
在一些实施方案中，所述CD47结合结构域包括可溶性SIRPα或其CD47结合片段或变体、或抗CD47抗体或其抗原结合片段。
在一些实施方案中，所述可溶性SIRPα包括所述SIRPα的胞外结构域(ECD)或其CD47结合片段或变体。
在一些实施方案中，所述可溶性SIRPα包括SEQ ID NO:84的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对CD47的结合特异性的氨基酸序列。
在一些实施方案中，所述SIRPα结合结构域包括可溶性CD47或其SIRPα结合片段或变体、或抗SIRPα抗体或其抗原结合片段。
在一些实施方案中，所述可溶性CD47包括所述CD47的胞外结构域(ECD)或其SIRPα结合片段或变体、抗SIRPα抗体或其抗原结合片段。
在一些实施方案中，所述双功能分子进一步包括连接所述第一部分和所述第二部分的接头。
在一些实施方案中，所述接头选自由以下组成的组：可切割接头、不可切割接头、肽接头、柔性接头、刚性接头、螺旋接头和非螺旋接头。
在一些实施方案中，所述接头包括((G)nS)m的氨基酸序列，其中m和n独立地是选自0到30的整数(例如，1、2、3、4、5、6、7、8、9、10)。在一些实施方案中，n是2、3、4或5，并且m是1、2、3、4、5、6、7、8、9或10。在一些实施方案中，所述接头包括SEQ ID NO:68的氨基酸序列。
在一些实施方案中，所述双功能分子包括所述第二部分中的一个或多个第二部分。
在一些实施方案中，所述多个第二部分中的至少一个第二部分连接到所述第一部分的多肽链的N端或C端。
在一些实施方案中，所述多个第二部分中的至少一个第二部分连接到：a)所述第一部分的重链的N端或C端，或b)所述第一部分的轻链的N端或C端。
在一些实施方案中，所述多个第二部分中的至少一个第二部分连接到所述第一部分的重链恒定区的C端。
在一些实施方案中，所述多个第二部分中的每个第二部分分别连接到所述第一部分的每个重链恒定区的C端。
在一些实施方案中，所述双功能分子包括所述第二部分中的多于一个第二部分，所述多于一个第二部分分别连接到：所述第一部分的重链的N端、所述第一部分的重链的C端、所述第一部分的轻链的N端、所述第一部分的轻链的C端或其任何组合。
在一些实施方案中，所述双功能分子包括同二聚体重链或异二聚体重链。
在一些实施方案中，所述重链就所述第二部分的存在或位置而言是异二聚体的。
在一些实施方案中，所述异二聚体重链包括具有所述第二部分的一条重链，而另一条重链不具有所述第二部分。
在一些实施方案中，所述异二聚体重链进一步包括以阻碍同二聚体化和/或有利于异二聚体化的方式缔合的异二聚体Fc区。
在一些实施方案中，第一和所述异二聚体Fc区能够通过杵臼结构(knobs-into-holes)、疏水相互作用、静电相互作用、亲水相互作用或增加的柔性缔合成异二聚体。
在一些实施方案中，所述异二聚体Fc区在一个Fc区中包括Y349C、T366S、L368A或Y407V或其任何组合并且在另一个Fc区中包括S354C或T366W或其组合，其中所述Fc区中的残基的编号为如Kabat中的EU索引的编号。
在一些实施方案中，所述双功能分子进一步连接到一个或多个缀合物部分。
在一些实施方案中，所述缀合物部分包括清除修饰剂、化学治疗剂、毒素、放射性同位素、镧系元素、发光标记、荧光标记、酶-底物标记、DNA烷化剂、拓扑异构酶抑制剂、微管蛋白结合剂或其它抗癌药物，如雄激素受体抑制剂。
在另一方面，本公开进一步提供了一种包含本文提供的双功能分子和药学上可接受的载体的药物组合物或试剂盒。
在另一方面，本公开进一步提供了一种编码本文提供的双功能分子的分离的多核苷酸。
在另一方面，本公开进一步提供了一种包括本文提供的分离的多核苷酸的载体。
在另一方面，本公开进一步提供了一种包括本文提供的载体的宿主细胞。
在另一方面，本公开进一步提供了一种表达本文提供的双功能分子的方法，所述方法包括在表达载体的条件下培养本文提供的宿主细胞。
在另一方面，本公开进一步提供了一种治疗、预防或减轻受试者的PD-L1相关疾病的方法，所述方法包括向所述受试者施用治疗有效量的本文提供的双功能分子和/或本文提供的药物组合物或试剂盒。
在一些实施方案中，所述疾病是免疫相关疾病或病症、癌症、自身免疫疾病或传染病。
在一些实施方案中，所述癌症选自由以下组成的组：肺癌(例如，非小细胞肺癌)、肝癌、胰腺癌、乳腺癌、支气管癌、骨癌、肝和胆管癌、卵巢癌、睾丸癌、肾癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、前列腺癌、胃食管癌、直肠癌、肛门癌、胃肠癌、皮肤癌、垂体癌、胃癌、阴道癌、甲状腺癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、胶质瘤和腺癌。
在一些实施方案中，所述受试者已被鉴定为具有表达PD-L1的癌细胞。
在一些实施方案中，受试者是人。
在一些实施方案中，所述方法进一步包括施用治疗有效量的第二治疗剂。
在一些实施方案中，所述第二治疗剂选自化学治疗剂、抗癌药物、放射疗法、免疫治疗剂、抗血管生成剂、靶向治疗剂、细胞治疗剂、基因治疗剂、激素治疗剂或细胞因子。
在另一方面，本公开提供了一种本文提供的双功能分子在制造用于治疗受试者的PD-L1相关疾病或病状的药物中的用途。
在另一方面，本公开提供了一种治疗、预防或减轻受试者的疾病或病状的方法，所述疾病或病状将受益于免疫抑制性细胞因子的抑制、持续免疫应答的诱导或抗肿瘤免疫的刺激，所述方法包括施用有效量的本文提供的双功能分子。
在一些实施方案中，所述免疫抑制性细胞因子是TGFβ。
在一些实施方案中，所述疾病或病状是TGFβ相关疾病或病状。
在一些实施方案中，所述TGFβ相关疾病是癌症、纤维化疾病或肾病。
在一些实施方案中，所述免疫抑制性细胞因子是IL-1。
在一些实施方案中，所述疾病或病状是IL-1相关疾病或病状。
在一些实施方案中，所述疾病或病状将受益于通过抑制免疫抑制性受体信号传导(例如，SIRPa信号传导)而刺激抗肿瘤免疫。
附图说明
图1示出了通过ELISA测量的人源化4B6抗体与人PD-L1的结合。
图2示出了通过ELISA测量的Hu4B6_HgLa与人PD-L1的结合。
图3A-图3C示出了通过ELISA测量的AM-4B6-IgG1-TGFβRII变体与PD-L1的结合。
图4示出了使用流式细胞术对AM-4B6-IgG1-TGFβRII变体的亲和力分级。
图5A和5B示出了AM-4B6-IgG1-TGFβRII变体对PD-L1/PD-1或PD-L1/B7-1的阻断。
图6示出了使用基于细胞的测定，AM-4B6-IgG1-TGFβRII变体对PD-L1/PD-1的阻断。
图7示出了用稳定细胞系表达的AM4B6_hIgG1_TBRII(20-136)的SDS-PAGE。
图8A和图8B示出了通过ELISA分析测量的与人PD-L1或食蟹猴PD-L1的结合。
图9A-图9C示出了通过ELISA分析测量的与人PD-L1和B7家族其它成员和TGFβ超家族其它成员的结合。
图10A-图10F示出了通过FACS分析测量的与PD-L1表达性细胞的结合。
图11示出了通过FACS分析测量的与激活的人T细胞上的人PD-L1的结合。
图12A-图12B示出了通过ELISA分析测量的对人PD-L1与人PD-1结合或食蟹猴PD-L1与食蟹猴PD-1结合的阻断。
图13示出了通过ELISA分析测量的与hPD-L1和TGFb1的同时结合。
图14示出了使用报告基因测定的阻断hPD-L1/hPD-1。
图15示出了使用TGF-β报告基因HEK-293细胞系阻断TGFβ1信号传导。
图16示出了AM4B6-hIgG1-TGFβRII'对由结核菌素(TB)刺激的PBMC的IFNγ释放的影响。
图17A-图17B示出了在MC38-hPD-L1肿瘤模型中的抗肿瘤活性。
图18A-图18B示出了在H460肿瘤模型中的抗肿瘤活性。
图19A-图19B示出了在EMT6-hPD-L1肿瘤模型中的抗肿瘤活性。
图20A-图20C示出了AM4B6-hIgG1-TGFβRII的体内药代动力学和药效学研究。
图21示出了通过ELISA测量的AM4B6-hIgG1-IL-1RA与人PD-L1的结合活性。
图22示出了通过FACS分析测量的AM4B6-hIgG1-IL-1RA与人PD-L1的结合活性。
图23示出了使用基于细胞的测定，AM4B6-hIgG1-IL-1RA对PD-L1/PD-1的阻断。
图24示出了通过ELISA测量的AM4B6-IgG1-IL-1RA对人IL-1β的阻断活性。
图25示出了AM4B6-hIgG1-IL-1RA对报告细胞上的人IL-1β的阻断活性。
图26示出了不对称双功能抗体的SEC-HPLC纯度。
图27示出了通过ELISA测量的双功能分子与人PD-L1的结合。
图28示出了通过ELISA测量的双功能分子与人CD47的结合。
图29示出了IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6双特异性抗体对hIL-1β蛋白的ELISA结合活性。
图30示出了IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6双特异性抗体对hPD-L1蛋白的ELISA结合活性。
图31示出了通过FACS测量IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6双特异性抗体对表达PD-L1的293T细胞的结合。
图32示出了IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6的基于细胞的PD1/PD-L1阻断活性。
图33示出了IgG-scFv-XOMA052-AM4B6对HDF细胞上任IL-1β的阻断活性。
图34示出了IgG-scFv-ACZ885-AM4B6对报告子细胞上hIL-1β的阻断活性。
图35(40℃结果的分析图)示出了处方F1-F12的样品在40℃放置4周，经SEC和NR CE-SDS检测得到的△HMW％和△LMW％的JMP软件分析结果，显示了pH、PS80和其他辅料对蛋白质稳定性的影响。
图36(搅拌结果的分析图)示出了处方F1-F12的样品在25℃搅拌3小时，经SEC和NR CE-SDS检测得到的△HMW％和△LMW％的JMP软件分析结果，显示了pH、PS80和其他辅料对蛋白质稳定性的影响。
图37和图38(图37：40℃条件下聚合物变化；图38：40℃条件下小分子碎片变化)示出了处方F11和F13的样品，在40℃放置4周，经SEC和NR CE-SDS检测得到的HMW％和LMW％变化，显示了缓冲剂对蛋白质稳定性的影响。
图39和图40(图39：40℃条件下聚合物变化；图40：40℃条件下小分子碎片变化)示出了处方F14和F15的样品，在40℃放置6周，经SEC和NR CE-SDS检测得到的HMW％和LMW％变化，显示了缓冲剂对蛋白质稳定性的影响。
图41-图43(图41：光照条件下聚合物变化；图42：40℃条件下聚合物变化；图43：25℃条件下聚合物变化)分别示出了处方F16和F17的样品，在光照7天、在40℃放置6周或在25℃放置6周，经SEC检测得到的HMW％变化，显示了辅料对蛋白质稳定性的影响。
图44-图45(图44：40℃条件下PS80浓度变化；图45：25℃条件下PS80浓度变化)分别示出了处方F18-F21的样品，在40℃放置一个月或在25℃放置6个月条件下的PS80的降解结果，显示了依地酸二钠对PS80的保护作用。
图46-图49(图46：25℃条件下聚合物变化；图47：25℃条件下小分子碎片变化；图48：5℃条件下聚合物变化；图49：5℃条件下小分子碎片变化)分别示出了处方F18-F21的样品在25℃放置4周或5℃放置6个月，经SEC和NR CE-SDS检测得到的HMW％和LMW％变化，显示了辅料对蛋白质稳定性的影响。
图50(40℃条件下PS80浓度变化)示出了在配制过程中加入不同浓度的依地酸二钠或不加入依地酸二钠，所得到的制剂在40℃放置4周条件下的PS80降解对比结果。
定义
本公开中所使用的术语具有如下所列定义。如果在文中没有给出定义，则所使用的术语具有本领域所通常理解的含义。
在整个本公开中，本文使用的冠词“一个”、“一种”和“所述”是指冠词的语法宾语中的一个或多于一个(即，至少一个)。举例来说，“一种抗体”意指一种抗体或多于一种抗体。应当理解的是，本文所用的术语仅是为了描述具体的实施方案，并且不是限制性的。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或更多项。
如本文所用，术语“包含”、“包括”或“含有”意指包括所述的要素、数值或步骤，但是不排除任意其他要素、数值或步骤。在本文中，当使用术语“包含”、“包括”或“含有”时，除非另有指明，否则也涵盖由所述及的要素、数值或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
如本文所用，术语“制剂”和“药物制剂”可互换使用，是指适合于向动物优选哺乳动物(包括人)施用的包含至少一种活性成分和至少一种非活性成分的组合物或配制物。本公开的制剂例如为液体形式、冻干形式或由冻干形式重构的液体形式。“液体制剂”是指液体形式的制剂。本公开的制剂优选为液体制剂，例如注射剂、静脉注射剂、肌内注射剂或皮下注射剂。
如本文所用，“缓冲剂”或“缓冲液”是指稳定药物制剂pH的药学上可接受的辅料。例如，醋酸盐、组氨酸、谷氨酸盐、柠檬酸盐、琥珀酸盐、苹果酸盐、磷酸盐或乳酸盐，和/或其各自游离酸或碱，以及各种盐和/或其酸和碱的混合物。优选的药学上可接受的缓冲剂包括但不限于醋酸盐缓冲剂和组氨酸缓冲剂。“醋酸盐缓冲剂”或“醋酸盐缓冲液”包括醋酸-醋酸钠、组氨酸-醋酸盐、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。“组氨酸缓冲剂”或“组氨酸缓冲液”包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等。上述缓冲剂通常以约1-100mM，例如约5-100mM，约10-50mM、约15-30mM、或约20mM的浓度使用。所述缓冲剂能够使本公开的液体制剂的pH保持在约4.5-7.0的范围，例如约4.5-6.0、约5.0-5.6或约5.0-5.5，例如约5.3或5.3±5％。
如本文所用，“表面活性剂”是指具有表面活性的药学上可接受的辅料。优选地，使用非离子表面活性剂。药学上可接受的表面活性剂包括但不限于聚氧乙烯脱水山梨醇脂肪酸酯(Tween)，聚氧乙烯烷基醚(Brij)，烷基苯基聚氧乙烯醚(Triton X)，聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆(poloxamer)，Pluronic)，以及十二烷基硫酸钠(SDS)，例如聚山梨酯20(PS20)、聚山梨酯80(PS80)、泊洛沙姆188、Brij系列、Triton X。当使用聚山梨酯，例如PS80和PS20时，其浓度通常为约0.001-1％，例如约0.01-0.1％、约0.02％-0.1％、约0.025％-0.1％、或约0.05％。在本公开的制剂中，表面活性剂的浓度作为以重量/体积(w/v，g/100mL)表述的百分比描述。
如本文所用，“稳定剂”表示非上文所述表面活性剂的稳定剂，其是在制造、贮存和应用期间保护活性药学组分和/或制剂处于稳定或不变状态的药学上可接受的辅料，例如帮助防止聚集、氧化、颜色变化等。稳定剂包括但不限于糖(包括单糖，例如葡萄糖、果糖、半乳糖、甘露糖、山梨糖、核糖、脱氧核糖、神经氨酸；以及和寡糖，例如蔗糖、海藻糖、乳糖、麦芽糖和棉子糖)，氨基酸(包括但不限于精氨酸、甘氨酸、鸟氨酸、赖氨酸、组氨酸、谷氨酸、天冬氨酸、异亮氨酸、亮氨酸、丙氨酸、苯丙氨酸、酪氨酸、色氨酸、甲硫氨酸、丝氨酸、脯氨酸。在每种情况中，所采用的氨基酸优选处于L-形式。例如以它们的无机盐的形式，例如以盐酸盐的形式，例如盐酸精氨酸、盐酸组氨酸或盐酸赖氨酸。浓度例如为约20至约250mM，约100至约200mM，例如约150mM)，盐(例如，无机盐，例如氯化钠、氯化镁、氯化钙)，螯合剂(例如EDTA(依地酸)，EDTA盐，例如EDTA·2Na)，多元醇(例如山梨醇、甘露醇、木糖醇、右旋糖苷、甘油、阿拉伯糖醇、丙二醇、聚乙二醇)，环糊精(例如羟基丙基-β-环糊精、磺基丁基乙基-β-环糊精、β-环糊精)，聚乙二醇(例如PEG 3000、PEG 3350、PEG 4000、PEG 6000)，清蛋白(例如人血清清蛋白(HSA)，牛血清清蛋白(BSA))。稳定剂例如是盐酸精氨酸、NaCl、蔗糖或山梨醇，优选是盐酸精氨酸或NaCl，例如约150mM 酸精氨酸或约135mM NaCl。稳定剂可以以约1-500mM，例如约10-300mM、约100-300mM、约120-300mM、约100-200mM、约135-280mM、约135-200mM、约130-170mM、约140-160mM、135、150、260或280mM的浓度存在于制剂中。氨基酸或无机盐稳定剂、例如盐酸精氨酸或NaCl的浓度是约120-300mM、约100-200mM、约135-200mM、约130-170mM、约140-160mM、约135mM或约150mM。例如盐酸精氨酸的浓度是约120-300mM、约100-200mM、约135-200mM、约130-170mM、约140-160mM、或约150mM。螯合剂、例如EDTA·2Na的浓度是约30-350μM、例如约75-350μM，例如约75、约150或约300μM。本公开的制剂中可以存在多于一种选自相同或不同组的稳定剂。本公开的制剂中的稳定剂还可以同时起到渗透压调节剂的作用。
如本文所用，“渗透压调节剂”是指用于调节溶液渗透压的药学上可接受的辅料。渗透压调节剂的实例包括但不限于糖类(包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖，例如葡萄糖，蔗糖，海藻糖)，氨基酸(包括精氨酸、甘氨酸、半胱氨酸、组氨酸)，盐(例如，无机盐，例如氯化钠、氯化镁、氯化钙)，以及多元醇(例如山梨醇、甘露醇、木糖醇、右旋糖苷、甘油、阿拉伯糖醇、丙二醇、聚乙二醇)。
稳定剂的一个亚组是冻干保护剂，其包括但不限于糖、多元醇(诸如例如糖醇)和氨基酸。优选的冻干保护剂可以选自糖，诸如蔗糖，海藻糖，乳糖，葡萄糖，甘露糖，麦芽糖，半乳糖，果糖，山梨糖，棉子糖，神经氨酸，氨基糖，诸如葡糖胺，半乳糖胺，葡甲胺，多元醇，诸如甘露醇和山梨醇，和氨基酸，诸如精氨酸和甘氨酸或其混合物。冻干保护剂一般以约10至500mM的量，优选以约10至约300mM的量和更优选以约100至约300mM的量使用。
稳定剂的一个亚组是抗氧化剂，其包括但不限于抗坏血酸、谷胱甘肽、半胱氨酸、甲硫氨酸、柠檬酸、EDTA。抗氧化剂可以以约0.01至约100mM的量，优选以约5至约50mM的量和更优选以约5至约25mM的量使用。
如文中所用，术语“约”在与数值结合使用时意为涵盖具有比指定数值小10％的下限和比指定数字数值大10％的上限的范围内的数值，即±10％的范围、例如±5％、例如±3％。
如文中所用，“w/v”是指“重量/体积”，单位为g/100mL。
本文所有的数值范围应当被理解为公开了在该范围内的每个数值和数值子集、而不论其是否被具体另外公开。例如，提及任何一个数值范围时，应当视为提及了该数值范围内的每一个数值，例如该数值范围内的每一个整数。本公开涉及落入这些范围的所有值，所有更小的范围以及数值的范围的上限或下限。
本文中使用的术语“抗体”包括与特定抗原结合的任何免疫球蛋白、单克隆抗体、多克隆抗体、多价抗体、二价抗体、单价抗体、多特异性抗体或双特异性抗体。天然的完整抗体包括两条重(H)链和两条轻(L)链。哺乳动物的重链分为α、δ、ε、γ和μ，每条重链由可变区(VH)以及第一、第二、第三和任选的第四恒定区(分别为CH1、CH2、CH3、CH4)组成；哺乳动物的轻链分为λ或κ，而每条轻链由可变区(VL)以及恒定区组成。抗体呈“Y”型，其中Y型结构的茎部由通过二硫键结合在一起的两条重链的第二和第三恒定区组成。Y的每个臂包括单条重链的与单个轻链的可变区和恒定区结合的可变区和第一恒定区。轻链和重链的可变区负责抗原结合。每条链的可变区通常含有三个高变区，称为互补决定区(CDR)(轻链CDR包括LCDR1、LCDR2、LCDR3，重链CDR包括HCDR1、HCDR2、HCDR3)。本文公开的抗体和抗原结合片段的CDR边界可以通过Kabat、IMGT、 Chothia或Al-Lazikani规则来定义或鉴定(Al-Lazikani,B.,Chothia,C.,Lesk,A.M.,《分子生物学杂志(J.Mol.Biol.)》,273(4),927(1997)；Chothia,C.等人,《分子生物学杂志(J Mol Biol.)》12月5日；186(3):651-63(1985)；Chothia,C.和Lesk,A.M.,《分子生物学杂志》,196,901(1987)；Chothia,C.等人,《自然》.12月21-28日；342(6252):877-83(1989)；Kabat E.A.等人,《具有免疫学意义的蛋白质序列(Sequences of Proteins of immunological Interest)》,第5版公共卫生署(Public Health Service),国立卫生研究院(National Institutes of Health),马里兰州贝塞斯达(Bethesda,Md.)(1991)；Marie-Paule Lefranc等人,《发育与比较免疫学(Developmental and Comparative Immunology)》,27:55-77(2003)；Marie-Paule Lefranc等人,《免疫组研究(Immunome Research)》,1(3),(2005)；Marie-Paule Lefranc,《B细胞的分子生物学(Molecular Biology of B cells)》(第二版),第26章,481-514,(2015))。三个CDR由被称为框架区(FR)(轻链FR包括LFR1、LFR2、LFR3和LFR4，重链FR包括HFR1、HFR2、HFR3和HFR4)的侧翼段间隔开，所述框架区比CDR更加高度保守并形成支架以支撑高度可变环。重链和轻链的恒定区与抗原结合无关，但表现出多种效应子功能。如本文所使用的，术语“效应子功能”是指由抗体的Fc区与免疫细胞上的C1q补体蛋白或Fc受体(FcR)之间的相互作用引起的细胞介导或补体介导的细胞毒性作用。示例性效应子功能包括但不限于抗体依赖性细胞毒性(ADCC)、抗体依赖性细胞介导的吞噬作用(ADCP)和补体依赖性细胞毒性(CDC)效应。抗体基于其重链恒定区的氨基酸序列可以分成几类。抗体的五个主要类别或同种型是IgA、IgD、IgE、IgG和IgM，它们的特征分别在于存在α、δ、ε、γ和μ重链。几个主要的抗体类别被分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)。
在一些实施方案中，本文提供的抗体涵盖其任何抗原结合片段。如本文所使用的，术语“抗原结合片段”是指由包括一个或多个CDR的抗体的一部分形成的抗体片段，或与抗原结合但不包括完整天然抗体结构的任何其它抗体片段。抗原结合片段的实例包括但不限于双抗体、Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双抗体(ds双抗体)、单链抗体分子(scFv)、scFv二聚体(二价双抗体)、双特异性抗体、多特异性抗体、骆驼化单结构域抗体、纳米抗体、结构域抗体和二价结构域抗体。抗原结合片段能够与亲本抗体所结合的相同抗原结合。
抗体的“Fab”是指由单条轻链(包括可变区和恒定区)和单条重链的可变区和第一恒定区经二硫键结合起来组成的抗体的一部分。
“Fab'”是指包括铰链区的一部分的Fab片段。
“F(ab')2”是指Fab'的二聚体。
抗体(例如，IgG、IgA或IgD同种型)的“Fc”是指由第一重链的第二和第三恒定结构域经由二硫键与第二重链的第二和第三恒定结构域结合组成的抗体的一部分。IgM和IgE同种型抗体的Fc进一步包括第四恒定结构域。抗体的Fc部分负责多种不同的效应子功能，如抗体依赖性细胞介导的细胞毒性(ADCC)和补体依赖性细胞毒性(CDC)，但在抗原结合中不起作用。
抗体的“Fv”是指含有完整抗原结合位点的最小抗体片段。Fv片段由单条轻链的可变区与单条重链的可变区结合组成。
“单链Fv抗体”或“scFv”是指由轻链可变区与重链可变区直接相互连接或通过肽接头序列连接而成的经工程化的抗体(Huston JS等人《美国国家科学院院刊(Proc Natl Acad Sci USA)》,85:5879(1988))。
“单链Fv-Fc抗体”或“scFv-Fc”是指由与抗体Fc部分连接的scFv组成的经工程化的抗体。
“骆驼化单结构域抗体”、“重链抗体”或“HCAb”是指含有两个VH结构域而不含有轻链的抗体(Riechmann L.和Muyldermans S.,《免疫学方法杂志(J Immunol Methods)》.12月10日；231(1-2):25-38(1999)；Muyldermans S.,《生物技术杂志(J Biotechnol.)》6月；74(4):277-302(2001)；WO94/04678；WO94/25591；美国专利第6,005,079号)。重链抗体最初来源于驼科(骆驼、单峰驼和美洲驼)。虽然缺失轻链，但是骆驼化抗体有确证的抗原结合全部功能(Hamers-Casterman C.等人,《自然》.6月3日；363(6428):446-8(1993)；Nguyen VK.等人《免疫遗传学(Immunogenetics)》.4月；54(1):39-47(2002)；Nguyen VK.等人《免疫学(Immunology)》.5月；109(1):93-101(2003))。重链抗体的可变区(VHH结构域)表示由适应性免疫应答产生的最小已知抗原结合单位(Koch-Nolte F.等人,《美国实验生物学会联合会杂志(FASEB J.)》11月；21(13):3490-8.Epub 2007年6月15日(2007))。
“纳米抗体”是指由来自重链抗体的VHH结构域以及两个恒定结构域CH2和CH3组成的抗体片段。
“双抗体”或“dAb”包括具有两个抗原结合位点的小抗体片段，其中所述片段包括在同一条多肽链上相连的VH结构域和VL结构域(VH-VL或VL-VH)(参见例如，Holliger P.等人,《美国国家科学院院刊(Proc Natl Acad Sci USA)》.7月15日；90(14):6444-8(1993)；EP404097；WO93/11161)。通过使用太短以至于不允许在同一条链上的两个结构域之间配对的接头，结构域被迫与另一条链的互补结构域配对，从而产生两个抗原结合位点。这两个抗原结合位点可靶向相同或不同的抗原(或表位)。在一些实施方案中，“双特异性ds双抗体”是靶向两种不同抗原(或表位)的双抗体。
“结构域抗体”是指仅含有重链可变区或轻链可变区的抗体片段。在某些情况下，两个或更多个VH结构域由肽接头共价连接形成二价或多价结构域抗体。二价结构域抗体的两个VH结构域可以靶向相同或不同的抗原。
本文使用的术语“价”是指给定分子中存在指定数量的抗原结合位点。术语“单价”是指仅具有一个抗原结合位点的抗体或抗原结合片段；并且术语“多价”是指具有多个抗原结合位点的抗体或抗原结合片段。如此，术语“二价”、“四价”和“六价”分别表示抗原结合分子中存在两个结合位点、四个结合位点和六个结合位点。在一些实施方案中，所述抗体或其抗原结合片段是二价的。
如本文所使用的，“双特异性”抗体是指具有来源于两个不同的单克隆抗体的片段并且能够与两个不同的表位结合的人工抗体。所述两个表位可以存在于同一个抗原上，或者其可以存在于两个不同的抗原上。
在一些实施方案中，“scFv二聚体”是二价双抗体或双特异性scFv(BsFv)，其包括二聚化的两个VH-VL(由肽接头连接)部分，使得一个部分的VH与另一个部分的VL协作 形成两个结合位点，所述两个结合位点可以靶向相同抗原(或表位)或不同抗原(或表位)。在其它实施方案中，“scFv二聚体”是双特异性双抗体，所述双特异性双抗体包括相互缔合的VH1-VL2(由肽接头连接)和VL1-VH2(也由肽接头连接)，使得VH1和VL1协作，VH2和VL2协作，并且每个协作的配对具有不同的抗原特异性。
“dsFv”是指二硫键稳定的Fv片段，其单条轻链的可变区与单条重链的可变区之间的连接是二硫键。在一些实施方案中，“(dsFv)2”或“(dsFv-dsFv')”包括三条肽链：两个VH部分通过肽接头(例如，长的柔性接头)相连，并通过二硫键分别与两个VL部分结合。在一些实施方案中，dsFv-dsFv'具有双特异性，其中每对通过二硫键配对的重链和轻链具有不同的抗原特异性。
本文使用的术语“嵌合”是指具有来源于一种物种的重链和/或轻链的一部分并且所述重链和/或轻链的其余部分来源于另一不同物种的抗体或抗原结合片段。在说明性实例中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物(如来源于小鼠)的可变区。在一些实施方案中，所述非人动物是哺乳动物，例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。
本文使用的术语“人源化”是指包括来源于非人动物的CDR、来源于人的FR区以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。
本文使用的术语“亲和力”是指免疫球蛋白分子(即，抗体)或其片段与抗原之间非共价相互作用的强度。
本文使用的“特异性结合(specific binding)”或“特异性地结合(specifically binds)”是指两分子间的非随机结合反应，例如，抗体和抗原间的反应。特异性结合的特征可以在于结合亲和力，例如由KD值表示，即，当抗原与抗原结合分子之间的结合达到平衡时解离速率与缔合速率的比率(koff/kon)。可以通过使用本领域已知的任何常规方法测定KD，包括但不限于，表面等离子共振法、Octet方法、微量热泳法、HPLC-MS方法和FACS测定方法。≤10-6M(例如≤5×10-7M、≤2×10-7M、≤10-7M、≤5×10-8M、≤2×10-8M、≤10-8M、≤5×10-9M、≤4×10-9M、≤3×10-9M、≤2×10-9M或≤10-9M)的KD值可以表示抗体或其抗原结合片段与PD-L1(例如人PD-L1或食蟹猴PD-L1)之间的特异性结合。
本文使用的“竞争结合PD-L1”的能力是指第一抗体或其抗原结合片段抑制PD-L1与第二抗PD-L1抗体之间结合的相互作用到任何可检测的程度的能力。在一些实施方案中，竞争结合PD-L1的抗体或抗原结合片段将PD-L1与第二抗PD-L1抗体之间结合的相互作用抑制至少85％或至少90％。在一些实施方案中，此抑制可以大于95％或大于99％。
本文使用的术语“氨基酸”是指含有氨基(-NH2)和羧基(-COOH)官能团以及每个氨基酸特有的侧链的有机化合物。氨基酸名称在本公开中也以标准的单字母或三字母代码表示，总结如下：

术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物。所述术语还适用于其中一个或多个氨基酸残基是对应天然存在的氨基酸的人造化学模拟物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。
当“保守取代”用于氨基酸序列时，是指将氨基酸残基用不同的具有相似理化性质的侧链的氨基酸残基替代。例如，可以在具有疏水侧链的氨基酸残基(例如Met、Ala、Val、Leu和Ile)之间、具有中性亲水侧链的氨基酸残基(例如Cys、Ser、Thr、Asn和Gln)之间、具有酸性侧链的氨基酸残基(例如Asp、Glu)之间、具有碱性侧链的氨基酸残基(例如His、Lys和Arg)之间或具有芳香族侧链的氨基酸残基(例如Trp、Tyr和Phe)之间进行保守取代。本领域已知，保守取代通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。
“百分比(％)序列同一性”在用于氨基酸序列(或核酸序列)时被定义为在进行序列比对，并且必要时引入空位以实现最大的对应性后，在候选序列中，与参考序列中的氨基酸(或核酸)残基相同的氨基酸(或核酸)残基的百分比。例如，可以使用公开可用的工具如BLASTN、BLASTp(可在美国国家生物技术信息中心(U.S.National Center for Biotechnology Information，NCBI)的网站上获得，还参见Altschul S.F.等人,《分子生物学杂志》,215:403–410(1990)；Stephen F.等人,《核酸研究(Nucleic Acids Res.)》,25:3389–3402(1997))、ClustalW2(可在欧洲生物信息研究所(European Bioinformatics Institute)的网站上获得，还参见Higgins D.G.等人,《酶学方法(Methods in Enzymology)》,266:383-402(1996)；Larkin M.A.等人,《生物信息学(Bioinformatics)》(英国牛津(Oxford,England)),23(21):2947-8(2007))和ALIGN或Megalign(DNASTAR)软件来实现比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用由所述工具提供的默认参数或可以根据比对的需要适当定制参数，例如通过挑选合适的算法。在一些实施方案中，不同的残基位置可能因保守氨基酸取代而不同。“保守氨基酸取代”是一个氨基酸残基被具有类似化学性质(例如，电荷或疏水性)的侧链(R基团)的另一个氨基酸残基取代的氨基酸取代。总体而言，保守氨基酸取代将不会实质上改变蛋白质的功能性质。在两个或更多个氨基酸序列因保守取代而彼此不同的情况下，百分比或类似性程度可以向上调整以校 正取代的保守性质。用于作出此调整的方法是本领域技术人员所熟知的。参见例如，Pearson(1994)《分子生物学方法(Methods Mol.Biol.)》24:307-331，所述文献通过引用并入本文。
如本文所使用的，“同源序列”是指这样的多核苷酸序列(或其互补链)或氨基酸序列，其与另一个序列在任选地比对时具有至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性。
“分离的”物质已经经人工由自然状态改变。如果自然界中出现某种“分离的”组合物或物质，那么其已经被改变或脱离其原始环境，或二者均有发生。例如，某一活体动物体内天然存在的多核苷酸或多肽不是“分离的”，但如果所述多核苷酸或多肽与其在天然状态下共存的物质充分分离并以基本上纯的状态存在，则可以认为是“分离的”。分离的“核酸”或“多核苷酸”可互换使用并指分离的核酸分子的序列。在一些实施方案中，“分离的抗体或其抗原结合片段”是指纯度为至少60％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗体或抗原结合片段，其中纯度由电泳方法(如SDS-PAGE、等电聚焦、毛细管电泳)或色谱方法(如离子交换色谱法或反相HPLC)确定。
术语“受试者”包括人和非人动物。非人动物包括所有脊椎动物，例如哺乳动物和非哺乳动物，如非人灵长类动物、小鼠、大鼠、猫、兔、绵羊、狗、牛、鸡、两栖动物和爬行动物。除在指出时之外，术语“患者”或“受试者”在本文中可互换使用。
如本文所使用的，“治疗(Treating或treatment)”病状包括预防或减轻病状、减缓病状的发作或发展速率、降低罹患病状的风险、预防或延迟与病状相关的症状的发展、减轻或结束与病状相关的症状、产生病状的完全或部分消退、治愈病状或其某种组合。
本文使用的术语“载体”是指可以将遗传元件可操作地插入其中并使所述遗传元件获得表达以便产生由所述遗传元件编码的蛋白质、RNA或DNA或复制所述遗传元件的一种运载工具。载体可以用于转化、转导或转染宿主细胞，使其携带的遗传元件在宿主细胞内得以表达。载体的实例包括质粒、噬菌粒、粘粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)、如λ噬菌体或M13噬菌体等噬菌体以及动物病毒。载体可以含有多种用于控制表达的元件，包括启动子序列、转录起始序列、增强子序列、可选择元件和报告基因。另外，载体还可以含有复制起点。载体还可以包括协助其进入细胞的材料，包括但不限于，病毒颗粒、脂质体或蛋白质涂层。载体可以是表达载体或克隆载体。本公开提供的载体(例如表达载体)含有本文提供的编码抗体或其抗原结合片段的核酸序列、至少一个可操作地连接到所述核酸序列的启动子(例如，SV40、CMV、EF-1α)以及至少一个选择标志物。
如本文所使用的，“宿主细胞”是指其中已引入有外源多核苷酸和/或载体的细胞。
如本文所使用的，术语“可溶性”是指分子(例如，蛋白质)溶解在溶剂(如液体和水性环境)中的能力。
如本文所使用的，术语“转化生长因子β”和“TGFβ”是指具有来自受试者(例如，人)的任何TGF-β的全长、天然氨基酸序列的任何TGFβ家族蛋白，包括前体和成熟TGFβ的潜在形式和相关或非相关复合物(“潜在TGFβ”)。在本文中提及此类TGFβ将被理解 为提及任何一种当前鉴定的形式，包括TGFβ1、TGFβ2、TGFβ3同种型及其潜在型式，以及未来鉴定的人TGFβ种类，包括衍生自任何已知TGFβ的序列并且与所述序列至少约75％、优选地至少约80％、更优选地至少约85％、仍更优选地至少约90％并且甚至更优选地至少约95％同源的多肽。具体术语“TGFβ1”、“TGFβ2”和“TGFβ3”是指文献中定义的TGF-β，例如Derynck等人,《自然·癌症研究(Nature,Cancer Res.)》,47:707(1987)；Seyedin等人,《生物化学杂志》,261:5693-5695(1986)；deMartin等人,《欧洲分子生物学学会杂志(EMBO J.)》,6:3673(1987)；Kuppner等人,《国际癌症杂志(Int.J.Cancer)》,42:562(1988)。术语“转化生长因子β”、“TGFβ”、“TGFbeta”、“TGF-β”和“TGF-beta”在本公开中可互换使用。
如本文所使用的，术语“人TGFβ1”是指由人TGFB1基因(例如，野生型人TGFB1基因)编码的TGFβ1蛋白。示例性野生型人TGFβ1蛋白由GenBank登录号NP_000651.3提供。如本文所使用的，术语“人TGFβ2”是指由人TGFB2基因(例如，野生型人TGFB2基因)编码的TGFβ2蛋白。示例性野生型人TGFβ2蛋白由GenBank登录号NP_001129071.1和NP_003229.1提供。如本文所使用的，术语“人TGFβ3”是指由人TGFB3基因(例如，野生型人TGFB3基因)编码的TGFβ3蛋白。示例性野生型人TGFβ3蛋白由GenBank登录号NP_003230.1、NP_001316868.1和NP_001316867.1提供。
如本文所使用的，术语“小鼠TGFβ1”、“小鼠TGFβ2”和“小鼠TGFβ3”是指分别由小鼠TGFB1基因(例如，野生型小鼠TGFB1基因)、小鼠TGFB2基因(例如，野生型小鼠TGFB2基因)和小鼠TGFB3基因(例如，野生型小鼠TGFB3基因)编码的TGFβ1蛋白、TGFβ2蛋白和TGFβ3蛋白。示例性野生型小鼠(小家鼠(Mus musculus))TGFβ1蛋白由GenBank登录号NP_035707.1和CAA08900.1提供。示例性野生型小鼠TGFβ2蛋白由GenBank登录号NP_033393.2提供。示例性野生型小鼠TGFβ3蛋白由GenBank登录号AAA40422.1提供。
如本文所使用的，术语“TGFβ受体”是指结合至少一种TGFβ同种型的任何受体。通常，TGFβ受体包括TGFβ受体I(TGFβRI)、TGFβ受体II(TGFβRII)或TGFβ受体III(TGFβRIII)。
关于人，术语“TGFβ受体I”或“TGFβRI”是指人TGFβ受体1型序列，包括野生型TGFβRI以及其已知能够与至少一种TGFβ同种型结合的所有同种型和变体。野生型TGFβRI的示例性氨基酸序列可在GenBank登录号ABD46753.1或UniProtKB-P36897下获得，也包括在本文中作为SEQ ID NO:69。变体TGFβRI可以具有与SEQ ID NO:69的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性的序列并保留野生型序列(例如SEQ ID NO:69)的TGFβ结合活性的至少25％、35％、50％、75％、90％、95％或99％。
关于人，术语“TGFβ受体II”或“TGFβRII”是指人TGFβ受体2型同种型A序列，包括野生型TGFβRII以及其已知能够与至少一种TGFβ同种型结合的所有同种型和变体。野生型TGFβRII同种型A或同种型1的示例性氨基酸序列可在GenBank登录号NP_001020018.1或UniProtKB-P37173-1下获得，也包括在本文中作为SEQ ID NO:70，并且野生型TGFβRII同种型B可在GenBank登录号NP_003233.4或UniProtKB-P37173-2下获得，也包括在本文中作为SEQ ID NO:71。变体TGFβRII可以具有与SEQ ID NO:70或71具有 至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性的序列并保留野生型序列(例如SEQ ID NO:70或71)的TGFβ结合活性的至少25％、35％、50％、75％、90％、95％或99％。
关于人，术语“TGFβ受体III”或“TGFβRIII”是指人TGFβ受体3型序列，包括野生型TGFβRII以及所有同种型和变体。野生型TGFβRIII的示例性氨基酸序列可在GenBank登录号NP_003234.2或UniProtKB-Q03167下获得，也包括在本文中作为SEQ ID NO:72。
如本文所使用的，关于某种参考蛋白或肽的术语“变体”是指参考蛋白或肽的经修饰型式，例如其功能等效物、片段、融合体、衍生物、模拟物或任何组合，所述经修饰型式具有与参考序列具有至少70％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列同一性的氨基酸序列并保留参考序列(例如野生型序列)的生物活性或结合活性的至少25％(例如35％、50％、75％、90％、95％或99％)。所述变体可以是参考蛋白或肽的片段、突变体、融合体、截短体或其任何组合。
如本文所使用的，术语“白细胞介素-1”或“IL-1”包括IL-1α和IL-1β、它们的前体(例如pro-IL-1α和pro-IL-1β)、同种型和变体。
如本文所使用的，术语“人IL-1α”是指由人IL1A基因(例如，野生型人IL1A基因)编码的IL-1α蛋白以及同种型和变体。UniProtKB-P01583提供了示例性野生型人IL1α蛋白。
如本文所使用的，术语“人IL-1β”是指由人IL1B基因(例如，野生型人IL1B基因)编码的IL-1β蛋白。示例性野生型人IL1β蛋白由GenBank登录号NP_000567.1或UniProtKB-C9JVK0提供。
如本文所使用的，术语“IL-1受体”或“IL-1R”是指可以与IL-1结合的受体，包括其能够与IL-1结合的所有野生型受体、同种型和变体。通常，存在两种类型的IL-1受体，即IL-1受体I(IL-1RI)和IL-1受体II(IL-1RII)。IL-1RII充当与配体结合而不转导信号的诱饵受体。IL-1RII的蛋白水解切割引起可溶性受体，例如IL-1sRI和IL-1sRII的形成，所述可溶性受体与配体结合而不转导信号(细节参见Thomas G.Kennedy,《激素百科全书(Encyclopedia of Hormones)》中的第V.B.2.章,2003)。IL-1sRI和IL-1sRII是IL-1RII的蛋白水解切割产物并且可以是一组IL-1RII的胞外结构域片段。术语IL-1R还旨在涵盖辅助受体IL-1RAP，所述辅助受体可以与结合到IL-1β的IL-1RI缔合，以形成高亲和力的白细胞介素1受体复合物，所述复合物介导NF-κ-B的白细胞介素1依赖性激活和其它通路。
如本文所使用的，术语“IL-1RI”包括野生型IL-1RI以及其能够与IL-1α和/或IL-1β结合的所有同种型和变体。野生型IL-1RI的示例性氨基酸序列可在UniProtKB-P14778下获得，也包括在本文中作为SEQ ID NO:73。
如本文所使用的，术语“IL-1RII”包括野生型IL-1RII以及其能够与IL-1α和/或IL-1β结合的所有同种型和变体。野生型IL-1RII的示例性氨基酸序列可在UniProtKB-P27930下获得，也包括在本文中作为SEQ ID NO:75。
如本文所使用的，术语“IL-1RAP”包括野生型IL-1RAP以及其能够与结合到IL-1β的IL-1R结合的所有同种型和变体。野生型IL-1RAP的示例性氨基酸序列可在UniProtKB-Q9NPH3下获得，也包括在本文中作为SEQ ID NO:74。
如本文所使用的，术语“IL-1sRI”包括可以通过涉及金属蛋白酶的蛋白水解切割产生的所有可溶形式的IL-1RI。天然存在的IL-1sRI的分子量可以在约45kDa到60Kda的范围内。此术语还涵盖IL-1sRI的能够与IL-1α和/或IL-1β结合的所有同种型和变体。
如本文所使用的，术语“IL-1sRII”包括可以通过涉及金属蛋白酶的蛋白水解切割产生的所有可溶形式的IL-1RII。天然存在的IL-1sRII的分子量可以在约45kDa到60Kda的范围内。此术语还涵盖IL-1sRII的能够与IL-1α和/或IL-1β结合的所有同种型和变体。
如本文所使用的，术语“IL-1受体拮抗剂”通常包括可以与IL-1α或IL-1β竞争结合IL-1受体并抑制IL-1α或IL-1β活性的任何蛋白。IL-1受体拮抗剂可以包括天然存在的拮抗剂，如IL-1Ra、IL-1sRI和IL-1sRII，以及其它可以阻断IL-1α或IL-1β的结合以结合到IL-1受体，具体地IL-1RI的人工拮抗剂。
如本文所使用的，术语“IL-1Ra”包括野生型IL-1Ra以及其能够与IL-1α和/或IL-1β结合的所有同种型和变体。野生型IL-1Ra的示例性氨基酸序列可在UniProtKB-P18510下获得，也包括在本文中作为SEQ ID NO:76。
如本文所使用的，“癌症”是指以恶性细胞生长或赘生物、异常增殖、浸润或转移为特征的任何医学病状，并且可以是良性或恶性的，并且包括实体瘤和非实体癌(例如血液系统恶性肿瘤)如白血病。如本文所使用的，“实体瘤”是指赘生性和/或恶性细胞的实体团块。
术语“药学上可接受的”表示指定的载体、媒剂、稀释剂、赋形剂和/或盐通常与构成调配物的其它成分在化学和/或物理上相容，并且与其接受者在生理上相容。
当关于氨基酸序列(例如，肽、多肽或蛋白质)使用时，术语“融合”或“融合的”是指例如通过化学键合或重组手段将两个或更多个氨基酸序列组合成天然不存在的单一氨基酸序列。融合氨基酸序列可以通过两个编码多核苷酸序列的基因重组产生，并且可以通过将含有重组多核苷酸的构建体引入到宿主细胞中的方法表达。
本公开实施方案的进一步详细描述
本公开上下文所述的各个实施方案以及各个实施方案中的特征应当被理解为可以以任何方式进行相互组合，这些相互组合得到的各个方案均包括在本公开的范围内、就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。
本公开的以下描述仅旨在说明本公开的各个实施方案。如此，所讨论的具体变化之处不应被解释为对本公开的范围的限制。本领域技术人员显然知晓，在不脱离本公开范围的情况下，可以做出各种等同物、改变和修改，并且应当理解，此类等同实施方案将被包括在本文中。在本文中引用的所有文献，包括公开出版物、专利和专利申请都通过引用的方式全文并入。
I.靶向免疫检查点分子并阻断IL-1活性的双功能分子
本公开提供了一种双功能分子，其包括与免疫检查点分子结合的第一部分和阻断白细胞介素-1(IL-1)的活性的第二部分。本文提供的双功能分子通过用IL-1结合部分或IL-1受体(IL-1R)结合部分(即，双功能分子的第二部分)阻断IL-1与IL-1受体之间的相互作用，允许阻断和/或降低肿瘤微环境中的IL-1活性。IL-1结合部分和/或IL-1R结合部分可以连接 到靶向免疫检查点分子的部分(即，双功能分子的第一部分)，所述免疫检查点分子可以在某些肿瘤细胞或免疫细胞的表面上发现。
IL-1是炎性细胞因子。炎症是肿瘤微环境的重要组成部分，并且IL-1在致癌作用和肿瘤进展中起着关键作用(A.Mantovani等人,《免疫学评论(Immunol Rev.)》2018年1月；281(1):57–61.)。IL-1在肿瘤起始和进展中在不同水平上起作用，包括驱动慢性非可控性炎症、肿瘤血管生成、IL-17通路的激活、骨髓源性抑制细胞(MDSC)的诱导和巨噬细胞募集、侵袭和转移(出处同上)。
免疫检查点分子在某些免疫细胞如T细胞、自然杀伤细胞等上表达。一些癌细胞也可以表达某些免疫检查点分子，这可能阻断免疫检查点的激活，从而使癌细胞避开免疫系统的监视。
通过减少肿瘤微环境中的IL-1并减少检查点阻断，本公开提供了一种新型双功能分子，所述新型双功能分子可以用于治疗免疫检查点相关疾病，如癌症、自身免疫疾病、传染病等。
在一些实施方案中，第一部分包括具有免疫刺激或共刺激活性的检查点分子的激动剂。此类免疫刺激性检查点分子可包括但不限于CD27、CD70、CD28、CD80(B7-1)、CD86(B7-2)、CD40、CD40L(CD154)、CD122、CD137、CD137L、OX40(CD134)、OX40L(CD252)、GITR、ICOS(CD278)和ICOSLG(CD275)、CD2、ICAM-1、LFA-1(CD11a/CD18)、CD30、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160和CD83。
在一些实施方案中，第一部分包括具有免疫抑制或共抑制活性的检查点分子的抑制剂。此类免疫抑制性检查点分子可以包括但不限于A2AR、B7-H3(CD276)、B7-H4(VTCN1)、BTLA(CD272)、CTLA-4(CD152)、IDO1、IDO2、TDO、KIR、LAG3、NOX2、PD-1、PD-L1、PD-L2、TIM-3、VISTA、SIGLEC7(CD328)、TIGIT、PVR(CD155)、SIGLEC9(CD329)、CD160、LAIR1、2B4(CD244)、CD47和B7-H5。
在一些实施方案中，所述免疫检查点分子是PD-L1。在一些实施方案中，第一部分包括针对PD-L1的抗体部分或其抗原结合片段。在一些实施方案中，第一部分包括针对PD-L1的拮抗剂抗体部分或其抗原结合片段。
在一些实施方案中，所述第二部分包括IL-1结合部分或IL-1受体(IL-1R)结合部分。
IL-1α和IL-1β都是促炎的并且与IL-1R结合。在与IL-1α或IL-1β结合后，IL-1R可以将IL-1R辅助蛋白和衔接蛋白MyD88募集到受体复合物，从而引起下游信号传导级联的激活，并最终激活大量免疫和炎症基因。本发明人发现，阻断IL-1的活性或其与IL-1R的结合与免疫检查点分子的调节的组合将是有用的。
在一些实施方案中，IL-1是IL-1α或IL-1β。在一些实施方案中，IL-1β是人IL-1β。
在一些实施方案中，所述第二部分包括IL-1结合部分。在一些实施方案中，所述IL-1结合部分特异性结合到IL-1α或IL-1β。在一些实施方案中，所述IL-1结合部分包括可溶性IL-1R、IL-1R的IL-1结合片段或变体、或针对IL-1的抗体或其抗原结合片段。
可溶性IL-1R可以是IL-1R的结构域或片段，例如IL-1R的胞外结构域(ECD)。可替代地，可溶性IL-1R也可以是IL-1sRI或IL-1sRII，它们是天然可溶性并能够与IL-1结合的同种型。
技术人员将理解，IL-1RI或IL-1RI的ECD、或IL-1RII或IL-1RII的ECD、或IL-1RAP或IL-1RAP的ECD、或IL-1sRI或IL-1sRII的缩短片段可能足以结合到IL-1(例如，IL-1α或IL-1β)，只要此类片段含有IL-1结合结构域。因此，本公开还涵盖IL-1RI、IL-1RI的ECD、或IL-1RII、或IL-1RII的ECD、或IL-1RAP、或IL-1RAP的ECD、IL-1sRI和IL-1sRII中的任一者的所有IL-1结合片段和变体。在一些实施方案中，IL-1结合部分包括SEQ ID NO:73、74或75的氨基酸序列或其IL-1结合片段或变体。在一些实施方案中，IL-1结合部分包括与SEQ ID NO:73、74和75中的任一者具有至少80％序列同一性的氨基酸序列或其IL-1结合片段或变体。
在一些实施方案中，IL-1结合部分包括针对IL-1的抗体或其抗原结合片段。也可以使用针对IL-1的抗体或其抗原结合片段，只要此类抗体或抗原结合片段可以阻断IL-1(例如IL-1α或IL-1β)与IL-1R的结合即可。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104或SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:105或SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:106或SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107或SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:108或SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:109或SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104的序列的HCDR1，包括SEQ ID NO:105的序列的HCDR2，和包括SEQ ID NO:106的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107的序列的LCDR1，包括SEQ ID NO:108的序列的LCDR2，和包括SEQ ID NO:109的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102,SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103,SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述第二部分包括IL-1R结合部分。
在一些实施方案中，IL-1R结合部分包括IL-1Ra或其IL-1R结合片段或变体。IL-1Ra是IL-1R的拮抗剂并且可以与IL-1α或IL-1β竞争结合IL-1R。类似地，技术人员将理解IL-1Ra的缩短片段可能足以用于结合IL-1R和/或与IL-1α或IL-1β竞争。在一些实施方案中，IL-1R结合部分包括截短形式的IL-1Ra。在一些实施方案中，IL-1R结合部分包括SEQ ID NO:67或76的氨基酸序列或其任何IL-1结合片段或变体。在一些实施方案中，IL-1R结合部分包括与SEQ ID NO:67或76具有至少80％序列同一性的氨基酸序列或其任何IL-1结合片段或变体。技术人员将理解，野生型IL-1Ra的变体也可用于本公开，只要此类变体能够与IL-1α或IL-1β竞争与IL-1R结合即可。
在一些实施方案中，IL-1R结合部分包括针对IL-1R的抗体或其抗原结合片段。也可以使用针对IL-1R的抗体或其抗原结合片段，只要此类抗体或抗原结合片段可以与IL-1α或IL-1β竞争与IL-1R结合即可。
II.靶向PD-L1的双功能分子和第二部分
当肿瘤微环境(“TME”)富集有免疫抑制性细胞因子时，PD-1/PD-L1轴检查点抑制剂(例如，PD-L1抗体)的治疗功效可能受到限制。局部微环境中的此类免疫抑制性细胞因子的信号传导可以减少肿瘤浸润性T细胞，并使它们偏向Tregs并减弱免疫效应细胞的激活。
在一个方面，本公开内容提供了一种新型双功能分子，其包括与PD-L1结合的第一部分和a)阻断免疫抑制性细胞因子的活性或b)刺激抗肿瘤免疫的第二部分。所述分子可以是化合物、肽、多肽、蛋白质或其任何组合。第二部分可以通过阻断免疫抑制活性或细胞因子或增加或刺激免疫来恢复肿瘤微环境中的免疫应答。
在一些实施方案中，本文提供的双功能分子包括与PD-L1结合的第一部分(即，PD-L1结合部分)和阻断免疫抑制性细胞因子的活性的第二部分。
在一些实施方案中，免疫抑制性细胞因子包括转化生长因子β(TGF-β)超家族中的细胞因子、IL-1或血管内皮生长因子(VEGF)。在一些实施方案中，所述TGF-β超家族中的免疫抑制性细胞因子包括骨形态发生蛋白(BMP)、激活素、NODAL和生长和分化因子(GDF)。
在一些实施方案中，所述免疫抑制性细胞因子是TGF-β。在一些实施方案中，所述免疫抑制性细胞因子是IL-1。
在一些实施方案中，所述第二部分包括TGFβ结合部分。在一些实施方案中，所述第二部分包括IL-1结合部分。如本文所使用的，术语“结合部分”、“结合片段”是指具有与靶分子或复合物特异性结合的能力的部分或片段。术语“TGFβ结合部分”是指具有与TGFβ家族的一个或多个家族成员或同种型(例如，TGFβ1、TGFβ2或TGFβ3)特异性结合 的能力的部分。类似地，术语“IL-1结合部分”是指具有与IL-1家族的一个或多个家族成员(例如，IL-1α、IL-1β)特异性结合的能力的部分。
在一些实施方案中，本文提供的双功能分子包括与PD-L1结合的第一部分(即，PD-L1结合部分)和刺激抗肿瘤免疫的第二部分。在一些实施方案中，所述第二部分包括免疫刺激性多肽或其功能等效物或其变体。在一些实施方案中，所述免疫刺激性多肽是白细胞介素(IL)-2(IL-2)、IL-15、IL-21、IL-10、IL-12、IL-23、IL-27、IL-35、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、可溶性CD4、可溶性LAG-3或IFN-α或其功能等效物。
在一些实施方案中，所述第二部分包括免疫抑制性受体信号传导的拮抗剂。在一些实施方案中，所述免疫抑制性受体是SIRPα。
在一些实施方案中，所述双功能分子包括所述第二部分中的一个或多个第二部分。在一些实施方案中，所述第二部分中的所述一个或多个第二部分可以具有相同的类型，例如，所述一个或多个第二部分中的每个第二部分可以阻断免疫抑制性细胞因子的活性，或者所述一个或多个第二部分中的每个第二部分可以刺激抗肿瘤免疫。在一些实施方案中，所述第二部分中的所述一个或多个第二部分可以具有不同的类型。在一些实施方案中，所述多个第二部分中的每个第二部分可以具有相同的序列或者可以具有不同的氨基酸序列。
i.TGFβ结合部分
在一些实施方案中，所述TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体、或针对TGFβ的抗体和其抗原结合片段。
“TGFβ结合部分”在本公开中也可以称为“TGFβ陷阱(TGFβTrap)”。因此，靶向PD-L1和TGFβ两者的蛋白质在本公开中也可以称为“抗PD-L1/TGFβ陷阱”。
在一些实施方案中，TGFβ结合部分与人和/或小鼠TGFβ结合。在一些实施方案中，TGFβ结合部分能够拮抗和/或抑制TGFβ信号传导通路。在一些实施方案中，TGFβ结合部分能够拮抗和/或抑制TGFβ。
在本公开中，TGFβ结合部分可以包括与TGFβ家族的一个或多个家族成员或同种型特异性结合的任何部分。在一些实施方案中，TGFβ结合部分包括与TGFβ1(例如，人TGFβ1)、TGFβ2(例如，人TGFβ2)和/或TGFβ3(例如，人TGFβ3)结合的部分或其具有相似或改善的TGFβ结合亲和力的变体。在一些实施方案中，TGFβ结合部分包括与TGFβ1(例如，人TGFβ1)结合的部分。在一些实施方案中，TGFβ结合部分包括与TGFβ2(例如，人TGFβ2)结合的部分。在一些实施方案中，TGFβ结合部分包括与TGFβ3(例如，人TGFβ3)结合的部分。在一些实施方案中，TGFβ结合部分包括与TGFβ1(例如，人TGFβ1)和TGFβ2(例如，人TGFβ2)两者特异性结合的部分。在一些实施方案中，TGFβ结合部分包括与TGFβ1(例如，人TGFβ1)和TGFβ3(例如，人TGFβ3)两者特异性结合的部分。在一些实施方案中，TGFβ结合部分包括与TGFβ2(例如，人TGFβ2)和TGFβ3(例如，人TGFβ3)两者特异性结合的部分。在一些实施方案中，TGFβ结合部分包括与TGFβ1(例如，人TGFβ1)、TGFβ2(例如，人TGFβ2)和TGFβ3(例如，人TGFβ3)中的每一者特异性结合的部分。本领域技术人员将理解，与TGFβ家族的一个家族成员或同种型结合的TGFβ结合部分可能能够以相似或更高的亲和力与TGFβ家族的一个或多个其它家族成员或同种型结合。
在一些实施方案中，TGFβ结合部分包括相对于TGFβ2和/或TGFβ3而选择性地结合到TGFβ1的部分。
在一些实施方案中，TGFβ结合部分包括以相似的亲和力与人TGFβ1和小鼠TGFβ1特异性结合的部分。
在一些实施方案中，本公开的TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体。
示例性TGFβ受体包括TGFβRI、TGFβRII和TGFβRIII。在一些实施方案中，TGFβ受体选自由以下组成的组：TGFβ受体I(TGFβRI)、TGFβ受体II(TGFβRII)、TGFβ受体III(TGFβRIII)和其任何组合。在一些实施方案中，TGFβ受体是TGFβRI(例如，人TGFβRI)。在一些实施方案中，TGFβ受体是TGFβRII(例如，人TGFβRII)。在一些实施方案中，TGFβ受体是TGFβRIII(例如，人TGFβRIII)。
在一些实施方案中，TGFβ结合部分包括TGFβ受体(例如，人TGFβ受体)的胞外结构域(ECD)或其TGFβ结合片段或变体。在一些实施方案中，TGFβ受体的ECD包括TGFβRI(例如，人TGFβRI)的ECD、TGFβRII(例如，人TGFβRII)的ECD、TGFβRIII(例如，人TGFβRIII)的ECD或其任何组合。在一些实施方案中，TGFβRII的ECD包括SEQ ID NO:66、79的氨基酸序列或与所述氨基酸序列具有至少80％(例如，至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性但仍保留对TGFβ的结合特异性的氨基酸序列。在一些实施方案中，TGFβRI的ECD包括SEQ ID NO:77的氨基酸序列或与所述氨基酸序列具有至少80％(例如，至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性但仍保留对TGFβ的结合特异性的氨基酸序列。在一些实施方案中，TGFβRIII的ECD包括SEQ ID NO:78的氨基酸序列或与所述氨基酸序列具有至少80％(例如，至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性但仍保留对TGFβ的结合特异性的氨基酸序列。
在一些实施方案中，TGFβ结合部分包括针对TGFβ的抗体和其抗原结合片段。示例性抗TGFβ抗体包括非苏木单抗(fresolimumab)和美替木单抗(metelimumab)以及在以下中描述的抗TGFβ抗体或其抗原结合片段：例如，US7494651B2、US8383780B2、US8012482B2、WO 2017141208A1，所述文献中的每个文献通过引用整体并入本文。
在一些实施方案中，TGFβ结合部分包括一种或多种TGFβ受体的一个或多个ECD和/或一种或多种抗TGFβ抗体或其抗原结合片段的组合。
所述一个或多个ECD可以相同或不同。例如，TGFβ结合部分可以包括TGFβ受体的ECD的相同重复序列，或者可替代地可以包括同一TGFβ受体的不同ECD序列的组合，或者可替代地可以包括来自不同TGFβ受体的不同ECD的组合。类似地，所述一种或多种抗TGFβ抗体可以相同或不同。
在一些实施方案中，TGFβ结合部分包括选自由以下组成的组的ECD的组合(或融合体)：TGFβRI(例如，人TGFβRI)的ECD、TGFβRII(例如，人TGFβRII)的ECD、TGFβRIII(例如，人TGFβRIII)的ECD或其任何组合。
在一些实施方案中，TGFβ结合部分包括一种或多种抗TGFβ抗体或其抗原结合片段的组合(或融合体)。
在一些实施方案中，TGFβ结合部分包括以下的组合(或融合体)：ECD，所述ECD选自由以下组成的组：TGFβRI(例如，人TGFβRI)的ECD、TGFβRII(例如，人TGFβRII)的ECD、TGFβRIII(例如，人TGFβRIII)的ECD；一种或多种抗TGFβ抗体或其抗原结合片段；或其任何组合。
ii.IL-1结合部分
在一些实施方案中，所述第二部分包括IL-1结合部分。在一些实施方案中，IL-1是IL-1α或IL-1β。在一些实施方案中，IL-1β是人IL-1β。
在一些实施方案中，所述IL-1结合部分特异性结合到IL-1α或IL-1β。在一些实施方案中，所述IL-1结合部分包括相对于IL-1α而选择性地结合到IL-1β的部分或相对于IL-1β而选择性地结合到IL-1α的部分。
在一些实施方案中，所述IL-1结合部分包括可溶性IL-1R、IL-1R的IL-1结合片段或变体、或针对IL-1的抗体或其抗原结合片段。
可溶性IL-1R可以包括IL-1R的结构域或片段或变体，例如，IL-1R的胞外结构域(ECD)。可替代地，可溶性IL-1R也可以包括IL-1sRI或IL-1sRII，它们是天然可溶性并能够与IL-1结合的同种型。
技术人员将理解，IL-1R或IL-1R的ECD、或IL-1sRI或IL-1sRII的缩短片段可能足以结合到IL-1(例如，IL-1α或IL-1β)，只要此类片段含有IL-1结合结构域。因此，本文提供的IL-1结合部分还可以包括IL-1R、IL-1R的ECD、IL-1sRI和IL-1sRII中的任一者的IL-1结合片段。在一些实施方案中，IL-1结合部分包括SEQ ID NO:73、74或75的氨基酸序列或其IL-1结合片段或变体。在一些实施方案中，IL-1结合部分包括与SEQ ID NO:73、74和75中的任一者具有至少80％序列同一性的氨基酸序列或其IL-1结合片段或变体。
在一些实施方案中，IL-1结合部分包括针对IL-1的抗体或其抗原结合片段。也可以使用针对IL-1的抗体或其抗原结合片段，只要此类抗体或抗原结合片段可以阻断IL-1(例如IL-1α或IL-1β)与IL-1R的结合即可。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104或SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:105或SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:106或SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107或SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:108或SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:109或SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:104的序列的HCDR1，包括SEQ ID NO:105的序列的HCDR2，和包括SEQ ID NO:106的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:107的序列的LCDR1，包括SEQ ID NO:108的序列的LCDR2，和包括SEQ ID NO:109的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含包括SEQ ID NO:112的序列的HCDR1，包括SEQ ID NO:113的序列的HCDR2，和包括SEQ ID NO:114的序列的HCDR3，所述轻链可变区包含包括SEQ ID NO:115的序列的LCDR1，包括SEQ ID NO:116的序列的LCDR2，和包括SEQ ID NO:117的序列的LCDR3。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102,SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103,SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:102和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:103和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，所述针对IL-1的抗体或其抗原结合片段包含重链可变区和/或轻链可变区，所述重链可变区包含选自由以下组成的组的序列：SEQ ID NO:110,和与其具有至少80％序列同一性的同源序列，所述轻链可变区包含选自由以下组成的组的序列：SEQ ID NO:111,和与其具有至少80％序列同一性的同源序列。
在一些实施方案中，IL-1结合部分包括选自由以下组成的组的一个或多个部分的组合：IL-1R、IL-1R的ECD、IL-1sRI、IL-1sRII、针对IL-1的抗体、其任何IL-1结合片段和其任何组合。此类一个或多个部分可以通过直接键连接或可以通过合适的接头连接。
在一些实施方案中，IL-1R结合部分包括IL-1Ra或其IL-1R结合片段或变体。IL-1Ra是IL-1R的拮抗剂并且可以与IL-1α或IL-1β竞争结合IL-1R。类似地，技术人员将理解IL-1Ra的缩短片段可能足以用于结合IL-1R和/或与IL-1α或IL-1β竞争。在一些实施方案中，IL-1R结合部分包括截短形式的IL-1Ra。在一些实施方案中，IL-1R结合部分包括SEQ ID NO:67的氨基酸序列或其任何IL-1结合片段或变体。在一些实施方案中，IL-1R结合部分包括与SEQ ID NO:67具有至少80％序列同一性的氨基酸序列或其任何IL-1结合片段或变体。技术人员将理解，野生型IL-1Ra的变体也可用于本公开，只要此类变体能够与IL-1α或IL-1β竞争与IL-1R结合即可。
在一些实施方案中，IL-1R结合部分包括针对IL-1R的抗体或其抗原结合片段。也可以使用针对IL-1R的抗体或其抗原结合片段，只要此类抗体或抗原结合片段可以与IL-1α或IL-1β竞争与IL-1R结合即可。
在一些实施方案中，IL-1R结合部分包括选自由以下组成的组的一个或多个部分的组合：IL-1Ra、针对IL-1R的抗体、其任何IL-1R结合片段或变体和其任何组合。此类一个或多个部分可以通过直接键连接或可以通过合适的接头连接。
iii.免疫刺激性多肽
在一些实施方案中，所述第二部分包括免疫刺激性多肽或其功能等效物或其变体。在一些实施方案中，免疫刺激性多肽是可溶性CD4、可溶性LAG-3或其功能等效物。
在一些实施方案中，可溶性LAG-3包括LAG-3的胞外结构域(ECD)或其MHC II类(MHCII)结合片段或变体。
LAG-3(Uniprot编号：Q61790)属于免疫球蛋白(Ig)超家族，是一种包括503个氨基酸的I型跨膜蛋白。Lag-3包括胞内结构域(ICD)、跨膜结构域(TMD)和胞外结构域(ECD)。ECD包括四个Ig样结构域，即D1到D4，其中D1包括9条β-链：A、B、C、C'、C”、D、E、F和G链。在C链与C'链之间，存在另外的序列，所述另外的序列具有形成“额外环”的约30个氨基酸。据报道，此类“额外环”涉及LAG-3与MHCII之间的相互作用。在一些实施方案中，可溶性LAG-3包括额外环、D1结构域、D1加D2结构域或其任何MHC II结合片段或变体的氨基酸序列。在一些实施方案中，可溶性LAG-3包括SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101的氨基酸序列或其任何MHC II结合片段或变体。
LAG-3在激活的T细胞、天然杀伤细胞、B细胞和浆细胞样树突状细胞上表达。LAG-3的主要配体是MHC II类，与CD4相比，LAG-3以更高的亲和力与MHC II类结合。连接肽(CP)存在于LAG-3的D4与TMD之间，其中在金属蛋白酶ADAM10和/或ADAM17的存在下发生切割以产生经切割的可溶性LAG-3。参见例如Huard等人,《美国国家科学院院刊(Proc Natl Acad Sci U S A)》1997；94:5744-9.；Workman等人,《免疫学杂志(J Immunol)》2002；169:5392–5.doi:10.4049/jimmunol.169.10.5392；和Lawrence等人,《癌症免疫治疗杂志(J Immunother Cancer)》.2015；3(增刊2):P216，所述文献通过引用并入本文。
LAG-3还编码可被翻译成可溶形式的LAG-3的替代性剪接变体。可溶性LAG-3通过MHCII信号传导激活抗原呈递细胞(APC)，从而使得体内抗原特异性T细胞应答增加。例如，可溶性LAG-3激活树突状细胞(DC)，并且据报道参与细胞因子激活(如TNF-α和/或IL-12激活)的旁观者T细胞的促炎活动，并且它可以直接激活DC。参见例如Triebel,《免疫学趋势(Trends Immunol.)》,2003,24:619-622，其通过引用并入本文。
在一些实施方案中，可溶性LAG-3包括依替莫德α(Eftilagimod alpha)(IMP321)或其MHC II结合片段或变体。IMP321是LAG-3的可溶性二聚体重组形式。IMP321通过借助于MHCII分子刺激树突状细胞来诱导持续的免疫应答。MP321和抗PD-1抗体或抗PD-L1抗体的组合疗法已显示协同激活T细胞(具体地CD8+T细胞)。参见例如Luc等人,在NLM目录添加到搜索中的PubMed搜索中的未来肿瘤行动搜索(Future Oncol Actions Search in PubMed Search in NLM Catalog Add to Search).2019年6月；15(17):1963-1973.doi:10.2217/fon-2018-0807.Epub 2019年4月12日.；Julio等人,《临床肿瘤学杂志(Journal of Clinical Oncology)》,第37卷,第15期；和US10874713B，所述文献通过引用并入本文。
iv.免疫抑制性受体信号传导的拮抗剂
在一些实施方案中，所述第二部分包括免疫抑制性受体信号传导的拮抗剂。在一些实施方案中，所述免疫抑制性受体是SIRPα。
如本文所使用的，可与术语“信号调节蛋白α”互换的术语“SIRPa”是指主要在骨髓细胞和树突状细胞上表达的抑制性受体。SIRPα属于SIRPs家族，所述家族还包括其它几种跨膜糖蛋白，包括SIRPβ和SIRPγ。SIRPs家族的每个成员都含有3个相似的胞外Ig样结构域，而跨膜和细胞质结构域不同。
SIRPa可以与传递“不要吃我”信号以抑制吞噬作用的CD47结合，并且阻断CD47介导的SIRPa在吞噬细胞上的接合可以引起去除带有“吃我”信号的活细胞。CD47是一种广泛表达的跨膜糖蛋白，具有一个胞外N末端IgV结构域、五个跨膜结构域和一个短的C末端胞内尾。CD47作为SIRPα的细胞配体发挥作用。肿瘤细胞经常过度表达CD47以避开巨噬细胞介导的破坏。已证明CD47与SIRPα的相互作用参与巨噬细胞介导的吞噬作用的调节(Takenaka等人,《自然免疫学(Nature Immunol.)》,8(12):1313-1323,2007)。
在一些实施方案中，所述第二部分阻断CD47与SIRPα之间的相互作用。在各种临床前模型中，阻断CD47与SIRPα相互作用的疗法会刺激体外癌细胞的吞噬作用和体内抗肿瘤免疫应答。
所述第二部分可以包括CD47结合结构域或SIRPα结合结构域。在一些实施方案中，所述免疫抑制性受体是信号调节蛋白α(SIRPα)。在一些实施方案中，所述第二部分阻断CD47与SIRPα之间的相互作用。在一些实施方案中，所述第二部分包括CD47结合结构域或SIRPα结合结构域。在一些实施方案中，所述CD47结合结构域包括可溶性SIRPα或其CD47结合片段、或抗CD47抗体或其抗原结合片段。
在一些实施方案中，所述可溶性SIRPα包括所述SIRPα的胞外结构域(ECD)或其CD47结合片段或变体。在一些实施方案中，可溶性SIRPα包括SEQ ID NO:84的氨基酸序列或与所述氨基酸序列具有至少80％(例如，至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性但仍保留对CD47的结合特异性的氨基酸序列。任选地，可溶性SIRPα是经工程化的高亲和力SIRPα变体，所述变体有效地拮抗癌细胞上的CD47，但本身不会诱导巨噬细胞吞噬作用。在一些实施方案中，SIRPα变体包括选自由以下组成的组的相对于SEQ ID NO:98的一个或多个突变：L4V、L4I、V6I、V6L、A21V、V27I、V27L、I31T、I31S、I31F、E47V、E47L、K53R、E54Q、H56P、H56R、V63I、S66T、S66G、K68R、V92I、F94L、F94V和F103V。在一些实施方案中，SIRPa变体包括选自由以下组成的组的突变的组合：1)V27I、K53R、S66T、K68R、F103V；2)L4V、V27L、E47V、K53R、E54Q、S66G、K68R、V92I；3)L4V、V6I、A21V、V27I、I31T、E47L、K53R、H56P、S66T、K68R、F94L；4)V6I、V27I、I31S、E47V、K53R、E54Q、H56P、S66G、V92I、F94L；5)L4I、A21V、V27I、I31F、E47V、K53R、E54Q、H56R、S66G、F94V、F103V；6)L4V、V6I、V27I、I31F、E47V、K53R、H56R、S66G、K68R、V92I、F94L；7)L4V、V6L、I31F、E47V、K53R、H56P、S66G、V92I、F103V；8)V6I、V27I、I31F、E47L、K53R、E54Q、H56P、S66T；9)L4V、V6I、V27I、I31F、E47V、K53R、E54Q、H56P、V63、S66T、K68R、V92I；10)V6I、V27I、I31T、E47V、K53R、E54Q、H56P、S66G、K68R、V92I、F103V；和11)V6I、V27I、I31F、E47V、K53R、E54Q、H56P、S66T、V92I。参见例如Kipp Weiskopf等人《科学(Science)》341,88(2013)，其通过引用并入本文。
在一些实施方案中，所述SIRPα结合结构域包括可溶性CD47或其SIRPα结合片段、或抗SIRPα抗体或其抗原结合片段。在一些实施方案中，所述可溶性CD47包括所述CD47的胞外结构域(ECD)或其SIRPα结合片段、抗SIRPα抗体或其抗原结合片段。
在一些实施方案中，CD47结合结构域包括抗CD47抗体和其抗原结合片段。示例性抗CD47抗体包括但不限于人源化5F9抗体、B6H12抗体和ZF1抗体。参见Lu等人,《肿瘤指 标与疗法(OncoTargets and Therapy)》,第13卷,DOI https://doi.org/10.2147/OTT.S249822，其通过引用并入本文。在一些实施方案中，SIRPα结合结构域包括抗SIRPα抗体或其抗原结合片段。示例性抗SIRPa抗体包括但不限于BI765064和AL008。参见例如WO 2019073080A1、WO 2019175218A1和WO 2018107058A1，所述文献通过引用并入本文。
在一些实施方案中，CD47结合结构域包括一个或多个SIRPα、SIRPβ或SIRPγ的一个或多个ECD和/或一种或多种抗CD47抗体或其抗原结合片段的组合。
所述一个或多个ECD可以相同或不同。例如，CD47结合结构域可以包括SIRPα、SIRPβ或SIRPγ的ECD的相同重复序列，或者可替代地可以包括相同SIRPα、SIRPβ或SIRPγ的不同ECD序列的组合，或者可替代地可以包括来自不同SIRPα、SIRPβ或SIRPγ的不同ECD的组合。类似地，所述一种或多种抗CD47抗体可以相同或不同。
在一些实施方案中，CD47结合结构域包括选自由以下组成的组的ECD的组合(或融合体)：SIRPα的ECD、SIRPβ的ECD、SIRPγ的ECD或其任何组合。
在一些实施方案中，CD47结合结构域包括一种或多种抗CD47抗体或其抗原结合片段的组合(或融合体)。
在一些实施方案中，CD47结合结构域包括以下的组合(或融合体)：ECD，所述ECD选自由以下组成的组：SIRPα的ECD、SIRPβ的ECD、SIRPγ的ECD；一种或多种抗CD47抗体或其抗原结合片段；或其任何组合。
v.PD-L1结合部分
在一些实施方案中，本文提供的双功能分子包括第一部分，所述第一部分是PD-L1结合部分。
在一些实施方案中，本公开的PD-L1结合部分与PD-L1(例如，人PD-L1或食蟹猴PD-L1)结合。在一些实施方案中，本公开的PD-L1结合部分与人PD-L1结合。在一些实施方案中，本公开的PD-L1结合部分与食蟹猴PD-L1结合。
在一些实施方案中，本公开的PD-L1结合部分包括抗PD-L1抗体部分。在一些实施方案中，示例性抗PD-L1抗体在本公开的抗PD-L1抗体章节和说明性抗PD-L1抗体章节中公开。
在一些实施方案中，抗PD-L1抗体部分包括一个或多个CDR。在一些实施方案中，抗PD-L1抗体部分包括在本公开的说明性抗PD-L1抗体章节中描述的一个或多个CDR。在一些实施方案中，抗PD-L1抗体部分包括重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，抗PD-L1抗体部分包括如本公开的说明性抗PD-L1抗体章节中公开的抗PD-L1抗体的VH和VL。
在一些实施方案中，抗PD-L1抗体部分进一步包括附加到重链可变区的羧基端的重链恒定结构域。在一些实施方案中，重链恒定区来源于由IgA、IgG和IgM组成的组。在一些实施方案中，重链恒定区来源于人IgG1、IgG2、IgG3、IgG4、IgA1、IgA2或IgM。在一些实施方案中，抗PD-L1抗体部分进一步包括附加到轻链可变区的羧基端的轻链恒定结构域。在一些实施方案中，轻链恒定区来源于κ轻链或λ轻链。在一些实施方案中，重链恒定区包 括SEQ ID NO:80或81的氨基酸序列。在一些实施方案中，轻链恒定区包括SEQ ID NO:82的氨基酸序列。
vi.第一部分与第二部分之间的连接
在本公开中，第二部分可以连接到第一部分的任何部分。例如，如TGFβ结合部分或IL-1结合部分等第二部分可以连接到第一部分的任何合适的部分，所述第一部分如PD-L1结合部分(例如，抗PD-L1抗体部分)。
在一些实施方案中，PD-L1结合部分包括一条或多条多肽链，如抗体重链和轻链。
在一些实施方案中，所述双功能分子包括所述第二部分中的一个或多个第二部分。在一些实施方案中，所述多个第二部分中的至少一个第二部分连接到所述第一部分的多肽链的氨基端(N端)或羧基(C端)。在一些实施方案中，所述多个第二部分中的所述至少一个第二部分连接到所述第一部分的重链的N端或C端或连接到第一部分的轻链的N端或C端。
在一些实施方案中，所述多个第二部分中的所述至少一个第二部分连接到所述第一部分的重链恒定区的C端。在一些实施方案中，所述多个第二部分中的每个第二部分分别连接到所述第一部分的每个重链恒定区的C端。
在一些实施方案中，所述双功能分子包括所述第二部分中的至少两个第二部分，所述至少两个第二部分中的每个第二部分分别连接到所述第一部分的每条重链的C端，或者所述至少两个第二部分中的每个第二部分分别连接到所述第一部分的每条轻链的C端。在一些实施方案中，所述双功能分子包括所述第二部分中的至少两个第二部分，所述至少两个第二部分中的每个第二部分分别连接到所述第一部分的每条重链的N端，或者所述至少两个第二部分中的每个第二部分分别连接到所述第一部分的每条轻链的N端。
在一些实施方案中，所述双功能分子包括所述第二部分中的多于一个第二部分，所述多于一个第二部分分别连接到：所述第一部分的重链的N端、所述第一部分的重链的C端、所述第一部分的轻链的N端、所述第一部分的轻链的C端或其任何组合。例如，所述双功能分子可以包括所述第二部分中的至少两个第二部分，所述至少两个第二部分中的一个第二部分连接到所述第一部分的重链的C端，并且另一个第二部分连接到所述第一部分的轻链的C端。例如，所述双功能分子可以包括所述第二部分中的至少两个第二部分，所述至少两个第二部分中的一个第二部分连接到所述第一部分的重链的N端，并且另一个第二部分连接到所述第一部分的轻链的N端。
在一些实施方案中，一个或多个TGFβ结合部分、一个或多个IL-1结合部分、一种或多种免疫刺激性多肽(例如，可溶性LAG3或可溶性CD4)或一个或多个CD47结合部分在选自由以下组成的组的一个或多个位置处连接到抗PD-L1抗体部分：抗PD-L1抗体部分的1)重链可变区的N端，2)轻链可变区的N端，3)重链可变区的C端；4)轻链可变区的C端；5)重链恒定区的C端；6)轻链恒定区的C端；以及7)其任何组合。
在一些实施方案中，所述双功能分子包括同二聚体重链。在一些实施方案中，所述双功能分子包括异二聚体重链。所述重链就所述第二部分的存在或位置而言是异二聚体的。在一些实施方案中，所述异二聚体重链包括具有所述第二部分的一条重链，而另一条重链不具有所述第二部分。
vii.接头
所述第二部分可以直接或通过接头连接到所述第一部分。直接连接可以是化学连接(如共价键)。
在一些实施方案中，所述双功能分子进一步包括连接所述第一部分和所述第二部分的接头。如本文所使用的，术语“接头”可以是任何合适的双功能部分，所述双功能部分能够与至少两个要连接的实体反应，从而将实体键合以形成一个分子或保持实体足够紧密地缔合。所述接头可以整合到所得连接分子或结构中而具有或不具有其反应的官能团。
在一些实施方案中，所述接头选自由以下组成的组：可切割接头、不可切割接头、肽接头、柔性接头、刚性接头、螺旋接头和非螺旋接头。
在一些实施方案中，所述接头包括肽接头。肽接头可以由通过肽键连接在一起的氨基酸残基构成。在一些实施方案中，肽接头可以进一步包括一种或多种非天然氨基酸。在一些实施方案中，肽接头包括具有通过肽键连接的至少1、2、3、4、5、8、10、15、20、30、50或更多个氨基酸残基并且能够连接两个或更多个多肽的氨基酸序列。肽接头可以具有或可以不具有二级结构。
可以使用任何合适的肽接头。许多肽接头序列是本领域已知的，参见例如Holliger等人,《美国国家科学院院刊(Proc.Natl.Acad.Sci.USA)》90:6444-6448(1993)；Poljak等人,《结构(Structure)》2:1121-1123(1994)。在一些实施方案中，肽接头可以包括选自以下的氨基酸残基或由选自以下的氨基酸残基组成：甘氨酸、丝氨酸、丙氨酸、甲硫氨酸、天冬酰胺和谷氨酰胺。在一些实施方案中，肽接头可以由大部分无空间位阻的氨基酸如甘氨酸和丙氨酸构成。在一些实施方案中，接头是聚甘氨酸、聚丙氨酸、甘氨酸和丙氨酸的组合(如聚(Gly-Ala))或甘氨酸和丝氨酸的组合(如聚(Gly-Ser))。
在一些实施方案中，所述接头包括((G)nS)m的氨基酸序列，其中m和n独立地是选自以下的整数：0到30、1到29、2到28、3到27、4到26、5到25、6到24、7到23、8到22、9到21、10到20、11到19、12到18、13到17、14到16或5。在一些实施方案中，m是4，并且n是4。
在一些实施方案中，所述接头包括SEQ ID NO:68的氨基酸序列。在一些实施方案中，所述接头包括与SEQ ID NO:68具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性的氨基酸序列。
viii.抗PD-L1抗体和其抗原结合片段
在一些实施方案中，本文提供的双功能分子的PD-L1结合部分包括包含抗PD-L1抗体或其抗原结合片段的部分。在一些实施方案中，抗PD-L1抗体和其抗原结合片段能够特异性地结合到PD-L1。
在一些实施方案中，如通过Biacore测定所测量的，本文提供的抗PD-L1抗体和其抗原结合片段以不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM或不超过0.4nM的KD值特异性地结合到人PD-L1。Biacore测定基于表面等离子共振技术，参见例如Murphy,M.等人,《蛋白科学实验指南(Current protocols in protein science)》,第19章,单 元19.14,2006。在一些实施方案中，KD值是通过如在本公开的实例6中描述的方法来测量的。
本文提供的抗体或其抗原结合片段与人PD-L1的结合也可以用“半最大有效浓度”(EC50)值表示，其是指抗体的观察到其最大结合的50％的浓度。EC50值可以通过本领域已知的结合测定来测量，例如直接或间接结合测定，如酶联免疫吸附测定(ELISA)、荧光激活细胞分选(FACS)测定和其它结合测定。在一些实施方案中，如通过ELISA所测量的，本文提供的抗体和其抗原结合片段以不超过0.3nM、不超过0.2nM、不超过0.1nM或不超过0.09nM的EC50(即，50％结合浓度)特异性地结合到PD-L1。在一些实施方案中，如通过FACS测定所测量的，本文提供的抗体和其抗原结合片段以不超过1.4nM、不超过1.3nM、不超过1.2nM、不超过1.1nM、不超过1.0nM、不超过0.3nM、不超过0.25nM或不超过0.21nM的EC50(即，50％结合浓度)特异性地结合到PD-L1。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段特异性地结合到PD-L1。在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段不与B7家族的其它成员结合。
在一些实施方案中，本文提供的抗PD-L1抗体和其抗原结合片段能够阻断PD-L1与其结合配偶体(例如，PD-1和B7-1)之间的相互作用，如通过ELISA所测量的，所述相互作用的IC50不超过2.2、2.1、2.0、1.9、1.8或1.2ug/ml。
在一些实施方案中，本文提供的抗PD-L1抗体和其抗原结合片段能够阻断PD-L1与其结合配偶体(例如，PD-1)之间的相互作用，如通过基于细胞的测定所测量的，所述相互作用的EC50不超过1.3、1.2、1.1、1.0、0.9或0.8nM。
ix.说明性抗PD-L1抗体和其抗原结合片段
在一些实施方案中，本公开的抗PD-L1抗体(即，针对PD-L1的抗体)和其抗原结合片段包括一个或多个(例如，1、2、3、4、5或6个)CDR，所述一个或多个CDR包括选自由以下组成的组的序列：DYYMN(SEQ ID NO:1)、DINPNNX1X2TX3YNHKFKG(SEQ ID NO:19)、WGDGPFAY(SEQ ID NO:3)、KASQNVX4X5X6VA(SEQ ID NO:20)、SX7SX8RYT(SEQ ID NO:21)、QQYSNYPT(SEQ ID NO:6)，其中X1是G或A，X2是G或D或Q或E或L，X3是S或M或Q或L或V，X4是G或P或K，X5是A或G，X6是A或I，X7是A或N或R或V，X8是N或H或V或D。
在一些实施方案中，所述重链可变区包括：
a)HCDR1包括SEQ ID NO:1的序列，
b)HCDR2包括选自由以下组成的组的序列：SEQ ID NO:2、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17和SEQ ID NO:18，以及
c)HCDR3包括SEQ ID NO:3的序列，
和/或
轻链可变区，所述轻链可变区包括：
d)LCDR1包括选自由以下组成的组的序列：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9，
e)LCDR2包括选自由以下组成的组的序列：SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12，以及
f)LCDR3包括SEQ ID NO:6的序列。
在一些实施方案中，所述重链可变区选自由以下组成的组：
g)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:2的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
h)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:13的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
i)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:14的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；
j)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:15的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；以及
k)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:17的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3；以及
l)包括以下的重链可变区：包括SEQ ID NO:1的序列的HCDR1、包括SEQ ID NO:18的序列的HCDR2和包括SEQ ID NO:3的序列的HCDR3。
在一些实施方案中，所述轻链可变区选自由以下组成的组：
a)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
b)包括以下的轻链可变区：包括SEQ ID NO:9的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
c)包括以下的轻链可变区：包括SEQ ID NO:8的序列的LCDR1、包括SEQ ID NO:5的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；
d)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:12的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3；以及
e)包括以下的轻链可变区：包括SEQ ID NO:4的序列的LCDR1、包括SEQ ID NO:11的序列的LCDR2和包括SEQ ID NO:6的序列的LCDR3。
如本文所使用的，抗体“4B6”是指包括具有SEQ ID NO:46的序列的重链可变区和具有SEQ ID NO:47的序列的轻链可变区的单克隆抗体。
在一些实施方案中，本公开提供了包括抗体4B6或抗体4B6的变体的一个或多个(例如，1、2、3、4、5或6个)CDR序列的抗PD-L1抗体和其抗原结合片段。CDR边界通过Kabat规则定义或鉴定。
在一些实施方案中，本公开提供了抗PD-L1抗体和其抗原结合片段，所述抗体和其抗原结合片段包括：包括SEQ ID NO:1的氨基酸序列的HCDR1；包括选自由SEQ ID NO:2、13、14、15、17和18组成的组的氨基酸序列的HCDR2；和包括SEQ ID NO:3的氨基酸序列的HCDR3；和/或包括选自由SEQ ID NO:4、7、8-9组成的组的氨基酸序列的LCDR1；包括选自由SEQ ID NO:5、10、11-12组成的组的氨基酸序列的LCDR2；和包括SEQ ID NO:6的氨基酸序列的LCDR3。
表1.抗体4B6的CDR氨基酸序列。
表2.抗体4B6的可变区氨基酸序列。
已知CDR负责抗原结合。然而，已经发现并非所有的6个CDR都是不可缺少或不可改变的。换言之，可以替换或改变或修饰抗PD-L1抗体4B6中的一个或多个CDR，但仍基本上保留对PD-L1的特异性结合亲和力。
在一些实施方案中，本文提供的抗体和其抗原结合片段包括合适的框架区(FR)序列，只要所述抗体和其抗原结合片段可以特异性地结合到PD-L1即可。上表1中提供的CDR序列是从小鼠抗体获得的，但所述CDR序列可以使用如重组技术等本领域已知的合适方法移植到如小鼠、人、大鼠、兔等任何合适物种的任何合适FR序列。
在一些实施方案中，本文提供的抗体和其抗原结合片段是人源化的。人源化抗体或其抗原结合片段在其于人中降低的免疫原性方面是期望的。人源化抗体在其可变区是嵌合的，因为非人CDR序列被移植到人或基本上人的FR序列。抗体或抗原结合片段的人源化基本上可以通过用非人(如鼠)CDR基因取代人免疫球蛋白基因中的对应人CDR基因来进行(参见例如，Jones等人(1986)《自然》321:522-525；Riechmann等人(1988)《自然》332:323-327；Verhoeyen等人(1988)《科学》239:1534-1536)。
可以使用本领域已知的方法选择合适的人重链和轻链可变结构域以实现此目的。在说明性实例中，可以使用“最佳拟合”方法，其中针对已知人可变结构域序列的数据库筛选或BLAST非人(例如，啮齿动物)抗体可变结构域序列，并且最接近非人查询序列的人序列被鉴定并用作用于移植非人CDR序列的人支架(参见例如Sims等人,(1993)《免疫学杂志》151:2296；Chothia等人(1987)《分子生物学杂志(J.Mot.Biol.)》196:901)。可替代地， 来源于所有人抗体的共有序列的框架可以用于移植非人CDR(参见例如Carter等人(1992)《美国国家科学院院刊》,89:4285；Presta等人(1993)《免疫学杂志》,151:2623)。
在一些实施方案中，本公开提供了4B6的12种人源化抗体，它们分别被命名为Hu4B6_Hg.2La.1、Hu4B6_Hg.2La.2、Hu4B6_Hg.2La.4、Hu4B6_Hg.2La.6、Hu4B6_Hg.3La.1、Hu4B6_Hg.3La.2、Hu4B6_Hg.3La.4、Hu4B6_Hg.3La.6、Hu4B6_Hg.5La.1、Hu4B6_Hg.5La.2、Hu4B6_Hg.5La.4和Hu4B6_Hg.5La.6。4B6的每种人源化抗体的重链和轻链可变区的SEQ ID NO在表5中示出。4B6的12种人源化抗体中的每种人源化抗体的CDR在表5(加下划线的序列)中示出。CDR边界通过Kabat规则定义或鉴定。
下表3a示出了人源化4B6的变体CDR的氨基酸序列，下表3b示出了人源化4B6重链和轻链可变区的FR。下表4示出了嵌合抗体4B6的12种人源化抗体的每条重链和轻链的FR氨基酸序列，所述人源化抗体分别被命名为Hu4B6_Hg.2La.1、Hu4B6_Hg.2La.2、Hu4B6_Hg.2La.4、Hu4B6_Hg.2La.6、Hu4B6_Hg.3La.1、Hu4B6_Hg.3La.2、Hu4B6_Hg.3La.4、Hu4B6_Hg.3La.6、Hu4B6_Hg.5La.1、Hu4B6_Hg.5La.2、Hu4B6_Hg.5La.4和Hu4B6_Hg.5La.6。这12种人源化抗体的重链可变区和轻链可变区在表5中示出。
表3a.4B6的人源化抗体的CDR变体的氨基酸序列。
表3b.4B6和4B6的人源化抗体的FR序列的氨基酸序列。

表4. 4B6的人源化抗体的每个人源化重链和轻链可变区的FR氨基酸序列。
下表5示出了人源化4B6重链可变区的3种变体(即，Hu4B6_Hg.2、Hu4B6_Hg.3和Hu4B6_Hg.5)和人源化4B6轻链可变区的4种变体(即，AM4B6_La.1、AM4B6_La.2、AM4B6_La.4、AM4B6_La.6)。
表5. 4B6的人源化抗体的可变区的氨基酸序列。


在一些实施方案中，除了非人的CDR序列之外，本文提供的人源化抗PD-L1抗体或其抗原结合片段由基本上全人序列构成。在一些实施方案中，可变区FR和恒定区(如果存在)完全或基本上来自人免疫球蛋白序列。人FR序列和人恒定区序列可以源自不同的人免疫球蛋白基因，例如，FR序列源自一种人抗体并且恒定区源自另一种人抗体。在一些实施方案中，人源化抗体或其抗原结合片段包括人重链HFR1-4和/或轻链LFR1-4。
在一些实施方案中，源自人的FR区可以包括与其所源自的人免疫球蛋白相同的氨基酸序列。在一些实施方案中，人FR的一个或多个氨基酸残基被来自亲本非人抗体的对应残基取代。在一些实施方案中，这可能是期望的，以使人源化抗体或其片段非常接近非人亲本抗体结构，从而优化结合特性(例如，增加结合亲和力)。在一些实施方案中，本文提供的人源化抗体或其抗原结合片段在多个人FR序列中的每个人FR序列中包括不超过10、9、8、7、6、5、4、3、2或1个氨基酸残基取代，或者在重链或轻链可变结构域的所有FR序列中包括不超过10、9、8、7、6、5、4、3、2或1个氨基酸残基取代。在一些实施方案中，氨基酸残基的此类变化可以仅存在于重链FR区、仅存在于轻链FR区、或存在于两条链中。在一些实施方案中，使人FR序列的一个或多个氨基酸随机突变以增加结合亲和力。在一些实施方案中，使人FR序列的一个或多个氨基酸回复突变为亲本非人抗体的对应氨基酸以增加结合亲和力。
在一些实施方案中，本公开的人源化抗PD-L1抗体和其抗原结合片段包括：重链HFR1，所述重链HFR1包括QVQLVQSGAEVKKPGASVKVSCKASGYX9FT(SEQ ID NO:40)的序列或与所述序列具有至少80％序列同一性的同源序列；重链HFR2，所述重链HFR2包括WVRQAPGQX10LEWMG(SEQ ID NO:41)的序列或与所述序列具有至少80％序列同一性的同源序列；重链HFR3，所述重链HFR3包括RVTX16TVDX11SISTAYMELSRLRSDDTAVYYCX12X13(SEQ ID NO:42)的序列或与所述序列具有至少80％序列同一性的同源序列；和重链HFR4，所述重链HFR4包括WGQGTLVTVSS(SEQ ID NO:25)的序列或与所述序列具有至少80％序列同一性的同源序列，其中X9是T或V，X10是G或S，X11是T或K，X12是A或V，并且X13是R或K。
在一些实施方案中，本公开的人源化抗PD-L1抗体和其抗原结合片段包括：轻链LFR1，所述轻链LFR1包括DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:26)的序列或与所述序列具有至少 80％序列同一性的同源序列；轻链LFR2，所述轻链LFR2包括WYQQKPGKX14PKLLIY(SEQ ID NO:43)的序列或与所述序列具有至少80％序列同一性 的同源序列；轻链LFR3，所述轻链LFR3包括GVPX15RFSGSGSGTDFTX17TISSLQPEDIATYYC(SEQ ID NO:44)的序列或与所述序列具有至少80％序列同一性的同源序列；和轻链LFR4，所述轻链LFR4包括FGQGTKLEIK(SEQ ID NO:29)的序列或与所述序列具有至少80％序列同一性的同源序列，其中X14是A或S，X15是S或D，X16是M或V，并且X17是F或L。
在一些实施方案中，HFR1包括选自由SEQ ID NO:22和30组成的组的序列，HFR2包括选自由SEQ ID NO:23和31组成的组的序列，HFR3包括选自由SEQ ID NO:24和32-35组成的组的序列，HFR4包括SEQ ID NO:25的序列，LFR1包括选自由SEQ ID NO:26组成的组的序列，LFR2包括选自由SEQ ID NO:27和36组成的组的序列，LFR3包括选自由SEQ ID NO:28和37-38、39、45组成的组的序列，并且LFR4包括SEQ ID NO:29的序列。
在一些实施方案中，本公开的人源化抗PD-L1抗体和其抗原结合片段包括包含在重链可变区中的HFR1、HFR2、HFR3和/或HFR4序列，所述重链可变区选自由以下组成的组：Hu4B6_Hg.2(SEQ ID NO:58)、AM4B6_Hg.3(SEQ ID NO:59)、AM4B6_Hg.5(SEQ ID NO:60)。
在一些实施方案中，本公开的人源化抗PD-L1抗体和其抗原结合片段包括包含在轻链可变区中的LFR1、LFR2、LFR3和/或LFR4序列，所述轻链可变区选自由以下组成的组：AM4B6_La.1(SEQ ID NO:62)、AM4B6_La.2(SEQ ID NO:63)、AM4B6_La.4(SEQ ID NO:64)和AM4B6_La.6(SEQ ID NO:65)。
在一些实施方案中，所述重链可变区包括选自由以下组成的组的序列：SEQ ID NO:46、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60和与其具有至少80％序列同一性的其同源序列。在一些实施方案中，所述轻链可变区包括选自由以下组成的组的序列：SEQ ID NO:47、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65和与其具有至少80％序列同一性的其同源序列。在一些实施方案中，所述针对PD-L1的抗体或其抗原结合片段包括选自由以下组成的组的一对重链可变区和轻链可变区序列：SEQ ID NO:49/54、50/54、51/54、52/54、49/55、50/55、51/55、52/55、58/62、58/63、58/64、58/65、59/62、59/63、59/64、59/65、60/62、60/63、60/64和60/65。
这些示例性人源化抗PD-L1抗体保留了对PD-L1的特异性结合能力或亲和力，并且在所述方面优于亲本小鼠抗体4B6。
在一些实施方案中，本文提供的抗PD-L1抗体和抗原结合片段包括重链可变结构域的全部或一部分和/或轻链可变结构域的全部或一部分。在一个实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是由本文提供的重链可变结构域的全部或一部分组成的单结构域抗体。此类单结构域抗体的更多信息可在本领域中获得(参见例如，美国专利第6,248,516号)。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段进一步包括免疫球蛋白(Ig)恒定区，所述恒定区任选地进一步包括重链和/或轻链恒定区。在一些实施方案中，重链恒定区包括CH1、铰链和/或CH2-CH3区(或任选的CH2-CH3-CH4区)。在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段包括人IgG1、IgG2、IgG3、IgG4、 IgA1、IgA2或IgM的重链恒定区。在一些实施方案中，轻链恒定区包括Cκ或Cλ。本文提供的抗PD-L1抗体或其抗原结合片段的恒定区可以与野生型恒定区序列相同或在一个或多个突变中不同。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段对人PD-L1具有特异性结合亲和力，这足以提供诊断和/或治疗用途。
本文提供的抗PD-L1抗体或其抗原结合片段可以是单克隆抗体、多克隆抗体、人源化抗体、嵌合抗体、重组抗体、双特异性抗体、多特异性抗体、标记抗体、二价抗体、抗独特型抗体或融合蛋白。重组抗体是使用重组方法在体外而不是在动物体内制备的抗体。
在一些实施方案中，PD-L1结合部分包括抗PD-L1抗体或其抗原结合片段，所述抗体或其抗原结合片段与包括选自由以下组成的组的一对重链可变区和轻链可变区序列的抗体或其抗原结合片段竞争结合PD-L1：SEQ ID NO:49/54、50/54、51/54、52/54、49/55、50/55、51/55、52/55、58/62、58/63、58/64、58/65、59/62、59/63、59/64、59/65、60/62、60/63、60/64和60/65。
x.抗体变体
本文提供的抗PD-L1抗体和其抗原结合片段还涵盖本文提供的抗体序列的各种变体。
在一些实施方案中，所述抗体变体在以上的表1提供的多个CDR序列中的一个或多个CDR序列中、以上的表3a、3b和5提供的重链可变区或轻链可变区的多个非CDR序列中的一个或多个非CDR序列中、和/或恒定区(例如，Fc区)中包括一个或多个修饰或取代。此类变体保留了其亲本抗体对PD-L1的结合特异性，但具有一种或多种由修饰或取代赋予的期望性质。例如，抗体变体可以具有改善的抗原结合亲和力、改善的糖基化模式、降低的糖基化风险、减少的脱氨基作用、减少或耗竭的效应子功能、改善的FcRn受体结合、增加的药代动力学半衰期、pH敏感性和/或与缀合的相容性(例如，一个或多个引入的半胱氨酸残基)。
可以使用本领域已知的方法，例如“丙氨酸扫描诱变”，筛选亲本抗体序列以鉴定合适的或优选的待修饰或取代的残基(参见例如Cunningham和Wells(1989)《科学》,244:1081-1085)。简要地说，可以鉴定靶标残基(例如带电的残基，如Arg、Asp、His、Lys和Glu)并由中性或带负电的氨基酸(例如丙氨酸或聚丙氨酸)替代，并且产生经修饰的抗体，并且针对所关注性质对其进行筛选。如果在特定的氨基酸位置处的取代表现出所关注功能性改变，则所述位置可以被鉴定为潜在的用于修饰或取代的残基。可以通过用另一种残基(例如半胱氨酸残基、带正电荷的残基等)取代来进一步评估所述潜在的残基。
xi.亲和性变体
抗体的亲和性变体可以在上表1中提供的一个或多个CDR序列、上表3b和4中提供的一个或多个FR序列或上表5中提供的重链或轻链可变区序列中含有修饰或取代。本领域技术人员可以基于上表1中的CDR序列和上表5中的可变区序列容易地鉴定FR序列，因为本领域公知在可变区中，CDR区侧接两个FR区。亲和性变体保留了亲本抗体对PD-L1的特异性结合亲和力，或者甚至比亲本抗体具有更高的PD-L1特异性结合亲和力。在一些实施方案中，CDR序列、FR序列或可变区序列中的至少一个(或全部)取代包括保守取代。
本领域技术人员将理解，在上表1和3a中提供的CDR序列以及上表5中提供的可变区序列中，一个或多个氨基酸残基可以被取代，但所得抗体或抗原结合片段仍保留对PD-L1的结合亲和力或结合能力或者甚至具有改进的结合亲和力或结合能力。可以使用本领域已知的各种方法来实现此目的。例如，可以生成抗体变体(如Fab或scFv变体)的文库，并用噬菌体展示技术表达，随后针对与PD-L1结合的亲和力对其进行筛选。再例如，计算机软件可以用于虚拟模拟抗体与PD-L1的结合，并鉴定抗体上的形成结合界面的氨基酸残基。在取代中可以避开此类残基以防止结合亲和性的降低，或者可以作为取代的靶标以获得更强的结合。
在一些实施方案中，本文提供的人源化抗PD-L1抗体或其抗原结合片段在多个CDR序列中的一个或多个CDR序列和/或多个FR序列中的一个或多个FR序列中包括一个或多个氨基酸残基取代。在一些实施方案中，亲和性变体在CDR序列和/或FR序列中总共包括不超过20、15、10、9、8、7、6、5、4、3、2或1个取代。
在一些实施方案中，抗PD-L1抗体或其抗原结合片段包括1、2或3个CDR序列，所述CDR序列与以上表1和3a中所列出的一个(或多个)序列具有至少80％(例如，至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列同一性，但仍以相对于其亲本抗体而言相似或甚至更高的水平保留了对PD-L1的特异性结合亲和力。
在一些实施方案中，抗PD-L1抗体或其抗原结合片段包括一个或多个可变区序列，所述一个或多个可变区序列与以上表5中所列出的一个(或多个)序列具有至少80％(例如，至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列同一性，但仍以相对于其亲本抗体而言相似或甚至更高的水平保留了对PD-L1的特异性结合亲和力。在一些实施方案中，在上表5中列出的可变区序列中总共取代、插入或缺失了1到10个氨基酸。在一些实施方案中，取代、插入或缺失发生在CDR之外的区中(例如，在FR中)。
xii.糖基化变体
本文提供的抗PD-L1抗体或其抗原结合片段还涵盖糖基化变体，可以获得所述糖基化变体以增加或减少所述抗体或其抗原结合片段的糖基化程度。
抗PD-L1抗体或其抗原结合片段可以包括引入或去除糖基化位点的一个或多个修饰。糖基化位点是具有侧链的氨基酸残基，碳水化合物部分(例如，寡糖结构)可以连接到所述侧链。抗体的糖基化通常是N连接的或O连接的。N连接是指碳水化合物部分连接到天冬酰胺残基(例如，如天冬酰胺-X-丝氨酸和天冬酰胺-X-苏氨酸等三肽序列中的天冬酰胺残基)的侧链，其中X是除了脯氨酸之外的任何氨基酸。O连接的糖基化是指将N-乙酰半乳糖胺、半乳糖或木糖之一与羟基氨基酸连接，最常见的是与丝氨酸或苏氨酸连接。可以很方便地去除天然糖基化位点，例如通过改变氨基酸序列，使得存在于所述序列中的上述三肽序列(对于N连接的糖基化位点)或者丝氨酸或苏氨酸残基(对于O连接的糖基化位点)中的一个被取代。可以通过引入此类三肽序列或者丝氨酸或苏氨酸残基以相似的方式产生新的糖基化位点。
在一些实施方案中，本文提供的抗PD-L1抗体和抗原结合片段包括一个或多个突变以去除一个或多个脱酰胺位点。在一些实施方案中，本文提供的抗PD-L1抗体和抗原结合片 段在重链中的G55(例如，G55A)处包括突变。测试了这些突变并且据信这些突变不会负面影响本文提供的抗体的结合亲和力。
xiii.半胱氨酸工程化的变体
本文提供的抗PD-L1抗体或其抗原结合片段还涵盖半胱氨酸工程化的变体，所述变体包括一个或多个引入的游离半胱氨酸氨基酸残基。
游离半胱氨酸残基是不为二硫键的一部分的半胱氨酸残基。半胱氨酸工程化的变体可用于通过例如马来酰亚胺或卤乙酰基在经工程化的半胱氨酸的位点处与例如细胞毒性化合物和/或成像化合物、标记或放射性同位素等缀合。用于工程化抗体或其抗原结合片段以引入游离半胱氨酸残基的方法是本领域已知的，参见例如，WO2006/034488。
xiv.Fc变体
本文提供的抗PD-L1抗体或其抗原结合片段还涵盖Fc变体，所述Fc变体在Fc区和/或铰链区处包括一个或多个氨基酸残基修饰或取代，例如以提供改变的效应子功能，如ADCC和CDC。通过抗体工程改变ADCC活性的方法在本领域已有描述，参见例如Shields RL.等人,《生物化学杂志(J Biol Chem.)》2001.276(9):6591-604；Idusogie EE.等人,《免疫学杂志(J Immunol.)》2000.164(8):4178-84；Steurer W.等人,《免疫学杂志》1995,155(3):1165-74；Idusogie EE.等人,《免疫学杂志》2001,166(4):2571-5；Lazar GA.等人,《美国国家科学院院刊(PNAS)》,2006,103(11):4005-4010；Ryan MC.等人,《分子癌症治疗(Mol.Cancer Ther.)》,2007,6:3009-3018；Richards JO,.等人,《分子癌症治疗》2008,7(8):2517-27；Shields R.L.等人,《生物化学杂志》,2002,277:26733-26740；Shinkawa T.等人,《生物化学杂志》,2003,278:3466-3473。
本文提供的抗体或抗原结合片段的CDC活性也可以例如通过改善或减少C1q结合和/或CDC来改变(参见例如WO99/51642；Duncan和Winter《自然》322:738-40(1988)；美国专利第5,648,260号；美国专利第5,624,821号)；和关于Fc区变体的其它实例的WO94/29351。可以将选自Fc区的氨基酸残基329、331和322的一个或多个氨基酸替换为不同的氨基酸残基，以改变C1q结合和/或减少或消除补体依赖性细胞毒性(CDC)(参见Idusogie等人的美国专利第6,194,551号)。也可以引入一个或多个氨基酸取代，以改变抗体固定补体的能力(参见Bodmer等人的PCT公开WO 94/29351)。
在一些实施方案中，本文提供的Fc变体相对于野生型Fc(例如，IgG1的Fc)具有降低的效应子功能，并且在选自由以下组成的组的位置处包括一个或多个氨基酸取代：Fc区的220、226、228、229、233、234、235、236、237、238、267、268、269、270、297、309、318、320、322、325、328、329、330、331和332(参见WO2016/196228；Richards等人(2008)《分子癌症治疗学(Mol.Cancer Therap.)》7:2517；Moore等人(2010)《单克隆抗体(mAbs)》2:181；和Strohl(2009)《生物技术当前述评(Current Opinion in Biotechnology)》20:685-691)，其中Fc区中残基的编号为如Kabat中的EU索引的编号。减少的效应子功能的示例性取代包括但不限于220S、226S、228P、229S、233P、234V、234G、234A、234F、234A、235A、235G、235E、236E、236R、237A、237K、238S、267R、268A、268Q、269R、297A、297Q、297G、309L、318A、322A、325L、328R、330S、331S或其任何组合(参见WO2016/196228；和Strohl(2009)《生物技术当前述评》20:685-691)。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段具有降低的效应子功能，并且在IgG1中在选自由以下组成的组的位置处包括一个或多个氨基酸取代：234、235、237、238、268、297、309、330和331。在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是IgG1同种型并且包括选自由以下组成的组的一个或多个氨基酸取代：N297A、N297Q、N297G、L235E、L234A、L235A、L234F、L235E、P331S和其任何组合。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是IgG1同种型并且包括L234A和L235A突变。在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是IgG1同种型并且包括L234F、L235E和P331S。定位于Fc结构域的CH2区中的取代的L234F、L235E和P331S集合(也称为FES三重突变)可以消除FCγR和C1q结合，从而使得抗体无法引发ADCC或CDC(Oganesyan等人,《晶体学报D(Acta Crystallogr.D)》64:700-704(2008))。PCT/US2013/36872已经表明，与野生型亲本分子(例如，野生型IgG1Fc)相比，在变体Fc结构域(例如，抗体中的变体Fc结构域)中组合这些突变使Fc结构域具有降低的热稳定性。在一些实施方案中，所述Fc变体包括SEQ ID NO:81的氨基酸序列。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是IgG2同种型，并且包括选自由以下组成的组的一个或多个氨基酸取代：H268Q、V309L、A330S、P331S、V234A、G237A、P238S、H268A和其任何组合(例如，H268Q/V309L/A330S/P331S、V234A/G237A/P238S/H268A/V309L/A330S/P331S)。在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是IgG4同种型，并且包括选自由以下组成的组的一个或多个氨基酸取代：S228P、N297A、N297Q、N297G、L235E、F234A、L235A和其任何组合。在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是IgG2/IgG4交叉同种型。Rother RP等人,《自然生物技术(Nat Biotechnol)》25:1256–1264(2007)中描述了IgG2/IgG4交叉同种型的实例。
在一些实施方案中，抗PD-L1抗体或其抗原结合片段包括改善与新生儿Fc受体(FcRn)的pH依赖性结合的一个或多个氨基酸取代。此类变体可以具有延长的药代动力学半衰期，因为它在酸性pH下与FcRn结合，使其得以免于在溶酶体中降解，并且随后被转移并释放到细胞外。工程化抗体或其抗原结合片段以提高与FcRn的结合亲和力的方法是本领域众所周知的，参见例如Vaughn,D.等人,《结构》,6(1):63-73,1998；Kontermann,R.等人,《抗体工程(Antibody Engineering)》,第1卷,第27章:将Fc区工程化以改善PK,由施普林格(Springer)出版,2010年；Yeung,Y.等人,《癌症研究(Cancer Research)》,70:3269-3277(2010)；和Hinton,P.等人,《免疫学杂志(J.Immunology)》,176:346-356(2006)。
在一些实施方案中，抗PD-L1抗体或其抗原结合片段在Fc区的界面中包括一个或多个氨基酸取代以便于和/或促进异二聚体化。这些修饰包括将突起引入到第一Fc多肽中并且将空腔引入到第二Fc多肽中，其中突起可以定位于空腔中，以便促进第一Fc多肽与第二Fc多肽的相互作用以形成异二聚体或复合物。产生具有这些修饰的抗体的方法是本领域已知的，例如，如美国专利第5,731,168号中所述。
xv.抗原结合片段
本文提供的双功能分子中的PD-L1结合部分还涵盖抗PD-L1抗原结合片段。各种类型的抗原结合片段是本领域已知的并且可以基于本文提供的抗PD-L1抗体和其不同变体(如 亲和性变体、糖基化变体、Fc变体、半胱氨酸工程化的变体等)开发，所述抗体包括例如其CDR在上表1和3a中示出并且其可变序列在表2和5中示出的示例性抗体。
在一些实施方案中，本文提供的抗PD-L1抗原结合片段是双抗体、Fab、Fab'、F(ab')2、Fd、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双抗体(ds双抗体)、单链抗体分子(scFv)、scFv二聚体(二价双抗体)、多特异性抗体、骆驼化单结构域抗体、纳米抗体、结构域抗体和二价结构域抗体。
多种技术可以用于产生此类抗原结合片段。说明性方法包括完整抗体的酶消化(参见例如Morimoto等人,《生物化学与生物物理方法杂志(Journal of Biochemical and Biophysical Methods)》24:107-117(1992)；和Brennan等人,《科学》,229:81(1985))、通过如大肠杆菌(E.Coli)等宿主细胞进行的重组表达(例如，对于Fab、Fv和ScFv抗体片段)、如上文讨论的从噬菌体展示文库筛选(例如，对于ScFv)以及两个Fab'-SH片段的化学偶联以形成F(ab')2片段(Carter等人,《生物/技术(Bio/Technology)》10:163-167(1992))。用于产生抗体片段的其它技术对本领域技术人员来说是显而易见的。
在一些实施方案中，抗原结合片段是scFv。以下中描述了scFv的产生：例如WO93/16185；美国专利第5,571,894号；和第5,587,458号。ScFv可以在氨基端或羧基端与效应蛋白融合以提供融合蛋白(参见例如《抗体工程》,Borrebaeck编辑)。
在一些实施方案中，本文提供的抗PD-L1抗体或其抗原结合片段是二价的、四价的、六价的或多价的。任何超过二价的分子被认为是多价的，涵盖例如三价、四价、六价等。
如果两个结合位点都特异性地结合到同一抗原或同一表位，则二价分子可以是单特异性的。在一些实施方案中，这提供了比单价对应物更强的与抗原或表位的结合。类似地，多价分子也可以是单特异性的。在一些实施方案中，在二价或多价抗原结合部分中，结合位点的第一价和结合位点的第二价在结构上相同(即，具有相同的序列)或在结构上不同(即，具有不同的序列，但具有相同的特异性)。
如果两个结合位点对不同的抗原或表位具有特异性，则二价也可以是双特异性的。这也适用于多价分子。例如，当两个结合位点对第一抗原(或表位)是单特异性的并且第三结合位点对第二抗原(或表位)是特异性的时，三价分子可以是双特异性的。
xvi.双特异性抗体
在一些实施方案中，抗PD-L1抗体或其抗原结合片段是双特异性的。在一些实施方案中，除了本文提供的第二部分之外，PD-L1结合抗体或其抗原结合片段进一步连接到与所述抗PD-L1抗体或其抗原结合片段具有不同结合特异性的额外功能结构域。
在一些实施方案中，本文提供的双特异性抗体或其抗原结合片段能够特异性结合到除PD-L1之外(以及除第二部分结合的靶标之外)的第二抗原或PD-L1上的第二表位(或第二部分结合的靶标上的第二表位)。
xvii.双功能分子
在一些实施方案中，本文提供的双功能分子能够与PD-L1和由第二部分结合的靶标结合。在一些实施方案中，本文提供的双功能分子能够与PD-L1和TGFβ两者结合，或与PD- L1和IL-1两者结合，或与PD-L1和IL-1R两者结合，或与PD-L1和MHCII两者结合，或与PD-L1和CD47两者结合，或与PD-L1和SIRPα两者结合。
在一些实施方案中，如通过ELISA测定所测量的，本公开的靶向PD-L1和TGFβ的双功能分子以不超过2.0nM(例如，不超过2.0nM、不超过1.2nM、不超过1.1nM、不超过1.0nM、不超过0.9nM、不超过0.8nM)的EC50与人TGFβ1特异性地结合。在一些实施方案中，如通过ELISA测定所测量的，本公开的靶向PD-L1和TGFβ的蛋白质能够同时与PD-L1和TGFβ结合。在一些实施方案中，如通过Biacore测定所测量的，本公开的靶向PD-L1和TGFβ的双功能分子能够以不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM或不超过0.4nM的KD值与人PD-L1特异性地结合。在一些实施方案中，如通过ELISA测定所测量的，本公开的靶向PD-L1和TGFβ的双功能分子能够以不超过2.0nM(例如，不超过2.0nM、不超过1.2nM、不超过1.1nM、不超过1.0nM、不超过0.9nM、不超过0.8nM)的KD值与人TGFβ1特异性地结合。
在一些实施方案中，本公开的靶向PD-L1和TGFβ的双功能分子能够以剂量依赖性方式对肿瘤生长抑制表现出协同作用。
在一些实施方案中，与仅包括免疫检查点分子的分子相比，本公开的靶向PD-L1和TGFβ的双功能分子能够表现出抗肿瘤免疫细胞向肿瘤微环境中的浸润增强。
在一些实施方案中，本公开的靶向PD-L1和TGFβ的双功能分子能够选择性地降低血浆中至少90％(例如，至少80％、70％、60％、50％、40％、30％或20％)的TGFβ1并且此类降低可以维持至少10、14或21天。
在一些实施方案中，所述双功能分子包括异二聚体重链。所述重链就所述第二部分的存在或位置而言是异二聚体的。在一些实施方案中，所述异二聚体重链包括具有所述第二部分的一条重链，而另一条重链不具有所述第二部分，其中所述第二部分包括CD47结合结构域(例如，可溶性SIRPα)或SIRPα结合结构域。
在所述双功能分子中，所述异二聚体重链包括具有所述第二部分的一条重链，而另一条重链不具有所述第二部分。所述异二聚体重链可以进一步包括以阻碍同二聚体化和/或有利于异二聚体化的方式缔合的异二聚体Fc区。例如，可以选择异二聚体Fc区，使得它们不相同并且它们优先在彼此之间形成异二聚体而不是在它们自身内形成同二聚体。在一些实施方案中，所述异二聚体Fc区能够通过形成杵臼结构、疏水相互作用、静电相互作用、亲水相互作用或增加的柔性缔合成异二聚体。在一些实施方案中，异二聚体Fc区包括分别突变成能够形成杵臼结构的CH2和/或CH3结构域。杵结构可以通过将第一CH2/CH3多肽中的小氨基酸残基替换为较大的氨基酸残基来获得，并且臼结构可以通过将较大的残基替换为较小的残基来获得。在一些实施方案中，异二聚体Fc区包括含有S354C和T366W取代(SEQ ID NO:96，杵结构)的IgG1同种型的第一CH3结构域和含有Y349C、T366S、L368A和Y407V取代(SEQ ID NO:97，臼结构)的IgG1同种型的第二CH3结构域。
在一些实施方案中，所述双功能分子包含SEQ ID NO:118或SEQ ID NO:120的氨基酸序列的重链，和/或包含SEQ ID NO:119或SEQ ID NO:121的氨基酸序列的轻链。
xviii.缀合物
在一些实施方案中，双功能分子进一步包括一个或多个缀合物部分。缀合物部分可以连接到双功能分子。缀合物部分是可以连接到双功能分子的部分。设想多种缀合物部分可以连接到本文提供的双功能分子(参见例如，“缀合物疫苗(Conjugate Vaccines)”,对微生物学和免疫学的贡献(Contributions to Microbiology and Immunology),J.M.Cruse和R.E.Lewis,Jr.(编辑),纽约Carger出版社(Carger Press,New York),(1989))。这些缀合物部分可以通过共价结合、亲和结合、嵌入、配位结合、络合、缔合、共混或添加等方法与双功能分子连接。在一些实施方案中，双功能分子可以通过接头连接到一种或多种缀合物。
在一些实施方案中，本文提供的双功能分子可以被工程化成在表位结合部分之外含有特异性位点，所述特异性位点可以用于结合到一个或多个缀合物部分。例如，此类位点可以包括一个或多个反应性氨基酸残基，例如半胱氨酸或组氨酸残基，以促进与缀合物部分的共价连接。
在一些实施方案中，双功能分子可以间接地或通过另一缀合物部分连接到缀合物部分。例如，本文提供的双功能分子可以与生物素缀合，然后间接缀合到与抗生物素蛋白缀合的第二缀合物。在一些实施方案中，缀合物部分包括清除修饰剂(例如，延长半衰期的聚合物，如PEG)、化学治疗剂、毒素、放射性同位素、镧系元素、可检测标记(例如，发光标记、荧光标记、酶底物标记)、DNA烷基化剂、拓扑异构酶抑制剂、微管蛋白结合剂、纯化部分或其它抗癌药物。
“毒素”可以是对细胞有害或可以损伤或杀死细胞的任何药剂。毒素的实例包括但不限于紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙锭、吐根碱、丝裂霉素、依托泊苷(etoposide)、替尼泊苷(tenoposide)、长春新碱(vincristine)、MMAE、MMAF、DM1、长春碱(vinblastine)、秋水仙碱(colchicin)、阿霉素(doxorubicin)、柔红霉素(daunorubicin)、二羧基炭疽菌素二酮(dihydroxy anthracin dione)、米托蒽醌(mitoxantrone)、光神霉素(mithramycin)、放线菌素D(actinomycin D)、1-去氢睾酮、糖皮质激素、普鲁卡因(procaine)、丁卡因(tetracaine)、利多卡因(lidocaine)、普萘洛尔(propranolol)、嘌呤霉素(puromycin)及其类似物、抗代谢物(例如甲氨蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶达卡巴嗪)、烷化剂(例如氮芥、塞替派苯丁酸氮芥(thioepa chlorambucil)、美法仑(melphalan)、卡莫司汀(carmustine)(BSNU)和洛莫司汀(lomustine)(CCNU)、环磷酰胺、白消安(busulfan)、二溴甘露醇、链脲霉素(streptozotocin)、丝裂霉素C(mitomycin C)和二氯二胺铂(II)(DDP顺铂)、蒽环霉素(anthracycline)(例如柔红霉素(以前的道诺霉素(daunomycin)和阿霉素)、抗生素(例如更生霉素(dactinomycin)(以前的放线菌素)、博来霉素(bleomycin)、光神霉素和氨茴霉素(anthramycin)(AMC))、抗有丝分裂剂(例如长春新碱和长春碱)、拓扑异构酶抑制剂和微管蛋白结合剂。
可检测标记的实例可以包括荧光标记(例如，荧光素、罗丹明(rhodamine)、丹酰、藻红蛋白或德克萨斯红(Texas Red))、酶底物标记(例如，辣根过氧化物酶、碱性磷酸酶、荧光素酶、葡糖淀粉酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶)、放射性同位素(例如，123I、124I、125I、131I、35S、3H、111In、112In、14C、64Cu、67Cu、86Y、88Y、90Y、177Lu、211At、186Re、188Re、153Sm、212Bi和32P、其它镧系元素)、发光标记、发色团部分、地高辛(digoxigenin)、生物素/抗生物素蛋白、DNA分子或用于检测的金。
在一些实施方案中，缀合物部分可以是帮助增加双功能分子的半衰期的清除修饰剂。说明性实例包括水溶性聚合物，例PEG、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、乙二醇/丙二醇的共聚物等。聚合物可以具有任何分子量，并且可以是支化的或未支化的。连接到抗体的聚合物的数量可能会有所不同，并且如果连接的聚合物多于一种，则它们可以是相同或不同的分子。
在一些实施方案中，缀合物部分可以是纯化部分，如磁珠。
在一些实施方案中，本文提供的双功能分子用作缀合物的基础。
III.药物制剂
本公开的制剂还可包含其他药学上可接受的辅料、诸如溶媒(含水或非水)、渗透压调节剂、降低粘度的辅料、防腐剂、润湿剂、乳化剂和分散剂。可通过灭菌程序并且通过加入各种抗菌和抗真菌剂(例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸等)以确保无微生物存在。防腐剂通常以约0.001％(w/v)至约2％(w/v)的浓度使用。防腐剂包括但不限于乙醇、苯甲醇、苯酚、间甲酚、对氯间甲酚、对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、苯扎氯铵。
本公开的制剂表现出良好的稳定性和施用便利性。在本公开的一些实施方案中、双功能分子、尤其是PD-L1/TGF-β蛋白可以保持长期稳定、满足临床使用需求。此外、本公开提供的方案还解决了聚山梨酯80降解的问题、避免聚山梨酯80的降解产物可能对蛋白质量产生影响。
在一些实施方案中，本公开的制剂是液体制剂。例如所述液体制剂是注射剂。例如所述液体制剂包含注射用水。
在一些实施方案中、本公开的制剂、例如液体制剂在约-80℃至约45℃、例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约37℃、约42℃或约45℃的条件下储存至少14天、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月、至少36个月、或更长时间后、用SEC法检测、制剂中双功能蛋白质分子的纯度下降不超过10％、例如不超过5％、4％、3％、2％、1％、0.5％或0.1％。
在一个实施方案中、本公开的制剂、例如液体制剂在约-80℃至约45℃、例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约37℃、约42℃或约45℃的条件下储存至少14天、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月、至少36个月、或更长时间后、用非还原性CE-SDS法检测、双功能蛋白质分子的纯度下降不超过10％、例如不超过5％、4％、3％、2％、1％、0.5％或0.1％。
IV.治疗方法和用途
本公开提供了治疗、预防或减轻受试者的PD-L1相关疾病的方法，所述方法包括向所述受试者施用治疗有效量的本文提供的制剂。
在一些实施方案中，受试者是人。
PD-1相关病状和病症可以是免疫相关疾病或病症、癌症、自身免疫疾病或传染病。
在一些实施方案中，PD-1相关病状和病症包括癌症，例如，非小细胞肺癌、小细胞肺癌、肾细胞癌、结直肠癌、卵巢癌、乳腺癌、胰腺癌、胃癌、膀胱癌、食道癌、间皮瘤、黑色素瘤、头颈癌、甲状腺癌、肉瘤、前列腺癌、胶质母细胞瘤、宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、有真菌特征的真菌病(mycoses fungoids)、默克尔细胞癌(merkel cell cancer)和其它血液学恶性肿瘤，如经典霍奇金淋巴瘤(CHL)、原发性纵隔大B细胞淋巴瘤、富含T细胞/组织细胞的B细胞淋巴瘤、EBV阳性和阴性PTLD以及EBV相关的弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关的原发性渗出性淋巴瘤、霍奇金淋巴瘤、中枢神经系统(CNS)赘生物，如原发性CNS淋巴瘤、脊髓轴肿瘤、脑干胶质瘤。在一些实施方案中，肿瘤和癌症是转移性的，尤其是表达PD-L1的转移性肿瘤。
在一些实施方案中，PD-1相关病状和病症包括自身免疫疾病。自身免疫疾病包括但不限于获得性免疫缺陷综合征(AIDS，它是一种具有自身免疫组分的病毒性疾病)、斑秃、强直性脊柱炎、抗磷脂综合征、自身免疫性阿狄森氏病(autoimmune Addison's disease)、自身免疫性糖尿病、自身免疫性溶血性贫血、自身免疫性肝炎、自身免疫性内耳病(AIED)、自身免疫性淋巴增生综合征(ALPS)、自身免疫性血小板减少性紫癜(ATP)、白塞氏病(Behcet's disease)、心肌病、口炎性腹泻-疱疹样皮炎；慢性疲劳免疫功能障碍综合征(CFIDS)、慢性炎性脱髓鞘性多发性神经病(CIPD)、瘢痕性类天疱疮、冷凝集素病、肢端硬皮综合征(crest syndrome)、克罗恩氏病(Crohn's disease)、德戈斯病(Degos'disease)、幼年性皮肌炎、盘状狼疮、原发性混合性冷球蛋白血症、纤维肌痛-纤维肌炎、格雷夫斯氏病(Graves'disease)、吉兰-巴利综合征(Guillain-Barre syndrome)、桥本氏甲状腺炎(Hashimoto's thyroiditis)、特发性肺纤维化、特发性血小板减少性紫癜(ITP)、IgA肾病、胰岛素依赖型糖尿病、幼年型慢性关节炎(斯蒂尔氏病(Still's disease))、幼年型类风湿性关节炎、梅尼埃氏病(Meniere's disease)、混合性结缔组织病、多发性硬化症、重症肌无力、严重贫血(pemacious anemia)、结节性多动脉炎、多软骨炎、多腺体综合征、风湿性多肌痛、多肌炎和皮肌炎、原发性无丙种球蛋白血症、原发性胆汁性肝硬化、银屑病、银屑病关节炎、雷诺氏现象(Raynaud's phenomena)、莱特尔氏综合征(Reiter's syndrome)、风湿热、类风湿性关节炎、结节病、硬皮病(进行性系统性硬化症(PSS)，也称为系统性硬化症(SS))、干燥综合征(Sjogren's syndrome)、僵人综合征、系统性红斑狼疮、高安氏动脉炎(Takayasu arteritis)、颞动脉炎/巨细胞动脉炎、溃疡性结肠炎、葡萄膜炎、白癜风和韦格纳氏肉芽肿(Wegener's granulomatosis)。
在一些实施方案中，PD-1相关病状和病症包括传染病。传染病包括例如，慢性病毒感染，例如，真菌感染、寄生虫/原生动物感染或慢性病毒感染，例如，疟疾、粗球孢子菌病(coccidioiodmycosis immitis)、组织胞浆菌病、甲真菌病、曲霉菌病、芽生菌病、白色念珠菌病、副球孢子菌病(paracoccidioiomycosis)、微孢子虫病、棘阿米巴角膜炎(Acanthamoeba keratitis)、阿米巴病、蛔虫病、巴贝斯虫病、小袋虫病、浣熊贝蛔虫病(Baylisascariasis)、南美锥虫病(Chagas disease)、华支睾吸虫病、锥蝇属(Cochliomyia)、隐孢子虫病、裂头绦虫病、麦地那龙线虫病、包虫病、象皮病、蛲虫病、肝片吸虫病、姜片虫病、丝虫病、贾第虫病、颚口线虫病(Gnathostomiasis)、膜壳绦虫病、等孢球虫病、片山热(Katayama fever)、利什曼病、莱姆病(Lyme disease)、后殖吸虫病、 蝇蛆病、盘尾丝虫病、虱病、疥疮、血吸虫病、昏睡病、类圆线虫病、绦虫病、弓蛔虫病、弓形体病、旋毛虫病、鞭虫病、锥虫病、蠕虫感染、乙型肝炎(HBV)感染、丙型肝炎(HCV)感染、疱疹病毒感染、艾普斯登-巴尔病毒(Epstein-Barr virus)感染、HIV-1感染、HIV-2感染、巨细胞病毒感染、单纯疱疹病毒I型感染、单纯疱疹病毒II型感染、人乳头瘤病毒感染、腺病毒感染、卡波西西肉瘤相关疱疹病毒流行病感染、细环病毒(转矩特诺病毒(Torquetenovirus))感染、人T嗜淋巴细胞病毒I感染、人T嗜淋巴细胞病毒II感染、水痘带状疱疹病毒感染、JC病毒感染或BK病毒感染。
在一些实施方案中，PD-L1相关疾病是表达PD-L1的癌症或过度表达PD-L1的癌症。“表达PD-L1的癌症”是一种涉及癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白的癌症。“过度表达PD-L1的癌症”是与相同组织类型的非癌细胞相比，在癌症或肿瘤细胞的细胞表面具有显著更高水平的PD-L1的癌症。
PD-L1表达或过度表达可以在诊断或预后测定中通过评估存在于细胞表面上的PD-L1水平增加来确定(例如，通过免疫组织化学测定；IHC)。可替代地或另外地，可以例如通过荧光原位杂交(FISH；参见1998年10月出版的WO98/45479)、southern印迹或聚合酶链反应(PCR)技术，如实时定量PCR(RT-PCR)来测量细胞中编码PD-L1的核酸的水平。还可以通过测量如血清等生物流体中的脱落抗原(例如，PD-L1胞外结构域或可溶性PD-L1)来研究PD-L1过度表达。除了上述测定之外，熟练的从业者还可获得各种体内测定。例如，可以将患者体内的细胞暴露于抗PD-L1抗体，所述抗体任选地用可检测标记(例如，放射性同位素)进行标记，并且可以例如通过外部扫描放射性或通过分析取自先前暴露于抗体的患者的活组织检查评估抗体与患者细胞的结合。
在一些实施方案中，受试者已被鉴定为可能对PD-1拮抗剂有应答。所关注生物样品上PD-L1的存在或水平可以指示生物样品所源自的受试者是否可能对PD-1拮抗剂有应答。在一些实施方案中，测试样品源自癌细胞或组织、或肿瘤浸润免疫细胞。在一些实施方案中，测试生物样品中PD-L1的存在或上调水平指示应答的可能性。如本文所使用的，术语“上调”是指与使用相同抗体检测的参考样品中的PD-L1蛋白水平相比，测试样品中PD-L1的蛋白水平总体增加不少于10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％或更大。参考样品可以是从健康或非患病个体获得的对照样品，或者是从测试样品所获自的同一个体获得的健康或非患病样品。例如，参考样品可以是与测试样品(例如，肿瘤)相邻或在测试样品附近的非患病样品。
在一些实施方案中，PD-L1相关疾病对PD-L1/PD-1单一疗法具有抗性。如本文所使用的，“PD-L1/PD-1单一疗法”是指通过抑制或减少PD-L1和PD-1相互作用或信号传导而起作用的单一疗法。示例性PD-L1/PD-1单一疗法可以包括抗PD-L1抗体疗法、抗PD-1抗体疗法或涉及针对PD-1或PD-L1的小分子抑制剂的单一疗法。“抗性”意指所述疾病对PD-L1/PD-1单一疗法没有应答或敏感性或具有降低的应答或敏感性。降低的应答可以通过例如需要增加剂量以实现给定功效来指示。在一些实施方案中，所述疾病可能对PD-L1/PD-1单一疗法无应答。例如，尽管使用PD-L1/PD-1单一疗法治疗，癌细胞或肿瘤大小仍会增加，或者疾病显示回归到其以前的状态，例如，在部分康复后恢复先前的症状。对PD-L1/PD-1单一疗法的抗性可以是新发的或获得性的。
在另一方面，本公开提供了治疗、预防或减轻受试者的疾病或病状的方法，所述疾病或病状将受益于免疫抑制性细胞因子的抑制、持续免疫应答的诱导或抗肿瘤免疫的刺激，所述方法包括施用有效量的本文提供的制剂。
在一些实施方案中，所述免疫抑制性细胞因子是TGFβ或IL-1。在一些实施方案中，所述免疫抑制性细胞因子是TGFβ1或IL-1β。
在一些实施方案中，所述疾病或病状是TGFβ相关疾病或病状。在一些实施方案中，所述TGFβ相关疾病是癌症、纤维化疾病或肾病。
在一些实施方案中，所述TGFβ相关疾病是癌症。在一些实施方案中，所述癌症选自由以下组成的组：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤、淋巴瘤、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头颈癌、肝癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑色素瘤、基底细胞癌、皮肤癌、鳞状细胞皮肤癌、皮肤纤维肉瘤突起、默克尔细胞癌、胶质母细胞瘤、胶质瘤、肉瘤、间皮瘤和骨髓增生异常综合征。
在一些实施方案中，所述TGFβ相关疾病是纤维化疾病。纤维化疾病是涉及纤维化的疾病或病状。纤维化是一种瘢痕形成过程，是慢性器官(例如肺、肝脏、肾脏、皮肤、心脏、肠道或肌肉)损伤的常见特征。纤维化的特征在于转化生长因子-β(TGF-β)活性升高，从而导致胞外基质和其它纤维化相关蛋白的沉积增加和改变。
纤维化疾病可以包括肺、肝脏、肾脏、眼睛、皮肤、心脏、肠道或肌肉的纤维化疾病。肺的纤维化疾病的实例包括肺纤维化、囊性纤维化、肺动脉高压、进行性大块纤维化、闭塞性细支气管炎、与慢性哮喘或特发性肺相关的气道重塑。肝脏的纤维化疾病的实例包括肝硬化或非酒精性脂肪性肝炎。肾脏的纤维化疾病的实例包括如肾纤维化、缺血性肾损伤、肾小管间质纤维化、糖尿病肾病、肾硬化或肾毒性。眼睛的纤维化疾病的实例包括如角膜纤维化、视网膜下纤维化。皮肤的纤维化疾病的实例包括如肾源性系统性纤维化、瘢痕疙瘩或硬皮病。心脏的纤维化疾病的实例包括心内膜心肌纤维化或陈旧性心肌梗塞。
在一些实施方案中，所述疾病或病状是IL-1相关疾病或病状。在一些实施方案中，所述IL-1相关疾病是自身炎性疾病、代谢综合征、急性炎症、慢性炎症或恶性肿瘤。
在一些实施方案中，所述疾病或病状将受益于通过用免疫刺激性多肽(例如，可溶性LAG-3)刺激MHCII信号传导而诱导持续免疫应答。在一些实施方案中，所述疾病或病状是癌症、病毒感染、寄生虫感染或其组合。
在一些实施方案中，所述疾病或病状将受益于通过抑制免疫抑制性受体信号传导而刺激抗肿瘤免疫。在一些实施方案中，所述免疫抑制性受体是SIRPα。在一些实施方案中，所述疾病、病症或病状与SIRPa相关，如癌症、实体瘤、慢性感染、炎性疾病、多发性硬化、自身免疫疾病、神经系统疾病、脑损伤、神经损伤、红细胞增多症、血色病、外伤、感染性休克、纤维化、动脉粥样硬化、肥胖症、II型糖尿病、移植功能障碍或关节炎。在一些实施方案中，所述癌症是肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆囊癌、胃癌、肺癌、支气管癌、骨癌、肝和胆管癌、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、肾盂和输尿管癌、唾液腺癌、小肠癌、尿道癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、直肠癌、肛门癌、食道癌、胃肠癌、皮肤癌、前列腺癌、垂体 癌、阴道癌、甲状腺癌、喉癌、胶质母细胞瘤、黑色素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、慢性淋巴细胞白血病(CLL)、慢性髓性白血病(CML)、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、霍奇金淋巴瘤、非霍奇金淋巴瘤、多发性骨髓瘤、T或B细胞淋巴瘤、GI器官间质瘤、软组织肿瘤、肝细胞癌和腺癌。在一些实施方案中，所述癌症是表达CD47的癌症或过度表达CD47的癌症。
本文提供的双功能分子的治疗有效量将取决于本领域已知的各种因素，例如，体重、年龄、既往病史、目前的药物治疗、受试者的健康状况和交叉反应的潜力、过敏、敏感性和不良副作用，以及施用途径和疾病发展的程度。
在一些实施方案中，本文提供的双功能分子可以以约0.01mg/kg到约100mg/kg的治疗有效剂量施用。
本文提供的制剂可以通过本领域已知的途径施用，例如，肠胃外(例如，皮下、腹膜内、静脉内，包括静脉内输注、肌内或皮内注射)或非肠胃外(例如，口服、鼻内、眼内、舌下、直肠或局部)途径。
在一些实施方案中，本文提供的双功能分子可以单独施用或与治疗有效量的第二治疗剂组合施用。例如，本文公开的双功能分子可以与第二治疗剂组合施用，所述第二治疗剂例如化学治疗剂、抗癌药物、放射疗法、免疫治疗剂、抗血管生成剂、靶向疗法、细胞疗法、基因疗法、激素疗法、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子。
如本文所使用的，术语“免疫疗法”是指刺激免疫系统对抗如癌症等疾病或以一般方式增强免疫系统的疗法类型。免疫疗法的实例包括但不限于检查点调节剂、过继细胞转移、细胞因子、溶瘤病毒和治疗性疫苗。
“靶向疗法”是作用于与癌症相关的特定分子的疗法类型，所述特定分子如存在于癌细胞中但不存在于正常细胞中或在癌细胞中更丰富的特定蛋白质，或有助于癌症生长和存活的癌症微环境中的靶分子。靶向疗法将治疗剂靶向肿瘤，从而使正常组织免受治疗剂的影响。
在一些实施方案中，与一种或多种另外的治疗剂组合施用的本文提供的双功能分子可以与一种或多种另外的治疗剂同时施用，并且在这些实施方案中的一些实施方案中，所述双功能分子和所述另外的治疗剂可以作为相同药物组合物的一部分施用。然而，与另一种治疗剂“组合”施用的双功能分子不必与所述药剂同时施用或不必与所述药剂在同一组合物中施用。在另一种药剂之前或之后施用的双功能分子被认为是与所述药剂“组合”施用，如本文所使用的短语，即使抗体或抗原结合片段和第二药剂通过不同途径施用也是如此。
在另一方面，本公开还提供了本文提供的双功能分子和/或本文提供的药物组合物、药物制剂在制造用于治疗受试者的PD-L1相关疾病和/或TGF-β相关疾病和/或IL-1相关疾病和/或CD47相关疾病的药物中的用途。
提供以下实施例以更好地说明所要求保护的发明并且不应将所述实施例解释为限制本公开的范围。下文描述的所有具体配制物或制剂、材料和方法全部或部分地落入本公开的范围内。这些具体配制物或制剂、材料和方法不旨在限制本公开，而仅用于说明落入本公开范围内的具体实施方案。本领域技术人员可以在不需要创造性劳动和不脱离本公开范围的情况下开发出等效的组合物、材料和方法。将理解，可以在本文描述的方案中做出许多变化，此类变化仍然包括在本公开的范围内。
实施例
实施例A抗体、双功能蛋白的制备、表征及其活性
实施例1：人源化4B6抗体的产生、表达和纯化
源自专利WO 2017161976A1的包括如下所示的SEQ ID NO:46的VH序列和SEQ ID NO:47的VL序列的抗PD-L1 mAb 4B6是强效PD-1/PD-L1阻滞剂。此抗体由小鼠杂交瘤抗体产生，因此需要适当的人源化。小鼠抗体4B6的可变结构域的序列用于鉴定与其相应鼠框架具有最高同源性的种系序列。计算机建模用于设计具有互补决定区(CDR)移植和回复突变的人源化变体。
小鼠/嵌合重链可变区(SEQ ID NO:46)：
小鼠/嵌合轻链可变区(SEQ ID NO:47)：
注意：斜体部分表示框架(FR)，并且下划线部分表示CDR序列。顺序是FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
轻链的人种系框架序列VK/1-33和重链的VH/1-2分别用于CDR移植。
重链变体1、2、3和4(即，VH变体1、2、3和4)通过将三个CDR直接移植到VH种系序列(SEQ ID NO:48)以及另外对VH变体1(SEQ ID NO:49)的M69V、R71V、VH变体2(SEQ ID NO:50)的M69V、R71V、A93V、R94K、VH变体3(SEQ ID NO:51)的M69V、R71V、T73K、T28V和VH变体4(SEQ ID NO:52)的M69V、R71V、A93V、R94K、T73K、T28V、G44S分别进行回复突变来获得。
4B6VH的种系序列:
VH/1-2(4B6-VH种系，SEQ ID NO:48)：
VH/1-2变体1(4B6_Ha，SEQ ID NO:49)：
VH/1-2变体2(4B6_Hb，SEQ ID NO:50)：
VH/1-2变体3(Hu4B6_Hc，SEQ ID NO:51)：

VH/1-2变体4(Hu4B6_Hd，SEQ ID NO:52)：
轻链变体1和2(VL变体1和2)通过将三个CDR直接移植到种系序列(SEQ ID NO:53)以及另外对VL变体1(SEQ ID NO:54)的F73L突变和VL变体2(SEQ ID NO:55)的F73L、A43S、S60D分别进行回复突变获得。
4B6VL的种系序列：
VK/1-33(4B6-VL-种系，SEQ ID NO:53)：
VK/1-33变体1(Hu4B6_La,SEQ ID NO:54)：
VK/1-33变体2(Hu4B6_Lb,SEQ ID NO:55)：
合成上述重链和轻链可变区的cDNA，并且然后将所述cDNA与人IgG1和人κ的恒定区序列融合。将所得抗体基因序列克隆到表达载体中。使用来自凯杰公司(Qiagen)的Plasmid Maxiprep System制备大规模DNA，以用于人源化4B6变体表达，如表6所示，并使用来自英杰公司(Invitrogen)的ExpiFectamineTM CHO试剂根据制造商的方案进行细胞转染。当细胞存活率超过60％时收获上清液并通过0.22um过滤胶囊过滤以去除细胞碎片。随后将经过滤的上清液加载到预平衡的蛋白质-A亲和柱上。用平衡缓冲液(PBS)洗涤蛋白A树脂，并且然后使用25mM柠檬酸盐(pH 3.5)洗脱抗体。通过使用1M Tris-碱(pH 9.0)将纯化的抗体溶液调节到pH 6.0-7.0。内毒素控制在1EU/mg以下。最后，纯化的抗体通过SDS-PAGE进行表征。
表6.人源化4B6变体的表达

注意：此表示出了不同突变的各种序列组合。例如，Hu4B6_HaLa指示人源化鼠抗体Hu4B6_HaLa上存在两种突变(重链Hu4B6_Ha和轻链Hu4B6_La)，以此类推。Hu4B6_L0和Hu4B6_H0是CDR移植获得的，所述CDR移植缺少关键回复突变，因此其不用于表达。
实施例2：通过ELISA测定测量的与人PD-L1的结合
通过ELISA方法评估人源化抗体的结合。简而言之，将人PD-L1-His固定在板上。将表6中列出的人源化4B6抗体在PBS中连续稀释并添加温育1小时。接下来，添加山羊pAb对人IgG-HRP和TMB以检测OD450nm处的结合。
如图1所示，测试所有人源化变体以筛选最好的变体。所有变体都保留了它们的结合活性，并且Hu4B6_HdLa显示出比其它变体更好的结合活性。
分析了此抗体的CDR序列，并且发现重链的CDR2中存在NG基序。在表达和纯化过程中可能存在脱氨基的风险。为了去除脱酰胺热点，将G55A的突变(下方粗体和放大)引入到Hu4B6_Hd中。然后获得Hu4B6_Hg(SEQ ID NO:56)，并且对人PD-L1的亲和力不受影响，如图2所示。
Hu4B6_Hg(SEQ ID NO:56)：
实施例3：单克隆噬菌体ELISA和序列分析
尽管Hu4B6-HgLa保留了嵌合4B6的活性，但作为拮抗剂药物，更高的亲和力是优选的。基于Hu4B6-HgLa序列，CDR中的定点诱变和体外解离速率依赖性选择的几个淘选循环被进一步用于亲和力成熟。首先，将4B6-HgLa的VL和VH结构域通过重叠PCR扩增并通过肽接头(G4S)3连接形成scFv，然后亚克隆到噬菌粒载体pComb3x(武汉妙灵生物(Wuhan MiaoLingBio)，P0862)中，作为通过SfiI切割位点进行亲和力成熟的野生型序列。
为了研究重链和轻链的CDR1和CDR2对4B6亲和力成熟的单独的贡献，将构建上述每个CDR的一个SPM(小扰动诱变)噬菌体文库，因为两条链的抗体CDR3通常在抗原结合中起着重要的作用。将两条链的CDR1和CDR2序列与种系序列比对，并且重链和轻链的可变区的种系分别为IGHV1-2和IGKV1-33。种系CDR序列的生物信息学分析结果用于指导文库设计。
在确定氨基酸突变位点和取代序列后，设计简并引物以增加突变文库的多样性。扩增多样化的CDR片段，以构建4B6scFv基因突变文库。将scFv基因与pComb3XSS噬菌体展示载体连接以生成scFv文库。每个CDR(包括表2中列出的HCDR1、HCDR2、LCDR1和LCDR2)的基于密码子的引物建立为独立文库，并且将4B6亲和力成熟文库分为4个文 库。HCDR1的容量为1.76×108CFM，HCDR2的容量为1.81×108CFM，LCDR1的容量为2.34×108CFM，并且LCDR2的容量为2.00×108CFM。每个文库随机选取5或6个克隆进行菌落测序。结果表明，构建的文库的插入率为100％。
将10μg/ml hPD-L1(Acro Biosystems，PD1-H5229)抗原包被到ELISA板，并在37℃下与200μL噬菌体(1×1010pfu/ml噬菌体展示文库)反应1小时。洗涤后，将OD600为大约0.5的TG1(Lucigen，60502-2)直接添加到孔中进行感染，并与噬菌体一起温育15分钟。足够体积的M13KO7辅助噬菌体(NEB，N0315S)到对数中期培养用于文库噬菌粒拯救，并且将噬菌体生成和纯化以用于下一轮筛选。筛选过程重复3轮，并且第2轮抗原浓度下降到2.5μg/ml且第3轮抗原浓度下降到1μg/ml。
进行ELISA结合测定以检测这些多克隆噬菌体变体的效价。在3轮淘选之后，包括4B6-H-CDR2、4B6-L-CDR1和4B6-L-CDR2的3个文库明显富集。
对于这3个文库，挑取每个文库的96个克隆并进行噬菌体ELISA以检测它们的结合活性。简而言之，将1μg/ml hPD-L1(Acro Biosystems，PD1-H5229)抗原包被到ELISA板并在4℃下放置过夜。然后添加300μL的3％(w/v)脱脂牛奶，以在室温下封闭1小时。1小时后，将含有单克隆抗体片段噬菌体的100μl上清液与作为阴性对照的PBS一起添加并且在37℃下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联抗M13 mAb(1:20000，Sino Biological，11973-MM05T-H)。在室温下温育1小时后，添加混合的TMB(InnoReagents，TMB-S-003)底物试剂并将板在室温下温育5分钟。添加0.1M H2SO4终止反应，然后记录OD450nm。挑选阳性克隆以用于由Genewiz(中国苏州)进行DNA测序。序列在表7中示出。
表7. 4B6scFv噬菌体文库的阳性克隆的序列

注意：下划线序列是指CDR序列，并且加粗氨基酸是指突变氨基酸。
实施例4：用于检测结合亲和力的生物层干涉测量法(BLI)
生物层干涉测量法(BLI)用于测试4B6scFv变体对人PD-L1-Fc(Sino Biological，70110-D02H)抗原的结合亲和力。材料和程序分别在表8和表9中示出。结果在表10-12中示出。根据结合亲和力结果，4个轻链变体(L-CDR1-2、L-CDR1-3、L-CDR2-2、L-CDR2-3)和3个重链变体(H-CDR2-2、H-CDR2-3、H-CDR2-5)被选择用于未来的构建。
表8 BLI测定中使用的样品和材料
表9 BLI测定程序
表10. 4B6-L-CDR1突变变体的结合亲和力分级
表11. 4B6-L-CDR2突变变体的结合亲和力分级
表12. 4B6-H-CDR2突变变体的结合亲和力分级
实施例5：AM-4B6-hIgG1-TGFβRII(1-136)融合蛋白的构建和表达
将所选重链和轻链变体与hIgG1-TGFβRII(1-136)融合蛋白交叉组合并表达。TGFβRII(1-136)具有在SEQ ID NO:79中所示的氨基酸序列：
hIgG1的氨基酸序列如下(SEQ ID NO:80)：
TGFβRII(1-136)通过肽接头(G4S)4G(SEQ ID NO:68)连接到hIgG1的羧基端。
hκ的氨基酸序列如下(SEQ ID NO:82)：
完整抗体变体以AM(亲和力成熟)为前缀。重链和轻链的序列构建体在表13中示出，并且完整抗体的设计在表14中示出。例如，如表13和表14所示，双功能分子“AM-4B6-hIgG1-TGFβRII变体1”具有重链和轻链，其中从N端到C端的重链包括：Hu4B6_Hg.2-_hIgG1-(G4S)4G-TGFβRII(1-136)；并且从N端到C端的轻链包括：Hu4B6_La.1-hκ。相同的命名法适用于表14中的其它变体。
根据制造商的方案，使用来自英杰公司的ExpiFectamineTM CHO试剂(赛默(Thermo)，A29129)进行重链和轻链的共转染。在第10天收获上清液并通过亲和色谱法纯化。
表13 AM-4B6-hIgG1-TGFβRII重链和轻链变体的列表
表14.AM-4B6-hIgG1-TGFβRII抗体变体的列表

实施例6：AM-4B6-hIgG1-TGFβRII变体对hPD-L1的结合亲和力
通过ELISA测定测量的与hPD-L1的结合
将1μg/ml hPD-L1(Acro Biosystems，PD1-H5229)抗原包被到ELISA板并在4℃下放置过夜。然后添加300μl的3％(w/v)脱脂牛奶，以在室温下封闭1小时。在1小时后，将100μl的浓度范围为100nM到0.006nM(四倍连续稀释)的AM-4B6-hIgG1-TGFβRII变体或原始4B6-hIgG1-TGFβRII与作为阴性对照的PBS一起添加并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗(Abcam)，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，然后通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。
如图3A-图3C所示，当与原始4B6-hIgG1-TGFβRII比较时，所有变体都具有增强的结合信号和亲和力。鉴于这些变体之间的差异有限，表面等离子共振技术被用于进一步评估它们的结合亲和力。
通过Biacore测量的与hPD-L1的结合
将4μg/ml AM-4B6-hIgG1-TGFβRII变体或原始4B6-hIgG1-TGFβRII固定在S系列蛋白A芯片的表面上。将人PD-L1稀释到适当的浓度梯度(0nM、1.875nM、3.75nM、7.5nM、15nM、30nM、60nM)并注射到Biacore 2000的样品通道中。结果在表15中示出。AM-4B6-hIgG1-TGFβRII变体7与人PD-L1的结合亲和力比原始4B6-hIgG1-TGFβRII提高了 约15倍。
表15.通过Biacore测量的AM-4B6-hIgG1-TGFβRII与hPD-L1的结合亲和力
通过FACS测定测量的与细胞表面上表达的PD-L1的结合
293T-PD-L1-CD3L细胞由迈博斯生物科学(MabSpace Bioscience)产生，以用于表征PD-L1抗体。用人PD-L1和抗CD3scFv转染细胞。将AM-4B6-hIgG1-TGFβRII变体或原始4B6-hIgG1-TGFβRII连续稀释(5倍稀释)以在稀释缓冲液(含2％BSA的PBS)中获得8个浓度。收获并离心293T-PD-L1-CD3L细胞。将它们以2×106个细胞/ml的密度重悬于PBS中，然后以每孔100ul添加到板中。在离心并去除上清液后，将稀释的抗体添加到板并且在4℃下温育30分钟。在用稀释缓冲液洗涤两次后，将PE缀合的驴抗人IgG(H+L)(Jacksonimmuno，709-116-149)添加到板并在4℃下温育30分钟。在洗涤后，将细胞重悬于200μl PBS中并通过流式细胞术进行分析。
如图4所示，这5种变体以相似的EC50与在293T-PD-L1-CD3L细胞表面表达的PD-L1结合。变体7的EC50略低于其它变体，这与通过Biacore测量的结合亲和力结果一致。
实施例7：AM-4B6-hIgG1-TGFbRII变体的PD-1/PD-L1阻断活性
通过ELISA测定测量的PD-1/PD-L1和B7-1/PD-L1阻断
为了测试配体/受体阻断活性，将0.5μg/ml hPD-L1-Fc抗原包被到ELISA板并在4℃下放置过夜。添加300μL封闭缓冲液，以在室温下封闭1小时。在1小时后，添加50μl的浓度范围为100nM到0.024nM(四倍连续稀释)的AM-4B6-hIgG1-TGFβRII变体7或原始4B6-hIgG1-TGFβRII以及50μl的浓度为1μg/ml的PD-L1-his并且在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联的链霉亲和素(1:5000，艾博抗，目录号ab7403)。在室温下温育1.5小时后，添加混合的TMB底物试剂并在室温下温育5分钟，然后添加0.1M H2SO4终止。OD450nm由微板读数器记录。如图5A-图5B所示，两个样品都可以阻断PD-L1/PD-1或PD-L1/B7-1，而AM-4B6-hIgG1-TGFβRII变体7的IC50较低，这表明变体7具有更高的活性。
通过基于细胞的测定测量的PD-1/PD-L1阻断
在此测定中，293T-PD-L1-CD3L细胞表达PD-L1和抗CD3scFv，而Jurkat-NFAT-Luc-PD1细胞表达PD-1并携带可以用CD3刺激激活的NFAT信号。NFAT激活导致下游荧光素酶基因转录和表达，这可以通过其底物进行检测。所述两个细胞由迈博斯生物科学产生。
简而言之，收获293T-PD-L1-CD3L细胞并以2×106个细胞/ml的密度重悬。将每孔20μl细胞添加到半孔板中。将AM-4B6-hIgG1-TGFβRII变体或原始4B6-hIgG1-TGFβRII连续稀释(3倍稀释)以在含2％FBS的RPMI培养基中获得8个浓度。向半孔板中每孔添加20μl抗体并且将板在37℃、5％CO2下温育30分钟。收获Jurkat-NFAT-Luc-PD1细胞并以4×106个细胞/ml的密度重悬于含有2％FBS的RPMI培养基中。最后，将每孔20μl细胞和5ng/ml TGF-β(R&D，240-B-010)添加到半孔板中并在37℃、5％CO2下温育5小时。将60μl的OneGlo检测试剂(普洛麦格(Promega)，E6120)添加到每个孔中并在室温下温育5分钟。发光信号由微板读数器读取。数据通过GraphPad Prism分析。
如图6所示，与之前的ELISA结果一致，与其它变体相比，变体7在此基于细胞的测定中具有最强效的阻断活性。因此，将AM-4B6-hIgG1-TGFβRII变体7的4B6Fab部分简称为AM4B6，并在以下实验中进一步评估AM4B6-hIgG1-TGFβRII融合蛋白。
实施例8：AM4B6-hIgG1-TGFβRII'的体外生成和表征
AM4B6-hIgG1-TGFβRII的克隆和表达
据报道，截短的TGFβRII ECD_20-136是可溶性的并保留了结合TGFβ1的能力(Kim-Ming Lo等人,US9676863 B2,2017；Christian C.等人,《蛋白质表达和纯化(Protein Expression and Purification)》,2000,20:98–104)。接下来，将TGFβRII_1-136的胞外结构域的全长替换为截短的胞外结构域并评估可开发性和稳定性。来自稳定细胞系的SDS-PAGE结果表明，蛋白质表达对于截短和全长TGFβRII ECD都很好，但截短的TGFβRII ECD_20-136的蛋白质稳定性比全长TGFβRII ECD好得多(图7)。截短的TGFβRII ECD_20-136的序列如下：
稳定的TGF-β陷阱、TGF-βRII胞外结构域(20-136)的序列
包括截短的TGF-βRII(即TGF-βRII(20-136)，SEQ ID NO:66)的双功能分子用于此实施例以及实施例9-11。此类双功能分子的名称用TGFβRII'表示，以区别于TGF-βRII(1-136)。例如，AM4B6-hIgG1-TGFβRII'表示具有TGF-βRII(20-136)的分子。
通过ELISA测定测量的与来自不同物种的PD-L1的结合
将1μg/ml人PD-L1(Acro Biosystems，PD1-H5229)或食蟹猴PD-L1抗原包被到ELISA板上并在4℃下放置过夜。然后添加300μl的3％(w/v)脱脂牛奶，以在室温下封闭1小时。在1小时后，添加100μl的浓度范围为100nM到0.02nM(四倍连续稀释)的AM4B6-hIgG1-TGFβRII'或对照hIgG1-TGFβRII'并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗(Abcam)，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。
如图8A和图8B所示，AM4B6-hIgG1-TGFβRII'以与人PD-L1相似的EC50与食蟹猴 PD-L1交叉反应。
通过ELISA测量的与来自不同物种的TGFβ1、TGFβ2和TGFβ3的结合
根据来自Uniport网站(https://www.uniprot.org/)上公布的4种常见物种：人、食蟹猴、小鼠和大鼠的TGFβ1、TGFβ2和TGFβ3的序列，TGFβ成员是相当保守。人TGFβ1和食蟹猴TGFβ1的序列相同；小鼠TGFβ1和大鼠TGFβ1相同；人TGFβ2和食蟹猴TGFβ2相同；小鼠TGFβ2和大鼠TGFβ2相同；人TGFβ3、食蟹猴TGFβ3和小鼠TGFβ3相同。
对于TGFβ1和TGFβ3，程序如下：将0.5μg/ml人TGFβ1(Sino Biological，10804-HNAC)或小鼠TGFβ1(Novoprotein，CK33)或人TGFβ3(金斯瑞(Genscript)，Z03430)或大鼠TGFβ3(Novoprotein，CJ44)抗原包被到ELISA板并在4℃下放置过夜。然后添加300μl的3％(w/v)脱脂牛奶，以在室温下封闭1小时。在1小时后，添加100μl的浓度范围为100nM到0.006nM(四倍连续稀释)的AM4B6-hIgG1-TGFβRII'或对照hIgG1-TGFβRII'并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗(Abcam)，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。
对于TGFβ2，测试程序不同：将2μg/ml的AM4B6-hIgG1-TGFβRII'或对照hIgG1-TGFβRII'包被到ELISA板并在4℃下放置过夜。然后添加300μl的3％(w/v)脱脂牛奶，以在室温下封闭1小时。在1小时后，添加100μl的浓度范围为39.4nM到0.3nM(两倍连续稀释)的人TGFβ2或小鼠TGFβ2并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl的TGFβ2生物素化抗体(1:10000，R&D，BAF302)。在室温下温育1小时并洗涤后，添加100μl的HRP-链霉亲和素(1:5000，艾博抗，ab7403)并将板在室温下温育1小时。在洗涤后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。
结果总结在表16中。对于与TGFβ1的结合亲和力，不同物种之间的EC50值非常相似。此外，与TGFβ1和TGFβ3的结合亲和力显著高于TGFβ2，这表明对TGFβ1和TGFβ3的阻断活性可能比TGFβ2更有效。
表16.AM4B6-hIgG1-TGFβRII'与TGFβ1、TGFβ2和TGFβ3的结合
通过ELISA测定测量的与B7家族或TGFβ超家族内的其它成员的结合
对于B7家族，将0.5μg/ml hPD-L1(Acro Biosystems，PD1-H5229)或hPD-L2或B7-2或B7-1或B7-H2或B7-H3或B7-H4或VISTA包被到ELISA板并在4℃下放置过夜。对于TGFβ超家族，将0.5μg/ml人激活素A、BMP-2、LAP或TGFβ1在4℃下包被过夜。然后添加300μl的3％(w/v)脱脂牛奶，以在室温下封闭1小时。在1小时后，添加100μl的浓度 范围为100nM到0.006nM(连续稀释)的AM4B6-hIgG1-TGFβRII'并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗(Abcam)，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。
如图9A-图9B所示，AM4B6-hIgG1-TGFβRII'特异性结合到PD-L1而不是也属于B7家族的其它抗原。如图9C所示，AM4B6-hIgG1-TGFβRII'特异性结合到TGF-β1而不是也属于TGFβ超家族的其它抗原。
AM4B6-hIgG1-TGFβRII'与表达PD-L1的细胞的结合
MC38/hPD-L1是通过经由CRISPR-Cas9系统删除mPD-L1，然后使用慢病毒转导hPD-L1来产生的。此细胞系由中国医学科学院苏州系统医学研究所系统医学中心(Center of Systems Medicine,Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine)秦晓峰教授实验室提供(Huang,Anfei等人《科学报告(Scientific Reports)》7(2017):42687.)。将MC-38/hPD-L1细胞在RPMI1640+10％FBS中培养。EMT-6/hPD-L1是稳定表达经转染的人PD-L1基因的小鼠乳腺癌细胞系。将EMT-6/hPD-L1细胞在Waymouth的(1×)MB752/1+15％FBS中培养。NCI-H460细胞购自COBIOER有限公司(COBIOER Ltd)。它是一种具有PD-L1表达的人肺上皮肿瘤细胞系。将NCI-H460细胞在RPMI1640+10％FBS中培养。NCI-H292细胞购自COBIOER有限公司。它是一种具有PD-L1表达的人肺上皮肿瘤细胞系。将NCI-H292细胞在RPMI1640+10％FBS+1nM丙酮酸钠溶液中培养。FACS分析方案与实施例6第3节相同。
将人或食蟹猴PBMC(TPCS，目录号PB025C)从液氮中回收并重悬于具有10％FBS的RPMI1640中。添加5μg/ml的PHA(西格马(Sigma)，目录号L8902)以刺激PBMC激活并将细胞培养3天。收集激活的PBMC并且离心并以2×106个细胞/ml的密度重悬于PBS中，并且以每孔100ul添加到板中。将AM4B6-hIgG1-TGFβRII'或AM4B6或对照hIgG1-TGFβRII'连续稀释(5倍稀释)以在稀释缓冲液(含2％BSA的PBS)获得10个浓度。在离心并去除板中的上清液后，将稀释的抗体添加到具有激活的PBMC的板中并在4℃下温育1小时。用稀释缓冲液洗涤两次后，添加Alexa488标记的小鼠抗人CD3(Biolegend，目录号300320)和APC标记的抗人IgG二级抗体(BD，目录号550931)并在4℃下温育30分钟。在洗涤后，将细胞重悬于150μl PBS中并通过流式细胞术进行分析。
如图10A-图10F所示，AM4B6-hIgG1-TGFβRII'可以以与单独的AM4B6mAb相似的亲和力与在这些癌细胞系和激活的人或食蟹猴T细胞上表达的PD-L1结合。
与激活的人T细胞的结合
将人PBMC(TPCS，目录号PB025C)从液氮中回收并重悬于具有10％FBS的RPMI1640中。添加5μg/ml的PHA(西格马，目录号L8902)以刺激PBMC激活并将细胞培养3天。收集激活的PBMC并且离心并以2×106个细胞/ml的密度重悬于PBS中，并且以每孔100ul添加到板中。将AM4B6-hIgG1-TGFβRII'或AM4B6或对照hIgG1-TGFβRII'连续稀释(5倍稀释)以在稀释缓冲液(含2％BSA的PBS)获得10个浓度。在离心并去除板中的上清液后，将稀释的抗体添加到具有激活的PBMC的板中并在4℃下温育1小时。用稀释缓冲液洗涤两次后，添加Alexa488标记的小鼠抗人CD3(Biolegend，目录号300320) 和APC标记的抗人IgG二级抗体(BD，目录号550931)并在4℃下温育30分钟。在洗涤后，将细胞重悬于150μl PBS中并通过流式细胞术进行分析。
如图11所示，AM4B6-hIgG1-TGFβRII'可以结合到在激活的人T细胞上表达的PD-L1。
通过ELISA测定测量的对hPD-L1/hPD-1和食蟹猴PD-L1/食蟹猴PD-1的阻断
将0.5μg/ml的hPD-L1-Fc或0.5μg/ml的食蟹猴PD-L1-Fc包被到ELISA板并在4℃下放置过夜。添加300μL的3％(w/v)脱脂牛奶，以在室温下封闭1小时。在1小时后，添加100μl的浓度范围为100nM到0.02nM(四倍连续稀释)的AM4B6-hIgG1-TGFβRII'或AM4B6以及0.5μg/ml的hPD-1-Fc-生物素或食蟹猴PD-1-Fc-生物素并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且添加100μl HRP偶联的链霉亲和素(1:5000)。在室温下温育1小时后，添加混合的TMB底物试剂并在室温下温育5分钟，然后添加0.1M H2SO4终止。OD450nm由微板读数器记录。
如图12A-图12B所示，AM4B6-hIgG1-TGFβRII'还可以以与阻断人PD-L1/人PD-1的IC50相似的IC50完全阻断食蟹猴PD-L1/食蟹猴PD-1。
与hPD-L1和hTGFβ1的同时结合
将0.5μg/ml的hTGFβ-1包被到ELISA板并在4℃下放置过夜。然后添加300μl的封闭缓冲液，以在室温下封闭1小时。在1小时后，添加100μl的浓度范围为100nM到0.02nM(四倍连续稀释)的AM4B6-hIgG1-TGFβRII'或AM4B6或对照hIgG1-TGFβRII'并在室温下温育1小时。0.5％PBS+Tween-20用于洗涤3次，并且然后每孔添加0.5μg/ml的hPD-L1-生物素。在1小时后，添加100μl的HRP偶联的链霉亲和素(1:5000)。在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。
如图13所示，由抗PD-L1抗体AM4B6和TGFβRII'构成的AM4B6-hIgG1-TGFβRII'可以同时结合两个靶标，这表明其双特异性或双功能特性。
通过报告细胞测定测量的对hPD-L1/hPD-1的阻断
方案与实施例7第2节相同。如图14所示，AM4B6-hIgG1-TGFβRII可以阻断PD-L1/PD-1的抑制并随后逆转信号传导激活，这与单独的AM4B6mAb相同。
通过报告细胞测定测量的对TGFβ1信号传导的阻断
TGFβ报告基因HEK-293细胞系购自Genomeditech(目录：GM-C05346)并于具有5％二氧化碳的37℃温育器中在含有10％FBS、4μg/ml杀稻瘟素、400μg/ml新霉素、125μg/ml潮霉素、0.75μg/ml嘌呤霉素和1％Pen/Strep的DMEM培养基中培养。
在对数生长期收集细胞并重悬于DMEM培养基中并以2×10^4个细胞/100μl/孔的密度种植在96孔板中。在细胞培养过夜后，将培养基更换为75μl含有10ng/ml人TGFβ1的培养基。以最终浓度范围为100nM到0.02nM(三倍连续稀释)添加75μl的AM4B6-hIgG1-TGFβRII'或AM4B6，。将板在37℃温育器中培养16小时。ONE-GloTM荧光素酶检测系统以150μl/孔添加，并且在室温下温育10分钟后，用微板读数器读取板。
如图15所示，AM4B6-hIgG1-TGFβRII'对TGFβ1信号传导显示出强效的阻断活性，其 中IC50为0.35nM，而单独的AM4B6mAb没有阻断活性，这表明阻断活性是TGFβ1特异性的。
AM4B6-hIgG1-TGFβRII'对由结核菌素(TB)刺激的PBMC的IFN-γ释放的影响
从液氮中回收人PBMC并以2×10^6/mL的密度重悬细胞。添加TB到终浓度为1.33μg/mL；在37℃下培养5天。在第六天，收集诱导的PBMC并且离心，用PBS洗涤一次，重悬于新鲜培养基中，调整到1×10^6/ml的密度，并且以180μL/孔接种到96孔细胞板中。将稀释的抗体以20μL/孔添加到96孔细胞培养板中。对照组和空白组添加有20μL PBS。将细胞培养板在37℃下在5％CO2温育器中温育3天。在温育结束时，将细胞上清液稀释10倍并且用IFN-γELISA检测试剂盒(R&D，DY285B)检测IFNγ的分泌水平。
如图16所示，AM4B6-hIgG1-TGFβRII'诱导的IFN-γ释放水平显著高于单独的AM4B6mAb，这表明其激活活性由于其双特异性结合和阻断活性而更强效。
AM4B6-hIgG1-TGFβRII'的ADCC/CDC活性
对于ADCC测定，效应细胞：Jurkat-NFAT Luc-FcγRIIIa-158V细胞系由(苏州)迈博斯生物科学有限公司(Mabspace Biosciences(Suzhou)Co.,Limited)构建。靶细胞：HEK-293T-hPD-L1细胞(购自冠科生物技术公司(Crown Biosciences Inc.)，目录：2005)。
将HEK-293T-hPD-L1细胞以10,000个细胞/12.5μl/孔添加细胞培养板。然后以12.5μl/孔添加最终浓度范围为200nM到0.003nM的AM4B6-hIgG1-TGFβRII'稀释液。然后将板放置于37℃的温育器中，以使抗体和细胞温育30分钟。然后将Jurkat-NFAT Luc-FcγRIIIa-158V细胞以60,000个细胞/25μl/孔添加到孔中。然后将板放置于37℃的温育器中持续6小时。ONE-GloTM荧光素酶检测系统以50μl/孔添加，并且在室温下温育10分钟后，用微板读数器读取板。
对于CDC测定，靶细胞也是HEK-293T-hPD-L1细胞。将HEK-293T-hPD-L1细胞以10,000个细胞/25孔添加细胞培养板。然后以12.5μl/孔添加最终浓度范围为200nM到0.3nM的AM4B6-hIgG1-TGFβRII'稀释液。然后将板放置于37℃的温育器中，以使抗体和细胞温育30分钟。将HEK-293T-hPD-L1细胞用40％补体以50μl/孔(最终浓度为20％)处理，然后在37℃下温育80分钟。ONE-GloTM荧光素酶检测系统以100μl/孔添加，并且在室温下温育10分钟后，用微板读数器读取板。
结果表明AM4B6-hIgG1-TGFβRII'对HEK-293T-hPD-L1细胞既没有ADCC也没有CDC活性(数据未示出)。
实施例9：AM4B6-hIgG1-TGFβRII'的体内功效
C57BL/6小鼠上的MC38-hPD-L1肿瘤模型
使用最近开发的高效CRISPR/Cas9系统敲除小鼠肿瘤细胞系MC38(ATCC)中的内源性小鼠PD-L1。简而言之，设计靶向小鼠PD-L1基因的第一编码外显子的sgRNA，并且将细胞通过“打了就跑”(hit-and-run)CRISPR/Cas9+sgRNA构建体转染并从中选择敲除细胞。完全敲除小鼠内源性PD-L1的细胞通过FACS分析鉴定PD-L1在稳态或干扰素γ刺激下的细胞表面表达，随后通过TA克隆和靶向基因组区测序进行验证。为了生成人PD-L1替代细胞系，将人PD-L1cDNA的编码序列克隆到FG12衍生的慢病毒载体中。然后用表达人PD- L1的慢病毒感染小鼠PD-L1敲除细胞，并且FACS分析证实了在建立的细胞系中人PD-L1的高水平和稳定表达。MC38的此经工程化的细胞被命名为MC38-hPD-L1。
将MC38-hPD-L1细胞作为单层培养物在补充有10％热灭活胎牛血清的RPMI1640培养基中于37℃下在空气中含有5％CO2的气氛中体外维持。收获在指数生长期中生长的肿瘤细胞并计数以用于肿瘤接种。向每只雌性SPF级C57BL/6小鼠接种混合的2×106MC38-hPD-L1细胞和50％基质凝胶。当肿瘤大小为大约90mm^3时，选择荷瘤小鼠并将其随机分到5个组(n＝8)。用2.5mg/kg同种型对照、3mg/kg同种型对照TGFβRII'、2.5mg/kg AM4B6、0.3mg/kg AM4B6-hIgG1-TGFβRII'、1mg/kg AM4B6-hIgG1-TGFβRII'和3mg/kg AM4B6-hIgG1-TGFβRII'处理动物。将所有抗体通过腹膜内注射每周两次施用，持续4周。使用卡尺(INSIZE)在两个维度上每周测量两次或三次肿瘤大小，并且使用以下公式以mm^3为单位表示体积：V＝0.5a×b^2，其中a和b分别为肿瘤的长直径和短直径。使用Prism GraphPad分析结果并表示为平均值±S.E.M.。通过T检验进行两组之间的比较，并且如果p为*<0.05并且**<0.01，则认为差异是显著的。
如图17A-图17B所示，3mg/kg同种型对照TGFβRII'根本不抑制肿瘤生长，这表明单独的TGFβRII'几乎没有功效。2.5mg/kg AM4B6只有部分抑制作用，这与0.3mg/kg AM4B6-hIgG1-TGFβRII'相似，似乎不足以控制肿瘤生长。随着剂量的增加，肿瘤体积越来越小。3mg/kg AM4B6-hIgG1-TGFβRII'以84％的TGI几乎使肿瘤生长完全停止(表17)。
表17.第32天MC38-hPD-L1肿瘤模型中抗体的肿瘤生长抑制(TGI)(平均值±S.E.M.，n＝8)
NOD-SCID小鼠上的H460肿瘤和人PBMC共接种模型
H460细胞购自COBIOER有限公司。将H460细胞作为单层培养物在补充有10％热灭活胎牛血清的RPMI1640培养基中于37℃下在空气中含有5％CO2的气氛中体外维持。收获在指数生长期中生长的肿瘤细胞并计数以用于肿瘤接种。向每只雌性SPF级NOD-SCID小鼠接种混合的3×10^6 H460细胞(模型组)或与1.5×10^6人PBMC混合的3×10^6 H460细胞。接种前将所有细胞悬浮液与基质胶以1:1的比率充分混合。在接种后大约4小时，将动物分组成7组(n＝10)并不同地给药。将动物用16.7mg/kg对照hIgG1、或20mg/kg对照hIgG1-TGFβRII'、或16.7mg/kg AM4B6、或5mg/kg AM4B6-hIgG1-TGFβRII'、或10mg/kg AM4B6-hIgG1-TGFβRII'、或20mg/kg AM4B6-hIgG1-TGFβRII'处理。模型组不做任何处理。将所有抗体通过腹膜内注射每周两次施用，持续5周。使用卡尺(INSIZE)在两个维度上每 周测量两次或三次肿瘤大小，并且使用以下公式以mm^3为单位表示体积：V＝0.5a×b^2，其中a和b分别为肿瘤的长直径和短直径。使用GraphPad Prism分析结果并表示为平均值±S.E.M.。通过T检验进行两组之间的比较，并且如果p为*<0.05并且**<0.01，则认为差异是显著的。
如图18A-图18B所示，20mg/kg同种型对照TGFβRII'根本不抑制肿瘤生长。16.7mg/kg AM4B6仅具有部分抑制作用，而5mg/kg AM4B6-hIgG1-TGFβRII'已经具有明显更好的肿瘤抑制作用，这表明TGFβRII融合增加了单独AM4B6mAb的抗肿瘤功效。另外，在此模型中观察到AM4B6-hIgG1-TGFβRII'的明显剂量应答，再次表明功效取决于AM4B6-hIgG1-TGFβRII'。
C57BL/6小鼠上的EMT6-hPD-L1肿瘤模型
敲除小鼠肿瘤细胞系EMT6(ATCC)中的内源性小鼠PD-L1并敲除细胞中的人PD-L1，将EMT6的经工程化的细胞命名为EMT6-hPD-L1。
向小鼠皮下接种EMT6/hPD-L1肿瘤细胞，并且然后根据肿瘤体积随机分为7组，每组10只小鼠。在分组后，向第1组到第7组的动物每周两次通过腹膜内注射分别施用24.9mg/kg对照hIgG1、30mg/kg对照hIgG1-TGFβRII'、24.9mg/kg AM4B6、3mg/kg AM4B6-hIgG1-TGFβRII'、10mg/kg AM4B6-hIgG1-TGFβRII'或30mg/kg AM4B6-hIgG1-TGFβRII'，持续4周。每周两次观察荷瘤小鼠的肿瘤体积和体重。如图19A-图19B所示，AM4B6-hIgG1-TGFβRII'在给药后第29天在3、10和30mg/kg下分别以21.43％、46.83％和79.39％的TGI剂量依赖性地抑制肿瘤生长。在等摩尔量下，AM4B6-hIgG1-TGFβRII'在30mg/kg下的抗肿瘤活性比AM4B6在24.9mg/kg下更明显，在所述组中，第29天的TGI为29.67％。
实施例10：AM4B6-hIgG1-TGFβRII'处理对MC38-hPD-L1肿瘤模型中肿瘤浸润淋巴细胞(TIL)的影响
将MC38-hPD-L1肿瘤细胞按照与实施例9相同的过程进行培养和接种。当肿瘤大小为250-300mm^3,时，选择荷瘤小鼠并将其随机分到4个组(n＝6)。将动物用PBS、或3mg/kg同种型对照TGFβRII'、或2.5mg/kg AM4B6、或3mg/kg AM4B6-hIgG1-TGFβRII'处理。将所有抗体通过静脉内注射每周两次施用，持续1或2周。分别在第2次给药后24小时和第4次给药后24小时收获肿瘤，然后用温和的MACS离解剂(MACS Dissociator)(美天旎生物技术(Miltenyi Biotec)，130-093-235)离解并用小鼠肿瘤离解试剂盒(美天旎生物技术，130-096-730)在37℃下消化40分钟。在通过PE抗小鼠CD45(BD生物科学(BD bioscience)，目录号553081)、APC抗小鼠CD8a(Biolegend，目录号100712)、APC抗小鼠NK1.1(Biolegend，目录号108710)、FITC抗小鼠颗粒酶B(Biolegend，目录号515403)和FITC抗小鼠IFNγ(英杰公司，目录号11-7311-82)染色后，使用FACS分析每组分离的单肿瘤细胞悬浮液的TIL亚群百分比，如表18和表19所示。
表18.第2次给药后24小时对MC38-hPD-L1肿瘤模型的TIL分析。

表19.第4次给药后24小时对MC38-hPD-L1肿瘤模型的TIL分析。
在第2次给药后，不同处理组中TIL亚群的百分比没有显著变化(表18)。但在第4次给药后，与同种型对照TGFβRII'组相比，AM4B6组和AM4B6-hIgG1-TGFβRII'组的CD8+/CD45+％显著增加(表19)。与同种型对照TGFβRII'组相比，CD8+GZMB+％和NK1.1+％也增加了很多。这些发现表明CD8+T细胞和NK1.1T细胞可能受到AM4B6或AM4B6-hIgG1-TGFβRII'的刺激而激活和增殖，并且在肿瘤微环境中富集以促进肿瘤细胞杀伤。与AM4B6相比，AM4B6-hIgG1-TGFβRII'具有更高的CD8+GZMB+％和NK1.1+％，这与其通过上述TGI测量的更有效的抗肿瘤活性相关。
实施例11：AM4B6-hIgG1-TGFβRII'的体内药代动力学和药效学研究
将C57BL/6雌性小鼠随机分为6组(n＝3)。将动物用3mg/kg同种型对照TGFβRII'、或2.5mg/kg AM4B6、或0.3mg/kg AM4B6-hIgG1-TGFβRII'、或1mg/kg AM4B6-hIgG1-TGFβRII'、或3mg/kg AM4B6-hIgG1-TGFβRII'、或3mg/kg M7824类似物处理。M7824类似物由迈博斯生物科学根据US9676863中公开的序列产生。所有抗体均通过静脉单次注射施用。注射后，在以下不同时间点收集每只小鼠的200μl血液：给药前、注射后30分钟、2小时、8小时、24小时、48小时、第4天、第7天、第10天、第14天、第21天。收集每只小鼠的80μl血浆并测试抗体浓度。
为了测量血浆中的抗体浓度，使用两种方法。第一种方法是检测整个双功能分子，包括AM4B6和TGFβRII'臂。通常，将1μg/ml的人PD-L1-his在室温下包被在96孔ELISA板上2小时。在封闭后，添加连续稀释的标准物和血浆样品并温育1.5小时。洗涤后添加0.1μg/ml生物素化抗人TGFβRII'，然后在洗涤后添加链霉亲和素-HRP。最后，添加TMB以显色，用稀硫酸终止显色。通过微板读数器读取板的OD450nm和OD620nm。通过OD450nm-OD620nm分析数据。
第二种方法是仅检测AM4B6抗体臂。与上述程序类似，包被1μg/ml的人PD-L1-his，并且添加连续稀释的标准物和血浆样品并温育1.5小时。洗涤后，添加稀释的山羊HRP偶联的抗人IgG Fc抗体。最后，添加TMB以显色，用稀硫酸终止显色。通过微板读数器读取板的OD450nm和OD620nm。通过OD450nm-OD620nm分析数据。
为了评估抗体浓度与血浆中TGFβ变化之间的相关性，测试了血浆中TGFβ1和TGFβ2的浓度变化。简而言之，将4μg/ml的小鼠TGF-β1捕获抗体或2μg/ml的小鼠TGF-β2捕获抗体在室温下包被在96孔ELISA板上2小时。将10μl的1N HCl添加到50μl每种血浆样品中，并在室温下温育10分钟。通过添加10μl的1.2N NaOH/0.5M HEPES中和酸化样品，以确保最终pH值在7.2-7.6内。在封闭后，添加连续稀释的标准物和血浆样品并温育1.5小时。洗涤后添加TGF-β1或TGF-β2检测抗体，然后在洗涤后添加链霉亲和素-HRP。最后，添加TMB以显色，用稀硫酸终止显色。通过微板读数器读取板的OD450nm和OD620nm。通过OD450nm-OD620nm分析数据。
图20A示出了血浆中的抗体浓度变化。使用两种方法的PK曲线没有显著差异，这表明整个双功能分子AM4B6-hIgG1-TGFβRII'非常稳定，而没有像AM4B6mAb那样在体内异常切割和清除。同时，AM4B6-hIgG1-TGFβRII'在静脉内注射后30分钟内耗竭TGF-β1，甚至在0.3mg/kg的最低剂量下也是如此，如图20B所示。M7824类似物和同种型对照TGFβRII'也耗竭TGF-β1，但M7824类似物不能从第2天起保持所述作用，而AM4B6-hIgG1-TGFβRII'可以保持低水平的TGF-β1到第21天。这个结果也对应于它们的PK暴露(图20C)，这表明TGF-β1可以作为AM4B6-hIgG1-TGFβRII'靶标参与血浆的良好药效标志物。未检测到小鼠血浆中TGF-β2的明显耗竭(现在示出数据)。
实施例12：AM4B6-hIgG1-IL-1RA融合蛋白的构建和表达
将所选重链和轻链变体与hIgG1-IL-1RA(34-177)(UniProtKB，P18510)融合蛋白交叉组合并表达。重链和轻链的序列在表20中示出。
表20.AM4B6-hIgG1-IL-1RA重链和轻链变体的列表
类似于AM4B6-hIgG1-TGFβRII'双功能分子，截短的人IL-1RA_34-177与AM4B6融合以获得更好的活性和稳定性。AM4B6-hIgG1-IL-1RA是AM4B6-hIgG1-IL-1RA(34-177)的简称。截短的人IL-1RA_34-177的序列如下：(SEQ ID NO:67)
根据制造商的方案，使用来自英杰公司的ExpiFectamineTM CHO试剂(赛默(Thermo)，A29129)进行重链和轻链的共转染。在第10天收获上清液并通过亲和色谱法 纯化。
实施例13：AM4B6-hIgG1-IL-1RA双功能分子与hPD-L1的亲和力
基于ELISA测定的与人PD-L1的结合
将1μg/ml hPD-L1(Acro Biosystems，PD1-H5229)抗原包被到ELISA板并在4℃下包被过夜。然后添加300μl的2％(w/v)BSA，以在室温下封闭1小时。在温育1小时后，将100μl的浓度范围为10nM到0.00017nM(三倍连续稀释)的AM4B6-hIgG1-IL-1RA双功能分子或AM4B6-hIgG1单克隆抗体与作为阴性对照的PBST一起添加并在室温下温育1小时。具有0.5％Tween-20的PBS用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。数据通过Graphpad prism分析。
如图21所示，与AM4B6单克隆抗体相比，AM4B6-hIgG1-IL-1RA双功能分子具有相似的结合信号和亲和力。
通过FACS测定测量的AM4B6-hIgG1-IL-1RA与在细胞表面上表达的PD-L1的结合
293T-PD-L1-CD3L细胞由迈博斯生物科学产生，以用于表征PD-L1抗体。用人PD-L1和抗CD3scFv转染细胞。将AM4B6-hIgG1-IL-1RA双功能分子或AM4B6单克隆抗体连续稀释3倍以在稀释缓冲液(含2％BSA的PBS)中获得11个浓度。收获并离心293T-PD-L1-CD3L细胞。然后将它们以2×106个细胞/ml的密度重悬于PBS中，并以每孔100μl添加到板中。在离心并去除上清液后，将稀释的抗体添加到板中并在4℃下温育30分钟。在用稀释缓冲液洗涤两次后，将PE缀合的驴抗人IgG(H+L)(Jacksonimmuno，709-116-149)添加到板并在4℃下温育30分钟。在洗涤后，将细胞重悬于200μl PBS中并通过流式细胞术进行分析。数据通过Graphpad prism分析。
如图22所示，AM4B6-hIgG1-IL-1RA双功能分子和AM4B6-hIgG1可以以相似的EC50结合到细胞表面上表达的PD-L1，这与通过ELISA测量的亲和力结果一致。
实施例14：AM4B6-hIgG1-IL-1RA的PD1/PD-L1阻断活性
在此测定中，293T-PD-L1-CD3L细胞表达PD-L1和抗CD3scFv，而Jurkat-NFAT-Luc-PD1细胞表达PD-1并携带可以被CD3刺激激活的NFAT信号。NFAT激活导致荧光素酶基因转录和表达，这可以通过其底物进行检测。两个细胞均由迈博斯生物科学产生。
简而言之，收获293T-PD-L1-CD3L细胞并以2×106个细胞/ml的密度重悬。将每孔20μl细胞添加到半孔板中。将AM4B6-hIgG1-IL-1RA双功能分子和AM4B6-hIgG1连续稀释(3倍稀释)以在含2％FBS的RPMI培养基中获得8个浓度。向半孔板中每孔添加20μl抗体并且将板在37℃、5％CO2下温育30分钟。收获Jurkat-NFAT-Luc-PD1细胞并以4×106个细胞/ml的密度重悬于含有2％FBS的RPMI培养基中。最后，将每孔20μl细胞添加到半孔板中并在37℃、5％CO2下温育5小时。将60μl的OneGlo检测试剂(普洛麦格(Promega)，E6120)添加到每个孔中并在室温下温育5分钟。发光信号由微板读数器读取。数据通过GraphPad Prism分析。
如图23所示，AM4B6-hIgG1-IL-1RA双功能分子和AM4B6-hIgG1在此基于细胞的测定 中具有相似的对PD-L1的阻断活性。
实施例15：AM4B6-hIgG1-IL-1RA对人IL-1β的阻断活性
基于ELISA的AM4B6-hIgG1-IL-1RA BsAb对hIL-1β的阻断活性
为了测试配体/受体阻断活性，将5μg/ml人IL-1β蛋白(Sino Biological，目录号10139)包被到ELISA板上并在4℃下温育过夜。添加300μl封闭缓冲液，以在室温下封闭1小时。在1小时后，将50μl的连续浓度范围为200nM到0.03nM(三倍连续稀释)的AM4B6-hIgG1-IL-1RA BsAb或IL-1RA蛋白(Sino Biological，目录号10123-HNAE)以及50μl的10nM人IL-1RI-his(Sino Biological，目录号10126-H08H)添加到孔中并在室温下温育1小时。具有0.5％Tween-20的PBS用于洗涤3次，并且添加100μl的HRP偶联的his标签抗体(1:2000稀释，Genscript，目录号A00612)，在室温下温育1小时。然后，添加混合的TMB底物试剂(InnoReagents，目录号：TMB-S-003)并在室温下温育5分钟，然后通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。数据通过Graphpad prism分析。
如图24所示，AM4B6-hIgG1-IL-1RA可以剂量依赖性地阻断IL-1β，并且AM4B6-hIgG1-IL-1RA对IL-1RI的阻断活性优于IL-1RA蛋白。
AM4B6-hIgG1-IL-1RA双功能分子对报告细胞上的hIL-1β的阻断活性
在此测定中，HEK-BlueTM CD40L细胞购自Invivogen(目录号hkb-cd40)，这些细胞通过用人CD40基因和NF-kB诱导型SEAP构建体稳定转染HEK293细胞产生。CD40L与其受体CD40的结合触发级联反应，从而引起NF-kB的激活和SEAP的后续产生，这可以通过QUANTI-Blue进行监测。HEK293细胞内源性地表达与CD40L共享共同信号通路的细胞因子IL-1β的受体。因此，可以使用中和抗体阻断IL-1b介导的SEAP产生。
简而言之，收集对数期细胞的HEK293-CD40L细胞并且以5×10^4/孔(100μl/孔)的密度接种细胞到96孔板中以粘附过夜。将AM4B6-hIgG1-IL-1RA双特异性抗体和IL-1RA蛋白连续稀释(5倍稀释)以在完全培养基中获得10个浓度。向细胞中添加50μl/孔稀释的抗体(或IL-1RA蛋白)和50μl/孔人IL-1β，在37℃下温育24小时。第二天向新板中添加160μl的QUANTI-Blue TM溶液(Invivogen，目录号rep-qbs)，并且向板中添加40μl细胞培养上清液。将板在37℃下温育2小时。使用分光光度计在620nm处确定SEAP水平。数据通过Graphpad prism分析。
如图25所示，AM4B6-hIgG1-IL-1RA可以以剂量依赖性方式阻断IL-1β，并且AM4B6-hIgG1-IL-1RA对IL-1β的阻断活性强于IL-1RA蛋白，这与通过ELISA测量的阻断结果一致。
实施例16：AM4B6-SIRPα双功能抗体的构建、表达、纯化
SIRPα_CV1是经工程化的高亲和力SIRPα变体，所述变体有效地拮抗癌细胞上的CD47，但自身不会诱导巨噬细胞吞噬作用(KippWeiskopf等人,《科学》341,88(2013))。发明了针对PD-L1和CD47两者的双功能抗体，包括对称抗体(AM4B6-hIgG1-SIRPα和3280A-hIgG1-SIRPα)和不对称抗体(AM4B6-hIgG1-SIRPα(KIH)和3280A-hIgG1-SIRPα(KIH))，其中KIH是杵臼结构的简称。这些分子的构建在表21中描述，并且测序的 SIRPα_CV1也在下面列出。
SIRPa_CV1序列(SEQ ID NO:84)：
表21抗PD-L1-SIRPa双功能抗体的构建
根据制造商的方案，用Expi-CHO细胞表达所有4种双功能抗体。对于两种对称双功能抗体，通过一步Mabselect SuRe纯化获得高纯度抗体，但对于不对称双功能抗体，无法通过常规一步Mabselect SuRe纯化获得高纯度抗体。为获得高纯度的不对称抗体，使用HiTrap PrismA树脂对抗体进行精制纯化，并且不对称抗体的纯度优于95％。精制纯化程序描述如下。
使用的缓冲液：
平衡缓冲液：50mM Tris-HAc、150mM NaCl、pH 7.4。
洗涤缓冲液：50mM NaAc/HAc、500mM NaCl、5％PEG、pH 5.5。
洗脱缓冲液：50mM HAc、500mM NaCl、5％PEG、pH 3.0。
用平衡缓冲液平衡HiTrap PrismA柱至少5个柱体积(CV)，直到UV基线、洗脱液 pH值和电导率不变。
将样品加载到预平衡的HiTrap PrismA柱上。
用5到10CV洗涤缓冲液洗涤，直到UV迹线返回到基线。
用0-100％洗脱缓冲液在10-20CV中洗脱，并分别用几个管收集5-10个级分。用1M Tris-碱(pH 9.0)将pH调整到约6.0-7.0。
然后通过SEC-HPLC表征级分。图26示出了3280A-hIgG1-SIRPα(KIH)和AM4B6-hIgG1-SIRPα(KIH)的纯度分别为95.33％和96.5％。
用ELISA测试对人PD-L1或人CD47的亲和力。图27和28示出了双功能抗体对抗原的亲和力与亲本单克隆抗体(4B6mAb对照)或融合蛋白(SIRPα-Fc(FES))相当。
实施例17：IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6双特异性抗体的构建和表达
AM4B6的单链片段(scFv)的序列如下表所示。抗-IL-1β抗体Gevokizumab(XOMA052)和Canakinumab(ACZ885)来自诺华。AM4B6的scFv连接至抗-IL-1β抗体重链C末端以获得更好的活性和稳定性。该scFv具有将VH连接至VL的GS连接子(GGGGSGGGGSGGGGSGGGGS)，并且在H44C和L100C(Kabat编号)残基之间包含一个结构域间的双硫键。抗-IL-1β抗体(XOMA052和ACZ885)的序列如表22所示。所构建的双特异性抗体分别被命名为IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6。
根据制造商的方案，使用来自英杰公司的ExpiFectamineTM CHO试剂(赛默(Thermo)，A29129)进行该双特异性抗体的重链和轻链的共转染。在第10天收获上清液并通过亲和色谱法纯化。
实施例18：IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6双特异性抗体对hIL-1β的结合活性
基于ELISA测定的与人IL-1β的结合
将100μL的1μg/ml hIL-1β蛋白(SinoBiological,Cat#10139-HNAE)抗原包被到ELISA板并在4℃下包被过夜。然后添加200μl的2％(w/v)BSA，以在室温下封闭2小时。在温育后，将100μl的浓度范围为20nM到0.000339nM(三倍连续稀释)的IgG-scFv-ACZ885-AM4B6,IgG-scFv-XOMA052-AM4B6双特异性抗体,ACZ885或XOMA052与作为阴性对照的PBST一起添加并在室温下温育1小时。具有0.5％Tween-20的PBS用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。数据通过Graphpad prism分析。
如图29所示，与ACZ885和XOMA052单克隆抗体相比，IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6bsAbs双功能分子分别具有相似的结合hIL-1β蛋白的活性。
实施例19：IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6双特异性抗体与hPD-L1的结合活性
基于ELISA测定的与人PD-L1的结合
将100μL的1μg/ml hPD-L1(Acro Biosystems，PD1-H5229)抗原包被到ELISA板并在4℃下包被过夜。然后添加300μl的2％(w/v)BSA，以在室温下封闭1小时。在温育1小时后，将100μl的浓度范围为20nM到0.000339nM(三倍连续稀释)的IgG-scFv-ACZ885-AM4B6,IgG-scFv-XOMA052-AM4B6双特异性抗体或AM4B6-hIgG1单克隆抗体(AM4B6mAb)与作为阴性对照的PBST一起添加并在室温下温育1小时。具有0.5％Tween-20的PBS用于洗涤3次，并且添加100μl HRP偶联的抗人Fc抗体(1:20000，艾博抗，ab98624)，在室温下温育1小时后，添加混合的TMB底物试剂(InnoReagents，TMB-S-003)并在室温下温育5分钟，并且通过添加0.1M H2SO4终止。OD450nm由微板读数器记录。数据通过Graphpad prism分析。
如图30所示，与AM4B6单克隆抗体相比，IgG-scFv-ACZ885-AM4B6,IgG-scFv-XOMA052-AM4B6双特异性抗体具有相似的结合hPD-L1蛋白的活性。
通过FACS测定测量的IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6与表达PD-L1的293T细胞的结合
293T-PD-L1-CD3L细胞由迈博斯生物科学产生，以用于表征PD-L1抗体。用人PD-L1和抗CD3scFv转染细胞。将IgG-scFv-ACZ885-AM4B6,IgG-scFv-XOMA052-AM4B6双特异性抗体AM4B6mAb连续稀释4倍以在稀释缓冲液(含2％BSA的PBS)中获得9个浓度。收获并离心293T-PD-L1-CD3L细胞。然后将它们以2×106个细胞/ml的密度重悬于PBS中，并以每孔100μl添加到板中。在离心并去除上清液后，将稀释的抗体添加到板中并在4℃下温育30分钟。在用稀释缓冲液洗涤两次后，将PE缀合的驴抗人IgG(H+L)(Jacksonimmuno，709-116-149)添加到板并在4℃下温育30分钟。在洗涤后，将细胞重悬于200μl PBS中并通过流式细胞术进行分析。数据通过Graphpad prism分析。
如图31所示，IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6可以以相似的EC50结合到细胞表面上表达的PD-L1，这与通过ELISA测量的亲和力结果一致。
实施例20：IgG-scFv-ACZ885-AM4B6和IgG-scFv-XOMA052-AM4B6的PD1/PD-L1阻断活性
在此测定中，293T-PD-L1-CD3L细胞表达PD-L1和抗CD3scFv，而Jurkat-NFAT-Luc-PD1细胞表达PD-1并携带可以被CD3刺激激活的NFAT信号。NFAT激活导致荧光素酶基因转录和表达，这可以通过其底物进行检测。两个细胞均由迈博斯生物科学产生。
简而言之，收获293T-PD-L1-CD3L细胞并以2×106个细胞/ml的密度重悬。将每孔20μl细胞添加到半孔板中。将IgG-scFv-ACZ885-AM4B6,IgG-scFv-XOMA052-AM4B6双特异性抗体和AM4B6-hIgG1连续稀释(3倍稀释)以在含2％FBS的RPMI培养基中获得9个浓度。向半孔板中每孔添加20μl抗体并且将板在37℃、5％CO2下温育30分钟。收获Jurkat-NFAT-Luc-PD1细胞并以4×106个细胞/ml的密度重悬于含有2％FBS的RPMI培养基中。最后，将每孔20μl细胞添加到半孔板中并在37℃、5％CO2下温育5小时。将60μl的OneGlo检测试剂(普洛麦格(Promega)，E6120)添加到每个孔中并在室温下温育5分钟。发光信号由微板读数器读取。数据通过GraphPad Prism分析。
如图32所示，IgG-scFv-ACZ885-AM4B6、IgG-scFv-XOMA052-AM4B6和AM4B6- hIgG1在此基于细胞的测定中具有相似的对PD-L1的阻断活性。
实施例21：IgG-scFv-XOMA052-AM4B6对人真皮成纤维(HDF)细胞上的人IL-1β的阻断活性
IgG-scFv-XOMA052-AM4B6对HDF细胞上的人IL-1β的阻断活性
为了测试配体/受体阻断活性，用50pg/ml的重组人IL-1β蛋白(Sino Biological，目录号10139)刺激100uL/孔的4x104/mL的HDF细胞，并用没有IL-1β刺激的细胞作为阴性对照。然后，将100uL/孔的连续浓度范围为100nM到0.00038nM(四倍连续稀释)的IgG-scFv-XOMA052-AM4B6和XOMA052添加到培养液中并在室温下温育过夜(16-17小时)。刺激之后，根据试剂盒的指导，用IL-6ELISA试剂盒(R&D,DY206,P209026)来检测细胞培养的悬浮液中的IL-6释放。
如图33所示，IgG-scFv-XOMA052-AM4B6和XOMA052可以剂量依赖性地阻断IL-1β，并且IgG-scFv-XOMA052-AM4B6对HDF细胞的阻断活性与XOMA052相似。
IgG-scFv-ACZ885-AM4B6对报告细胞上的hIL-1β的阻断活性
在此测定中，HEK-BlueTM CD40L细胞购自Invivogen(目录号hkb-cd40)，这些细胞通过用人CD40基因和NF-kB诱导型SEAP构建体稳定转染HEK293细胞产生。CD40L与其受体CD40的结合触发级联反应，从而引起NF-kB的激活和SEAP的后续产生，这可以通过QUANTI-Blue进行监测。HEK293细胞内源性地表达与CD40L共享共同信号通路的细胞因子IL-1β的受体。因此，可以使用中和抗体阻断IL-1β介导的SEAP产生。
简而言之，收集对数期细胞的HEK293-CD40L细胞并且以5×10^4/孔(100μl/孔)的密度接种细胞到96孔板中以粘附过夜。将IgG-scFv-ACZ885-AM4B6和ACZ885从100nM连续稀释(4倍稀释)以在完全培养基中获得9个浓度。向细胞中添加50μl/孔稀释的抗体和50μl/孔人IL-1β(1ng/mL)，在37℃下温育24小时。第二天向新板中添加160μl的QUANTI-Blue TM溶液(Invivogen，目录号rep-qbs)，并且向板中添加40μl细胞培养上清液。将板在37℃下温育2小时。使用分光光度计在620nm处确定SEAP水平。数据通过Graphpad prism分析。
如图34所示，IgG-scFv-ACZ885-AM4B6和ACZ885(Canakinumab)可以以剂量依赖性方式阻断IL-1β，并且IgG-scFv-ACZ885-AM4B6对HEK293-CD40L报告细胞的阻断活性与ACZ885(Canakinumab)相似。
表22.本公开提到的氨基酸序列













实施例B：制剂及其效果
本实施例中涉及的分析方法、试剂相关信息以及测试样品配制方法如下：
1.动态光散射DLS
蛋白粒径及分布通过动态光散射仪(DLS，Wyatt，型号WP2-09)进行测定，方法参数如下：采集时间5s，每次测量采集20次，测量温度25℃。
2.分子排阻色谱SEC
蛋白聚集情况使用Agilent 1260系统和TSKgel G3000SWXL柱(300×7.8mm，5μm)通过SEC方法测定。流动相为50mM磷酸钠缓冲液，300mM NaCl，pH 6.8±0.1。流速为1.0mL/min。将样品稀释至10mg/mL，检测体积10μL，检测波长为280nm。
通过SEC测定样品中的高分子量聚合物(至少是二聚体)HMW％
3.非还原性毛细管电泳NR CE-SDS
蛋白碎片情况通过CE-SDS(NR)方法进行测定。将标准品或供试样品用磷酸-柠檬酸缓冲液稀释至4mg/mL，然后取样25μL与75μL SDS样品缓冲液和5μL NEM(100mM N-乙基马来酰亚胺)涡旋混合进行变性处理。变性的样品经离心后，在70±2℃下孵育10±2min，在室温下冷却，然后再次离心。使用SDS分离凝胶试剂盒和未涂层的熔融石英毛细管在 PA800plus上进行分离。
通过NR CE-SDS测定样品中的小分子碎片(分子量低于单体)LMW％
4.结合活性
包被抗原为TGFβ1，检测抗体为Goat anti-Human IgG-Fc HRP conjugated，酶反应的底物为TMB。将抗原包被吸附在96孔高吸附板上，洗涤封闭后加入供试品与之结合，孵育后洗涤，加入检测抗体，孵育洗涤除去未结合的检测抗体，再加入底物显色，最后加入反应终止液(1M硫酸)，于酶标仪上读取吸光值，其检测波长为450nm，参比波长为650nm。
5.细胞活性
细胞铺板在96孔检测板中，参比品和样品以浓度10μg/mL为首孔、2倍梯度稀释至0.0195μg/mL，加入96孔检测板中。加入梯度稀释样品的细胞置于37.0℃±1.0℃，5.0±1.0％CO2培养箱中培养4-5小时。通过Bio-LiteTM化学发光试剂盒或等效试剂检测以上实施例制备的双功能蛋白质分子、例如AM4B6-hIgG1-TGFβRII'是否抑制PD-1和PD-L1细胞间的交互作用。化学发光的强度与每个孔的抑制程度成线性相关。对剂量依赖曲线进行四参数模型拟合，最终以参比品与样品之半数有效浓度比值，计算样品的相对活性。
6.流动注射HPLC荧光分析法
聚山梨酯80浓度通过流动注射HPLC荧光分析法进行测定。使用Agilent 1260系统和荧光检测器，激发波长为350nm，发射波长为420nm，流动相为0.15M氯化钠，0.025M四硼酸钠，5.0％乙腈，5.0μM奈氨基苯，2.5ppm聚山梨酯80。
7.试剂相关信息如下：
8.如无特别说明，本实施例的测试制剂样品采用的配制方法如下：
(1).采用透析的方法将蛋白质原液(即，PDL1/TGFβ蛋白(AM4B6-hIgG1-TGFβRII'))的缓冲液置换为目标缓冲体系：将一定体积的样品置于Snake透析袋内，密封后，置于≧100倍体积的目标缓冲液中，持续搅拌以促进置换。透析进行3次，持续时间分别为4h，4h并且过夜，搅拌速度为300rpm。
(2).透析结束后，蛋白原液中加入所需量的稳定剂、表面活性剂母液等辅料，并用目标缓冲液稀释至目标蛋白浓度，然后除菌过滤、无菌灌装，得到测试制剂溶液，制剂的具体参 数分别如下文处方设计表格所示。
实施例B-1：pH、表面活性剂和其他辅料对制剂性质的影响研究
1.材料和方法
采用JMP软件的定制设计进行DOE(Design of expriment)处方设计。筛选的处方包含20mg/mL PDL1/TGFβ蛋白(AM4B6-hIgG1-TGFβRII')，pH为4.5～5.5的20mM醋酸-醋酸钠缓冲液，135mM～280mM其他辅料，0.025～0.1％(w/v,g/100mL)聚山梨酯80。具体处方设计见表23。
如下表所示，考察所有处方测试样品初始时刻T0的粒径，40℃4周及在25℃搅拌3小时条件下的稳定性。
表23处方设计
2.实验结果和分析
2.1各个测试样品初始时刻T0的粒径
通过动态光散射仪，检测样品的粒径。检测条件，如上文所述。
T0时的粒径(DLS)结果见表24。从实验结果可知随着pH增加，含蔗糖和山梨醇的处 方，粒径明显增加(F2，F4，F6，F8，F10，F12，从～7nm增加到～15nm)，特别是在pH5.0和pH5.5时，含蔗糖或山梨醇的处方(F6，F8，F10，F12)，粒径明显大于含盐酸精氨酸或NaCl的处方。实验结果显示，含盐酸精氨酸或NaCl的处方在不同pH下T0粒径没有明显差异。这显示，渗透压调节剂盐酸精氨酸或NaCl还可以减少蛋白聚合物的产生，有利于蛋白质稳定。
表24 DLS结果
2.2各个测试样品40℃放置4周的稳定性
将各个处方样品在40℃放置4周(T4w)。采用SEC和NR CE-SDS进行检测，得到样品T0(初始时刻)和T4w的高分子量聚合物HMW％(采用SEC检测)和小分子碎片LMW％(采用NR CE-SDS检测)数据。高分子量聚合物HMW％和小分子碎片LMW％的变化值(△HMW％和△LMW％)为T4w与T0的差异值。
将△HMW％和△LMW％作为响应值输入JMP软件，采用最小二乘法对回归模型进行拟合。HMW和LMW的模型拟合R2均为0.96，数据拟合度很高。图35展示了JMP软件对数据进行分析后的结果。在0.025％～0.1％(w/v)的范围内，PS80的浓度对HMW和LMW几乎没有影响。辅料种类对HMW的影响在于，含蔗糖和山梨醇的处方，HMW的增加较多；与含蔗糖和山梨醇的处方相比，含盐酸精氨酸和氯化钠的处方，HMW的增加明显减少。这显示渗透压调节剂盐酸精氨酸或NaCl还可以减少蛋白聚合物的产生，有利于蛋白质稳定。此外，从图35中可见，在pH 4.5～5.5的范围内，pH越高，越有利于PD-L1/TGF-β蛋白稳定，HMW和LMW的增加越缓慢，即pH 5.0～5.5是比较优异的pH区间范围。
2.3搅拌条件的稳定性
将各个处方样品在25℃搅拌3小时(T3h)。采用SEC和NR CE-SDS进行检测，得到样 品T0(初始时刻)和T3h的高分子量聚合物HMW％(采用SEC检测)和小分子碎片LMW％(采用NR CE-SDS检测)数据。高分子量聚合物HMW％和小分子碎片LMW％的变化值(△HMW％和△LMW％)为T3h与T0的差异值。
将△HMW％和△LMW％作为响应值输入JMP软件，采用最小二乘法对回归模型进行拟合。HMW和LMW的模型拟合R2分别为0.91和0.93，数据拟合度很高。图36展示了JMP软件对数据进行分析后的结果。从图36中可以看出，F1-F12：pH，PS80浓度和辅料种类在测试条件下对HMW和LMW都没有影响。
综上，T0粒径、40℃稳定性以及搅拌实验显示，在pH 5.0～5.5的范围内时，HMW和LWM的增加比较缓慢，并且pH对HMW和LMW的变化几乎没有影响，因此优选的pH范围是pH 5.0～5.5。盐酸精氨酸或NaCl不仅可以起到渗透压调节剂的作用，还可以减少蛋白聚合物的产生，有利于蛋白质稳定，因此优选的辅料种类为盐酸精氨酸或氯化钠。在0.025％～0.1％(w/v)的范围内，PS80的浓度对HMW和LMW几乎没有影响。
实施例B-2：不同缓冲体系的比较实验
1.材料和方法
在pH5.5的条件下，比较醋酸缓冲体系和组氨酸缓冲体系对蛋白质稳定性的影响。具体的处方设计见表25。
表25比较不同缓冲体系的处方设计
2.不同缓冲体系的结果与分析
将两个处方样品在40℃放置4周(T4w)。采用SEC和NR CE-SDS进行检测，得到样品T0(初始时刻)、T2w和T4w的高分子量聚合物HMW％(采用SEC检测)和小分子碎片LMW％(采用NR CE-SDS检测)数据。如图37和图38所示，在醋酸缓冲体系和组氨酸缓冲体系中，PD-L1/TGF-β蛋白的高分子量聚合物HMW和小分子碎片LMW的变化均较小，说明缓冲盐的种类对蛋白质稳定性没有影响，即这两种缓冲体系均可满足蛋白质稳定性要求。
实施例B-3：不同蛋白质浓度的的比较实验
1.材料和方法
在本实施例中，对蛋白质浓度进行了筛选。根据实施例B-1中的结果图35，优选的pH范围是pH 5.0～5.5。当pH为5.3时，如图35所示，HWM在40℃条件下几乎没有增加，LMW仅有少量增加。因此，本实施例中选择pH5.3的缓冲液作为缓冲体系。同时根据实施例B-1中的结果图35，聚山梨酯80的浓度0.025-0.1％对蛋白质稳定性没有影响，因此，选择一个中间值0.05％(w/v)的聚山梨酯80作为本实验表面活性剂的浓度进行实验。具体的 处方设计和测试条件见表26。
表26不同蛋白浓度的处方设计
2.结果与分析
将两个处方样品在40℃放置6周(T6w)。采用SEC和NR CE-SDS进行检测，得到样品T0(初始时刻)、T2w、T4w和T6w的高分子量聚合物HMW％(采用SEC检测)和小分子碎片LMW％(采用NR CE-SDS检测)数据。如图39和图40所示，在蛋白浓度分别为20mg/ml和30mg/ml时，PD-L1/TGF-β蛋白的高分子量聚合物HMW和小分子碎片LMW的变化趋势很接近，说明在20mg/ml～30mg/ml的范围内，PD-L1/TGF-β蛋白的稳定性相似，满足稳定性要求。
实施例B-4：不同辅料的比较实验
1.材料和方法
在本实施例中，对辅料种类进行了进一步筛选。根据实施例B-1的结果，选择pH5.3的20mM醋酸-醋酸钠缓冲液作为缓冲体系，0.05％(w/v)的聚山梨酯80作为表面活性剂，盐酸精氨酸或氯化钠作为稳定剂和渗透压调节剂。具体的处方设计和测试条件见表27。
表27进一步筛选辅料的处方设计
2.结果与分析
2.1冻融和振摇结果
在冻融和振摇条件下，F16和F17在外观，蛋白质粒径，HMW和LMW方面均无明显变化，且与T0相比这些方面亦无明显变化。这显示F16和F17在冻融和振摇条件下满足药物制剂稳定性要求。
2.2光照结果
如图41所示，在光照条件下，与氯化钠相比，盐酸精氨酸在抑制HMW方面有明显的优势，显示其特别有利于维持蛋白质稳定。在外观、蛋白质粒径、LMW及活性方面，F16和F17均未发生明显变化，且与T0相比这些方面亦无明显变化。这显示F16和F17在光照条件下均满足药物制剂稳定性要求，而F16显示出更加优异的稳定性。
2.3 40℃结果
如图42所示，在40℃条件下，与氯化钠相比，盐酸精氨酸在抑制HMW方面有明显的优势，显示其特别有利于维持蛋白质稳定。在外观、蛋白质粒径及活性方面，F16和F17均未发生明显变化；F17的LMW增加比F16略少，优势不明显；且与T0相比这些方面亦无明显变化。这显示F16和F17在40℃条件下均满足药物制剂稳定性要求，而F16显示出更加优异的稳定性。
2.4 25℃结果
如图43示，在25℃条件下，与氯化钠相比，盐酸精氨酸在抑制HMW方面有明显的优势。在外观、蛋白质粒径及LMW方面，F16和F17均未发生明显变化，且与T0相比这些方面亦无明显变化。这显示F16和F17在光照条件下均满足药物制剂稳定性要求，而F16显示出更加优异的稳定性。
综合考虑各条件的结果，在光照、高温强制降解(40℃)、加速(25℃)的条件下，盐酸精氨酸可以更好地减缓HMW的增加。在LMW及活性方面，盐酸精氨酸和氯化钠的作用无明显差异。因此，在本实施例中，制剂稳定性最佳的处方为：30mg/ml的PD-L1/TGF-β蛋白，pH为5.3的20mM醋酸-醋酸钠缓冲液，150mM的盐酸精氨酸，0.05％(w/v)的聚山梨酯80。
实施例B-5：考察依地酸二钠的保护作用
1.材料和方法
在实施例B-4中优选处方的基础上，进一步考察加入依地酸二钠后对蛋白质和聚山梨酯80稳定性的影响。具体的处方设计和测试条件见表28。
表28处方设计

2.结果与分析
如图44和图45所示，在所测试的75μM～300μM的范围内，依地酸二钠可以为PD-L1/TGF-β蛋白制剂中的聚山梨酯80提供良好的保护作用，在40℃储存一个月及储存25℃6个月后，聚山梨酯80几乎没有发生降解。
如图46、图47、图48和图49所示，F19、F20和F21在防止HMW和LWM增加方面的效果与F18相似，说明加入依地酸二钠不影响蛋白质的稳定性。依地酸二钠的作用主要在于防止聚山梨酯80降解，从而提高制剂的安全性。
实施例B-6：考察处方制备工艺中加入依地酸二钠的作用
1.材料和方法
在实施例B-5的基础上，考察在工艺中加入依地酸二钠，是否仍然可以防止聚山梨酯80降解。工艺描述如下：
在pH为5.3的20mM醋酸-醋酸钠缓冲液的40mg/ml蛋白质(即，PDL1/TGFβ蛋白(AM4B6-hIgG1-TGFβRII'))溶液中，先加入依地酸二钠母液，使其终浓度为0.5mM和5mM，所得溶液分别在室温下放置过夜。
将上述步骤中的蛋白溶液置于Thermo的透析袋(货号：88245)中，用pH为5.3的20mM醋酸-醋酸钠缓冲液透析三次，每次透析时间≥4小时，将依地酸二钠除去。
将透析后的蛋白溶液采用实施例B中的通用方法的第(2)部分配制成处方溶液(30mg/ml的PD-L1/TGF-β蛋白，pH为5.3的20mM醋酸-醋酸钠缓冲液，150mM的盐酸精氨酸，0.05％(w/v)的聚山梨酯80)，并考察在40℃条件下聚山梨酯80降解的情况。
同时，还采用类似方法制备了对照溶液，区别仅在于蛋白质溶液未经依地酸二钠处理。
2.结果与分析
如图50所示，与未经依地酸二钠处理的处方相比，经0.5mM或5mM依地酸二钠处理后的处方，均可以在40℃下防止聚山梨酯80的降解，减少降解副产物的产生，从而提高制剂的安全性。

Claims (18)

  1. 蛋白质制剂，其包括：
    (1)双功能分子，其包括：
    与免疫检查点分子结合的第一部分，所述第一部分优选是针对PD-L1的抗体或其抗原结合片段，和
    i)阻断免疫抑制性细胞因子的活性或ii)刺激免疫的第二部分，所述第二部分优选是TGFβ结合部分、IL-1结合部分、LAG-3结合部分或Sirpa结合部分，其中所述TGFβ结合部分优选是可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体、或针对TGFβ的抗体和其抗原结合片段；
    (2)缓冲剂；和
    (3)表面活性剂。
  2. 根据权利要求1的蛋白质制剂，其包括：
    (1)双功能分子，其包括与PD-L1结合的第一部分和a)阻断免疫抑制性细胞因子的活性或b)刺激免疫的第二部分，其中所述第一部分包括针对PD-L1的抗体或其抗原结合片段，所述抗体或其抗原结合片段包括重链可变(VH)区和/或轻链可变(VL)区，其中所述重链可变区包括：
    a)HCDR1，所述HCDR1包括DYYMN(SEQ ID NO:1)或与DYYMN具有至少80％序列同一性的同源序列，
    b)HCDR2，所述HCDR2包括DINPNNX1X2TX3YNHKFKG(SEQ ID NO:19)或与DINPNNX1X2TX3YNHKFKG具有至少80％序列同一性的同源序列，以及
    c)HCDR3，所述HCDR3包括WGDGPFAY(SEQ ID NO:3)或与WGDGPFAY具有至少80％序列同一性的同源序列，和/或
    其中所述轻链可变区包括：
    d)LCDR1包括选自由KASQNVX4X5X6VA(SEQ ID NO:20)或与KASQNVX4X5X6VA具有至少80％序列同一性的同源序列组成的组的序列，
    e)LCDR2包括选自由SX7SX8RYT(SEQ ID NO:21)或与SX7SX8RYT具有至少80％序列同一性的同源序列组成的组的序列，以及
    f)LCDR3包括选自由QQYSNYPT(SEQ ID NO:6)或与QQYSNYPT具有至少80％序列同一性的同源序列组成的组的序列；
    其中X1是G或A，X2是G或D或Q或E或L，X3是S或M或Q或L或V，X4是G或P或K，X5是A或G，X6是A或I，X7是A或N或R或V，并且X8是N或H或V或D；
    (2)缓冲剂；和
    (3)表面活性剂。
  3. 根据前述权利要求中任一项的蛋白质制剂，其中所述双功能分子第一部分包括针对 PD-L1的抗体或其抗原结合片段，且第二部分包括TGFβ结合部分，
    其中所述针对PD-L1的抗体或其抗原结合片段包括选自由以下组成的组的一对重链可变区和轻链可变区序列：SEQ ID NO:49/54、50/54、51/54、52/54、49/55、50/55、51/55、52/55、58/62、58/63、58/64、58/65、59/62、59/63、59/64、59/65、60/62、60/63、60/64和60/65及与其具有至少80％序列同一性的其同源序列；和/或
    其中所述TGFβ结合部分包括可溶性TGFβ受体(TGFβR)或其TGFβ结合片段或变体，其中所述可溶性TGFβR包括所述TGFβR的胞外结构域(ECD)或其TGFβ结合片段或变体，且TGFβR的所述ECD包括SEQ ID NO:66、79、78、77的氨基酸序列或与所述氨基酸序列具有至少80％序列同一性但仍保留对TGF-β的特定结合特异性和/或亲和力的序列。
  4. 根据前述权利要求中任一项的蛋白质制剂，其中所述双功能分子的浓度是约1-150mg/mL，例如约10-50mg/mL或约20-30mg/mL。
  5. 根据前述权利要求中任一项的蛋白质制剂，其中所述缓冲剂是醋酸盐、组氨酸、柠檬酸盐、琥珀酸盐、苹果酸盐、谷氨酸盐、磷酸盐或乳酸盐缓冲剂，例如醋酸-醋酸钠缓冲剂或组氨酸-盐酸缓冲剂。
  6. 根据前述权利要求中任一项的蛋白质制剂，其中所述缓冲剂的浓度是约5-100mM，例如约10-50mM、约15-30mM或约20mM。
  7. 根据前述权利要求中任一项的蛋白质制剂，其中所述制剂的pH在约4.5-6.0的范围，例如约5.0-5.6、约5.0-5.5或约5.3。
  8. 根据前述权利要求中任一项的蛋白质制剂，其中所述表面活性剂是非离子表面活性剂，优选聚山梨酯，例如聚山梨酯80或聚山梨酯20。
  9. 根据前述权利要求中任一项的蛋白质制剂，其中所述表面活性剂浓度是约0.01-0.1％(w/v)，例如约0.025％-0.1％(w/v)或约0.05％(w/v)。
  10. 根据前述权利要求中任一项的蛋白质制剂，其中所述制剂包含稳定剂，所述稳定剂例如选自氨基酸、无机盐、糖、多元醇和螯合剂中的一种或多种，优选盐酸精氨酸或NaCl。
  11. 根据前述权利要求中任一项的蛋白质制剂，其中所述制剂包含稳定剂，所述稳定剂的浓度是约10-300mM，例如约100-300mM、约120-300mM、约100-200mM、约130-170mM或约150mM。
  12. 根据前述权利要求中任一项的蛋白质制剂，其中所述制剂包含盐酸精氨酸，其浓度优选为约100-200mM、约130-170mM或约150mM。
  13. 根据前述权利要求中任一项的蛋白质制剂，其中所述制剂任选地包含螯合剂，例如EDTA·2Na，其浓度优选为约30-350μM。
  14. 根据前述权利要求中任一项的蛋白质制剂，其包括：
    10-50mg/mL的所述双功能分子；
    10-50mM的醋酸盐缓冲剂或组氨酸缓冲剂；
    0.025％-0.1％(w/v)的聚山梨酯80或聚山梨酯20；
    100-200mM的盐酸精氨酸；以及
    任选地30-350μM的EDTA·2Na；
    pH是5.0-5.6；
    或者包含：
    20-30mg/mL的所述双功能分子；
    20-30mM的醋酸盐缓冲剂或组氨酸缓冲剂；
    0.025％-0.1％(w/v)的聚山梨酯80；以及
    140-160mM的盐酸精氨酸；
    pH是5.3±5％。
  15. 根据前述权利要求中任一项的蛋白质制剂，其中所述双功能分子在配制过程中用螯合剂，例如EDTA·2Na(例如约0.3-10mM的EDTA·2Na)进行处理，然后将螯合剂除去。
  16. 根据前述权利要求中任一项的蛋白质制剂，其为液体形式，冻干形式或由冻干形式重构的液体形式，例如注射剂，例如静脉注射剂、肌内注射剂或皮下注射剂。
  17. 根据权利要求1-16中任一项的蛋白质制剂，其用于治疗、预防或减轻受试者的PD-L1相关疾病，例如所述疾病是免疫相关疾病或病症、癌症或传染病，例如所述癌症选自由以下组成的组：肺癌(例如非小细胞肺癌)、肝癌、胰腺癌、乳腺癌、支气管癌、骨癌、肝和胆管癌、卵巢癌、睾丸癌、肾癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、前列腺癌、胃食管癌、直肠癌、肛门癌、胃肠癌、皮肤癌、垂体癌、胃癌、阴道癌、甲状腺癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、胶质瘤和腺癌，例如所述受试者已被鉴定为具有表达PD-L1的癌细胞，例如其中所述PD-L1相关疾病对PD-L1/PD-1单一疗法具有抗性。
  18. 根据权利要求1-16中任一项的蛋白质制剂，其用于治疗、预防或减轻将受益于免疫抑制性细胞因子的抑制、持续免疫应答的诱导或抗肿瘤免疫的刺激的疾病或病状；
    其中，例如所述免疫抑制性细胞因子是TGFβ；例如所述疾病或病状是TGFβ相关疾病或病状；例如所述TGFβ相关疾病是癌症，纤维化疾病或肾病；
    其中，例如所述免疫抑制性细胞因子是IL-1；例如所述疾病或病状是IL-1相关疾病或病状；
    其中，例如所述疾病或病状将受益于通过用免疫刺激性多肽刺激MHCII信号传导而诱导持续免疫应答；例如所述免疫刺激性多肽是可溶性LAG-3；或
    其中，例如所述疾病或病状将受益于通过抑制免疫抑制性受体信号传导而刺激抗肿瘤免疫；例如所述免疫抑制性受体是SIRPα。
PCT/CN2023/094267 2022-05-17 2023-05-15 双功能蛋白质及其制剂和用途 WO2023221936A1 (zh)

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