Bifunctional protein as well as preparation and application thereof

Abstract

Provided are proteins comprising a PD-L1 binding moiety linked to a TGF [beta] binding moiety, an IL-1 binding moiety, an immunostimulatory polypeptide (e.g., soluble LAG3 or soluble CD4), or a CD47 binding moiety, pharmaceutical formulations, methods of preparation, and uses thereof.

Description

Bifunctional proteins and their preparations and uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to patent application CN202210541662.4 filed on May 17, 2022, the disclosure of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to bifunctional protein molecules that target immune checkpoint molecules (e.g., PD-L1) and block the activity of anti-tumor immunosuppressive (ATIS) cytokines (e.g., IL-1 or TGFβ) or stimulate immunity, as well as pharmaceutical preparations, preparation methods, and uses thereof. Background Art

[0004] PD-L1 is often 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 MK 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 of this approach represents a breakthrough in the fight against human cancer, particularly solid tumors. Although an association has been shown between the expression of PD-L1 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., Nature 515, 563–567 (2014); Taube JM et al., Clinical Cancer Research, 2014, 20(19):5064-5074). Only a minority of PD-L1-positive tumors respond to these treatments, whereas some PD-L1-negative tumors still respond to treatment. This raises the possibility that additional factors control a patient's response to PD-1 / PD-L1 targeted therapies, and that additional predictive biomarkers must be identified to improve the clinical use of these agents.

[0005] Mariathasan S et al. found that the lack of response was associated with the characteristics of transforming growth factor beta (TGF-β, TGFβ) signaling in fibroblasts (Mariathasan S et al., Nature, 2018, 554(7693):544-548). David JM et al. also found that TGF-β, a pleiotropic cytokine known to induce epithelial-mesenchymal transition (EMT) and inhibit anti-tumor immunity, can upregulate tumor PD-L1 expression in multiple epithelial NSCLC cell lines, and the upregulation is associated with the phosphorylation of Smad2, a key downstream effector of TGF-β signaling (David JM et al., Oncoimmunology, 2017, 6(10):e1349589). In mice, therapeutic administration of a TGF-β blocking antibody and anti-PD-L1 reduced TGF-β signaling in stromal cells, promoted T cell infiltration into the tumor center, and stimulated strong antitumor immunity and tumor regression (Mariathasan S et al., Nature, 2018, 554(7693):544-548).

[0006] However, the low affinity of anti-PD-L1 antibodies still poses a challenge to achieving high therapeutic efficacy and low toxic side effects.

[0007] Therefore, there is a need for therapeutic molecules and pharmaceutical formulations thereof with high binding affinity to PD-L1, improved therapeutic efficacy, and reduced toxic side effects.

[0008] Contents of this Disclosure

[0009] The present disclosure provides proteins comprising a PD-L1 binding portion linked to a TGFβ binding portion, an IL-1 binding portion, an immunostimulatory polypeptide (e.g., soluble LAG3 or soluble CD4), or a CD47 binding portion, and pharmaceutical formulations, methods of preparation, and uses thereof.

[0010] The present disclosure provides a protein formulation comprising:

[0011] (1) A bifunctional molecule comprising:

[0012] a first portion that binds to an immune checkpoint molecule, preferably an antibody or antigen-binding fragment thereof directed against PD-L1, and

[0013] i) blocks the activity of immunosuppressive cytokines or ii) stimulates immunity, wherein the second moiety is preferably a TGFβ binding portion, an IL-1 binding portion, a LAG-3 binding portion or a Sirpa binding portion, wherein the TGFβ binding portion is preferably a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, or an antibody to TGFβ and an antigen-binding fragment thereof;

[0014] (2) buffer; and

[0015] (3) Surfactants

[0016] More specifically, the present disclosure also provides a protein formulation comprising:

[0017] (1) A bifunctional molecule comprising a first portion that binds to PD-L1 and a second portion that i) blocks the activity of immunosuppressive cytokines or ii) stimulates immunity; wherein the first portion comprises an antibody or antigen-binding fragment thereof directed against PD-L1;

[0018] (2) buffer; and

[0019] (3) Surfactant. In this regard, the present disclosure also provides the following more specific embodiments:

[0020] Embodiment 1. A protein formulation comprising:

[0021] (1) a bifunctional molecule comprising a first portion that binds to an immune checkpoint molecule and a second portion that blocks interleukin-1 (IL-1) activity;

[0022] (2) buffer; and

[0023] (3) Surfactants

[0024] Embodiment 2. The protein formulation of embodiment 1, wherein the first portion comprises an agonist of an immune stimulatory checkpoint molecule, optionally 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.

[0025] Embodiment 3. The protein formulation of embodiment 1, wherein the first portion comprises an antagonist of an immunoinhibitory checkpoint molecule, optionally 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.

[0026] Embodiment 4. The protein formulation of embodiment 3, wherein the immune checkpoint molecule is PD-L1.

[0027] Embodiment 5. The protein formulation of any one of the preceding embodiments, wherein the first portion comprises an antibody or antigen-binding fragment thereof against PD-L1, and the second portion comprises an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion.

[0028] Embodiment 6. The protein formulation of embodiment 5, wherein the IL-1 binding portion comprises IL-1R or a fragment or variant thereof, or an antibody or antigen-binding fragment thereof directed against IL-1.

[0029] Embodiment 7. The protein formulation of embodiment 6, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region from an anti-IL-1α antibody or an anti-IL-1β antibody, wherein the anti-IL-1α antibody is selected from the group consisting of XB2001, lutikizumab, LY2189102, and bermekimab, or the anti-IL-1β antibody is selected from the group consisting of SSGJ-613, CDP484, canakinumab, and gevokizumab.

[0030] Embodiment 8. The protein formulation of embodiment 6, wherein the antibody or antigen-binding fragment thereof directed against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104 or SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 105 or SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 106 or SEQ ID NO: 114, and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 109 or SEQ ID NO: 117.

[0031] Embodiment 9. The protein formulation of embodiment 6, wherein the antibody or antigen-binding fragment thereof directed against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104, a HCDR2 comprising the sequence of SEQ ID NO: 105, and a HCDR3 comprising the sequence of SEQ ID NO: 106 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107, a LCDR2 comprising the sequence of SEQ ID NO: 108, and a LCDR3 comprising the sequence of SEQ ID NO: 109.

[0032] Embodiment 10. The protein formulation of embodiment 6, wherein the antibody or antigen-binding fragment thereof directed against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 114, and the light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 117.

[0033] Embodiment 11. The protein formulation of embodiment 6, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 110, and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 111, and a homologous sequence thereof having at least 80% sequence identity.

[0034] Embodiment 12. The protein formulation of embodiment 6, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102 and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103 and a homologous sequence thereof having at least 80% sequence identity.

[0035] Embodiment 13. A protein formulation according to embodiment 6, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 110, and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 111, and a homologous sequence thereof having at least 80% sequence identity.

[0036] Embodiment 14. The protein formulation of embodiment 5, wherein the IL-1R binding portion comprises an interleukin-1 receptor antagonist or a fragment or variant thereof, or an antibody or antigen-binding fragment thereof directed against IL-1R.

[0037] Embodiment 15. The protein formulation of embodiment 14, wherein the antibody or antigen-binding fragment thereof against IL-1R comprises a heavy chain variable region and / or a light chain variable region from an antibody selected from spesolimab, astegolimab, imsidolimab, AMG 108, melrilimab, nidanilimab, MEDI8968, REGN6490, HB0034, and CSC012.

[0038] Embodiment 16. A protein formulation comprising:

[0039] (1) A bifunctional molecule comprising 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 the first portion comprises an antibody or antigen-binding fragment thereof directed against PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region comprises:

[0040] a) HCDR1 comprising DYYMN (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity to DYYMN,

[0041] b) a HCDR2 comprising DINPNNX1X2TX3YNHKFKG (SEQ ID NO: 19) or a homologous sequence having at least 80% sequence identity to DINPNNX1X2TX3YNHKFKG, and

[0042] c) a HCDR3 comprising WGDGPFAY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity to WGDGPFAY, and / or

[0043] wherein the light chain variable region comprises:

[0044] d) LCDR1 comprises a sequence selected from the group consisting of KASQNVX4X5X6VA (SEQ ID NO: 20) or a homologous sequence having at least 80% sequence identity thereto,

[0045] e) LCDR2 comprises a sequence selected from the group consisting of SX7SX8RYT (SEQ ID NO: 21) or a homologous sequence having at least 80% sequence identity thereto, and

[0046] f) LCDR3 comprises a sequence selected from the group consisting of QQYSNYPT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity to QQYSNYPT;

[0047] wherein X1 is G or A, X2 is G or D or Q or E or L, X3 is S or M or Q or L or V, X4 is G or P or K, X5 is A or G, X6 is A or I, X7 is A or N or R or V, and X8 is N or H or V or D;

[0048] (2) buffer; and

[0049] (3) Surfactants

[0050] Embodiment 17. The protein formulation of embodiment 16, wherein the heavy chain variable region comprises:

[0051] a) HCDR1 comprises the sequence of SEQ ID NO: 1,

[0052] b) HCDR2 comprises 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

[0053] c) HCDR3 comprises the sequence of SEQ ID NO: 3,

[0054] and / or

[0055] A light chain variable region, comprising:

[0056] d) LCDR1 comprises 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,

[0057] e) LCDR2 comprises 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

[0058] f) LCDR3 comprises the sequence of SEQ ID NO: 6.

[0059] Embodiment 18. The protein formulation of embodiment 17, wherein the heavy chain variable region is selected from the group consisting of:

[0060] a) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0061] b) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 13, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0062] c) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 14, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0063] d) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 15, and a HCDR3 comprising the sequence of SEQ ID NO: 3; and

[0064] e) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 17, and a HCDR3 comprising the sequence of SEQ ID NO: 3.

[0065] Embodiment 19. The protein formulation of embodiment 17 or 18, wherein the light chain variable region is selected from the group consisting of:

[0066] a) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 4, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0067] b) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 9, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0068] c) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 8, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0069] d) 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

[0070] e) 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.

[0071] Embodiment 20. The protein formulation of any one of Embodiments 16 to 19, wherein the antibody or antigen-binding fragment thereof against PD-L1 further comprises one or more of the heavy chain HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chain LFR1, LFR2, LFR3, and LFR4, wherein:

[0072] a) the HFR1 comprises QVQLVQSGAEVKKPGASVKVSCKASGYX9FT (SEQ ID NO: 40) or a homologous sequence having at least 80% sequence identity to QVQLVQSGAEVKKPGASVKVSCKASGYX9FT,

[0073] b) The HFR2 includes WVRQAPGQX 10 LEWMG (SEQ ID NO: 41) or WVRQAPGQX 10 LEWMG homologous sequences with at least 80% sequence identity,

[0074] c) the HFR3 sequence includes RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 (SEQ ID NO: 42) or with RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 Homologous sequences having at least 80% sequence identity,

[0075] d) the HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 25) or a homologous sequence having at least 80% sequence identity to WGQGTLVTVSS,

[0076] e) the LFR1 comprises DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 26) or a homologous sequence having at least 80% sequence identity to DIQMTQSPSSLSASVGDRVTITC,

[0077] f) The LFR2 includes WYQQKPGKX 14 PKLLIY (SEQ ID NO: 43) or WYQQKPGKX 14 PKLLIY has a homologous sequence with at least 80% sequence identity,

[0078] g) The LFR3 includes GVPX 15 RFSGSGSGTDFTX 17 TISSLQPEDIATYYC (SEQ ID NO: 44) or GVPX 15 RFSGSGSGTDFTX 17 TISSLQPEDIATYYC has a homologous sequence with at least 80% sequence identity, and

[0079] h) the LFR4 comprises FGQGTKLEIK (SEQ ID NO: 29) or a homologous sequence having at least 80% sequence identity to FGQGTKLEIK,

[0080] Where X9 is T or V, X 10 Is G or S, X 11 Is T or K, X 12 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, and X 17 It is F or L.

[0081] Embodiment 21. The protein formulation of embodiment 20, wherein:

[0082] The HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 22 and 30,

[0083] The HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 23 and 31,

[0084] The HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 24 and 32-35,

[0085] The HFR4 comprises the sequence of SEQ ID NO: 25,

[0086] The LFR1 comprises a sequence from the group consisting of SEQ ID NO: 26,

[0087] The LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 27 and 36,

[0088] The LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 28 and 37-38, 39, 45, and

[0089] The LFR4 comprises the sequence of SEQ ID NO:29.

[0090] 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 homologous sequences thereof having at least 80% sequence identity thereto.

[0091] 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 thereto.

[0092] Embodiment 24. The protein formulation of embodiment 16, wherein the antibody or antigen-binding fragment thereof to PD-L1 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.

[0093] Embodiment 25. The protein formulation of any one of Embodiments 16 to 24, wherein the antibody or antigen-binding fragment thereof directed against PD-L1 further comprises one or more amino acid residue substitutions or modifications, but still retains specific binding specificity and / or affinity for PD-L1.

[0094] Embodiment 26. A protein formulation according to embodiment 25, wherein at least one of the substitutions or modifications is in one or more of the CDR sequences and / or in one or more of the non-CDR regions of the VH or VL sequences.

[0095] Embodiment 27. The protein formulation of any one of Embodiments 16 to 26, wherein the antibody or antigen-binding fragment thereof against PD-L1 further comprises an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.

[0096] Embodiment 28. The protein formulation of embodiment 27, wherein the constant region comprises the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0097] 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.

[0098] 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 that of the EU index as in Kabat.

[0099] 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) L23 4A 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 that of the EU index as in Kabat.

[0100] 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.

[0101] Embodiment 33. The protein formulation of any one of Embodiments 16 to 32, wherein the antibody or antigen-binding fragment thereof against PD-L1 is humanized.

[0102] Embodiment 34. The protein formulation of any one of embodiments 16 to 33, wherein the antigen-binding fragment is a diabody, a Fab, a Fab', a F(ab')2, a Fd, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.

[0103] 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.

[0104] Embodiment 36. The protein formulation of any one of Embodiments 1 to 15, wherein the first portion comprises an antibody or antigen-binding fragment thereof that competes for binding to PD-L1 with the antibody or antigen-binding fragment thereof of any one of Embodiments 16 to 35.

[0105] 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-β) superfamily, IL-1, or vascular endothelial growth factor (VEGF).

[0106] Embodiment 38. The protein formulation of embodiment 37, wherein the immunosuppressive cytokines in the TGF-β superfamily include TGF-β, bone morphogenetic protein (BMP), activin, NODAL, and growth and differentiation factor (GDF).

[0107] Embodiment 39. The protein formulation of any one of Embodiments 16 to 38, wherein the immunosuppressive cytokine is TGF-β.

[0108] Embodiment 40. The protein formulation of any of embodiments 16 to 39, wherein the second portion comprises a TGFβ binding portion.

[0109] Embodiment 41. A protein formulation according to embodiment 40, wherein the TGFβ binding portion comprises a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, or an antibody to TGFβ and an antigen-binding fragment thereof.

[0110] Embodiment 42. The protein formulation of embodiment 41, wherein the soluble TGFβR comprises the extracellular domain (ECD) of the TGFβR or a TGFβ-binding fragment or variant thereof.

[0111] Embodiment 43. The protein formulation of embodiment 42, wherein the TGFβR is selected from the group consisting of TGFβ receptor I (TGFβRI), TGFβ receptor II (TGFβRII), TGFβ receptor III (TGFβRIII), and any combination thereof.

[0112] Embodiment 44. The protein formulation of embodiment 42, wherein the TGFβR is TGFβRII.

[0113] Embodiment 45. The protein formulation of Embodiment 44, wherein the TGFβRII selectively binds to TGFβ1 relative to TGFβ2 and TGFβ3.

[0114] Embodiment 46. The protein formulation of embodiment 45, wherein the TGFβ1 is human TGFβ1 or mouse TGFβ1.

[0115] Embodiment 47. The protein formulation of any one of Embodiments 42 to 46, wherein the ECD of TGFβR comprises the amino acid sequence of SEQ ID NO: 66, 79, 78, 77, or a sequence having at least 80% sequence identity thereto but still retaining a specific binding specificity and / or affinity for TGF-β.

[0116] Embodiment 48. The protein formulation of embodiment 36, wherein the second portion comprises an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion.

[0117] Embodiment 49. The protein formulation of embodiment 48, wherein the IL-1 binding portion comprises a soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody to IL-1 or an antigen-binding fragment thereof.

[0118] Embodiment 50. The protein formulation of embodiment 49, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region from an anti-IL-1α antibody or an anti-IL-1β antibody, wherein the anti-IL-1α antibody is selected from the group consisting of XB2001, lutikizumab, LY2189102, and bermekimab, or the anti-IL-1β antibody is selected from the group consisting of SSGJ-613, CDP484, canakinumab, and gevokizumab.

[0119] Embodiment 51. The protein formulation of embodiment 49, wherein the antibody or antigen-binding fragment thereof directed against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104 or SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 105 or SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 106 or SEQ ID NO: 114, and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 109 or SEQ ID NO: 117.

[0120] Embodiment 52. The protein formulation of embodiment 49, wherein the antibody or antigen-binding fragment thereof directed against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104, a HCDR2 comprising the sequence of SEQ ID NO: 105, and a HCDR3 comprising the sequence of SEQ ID NO: 106 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107, a LCDR2 comprising the sequence of SEQ ID NO: 108, and a LCDR3 comprising the sequence of SEQ ID NO: 109.

[0121] Embodiment 53. The protein formulation of embodiment 49, wherein the antibody or antigen-binding fragment thereof directed against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 114 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 117.

[0122] Embodiment 54. A protein formulation according to embodiment 49, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 110, and homologous sequences thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences thereof having at least 80% sequence identity.

[0123] Embodiment 55. A protein formulation according to embodiment 49, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102 and a homologous sequence having at least 80% sequence identity thereto, and 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.

[0124] Embodiment 56. A protein formulation according to embodiment 49, wherein the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 110, and a homologous sequence having at least 80% sequence identity thereto, and 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 thereto.

[0125] Embodiment 57. The protein formulation of embodiment 49, wherein the bifunctional molecule comprises a heavy chain having an amino acid sequence of SEQ ID NO: 118 or SEQ ID NO: 120, and / or a light chain having an amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 121.

[0126] Embodiment 58. A protein formulation according to embodiment 49, wherein the IL-1 binding portion comprises: the extracellular domain (ECD) of IL-1RI; an IL-1 binding fragment or variant of any of IL-1RI, the ECD of IL-1RI, IL-1RII or the ECD of IL-1RII, or IL-1RAP or the ECD of IL-1RAP, IL-1sRI or IL-1sRII.

[0127] Embodiment 59. The protein formulation of embodiment 48, wherein the IL-1R binding portion comprises IL-1Ra or an IL-1 binding fragment or variant thereof, or an antibody or antigen-binding fragment thereof to IL-1R.

[0128] Embodiment 60. The protein formulation of embodiment 59, wherein the antibody or antigen-binding fragment thereof against IL-1R comprises a heavy chain variable region and / or a light chain variable region from an antibody selected from spesolimab, astegolimab, imsidolimab, AMG 108, melrilimab, nidanilimab, MEDI8968, REGN6490, HB0034, and CSC012.

[0129] 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 an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 67 or 76, or an IL-1 binding fragment or variant thereof.

[0130] Embodiment 62. The protein formulation of any one of Embodiments 48 to 49, wherein the IL-1 is IL-1α or IL-1β.

[0131] Embodiment 63. The protein formulation of embodiment 62, wherein the IL-1β is human IL-1β.

[0132] Embodiment 64. The protein formulation of any one of Embodiments 16 to 36, wherein the second portion stimulates anti-tumor immunity and comprises an immunostimulatory polypeptide.

[0133] 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-α, or a functional equivalent thereof.

[0134] Embodiment 66. The protein formulation of Embodiment 65, wherein the soluble LAG-3 comprises the extracellular domain (ECD) of LAG-3 or an MHC II binding variant thereof.

[0135] Embodiment 67. The protein formulation of any one of Embodiments 16 to 36, wherein the second portion stimulates anti-tumor immunity and comprises an antagonist of immunoinhibitory receptor signaling.

[0136] Embodiment 68. The protein formulation of embodiment 67, wherein the immunoinhibitory receptor is signal regulatory protein alpha (SIRPα).

[0137] Embodiment 69. The protein formulation of embodiment 68, wherein the second portion blocks the interaction between CD47 and SIRPα.

[0138] Embodiment 70. The protein formulation of embodiment 69, wherein the second portion comprises a CD47 binding domain or a SIRPα binding domain.

[0139] Embodiment 71. The protein formulation of embodiment 70, wherein the CD47 binding domain comprises soluble SIRPα or a CD47 binding fragment or variant thereof, or an anti-CD47 antibody or an antigen-binding fragment thereof.

[0140] Embodiment 72. The protein formulation of embodiment 71, wherein the soluble SIRPα comprises the extracellular domain (ECD) of the SIRPα or a CD47-binding variant thereof.

[0141] Embodiment 73. The protein formulation of embodiment 70, wherein the CD47 binding domain comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence having at least 80% sequence identity thereto but still retaining binding specificity for CD47.

[0142] Embodiment 74. The protein formulation of embodiment 70, wherein the SIRPα binding domain comprises soluble CD47 or a SIRPα binding fragment or variant thereof, or an anti-SIRPα antibody or an antigen-binding fragment thereof.

[0143] Embodiment 75. The protein formulation of embodiment 74, wherein the soluble CD47 comprises the extracellular domain (ECD) of CD47 or a SIRPα binding fragment or variant thereof, an anti-SIRPα antibody or an antigen-binding fragment thereof.

[0144] Embodiment 76. The protein formulation of any one of the preceding embodiments, further comprising a linker connecting the first portion and the second portion.

[0145] Embodiment 77. The protein formulation of embodiment 76, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0146] Embodiment 78. The protein formulation of Embodiment 77, wherein the linker comprises an amino acid sequence of ((G)nS)m, wherein m and n are independently integers selected from 0 to 30.

[0147] Embodiment 79. The protein formulation of any one of Embodiments 16 to 78, wherein the bifunctional molecule comprises one or more of the second moieties.

[0148] Embodiment 80. The protein formulation of embodiment 79, wherein at least one of the plurality of second moieties is linked to the N-terminus or the C-terminus of the polypeptide chain of the first moiety.

[0149] Embodiment 81. A protein formulation according to embodiment 79, wherein at least one of the plurality of second portions is linked to: a) the N-terminus or C-terminus of the heavy chain of the first portion, or b) the N-terminus or C-terminus of the light chain of the first portion.

[0150] Embodiment 82. A protein formulation according to embodiment 79, wherein 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.

[0151] Embodiment 83. A protein formulation according to embodiment 79, wherein each of the plurality of second portions is linked to the C-terminus of each heavy chain constant region of the first portion, respectively.

[0152] Embodiment 84. A protein formulation according to embodiment 79, wherein the bifunctional molecule comprises more than one second portion of the second portion, and the more than one second portion is respectively connected to: the N-terminus of the heavy chain of the first 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.

[0153] 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.

[0154] 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 portion.

[0155] Embodiment 87. A protein formulation according to embodiment 86, wherein the heterodimeric heavy chain comprises one heavy chain having the second portion and the other heavy chain does not have the second portion.

[0156] Embodiment 88. A protein formulation according to embodiment 86, wherein the heterodimeric heavy chain further comprises a heterodimeric Fc region that associates in a manner that hinders homodimerization and / or favors heterodimerization.

[0157] Embodiment 89. The protein formulation of embodiment 86, wherein the heterodimeric Fc regions are capable of associating into heterodimers through knob-in-hole structures, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

[0158] Embodiment 90. The protein formulation of any one of embodiments 86 to 89, wherein the heterodimeric Fc region comprises Y349C, T366S, L368A, or Y407V, or any combination thereof, in one Fc polypeptide chain and S354C or T366W, or a combination thereof, in the other Fc polypeptide chain, wherein the numbering of the residues in the Fc polypeptide chains is that of the EU index as in Kabat.

[0159] Embodiment 91. The protein formulation of any of the preceding embodiments, further linked to one or more conjugate moieties.

[0160] Embodiment 92. The protein formulation of embodiment 91, wherein the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent, or other anti-cancer drug, such as an androgen receptor inhibitor.

[0161] Embodiment 93. A protein formulation according to any one of embodiments 16 to 35, 37 to 47 and 76 to 92, wherein the first portion of the bifunctional molecule comprises an antibody or antigen-binding fragment thereof directed against PD-L1 and the second portion comprises a TGFβ binding portion,

[0162] wherein the antibody or antigen-binding fragment thereof against PD-L1 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; and / or

[0163] wherein the TGFβ binding portion comprises a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, wherein the soluble TGFβR comprises the extracellular domain (ECD) of the TGFβR or a TGFβ binding fragment or variant thereof, and the ECD of the TGFβR comprises the amino acid sequence of SEQ ID NO: 66, 79, 78, 77, or a sequence having at least 80% sequence identity thereto but still retaining a specific binding specificity and / or affinity for TGF-β.

[0164] Embodiment 94. The protein formulation according to embodiment 93, wherein the first portion of the bifunctional molecule comprises an antibody or antigen-binding fragment thereof directed against PD-L1, and the second portion comprises a TGFβ binding portion,

[0165] wherein the antibody or antigen-binding fragment thereof directed against PD-L1 comprises 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 homologous sequences thereof having at least 80% sequence identity thereto; and / or

[0166] wherein the TGFβ binding portion comprises a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, wherein the soluble TGFβR comprises the extracellular domain (ECD) of the TGFβR or a TGFβ binding fragment or variant thereof, and the ECD of the TGFβR comprises the amino acid sequence of SEQ ID NO: 66 or a sequence having at least 80% sequence identity thereto but still retaining a specific binding specificity and / or affinity for TGF-β.

[0167] Embodiment 95. A 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.

[0168] Embodiment 96. A protein formulation according to any one of the preceding embodiments, wherein the buffer is an acetate, histidine, citrate, succinate, malate, glutamate, phosphate or lactate buffer, such as an acetate buffer or a histidine buffer; the acetate buffer is, for example, an acetic acid-sodium acetate buffer; the histidine buffer is, for example, a histidine-hydrochloric acid buffer.

[0169] Embodiment 97. The protein formulation according to any one of the preceding embodiments, wherein the concentration of the buffer is about 5-100 mM, such as about 10-50 mM, about 15-30 mM, about 10-20 mM, about 25 mM or about 20 mM.

[0170] Embodiment 98. The protein formulation according to any one of the preceding embodiments, wherein the buffer is an acetate buffer of about 10-50 mM, about 15-30 mM, or about 20 mM, such as acetic acid-sodium acetate buffer.

[0171] Embodiment 99. The protein formulation according to any one of the preceding embodiments, wherein the buffer is about 10-50 mM, about 15-30 mM or about 20 mM histidine buffer, such as a histidine-HCl buffer.

[0172] 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%.

[0173] Embodiment 101. The protein formulation according to any one of the preceding embodiments, wherein the surfactant is a non-ionic surfactant, such as a polysorbate, a poloxamer, Brij or Triton X, such as polysorbate 80 or polysorbate 20.

[0174] Embodiment 102. A protein formulation according to any one of the preceding embodiments, wherein the surfactant concentration is about 0.01-0.5% (w / v), for example, 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).

[0175] Embodiment 103. A 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) of a polysorbate, such as polysorbate 80 or polysorbate 20.

[0176] Embodiment 104. The protein formulation according to any one of the preceding embodiments, wherein the surfactant is polysorbate 80 at 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).

[0177] Embodiment 105. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises a stabilizer.

[0178] Embodiment 106. A 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.

[0179] Embodiment 107. A protein formulation according to any one of the preceding embodiments, wherein the formulation comprises a stabilizer at a concentration of about 10-300 mM, such as about 100-300 mM, about 120-300 mM, about 100-200 mM, about 130-170 mM, about 140-160 mM or about 150 mM.

[0180] Embodiment 108. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises arginine hydrochloride or arginine.

[0181] Embodiment 109. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 100-200 mM, about 130-170 mM, about 140-160 mM, or about 150 mM arginine hydrochloride or arginine.

[0182] Embodiment 110. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 100-200 mM, about 130-170 mM, about 140-160 mM, or about 150 mM arginine hydrochloride.

[0183] Embodiment 111. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 130-170 mM, about 140-160 mM, or about 150 mM arginine hydrochloride.

[0184] Embodiment 112. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises NaCl.

[0185] Embodiment 113. The protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 100-200 mM, about 130-170 mM, or about 135 mM NaCl.

[0186] Embodiment 114. A protein formulation according to any one of the preceding embodiments, wherein the formulation optionally comprises a chelating agent, such as EDTA·2Na, for example at a concentration of about 30-350 μM, such as 75-300 μM.

[0187] Embodiment 115. A 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 μM, such as 75-300 μM EDTA·2Na.

[0188] Embodiment 116. A protein formulation according to any one of the preceding embodiments, wherein the formulation comprises about 0.025%-0.1% (w / v) (e.g., about 0.05% (w / v)) of polysorbate (e.g., polysorbate 80) and about 30-350 μM EDTA·2Na.

[0189] Embodiment 117. A protein formulation according to any one of the preceding embodiments, comprising:

[0190] 10-50 mg / mL of the bifunctional molecule;

[0191] 10-50 mM buffer;

[0192] 0.025%-0.1% (w / v) polysorbate 80 or polysorbate 20;

[0193] 100-200 mM arginine hydrochloride; and

[0194] Optionally 30-350 μM EDTA·2Na;

[0195] The pH is 5.0-5.6.

[0196] Embodiment 118. A protein formulation according to any one of the preceding embodiments, comprising:

[0197] 10-50 mg / mL of the bifunctional molecule;

[0198] 10-50 mM acetate buffer or histidine buffer;

[0199] 0.025%-0.1% (w / v) polysorbate 80 or polysorbate 20;

[0200] 100-200 mM arginine hydrochloride; and

[0201] Optionally 30-350 μM EDTA·2Na;

[0202] The pH is 5.0-5.6.

[0203] Embodiment 119. A protein formulation according to any one of the preceding embodiments, comprising:

[0204] 10-50 mg / mL of the bifunctional molecule;

[0205] 10-50 mM acetate buffer or histidine buffer;

[0206] 0.025%-0.1% (w / v) polysorbate 80 or polysorbate 20;

[0207] 130-170 mM arginine hydrochloride; and

[0208] Optionally 30-350 μM EDTA·2Na;

[0209] The pH is 5.0-5.6.

[0210] Embodiment 120. The protein formulation according to any one of the preceding embodiments, comprising:

[0211] 10-50 mg / mL of the bifunctional molecule;

[0212] 10-50 mM acetate buffer or histidine buffer;

[0213] 0.025%-0.1% (w / v) polysorbate 80 or polysorbate 20;

[0214] 140-160 mM arginine hydrochloride; and

[0215] Optionally 30-350 μM EDTA·2Na;

[0216] The pH was 5.3 ± 5%.

[0217] Embodiment 121. A protein formulation according to any one of the preceding embodiments, comprising:

[0218] 10-50 mg / mL of the bifunctional molecule;

[0219] 10-50 mM acetate buffer or histidine buffer;

[0220] 0.025%-0.1% (w / v) polysorbate 80 or polysorbate 20; and

[0221] 140-160 mM arginine hydrochloride;

[0222] The pH was 5.3 ± 5%.

[0223] Embodiment 122. The protein formulation according to any one of the preceding embodiments, comprising:

[0224] 20-30 mg / mL of the bifunctional molecule;

[0225] 20-30 mM acetate buffer or histidine buffer;

[0226] 0.025%-0.1% (w / v) polysorbate 80; and

[0227] 140-160 mM arginine hydrochloride;

[0228] The pH was 5.3 ± 5%.

[0229] Embodiment 123. A protein formulation according to any one of the preceding embodiments, comprising:

[0230] 30 mg / mL of the bifunctional molecule;

[0231] 20 mM acetate buffer or histidine buffer;

[0232] 0.05% (w / v) polysorbate 80; and

[0233] 150 mM arginine hydrochloride;

[0234] The pH is 5.3.

[0235] Embodiment 124. A protein formulation according to any one of the preceding embodiments, wherein the bifunctional molecule is treated with a chelating agent, such as EDTA·2Na (e.g., about 0.3-10 mM, about 0.1-20 mM, about 0.3-15 mM, about 0.3-10 mM, or about 0.5-5 mM EDTA·2Na) during formulation, and the chelating agent is then 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 against a buffer); optionally, other excipients are added after the chelating agent is removed to formulate the formulation.

[0236] Embodiment 125. A protein formulation according to any one of the preceding embodiments, wherein the bifunctional molecule is treated with a chelating agent, such as EDTA·2Na (e.g., about 0.3-10 mM, about 0.1-20 mM, about 0.3-15 mM, about 0.3-10 mM, or about 0.5-5 mM EDTA·2Na) in a buffer at room temperature (e.g., overnight), during formulation, and the chelating agent is then removed by dialysis against a buffer, ultrafiltration, or diafiltration.

[0237] Embodiment 126. A protein formulation according to any one of the preceding embodiments, wherein the bifunctional molecule is treated with a chelating agent, such as EDTA·2Na (e.g., about 0.3-10 mM, about 0.1-20 mM, about 0.3-15 mM, about 0.3-10 mM or about 0.5-5 mM EDTA·2Na) in a buffer at room temperature (e.g., overnight) during formulation, and the chelating agent is then removed by dialysis, ultrafiltration or diafiltration against a buffer, and then other pharmaceutically acceptable excipients are added to formulate the formulation.

[0238] Embodiment 127. The protein formulation according to any one of the preceding embodiments, which is in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form.

[0239] Embodiment 128. The protein formulation according to any one of the preceding embodiments, which is an injection, such as an intravenous injection, an intramuscular injection or a subcutaneous injection.

[0240] Embodiment 129. The protein formulation according to any one of the preceding embodiments, which is a liquid injection.

[0241] Embodiment 130. The protein formulation according to any one of embodiments 1-129, for use in treating, preventing or ameliorating a PD-L1-related disease in a subject.

[0242] 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.

[0243] 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, spinal 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, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma and adenocarcinoma.

[0244] Embodiment 133. A 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.

[0245] Embodiment 134. The protein formulation for use according to any one of embodiments 130-133, wherein the PD-L1-associated disease is resistant to PD-L1 / PD-1 monotherapy.

[0246] Embodiment 135. A protein formulation for use according to any one of embodiments 130-134, wherein the subject is a human.

[0247] Embodiment 136. A protein formulation according to any one of embodiments 1 to 129 for use in treating, preventing or ameliorating a disease or condition that would benefit from the suppression of immunosuppressive cytokines, the induction of a sustained immune response or the stimulation of anti-tumor immunity;

[0248] Wherein, for example, the immunosuppressive cytokine is TGFβ; for example, the disease or condition is a TGFβ-related disease or condition; for example, the TGFβ-related disease is cancer, fibrotic disease or renal disease;

[0249] wherein, for example, the immunosuppressive cytokine is IL-1; for example, the disease or condition is an IL-1-related disease or condition;

[0250] wherein, for example, the disease or condition would benefit from the induction of a sustained immune response by stimulation of MHC II signaling with an immunostimulatory polypeptide; for example, the immunostimulatory polypeptide is soluble LAG-3; or

[0251] Wherein, for example, the disease or condition would benefit from stimulation of anti-tumor immunity by inhibiting immunoinhibitory receptor signaling; for example, the immunoinhibitory receptor is SIRPα.

[0252] 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.

[0253] Embodiment 138. The protein formulation for use according to embodiment 137, wherein the second therapeutic agent is selected from a chemotherapeutic agent, an anti-cancer drug, a radiation therapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent or a cytokine.

[0254] Embodiment 139. A pharmaceutical combination comprising a protein formulation according to any one of embodiments 1-129 and a second therapeutic agent selected from a chemotherapeutic agent, an anticancer drug, a radiation therapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, or a cytokine.

[0255] 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 ameliorating a PD-L1-related disease in a subject.

[0256] Embodiment 141. The use according to embodiment 140, wherein the disease is an immune-related disease or disorder, cancer or an infectious disease.

[0257] Embodiment 142. The 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 bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal 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, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma and adenocarcinoma.

[0258] 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.

[0259] Embodiment 144. The use according to any one of embodiments 140-143, wherein the PD-L1-associated disease is resistant to PD-L1 / PD-1 monotherapy.

[0260] Embodiment 145. The use according to any one of embodiments 140-144, wherein the subject is a human.

[0261] Embodiment 146. Use of a protein formulation according to any one of embodiments 1-129 for the preparation of a medicament for treating, preventing or ameliorating a disease or condition that would benefit from the suppression of immunosuppressive cytokines, the induction of a sustained immune response or the stimulation of anti-tumor immunity;

[0262] Wherein, for example, the immunosuppressive cytokine is TGFβ; for example, the disease or condition is a TGFβ-related disease or condition; for example, the TGFβ-related disease is cancer, fibrotic disease or renal disease;

[0263] wherein, for example, the immunosuppressive cytokine is IL-1; for example, the disease or condition is an IL-1-related disease or condition;

[0264] wherein, for example, the disease or condition would benefit from the induction of a sustained immune response by stimulation of MHC II signaling with an immunostimulatory polypeptide; for example, the immunostimulatory polypeptide is soluble LAG-3; or

[0265] Wherein, for example, the disease or condition would benefit from stimulation of anti-tumor immunity by inhibiting immunoinhibitory receptor signaling; for example, the immunoinhibitory receptor is SIRPα.

[0266] Embodiment 147. The use according to any one of embodiments 140-146, wherein the medicament is administered in combination with a second therapeutic agent.

[0267] Embodiment 148. The use according to embodiment 147, wherein the second therapeutic agent is selected from a chemotherapeutic agent, an anticancer drug, a radiotherapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent or a cytokine.

[0268] Embodiment 149. A method for treating, preventing or ameliorating a PD-L1-associated 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.

[0269] Embodiment 150. The method according to embodiment 149, wherein the disease is an immune-related disease or disorder, cancer, or an infectious disease.

[0270] 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 bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal 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, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma and adenocarcinoma.

[0271] Embodiment 152. The method according to any one of embodiments 149-151, wherein the subject has been identified as having cancer cells that express PD-L1.

[0272] Embodiment 153. The method according to any one of embodiments 149-152, wherein the PD-L1-associated disease is resistant to PD-L1 / PD-1 monotherapy

[0273] Embodiment 154. The method according to any one of embodiments 149-153, wherein the subject is a human.

[0274] Embodiment 155. A method for treating, preventing, or ameliorating a disease or condition in a subject that would benefit from suppression of immunosuppressive cytokines, induction of a sustained immune response, or stimulation of anti-tumor immunity, the method comprising administering an effective amount of a protein formulation according to any one of embodiments 1-129;

[0275] Wherein, for example, the immunosuppressive cytokine is TGFβ; for example, the disease or condition is a TGFβ-related disease or condition; for example, the TGFβ-related disease is cancer, fibrotic disease or renal disease;

[0276] wherein, for example, the immunosuppressive cytokine is IL-1; for example, the disease or condition is an IL-1-related disease or condition;

[0277] wherein, for example, the disease or condition would benefit from the induction of a sustained immune response by stimulation of MHC II signaling with an immunostimulatory polypeptide; for example, the immunostimulatory polypeptide is soluble LAG-3; or

[0278] Wherein, for example, the disease or condition would benefit from stimulation of anti-tumor immunity by inhibiting immunoinhibitory receptor signaling; for example, the immunoinhibitory receptor is SIRPα.

[0279] Embodiment 156. The method according to any one of embodiments 149-155, further comprising administering a therapeutically effective amount of a second therapeutic agent.

[0280] Embodiment 157. The method of embodiment 156, wherein the second therapeutic agent is selected from a chemotherapeutic agent, an anticancer drug, a radiation therapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, or a cytokine.

[0281] Embodiment 158. A method for preparing a protein formulation according to any one of embodiments 1-129, comprising the following steps:

[0282] a. providing a bifunctional molecule according to any one of embodiments 1-129 in a buffer;

[0283] b. adding a surfactant according to any one of embodiments 1-129, optionally a stabilizer, and optionally other pharmaceutically acceptable excipients;

[0284] c. Optionally sterile filtered.

[0285] Embodiment 159. A preparation method according to embodiment 158, wherein the bifunctional molecule of step a is treated with a chelating agent, such as EDTA·2Na (e.g., about 0.3-10 mM, about 0.1-20 mM, about 0.3-15 mM, about 0.3-10 mM or about 0.5-5 mM EDTA·2Na), 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, ultrafiltration or diafiltration against a buffer).

[0286] 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 (e.g., about 0.3-10 mM, about 0.1-20 mM, about 0.3-15 mM, about 0.3-10 mM or about 0.5-5 mM EDTA·2Na) in a buffer at room temperature (e.g., overnight), and then the chelating agent is removed by dialysis against a buffer, ultrafiltration or diafiltration.

[0287] With respect to the bifunctional molecules described above, especially in the embodiments above, the present disclosure also provides the following embodiments:

[0288] In one aspect, 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).

[0289] In some embodiments, the first portion comprises an agonist of an immunostimulatory checkpoint molecule, optionally 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.

[0290] In some embodiments, the first portion comprises an antagonist of an immunoinhibitory checkpoint molecule, optionally 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.

[0291] In some embodiments, the immune checkpoint molecule is PD-L1.

[0292] In some embodiments, the first portion comprises an antibody or antigen-binding fragment thereof to PD-L1, and the second portion comprises an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion.

[0293] In some embodiments, the IL-1 binding portion comprises IL-1R or an IL-1 binding fragment or variant thereof, or an antibody to IL-1 or an antigen-binding fragment thereof.

[0294] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104 or SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 105 or SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 106 or SEQ ID NO: 114, and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 109 or SEQ ID NO: 117.

[0295] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104, a HCDR2 comprising the sequence of SEQ ID NO: 105, and a HCDR3 comprising the sequence of SEQ ID NO: 106 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107, a LCDR2 comprising the sequence of SEQ ID NO: 108, and a LCDR3 comprising the sequence of SEQ ID NO: 109.

[0296] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 114 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 117.

[0297] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 110, and homologous sequences thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences thereof having at least 80% sequence identity.

[0298] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102 and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103 and a homologous sequence thereof having at least 80% sequence identity.

[0299] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 110, and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 111, and a homologous sequence thereof having at least 80% sequence identity.

[0300] In some embodiments, the IL-1R binding moiety comprises an interleukin-1 receptor antagonist, or a fragment or variant thereof, or an antibody to IL-1R, or an antigen-binding fragment thereof.

[0301] In another aspect, a bifunctional molecule comprises 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 the first portion comprises an antibody or antigen-binding fragment thereof directed against PD-L1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region comprises:

[0302] a) HCDR1 comprising DYYMN (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity to DYYMN,

[0303] b) a HCDR2 comprising DINPNNX1X2TX3YNHKFKG (SEQ ID NO: 19) or a homologous sequence having at least 80% sequence identity to DINPNNX1X2TX3YNHKFKG, and

[0304] c) a HCDR3 comprising WGDGPFAY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity to WGDGPFAY, and / or

[0305] wherein the light chain variable region comprises:

[0306] d) LCDR1 comprises a sequence selected from the group consisting of KASQNVX4X5X6VA (SEQ ID NO: 20) or a homologous sequence having at least 80% sequence identity thereto,

[0307] e) LCDR2 comprises a sequence selected from the group consisting of SX7SX8RYT (SEQ ID NO: 21) or a homologous sequence having at least 80% sequence identity thereto, and

[0308] f) LCDR3 comprises a sequence selected from the group consisting of QQYSNYPT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity to QQYSNYPT;

[0309] wherein X1 is G or A, X2 is G or D or Q or E or L, X3 is S or M or Q or L or V, X4 is G or P or K, X5 is A or G, X6 is A or I, X7 is A or N or R or V, and X8 is N or H or V or D.

[0310] In some embodiments, the heavy chain variable region comprises:

[0311] a) HCDR1 comprises the sequence of SEQ ID NO: 1,

[0312] b) HCDR2 comprises 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

[0313] c) HCDR3 comprises the sequence of SEQ ID NO: 3,

[0314] and / or

[0315] A light chain variable region, comprising:

[0316] d) LCDR1 comprises 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,

[0317] e) LCDR2 comprises 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

[0318] f) LCDR3 comprises the sequence of SEQ ID NO: 6.

[0319] In some embodiments, the heavy chain variable region is selected from the group consisting of:

[0320] a) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0321] b) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 13, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0322] c) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 14, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0323] d) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 15, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0324] e) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 17, and a HCDR3 comprising the sequence of SEQ ID NO: 3; and

[0325] f) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 18, and a HCDR3 comprising the sequence of SEQ ID NO: 3.

[0326] In some embodiments, the light chain variable region is selected from the group consisting of:

[0327] a) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 4, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0328] b) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 9, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0329] c) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 8, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0330] d) 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

[0331] e) 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.

[0332] In some embodiments, the antibody or antigen-binding fragment thereof against PD-L1 further comprises one or more of heavy chain HFR1, HFR2, HFR3, and HFR4, and / or one or more of light chain LFR1, LFR2, LFR3, and LFR4, wherein:

[0333] a) the HFR1 comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYX9FT (SEQ ID NO: 40) or a homologous sequence having at least 80% sequence identity thereto,

[0334] b) the 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,

[0335] c) 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 thereto,

[0336] d) the HFR4 comprises the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 25) or a homologous sequence having at least 80% sequence identity thereto,

[0337] e) the LFR1 comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 26) or a homologous sequence having at least 80% sequence identity thereto,

[0338] f) the LFR2 comprises the amino acid sequence WYQQKPGKX 14 PKLLIY (SEQ ID NO: 43) or a homologous sequence having at least 80% sequence identity thereto,

[0339] g) the 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 thereto, and

[0340] h) the LFR4 comprises the amino acid sequence FGQGTKLEIK (SEQ ID NO: 29) or a homologous sequence having at least 80% sequence identity thereto,

[0341] Where X9 is T or V, X 10 Is G or S, X 11 Is T or K, X 12 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, and X 17 It is F or L.

[0342] In some embodiments,

[0343] The HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 22 and 30,

[0344] The HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 23 and 31,

[0345] The HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 24 and 32-35,

[0346] The HFR4 comprises the sequence of SEQ ID NO: 25,

[0347] The LFR1 comprises a sequence from the group consisting of SEQ ID NO: 26,

[0348] The LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 27 and 36,

[0349] The LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 28 and 37-38, 39, 45, and

[0350] The LFR4 comprises the sequence of SEQ ID NO:29.

[0351] In some embodiments, 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 homologous sequences thereof having at least 80% sequence identity thereto.

[0352] In some embodiments, 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 thereto.

[0353] In some embodiments, the antibody or antigen-binding fragment thereof against PD-L1 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.

[0354] In some embodiments, the antibody or antigen-binding fragment thereof directed against PD-L1 further comprises one or more amino acid residue substitutions or modifications, but still retains specific binding specificity and / or affinity for PD-L1.

[0355] In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or in one or more of the non-CDR regions of the VH or VL sequence.

[0356] In some embodiments, the antibody or antigen-binding fragment thereof against PD-L1 further comprises an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.

[0357] In some embodiments, the constant region comprises the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0358] In some embodiments, the Fc region of human IgG1 comprises SEQ ID NO: 80 or a variant thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 80.

[0359] In some embodiments, the constant region comprises an Fc variant having reduced effector function relative to a corresponding wild-type Fc region. In some embodiments, the Fc region comprises one or more amino acid residue modifications or substitutions such that effector function is reduced relative to SEQ ID NO: 80.

[0360] In some embodiments, the Fc region 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 that of the EU index as in Kabat.

[0361] In some embodiments, the Fc region 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); 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 that of the EU index as in Kabat.

[0362] In some embodiments, the Fc variant comprises the amino acid sequence of SEQ ID NO:81.

[0363] In some embodiments, the antibody or antigen-binding fragment thereof against PD-L1 is humanized.

[0364] In some embodiments, the antigen-binding fragment is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody and bivalent domain antibody.

[0365] In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to both human PD-L1 and cynomolgus monkey PD-L1.

[0366] In some embodiments, the first portion comprises an antibody or antigen-binding fragment thereof that competes for binding to PD-L1 with an antibody or antigen-binding fragment thereof provided herein.

[0367] In some embodiments, the immunosuppressive cytokine comprises a cytokine in the transforming growth factor beta (TGF-β) superfamily, IL-1, or vascular endothelial growth factor (VEGF).

[0368] In some embodiments, the immunosuppressive cytokines in the TGF-β superfamily include TGF-β, bone morphogenetic proteins (BMPs), activins, NODAL, and growth and differentiation factors (GDFs).

[0369] In some embodiments, the immunosuppressive cytokine is TGF-β.

[0370] In some embodiments, the second moiety comprises a TGFβ binding moiety.

[0371] In some embodiments, the TGFβ binding portion includes a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, or an antibody to TGFβ and an antigen-binding fragment thereof.

[0372] In some embodiments, the soluble TGFβR comprises the extracellular domain (ECD) of the TGFβR or a TGFβ-binding fragment or variant thereof.

[0373] In some embodiments, the TGFβR is selected from the group consisting of TGFβ receptor I (TGFβRI), TGFβ receptor II (TGFβRII), TGFβ receptor III (TGFβRIII), and any combination thereof.

[0374] In some embodiments, the TGFβR is TGFβRII.

[0375] In some embodiments, the TGFβRII selectively binds to TGFβ1 relative to TGFβ2 and TGFβ3.

[0376] In some embodiments, the TGFβ1 is human TGFβ1 or mouse TGFβ1.

[0377] In some embodiments, the ECD of TGFβR comprises the amino acid sequence of SEQ ID NO: 66, 79, 78, 77, or a sequence having at least 80% sequence identity thereto but still retaining a particular binding specificity and / or affinity for TGF-β.

[0378] In some embodiments, the second moiety comprises an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion.

[0379] In some embodiments, the IL-1 binding portion comprises a soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody to IL-1 or an antigen-binding fragment thereof.

[0380] In some embodiments, the IL-1 binding portion includes: the extracellular domain (ECD) of IL-1RI; an IL-1 binding fragment or variant of any of IL-1RI, the ECD of IL-1RI, IL-1RII or the ECD of IL-1RII, or IL-1RAP or the ECD of IL-1RAP, IL-1sRI or IL-1sRII.

[0381] In some embodiments, the IL-1R binding portion comprises IL-1Ra or an IL-1 binding fragment or variant thereof, or an antibody or antigen-binding fragment thereof to IL-1R.

[0382] In some embodiments, the IL-1R binding portion comprises the amino acid sequence of SEQ ID NO: 67 or 76, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 67 or 76, or an IL-1 binding fragment or variant thereof.

[0383] In some embodiments, the IL-1 is IL-1α or IL-1β.

[0384] In some embodiments, the IL-1β is human IL-1β.

[0385] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104 or SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 105 or SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 106 or SEQ ID NO: 114, and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 109 or SEQ ID NO: 117.

[0386] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104, a HCDR2 comprising the sequence of SEQ ID NO: 105, and a HCDR3 comprising the sequence of SEQ ID NO: 106 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107, a LCDR2 comprising the sequence of SEQ ID NO: 108, and a LCDR3 comprising the sequence of SEQ ID NO: 109.

[0387] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 114 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 117.

[0388] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 110, and homologous sequences thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences thereof having at least 80% sequence identity.

[0389] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102 and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103 and a homologous sequence thereof having at least 80% sequence identity.

[0390] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 110, and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 111, and a homologous sequence thereof having at least 80% sequence identity.

[0391] In some embodiments, the bifunctional molecule comprises a heavy chain comprising 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.

[0392] In some embodiments, the second moiety stimulates anti-tumor immunity and comprises an immunostimulatory polypeptide.

[0393] In some embodiments, 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 or IFN-α or a functional equivalent thereof.

[0394] In some embodiments, the soluble LAG-3 comprises the extracellular domain (ECD) of LAG-3 or an MHC II-binding fragment or variant thereof.

[0395] In some embodiments, the second moiety stimulates anti-tumor immunity and comprises an antagonist of immunoinhibitory receptor signaling.

[0396] In some embodiments, the immunoinhibitory receptor is signal regulatory protein alpha (SIRPα).

[0397] In some embodiments, the second moiety blocks the interaction between CD47 and SIRPα.

[0398] In some embodiments, the second portion comprises a CD47 binding domain or a SIRPα binding domain.

[0399] In some embodiments, the CD47 binding domain comprises soluble SIRPα or a CD47 binding fragment or variant thereof, or an anti-CD47 antibody or an antigen-binding fragment thereof.

[0400] In some embodiments, the soluble SIRPα comprises the extracellular domain (ECD) of SIRPα or a CD47-binding fragment or variant thereof.

[0401] In some embodiments, the soluble SIRPα comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having at least 80% sequence identity thereto but still retaining binding specificity for CD47.

[0402] In some embodiments, the SIRPα binding domain comprises soluble CD47 or a SIRPα binding fragment or variant thereof, or an anti-SIRPα antibody or an antigen-binding fragment thereof.

[0403] In some embodiments, the soluble CD47 comprises the extracellular domain (ECD) of CD47 or a SIRPα binding fragment or variant thereof, an anti-SIRPα antibody or an antigen-binding fragment thereof.

[0404] In some embodiments, the bifunctional molecule further comprises a linker connecting the first portion and the second portion.

[0405] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0406] In some embodiments, the linker comprises an 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 comprises the amino acid sequence of SEQ ID NO: 68.

[0407] In some embodiments, the bifunctional molecule includes one or more of the second moieties.

[0408] In some embodiments, at least one second moiety of the plurality of second moieties is linked to the N-terminus or the C-terminus of the polypeptide chain of the first moiety.

[0409] In some embodiments, at least one second moiety of the plurality of second moieties is linked to: a) the N-terminus or C-terminus of the heavy chain of the first moiety, or b) the N-terminus or C-terminus of the light chain of the first moiety.

[0410] In some embodiments, 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.

[0411] In some embodiments, each second portion of the plurality of second portions is linked to the C-terminus of each heavy chain constant region of the first portion, respectively.

[0412] In some embodiments, the bifunctional molecule comprises more than one second portion of the second portion, wherein the more than one second portion is respectively connected to: the N-terminus of the heavy chain of the first 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.

[0413] In some embodiments, the bifunctional molecule comprises a homodimeric heavy chain or a heterodimeric heavy chain.

[0414] In some embodiments, the heavy chain is heterodimeric with respect to the presence or position of the second moiety.

[0415] In some embodiments, the heterodimeric heavy chain includes one heavy chain having the second portion and the other heavy chain does not have the second portion.

[0416] In some embodiments, the heterodimeric heavy chain further comprises a heterodimeric Fc region that associates in a manner that hinders homodimerization and / or favors heterodimerization.

[0417] In some embodiments, 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.

[0418] In some embodiments, the heterodimeric Fc region comprises Y349C, T366S, L368A, or Y407V, or any combination thereof, in one Fc region and S354C or T366W, or a combination thereof, in the other Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.

[0419] In some embodiments, the bifunctional molecule is further linked to one or more conjugate moieties.

[0420] In some embodiments, 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, a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent, or other anti-cancer drug, such as an androgen receptor inhibitor.

[0421] In another aspect, the present disclosure further provides a pharmaceutical composition or kit comprising the bifunctional molecule provided herein and a pharmaceutically acceptable carrier.

[0422] In another aspect, the present disclosure further provides an isolated polynucleotide encoding a bifunctional molecule provided herein.

[0423] In another aspect, the present disclosure further provides a vector comprising the isolated polynucleotide provided herein.

[0424] In another aspect, the present disclosure further provides a host cell comprising the vector provided herein.

[0425] In another aspect, the present disclosure further provides a method for expressing a bifunctional molecule provided herein, the method comprising culturing a host cell provided herein under conditions of an expression vector.

[0426] In another aspect, the present disclosure further provides a method for treating, preventing, or alleviating a PD-L1-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the bifunctional molecule provided herein and / or the pharmaceutical composition or kit provided herein.

[0427] In some embodiments, the disease is an immune-related disease or disorder, cancer, an autoimmune disease, or an infectious disease.

[0428] In some embodiments, 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, spinal 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, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, and adenocarcinoma.

[0429] In some embodiments, the subject has been identified as having cancer cells that express PD-L1.

[0430] In some embodiments, the subject is a human.

[0431] In some embodiments, the method further comprises administering a therapeutically effective amount of a second therapeutic agent.

[0432] In some embodiments, the second therapeutic agent is selected from a chemotherapeutic agent, an anti-cancer drug, radiation therapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, or a cytokine.

[0433] In another aspect, the present disclosure provides a use of a bifunctional molecule provided herein in the manufacture of a medicament for treating a PD-L1-associated disease or condition in a subject.

[0434] In another aspect, the present disclosure provides a method for treating, preventing, or ameliorating 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 stimulation of anti-tumor immunity, comprising administering an effective amount of a bifunctional molecule provided herein.

[0435] In some embodiments, the immunosuppressive cytokine is TGFβ.

[0436] In some embodiments, the disease or condition is a TGFβ-related disease or condition.

[0437] In some embodiments, the TGFβ-related disease is cancer, a fibrotic disease, or a renal disease.

[0438] In some embodiments, the immunosuppressive cytokine is IL-1.

[0439] In some embodiments, the disease or condition is an IL-1 related disease or condition.

[0440] In some embodiments, the disease or condition would benefit from stimulation of anti-tumor immunity by inhibiting immunoinhibitory receptor signaling (eg, SIRPα signaling). BRIEF DESCRIPTION OF THE DRAWINGS

[0441] FIG1 shows the binding of humanized 4B6 antibody to human PD-L1 as measured by ELISA.

[0442] FIG2 shows the binding of Hu4B6_HgLa to human PD-L1 measured by ELISA.

[0443] 3A-3C show binding of AM-4B6-IgG1-TGFβRII variants to PD-L1 as measured by ELISA.

[0444] FIG4 shows affinity ranking of AM-4B6-IgG1-TGFβRII variants using flow cytometry.

[0445] Figures 5A and 5B show blockade of PD-L1 / PD-1 or PD-L1 / B7-1 by AM-4B6-IgG1-TGFβRII variants.

[0446] FIG6 shows blockade of PD-L1 / PD-1 by AM-4B6-IgG1-TGFβRII variants using a cell-based assay.

[0447] FIG. 7 shows SDS-PAGE of AM4B6_hIgG1_TBRII(20-136) expressed in a stable cell line.

[0448] Figures 8A and 8B show binding to human PD-L1 or cynomolgus monkey PD-L1 as measured by ELISA analysis.

[0449] 9A-9C show binding to human PD-L1 and other members of the B7 family and TGFβ superfamily as measured by ELISA analysis.

[0450] 10A-10F show binding to PD-L1 expressing cells as measured by FACS analysis.

[0451] FIG11 shows binding to human PD-L1 on activated human T cells measured by FACS analysis.

[0452] 12A-B show the blocking of human PD-L1 binding to human PD-1 or cynomolgus monkey PD-L1 binding to cynomolgus monkey PD-1 as measured by ELISA analysis.

[0453] FIG13 shows simultaneous binding to hPD-L1 and TGFb1 as measured by ELISA analysis.

[0454] FIG14 shows blocking of hPD-L1 / hPD-1 using a reporter gene assay.

[0455] Figure 15 shows blockade of TGFβ1 signaling using the TGFβ reporter HEK-293 cell line.

[0456] FIG. 16 shows the effect of AM4B6-hIgG1-TGFβRII′ on IFNγ release from PBMCs stimulated with tuberculin (TB).

[0457] 17A-17B show anti-tumor activity in the MC38-hPD-L1 tumor model.

[0458] 18A-18B show anti-tumor activity in the H460 tumor model.

[0459] 19A-19B show anti-tumor activity in the EMT6-hPD-L1 tumor model.

[0460] 20A-20C show in vivo pharmacokinetic and pharmacodynamic studies of AM4B6-hIgG1-TGFβRII.

[0461] FIG21 shows the binding activity of AM4B6-hIgG1-IL-1RA to human PD-L1 measured by ELISA.

[0462] FIG22 shows the binding activity of AM4B6-hIgG1-IL-1RA to human PD-L1 measured by FACS analysis.

[0463] FIG. 23 shows blockade of PD-L1 / PD-1 by AM4B6-hIgG1-IL-1RA using a cell-based assay.

[0464] FIG. 24 shows the blocking activity of AM4B6-IgG1-IL-1RA against human IL-1β measured by ELISA.

[0465] FIG. 25 shows the blocking activity of AM4B6-hIgG1-IL-1RA against human IL-1β on reporter cells.

[0466] FIG26 shows the SEC-HPLC purity of asymmetric bifunctional antibodies.

[0467] Figure 27 shows the binding of bifunctional molecules to human PD-L1 measured by ELISA.

[0468] Figure 28 shows binding of bifunctional molecules to human CD47 measured by ELISA.

[0469] FIG29 shows the ELISA binding activity of the IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6 bispecific antibodies to the hIL-1β protein.

[0470] FIG30 shows the ELISA binding activity of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6 bispecific antibodies to hPD-L1 protein.

[0471] FIG31 shows the binding of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6 bispecific antibodies to PD-L1-expressing 293T cells measured by FACS.

[0472] FIG32 shows the cell-based PD1 / PD-L1 blocking activity of IgG-scFv-ACZ885-AM4B6 and IgG-scFv-XOMA052-AM4B6.

[0473] FIG33 shows the blocking activity of IgG-scFv-XOMA052-AM4B6 against IL-1β on HDF cells.

[0474] FIG34 shows the blocking activity of IgG-scFv-ACZ885-AM4B6 on hIL-1β on reporter cells.

[0475] Figure 35 (analysis chart of the results at 40°C) shows the JMP software analysis results of △HMW% and △LMW% obtained by SEC and NR CE-SDS detection of samples of formulations F1-F12 after being placed at 40°C for 4 weeks, demonstrating the effects of pH, PS80 and other excipients on protein stability.

[0476] Figure 36 (Analysis diagram of stirring results) shows the JMP software analysis results of △HMW% and △LMW% obtained by SEC and NR CE-SDS detection of samples of formulations F1-F12 after stirring at 25°C for 3 hours, showing the effects of pH, PS80 and other excipients on protein stability.

[0477] Figures 37 and 38 (Figure 37: polymer changes at 40°C; Figure 38: small molecule fragment changes at 40°C) show the changes in HMW% and LMW% of samples of formulations F11 and F13 after being placed at 40°C for 4 weeks, as detected by SEC and NR CE-SDS, demonstrating the effect of the buffer on protein stability.

[0478] Figures 39 and 40 ( Figure 39 : polymer changes at 40°C; Figure 40 : small molecule fragment changes at 40°C) show the changes in HMW% and LMW% detected by SEC and NR CE-SDS for samples of formulations F14 and F15 after being placed at 40°C for 6 weeks, demonstrating the effect of the buffer on protein stability.

[0479] Figures 41-43 (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 HMW% changes of samples of formulations F16 and F17 after 7 days of light exposure, 6 weeks at 40°C or 6 weeks at 25°C, as detected by SEC, showing the effect of excipients on protein stability.

[0480] Figures 44-45 (Figure 44: Changes in PS80 concentration at 40°C; Figure 45: Changes in PS80 concentration at 25°C) respectively show the degradation results of PS80 for samples of prescriptions F18-F21 after being placed at 40°C for one month or at 25°C for six months, demonstrating the protective effect of disodium edetate on PS80.

[0481] Figures 46-49 (Figure 46: polymer changes at 25°C; Figure 47: small molecule fragment changes at 25°C; Figure 48: polymer changes at 5°C; Figure 49: small molecule fragment changes at 5°C) respectively show the changes in HMW% and LMW% detected by SEC and NR CE-SDS when the samples of formulations F18-F21 were placed at 25°C for 4 weeks or 5°C for 6 months, respectively, showing the effect of excipients on protein stability.

[0482] FIG50 (PS80 concentration change at 40° C.) shows the comparative results of PS80 degradation of formulations with or without the addition of edetate disodium at different concentrations at 40° C. for 4 weeks.

[0483] definition

[0484] The terms used in this disclosure have the definitions listed below. If no definition is given herein, the terms used have the meanings commonly understood in the art.

[0485] Throughout this disclosure, the articles "a," "an," and "the" as used herein refer to one or more than one (i.e., to at least one) of the grammatical object of the article. For example, "an antibody" means one antibody or more than one antibody. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0486] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0487] As used herein, the terms "comprise," "include," or "contain" mean including the stated elements, values, or steps, but not excluding any other elements, values, or steps. In this document, when the terms "comprise," "include," or "contain" are used, unless otherwise indicated, combinations of the stated elements, values, or steps are also encompassed. For example, when referring to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0488] As used herein, the terms "preparation" and "pharmaceutical preparation" are used interchangeably and refer to a composition or formulation comprising at least one active ingredient and at least one inactive ingredient suitable for administration to an animal, preferably a mammal (including a human). The preparation of the present disclosure is, for example, in liquid form, lyophilized form, or a liquid form reconstituted from a lyophilized form. "Liquid preparation" refers to a preparation in liquid form. The preparation of the present disclosure is preferably a liquid preparation, such as an injection, intravenous injection, intramuscular injection, or subcutaneous injection.

[0489] As used herein, "buffer" or "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. For example, acetate, histidine, glutamate, citrate, succinate, malate, phosphate or lactate, and / or their respective free acids or bases, as well as various salts and / or mixtures of acids and bases thereof. Preferred pharmaceutically acceptable buffers include, but are not limited to, acetate buffer and histidine buffer. "Acetate buffer" or "acetate buffer" includes acetate-sodium acetate, histidine-acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc. "Histidine buffer" or "histidine buffer" includes histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc. The above buffers are typically used at a concentration of about 1-100 mM, for example, about 5-100 mM, about 10-50 mM, about 15-30 mM, or about 20 mM. The buffering agent can maintain 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%.

[0490] As used herein, "surfactant" refers to a pharmaceutically acceptable excipient with surface activity. Preferably, a nonionic surfactant is used. Pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluronic), and sodium lauryl sulfate (SDS), such as polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188, Brij series, Triton X. When polysorbates, such as PS80 and PS20, are used, their 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%. In the formulations of the present disclosure, the concentration of surfactant is described as a percentage expressed as weight / volume (w / v, g / 100 mL).

[0491] As used herein, "stabilizer" refers to a stabilizer other than the surfactants described above, which is a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation in a stable or unchanged state during manufacture, storage and use, for example, to help prevent aggregation, oxidation, color change, 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. In each case, the amino acids employed are preferably in the L-form. For example, in the form of their inorganic salts, for example, in the form of hydrochlorides, for example Arginine hydrochloride, histidine hydrochloride or lysine hydrochloride (at a concentration of, for example, about 20 to about 250 mM, about 100 to about 200 mM, for example, about 150 mM), salts (e.g., inorganic salts such as sodium chloride, magnesium chloride, calcium chloride), chelating agents (e.g., EDTA (edetic acid), EDTA salts such as EDTA·2Na), polyols (e.g., sorbitol, mannitol, xylitol, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol), cyclodextrins (e.g., hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin), polyethylene glycols (e.g., PEG 3000, PEG 3350, PEG 4000, PEG 6000), albumins (e.g., human serum albumin (HSA), bovine serum albumin (BSA)). Stabilizers are, for example, arginine hydrochloride, NaCl, sucrose or sorbitol, preferably arginine hydrochloride or NaCl, for example, about 150 mM arginine hydrochloride or about 135 mM NaCl. Stabilizers can be present in the formulation at a concentration of about 1-500 mM, for example, about 10-300 mM, about 100-300 mM, about 120-300 mM, about 100-200 mM, about 135-280 mM, about 135-200 mM, about 130-170 mM, about 140-160 mM, 135, 150, 260 or 280 mM. The concentration of an amino acid or inorganic salt stabilizer, such as arginine hydrochloride or NaCl, is about 120-300 mM, about 100-200 mM, about 135-200 mM, about 130-170 mM, about 140-160 mM, about 135 mM, or about 150 mM. For example, the concentration of arginine hydrochloride is about 120-300 mM, about 100-200 mM, about 135-200 mM, about 130-170 mM, about 140-160 mM, or about 150 mM. The concentration of a chelating agent, such as EDTA·2Na, is about 30-350 μM, such as about 75-350 μM, for example, about 75, about 150, or about 300 μM.More than one stabilizer selected from the same or different groups may be present in the formulation of the present disclosure. The stabilizer in the formulation of the present disclosure may also simultaneously function as an osmotic pressure regulator.

[0492] As used herein, "osmotic pressure regulator" refers to a pharmaceutically acceptable excipient used to regulate the osmotic pressure of a solution. Examples of 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 arginine, glycine, cysteine, and histidine), salts (e.g., inorganic salts, such as sodium chloride, magnesium chloride, and calcium chloride), and polyols (e.g., sorbitol, mannitol, xylitol, dextran, glycerol, arabitol, propylene glycol, and polyethylene glycol).

[0493] A subgroup of stabilizers is a lyoprotectant, which includes but is not limited to sugars, polyols (such as, for example, sugar alcohols) and amino acids. Preferred lyoprotectants can 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 500 mM, preferably in an amount of about 10 to about 300 mM and more preferably in an amount of about 100 to about 300 mM.

[0494] A subgroup of stabilizers is antioxidants, which include but are not limited to ascorbic acid, glutathione, cysteine, methionine, citric acid, EDTA. Antioxidants can be used in an amount of about 0.01 to about 100 mM, preferably in an amount of about 5 to about 50 mM and more preferably in an amount of about 5 to about 25 mM.

[0495] As used herein, the term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values ​​having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value, i.e., a range of ±10%, such as ±5%, such as ±3%.

[0496] As used herein, "w / v" means "weight / volume" and the unit is g / 100 mL.

[0497] All numerical ranges herein should be understood to disclose every value and subset of values ​​within the range, regardless of whether they are specifically disclosed separately. For example, when any numerical range is mentioned, it should be considered to refer to every value within the numerical range, such as every integer within the numerical range. The present disclosure is intended to encompass all values ​​falling within these ranges, all smaller ranges, and upper or lower limits of numerical ranges.

[0498] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A natural complete antibody comprises two heavy (H) chains and two light (L) chains. The heavy chains of mammals are divided into α, δ, ε, γ and μ, each consisting of a variable region (VH) and a first, second, third and optional fourth constant region (CH1, CH2, CH3, CH4, respectively); the light chains of mammals are divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region. Antibodies are "Y"-shaped, in which the stem of the Y-shaped structure consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain combined with the variable region and constant region 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) (light chain CDRs include LCDR1, LCDR2, LCDR3, and heavy chain CDRs include HCDR1, HCDR2, HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, IMGT, Chothia, or Al-Lazikani rules (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest. Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015). The three CDRs are separated by flanking segments called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3, and HFR4), which are more highly conserved than CDRs and form a scaffold to support highly variable loops. The constant regions of the heavy and light chains are not related to antigen binding, but exhibit a variety of effector functions. As used herein, the term "effector function" refers to the cell-mediated or complement-mediated cytotoxic effects caused by the interaction between the Fc region of an antibody and the C1q complement protein or Fc receptor (FcR) on immune cells. Exemplary effector functions include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) effects.Antibodies can be divided into several classes based on the amino acid sequence of their heavy chain constant regions. The five major classes, or isotypes, of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0499] In some embodiments, provided herein are antibodies encompassing any antigen binding fragment thereof. As used herein, the term "antigen binding fragment" refers to an antibody fragment formed by a part of an antibody including one or more CDRs, or is bound to an antigen but does not include any other antibody fragment of a complete natural antibody structure. Examples of antigen binding fragments include, but are not limited to, diabody, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecules (scFv), scFv dimers (divalent diabodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and divalent domain antibodies. Antigen binding fragments can be combined with the same antigen bound by the parent antibody.

[0500] The "Fab" portion of an antibody is composed of a single light chain (including variable and constant regions) and the variable region and first constant region of a single heavy chain, joined together by disulfide bonds.

[0501] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0502] "F(ab')2" refers to a dimer of Fab'.

[0503] The "Fc" of an antibody (e.g., IgG, IgA, or IgD isotype) refers to a portion of an antibody consisting of the second and third constant domains of the first heavy chain bound to the second and third constant domains of the second heavy chain via a disulfide bond. 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.

[0504] The "Fv" of an antibody refers to the smallest antibody fragment that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0505] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region linked directly to the heavy chain variable region or connected by a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0506] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc portion of an antibody.

[0507] A "camelized single domain antibody," "heavy chain antibody," or "HCAb" is a H Heavy chain antibodies are antibodies that contain heavy chain domains but not light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies originally came from the Camelidae (camels, dromedaries, and llamas). Despite the absence of light chains, camelized antibodies have a well-established full complement of antigen-binding functions (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1):93-101 (2003)). The variable region (VHH domain) of a heavy-chain antibody represents the smallest known antigen-binding unit produced by an adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0508] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and the two constant domains CH2 and CH3.

[0509] "Diabodies" or "dAbs" include small antibody fragments with 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). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby generating two antigen-binding sites. The two antigen-binding sites can target the same or different antigens (or epitopes). In some embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0510] "Domain antibodies" refer to antibody fragments that contain only the variable region of a heavy chain or a variable region of a light chain. In some cases, 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.

[0511] As used herein, the term "valence" 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. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, the antibody or antigen-binding fragment thereof is bivalent.

[0512] As used herein, 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 can be present on the same antigen, or they can be present on two different antigens.

[0513] In some embodiments, the "scFv dimer" is a bivalent diabody or a bispecific scFv (BsFv), which includes two dimerized VH-VL (connected by a peptide linker) parts, so that the VH of one part cooperates with the VL of the other part to form two binding sites, and the two binding sites can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific diabody, which includes VH1-VL2 (connected by a peptide linker) and VL1-VH2 (also connected by a peptide linker) that associate with each other, so that VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperative pairing has a different antigen specificity.

[0514] "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain is connected to the variable region of a single heavy chain by a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: two VH portions are connected by a peptide linker (e.g., a long flexible linker), and are each bound to two VL portions by a disulfide bond. In some embodiments, dsFv-dsFv' has bispecificity, wherein each pair of heavy and light chains paired by disulfide bonds has a different antigenic specificity.

[0515] The term "chimeric" as used herein refers to an antibody or antigen-binding fragment 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 another different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal (e.g., from a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0516] The term "humanized" as used herein refers to 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).

[0517] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, antibody) or fragment thereof and an antigen.

[0518] As used herein, "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, for example, an antibody and an antigen. Specific binding can be characterized by binding affinity, for example, as measured by K. D The value represents, that is, the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on K can be determined by any conventional method known in the art. D , including but not limited to surface plasmon resonance, Octet method, microthermophoresis, HPLC-MS method and FACS assay. ≤10 -6 M (e.g. ≤5×10 -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M or ≤10 -9 M)K D The value can represent specific binding between the antibody or antigen-binding fragment thereof and PD-L1 (e.g., human PD-L1 or cynomolgus monkey PD-L1).

[0519] As used herein, the ability to "compete for binding to PD-L1" refers to the ability of a first antibody or antigen-binding fragment thereof to inhibit the binding interaction between PD-L1 and a second anti-PD-L1 antibody to any detectable extent. In some embodiments, 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 can be greater than 95% or greater than 99%.

[0520] As used herein, the term "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. Amino acid names are also represented in this disclosure using standard single-letter or three-letter codes, which are summarized below:

[0521] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0522] When "conservative substitution" is used for an amino acid sequence, it refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, 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.

[0523] "Percent (%) sequence identity" when applied to amino acid sequences (or nucleic acid sequences) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum correspondence. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997)), can be used. MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software are used to perform the alignment to determine the percent sequence identity of amino acid (or nucleic acid) sequences. One skilled in the art can use the default parameters provided by the tool or can customize the parameters as needed for the alignment, for example by selecting an appropriate algorithm. In some embodiments, different residue positions may differ due to 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 having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. 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.

[0524] As used herein, a "homologous sequence" refers to a polynucleotide sequence (or its complementary strand) or an amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optionally aligned.

[0525] "Isolated" material 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. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but it is considered "isolated" if the polynucleotide or polypeptide is sufficiently separated from coexisting materials in nature and is present in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In some embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that is 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% pure, wherein purity is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

[0526] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

[0527] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, alleviating or ending symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0528] The term "vector" as used herein refers to a vehicle into which a genetic element can be operably inserted and into which the genetic element is expressed to produce a protein, RNA or DNA encoded by the genetic element or to replicate the genetic element. A vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) from P1 sources, phages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector can also contain an origin of replication. A vector can also include materials that assist it in entering cells, including but not limited to viral particles, liposomes, or protein coatings. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0529] As used herein, a "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0530] As used herein, the term "soluble" refers to the ability of a molecule (eg, a protein) to dissolve in a solvent (eg, a liquid and aqueous environment).

[0531] As used herein, the terms "transforming growth factor β" and "TGFβ" refer to any TGFβ family protein having the full-length, native amino acid sequence of any TGF-β from a subject (e.g., a human), including latent forms of precursors and mature TGFβ and related or unrelated complexes ("latent TGFβ"). References to such TGFβ herein will be understood as references to any currently identified form, including TGFβ1, TGFβ2, TGFβ3 isoforms and latent forms thereof, as well as future identified human TGFβ species, including polypeptides derived from the sequence of any known TGFβ and at least about 75%, preferably at least about 80%, more preferably at least about 85%, still more preferably at least about 90%, and even more preferably at least about 95% homologous to said sequence. The specific terms "TGFβ1," "TGFβ2," and "TGFβ3" refer to TGF-β as defined in the literature, e.g., Derynck et al., Nature Cancer Res., 47:707 (1987); Seyedin et al., J. Biol. Chem., 261:5693-5695 (1986); deMartin et al., EMBO J., 6:3673 (1987); Kuppner et al., Int. J. Cancer, 42:562 (1988). The terms "transforming growth factor β," "TGFβ," "TGFbeta," "TGF-β," and "TGF-beta" are used interchangeably in this disclosure.

[0532] As used herein, the term "human TGFβ1" refers to a TGFβ1 protein encoded by the human TGFB1 gene (e.g., a wild-type human TGFB1 gene). Exemplary wild-type human TGFβ1 proteins are provided by GenBank Accession No. NP_000651.3. As used herein, the term "human TGFβ2" refers to a TGFβ2 protein encoded by the human TGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild-type human TGFβ2 proteins are provided by GenBank Accession Nos. NP_001129071.1 and NP_003229.1. As used herein, the term "human TGFβ3" refers to a TGFβ3 protein encoded by the human TGFB3 gene (e.g., a wild-type human TGFB3 gene). Exemplary wild-type human TGFβ3 proteins are provided by GenBank Accession Nos. NP_003230.1, NP_001316868.1, and NP_001316867.1.

[0533] As used herein, the terms "mouse TGFβ1," "mouse TGFβ2," and "mouse TGFβ3" refer to the TGFβ1 protein, TGFβ2 protein, and TGFβ3 protein encoded by the mouse TGFB1 gene (e.g., a wild-type mouse TGFB1 gene), the mouse TGFB2 gene (e.g., a wild-type mouse TGFB2 gene), and the mouse TGFB3 gene (e.g., a wild-type mouse TGFB3 gene), respectively. Exemplary wild-type mouse (Mus musculus) TGFβ1 proteins are provided by GenBank Accession Nos. NP_035707.1 and CAA08900.1. An exemplary wild-type mouse TGFβ2 protein is provided by GenBank Accession No. NP_033393.2. An exemplary wild-type mouse TGFβ3 protein is provided by GenBank Accession No. AAA40422.1.

[0534] As used herein, the term "TGFβ receptor" refers to any receptor that binds at least one TGFβ isoform. Typically, the TGFβ receptor includes TGFβ receptor I (TGFβRI), TGFβ receptor II (TGFβRII), or TGFβ receptor III (TGFβRIII).

[0535] With respect to humans, the term "TGFβ receptor 1" or "TGFβRI" refers to the human TGFβ receptor type 1 sequence, including wild-type TGFβRI and all isoforms and variants thereof known to bind to at least one TGFβ isoform. An exemplary amino acid sequence of wild-type TGFβRI is available under GenBank Accession No. ABD46753.1 or UniProtKB-P36897, also included herein as SEQ ID NO: 69. Variant TGFβRI may have a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 69 and retain at least 25%, 35%, 50%, 75%, 90%, 95% or 99% of the TGFβ binding activity of the wild-type sequence (e.g., SEQ ID NO: 69).

[0536] With respect to humans, the term "TGFβ receptor II" or "TGFβRII" refers to the sequence of human TGFβ receptor type 2 isoform A, including wild-type TGFβRII and all isoforms and variants thereof known to be capable of binding to at least one TGFβ isoform. An exemplary amino acid sequence of wild-type TGFβRII isoform A or isoform 1 is available under GenBank Accession No. NP_001020018.1 or UniProtKB-P37173-1, also included herein as SEQ ID NO: 70, and wild-type TGFβRII isoform B is available under GenBank Accession No. NP_003233.4 or UniProtKB-P37173-2, also included herein as SEQ ID NO: 71. The variant TGFβRII can have a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 70 or 71 and retain at least 25%, 35%, 50%, 75%, 90%, 95% or 99% of the TGFβ binding activity of the wild-type sequence (e.g., SEQ ID NO: 70 or 71).

[0537] With respect to humans, the term "TGFβ receptor III" or "TGFβRIII" refers to the human TGFβ receptor type 3 sequence, including wild-type TGFβRII and all isoforms and variants. An exemplary amino acid sequence of wild-type TGFβRIII is available under GenBank Accession No. NP_003234.2 or UniProtKB-Q03167, and is also included herein as SEQ ID NO: 72.

[0538] As used herein, the term "variant" with respect to a reference protein or peptide refers to a modified version of the reference protein or peptide, such as a functional equivalent, fragment, fusion, derivative, mimetic, or any combination thereof, wherein the modified version has an amino acid sequence that has at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the reference sequence and retains 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., wild-type sequence). The variant can be a fragment, mutant, fusion, truncation, or any combination thereof of the reference protein or peptide.

[0539] As used herein, the term "interleukin-1" or "IL-1" includes IL-1α and IL-1β, their precursors (eg, pro-IL-1α and pro-IL-1β), isoforms, and variants.

[0540] As used herein, the term "human IL-1α" refers to IL-1α proteins encoded by human IL1A genes (eg, wild-type human IL1A genes), as well as isoforms and variants. UniProtKB-P01583 provides an exemplary wild-type human IL1α protein.

[0541] As used herein, the term "human IL-1β" refers to the IL-1β protein encoded by the human IL1B gene (eg, the wild-type human IL1B gene). An exemplary wild-type human IL1β protein is provided by GenBank Accession No. NP_000567.1 or UniProtKB-C9JVK0.

[0542] As used herein, the term "IL-1 receptor" or "IL-1R" refers to a receptor that can bind to IL-1, including all wild-type receptors, isoforms, and variants thereof that can bind to IL-1. Typically, there are two types of IL-1 receptors, IL-1 receptor I (IL-1RI) and IL-1 receptor II (IL-1RII). IL-1RII acts as a decoy receptor that binds to a ligand without transducing a signal. Proteolytic cleavage of IL-1RII causes the formation of soluble receptors, such as IL-1sRI and IL-1sRII, which bind to a ligand without transducing a signal (for details, see Thomas G. Kennedy, Chapter VB2. in Encyclopedia of Hormones, 2003). IL-1sRI and IL-1sRII are proteolytic cleavage products of IL-1RII and can 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-1β to form a high-affinity interleukin 1 receptor complex that mediates interleukin 1-dependent activation of NF-κ-B and other pathways.

[0543] As used herein, the term "IL-1RI" includes wild-type IL-1RI and all isoforms and variants thereof that are capable of binding to IL-1α and / or IL-1β. An exemplary amino acid sequence of wild-type IL-1RI is available under UniProtKB-P14778 and is also included herein as SEQ ID NO: 73.

[0544] As used herein, the term "IL-1RII" includes wild-type IL-1RII and all isoforms and variants thereof that are capable of binding to IL-1α and / or IL-1β. An exemplary amino acid sequence of wild-type IL-1RII is available under UniProtKB-P27930 and is also included herein as SEQ ID NO: 75.

[0545] As used herein, the term "IL-1RAP" includes wild-type IL-1RAP and all isoforms and variants thereof that are capable of binding to IL-1R bound to IL-1β. An exemplary amino acid sequence of wild-type IL-1RAP is available under UniProtKB-Q9NPH3 and is also included herein as SEQ ID NO: 74.

[0546] As used herein, the term "IL-1sRI" includes all soluble forms of IL-1RI that can be produced by proteolytic cleavage involving metalloproteinases. 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 that are capable of binding to IL-1α and / or IL-1β.

[0547] As used herein, the term "IL-1sRII" includes all soluble forms of IL-1RII that can be produced by proteolytic cleavage involving metalloproteinases. 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 that are capable of binding to IL-1α and / or IL-1β.

[0548] As used herein, the term "IL-1 receptor antagonist" generally includes any protein that can compete with IL-1α or IL-1β for binding to the IL-1 receptor and inhibit the activity of IL-1α or IL-1β. IL-1 receptor antagonists can include naturally occurring antagonists such as IL-1Ra, IL-1sRI, and IL-1sRII, as well as other artificial antagonists that can block the binding of IL-1α or IL-1β to the IL-1 receptor, specifically IL-1RI.

[0549] As used herein, the term "IL-1Ra" includes wild-type IL-1Ra and all isoforms and variants thereof that are capable of binding to IL-1α and / or IL-1β. An exemplary amino acid sequence of wild-type IL-1Ra is available under UniProtKB-P18510 and is also included herein as SEQ ID NO: 76.

[0550] As used herein, "cancer" refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and can be benign or malignant, and includes solid tumors and non-solid cancers (e.g., hematological malignancies) such as leukemias. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0551] The term "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 physiologically compatible with the recipient thereof.

[0552] The term "fusion" or "fused" when used in reference to amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur in nature, for example, by chemical bonding or recombinant means. A fused 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.

[0553] Further detailed description of the disclosed embodiments

[0554] The various embodiments and features in the various embodiments described in the context of this disclosure should be understood to be capable of being combined with each other in any manner, and the various schemes obtained by these combinations are all included in the scope of this disclosure, just as the schemes obtained by these combinations are specifically and individually listed in this document, unless the context clearly indicates otherwise.

[0555] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Thus, the specific variations discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the present disclosure, and it will be understood that such equivalent embodiments are intended to be included herein. All documents cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0556] I. Bifunctional molecules targeting immune checkpoint molecules and blocking IL-1 activity

[0557] The present disclosure provides a bifunctional molecule, which includes a first portion bound to an immune checkpoint molecule and a second portion that blocks the activity of interleukin-1 (IL-1). The bifunctional molecule provided herein blocks the interaction between IL-1 and IL-1 receptors with an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion (i.e., the second portion of the bifunctional molecule), allowing blocking and / or reducing the IL-1 activity in the tumor microenvironment. The IL-1 binding portion and / or the IL-1R binding portion can be connected to the portion of the targeting immune checkpoint molecule (i.e., the first portion of the bifunctional molecule), which can be found on the surface of certain tumor cells or immune cells.

[0558] 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 macrophage recruitment, invasion, and metastasis (ibid.).

[0559] 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 the activation of immune checkpoints, thereby allowing cancer cells to evade the surveillance of the immune system.

[0560] By reducing IL-1 in the tumor microenvironment and reducing checkpoint blockade, the present disclosure provides a novel bifunctional molecule that can be used to treat immune checkpoint-related diseases, such as cancer, autoimmune diseases, infectious diseases, etc.

[0561] In some embodiments, the first portion includes an agonist of a checkpoint molecule with immunostimulatory or costimulatory activity. Such 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.

[0562] In some embodiments, the first portion includes an inhibitor of a checkpoint molecule with immunosuppressive or co-inhibitory activity. Such 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.

[0563] In some embodiments, the immune checkpoint molecule is PD-L1. In some embodiments, the first portion comprises an antibody portion or an antigen-binding fragment thereof directed against PD-L1. In some embodiments, the first portion comprises an antagonist antibody portion or an antigen-binding fragment thereof directed against PD-L1.

[0564] In some embodiments, the second moiety comprises an IL-1 binding portion or an IL-1 receptor (IL-1R) binding portion.

[0565] Both IL-1α and IL-1β are pro-inflammatory and bind to IL-1R. Upon binding to IL-1α or IL-1β, IL-1R recruits the IL-1R accessory protein and adaptor protein MyD88 to the receptor complex, leading to activation of downstream signaling cascades and ultimately activating a large number of immune and inflammatory genes. The inventors have discovered that combining blocking IL-1 activity or its binding to IL-1R with modulation of immune checkpoint molecules would be useful.

[0566] In some embodiments, IL-1 is IL-1α or IL-1β. In some embodiments, IL-1β is human IL-1β.

[0567] In some embodiments, the second portion comprises an IL-1 binding portion. In some embodiments, the IL-1 binding portion specifically binds to IL-1α or IL-1β. In some embodiments, the IL-1 binding portion comprises a soluble IL-1R, an IL-1 binding fragment or variant of an IL-1R, or an antibody or antigen-binding fragment thereof directed against IL-1.

[0568] The soluble IL-1R may be a domain or fragment of IL-1R, such as the extracellular domain (ECD) of IL-1R. Alternatively, the soluble IL-1R may be IL-1sRI or IL-1sRII, which are naturally soluble isoforms capable of binding to IL-1.

[0569] The skilled artisan will appreciate that shortened fragments of IL-1RI or IL-1RI's ECD, or IL-1RII or IL-1RII's ECD, or IL-1RAP or IL-1RAP's ECD, or IL-1sRI or IL-1sRII may be sufficient to bind to IL-1 (e.g., IL-1α or IL-1β) as long as such fragments contain an IL-1 binding domain. Thus, the present disclosure also encompasses all IL-1 binding fragments and variants of any of IL-1RI, IL-1RI's ECD, or IL-1RII, or IL-1RII's ECD, or IL-1RAP or IL-1RAP's ECD, IL-1sRI, and IL-1sRII. In some embodiments, the IL-1 binding portion comprises 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 comprises an amino acid sequence having 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.

[0570] In some embodiments, the IL-1 binding portion includes an antibody or antigen-binding fragment thereof directed against IL-1. Antibodies or antigen-binding fragments thereof directed against IL-1 may also be used, as long as such antibodies or antigen-binding fragments can block the binding of IL-1 (e.g., IL-1α or IL-1β) to IL-1R.

[0571] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104 or SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 105 or SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 106 or SEQ ID NO: 114, and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 109 or SEQ ID NO: 117.

[0572] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104, a HCDR2 comprising the sequence of SEQ ID NO: 105, and a HCDR3 comprising the sequence of SEQ ID NO: 106 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107, a LCDR2 comprising the sequence of SEQ ID NO: 108, and a LCDR3 comprising the sequence of SEQ ID NO: 109.

[0573] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 114 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 117.

[0574] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 110, and homologous sequences thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences thereof having at least 80% sequence identity.

[0575] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102 and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103 and a homologous sequence thereof having at least 80% sequence identity.

[0576] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 110, and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 111, and a homologous sequence thereof having at least 80% sequence identity.

[0577] In some embodiments, the second moiety comprises an IL-1R binding moiety.

[0578] In some embodiments, the IL-1R binding portion comprises IL-1Ra or an IL-1R binding fragment or variant thereof. IL-1Ra is an antagonist of IL-1R and can compete with IL-1α or IL-1β for binding to IL-1R. Similarly, one of skill will appreciate that shortened fragments of IL-1Ra may be sufficient for binding to IL-1R and / or competing with IL-1α or IL-1β. In some embodiments, the IL-1R binding portion comprises a truncated form of IL-1Ra. In some embodiments, the IL-1R binding portion comprises the amino acid sequence of SEQ ID NO: 67 or 76, or any IL-1 binding fragment or variant thereof. In some embodiments, the IL-1R binding portion comprises 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. One of skill will appreciate that 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-1α or IL-1β for binding to IL-1R.

[0579] In some embodiments, the IL-1R binding portion includes an antibody or antigen-binding fragment thereof directed against IL-1R. Antibodies or antigen-binding fragments thereof directed against IL-1R may also be used, as long as such antibodies or antigen-binding fragments can compete with IL-1α or IL-1β for binding to IL-1R.

[0580] II. Bifunctional Molecule Targeting PD-L1 and Second Part

[0581] The therapeutic efficacy of PD-1 / PD-L1 axis checkpoint inhibitors (e.g., PD-L1 antibodies) may be limited when the tumor microenvironment (TME) is enriched with immunosuppressive cytokines. Signaling by such immunosuppressive cytokines in the local microenvironment can reduce tumor-infiltrating T cells, skew them toward Tregs, and attenuate the activation of immune effector cells.

[0582] In one aspect, the present disclosure provides a novel bifunctional molecule comprising 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 can be a compound, a peptide, a polypeptide, a protein, or any combination thereof. The second portion can restore the immune response in the tumor microenvironment by blocking immunosuppressive activity or cytokines or by increasing or stimulating immunity.

[0583] In some embodiments, the bifunctional molecules provided herein include a first portion that binds to PD-L1 (ie, a PD-L1 binding portion) and a second portion that blocks the activity of an immunosuppressive cytokine.

[0584] In some embodiments, immunosuppressive cytokines include cytokines in the transforming growth factor beta (TGF-β) superfamily, IL-1, or vascular endothelial growth factor (VEGF). In some embodiments, immunosuppressive cytokines in the TGF-β superfamily include bone morphogenetic proteins (BMPs), activins, NODAL, and growth and differentiation factors (GDFs).

[0585] In some embodiments, the immunosuppressive cytokine is TGF-β. In some embodiments, the immunosuppressive cytokine is IL-1.

[0586] In some embodiments, the second portion includes a TGFβ binding portion. In some embodiments, the second portion includes an IL-1 binding portion. As used herein, the terms "binding portion", "binding fragment" refer to a portion or fragment with the ability to specifically bind to a target molecule or complex. The term "TGFβ binding portion" refers to a portion with the ability to specifically bind to one or more family members or isoforms of the TGFβ family (e.g., TGFβ1, TGFβ2 or TGFβ3). Similarly, the term "IL-1 binding portion" refers to a portion with the ability to specifically bind to one or more family members of the IL-1 family (e.g., IL-1α, IL-1β).

[0587] In some embodiments, the 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. In some embodiments, the second portion includes an immunostimulatory polypeptide or a functional equivalent thereof or a variant thereof. In some embodiments, 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-α or its functional equivalent.

[0588] In some embodiments, the second moiety comprises an antagonist of immunoinhibitory receptor signaling. In some embodiments, the immunoinhibitory receptor is SIRPα.

[0589] In some embodiments, the bifunctional molecule includes one or more second portions in the second portion. In some embodiments, the one or more second portions in the second portion can be of the same type, for example, each of the one or more second portions can block the activity of immunosuppressive cytokines, or each of the one or more second portions can stimulate anti-tumor immunity. In some embodiments, the one or more second portions in the second portion can be of different types. In some embodiments, each of the plurality of second portions can have the same sequence or can have a different amino acid sequence.

[0590] i. TGFβ binding part

[0591] In some embodiments, the TGFβ binding portion includes a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, or an antibody to TGFβ and an antigen-binding fragment thereof.

[0592] A “TGFβ binding moiety” may also be referred to as a “TGFβ Trap” in the present disclosure. Therefore, a protein that targets both PD-L1 and TGFβ may also be referred to as an “anti-PD-L1 / TGFβ trap” in the present disclosure.

[0593] In some embodiments, the TGFβ binding portion binds to human and / or mouse TGFβ. In some embodiments, the TGFβ binding portion is capable of antagonizing and / or inhibiting the TGFβ signaling pathway. In some embodiments, the TGFβ binding portion is capable of antagonizing and / or inhibiting TGFβ.

[0594] In the present disclosure, the TGFβ binding portion may include any portion that specifically binds to one or more family members or isoforms of the TGFβ family. In some embodiments, the TGFβ binding portion includes a portion that binds to TGFβ1 (e.g., human TGFβ1), TGFβ2 (e.g., human TGFβ2) and / or TGFβ3 (e.g., human TGFβ3) or a variant thereof that has similar or improved TGFβ binding affinity. In some embodiments, the TGFβ binding portion includes a portion that binds to TGFβ1 (e.g., human TGFβ1). In some embodiments, the TGFβ binding portion includes a portion that binds to TGFβ2 (e.g., human TGFβ2). In some embodiments, the TGFβ binding portion includes a portion that binds to TGFβ3 (e.g., human TGFβ3). In some embodiments, the TGFβ binding portion includes a portion that specifically binds to both TGFβ1 (e.g., human TGFβ1) and TGFβ2 (e.g., human TGFβ2). In some embodiments, the TGFβ binding portion includes a portion that specifically binds to both TGFβ1 (e.g., human TGFβ1) and TGFβ3 (e.g., human TGFβ3). In some embodiments, the TGFβ binding portion includes a portion that specifically binds to both TGFβ2 (e.g., human TGFβ2) and TGFβ3 (e.g., human TGFβ3). In some embodiments, the TGFβ binding portion includes a portion that specifically binds to each of TGFβ1 (e.g., human TGFβ1), TGFβ2 (e.g., human TGFβ2), and TGFβ3 (e.g., human TGFβ3). It will be understood by those skilled in the art that a TGFβ binding portion that binds to one family member or isoform of the TGFβ family may be able to bind to one or more other family members or isoforms of the TGFβ family with similar or higher affinity.

[0595] In some embodiments, the TGFβ binding portion comprises a portion that selectively binds to TGFβ1 relative to TGFβ2 and / or TGFβ3.

[0596] In some embodiments, the TGFβ binding portion includes a portion that specifically binds to human TGFβ1 and mouse TGFβ1 with similar affinity.

[0597] In some embodiments, the TGFβ binding portions of the present disclosure include a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof.

[0598] Exemplary TGFβ receptors include TGFβRI, TGFβRII, and TGFβRIII. In some embodiments, the TGFβ receptor is selected from the group consisting of: TGFβ receptor I (TGFβRI), TGFβ receptor II (TGFβRII), TGFβ receptor III (TGFβRIII), and any combination thereof. In some embodiments, the TGFβ receptor is TGFβRI (e.g., human TGFβRI). In some embodiments, the TGFβ receptor is TGFβRII (e.g., human TGFβRII). In some embodiments, the TGFβ receptor is TGFβRIII (e.g., human TGFβRIII).

[0599] In some embodiments, the TGFβ binding portion includes the extracellular domain (ECD) of a TGFβ receptor (e.g., a human TGFβ receptor) or a TGFβ binding fragment or variant thereof. In some embodiments, the ECD of a TGFβ receptor includes the ECD of TGFβRI (e.g., human TGFβRI), the ECD of TGFβRII (e.g., human TGFβRII), the ECD of TGFβRIII (e.g., human TGFβRIII), or any combination thereof. In some embodiments, the ECD of TGFβRII includes the amino acid sequence of SEQ ID NO: 66, 79, or an amino acid sequence having 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 thereto but still retains binding specificity for TGFβ. In some embodiments, the ECD of TGFβRI comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that has 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 thereto while retaining binding specificity to TGFβ. In some embodiments, the ECD of TGFβRIII comprises the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence that has 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 thereto while retaining binding specificity to TGFβ.

[0600] In some embodiments, the TGFβ binding portion includes antibodies to TGFβ and antigen-binding fragments thereof. Exemplary anti-TGFβ antibodies include fresolimumab and metelimumab and anti-TGFβ antibodies or antigen-binding fragments thereof described in: for example, US7494651B2, US8383780B2, US8012482B2, WO 2017141208A1, each of which is incorporated herein by reference in its entirety.

[0601] In some embodiments, the TGFβ binding portion comprises a combination of one or more ECDs of one or more TGFβ receptors and / or one or more anti-TGFβ antibodies or antigen-binding fragments thereof.

[0602] The one or more ECDs may be the same or different. For example, the TGFβ binding portion may include the same repeat sequence of the ECD of a TGFβ receptor, or alternatively may include a combination of different ECD sequences of the same TGFβ receptor, or alternatively may include a combination of different ECDs from different TGFβ receptors. Similarly, the one or more anti-TGFβ antibodies may be the same or different.

[0603] In some embodiments, the TGFβ binding portion includes a combination (or fusion) of ECDs selected from the group consisting of: the ECD of TGFβRI (e.g., human TGFβRI), the ECD of TGFβRII (e.g., human TGFβRII), the ECD of TGFβRIII (e.g., human TGFβRIII), or any combination thereof.

[0604] In some embodiments, the TGFβ binding portion comprises a combination (or fusion) of one or more anti-TGFβ antibodies or antigen-binding fragments thereof.

[0605] In some embodiments, the TGFβ binding portion comprises a combination (or fusion) of: an ECD selected from the group consisting of: the ECD of TGFβRI (e.g., human TGFβRI), the ECD of TGFβRII (e.g., human TGFβRII), the ECD of TGFβRIII (e.g., human TGFβRIII); one or more anti-TGFβ antibodies or antigen-binding fragments thereof; or any combination thereof.

[0606] ii. IL-1 binding portion

[0607] In some embodiments, the second portion comprises an IL-1 binding portion. In some embodiments, IL-1 is IL-1α or IL-1β. In some embodiments, IL-1β is human IL-1β.

[0608] In some embodiments, the IL-1 binding portion specifically binds to IL-1α or IL-1β. In some embodiments, the IL-1 binding portion includes a portion that selectively binds to IL-1β relative to IL-1α or a portion that selectively binds to IL-1α relative to IL-1β.

[0609] In some embodiments, the IL-1 binding portion comprises a soluble IL-1R, an IL-1 binding fragment or variant of IL-1R, or an antibody to IL-1 or an antigen-binding fragment thereof.

[0610] Soluble IL-1R may include a domain, fragment, or variant of IL-1R, for example, the extracellular domain (ECD) of IL-1R. Alternatively, soluble IL-1R may also include IL-1sRI or IL-1sRII, which are naturally soluble isoforms capable of binding to IL-1.

[0611] The skilled artisan will appreciate that shortened fragments of IL-1R or IL-1R ECD, or IL-1sRI or IL-1sRII may be sufficient to bind to IL-1 (e.g., IL-1α or IL-1β), as long as such fragments contain an IL-1 binding domain. Thus, the IL-1 binding moieties provided herein may also include IL-1 binding fragments of any one of IL-1R, IL-1R ECD, IL-1sRI, and IL-1sRII. In some embodiments, the IL-1 binding moiety comprises 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 moiety comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 73, 74, and 75, or an IL-1 binding fragment or variant thereof.

[0612] In some embodiments, the IL-1 binding portion includes an antibody or antigen-binding fragment thereof directed against IL-1. Antibodies or antigen-binding fragments thereof directed against IL-1 may also be used, as long as such antibodies or antigen-binding fragments can block the binding of IL-1 (e.g., IL-1α or IL-1β) to IL-1R.

[0613] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104 or SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 105 or SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 106 or SEQ ID NO: 114, and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107 or SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 108 or SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 109 or SEQ ID NO: 117.

[0614] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 104, a HCDR2 comprising the sequence of SEQ ID NO: 105, and a HCDR3 comprising the sequence of SEQ ID NO: 106 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 107, a LCDR2 comprising the sequence of SEQ ID NO: 108, and a LCDR3 comprising the sequence of SEQ ID NO: 109.

[0615] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 112, a HCDR2 comprising the sequence of SEQ ID NO: 113, and a HCDR3 comprising the sequence of SEQ ID NO: 114 and / or a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 115, a LCDR2 comprising the sequence of SEQ ID NO: 116, and a LCDR3 comprising the sequence of SEQ ID NO: 117.

[0616] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 110, and homologous sequences thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 111, and homologous sequences thereof having at least 80% sequence identity.

[0617] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 102 and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 103 and a homologous sequence thereof having at least 80% sequence identity.

[0618] In some embodiments, the antibody or antigen-binding fragment thereof against IL-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 110, and a homologous sequence thereof having at least 80% sequence identity, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 111, and a homologous sequence thereof having at least 80% sequence identity.

[0619] In some embodiments, the IL-1 binding portion comprises a combination of one or more moieties selected from the group consisting of IL-1R, the ECD of IL-1R, IL-1sRI, IL-1sRII, an antibody to IL-1, any IL-1 binding fragment thereof, and any combination thereof. Such one or more moieties may be linked by a direct bond or may be linked by a suitable linker.

[0620] In some embodiments, the IL-1R binding portion comprises IL-1Ra or an IL-1R binding fragment or variant thereof. IL-1Ra is an antagonist of IL-1R and can compete with IL-1α or IL-1β for binding to IL-1R. Similarly, one of skill will appreciate that shortened fragments of IL-1Ra may be sufficient for binding to IL-1R and / or competing with IL-1α or IL-1β. In some embodiments, the IL-1R binding portion comprises a truncated form of IL-1Ra. In some embodiments, the IL-1R binding portion comprises the amino acid sequence of SEQ ID NO: 67 or any IL-1 binding fragment or variant thereof. In some embodiments, the IL-1R binding portion comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 67 or any IL-1 binding fragment or variant thereof. One of skill will appreciate that 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-1α or IL-1β for binding to IL-1R.

[0621] In some embodiments, the IL-1R binding portion includes an antibody or antigen-binding fragment thereof directed against IL-1R. Antibodies or antigen-binding fragments thereof directed against IL-1R may also be used, as long as such antibodies or antigen-binding fragments can compete with IL-1α or IL-1β for binding to IL-1R.

[0622] In some embodiments, the IL-1R binding portion comprises a combination of one or more moieties selected from the group consisting of IL-1Ra, an antibody to IL-1R, any IL-1R binding fragment or variant thereof, and any combination thereof. Such one or more moieties may be linked by a direct bond or may be linked by a suitable linker.

[0623] iii. Immunostimulatory peptides

[0624] In some embodiments, the second portion comprises an immunostimulatory polypeptide or a functional equivalent thereof or a variant thereof. In some embodiments, the immunostimulatory polypeptide is soluble CD4, soluble LAG-3 or a functional equivalent thereof.

[0625] In some embodiments, soluble LAG-3 includes the extracellular domain (ECD) of LAG-3 or an MHC class II (MHCII) binding fragment or variant thereof.

[0626] LAG-3 (Uniprot No.: Q61790) belongs to the immunoglobulin (Ig) superfamily and is a type I transmembrane protein consisting of 503 amino acids. Lag-3 consists of an intracellular domain (ICD), a transmembrane domain (TMD), and an extracellular domain (ECD). The ECD comprises four Ig-like domains, D1 to D4, of which D1 comprises nine β-strands: A, B, C, C', C", D, E, F, and G. Between the C and C' strands, there is an additional sequence of approximately 30 amino acids that forms an "extra loop." This "extra loop" has been reported to be involved in the interaction between LAG-3 and MHC II. In some embodiments, soluble LAG-3 comprises the amino acid sequence of the extra loop, the D1 domain, the D1 plus D2 domains, or any MHC II-binding fragment or variant thereof. In some embodiments, the soluble LAG-3 comprises the amino acid sequence of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or any MHC II-binding fragment or variant thereof.

[0627] LAG-3 is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. The primary ligand for LAG-3 is MHC class II, to which it binds with higher affinity than CD4. A connecting peptide (CP) exists between D4 and the TMD of LAG-3, which is cleaved in the presence of the metalloproteases ADAM10 and / or ADAM17 to produce cleaved, soluble LAG-3. See, e.g., Huard et al., Proc Natl Acad Sci USA 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.

[0628] LAG-3 also encodes an alternative splice variant that can be translated into a soluble form of LAG-3. Soluble LAG-3 activates antigen-presenting cells (APCs) through MHC II signaling, thereby increasing antigen-specific T cell responses in vivo. For example, soluble LAG-3 activates dendritic cells (DCs) and is reported to participate in the pro-inflammatory activity of bystander T cells activated by cytokines such as TNF-α and / or IL-12, and it can directly activate DCs. See, for example, Triebel, Trends Immunol., 2003, 24:619-622, which is incorporated herein by reference.

[0629] In some embodiments, soluble LAG-3 includes Eftilagimod alpha (IMP321) or its MHC II binding fragment or variant. IMP321 is a soluble dimeric recombinant form of LAG-3. IMP321 induces a sustained immune response by stimulating dendritic cells via MHC II molecules. Combination therapy of IMP321 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. 2019 Jun;15(17):1963-1973. doi:10.2217 / fon-2018-0807. Epub 2019 Apr 12.; Julio et al., Journal of Clinical Oncology, Vol. 37, No. 15; and US10874713B, which are incorporated herein by reference.

[0630] iv. Antagonists of immunosuppressive receptor signaling

[0631] In some embodiments, the second moiety comprises an antagonist of immunoinhibitory receptor signaling. In some embodiments, the immunoinhibitory receptor is SIRPα.

[0632] As used herein, the term "SIRPa," which is interchangeable with the term "signal regulatory protein alpha," refers to an inhibitory receptor expressed primarily on myeloid and dendritic cells. SIRPα belongs to the SIRPs family, which also includes several other transmembrane glycoproteins, including SIRPβ and SIRPγ. Each member of the SIRPs family contains three similar extracellular Ig-like domains, while the transmembrane and cytoplasmic domains differ.

[0633] SIRPα can bind to CD47, which transmits the "don't eat me" signal to inhibit phagocytosis, and blocking CD47-mediated SIRPα binding on phagocytes can lead to the removal of viable cells carrying the "eat me" signal. CD47 is a widely expressed transmembrane glycoprotein with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. CD47 acts as a cellular ligand for SIRPα. Tumor cells often overexpress CD47 to avoid macrophage-mediated destruction. The interaction between CD47 and SIRPα has been shown to be involved in the regulation of macrophage-mediated phagocytosis (Takenaka et al., Nature Immunol., 8(12):1313-1323, 2007).

[0634] In some embodiments, the second portion blocks the interaction between CD47 and SIRPα.In various preclinical models, therapies that block the interaction between CD47 and SIRPα stimulate phagocytosis of cancer cells in vitro and anti-tumor immune responses in vivo.

[0635] The second portion may include a CD47 binding domain or a SIRPα binding domain. In some embodiments, the immunoinhibitory receptor is signal regulatory protein α (SIRPα). In some embodiments, the second portion blocks the interaction between CD47 and SIRPα. In some embodiments, the second portion includes a CD47 binding domain or a SIRPα binding domain. In some embodiments, the CD47 binding domain includes a soluble SIRPα or a CD47 binding fragment thereof, or an anti-CD47 antibody or an antigen-binding fragment thereof.

[0636] In some embodiments, the soluble SIRPα includes the extracellular domain (ECD) of the SIRPα or its CD47 binding fragment or variant. In some embodiments, the soluble SIRPα includes the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence having 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 thereto but still retaining binding specificity to CD47. Optionally, the soluble SIRPα is an engineered high-affinity SIRPα variant that effectively antagonizes CD47 on cancer cells but does not itself induce macrophage phagocytosis. In some embodiments, the SIRPα variant comprises 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.In some embodiments, the SIRPα variant comprises a combination 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, F94L; 7) L4V, V6L, I31F, E47V, K53R, H56P, S66G, V 92I, F103V; 8) V6I, V27I, I31F, E47L, K53R, E54Q, H56P, S66T; 9) L4V, V6I, V27I, I31F, E , E54Q, H56P, S66T, K68R, V92I; 10) V6I, V27I, I31T, E47V, K53R, E54Q, H56P, S66G, K68R, V92I, F103V; and 11) V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T, V92I. See, e.g., Kipp Weiskopf et al., Science, 341, 88 (2013), which is incorporated herein by reference.

[0637] In some embodiments, the SIRPα binding domain comprises soluble CD47 or a SIRPα binding fragment thereof, or an anti-SIRPα antibody or an antigen-binding fragment thereof. In some embodiments, the soluble CD47 comprises the extracellular domain (ECD) of CD47 or a SIRPα binding fragment thereof, or an anti-SIRPα antibody or an antigen-binding fragment thereof.

[0638] In some embodiments, 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 antibodies, B6H12 antibodies, and ZF1 antibodies. See Lu et al., OncoTargets and Therapy, Vol. 13, DOI https: / / doi.org / 10.2147 / OTT.S249822, which is incorporated herein by reference. In some embodiments, the SIRPα binding domain includes an anti-SIRPα antibody or an antigen-binding fragment thereof. Exemplary anti-SIRPα 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.

[0639] In some embodiments, the CD47 binding domain comprises a combination of one or more ECDs of one or more SIRPα, SIRPβ, or SIRPγ and / or one or more anti-CD47 antibodies or antigen-binding fragments thereof.

[0640] The one or more ECDs may be the same or different. For example, the CD47 binding domain may include the same repeat sequence of the ECD of SIRPα, SIRPβ or SIRPγ, or alternatively may include a combination of different ECD sequences of the same SIRPα, SIRPβ or SIRPγ, or alternatively may include a combination of different ECDs from different SIRPα, SIRPβ or SIRPγ. Similarly, the one or more anti-CD47 antibodies may be the same or different.

[0641] In some embodiments, the CD47 binding domain includes a combination (or fusion) of ECDs selected from the group consisting of the ECD of SIRPα, the ECD of SIRPβ, the ECD of SIRPγ, or any combination thereof.

[0642] In some embodiments, the CD47 binding domain comprises a combination (or fusion) of one or more anti-CD47 antibodies or antigen-binding fragments thereof.

[0643] In some embodiments, the CD47 binding domain comprises a combination (or fusion) of: an ECD selected from the group consisting of: an ECD of SIRPα, an ECD of SIRPβ, an ECD of SIRPγ; one or more anti-CD47 antibodies or antigen-binding fragments thereof; or any combination thereof.

[0644] v.PD-L1 binding portion

[0645] In some embodiments, the bifunctional molecules provided herein include a first moiety that is a PD-L1 binding moiety.

[0646] In some embodiments, the PD-L1 binding portions of the present disclosure bind to PD-L1 (e.g., human PD-L1 or cynomolgus monkey 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 portions of the present disclosure bind to cynomolgus monkey PD-L1.

[0647] In some embodiments, the PD-L1 binding portion of the present disclosure includes an anti-PD-L1 antibody portion. In some embodiments, exemplary anti-PD-L1 antibodies are disclosed in the Anti-PD-L1 Antibody Section and the Illustrative Anti-PD-L1 Antibody Section of the present disclosure.

[0648] In some embodiments, the anti-PD-L1 antibody portion includes one or more CDRs. In some embodiments, the anti-PD-L1 antibody portion includes one or more CDRs described in the illustrative anti-PD-L1 antibody section of the present disclosure. In some embodiments, the 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 the anti-PD-L1 antibody disclosed in the illustrative anti-PD-L1 antibody section of the present disclosure.

[0649] In some embodiments, the anti-PD-L1 antibody portion further includes a heavy chain constant domain appended to the carboxyl terminus of the heavy chain variable region. In some embodiments, the heavy chain constant region is derived from the group consisting of IgA, IgG, and IgM. In some embodiments, the heavy chain constant region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. In some embodiments, the anti-PD-L1 antibody portion further includes a light chain constant domain appended to the carboxyl terminus of the light chain variable region. In some embodiments, the light chain constant region is derived from a kappa light chain or a lambda light chain. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 80 or 81. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 82.

[0650] vi. Connection between the first and second parts

[0651] In the present disclosure, the second moiety can be linked to any portion of the first moiety. For example, a second moiety such as a TGFβ binding moiety or an IL-1 binding moiety can be linked to any suitable portion of a first moiety, such as a PD-L1 binding moiety (e.g., an anti-PD-L1 antibody moiety).

[0652] In some embodiments, the PD-L1 binding portion includes one or more polypeptide chains, such as antibody heavy and light chains.

[0653] In some embodiments, the bifunctional molecule comprises one or more second moieties in the second moiety. In some embodiments, at least one second moiety in the plurality of second moieties is connected to the amino terminus (N-terminus) or carboxyl terminus (C-terminus) of the polypeptide chain of the first moiety. In some embodiments, at least one second moiety in the plurality of second moieties is connected to the N-terminus or C-terminus of the heavy chain of the first moiety or to the N-terminus or C-terminus of the light chain of the first moiety.

[0654] In some embodiments, at least one of the plurality of second moieties is linked to the C-terminus of the heavy chain constant region of the first moiety. In some embodiments, each of the plurality of second moieties is linked to the C-terminus of each heavy chain constant region of the first moiety.

[0655] In some embodiments, the bifunctional molecule comprises at least two second portions in the second portion, each of the at least two second portions being connected to the C-terminus of each heavy chain of the first portion, or each of the at least two second portions being connected to the C-terminus of each light chain of the first portion. In some embodiments, the bifunctional molecule comprises at least two second portions in the second portion, each of the at least two second portions being connected to the N-terminus of each heavy chain of the first portion, or each of the at least two second portions being connected to the N-terminus of each light chain of the first portion.

[0656] In some embodiments, the bifunctional molecule includes more than one second portion in the second portion, and the more than one second portion is respectively connected to: the N-terminus of the heavy chain of the first 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. For example, the bifunctional molecule can include at least two second portions in the second portion, one of the at least two second portions is connected to the C-terminus of the heavy chain of the first portion, and the other second portion is connected to the C-terminus of the light chain of the first portion. For example, the bifunctional molecule can include at least two second portions in the second portion, one of the at least two second portions is connected to the N-terminus of the heavy chain of the first portion, and the other second portion is connected to the N-terminus of the light chain of the first portion.

[0657] In some embodiments, one or more TGFβ 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 are linked 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, 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 of the anti-PD-L1 antibody portion.

[0658] In some embodiments, the bifunctional molecule comprises a homodimeric heavy chain. In some embodiments, the bifunctional molecule comprises a heterodimeric heavy chain. The heavy chain is heterodimeric with respect to the presence or position of the second portion. In some embodiments, the heterodimeric heavy chain comprises one heavy chain having the second portion, while the other heavy chain does not have the second portion.

[0659] vii. Connectors

[0660] The second part can be linked to the first part directly or through a linker. Direct linkage can be a chemical linkage (such as a covalent bond).

[0661] In some embodiments, the bifunctional molecule further comprises a linker connecting the first portion and the second portion. As used herein, the term "linker" can be any suitable bifunctional moiety that is capable of reacting with at least two entities to be connected, thereby bonding the entities to form a single molecule or maintaining a sufficiently tight association of the entities. The linker can be incorporated into the resulting connected molecule or structure with or without its reactive functional group.

[0662] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0663] In some embodiments, the linker comprises a peptide linker. The peptide linker can be composed of amino acid residues linked together by peptide bonds. In some embodiments, the peptide linker can further include one or more non-natural amino acids. In some embodiments, the peptide linker comprises an amino acid sequence having at least 1, 2, 3, 4, 5, 8, 10, 15, 20, 30, 50 or more amino acid residues linked by peptide bonds and capable of connecting two or more polypeptides. The peptide linker may have or may not have a secondary structure.

[0664] 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 2:1121-1123 (1994). In some embodiments, the peptide linker may include or be composed of an amino acid residue selected from the following: glycine, serine, alanine, methionine, asparagine, and glutamine. In some embodiments, the peptide linker may be composed of a majority of sterically unhindered amino acids such as glycine and alanine. In some embodiments, the linker is a combination of polyglycine, polyalanine, glycine, and alanine (such as poly (Gly-Ala)) or a combination of glycine and serine (such as poly (Gly-Ser)).

[0665] In some embodiments, the linker comprises an amino acid sequence of ((G)nS)m, wherein m and n are independently an integer selected from the group consisting of 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. In some embodiments, m is 4 and n is 4.

[0666] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the linker comprises an amino acid sequence having 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 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 68.

[0667] viii. Anti-PD-L1 Antibodies and Antigen-Binding Fragments Thereof

[0668] In some embodiments, the PD-L1 binding portion of the bifunctional molecules provided herein includes a portion comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody and antigen-binding fragment thereof are capable of specifically binding to PD-L1.

[0669] In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein have a K of 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 0.4 nM as measured by a Biacore assay. DThe Biacore assay is based on surface plasmon resonance technology, see, for example, Murphy, M. et al., Current protocols in protein science, Chapter 19, Unit 19.14, 2006. In some embodiments, K D The values ​​were measured by the method as described in Example 6 of the present disclosure.

[0670] The binding of the antibodies or antigen-binding fragments thereof provided herein to human PD-L1 can also be expressed as a "half maximal effective concentration" (EC 50 ) value, which refers to the concentration of the antibody at which 50% of its maximum binding is observed. 50 The value can be measured by binding assays known in the art, such as direct or indirect binding assays, such as enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS) assays, and other binding assays. In some embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC value of no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM, or no more than 0.09 nM as measured by ELISA. 50 In some embodiments, the antibodies and antigen-binding fragments thereof provided herein bind specifically to PD-L1 at an EC no greater than 1.4 nM, no greater than 1.3 nM, no greater than 1.2 nM, no greater than 1.1 nM, no greater than 1.0 nM, no greater than 0.3 nM, no greater than 0.25 nM, or no greater than 0.21 nM as measured by a FACS assay. 50 (i.e., 50% binding concentration) specifically binds to PD-L1.

[0671] In some embodiments, the 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.

[0672] In some embodiments, the 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) with an IC50 of no more than 2.2, 2.1, 2.0, 1.9, 1.8, or 1.2 ug / ml as measured by ELISA.

[0673] In some embodiments, the 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) with an EC50 of no more than 1.3, 1.2, 1.1, 1.0, 0.9, or 0.8 nM as measured by a cell-based assay.

[0674] ix. Illustrative Anti-PD-L1 Antibodies and Antigen-Binding Fragments Thereof

[0675] In some embodiments, the anti-PD-L1 antibodies (i.e., antibodies to PD-L1) and antigen-binding fragments thereof of the present disclosure include one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs comprising a sequence selected from the group consisting of: 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), wherein X1 is G or A, X2 is G or D or Q or E or L, X3 is S or M or Q or L or V, X4 is G or P or K, X5 is A or G, X6 is A or I, X7 is A or N or R or V, and X8 is N or H or V or D.

[0676] In some embodiments, the heavy chain variable region comprises:

[0677] a) HCDR1 comprises the sequence of SEQ ID NO: 1,

[0678] b) HCDR2 comprises 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

[0679] c) HCDR3 comprises the sequence of SEQ ID NO: 3,

[0680] and / or

[0681] A light chain variable region, comprising:

[0682] d) LCDR1 comprises 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,

[0683] e) LCDR2 comprises 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

[0684] f) LCDR3 comprises the sequence of SEQ ID NO: 6.

[0685] In some embodiments, the heavy chain variable region is selected from the group consisting of:

[0686] g) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0687] h) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 13, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0688] i) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 14, and a HCDR3 comprising the sequence of SEQ ID NO: 3;

[0689] j) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 15, and a HCDR3 comprising the sequence of SEQ ID NO: 3; and

[0690] k) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 17, and a HCDR3 comprising the sequence of SEQ ID NO: 3; and

[0691] 1) a heavy chain variable region comprising: a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 18, and a HCDR3 comprising the sequence of SEQ ID NO: 3.

[0692] In some embodiments, the light chain variable region is selected from the group consisting of:

[0693] a) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 4, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0694] b) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 9, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0695] c) a light chain variable region comprising: a LCDR1 comprising the sequence of SEQ ID NO: 8, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;

[0696] d) 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

[0697] e) 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.

[0698] As used herein, antibody "4B6" refers to a monoclonal antibody comprising 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.

[0699] In some embodiments, the present disclosure provides anti-PD-L1 antibodies and antigen-binding fragments thereof comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of antibody 4B6 or a variant of antibody 4B6. CDR boundaries are defined or identified by Kabat's rules.

[0700] In some embodiments, the present disclosure provides anti-PD-L1 antibodies and antigen-binding fragments thereof, comprising: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 13, 14, 15, 17, and 18; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and / or a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 7, 8-9; a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 11-12; and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0701] Table 1. CDR amino acid sequences of antibody 4B6.

[0702] Table 2. Variable region amino acid sequences of antibody 4B6.

[0703] It is known that CDRs are responsible for antigen binding. However, it has been found that not all six CDRs are indispensable or unchangeable. In other words, one or more CDRs in the anti-PD-L1 antibody 4B6 can be replaced, changed, or modified while still substantially retaining specific binding affinity for PD-L1.

[0704] In some embodiments, the antibodies and antigen-binding fragments thereof provided herein include suitable framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can specifically bind to PD-L1. The CDR sequences provided in Table 1 above are obtained from mouse antibodies, but the CDR sequences can be transplanted to any suitable FR sequence of any suitable species such as mouse, human, rat, rabbit, etc. using suitable methods known in the art, such as recombinant technology.

[0705] In some embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or their antigen-binding fragments are desirable in terms of their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are transplanted to human or substantially human FR sequences. The humanization of antibodies or antigen-binding fragments can be carried out by replacing the corresponding human CDR genes in human immunoglobulin genes with non-human (such as mouse) CDR genes (see, for example, Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536).

[0706] Suitable human heavy chain and light chain variable domains can be selected using methods known in the art to achieve this purpose.In an illustrative example, "best fit" method can be used, wherein for the database screening of known human variable domain sequences or BLAST non-human (for example, rodent) antibody variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as the people's support for transplanting non-human CDR sequences (see, for example, Sims et al., (1993) "Journal of Immunology" 151:2296; Chothia et al. (1987) "Journal of Molecular Biology (J.Mot.Biol.)" 196:901). Alternatively, The framework derived from the consensus sequence of all human antibodies can be used for transplanting non-human CDR (see, for example, Carter et al. (1992) "Proceedings of the National Academy of Sciences of the United States", 89:4285; Presta et al. (1993) "Journal of Immunology", 151:2623).

[0707] In some embodiments, the present disclosure provides 12 humanized antibodies of 4B6, which are respectively designated as 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. The SEQ ID NOs of the heavy and light chain variable regions of each humanized antibody of 4B6 are shown in Table 5. The CDRs of each of the 12 humanized antibodies of 4B6 are shown in Table 5 (underlined sequences). CDR boundaries are defined or identified by the Kabat rules.

[0708] Table 3a below shows the amino acid sequences of variant CDRs of humanized 4B6, and Table 3b below shows the FRs of the humanized 4B6 heavy and light chain variable regions. Table 4 below shows the FR amino acid sequences of each heavy and light chain of 12 humanized antibodies of chimeric antibody 4B6, which are designated as 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, respectively. The heavy chain variable regions and light chain variable regions of these 12 humanized antibodies are shown in Table 5.

[0709] Table 3a. Amino acid sequences of CDR variants of the humanized antibody 4B6.

[0710] Table 3b. Amino acid sequences of FR sequences of 4B6 and humanized antibodies of 4B6.

[0711] Table 4. FR amino acid sequences of each humanized heavy and light chain variable region of the humanized antibody 4B6.

[0712] Table 5 below shows three variants of the humanized 4B6 heavy chain variable region (i.e., Hu4B6_Hg.2, Hu4B6_Hg.3, and Hu4B6_Hg.5) and four variants of the humanized 4B6 light chain variable region (i.e., AM4B6_La.1, AM4B6_La.2, AM4B6_La.4, AM4B6_La.6).

[0713] Table 5. Amino acid sequences of the variable regions of the humanized antibody 4B6.

[0714] In some embodiments, in addition to non-human CDR sequences, the humanized anti-PD-L1 antibodies or their antigen-binding fragments provided herein are composed of substantially all human sequences. In some embodiments, the variable region FR and constant region (if present) are completely or substantially derived from human immunoglobulin sequences. Human FR sequences and human constant region sequences can be derived from different human immunoglobulin genes, for example, FR sequences are derived from one human antibody and constant regions are derived from another human antibody. In some embodiments, the humanized antibody or its antigen-binding fragment includes human heavy chain HFR1-4 and / or light chain LFR1-4.

[0715] In some embodiments, the FR district derived from people can include the same amino acid sequence of the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of people FR are replaced by corresponding residues from parent non-human antibody. In some embodiments, this may be desirable, so that humanized antibody or its fragment is very close to non-human parent antibody structure, so as to optimize binding properties (for example, increase binding affinity). In some embodiments, humanized antibody provided herein or its Fab comprises no more than 10,9,8,7,6,5,4,3,2 or 1 amino acid residue replacements in each FR sequence in multiple people FR sequences, or comprises no more than 10,9,8,7,6,5,4,3,2 or 1 amino acid residue replacements in all FR sequences of heavy chain or light chain variable domains. In some embodiments, this type of change of amino acid residue can be only present in heavy chain FR district, only present in light chain FR district or present in two chains. In some embodiments, one or more amino acid random mutations of people FR sequence are made 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.

[0716] In some embodiments, the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure comprise: a heavy chain HFR1 comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYX9FT (SEQ ID NO: 40) or a homologous sequence having at least 80% sequence identity thereto; a heavy chain HFR2 comprising WVRQAPGQX 10 LEWMG (SEQ ID NO: 41) or a homologous sequence having at least 80% sequence identity thereto; a heavy chain HFR3 comprising RVTX 16 TVDX 11 SISTAYMELSRLRSDDTAVYYCX 12 X 13 (SEQ ID NO: 42) or a homologous sequence having at least 80% sequence identity thereto; and a heavy chain HFR4 comprising the sequence of WGQGTLVTVSS (SEQ ID NO: 25) or a homologous sequence having at least 80% sequence identity thereto, wherein X9 is T or V, X 10 Is G or S, X 11 Is T or K, X 12 is A or V, and X 13 It is R or K.

[0717] In some embodiments, the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include: a light chain LFR1 comprising the sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 26) or a homologous sequence having at least 80% sequence identity thereto; a light chain LFR2 comprising WYQQKPGKX 14 PKLLIY (SEQ ID NO: 43) or a homologous sequence having at least 80% sequence identity thereto; a light chain LFR3 comprising GVPX 15 RFSGSGSGTDFTX 17 TISSLQPEDIATYYC (SEQ ID NO: 44) or a homologous sequence having at least 80% sequence identity thereto; and a light chain LFR4 comprising the sequence of FGQGTKLEIK (SEQ ID NO: 29) or a homologous sequence having at least 80% sequence identity thereto, wherein X 14 Is A or S, X 15 Is S or D, X 16 is M or V, and X 17It is F or L.

[0718] In some embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 22 and 30, HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 23 and 31, HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 24 and 32-35, HFR4 comprises a sequence of SEQ ID NO: 25, LFR1 comprises a sequence selected from the group consisting of SEQ ID NO: 26, LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 27 and 36, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 28 and 37-38, 39, 45, and LFR4 comprises a sequence of SEQ ID NO: 29.

[0719] In some embodiments, the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include HFR1, HFR2, HFR3 and / or HFR4 sequences contained in the heavy chain variable region selected 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).

[0720] In some embodiments, the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof of the present disclosure include LFR1, LFR2, LFR3 and / or LFR4 sequences contained in the light chain variable region selected 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).

[0721] In some embodiments, 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 homologous sequences thereof having at least 80% sequence identity thereto. In some embodiments, 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 thereto. In some embodiments, the antibody or antigen-binding fragment thereof against PD-L1 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.

[0722] These exemplary humanized anti-PD-L1 antibodies retain specific binding ability or affinity for PD-L1 and are superior to the parental mouse antibody 4B6 in this regard.

[0723] In some embodiments, 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. In one embodiment, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are single domain antibodies consisting of all or a portion of the heavy chain variable domain provided herein. More information on such single domain antibodies is available in the art (see, e.g., U.S. Patent No. 6,248,516).

[0724] In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein further include an immunoglobulin (Ig) constant region, which optionally further includes a heavy chain and / or light chain constant region. In some embodiments, the heavy chain constant region includes CH1, a hinge and / or a CH2-CH3 region (or an optional CH2-CH3-CH4 region). In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein include a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In some embodiments, the light chain constant region includes Cκ or Cλ. The constant region of the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein may be identical to the wild-type constant region sequence or different in one or more mutations.

[0725] In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein have specific binding affinity for human PD-L1 that is sufficient to provide diagnostic and / or therapeutic use.

[0726] 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 in vitro using recombinant methods rather than in animals.

[0727] In some embodiments, the PD-L1 binding portion comprises an anti-PD-L1 antibody or antigen-binding fragment thereof that competes for PD-L1 binding with an antibody or antigen-binding fragment thereof comprising 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.

[0728] x. Antibody variants

[0729] The anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0730] In some embodiments, the antibody variants include one or more modifications or substitutions in one or more of the multiple CDR sequences provided in Table 1 above, in one or more of the multiple non-CDR sequences of the heavy chain variable region or light chain variable region provided in Tables 3a, 3b, and 5 above, and / or in a constant region (e.g., Fc region). Such variants retain the binding specificity of their parent antibody to PD-L1, but have one or more desired properties conferred by the modification or substitution. For example, the antibody variants 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 (e.g., one or more introduced cysteine ​​residues).

[0731] Methods known in the art, such as "alanine scanning mutagenesis", can be used to screen the parent antibody sequence to identify suitable or preferred residues to be modified or replaced (see, for example, 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 modified antibodies are generated and screened for properties of interest. If the substitution at a specific amino acid position shows a functional change of interest, the position can be identified as a potential residue for modification or replacement. The potential residue can be further evaluated by replacing it with another residue (e.g., a cysteine ​​residue, a positively charged residue, etc.).

[0732] xi. Affinity variants

[0733] The affinity variants of the antibodies may contain modifications or substitutions in one or more CDR sequences provided in Table 1 above, one or more FR sequences provided in Tables 3b and 4 above, or the heavy chain or light chain variable region sequences provided in Table 5 above. Those 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, as it is well known in the art that in the variable region, the CDR region is flanked by two FR regions. The affinity variants retain the specific binding affinity of the parent antibody for PD-L1, or even have a higher specific binding affinity for PD-L1 than the parent antibody. In some embodiments, at least one (or all) substitutions in the CDR sequence, FR sequence, or variable region sequence include conservative substitutions.

[0734] Those skilled in the art will understand that in the CDR sequences provided in Tables 1 and 3a above and the variable region sequences provided in Table 5 above, one or more amino acid residues may be substituted, but the resulting antibody or antigen-binding fragment still retains binding affinity or binding ability to PD-L1 or even has improved binding affinity or binding ability. Various methods known in the art can be used to achieve this. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology, and then screened for affinity for binding to PD-L1. For another example, computer software can be used to virtually simulate the binding of antibodies to PD-L1 and identify amino acid residues on the antibody that form the binding interface. Such residues can be avoided in substitution to prevent a decrease in binding affinity, or can be targeted for substitution to obtain stronger binding.

[0735] In some embodiments, the humanized anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences in a plurality of CDR sequences and / or one or more FR sequences in a plurality of FR sequences. In some embodiments, the affinity variants comprise no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a total of CDR sequences and / or FR sequences.

[0736] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof includes 1, 2, or 3 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to one (or more) of the sequences listed in Tables 1 and 3a above, but still retain specific binding affinity for PD-L1 at a similar or even higher level relative to its parent antibody.

[0737] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to one (or more) of the sequences listed in Table 5 above, but still retaining specific binding affinity for PD-L1 at a similar or even higher level relative to its parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Table 5 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., in the FRs).

[0738] xii. Glycosylation variants

[0739] The anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the extent of glycosylation of the antibody or antigen-binding fragment thereof.

[0740] Anti-PD-L1 antibodies or antigen-binding fragments thereof may include one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) may be attached. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine), where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Natural glycosylation sites can be conveniently removed, for example, by altering the amino acid sequence such that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can be generated in a similar manner by introducing such a tripeptide sequence or a serine or threonine residue.

[0741] In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments provided herein include one or more mutations to remove one or more deamidation sites. In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments provided herein include a mutation at G55 (e.g., G55A) in the heavy chain. These mutations have been tested and are believed not to negatively affect the binding affinity of the antibodies provided herein.

[0742] xiii. Cysteine-engineered variants

[0743] The anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein also encompass cysteine ​​engineered variants comprising one or more introduced free cysteine ​​amino acid residues.

[0744] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bond. Cysteine ​​engineered variants can be used to conjugate, for example, cytotoxic compounds and / or imaging compounds, labels, or radioisotopes at the site of the engineered cysteine ​​via, for example, maleimide or haloacetyl groups. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0745] xiv. Fc variants

[0746] The anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein also encompass Fc variants comprising 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 for altering ADCC activity by 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., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., PNAS, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards J. et al., J Immunol. 2008, 6:3010-3019; JO, et al., Mol Cancer Therapeutics 2008, 7(8):2517-27; Shields RL et al., J Biol Chem, 2002, 277:26733-26740; Shinkawa T. et al., J Biol Chem, 2003, 278:3466-3473.

[0747] 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., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821); and WO 94 / 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 a different amino acid residue to alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see U.S. Patent No. 6,194,551 to Idusogie et al.). One or more amino acid substitutions can also be introduced to alter the ability of an antibody to fix complement (see PCT Publication WO 94 / 29351 to Bodmer et al.).

[0748] In some embodiments, the Fc variants provided herein have reduced effector function relative to wild-type Fc (e.g., Fc of IgG1) and include one or more amino acid substitutions at positions 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, and 332 of the Fc region (see WO2016 / 196228; Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010) mAbs 2:181; and Strohl (2009) Current Opinion in Biotechnology. Biotechnology) 20: 685-691), wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. Exemplary 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 WO 2016 / 196228; and Strohl (2009) Biotechnol Current Review 20:685-691).

[0749] In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein have reduced effector function and comprise 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. In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and comprise 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.

[0750] In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of IgG1 isotype and include L234A and L235A mutations. In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of IgG1 isotype and include L234F, L235E and P331S. The L234F, L235E and P331S set of substitutions located in the CH2 region of the Fc domain (also known as the FES triple mutation) can eliminate FCγR and C1q binding, thereby rendering the antibody unable to induce ADCC or CDC (Oganesyan et al., 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 a reduced thermal stability of the Fc domain compared to a wild-type parent molecule (e.g., a wild-type IgG1 Fc). In some embodiments, the Fc variant comprises the amino acid sequence of SEQ ID NO:81.

[0751] In some embodiments, 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 (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In some embodiments, 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. In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are of the IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256–1264 (2007).

[0752] In some embodiments, the 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). Such variants can have a prolonged pharmacokinetic half-life because they bind to FcRn at acidic pH, thereby protecting them from degradation in lysosomes and subsequently being transported 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. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering the Fc Region to Improve PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0753] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions in the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into the first Fc polypeptide and introducing a cavity into the second Fc polypeptide, wherein the protrusion can be positioned in the cavity to promote the interaction of the first Fc polypeptide with the second Fc polypeptide to form a heterodimer or complex. Methods for producing antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0754] xv. Antigen-binding fragments

[0755] The PD-L1 binding portion of the bifunctional molecules provided herein also encompasses anti-PD-L1 antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-PD-L1 antibodies provided herein and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.), including, for example, the exemplary antibodies whose CDRs are shown in Tables 1 and 3a above and whose variable sequences are shown in Tables 2 and 5.

[0756] In some embodiments, the 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-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

[0757] A variety of techniques can be used to produce such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science 229: 81 (1985)), by recombinant expression (for example, for Fab, Fv and ScFv antibody fragments) carried out by host cells such as Escherichia coli (E.Coli), screening (for example, for ScFv) from phage display libraries as discussed above, and chemical coupling of two Fab'-SH fragments to form F (ab') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0758] In some embodiments, the antigen-binding fragment is a scFv. The production of scFvs is described in, for example, WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. ScFvs can be fused to effector proteins at the amino or carboxyl termini to provide fusion proteins (see, for example, Antibody Engineering, ed. Borrebaeck).

[0759] In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0760] If both binding sites specifically bind to the same antigen or the same epitope, the bivalent molecule can be monospecific. In some embodiments, this provides stronger binding to the antigen or epitope than a monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In some embodiments, in a bivalent or multivalent antigen binding portion, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences but have the same specificity).

[0761] A bivalent molecule 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 the third binding site is specific for a second antigen (or epitope).

[0762] xvi. Bispecific Antibodies

[0763] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is bispecific. In some embodiments, in addition to the second moiety provided herein, the PD-L1 binding antibody or antigen-binding fragment thereof is further linked to an additional functional domain having a different binding specificity than the anti-PD-L1 antibody or antigen-binding fragment thereof.

[0764] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second antigen other than PD-L1 (and other than the target to which the second moiety binds) or a second epitope on PD-L1 (or a second epitope on the target to which the second moiety binds).

[0765] xvii. Bifunctional molecules

[0766] In some embodiments, the bifunctional molecules provided herein are capable of binding to PD-L1 and a target bound by a second moiety. In some embodiments, the bifunctional molecules provided herein are capable of binding to both PD-L1 and TGFβ, or to both PD-L1 and IL-1, or to both PD-L1 and IL-1R, or to both PD-L1 and MHCII, or to both PD-L1 and CD47, or to both PD-L1 and SIRPα.

[0767] In some embodiments, the bifunctional molecules targeting PD-L1 and TGFβ of the present disclosure have an EC no greater than 2.0 nM (e.g., no greater than 2.0 nM, no greater than 1.2 nM, no greater than 1.1 nM, no greater than 1.0 nM, no greater than 0.9 nM, no greater than 0.8 nM) as measured by an ELISA assay. 50Specifically binds to human TGFβ1. In some embodiments, the proteins targeting PD-L1 and TGFβ of the present disclosure are capable of binding to PD-L1 and TGFβ simultaneously as measured by ELISA assay. In some embodiments, the bifunctional molecules targeting PD-L1 and TGFβ of the present disclosure are capable of binding to PD-L1 and TGFβ simultaneously as measured by Biacore assay with a K of 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 0.4 nM. D In some embodiments, the bifunctional molecules targeting PD-L1 and TGFβ of the present disclosure are capable of specifically binding to human PD-L1 with a K of 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, no more than 1.0 nM, no more than 0.9 nM, no more than 0.8 nM) as measured by ELISA assay. D The value specifically binds to human TGFβ1.

[0768] In some embodiments, the bifunctional molecules targeting PD-L1 and TGFβ of the present disclosure can exhibit synergistic effects on tumor growth inhibition in a dose-dependent manner.

[0769] In some embodiments, the bifunctional molecules of the present disclosure targeting PD-L1 and TGFβ can exhibit enhanced infiltration of anti-tumor immune cells into the tumor microenvironment compared to molecules comprising only immune checkpoint molecules.

[0770] In some embodiments, the bifunctional molecules targeting PD-L1 and TGFβ of the present disclosure are capable of selectively reducing TGFβ1 in plasma by at least 90% (e.g., at least 80%, 70%, 60%, 50%, 40%, 30% or 20%) and such reduction can be maintained for at least 10, 14 or 21 days.

[0771] In some embodiments, the bifunctional molecule includes a heterodimeric heavy chain. The heavy chain is heterodimeric with respect to the presence or position of the second portion. In some embodiments, the heterodimeric heavy chain includes a heavy chain having the second portion, while the other heavy chain does not have the second portion, wherein the second portion includes a CD47 binding domain (e.g., soluble SIRPα) or a SIRPα binding domain.

[0772] In the bifunctional molecule, the heterodimeric heavy chain includes a heavy chain having the second portion, while the other heavy chain does not have the second portion. The heterodimeric heavy chain may further include a heterodimeric Fc region that associates in a manner that hinders homodimerization and / or facilitates heterodimerization. For example, the heterodimeric Fc regions may be selected so that they are different and they preferentially form heterodimers between each other rather than forming homodimers within themselves. In some embodiments, the heterodimeric Fc region is capable of associating into heterodimers by forming a knob-in-hole structure, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility. In some embodiments, the heterodimeric Fc region includes a CH2 and / or CH3 domain that is mutated to form a knob-in-hole structure. The knob structure can be obtained by replacing a small amino acid residue in the first CH2 / CH3 polypeptide with a larger amino acid residue, and the hole structure can be obtained by replacing a larger residue with a smaller residue. In some embodiments, the heterodimeric Fc region comprises a first CH3 domain of an IgG1 isotype comprising S354C and T366W substitutions (SEQ ID NO: 96, knob structure) and a second CH3 domain of an IgG1 isotype comprising Y349C, T366S, L368A, and Y407V substitutions (SEQ ID NO: 97, hole structure).

[0773] In some embodiments, the bifunctional molecule comprises a heavy chain comprising 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.

[0774] xviii. Conjugates

[0775] In some embodiments, the bifunctional molecule further includes one or more conjugate moieties. The conjugate moieties can be connected to the bifunctional molecule. The conjugate moieties are parts that can be connected to the bifunctional molecule. It is envisioned that a variety of conjugate moieties can be connected to the bifunctional molecule provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be connected to the bifunctional molecule by methods such as covalent bonding, affinity bonding, embedding, coordination bonding, complexing, association, blending or adding. In some embodiments, the bifunctional molecule can be connected to one or more conjugates by a joint.

[0776] In some embodiments, the 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. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate moiety.

[0777] In some embodiments, the bifunctional molecule can be connected to the conjugate portion indirectly or through another conjugate portion. For example, the bifunctional molecule provided herein can be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate portion includes a scavenging modifier (e.g., a polymer that extends half-life, such as PEG), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent, a purification moiety or other anticancer drugs.

[0778] A "toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, and d-actinomycin. D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C ( C) and dichlorodiamine platinum (II) (DDP cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotics (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0779] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-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 lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0780] In some embodiments, 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, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol, and the like. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules.

[0781] In some embodiments, the conjugate moiety can be a purification moiety, such as a magnetic bead.

[0782] In some embodiments, the bifunctional molecules provided herein serve as the basis for conjugates.

[0783] III. Pharmaceutical Preparations

[0784] The preparation of the present disclosure may also include other pharmaceutically acceptable excipients, such as solvents (aqueous or non-aqueous), osmotic pressure regulators, excipients that reduce viscosity, preservatives, wetting agents, emulsifiers, and dispersants. Sterilization procedures and the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) can be used to ensure that no microorganisms are present. 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, metacresol, parachlorometracresol, methylparaben or propylparaben, and benzalkonium chloride.

[0785] The formulations disclosed herein exhibit excellent stability and ease of administration. In some embodiments of the present disclosure, bifunctional molecules, particularly PD-L1 / TGF-β proteins, can maintain long-term stability, meeting clinical application requirements. Furthermore, the solutions provided herein address the issue of polysorbate 80 degradation, preventing the potential impact of polysorbate 80 degradation products on protein quality.

[0786] In some embodiments, the formulation of the present disclosure is a liquid formulation. For example, the liquid formulation is an injection. For example, the liquid formulation comprises water for injection.

[0787] In some embodiments, the purity of the bifunctional protein molecule in a formulation, e.g., a liquid formulation of the present disclosure, decreases by no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%, after storage at about -80°C to about 45°C, e.g., -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 37°C, about 42°C, or about 45°C for at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, as determined by SEC.

[0788] In one embodiment, the purity of the bifunctional protein molecule of a formulation, e.g., a liquid formulation of the present disclosure, as measured by non-reducing CE-SDS, decreases by no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%, after storage at about -80°C to about 45°C, e.g., -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 37°C, about 42°C, or about 45°C for at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer.

[0789] IV. Methods of Treatment and Use

[0790] The present disclosure provides a method for treating, preventing, or ameliorating a PD-L1-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of a formulation provided herein.

[0791] In some embodiments, the subject is a human.

[0792] PD-1-associated conditions and disorders may be immune-related diseases or disorders, cancer, autoimmune diseases, or infectious diseases.

[0793] In some embodiments, 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, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma, Cancer) and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) neoplasms, such as primary CNS lymphoma, spinal cord axis tumors, brainstem gliomas. In some embodiments, the tumors and cancers are metastatic, especially metastatic tumors expressing PD-L1.

[0794] In some embodiments, PD-1-associated conditions and disorders include autoimmune diseases. Autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, sprue sprue-dermatitis herpetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, disease), Degos' disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, severe anemia anemia), polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, essential agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff-person syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0795] In some embodiments, PD-1 related conditions and disorders include infectious diseases. Infectious diseases include, for example, chronic viral infections, for example, fungal infections, parasitic / protozoal infections, or chronic viral infections, for example, malaria, coccidioidomycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candidiasis, paracoccidioidomycosis, microsporidiosis, Acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiasis, Baylisascariasis, Chagas disease, disease), clonorchiasis, Cochliomyiasis, cryptosporidiosis, diphyllobothrium, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenolepis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, metagonomiasis, myiasis, onchocerciasis, lice, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocaraosis, toxoplasmosis, trichinosis, whipworm disease, trypanosomiasis, helminth infections, hepatitis B (HBV) infection, hepatitis C (HCV) infection, herpes virus infection, Epstein-Barr virus virus infection, HIV-1 infection, HIV-2 infection, cytomegalovirus infection, herpes simplex virus type 1 infection, herpes simplex virus type 2 infection, human papillomavirus infection, adenovirus infection, Kaposi's sarcoma-associated herpesvirus epidemic infection, parvovirus (Torquetenovirus) infection, human T-lymphotropic virus I infection, human T-lymphotropic virus II infection, varicella-zoster virus infection, JC virus infection, or BK virus infection.

[0796] In some embodiments, the PD-L1-associated disease is a PD-L1-expressing cancer or a PD-L1-overexpressing cancer. A "PD-L1-expressing cancer" is a cancer involving cancer cells or tumor cells that have the PD-L1 protein on their cell surface. A "PD-L1-overexpressing cancer" is a cancer that has significantly higher levels of PD-L1 on the cell surface of cancer or tumor cells compared to non-cancerous cells of the same tissue type.

[0797] PD-L1 expression or overexpression can be determined by assessing the increased level of PD-L1 present on the cell surface in a diagnostic or prognostic assay (e.g., by immunohistochemistry; IHC). Alternatively or additionally, the level of nucleic acid encoding PD-L1 in the cell can be measured, for example, by fluorescence in situ hybridization (FISH; see WO98 / 45479 published in October 1998), southern blot, or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). PD-L1 overexpression can also be studied by measuring shed antigen (e.g., PD-L1 extracellular domain or soluble PD-L1) in biological fluids such as serum. In addition to the above assays, skilled practitioners can also obtain various in vivo assays. For example, cells in a patient's body can be exposed to an anti-PD-L1 antibody, which is optionally labeled with a detectable label (e.g., a radioisotope), and the binding of the antibody to the patient's cells can be assessed, for example, by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.

[0798] In some embodiments, the subject has been identified as being likely to respond to a PD-1 antagonist. The presence or level of PD-L1 on the biological sample of interest may indicate whether the subject from which the biological sample is derived is likely to respond to a PD-1 antagonist. In some embodiments, the test sample is derived from cancer cells or tissues, or tumor-infiltrating immune cells. In some embodiments, the presence or upregulated level of PD-L1 in the test biological sample indicates the possibility of a response. As used herein, the term "upregulated" refers to an overall increase in the protein level of PD-L1 in the test sample by no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or greater compared to the PD-L1 protein level in a reference sample detected using the same antibody. 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 is obtained. For example, a reference sample can be a non-diseased sample adjacent to or near a test sample (eg, a tumor).

[0799] In some embodiments, a PD-L1-associated disease is resistant to PD-L1 / PD-1 monotherapy. As used herein, "PD-L1 / PD-1 monotherapy" refers to a monotherapy that works by inhibiting or reducing the interaction or signaling between PD-L1 and PD-1. Exemplary PD-L1 / PD-1 monotherapy may include anti-PD-L1 antibody therapy, anti-PD-1 antibody therapy, or a monotherapy involving a small molecule inhibitor directed against PD-1 or PD-L1. "Resistance" means that the disease is unresponsive or sensitive to PD-L1 / PD-1 monotherapy or has a reduced response or sensitivity. A reduced response can be indicated, for example, by the need to increase the dose to achieve a given efficacy. In some embodiments, the disease may be unresponsive to PD-L1 / PD-1 monotherapy. For example, despite treatment with PD-L1 / PD-1 monotherapy, cancer cells or tumor size may increase, or the disease may show a regression to its previous state, for example, a return of previous symptoms after partial recovery. Resistance to PD-L1 / PD-1 monotherapy may be de novo or acquired.

[0800] In another aspect, the present disclosure provides a method for treating, preventing, or ameliorating 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 stimulation of anti-tumor immunity, comprising administering an effective amount of a formulation provided herein.

[0801] In some embodiments, the immunosuppressive cytokine is TGFβ or IL- 1. In some embodiments, the immunosuppressive cytokine is TGFβ1 or IL-1β.

[0802] In some embodiments, the disease or condition is a TGFβ-associated disease or condition. In some embodiments, the TGFβ-associated disease is cancer, a fibrotic disease, or a kidney disease.

[0803] In some embodiments, the TGFβ-related disease is cancer. In some embodiments, 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 skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

[0804] In some embodiments, the TGFβ-related disease is a fibrotic disease. A fibrotic disease is a disease or condition involving fibrosis. Fibrosis is a scarring process that is a common feature of chronic organ (e.g., lung, liver, kidney, skin, heart, intestine, or muscle) damage. Fibrosis is characterized by elevated transforming growth factor-β (TGF-β) activity, which leads to increased deposition and alterations in the extracellular matrix and other fibrosis-related proteins.

[0805] Fibrotic diseases can include fibrotic diseases of the lungs, liver, kidneys, eyes, skin, heart, intestines or muscles. Examples of fibrotic diseases of the lungs include pulmonary fibrosis, cystic fibrosis, pulmonary hypertension, progressive massive fibrosis, bronchiolitis obliterans, and airway remodeling associated with chronic asthma or idiopathic lung disease. Examples of fibrotic diseases of the liver include cirrhosis or nonalcoholic steatohepatitis. Examples of fibrotic diseases of the kidneys include renal fibrosis, ischemic renal injury, tubulointerstitial fibrosis, diabetic nephropathy, nephrosclerosis or nephrotoxicity. Examples of fibrotic diseases of the eyes include corneal fibrosis and subretinal fibrosis. Examples of fibrotic diseases of the skin include nephrogenic systemic fibrosis, keloids or scleroderma. Examples of fibrotic diseases of the heart include endomyocardial fibrosis or old myocardial infarction.

[0806] In some embodiments, the disease or condition is an IL-1 related disease or condition. In some embodiments, the IL-1 related disease is an autoinflammatory disease, metabolic syndrome, acute inflammation, chronic inflammation, or malignancy.

[0807] In some embodiments, the disease or condition would benefit from inducing a sustained immune response by stimulating MHCII signaling with an immunostimulatory polypeptide (e.g., soluble LAG-3). In some embodiments, the disease or condition is cancer, a viral infection, a parasitic infection, or a combination thereof.

[0808] In some embodiments, the disease or condition would benefit from stimulating anti-tumor immunity by inhibiting immunoinhibitory receptor signaling. In some embodiments, the immunoinhibitory receptor is SIRPα. In some embodiments, the disease, disorder, or condition is associated with SIRPα, such as cancer, solid tumors, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction, or arthritis. In some embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal 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 cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T or B cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma and adenocarcinoma. In some embodiments, the cancer is a cancer that expresses CD47 or a cancer that overexpresses CD47.

[0809] The therapeutically effective amount of the bifunctional molecules provided herein will depend on various factors known in the art, for example, weight, age, past medical history, current medications, the subject's health status and potential for cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease progression.

[0810] In some embodiments, the bifunctional molecules provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg.

[0811] The formulations provided herein can be administered by routes known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0812] In some embodiments, the bifunctional molecules provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the bifunctional molecules disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent, an anticancer drug, radiotherapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy, a cell therapy, a gene therapy, a hormone therapy, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine.

[0813] As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or generally enhances the immune system. Examples of immunotherapy include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0814] "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 target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapy targets the therapeutic agent to the tumor, sparing normal tissues from the therapeutic's effects.

[0815] In some embodiments, the bifunctional molecules provided herein that are administered in combination with one or more additional therapeutic agents can be administered simultaneously with the one or more additional therapeutic agents, and in some of these embodiments, the bifunctional molecules and the additional therapeutic agents can be administered as part of the same pharmaceutical composition. However, a bifunctional molecule that is administered "in combination" with another therapeutic agent need not be administered simultaneously with 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 the agent, as the phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered by different routes.

[0816] In another aspect, the present disclosure also provides use of the bifunctional molecules provided herein and / or the pharmaceutical compositions and pharmaceutical formulations provided herein in the manufacture of a medicament for treating a PD-L1-related disease and / or a TGF-β-related disease and / or an IL-1-related disease and / or a CD47-related disease in a subject.

[0817] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the present disclosure. All specific formulations or preparations, materials and methods described below fall within the scope of the present disclosure in whole or in part. These specific formulations or preparations, materials and methods are not intended to limit the present disclosure, but are only used to illustrate specific embodiments that fall within the scope of the present disclosure. Those skilled in the art can develop equivalent compositions, materials and methods without the need for creative work and without departing from the scope of the present disclosure. It will be understood that many changes can be made in the scheme described herein, and such changes are still included within the scope of the present disclosure. Example

[0818] Example A Preparation, Characterization and Activity of Antibodies and Bifunctional Proteins

[0819] Example 1: Production, expression and purification of humanized 4B6 antibody

[0820] The anti-PD-L1 mAb 4B6, derived from patent WO 2017161976A1 and comprising 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 by a mouse hybridoma, thus requiring appropriate humanization. The sequence of the variable domain of the mouse antibody 4B6 was used to identify germline sequences with the highest homology to the corresponding murine framework. Computational modeling was used to design humanized variants with complementarity-determining region (CDR) grafting and backmutations.

[0821] Mouse / chimeric heavy chain variable region (SEQ ID NO: 46):

[0822] Mouse / chimeric light chain variable region (SEQ ID NO: 47):

[0823] Note: The italic part represents the framework (FR), and the underlined part represents the CDR sequence. The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0824] The human germline framework sequences VK / 1-33 of the light chain and VH / 1-2 of the heavy chain were used for CDR grafting, respectively.

[0825] Heavy chain variants 1, 2, 3 and 4 (i.e., VH variants 1, 2, 3 and 4) were obtained by directly grafting the three CDRs into the VH germline sequence (SEQ ID NO: 48) and additionally backmutating M69V, R71V of VH variant 1 (SEQ ID NO: 49), M69V, R71V, A93V, R94K of VH variant 2 (SEQ ID NO: 50), M69V, R71V, T73K, T28V of VH variant 3 (SEQ ID NO: 51) and M69V, R71V, A93V, R94K, T73K, T28V, G44S of VH variant 4 (SEQ ID NO: 52), respectively.

[0826] Germline sequence of 4B6VH:

[0827] VH / 1-2 (4B6-VH germline, SEQ ID NO: 48):

[0828] VH / 1-2 variant 1 (4B6_Ha, SEQ ID NO: 49):

[0829] VH / 1-2 variant 2 (4B6_Hb, SEQ ID NO: 50):

[0830] VH / 1-2 variant 3 (Hu4B6_Hc, SEQ ID NO: 51):

[0831] VH / 1-2 variant 4 (Hu4B6_Hd, SEQ ID NO: 52):

[0832] Light chain variants 1 and 2 (VL variants 1 and 2) were obtained by directly grafting the three CDRs into the germline sequence (SEQ ID NO: 53) and additionally backmutating the F73L mutation of VL variant 1 (SEQ ID NO: 54) and F73L, A43S, and S60D of VL variant 2 (SEQ ID NO: 55), respectively.

[0833] Germline sequence of 4B6VL:

[0834] VK / 1-33 (4B6-VL-germline, SEQ ID NO: 53):

[0835] VK / 1-33 variant 1 (Hu4B6_La, SEQ ID NO: 54):

[0836] VK / 1-33 variant 2 (Hu4B6_Lb, SEQ ID NO: 55):

[0837] The cDNAs for the heavy and light chain variable regions were synthesized and then fused to the constant region sequences of human IgG1 and human kappa. The resulting antibody gene sequences were cloned into expression vectors. Large-scale DNA was prepared using the Plasmid Maxiprep System from Qiagen for expression of humanized 4B6 variants, as shown in Table 6, and the ExpiFectamine 5-D-100 from Invitrogen was used. TM CHO reagent is carried out cell transfection according to the manufacturer's protocol. When the cell viability exceeds 60%, the supernatant is harvested and filtered through a 0.22um filter capsule to remove cell debris. The filtered supernatant is subsequently loaded onto a pre-equilibrated protein-A affinity column. The protein A resin is washed with equilibration buffer (PBS), and the antibody is then eluted using 25mM citrate (pH 3.5). The purified antibody solution is adjusted to pH 6.0-7.0 using 1M Tris-base (pH 9.0). Endotoxin is controlled to below 1EU / mg. Finally, the purified antibody is characterized by SDS-PAGE.

[0838] Table 6. Expression of humanized 4B6 variants

[0839] Note: This table shows various sequence combinations of different mutations. For example, Hu4B6_HaLa indicates the presence of two mutations (heavy chain Hu4B6_Ha and light chain Hu4B6_La) in the humanized murine antibody Hu4B6_HaLa, and so on. Hu4B6_L0 and Hu4B6_H0 are CDR-grafted products that lack key back mutations and are therefore not used for expression.

[0840] Example 2: Binding to human PD-L1 measured by ELISA assay

[0841] The binding of humanized antibodies was assessed by ELISA. Briefly, human PD-L1-His was immobilized on the plate. The humanized 4B6 antibodies listed in Table 6 were serially diluted in PBS and added for 1 hour. Next, goat pAbs were added to human IgG-HRP and TMB to detect binding at OD450nm.

[0842] All humanized variants were tested to screen for the best variant as shown in Figure 1. All variants retained their binding activity, and Hu4B6_HdLa showed better binding activity than the other variants.

[0843] The CDR sequence of this antibody was analyzed, and an NG motif was found in the CDR2 of the heavy chain. There may be a risk of deamination during expression and purification. To remove the deamidation hotspot, a G55A mutation (bold and enlarged below) was introduced into Hu4B6_Hd. Hu4B6_Hg (SEQ ID NO: 56) was then obtained, and its affinity for human PD-L1 was not affected, as shown in Figure 2.

[0844] Hu4B6_Hg (SEQ ID NO: 56):

[0845] Example 3: Monoclonal phage ELISA and sequence analysis

[0846] Although Hu4B6-HgLa retains the activity of chimeric 4B6, as an antagonist drug, higher affinity is preferred. Based on the Hu4B6-HgLa sequence, several panning cycles of site-directed mutagenesis in the CDR and in vitro dissociation rate dependency selection are further used for affinity maturation. First, the VL and VH domains of 4B6-HgLa are amplified by overlapping PCR and connected to form scFv by a peptide linker (G4S)3, and then subcloned into the phagemid vector pComb3x (Wuhan MiaoLingBio, P0862) as the wild-type sequence for affinity maturation by the SfiI cleavage site.

[0847] To investigate the individual contributions of the heavy and light chain CDR1 and CDR2 to 4B6 affinity maturation, a SPM (small perturbation mutagenesis) phage library was constructed for each of the CDRs mentioned above, as the antibody CDR3s of both chains typically play an important role in antigen binding. The CDR1 and CDR2 sequences of both chains were aligned with the germline sequences, and the germline sequences for the variable regions of the heavy and light chains were IGHV1-2 and IGKV1-33, respectively. Bioinformatics analysis of the germline CDR sequences was used to guide library design.

[0848] After determining the amino acid mutation sites and substitution sequences, degenerate primers were designed to increase the diversity of the mutation library. Diverse CDR fragments were amplified to construct the 4B6scFv gene mutation library. The scFv gene was ligated to the pComb3XSS phage display vector to generate the scFv library. 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 four libraries. The capacity of HCDR1 was 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, and the capacity of LCDR2 is 2.00×10 8 CFM. Five or six clones were randomly selected from each library for colony sequencing. The results showed that the insertion rate of the constructed library was 100%.

[0849] 10 μg / ml hPD-L1 (Acro Biosystems, PD1-H5229) antigen was coated onto an ELISA plate and incubated with 200 μL phage (1×10 10 pfu / ml phage display library) 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-logarithmic phase for library phagemid rescue, and phage was generated and purified for the next round of screening. The screening process was repeated 3 times, and the second round antigen concentration dropped to 2.5 μg / ml and the third round antigen concentration dropped to 1 μg / ml.

[0850] ELISA binding assays were performed to detect the titers of these polyclonal phage variants.After three rounds of panning, three libraries including 4B6-H-CDR2, 4B6-L-CDR1 and 4B6-L-CDR2 were significantly enriched.

[0851] For these 3 libraries, 96 clones of each library were picked and phage ELISA was performed to detect their binding activity. Briefly, 1 μg / ml hPD-L1 (Acro Biosystems, PD1-H5229) antigen was coated onto an ELISA plate and placed at 4°C overnight. 300 μL of 3% (w / v) skim milk was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of supernatant containing monoclonal antibody fragment phage was added together with PBS as a negative control and incubated at 37°C for 1 hour. 0.5% PBS+Tween-20 was used for washing 3 times, and 100 μl of HRP-coupled anti-M13 mAb (1:20000, Sino Biological, 11973-MM05T-H) was added. After incubation at room temperature for 1 hour, mixed TMB (InnoReagents, TMB-S-003) substrate reagent was added and the plate was incubated at room temperature for 5 minutes. The reaction was terminated by adding 0.1 M H2SO4 and the OD450 nm was recorded. Positive clones were selected for DNA sequencing by Genewiz (Suzhou, China). The sequences are shown in Table 7.

[0852] Table 7. Sequences of positive clones from the 4B6scFv phage library

[0853] Note: The underlined sequences refer to the CDR sequences, and the bold amino acids refer to the mutated amino acids.

[0854] Example 4: Biolayer Interferometry (BLI) for detecting binding affinity

[0855] Biolayer interferometry (BLI) 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 Tables 8 and 9, respectively. The results are shown in Tables 10-12. Based on the binding affinity results, four light chain variants (L-CDR1-2, L-CDR1-3, L-CDR2-2, L-CDR2-3) and three heavy chain variants (H-CDR2-2, H-CDR2-3, H-CDR2-5) were selected for future construction.

[0856] Table 8 Samples and materials used in BLI assay

[0857] Table 9 BLI assay procedure

[0858] Table 10. Binding affinity ranking of 4B6-L-CDR1 mutant variants

[0859] Table 11. Binding affinity ranking of 4B6-L-CDR2 mutant variants

[0860] Table 12. Binding affinity ranking of 4B6-H-CDR2 mutant variants

[0861] Example 5: Construction and expression of AM-4B6-hIgG1-TGFβRII (1-136) fusion protein

[0862] The selected heavy and light chain variants were cross-combined and expressed with the hIgG1-TGFβRII(1-136) fusion protein. TGFβRII(1-136) has the amino acid sequence shown in SEQ ID NO: 79:

[0863] The amino acid sequence of hIgG1 is as follows (SEQ ID NO: 80):

[0864] TGFβRII(1-136) was linked to the carboxyl terminus of hIgG1 via a peptide linker (G4S)4G (SEQ ID NO: 68).

[0865] The amino acid sequence of hκ is as follows (SEQ ID NO: 82):

[0866] The complete antibody variants are prefixed with AM (affinity mature). The sequence constructs of the heavy and light chains are shown in Table 13, and the design of the complete antibodies is shown in Table 14. For example, as shown in Tables 13 and 14, the bifunctional molecule "AM-4B6-hIgG1-TGFβRII variant 1" has a heavy chain and a light chain, wherein the heavy chain from N-terminus to C-terminus includes: Hu4B6_Hg.2-_hIgG1-(G4S)4G-TGFβRII (1-136); and the light chain from N-terminus to C-terminus includes: Hu4B6_La.1-hκ. The same nomenclature applies to the other variants in Table 14.

[0867] ExpiFectamine from Invitrogen was used according to the manufacturer's protocol. TM Co-transfection of heavy and light chains was performed using CHO reagent (Thermo, A29129). Supernatants were harvested on day 10 and purified by affinity chromatography.

[0868] Table 13 List of AM-4B6-hIgG1-TGFβRII heavy chain and light chain variants

[0869] Table 14. List of AM-4B6-hIgG1-TGFβRII antibody variants

[0870] Example 6: Binding affinity of AM-4B6-hIgG1-TGFβRII variant to hPD-L1

[0871] Binding to hPD-L1 measured by ELISA assay

[0872] 1 μg / ml hPD-L1 (Acro Biosystems, PD1-H5229) antigen was coated onto an ELISA plate and placed overnight at 4 ° C. 300 μl of 3% (w / v) skim milk was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of AM-4B6-hIgG1-TGFβRII variants or original 4B6-hIgG1-TGFβRII at concentrations ranging from 100 nM to 0.006 nM (fourfold serial dilutions) were added together with PBS as a negative control and incubated at room temperature for 1 hour. The plate was washed three times with 0.5% PBS + Tween-20, and 100 μl of HRP-conjugated anti-human Fc antibody (1:20,000, Abcam, ab98624) was added. After incubation at room temperature for 1 hour, mixed TMB substrate reagent (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes, followed by termination by addition of 0.1 M HSO. OD450 nm was recorded on a microplate reader.

[0873] As shown in Figures 3A-3C, all variants had enhanced binding signals and affinities when compared to the original 4B6-hIgG1-TGFβRII. Given the limited differences between these variants, surface plasmon resonance was used to further evaluate their binding affinities.

[0874] Binding to hPD-L1 measured by Biacore

[0875] 4 μg / ml AM-4B6-hIgG1-TGFβRII variant or original 4B6-hIgG1-TGFβRII was immobilized on the surface of an S-series protein A chip. Human PD-L1 was diluted to an 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 the Biacore 2000. The results are shown in Table 15. The binding affinity of AM-4B6-hIgG1-TGFβRII variant 7 to human PD-L1 was increased by approximately 15 times compared to the original 4B6-hIgG1-TGFβRII.

[0876] Table 15. Binding affinity of AM-4B6-hIgG1-TGFβRII to hPD-L1 measured by Biacore

[0877] Binding to PD-L1 expressed on the cell surface measured by FACS assay

[0878] 293T-PD-L1-CD3L cells were generated by MabSpace Bioscience for the characterization of PD-L1 antibodies. The cells were transfected with human PD-L1 and anti-CD3 scFv. AM-4B6-hIgG1-TGFβRII variants or original 4B6-hIgG1-TGFβRII were serially diluted (5-fold dilution) to obtain 8 concentrations in dilution buffer (PBS containing 2% BSA). 293T-PD-L1-CD3L cells were harvested and centrifuged. They were plated at 2×10 6 The cells were resuspended in PBS at a density of 100 cells / ml and then added to the plate with 100ul per well. After centrifugation and removal of the supernatant, the diluted antibody was 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, the cells were resuspended in 200 μl PBS and analyzed by flow cytometry.

[0879] As shown in Figure 4, these five variants bound to PD-L1 expressed on the surface of 293T-PD-L1-CD3L cells with similar EC50s. The EC50 of variant 7 was slightly lower than that of the other variants, which was consistent with the binding affinity results measured by Biacore.

[0880] Example 7: PD-1 / PD-L1 blocking activity of AM-4B6-hIgG1-TGFbRII variant

[0881] PD-1 / PD-L1 and B7-1 / PD-L1 blockade measured by ELISA assay

[0882] To test ligand / receptor blocking activity, 0.5 μg / ml hPD-L1-Fc antigen was coated onto an ELISA plate and placed at 4°C overnight. 300 μL of blocking buffer was added to block for 1 hour at room temperature. After 1 hour, 50 μl of AM-4B6-hIgG1-TGFβRII variant 7 or original 4B6-hIgG1-TGFβRII with a concentration range of 100 nM to 0.024 nM (fourfold serial dilution) and 50 μl of PD-L1-his at a concentration of 1 μg / ml were added and incubated at room temperature for 1 hour. 0.5% PBS+Tween-20 was used for washing 3 times, and 100 μl of HRP-coupled streptavidin (1:5000, Abcam, catalog number ab7403) was added. After incubation at room temperature for 1.5 hours, the mixed TMB substrate reagent was added and incubated at room temperature for 5 minutes, and then 0.1 M H2SO4 was added to stop. OD450nm was recorded by a microplate reader. As shown in Figures 5A-5B, both samples could block PD-L1 / PD-1 or PD-L1 / B7-1, but AM-4B6-hIgG1-TGFβRII variant 7 had a lower IC50, indicating that variant 7 had higher activity.

[0883] PD-1 / PD-L1 blockade measured by cell-based assays

[0884] In this assay, 293T-PD-L1-CD3L cells express PD-L1 and anti-CD3 scFv, while Jurkat-NFAT-Luc-PD1 cells express PD-1 and carry a NFAT signaling pathway that can be activated by CD3 stimulation. NFAT activation leads to downstream luciferase gene transcription and expression, which can be detected using its substrate. Both cells were generated by Mabspace Biosciences.

[0885] Briefly, 293T-PD-L1-CD3L cells were harvested and cultured at 2 × 10 6 Resuspend at a density of 10 cells / ml. 20 μl of cells were added to each well of the half-well plate. AM-4B6-hIgG1-TGFβRII variant or original 4B6-hIgG1-TGFβRII was serially diluted (3-fold dilution) to obtain 8 concentrations in RPMI medium containing 2% FBS. 20 μl of antibody was added to each well of the half-well plate and the plate was incubated at 37°C, 5% CO2 for 30 minutes. Jurkat-NFAT-Luc-PD1 cells were harvested and plated at 4×10 6The cells were resuspended in RPMI medium containing 2% FBS at a density of 10 cells / ml. Finally, 20 μl of cells and 5 ng / ml TGF-β (R&D, 240-B-010) were added to a half-well plate and incubated at 37°C, 5% CO2 for 5 hours. 60 μl of OneGlo detection reagent (Promega, E6120) was added to each well and incubated at room temperature for 5 minutes. The luminescent signal was read by a microplate reader. Data were analyzed by GraphPad Prism.

[0886] As shown in Figure 6, consistent with the previous ELISA results, variant 7 had the most potent blocking activity in this cell-based assay compared to the other variants. Therefore, the 4B6 Fab portion of AM-4B6-hIgG1-TGFβRII variant 7 was abbreviated as AM4B6, and the AM4B6-hIgG1-TGFβRII fusion protein was further evaluated in the following experiments.

[0887] Example 8: In vitro generation and characterization of AM4B6-hIgG1-TGFβRII′

[0888] Cloning and expression of AM4B6-hIgG1-TGFβRII

[0889] It has been reported that the truncated TGFβRII ECD_20-136 is soluble and retains the ability to bind to TGFβ1 (Kim-Ming Lo et al., US9676863 B2, 2017; Christian C. et al., Protein Expression and Purification, 2000, 20:98–104). Next, the full length of the extracellular domain of TGFβRII_1-136 was replaced with the truncated extracellular domain and the developability and stability were evaluated. SDS-PAGE results from stable cell lines showed that protein expression was good for both truncated and full-length TGFβRII ECD, but the protein stability of the truncated TGFβRII ECD_20-136 was much better than that of the full-length TGFβRII ECD (Figure 7). The sequence of the truncated TGFβRII ECD_20-136 is as follows:

[0890] Sequence of the stable TGF-β trap, TGF-βRII extracellular domain (20-136)

[0891] Bifunctional molecules comprising a truncated TGF-βRII (i.e., TGF-βRII (20-136), SEQ ID NO: 66) were used in this example and in Examples 9-11. These bifunctional molecules are designated TGFβRII' to distinguish them from TGF-βRII (1-136). For example, AM4B6-hIgG1-TGFβRII' represents a molecule comprising TGF-βRII (20-136).

[0892] Binding to PD-L1 from different species measured by ELISA assay

[0893] 1 μg / ml human PD-L1 (Acro Biosystems, PD1-H5229) or cynomolgus monkey PD-L1 antigen was coated onto an ELISA plate and placed overnight at 4°C. 300 μl of 3% (w / v) skim milk was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of AM4B6-hIgG1-TGFβRII' or control hIgG1-TGFβRII' at concentrations ranging from 100 nM to 0.02 nM (fourfold serial dilutions) was added and incubated at room temperature for 1 hour. 0.5% PBS + Tween-20 was used for washing three times, and 100 μl of HRP-conjugated anti-human Fc antibody (1:20,000, Abcam, ab98624) was added. After incubation at room temperature for 1 hour, mixed TMB substrate reagent (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes, and stopped by adding 0.1M H2SO4. OD450nm was recorded by a microplate reader.

[0894] As shown in Figures 8A and 8B, AM4B6-hIgG1-TGFβRII' cross-reacted with cynomolgus monkey PD-L1 with an EC50 similar to that of human PD-L1.

[0895] Binding to TGFβ1, TGFβ2, and TGFβ3 from different species measured by ELISA

[0896] According to the sequences of TGFβ1, TGFβ2, and TGFβ3 from four common species published on the Uniport website (https: / / www.uniprot.org / ): human, cynomolgus monkey, mouse, and rat, TGFβ members are quite conserved. The sequences of human TGFβ1 and cynomolgus monkey TGFβ1 are identical; mouse TGFβ1 and rat TGFβ1 are identical; human TGFβ2 and cynomolgus monkey TGFβ2 are identical; mouse TGFβ2 and rat TGFβ2 are identical; and human TGFβ3, cynomolgus monkey TGFβ3, and mouse TGFβ3 are identical.

[0897] For TGFβ1 and TGFβ3, the procedure was as follows: 0.5 μg / ml human TGFβ1 (Sino Biological, 10804-HNAC) or mouse TGFβ1 (Novoprotein, CK33) or human TGFβ3 (Genscript, Z03430) or rat TGFβ3 (Novoprotein, CJ44) antigen was coated onto an ELISA plate and placed overnight at 4°C. 300 μl of 3% (w / v) skim milk was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of AM4B6-hIgG1-TGFβRII' or control hIgG1-TGFβRII' at a concentration range of 100 nM to 0.006 nM (fourfold serial dilution) was added and incubated at room temperature for 1 hour. 0.5% PBS + Tween-20 was used for washing three times, and 100 μl of HRP-conjugated anti-human Fc antibody (1:20,000, Abcam, ab98624) was added. After incubation at room temperature for 1 hour, mixed TMB substrate reagent (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes, and stopped by adding 0.1M H2SO4. OD450nm was recorded by a microplate reader.

[0898] For TGFβ2, the test procedure was different: 2 μg / ml of AM4B6-hIgG1-TGFβRII' or control hIgG1-TGFβRII' was coated onto an ELISA plate and placed at 4°C overnight. 300 μl of 3% (w / v) skim milk was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of human TGFβ2 or mouse TGFβ2 with a concentration range of 39.4 nM to 0.3 nM (two-fold serial dilution) was added and incubated at room temperature for 1 hour. 0.5% PBS+Tween-20 was used for washing 3 times, and 100 μl of TGFβ2 biotinylated antibody (1:10000, R&D, BAF302) was added. After incubation at room temperature for 1 hour and washing, 100 μl of HRP-streptavidin (1:5000, Abcam, ab7403) was added and the plate was incubated at room temperature for 1 hour. After washing, mixed TMB substrate reagent (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes and stopped by adding 0.1 M H 2 SO 4 OD 450 nm was recorded by a microplate reader.

[0899] The results are summarized in Table 16. For the binding affinity to TGFβ1, the EC50 values ​​were very similar between different species. In addition, the binding affinity to TGFβ1 and TGFβ3 was significantly higher than that to TGFβ2, suggesting that the blocking activity against TGFβ1 and TGFβ3 may be more effective than that against TGFβ2.

[0900] Table 16. Binding of AM4B6-hIgG1-TGFβRII' to TGFβ1, TGFβ2, and TGFβ3

[0901] Binding to other members within the B7 family or TGFβ superfamily measured by ELISA assay

[0902] For the B7 family, 0.5 μg / ml 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 onto an ELISA plate and placed at 4°C overnight. For the TGFβ superfamily, 0.5 μg / ml human activin A, BMP-2, LAP or TGFβ1 was coated overnight at 4°C. 300 μl of 3% (w / v) skim milk was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of AM4B6-hIgG1-TGFβRII' with a concentration range of 100 nM to 0.006 nM (serial dilutions) was added and incubated at room temperature for 1 hour. 0.5% PBS + Tween-20 was used for washing three times, and 100 μl of HRP-conjugated anti-human Fc antibody (1:20,000, Abcam, ab98624) was added. After incubation at room temperature for 1 hour, mixed TMB substrate reagent (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes, and stopped by adding 0.1M H2SO4. OD450nm was recorded by a microplate reader.

[0903] As shown in Figures 9A-9B, AM4B6-hIgG1-TGFβRII' specifically binds to PD-L1 rather than other antigens belonging to the B7 family. As shown in Figure 9C, AM4B6-hIgG1-TGFβRII' specifically binds to TGF-β1 rather than other antigens belonging to the TGFβ superfamily.

[0904] Binding of AM4B6-hIgG1-TGFβRII′ to cells expressing PD-L1

[0905] MC38 / hPD-L1 was generated by deleting mPD-L1 via the CRISPR-Cas9 system and then transducing hPD-L1 using lentiviral transduction. This cell line was provided by Professor Qin Xiaofeng's laboratory at the Center of Systems Medicine, Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine (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 stably expressing the transfected human PD-L1 gene. EMT-6 / hPD-L1 cells were cultured in Waymouth (1×) MB752 / 1 + 15% FBS. NCI-H460 cells were purchased from 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., a human lung epithelial tumor cell line that expresses PD-L1. NCI-H292 cells were cultured in RPMI1640 + 10% FBS + 1 nM sodium pyruvate. The FACS analysis protocol was the same as in Example 6, Section 3.

[0906] Human or cynomolgus monkey PBMC (TPCS, catalog number PB025C) were recovered from liquid nitrogen and resuspended in RPMI1640 with 10% FBS. 5 μg / ml of PHA (Sigma, catalog number L8902) was added to stimulate PBMC activation and the cells were cultured for 3 days. The activated PBMCs were collected and centrifuged and the cells were plated at 2×10 6The cells were resuspended in PBS at a density of 10 cells / ml and added to the plate at 100ul per well. AM4B6-hIgG1-TGFβRII' or AM4B6 or control hIgG1-TGFβRII' were serially diluted (5-fold dilution) to obtain 10 concentrations in dilution buffer (PBS containing 2% BSA). After centrifugation and removal of the supernatant in the plate, the diluted antibody was added to the plate with activated PBMC and incubated at 4°C for 1 hour. After washing twice with dilution buffer, Alexa488-labeled mouse anti-human CD3 (Biolegend, catalog number 300320) and APC-labeled anti-human IgG secondary antibody (BD, catalog number 550931) were added and incubated at 4°C for 30 minutes. After washing, the cells were resuspended in 150μl PBS and analyzed by flow cytometry.

[0907] As shown in FIG. 10A to FIG. 10F , AM4B6-hIgG1-TGFβRII′ can bind to PD-L1 expressed on these cancer cell lines and activated human or cynomolgus monkey T cells with similar affinity as AM4B6 mAb alone.

[0908] Binding to activated human T cells

[0909] Human PBMC (TPCS, catalog number PB025C) were recovered from liquid nitrogen and resuspended in RPMI1640 with 10% FBS. 5 μg / ml of PHA (Sigma, catalog number L8902) was added to stimulate PBMC activation and the cells were cultured for 3 days. The activated PBMCs were collected and centrifuged and the cells were plated at 2×10 6 The cells were resuspended in PBS at a density of 10 cells / ml and added to the plate at 100ul per well. AM4B6-hIgG1-TGFβRII' or AM4B6 or control hIgG1-TGFβRII' were serially diluted (5-fold dilution) to obtain 10 concentrations in dilution buffer (PBS containing 2% BSA). After centrifugation and removal of the supernatant in the plate, the diluted antibody was added to the plate with activated PBMC and incubated at 4°C for 1 hour. After washing twice with dilution buffer, Alexa488-labeled mouse anti-human CD3 (Biolegend, catalog number 300320) and APC-labeled anti-human IgG secondary antibody (BD, catalog number 550931) were added and incubated at 4°C for 30 minutes. After washing, the cells were resuspended in 150μl PBS and analyzed by flow cytometry.

[0910] As shown in FIG11 , AM4B6-hIgG1-TGFβRII′ can bind to PD-L1 expressed on activated human T cells.

[0911] Blockade of hPD-L1 / hPD-1 and cynomolgus PD-L1 / cynomolgus PD-1 measured by ELISA assay

[0912] 0.5 μg / ml of hPD-L1-Fc or 0.5 μg / ml of cynomolgus monkey PD-L1-Fc was coated onto an ELISA plate and placed at 4°C overnight. 300 μL of 3% (w / v) skim milk was added to block for 1 hour at room temperature. After 1 hour, 100 μl of AM4B6-hIgG1-TGFβRII' or AM4B6 with a concentration range of 100 nM to 0.02 nM (fourfold serial dilution) and 0.5 μg / ml of hPD-1-Fc-biotin or cynomolgus monkey PD-1-Fc-biotin were added and incubated at room temperature for 1 hour. 0.5% PBS + Tween-20 was used for washing 3 times, and 100 μl of HRP-coupled streptavidin (1:5000) was added. After incubation at room temperature for 1 hour, the mixed TMB substrate reagent was added and incubated at room temperature for 5 minutes, and then 0.1 M H2SO4 was added to stop. OD450nm was recorded by a microplate reader.

[0913] As shown in FIG. 12A-B , AM4B6-hIgG1-TGFβRII′ can also completely block cynomolgus monkey PD-L1 / cynomolgus monkey PD-1 with an IC50 similar to that of blocking human PD-L1 / human PD-1.

[0914] Simultaneous binding to hPD-L1 and hTGFβ1

[0915] 0.5 μg / ml of hTGFβ-1 was coated onto an ELISA plate and placed at 4°C overnight. 300 μl of blocking buffer was then added to block for 1 hour at room temperature. After 1 hour, 100 μl of AM4B6-hIgG1-TGFβRII' or AM4B6 or control hIgG1-TGFβRII' with a concentration range of 100 nM to 0.02 nM (fourfold serial dilution) was added and incubated at room temperature for 1 hour. 0.5% PBS+Tween-20 was used to wash 3 times, and then 0.5 μg / ml of hPD-L1-biotin was added to each well. After 1 hour, 100 μl of HRP-coupled streptavidin (1:5000) was added. After incubation at room temperature for 1 hour, mixed TMB substrate reagent (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes and terminated by adding 0.1 M H2SO4. OD450nm was recorded by a microplate reader.

[0916] As shown in FIG13 , AM4B6-hIgG1-TGFβRII′, composed of the anti-PD-L1 antibody AM4B6 and TGFβRII′, can bind to two targets simultaneously, indicating its bispecific or bifunctional properties.

[0917] Blockade of hPD-L1 / hPD-1 measured by reporter cell assay

[0918] The protocol was the same as in Example 7, Section 2. As shown in Figure 14, AM4B6-hIgG1-TGFβRII could block the inhibition of PD-L1 / PD-1 and subsequently reverse signaling activation, which was the same as AM4B6 mAb alone.

[0919] Blockade of TGFβ1 signaling measured by reporter cell assay

[0920] The TGFβ reporter gene HEK-293 cell line was purchased from Genomeditech (catalog: GM-C05346) and cultured in DMEM medium containing 10% FBS, 4 μg / ml blasticidin, 400 μg / ml neomycin, 125 μg / ml hygromycin, 0.75 μg / ml puromycin and 1% Pen / Strep in a 37°C incubator with 5% carbon dioxide.

[0921] The cells were collected in the logarithmic growth phase and resuspended in DMEM medium and seeded in a 96-well plate at a density of 2×10^4 cells / 100μl / well. After the cells were cultured overnight, the culture medium was replaced with 75μl of culture medium containing 10ng / ml human TGFβ1. 75μl of AM4B6-hIgG1-TGFβRII' or AM4B6 were added at a final concentration range of 100nM to 0.02nM (three-fold serial dilution). The plate was cultured in a 37°C incubator for 16 hours. The ONE-GloTM luciferase detection system was added at 150μl / well and after incubation at room temperature for 10 minutes, the plate was read with a microplate reader.

[0922] As shown in FIG15 , AM4B6-hIgG1-TGFβRII′ exhibited potent blocking activity on TGFβ1 signaling with an IC50 of 0.35 nM, whereas AM4B6 mAb alone had no blocking activity, indicating that the blocking activity was specific to TGFβ1.

[0923] Effect of AM4B6-hIgG1-TGFβRII' on IFN-γ release from PBMCs stimulated with tuberculin (TB)

[0924] Human PBMCs were recovered from liquid nitrogen and resuspended at a density of 2×10^6 / mL. TB was added to a final concentration of 1.33 μg / mL; cultured at 37°C for 5 days. On the sixth day, the induced PBMCs were collected and centrifuged, washed once with PBS, resuspended in fresh culture medium, adjusted to a density of 1×10^6 / ml, and seeded into 96-well cell plates at 180 μL / well. The diluted antibodies were added to 96-well cell culture plates at 20 μL / well. The control group and the blank group were added with 20 μL PBS. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 3 days. At the end of incubation, the cell supernatant was diluted 10 times and the secretion level of IFNγ was detected using an IFN-γ ELISA detection kit (R&D, DY285B).

[0925] As shown in FIG16 , AM4B6-hIgG1-TGFβRII′ induced significantly higher levels of IFN-γ release than AM4B6 mAb alone, indicating that its activation activity was more potent due to its bispecific binding and blocking activities.

[0926] ADCC / CDC activity of AM4B6-hIgG1-TGFβRII'

[0927] For ADCC assay, effector cells: Jurkat-NFAT Luc-FcγRIIIa-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).

[0928] HEK-293T-hPD-L1 cells were added to the cell culture plate at 10,000 cells / 12.5 μl / well. AM4B6-hIgG1-TGFβRII' dilutions with a final concentration range of 200 nM to 0.003 nM were then added at 12.5 μl / well. The plate was then placed in an incubator at 37°C to allow the antibody and cells to incubate for 30 minutes. Jurkat-NFAT Luc-FcγRIIIa-158V cells were then added to the wells at 60,000 cells / 25 μl / well. The plate was then placed in an incubator at 37°C for 6 hours. The ONE-Glo™ luciferase assay system was added at 50 μl / well and, after incubation at room temperature for 10 minutes, the plate was read using a microplate reader.

[0929] For the CDC assay, the target cells were also HEK-293T-hPD-L1 cells. HEK-293T-hPD-L1 cells were added to a cell culture plate at 10,000 cells / 25 wells. AM4B6-hIgG1-TGFβRII' dilutions with a final concentration range of 200nM to 0.3nM were then added at 12.5μl / well. The plate was then placed in an incubator at 37°C to incubate the antibody and cells for 30 minutes. HEK-293T-hPD-L1 cells were treated with 40% complement at 50μl / well (final concentration of 20%) and then incubated at 37°C for 80 minutes. The ONE-GloTM luciferase detection system was added at 100μl / well, and after incubation at room temperature for 10 minutes, the plate was read with a microplate reader.

[0930] The results showed that AM4B6-hIgG1-TGFβRII′ had neither ADCC nor CDC activity against HEK-293T-hPD-L1 cells (data not shown).

[0931] Example 9: In vivo efficacy of AM4B6-hIgG1-TGFβRII′

[0932] MC38-hPD-L1 tumor model in C57BL / 6 mice

[0933] The endogenous mouse PD-L1 in the mouse tumor cell line MC38 (ATCC) was knocked out using the recently developed eff...

Claims

1. A protein preparation comprising: (1) A bifunctional molecule comprising: a first portion that binds to an immune checkpoint molecule, preferably an antibody or antigen-binding fragment thereof directed against PD-L1, and i) blocks the activity of immunosuppressive cytokines or ii) stimulates immunity, wherein the second moiety is preferably a TGFβ binding portion, an IL-1 binding portion, a LAG-3 binding portion or a Sirpa binding portion, wherein the TGFβ binding portion is preferably a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, or an antibody to TGFβ and an antigen-binding fragment thereof; (2) buffer; and (3) Surfactants 2. The protein formulation according to claim 1, comprising: (1) A bifunctional molecule comprising 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 the first portion comprises an antibody or antigen-binding fragment thereof directed against PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region comprises: a) HCDR1 comprising DYYMN (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity to DYYMN, b) HCDR2 comprising DINPNNX1X2TX3YNHKFKG (SEQ ID NO: 19) or a homologous sequence having at least 80% sequence identity to DINPNNX1X2TX3YNHKFKG, and c) a HCDR3 comprising WGDGPFAY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity to WGDGPFAY, and / or wherein the light chain variable region comprises: d) LCDR1 comprises a sequence selected from the group consisting of KASQNVX4X5X6VA (SEQ ID NO: 20) or a homologous sequence having at least 80% sequence identity thereto, e) LCDR2 comprises a sequence selected from the group consisting of SX7SX8RYT (SEQ ID NO: 21) or a homologous sequence having at least 80% sequence identity thereto, and f) LCDR3 comprises a sequence selected from the group consisting of QQYSNYPT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity to QQYSNYPT; wherein X1 is G or A, X2 is G or D or Q or E or L, X3 is S or M or Q or L or V, X4 is G or P or K, X5 is A or G, X6 is A or I, X7 is A or N or R or V, and X8 is N or H or V or D; (2) buffer; and (3) Surfactants 3. The protein formulation according to any one of the preceding claims, wherein the first portion of the bifunctional molecule comprises an antibody or antigen-binding fragment thereof directed against PD-L1, and the second portion comprises a TGFβ binding portion, wherein the antibody or antigen-binding fragment thereof against PD-L1 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; and / or wherein the TGFβ binding portion comprises a soluble TGFβ receptor (TGFβR) or a TGFβ binding fragment or variant thereof, wherein the soluble TGFβR comprises the extracellular domain (ECD) of the TGFβR or a TGFβ binding fragment or variant thereof, and the ECD of the TGFβR comprises the amino acid sequence of SEQ ID NO: 66, 79, 78, 77, or a sequence having at least 80% sequence identity thereto but still retaining a specific binding specificity and / or affinity for TGF-β.

4. The protein formulation according to any one of the preceding claims, wherein the concentration of the bifunctional molecule is about 1-150 mg / mL, such as about 10-50 mg / mL or about 20-30 mg / mL.

5. The protein formulation according to any one of the preceding claims, wherein the buffer is an acetate, histidine, citrate, succinate, malate, glutamate, phosphate or lactate buffer, such as an acetic acid-sodium acetate buffer or a histidine-hydrochloric acid buffer.

6. The protein formulation according to any one of the preceding claims, wherein the concentration of the buffer is about 5-100 mM, such as about 10-50 mM, about 15-30 mM or about 20 mM.

7. The protein formulation according to any one of the preceding claims, 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 or about 5.

3.

8. The protein formulation according to any one of the preceding claims, wherein the surfactant is a non-ionic surfactant, preferably a polysorbate, such as polysorbate 80 or polysorbate 20.

9. The protein formulation according to any one of the preceding claims, wherein the surfactant concentration is about 0.01-0.1% (w / v), such as about 0.025%-0.1% (w / v) or about 0.05% (w / v).

10. The protein formulation according to any one of the preceding claims, wherein the formulation comprises a stabilizer, for example selected from one or more of amino acids, inorganic salts, sugars, polyols and chelating agents, preferably arginine hydrochloride or NaCl.

11. The protein formulation according to any one of the preceding claims, wherein the formulation comprises a stabilizer at a concentration of about 10-300 mM, such as about 100-300 mM, about 120-300 mM, about 100-200 mM, about 130-170 mM or about 150 mM.

12. The protein formulation according to any one of the preceding claims, wherein the formulation comprises arginine hydrochloride, preferably at a concentration of about 100-200 mM, about 130-170 mM or about 150 mM.

13. The protein formulation according to any one of the preceding claims, wherein the formulation optionally comprises a chelating agent, such as EDTA.2Na, preferably at a concentration of about 30-350 μM.

14. A protein formulation according to any one of the preceding claims, comprising: 10-50 mg / mL of the bifunctional molecule; 10-50 mM acetate buffer or histidine buffer; 0.025%-0.1% (w / v) polysorbate 80 or polysorbate 20; 100-200 mM arginine hydrochloride; and Optionally 30-350 μM EDTA·2Na; pH is 5.0-5.6; or contain: 20-30 mg / mL of the bifunctional molecule; 20-30 mM acetate buffer or histidine buffer; 0.025%-0.1% (w / v) polysorbate 80; and 140-160 mM arginine hydrochloride; The pH was 5.3 ± 5%.

15. The protein formulation according to any one of the preceding claims, wherein the bifunctional molecule is treated with a chelating agent, such as EDTA.2Na (eg about 0.3-10 mM EDTA.2Na) during formulation and the chelating agent is subsequently removed.

16. The protein formulation according to any one of the preceding claims, which is in liquid form, lyophilized form or liquid form reconstituted from a lyophilized form, such as an injection, such as an intravenous injection, an intramuscular injection or a subcutaneous injection.

17. A protein formulation according to any one of claims 1 to 16, for treating, preventing or ameliorating a PD-L1-associated disease in a subject, e.g., the disease is an immune-related disease or disorder, a cancer or an infectious disease, e.g., 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, spinal 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, e.g., the subject has been identified as having cancer cells expressing PD-L1, e.g., wherein the PD-L1-associated disease is resistant to PD-L1 / PD-1 monotherapy.

18. A protein formulation according to any one of claims 1 to 16 for use in treating, preventing or ameliorating a disease or condition that would benefit from the suppression of immunosuppressive cytokines, the induction of a sustained immune response or the stimulation of anti-tumor immunity; in, For example, the immunosuppressive cytokine is TGFβ; for example, the disease or condition is a TGFβ-related disease or condition; for example, the TGFβ-related disease is cancer, fibrotic disease or renal disease; wherein, for example, the immunosuppressive cytokine is IL-1; for example, the disease or condition is an IL-1-related disease or condition; wherein, for example, the disease or condition would benefit from the induction of a sustained immune response by stimulation of MHC II signaling with an immunostimulatory polypeptide; for example, the immunostimulatory polypeptide is soluble LAG-3; or Wherein, for example, the disease or condition would benefit from stimulation of anti-tumor immunity by inhibiting immunoinhibitory receptor signaling; for example, the immunoinhibitory receptor is SIRPα.

Images