Antibodies targeting PDL1 and methods of using the same

Abstract

The present invention relates to an isolated antibody that specifically binds to human PDL1, and a pharmaceutical composition and a method of using the same. The present invention also relates to a nucleic acid encoding the antibody, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector, and a method for producing the antibody.

Description

Technical Field

[0001] The present invention relates to an isolated antibody that specifically binds to human PDL1, and a pharmaceutical composition and a method of using the same. The present invention also relates to a nucleic acid encoding the antibody, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector, and a method for producing the antibody. Background Art

[0002] PDL1 (CD274, B7-H1) is a 40 kDa type I transmembrane protein. PDL1 is the surface glycoprotein ligand for PD-1, a key immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PDL1 has been implicated in the suppression of immune system responses during chronic infection, pregnancy, allogeneic transplantation, autoimmune diseases, and cancer. PDL1 has been found on antigen presenting cells and human cancer cells, such as head and neck squamous cell carcinoma, melanoma and brain tumors, thyroid, thymus, esophagus, lung, breast, gastrointestinal tract, colorectal, liver, pancreas, kidney, adrenal cortex, bladder, urothelium, ovary and skin (Katsuya Y et al., Lung Cancer. 88(2):154–159 (2015); Nakanishi J et al., Cancer Immunol Immunother. 56(8):1173–1182 (2007); Nomi T et al., Clin Cancer Res. 13(7):2151–2157 (2007); Fay AP et al., J Immunother Cancer. 3:3 (2015); Strome SE et al., Cancer Res. 63(19):6501–6505 (2003); Jacobs JF et al., Neurol. Oncol.11(4):394–402(2009); Wilmotte R et al. Neuroreport.16(10):1081–1085(2005)). PDL1 is rarely expressed in normal tissues, but can be induced in tumor sites (Dong H et al., Nat Med.8(8):793–800(2002); Wang et al., OncoTargets Ther.9:5023–5039(2016)). PDL1 downregulates T cell activation and cytokine secretion by binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). PD-1 activated by PDL1 may provide an immune tolerance environment for tumor development and growth. PDL1 also negatively regulates T cell function by interacting with another receptor B7.1 (B7-1, CD80).

[0003] Inhibition of the PDL1 / PD-1 interaction can provide effective anti-tumor activity. A variety of anti-PDL1 antibodies are known (e.g., see WO 2013 / 079174 and WO 2017 / 118321), and many antibodies that disrupt PD-1 signaling have entered clinical development. These antibodies belong to two main categories: antibodies targeting PD-1 (nivolumab, Bristol-Myers Squibb; pembrolizumab, Merck, Whitehouse Station, NJ; pidilizumab, CureTech, Yavne, Israel) and antibodies targeting PDL1 (MPDL3280A, Genentech, South San Francisco, CA; MEDI4736, MedImmune / AstraZeneca; BMS-936559, Bristol-Myers Squibb; MSB0010718C, EMD Serono, Rockland, MA) (see Postow MA et al., J Clin Oncol. Jun 10; 33(17): 1974-82 (2015)). Targeting PDL1 may lead to different biological effects than targeting PD-1. PD-1 antibodies can prevent PD-1 from interacting with its two ligands, PDL1 and PDL2. Although the effect of PD-1 on the interaction with PDL2 remains unknown, PDL1 antibodies do not prevent PD-1 from interacting with PDL2. However, PDL1 antibodies not only prevent the interaction of PDL1 with PD-1, but also prevent the interaction with B7-1 (Butte MJ et al., Immunity 27: 111-122, (2007)), which is thought to produce negative signals for T cells. Blocking PDL1 has shown encouraging early data, and four clinical anti-PDL1 mAbs are currently being tested: atezolizumab and MEDI4736 (both Fc null variants of human IgG1), MSB001078C (IgG1) and BMS-936559 (IgG4) (Chester C et al., Cancer Immunol Immunother Oct; 65 (10): 1243-8 (2016)).

[0004] To date, no satisfactory methods have been demonstrated to induce effective immune responses in cancer patients. There is a need in the art to create improved therapeutic modulators of the PDL1 / PD-1 interaction and methods to overcome the immunosuppressive mechanisms observed in cancer patients. Summary of the invention

[0005] The object of the present invention is to provide an antibody that specifically binds to human PDL1 protein and has beneficial properties for treatment, such as higher affinity, improved efficacy, and improved biophysical properties, such as solubility, developability, and stability.

[0006] In one aspect, the present invention relates to a novel PDL1 antibody.

[0007] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.

[0008] In another aspect, the invention relates to an antibody of the invention or a composition of the invention for use as a medicament.

[0009] In one aspect, the invention relates to an antibody of the invention or a composition of the invention for use in treating cancer in a subject in need thereof.

[0010] In one aspect, the invention relates to the use of an antibody of the invention or a composition of the invention in the preparation of a medicament for treating cancer in a subject in need thereof.

[0011] In another aspect, the invention relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody of the invention or a composition of the invention.

[0012] In another aspect, the present invention relates to a nucleic acid encoding an antibody of the present invention. In another aspect, the present invention relates to a vector comprising said nucleic acid. In another aspect, the present invention relates to a host cell comprising said nucleic acid or said vector.

[0013] In another aspect, the present invention relates to a method for producing the antibody of the present invention, the method comprising the following steps: culturing a host cell comprising the nucleic acid or vector of the present invention.

[0014] The aspects, advantageous features and preferred embodiments of the invention which are outlined in the following, respectively alone or in combination, further contribute to solving the objects of the invention:

[0015] 1. An isolated antibody with binding specificity to human PDL1, comprising: (a) a heavy chain variable region CDR1 comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, preferably SEQ ID NOs: 1 or 32, more preferably SEQ ID NO: 1; (b) a heavy chain variable region CDR2 comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NOs: 2 or 33, more preferably SEQ ID NO: 2; (c) a heavy chain variable region CDR3 comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 3, 7, 10, 13, 34, 38, 41 and 44, preferably SEQ ID NOs: 3 or 34, more preferably SEQ ID NO: 3; (d) a light chain variable region CDR1 comprising, preferably consisting of, an amino acid sequence selected from SEQ ID NOs: NO: 17, 20, 23, 48, 51 and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17 amino acid sequence, preferably consisting of thereof; (e) a light chain variable region CDR2, which comprises an amino acid sequence selected from any one of SEQ ID NO: 18, 21, 24, 49, 52 and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18 amino acid sequence, preferably consisting of thereof; and (f) a light chain variable region CDR3, which comprises an amino acid sequence selected from any one of SEQ ID NO: 19, 22, 25, 50, 53 and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19 amino acid sequence, preferably consisting of thereof.

[0016] 2. The antibody according to item 1, wherein the antibody comprises: (a) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; (b) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively; (c) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 5, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively; (d) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 8, 9 and 10, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; (e) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 8, 9 and 10, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; (f) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; (g) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (h) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (h) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (f) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; NO: 39, 40 and 41, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 48, 49 and 50, respectively; (j) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 42, 43 and 44, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 54, 55 and 56, respectively.

[0017] 3. The antibody according to item 1, comprising: (a) HCDR1, comprising the amino acid sequence of SEQ ID NO: 1, preferably consisting of it; (b) HCDR2, comprising the amino acid sequence of SEQ ID NO: 2, preferably consisting of it; (c) HCDR3, comprising the amino acid sequence of SEQ ID NO: 3, preferably consisting of it; (d) LCDR1, comprising the amino acid sequence of SEQ ID NO: 17, preferably consisting of it; (e) LCDR2, comprising the amino acid sequence of SEQ ID NO: 18, preferably consisting of it; and (f) LCDR3, comprising the amino acid sequence of SEQ ID NO: 19, preferably consisting of it.

[0018] 4. The antibody according to item 1, comprising: (a) HCDR1, comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, preferably consisting thereof; (b) HCDR2, comprising the amino acid sequence of SEQ ID NO: 6, preferably consisting thereof; (c) HCDR3, comprising the amino acid sequence of SEQ ID NO: 7, preferably consisting thereof; (d) LCDR1, comprising the amino acid sequence of SEQ ID NO: 20, preferably consisting thereof; (e) LCDR2, comprising the amino acid sequence of SEQ ID NO: 21, preferably consisting thereof; and (f) LCDR3, comprising the amino acid sequence of SEQ ID NO: 22, preferably consisting thereof.

[0019] 5. The antibody according to item 1, comprising: (a) HCDR1, comprising the amino acid sequence of SEQ ID NO: 32, preferably consisting of it; (b) HCDR2, comprising the amino acid sequence of SEQ ID NO: 33, preferably consisting of it; (c) HCDR3, comprising the amino acid sequence of SEQ ID NO: 34, preferably consisting of it; (d) LCDR1, comprising the amino acid sequence of SEQ ID NO: 48, preferably consisting of it; (e) LCDR2, comprising the amino acid sequence of SEQ ID NO: 49, preferably consisting of it; and (f) LCDR3, comprising the amino acid sequence of SEQ ID NO: 50, preferably consisting of it.

[0020] 6. The antibody according to item 1, comprising: (a) HCDR1, comprising the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, preferably consisting thereof; (b) HCDR2, comprising the amino acid sequence of SEQ ID NO: 37, preferably consisting thereof; (c) HCDR3, comprising the amino acid sequence of SEQ ID NO: 38, preferably consisting thereof; (d) LCDR1, comprising the amino acid sequence of SEQ ID NO: 51, preferably consisting thereof; (e) LCDR2, comprising the amino acid sequence of SEQ ID NO: 52, preferably consisting thereof; and (f) LCDR3, comprising the amino acid sequence of SEQ ID NO: 53, preferably consisting thereof.

[0021] 7. The antibody according to any one of the preceding items, wherein the antibody comprises: a heavy chain variable region (VH), wherein the VH is VH1, VH3 or VH4, preferably VH3 or VH4, more preferably VH3.

[0022] 8. The antibody according to any one of the preceding items, wherein the antibody comprises: a light chain variable region (VL), wherein the VL comprises Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and a framework FR4, wherein the framework FR4 is selected from Vκ FR4, in particular Vκ1 FR4, Vκ3 FR4 and Vλ FR4, in particular a Vλ FR4 comprising an amino acid sequence having at least 60, 70, 80, 90% identity with an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably a Vλ FR4 as shown in any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably a Vλ FR4 as shown in SEQ ID NO: 64 or 65, more preferably a Vλ FR4 as shown in SEQ ID NO: 64.

[0023] 9. The antibody according to any of the preceding items, wherein the antibody comprises: a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16; and a light chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 126, 27, 57 and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27.

[0024] 10. The antibody according to any of the preceding items, wherein the antibody comprises: (a) a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 26; (b) a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 26; (c) a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 27; (d) a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 57; (f) a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: NO:46; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:58; (g) a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:47; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:57;

[0025] 11. The antibody according to any one of the preceding items, wherein the antibody comprises: a heavy chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16; and a light chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 57 and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27.

[0026] 12. The antibody according to any one of the preceding items, comprising: (a) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26; (b) a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 26; (c) a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 27; (d) a VH sequence of SEQ ID NO: 45 and a VL sequence of SEQ ID NO: 57; (f) a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 58; or (g) a VH sequence of SEQ ID NO: 47 and a VL sequence of SEQ ID NO: 57.

[0027] 13. The antibody according to any one of the preceding items, wherein the antibody:

[0028] (i) binds to human PDL1 with a dissociation constant (KD) of less than 5 nM, particularly less than 1 nM, particularly less than 500 pM, more particularly less than 100 pM, preferably less than 50 pM, more preferably less than 10 pM, as measured by surface plasmon resonance (SPR), in particular, wherein the antibody is a scFv (monovalent affinity);

[0029] (ii) K of binding to human PDL1 off The rate is 10 -3 s -1 or lower, or 10 -4 s -1 or lower, or 10 -5 s -1 or lower, as measured by SPR, in particular, wherein the antibody is a scFv;

[0030] (iii) K of binding to human PDL1 on The rate is at least 10 3 M -1 s -1 or higher, at least 10 4 M -1 s -1 or higher, at least 10 5 M -1 s -1 or higher, at least 10 6 M -1 s -1 or higher, as measured by SPR, in particular, wherein the antibody is a scFv;

[0031] (iv) cross-reacts with cynomolgus monkey (Macaca fascicularis) PDL1, in particular binds to cynomolgus monkey PDL1 with a KD of less than 5 nM, in particular less than 1 nM, in particular less than 500 pM, more in particular less than 100 pM, preferably less than 10 pM, as measured by SPR, in particular, wherein the antibody is a scFv;

[0032] (v) does not cross-react with Mus musculus PDL1, particularly as measured by SPR; and / or

[0033] (vi) does not bind to human PDL2, particularly as measured by SPR.

[0034] 14. The antibody according to any one of the preceding items, wherein the antibody has the following characteristics:

[0035] (i) having the ability to neutralize the PDL1 / PD-1 interaction, with a potency relative to the potency of avelumab (relative potency) (measured in an ELISA assay) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4; and wherein the relative potency is the IC of avelumab 50 The values ​​(in ng / mL, as measured in an ELISA assay) are related to the IC 50 values ​​(in ng / mL as measured in an ELISA assay), particularly wherein the antibody is a scFv; and / or

[0036] (ii) optionally, having an ability to neutralize the PDL1 / PD-1 interaction, with a potency relative to the potency of avelumab (relative potency) (measured in a NFAT reporter gene assay) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4; and wherein the relative potency is the IC of avelumab 50 The values ​​(in ng / mL, as measured in the NFAT reporter gene assay) were compared with the IC 50 values ​​(in ng / mL as measured in a NFAT reporter gene assay), particularly wherein the antibody is a scFv; and / or

[0037] (iii) having an ability to neutralize the PDL1 / B7-1 interaction with a potency relative to the potency of avelumab (relative potency) (measured in an ELISA assay) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4; and wherein the relative potency is the IC of avelumab 50 The values ​​(in ng / mL, as measured in an ELISA assay) are related to the IC 50The ratio of the values ​​(in ng / mL as measured in an ELISA assay) is particularly wherein the antibody is a scFv.

[0038] 15. The antibody according to any one of the preceding items, wherein the antibody:

[0039] (i) when in scFv format, has a melting temperature (Tm) (determined by differential scanning fluorimetry) of at least 60°C, preferably at least 65°C, more preferably at least 70°C, in particular, wherein the antibody is formulated in 50 mM phosphate citrate buffer, pH 6.4, 150 mM NaCl;

[0040] (ii) when in scFv format, when the starting concentration of the antibody of the invention is 10 mg / ml, and in particular, wherein the antibody is formulated in 50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.4, after 5 consecutive freeze-thaw cycles, the antibody loses less than 5%, preferably less than 3%, more preferably less than 1% of its monomer content; and / or

[0041] (iii) when in scFv format, when the starting concentration of the antibody of the invention is 10 mg / ml, and in particular wherein the antibody is formulated in 50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.4, the antibody loses less than 15% of its monomer content, such as less than 12%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1% after storage at 4°C for at least 2 weeks, in particular at least 4 weeks.

[0042] 16. The antibody according to any of the preceding items, wherein the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, Fab, Fv, scFv, dsFv, scAb and binding domains based on other scaffolds, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectin and binding sites constructed in antibody constant regions (e.g., Modular Antibody Technology of F-star). TM ).

[0043] 17. The antibody according to any one of the preceding items, wherein the antibody is a single chain variable fragment (scFv) or Fv.

[0044] 18. The antibody according to item 17, wherein the scFv has an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, preferably SEQ ID NO: 29 and SEQ ID NO: 31, more preferably SEQ ID NO: 31.

[0045] 19. The antibody according to item 16, wherein the antibody is an IgG selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, preferably, wherein the antibody is IgG1.

[0046] 20. The antibody according to any of the preceding items, wherein the antibody is chimeric or humanized.

[0047] 21. An antibody that binds to substantially the same epitope as the antibody of any one of items 1 to 20.

[0048] 22. The antibody according to any one of the preceding items, which is a multispecific molecule, in particular a multispecific molecule having at least a second functional molecule.

[0049] 23. The antibody of item 22, wherein the form of the antibody is selected from the group consisting of: single-chain diabody (scDb), tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), bispecific T cell engager (BiTE; tandem bi-scFv), tandem tri-scFv, triabody (Fab-(scFv)2) or diabody (Fab-(scFv)1), Fab, Fab-Fv2, Morrison (IgG CH3-scFv fusion (Morrison L) or IgG CL-scFv fusion (Morrison H)), triabodies, scDb-scFv, bispecific Fab2, diabodies, tetrabodies, scFv-Fc-scFv fusions, scFv-HSA-scFv fusions, diabodies, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions such as bsAb (scFv linked to the C-terminus of the light chain), Bs1Ab (scFv linked to the N-terminus of the light chain), Bs2Ab (scFv linked to the N-terminus of the heavy chain), Bs3Ab (scFv linked to the C-terminus of the heavy chain), Ts1Ab (scFv linked to the N-terminus of the heavy and light chains), Ts2Ab (dsscFv linked to the C-terminus of the heavy chain), bispecific antibodies based on heterodimeric Fc domains such as Knob-into-Hole antibodies (KiH); Fv, scFv, scDb, tandem bi-scFv, tandem tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD fused to the N- and / or C-terminus of any one chain of a heterodimeric Fc domain or any other heterodimeric domain; MATCH and DuoBody.

[0050] 24. A pharmaceutical composition comprising the antibody according to any one of items 1 to 23 and a pharmaceutically acceptable carrier.

[0051] 25. The antibody according to any one of items 1 to 23, or the composition according to item 24, for use as a medicament.

[0052] 26. The antibody of any one of items 1 to 23, or the composition of item 24, for use in treating cancer in a subject in need thereof.

[0053] 27. Use of the antibody of any one of items 1 to 23 or the composition of item 24 for treating cancer in a subject in need thereof.

[0054] 28. Use of the antibody of any one of items 1 to 23 or the composition of item 24 in the preparation of a medicament for treating cancer in a subject in need thereof.

[0055] 29. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of items 1 to 23 or the composition of item 24.

[0056] 30. A nucleic acid encoding the antibody according to any one of items 1 to 23.

[0057] 31. A vector comprising the nucleic acid of item 31.

[0058] 32. A host cell comprising the nucleic acid according to item 31 or the vector according to item 32.

[0059] 33. A method for producing the antibody according to any one of items 1 to 23, the method comprising the following steps: culturing a host cell comprising the nucleic acid according to item 31 or the vector according to item 31.

[0060] 34. A kit comprising the antibody according to any one of items 1 to 23, or the composition according to item 24. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Neutralization of PDL1 / PD-1 interaction by rabbit IgG clones with the best affinity for PDL1. Absorbance measured by ELISA is expressed as a function of 33-03-G02 (A) or 37-20-B03 (B) molecular concentration (in ng / ml). Avelumab was used as reference.

[0062] Figure 2 Neutralization of PDL1 / PD-1 interaction by selected rabbit IgG clones 33-03-G02 (A) and 37-20-B03 (B) with the best affinity to PDL1. The absorbance measured by ELISA is expressed as a function of the molecular concentration in ng / ml. Avelumab was used as a reference.

[0063] Figure 3 Neutralization of PDL1 / PD-1 interaction by selected rabbit IgG clones with the best affinity for PDL1 in a cell-based reporter gene assay. The % inhibition proportional to the luminescence signal obtained in the assay is expressed as a function of the molecular concentration in ng / ml. Avelumab was used as a reference.

[0064] Figure 4 Figure 4A shows the effect of CDR set and framework selection on the neutralization of PDL1 / PD-1 interaction in the NFAT-luciferase reporter assay. The % inhibition proportional to the luminescence signal obtained in the assay is expressed as a function of the scFvs concentration (in ng / ml). Avelumab was used as a reference. Figure 4 B shows the effect of domain optimization on the neutralization potency of PDL1 / PD-1 interaction in NFAT-luciferase reporter assay. The % inhibition proportional to the luminescence signal obtained in the assay is expressed as a function of scDbs concentration (in ng / ml). Avelumab was used as reference.

[0065] Figure 5 Neutralization potency of scDb-scFvs PRO963 and PRO1057 (A), PRO1186 and PRO1430 (B), PRO1431 and PRO1432 (C), PRO1473 (D), PRO1476 (E), PRO1479 (F) and PRO1482 (G) on PDL1 / PD-1 interaction in the presence of recombinant human serum albumin in a reporter gene assay. The % inhibition proportional to the luminescence signal obtained in the assay is expressed as a function of the molecular concentration (in ng / ml). Avelumab was used as a reference.

[0066] Figure 6 Effect of the potency of bivalent molecules and the neutralization potency of LC or HC scFv fusions in Morrison format on the PDL1 / PD-1 interaction in the NFAT-luciferase reporter assay. The % inhibition proportional to the luminescence signal obtained in the assay is expressed as a function of the molecule concentration (in ng / ml). Avelumab was used as a reference.

[0067] Figure 7 PD-1 / PDL1 competition ELISA. All molecules effectively blocked the interaction between PD-1 and PDL1, IC 50 Values ​​were similar or smaller than those of the reference IgG avelumab.

[0068] Figure 8 B7-1 / PDL1 competition ELISA. Similar to avelumab, all molecules effectively blocked the interaction between B7-1 and PDL1.

[0069] Fig. 9In vitro T cell activation assay. PBMCs were stimulated with 10 ng / ml SEA and treated with serially diluted scFvPRO997 or scDb PRO885 for 96 hours. T cell activation was assessed by ELISA quantification of IL-2 in the harvested supernatant. Treatment with PRO885 and PRO997 resulted in significant IL-2 secretion. PRO997 showed higher potency than Avelumab. The effect size of PRO885 was greatly increased compared to Avelumab. Data were fitted using S-curve 4PL fitting (GraphPad Prism).

[0070] Fig.10 Antitumor activity of anti-PDL1 IgG1 (PRO1137) treatment was performed in human HCC827 NSCLC xenografts using the immunodeficient NOG mouse strain and allogeneic human peripheral blood mononuclear cells (hPBMCs). Mice were treated with anti-PDL1 IgG1 (PRO1137) or vehicle intravenously on days 0, 3, 7, and 10. Tumor volume was measured twice a week until mice were sacrificed on days 17 and 18. Tumor volume was normalized to the tumor volume at the start of treatment (relative tumor volume). (A) Average relative tumor volume of mice reconstituted with PBMCs from two donors (n = 8 mice per group). The dotted line indicates the treatment time. (B) Average relative tumor volume of mice reconstituted with PBMCs from donor B (n = 4 mice per group). (C) Individual relative tumor volume of mice reconstituted with PBMCs from two donors. Each symbol represents a single animal within the same treatment group. (D) Individual relative tumor volume of mice reconstituted with PBMCs from donor B. Each symbol represents an individual animal within the same treatment group.

[0071] Fig.11 HCC827 xenografts in hPBMC-replaced NOG mice. Body weights of HCC827-challenged NOG mice after treatment with anti-PDL1 IgG1 (PRO1137). Body weights were measured twice a week until mice were sacrificed on days 17 and 18. Body weights were normalized to the body weight at the start of treatment (relative body weight).

[0072] Fig.12Evaluation of the anti-tumor efficacy of anti-PDL1 IgG1 (PRO1196) in human HCC827 NSCLC xenografts in NOG mice implanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs). The anti-tumor activity of PRO1196 (0.1 mg) was compared with avelumab (0.1 mg) or vehicle treatment (palivizumab, 0.1 mg). Mice were treated on days 0, 5, 10, 15, and 20 (dashed lines). Tumor growth and body weight were recorded twice a week. Tumor volume was normalized to the tumor volume at the start of treatment (relative tumor volume). DETAILED DESCRIPTION

[0073] The present invention provides antibodies that specifically bind to human PDL1 protein, as well as pharmaceutical compositions, production methods, and methods of using such antibodies and compositions.

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0075] Unless otherwise indicated, the terms "including" and "comprising" are used herein in an open and non-limiting sense. With respect to these latter embodiments, the term "comprising" therefore encompasses the narrower term "consisting of."

[0076] Unless otherwise indicated herein or clearly contradicted by context, in the context of describing the invention (particularly in the context of the following claims), the terms "a" and "an" and "the" and similar references should be construed to cover both the singular and the plural. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. When compounds, salts, etc. are used in the plural, this is also considered to refer to a single compound, salt, etc.

[0077] In a first aspect, the present invention relates to an antibody that specifically binds to human PDL1.

[0078] As used herein, the term "antibody" and the like include: complete antibodies or single chains thereof; any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains; and molecules (including but not limited to multispecific antibodies) comprising antibody CDRs, VH regions, or VL regions. A naturally occurring "complete antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0079] As used herein, the terms "antigen-binding fragment", "antigen-binding fragment thereof", "antigen-binding portion" and the like refer to one or more fragments of an intact, complete antibody that retains the ability to specifically bind to a given antigen (e.g., PDL1). Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; a F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; a Fd fragment, which consists of VH and CH1 domains; a Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; and binding domains based on other scaffolds, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectin, and binding sites constructed in antibody constant regions (e.g., Modular Antibody Technology of F-star). TM ).

[0080] The term "complementarity determining region" ("CDR") is an amino acid sequence having boundaries defined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme), and Honegger & Plückthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering scheme). For example, for the classical form, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 5 The amino acid residues in the VL are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-3 4 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2) and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2) and 89-97 (LCDR3) (according to "Kabat" numbering).Under IMGT, the CDRs of antibodies can be determined using the program IMGT / DomainGap Align. In the context of the present invention, unless otherwise specifically indicated, the numbering system suggested by Honegger & Plückthun ("AHo") is used (Honegger & Plückthun, J. Mol. Biol. 309 (2001) 657-670). In addition, according to the AHo numbering scheme, the following residues are defined as CDRs: LCDR1 (also referred to as CDR-L1): L24-L42; LCDR2 (also referred to as CDR-L2): L58-L72; LCDR3 (also referred to as CDR-L3): L107-L138; HCDR1 (also referred to as CDR-H1): H27-H42; HCDR2 (also referred to as CDR-H2): H57-H76; HCDR3 (also referred to as CDR-H3): H108-H138. For the sake of clarity, the numbering system according to Honegger & Plückthun takes into account the length diversity found in naturally occurring antibodies in both different VH and VL subfamilies, particularly in the CDRs, and provides for gaps in the sequence. Thus, in a given antibody variable domain, generally not all positions 1 to 149 are occupied by amino acid residues.

[0081] The antigen binding portion can also be incorporated into a maxibody, a minibody, an intrabody, a diabody, a triabody, a tetrabody, a scDb-scFv, a v-NAR, and a bi-scFv (see, e.g., Holliger and Hudson, 2005, Nature Biotechnology, 23, 1126-36). The antigen binding portion of an antibody can be transplanted into a scaffold based on a polypeptide, such as type III fibronectin (Fn3) (see U.S. Pat. No. 6,703,199, which describes a fibronectin polypeptide monoclonal antibody). The antigen binding portion can be incorporated into a single-chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8 (10): 1057-1062; and U.S. Pat. No. 5,641,870).

[0082] As used herein, the term "binding specificity" refers to the ability of a single antibody to react with a kind of antigenic determinant but not with different antigenic determinants. As used herein, the term "with ... specific binding" or "specific for" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which is a determinant of the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that has a greater affinity, greater avidity, easier and / or longer duration than that of binding to the target than to other targets. In its most general form (and when the defined standard is not mentioned), "specific binding" refers to the ability of an antibody to distinguish between a target and an unrelated molecule, such as determined by a specific determination method known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) tests and peptide scanning. For example, standard ELISA assays can be performed. Scoring can be performed by standard color development (e.g., a secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). The reactions in certain wells are scored by optical density (e.g. at 450 nm). A typical background (= negative reaction) may be about 0.1 OD; a typical positive reaction may be about 1 OD. This means that the ratio between positive and negative scores can be 10 times or higher. In another example, an SPR assay can be performed, in which a difference of at least 10 times, preferably at least 100 times, between background and signal indicates specific binding. Typically, a group of about three to five unrelated molecules, such as milk powder, transferrin, etc., is used, rather than a single reference molecule, to determine binding specificity. The antibodies of the present invention have binding specificity to human PDL1. In a specific embodiment, the antibodies of the present invention have binding specificity to human PDL1 and do not bind to human PDL2, particularly as determined by SPR.

[0083] Suitably, the antibody of the present invention is an isolated antibody. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to PDL1 is substantially free of antibodies that specifically bind to antigens other than PDL1). However, an isolated antibody that specifically binds to PDL1 may have cross-reactivity with other antigens (e.g., PDL1 molecules from other species). Therefore, in one embodiment, the antibody of the present invention has binding specificity to human PDL1 and cynomolgus monkey (Macaca fascicularis) (also referred to as cynomolgus monkey or "cynomolgus") PDL1. In addition, the isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0084] Suitably, the antibodies of the present invention are monoclonal antibodies. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to antibodies having substantially identical amino acid sequences or derived from the same genetic source. A monoclonal antibody composition exhibits binding specificity and affinity for one specific epitope, or exhibits binding specificity and affinity for multiple specific epitopes.

[0085] Antibodies of the present invention include, but are not limited to, chimeric and humanized.

[0086] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or type, or to a completely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is altered, replaced or exchanged for a variable region with a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with that of a human immunoglobulin. Due to replacement with a human constant region, a chimeric antibody can retain its specificity for recognizing an antigen while having reduced antigenicity to humans compared to the original mouse antibody.

[0087] As used herein, a "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having low immunogenicity to humans. For example, this can be achieved by retaining the non-human CDR region and replacing the remainder of the antibody with a human counterpart (i.e., the framework portion of the constant region and the variable region). Additional framework region modifications can be made within the human framework sequence and the CDR sequence derived from the germline of another mammalian species. The humanized antibodies of the present invention may include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo, or conservative substitutions to promote stability or preparation). See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of human engineering technology include, but are not limited to, the Xoma technology disclosed in U.S. Pat. No. 5,766,886.

[0088] As used herein, the term "recombinant humanized antibody" includes all human antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from host cells transformed to express humanized antibodies, such as antibodies isolated from transfectomas, as well as antibodies prepared, expressed, created or isolated by any other means involving splicing all or part of human immunoglobulin genes to other DNA sequences.

[0089] The term "PDL1" particularly refers to human PDL1 with UniProt ID number Q9NZQ7, reproduced herein as SEQ ID NO: 63. Suitably, the antibodies of the invention target PDL1, particularly human PDL1, as shown in UniProt ID number Q9NZQ7, which is reproduced herein as SEQ ID NO: 63. Suitably, the antibodies of the invention target human and cynomolgus (Macaca fascicularis) PDL1, and preferably do not cross-react with Mus musculus PDL1, particularly as measured by surface plasmon resonance (SPR). Suitably, the antibodies of the invention have binding specificity for human PDL1. In particular, the antibodies of the invention do not bind to human PDL2, particularly as measured by SPR.

[0090] The antibodies of the present invention are PDL1 inhibitors. The term "blocker" or "blocking antibody" or "inhibitor" or "inhibiting antibody" or "antagonist" or "antagonist antibody" refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The antibodies of the present invention target, reduce, and inhibit the binding ability of PDL1 to its binding partner, thereby interfering with the function of PDL1. In particular, the antibodies of the present invention block the interaction of PDL1 with PD-1. In some embodiments, the antibodies of the present invention block the interaction of PDL1 with PD-1 and B7-1.

[0091] Antibodies of the invention include, but are not limited to, isolated humanized monoclonal antibodies as described herein, including in the Examples. Examples of such anti-human PDL1 antibodies are antibodies whose sequences are listed in Table 1. Additional details on the generation and characterization of the antibodies described herein are provided in the Examples.

[0092] The isolated antibody of the present invention having binding specificity to human PDL1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 in sequence, and (b) the VL comprises three complementarity determining regions LCDR1, LCDR2 and LCDR3 in sequence.

[0093] The present invention provides an antibody that specifically binds to a PDL1 protein, the antibody comprising a VH CDR, the VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 1. In particular, the present invention provides an antibody that specifically binds to a PDL1 protein VH CDR, the antibody comprising one, two, three or more VH CDRs, the VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 1.

[0094] The present invention also provides an antibody that specifically binds to a PDL1 protein, the antibody comprising a VL CDR, and the VL CDR has an amino acid sequence of any one of the VL CDRs listed in Table 1. In particular, the present invention provides an antibody that specifically binds to a PDL1 protein VLCDR, the antibody comprising one, two, three or more VL CDRs, and the VL CDR has an amino acid sequence of any one of the VLCDRs listed in Table 1.

[0095] Other antibodies of the present invention include amino acid sequences that have been mutated but still specifically bind to the PDL1 protein and have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity in the CDR region with the CDR region described in the sequence described in Table 1. In one aspect, other antibodies of the present invention include mutant amino acid sequences that specifically bind to the PDL1 protein, wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR region when compared to the CDR region described in the sequence described in Table 1.

[0096] In the context of two or more nucleic acids or polypeptide sequences, the term "same" or "identity" refers to two or more sequences or subsequences that are identical. "Percentage (%) of sequence identity" and "homology" with respect to nucleic acids, peptides, polypeptides or antibody sequences are defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to the nucleotides or amino acid residues in a particular nucleic acid, peptide or polypeptide sequence, after alignment of the sequences and introduction of gaps, if necessary, to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using disclosed computer software, such as BLAST, BLAST-2 or ALIGN software. One skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0097] To perform sequence comparison, one sequence is typically used as a reference sequence to which a test sequence is compared. When a sequence comparison algorithm is used, the test and reference sequences are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters may be used, or other parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0098] Two examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988), which has been incorporated into the ALIGN program (version 2.0), which uses the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0099] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0100] The present invention provides an isolated antibody with binding specificity to human PDL1, comprising (a) a heavy chain variable region CDR1 (HCDR1), comprising an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, preferably SEQ ID NOs: 1 or 32, more preferably SEQ ID NO: 1, and preferably consisting thereof; (b) a heavy chain variable region CDR2 (HCDR2), comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NOs: 2 or 33, more preferably SEQ ID NO: 2, and preferably consisting thereof; (c) a heavy chain variable region CDR3 (HCDR3), comprising an amino acid sequence selected from any one of SEQ ID NOs: 3, 7, 10, 13, 34, 38, 41 and 44, preferably SEQ ID NOs: 3 or 34, more preferably SEQ ID NOs: NO:3, preferably consisting of the amino acid sequence; (d) a light chain variable region CDR1 (LCDR1) comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 17, 20, 23, 48, 51 and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17; (e) a light chain variable region CDR2 (LCDR2) comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 18, 21, 24, 49, 52 and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18; and (f) a light chain variable region CDR3 (LCDR3) comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 19, 22, 25, 50, 53 and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19.

[0101] Suitably, the isolated antibody of the present invention having binding specificity to human PDL1 comprises: (a) a heavy chain variable region CDR1 (HCDR1) comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, preferably SEQ ID NOs: 1 or 32, more preferably SEQ ID NO: 1; (b) a heavy chain variable region CDR2 (HCDR2) comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NOs: 2 or 33, more preferably SEQ ID NO:2 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity, preferably consisting of the same; (c) a heavy chain variable region CDR3 (HCDR3) comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one of SEQ ID NO:3, 7, 10, 13, 34, 38, 41 and 44, preferably SEQ ID NO:3 or 34, more preferably SEQ ID NO:3; (d) a light chain variable region CDR1 (LCDR1) comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one of SEQ ID NO:17, 20, 23, 48, 51 and 54, preferably SEQ ID NO:17 or 48, more preferably SEQ ID NO:17 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity, preferably consisting of the same; (e) a light chain variable region CDR2 (LCDR2) comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one of SEQ ID NO:18, 21, 24, 49, 52 and 55, preferably SEQ ID NO:18 or 49, more preferably SEQ ID NO:18; and (f) a light chain variable region CDR3 (LCDR3) comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one of SEQ ID NO:9, 22, 25, 50, 53 and 56, preferably SEQ ID NO:19 or 50, more preferably SEQ ID NO:19 has, preferably consists of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical.

[0102] In one embodiment, an antibody of the invention having binding specificity to human PDL1 comprises: (a) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, respectively; (b) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 4, 6, and 7, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 20, 21, and 22, respectively; (c) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 5, 6, and 7, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 20, 21, and 22, respectively; (d) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 8, 9, and 10, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, respectively; (e) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 8, 9, and 10, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, respectively; (f) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; (g) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (h) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (h) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (f) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; NO: 39, 40 and 41, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 48, 49 and 50, respectively; (j) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 42, 43 and 44, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 54, 55 and 56, respectively.In one embodiment, an antibody of the invention having binding specificity to human PDL1 comprises HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, respectively. In another embodiment, an antibody of the invention having binding specificity to human PDL1 comprises HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 48, 49, and 50, respectively.

[0103] Suitably, an antibody of the invention having binding specificity for human PDL1 comprises: (a) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NOs: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NOs: 17, 18 and 19, respectively; (b) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NOs: 4, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NOs: NO: 20, 21 and 22 have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity LCDR1, LCDR2 and LCDR3 sequences; (c) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 5, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 20, 21 and 22, respectively; (d) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: NO: 8, 9 and 10 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity, and SEQ ID NO: 17, 18 and 19 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity LCDR1, LCDR2 and LCDR3 sequences, respectively; (e) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 11, 12 and 13, respectively, and SEQ ID NO: 14, 15 and 16 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: NOs: 23, 24 and 25 have LCDR1, LCDR2 and LCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical;(f) HCDR1, HCDR2 and HCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 48, 49 and 50, respectively; (g) HCDR1, HCDR2 and HCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 35, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: NO: 51, 52 and 53 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity LCDR1, LCDR2 and LCDR3 sequences; (h) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 36, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 51, 52 and 53, respectively; (i) HCDR1, HCDR2 and HCDR3 sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 39, 40, and 41, and LCDR1, LCDR2, and LCDR3 sequences at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 48, 49, and 50, respectively;(j) HCDR1, HCDR2 and HCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 42, 43 and 44, respectively, and LCDR1, LCDR2 and LCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NOs: 54, 55 and 56, respectively. In one embodiment, an antibody of the invention having binding specificity to human PDL1 comprises HCDR1, HCDR2, and HCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 17, 18, and 19, respectively. In another embodiment, an antibody of the invention having binding specificity to human PDL1 comprises HCDR1, HCDR2, and HCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 48, 49, and 50, respectively. ;

[0104] Suitably, the antibody of the present invention having binding specificity to human PDL1 comprises: (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1; (b) HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 2; (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 3; (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 17; (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 18; and (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 19. Suitably, the antibody of the present invention having binding specificity for human PDL1 comprises: (a) HCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 1; (b) HCDR2 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 2; (c) HCDR3 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 3; (d) LCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: NO:17 having an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto; (e) LCDR2 comprising, preferably consisting of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto; and (f) LCDR3 comprising, preferably consisting of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0105] In another embodiment, the antibody of the present invention having binding specificity to human PDL1 comprises: (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20; (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 21; and (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 22. Suitably, the antibody of the invention having binding specificity for human PDL1 comprises: (a) HCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 4 or SEQ ID NO: 5; (b) HCDR2 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 6; (c) HCDR3 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 7; (d) LCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: NO:20 having an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto; (e) LCDR2 comprising, preferably consisting of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto; and (f) LCDR3 comprising, preferably consisting of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0106] Suitably, the antibody of the present invention having binding specificity to human PDL1 comprises: (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 32; (b) HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 33; (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 34; (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 48; (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 49; and (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 50. Suitably, an antibody of the invention having binding specificity for human PDL1 comprises: (a) a HCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 32; (b) a HCDR2 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 33; (c) a HCDR3 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 34; (d) a LCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: NO:48 having an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto, (e) LCDR2 comprising, preferably consisting of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto, and (f) LCDR3 comprising, preferably consisting of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0107] In another embodiment, the antibody of the present invention having binding specificity to human PDL1 comprises: (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36; (b) HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 37; (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 38; (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 51; (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 52; and (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 53. Suitably, an antibody of the invention having binding specificity for human PDL1 comprises: (a) a HCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 35 or SEQ ID NO: 36; (b) a HCDR2 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 37; (c) a HCDR3 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 38; (d) a LCDR1 comprising, preferably consisting of, an amino acid sequence having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: NO:51 has an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto, (e) LCDR2 comprises, preferably consists of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto, and (f) LCDR3 comprises, preferably consists of, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0108] In another embodiment, the present invention provides an antibody that specifically binds to PDL1 (e.g., human PDL1 protein), wherein the antibody comprises a VH domain and a VL domain. In the context of the present invention, the terms "VH" (variable heavy chain), "VL" (variable light chain), "Vκ" and "Vλ" refer to a family of antibody heavy and light chain sequences grouped according to sequence identity and homology. Methods for determining sequence homology, such as by using a homology search matrix such as BLOSUM (HeniKoff, S. & HeniKoff, JG, Proc. Natl. Acad. Sci. USA 89 (1992) 10915-10919), and methods for grouping sequences according to homology are well known to those of ordinary skill in the art. For VH, Vκ and Vλ, different subfamilies can be identified, such as shown in Knappik et al., J.Mol.Biol.296 (2000) 57-86, which groups VH in VH1A, VH1B and VH2 into VH6, Vκ in Vκ1 into Vκ4, and Vλ in Vλ1 into Vλ3. In vivo, antibody Vκ chains, Vλ chains and VH chains are the result of random rearrangements of germline κ chain V and J segments, germline λ chain V and J segments, and heavy chain V, D and J segments, respectively. Which subfamily a given antibody variable chain belongs to is determined by the corresponding V segment, in particular by the framework region FR1 to FR3. Therefore, any VH sequence characterized only by a specific set of framework regions HFR1 to HFR3 in the present application can be combined with any HFR4 sequence, such as an HFR4 sequence obtained from one of the heavy chain germline J segments, or an HFR4 sequence obtained from a rearranged VH sequence.

[0109] Suitably, the present invention provides an isolated antibody that specifically binds to PDL1 (eg, human PDL1 protein), wherein the antibody comprises VH1A, VH1B, VH3 or VH4.

[0110] A specific example of a VH belonging to the VH1 family is represented by SEQ ID NO: 15. In particular, the framework regions FR1 to FR4 taken from SEQ ID NO: 15 belong to the VH1 family (Table 1, non-bold marked regions). Suitably, the VH belonging to the VH1 family used herein comprises FR1 to FR4 having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with FR1 to FR4 of SEQ ID NO: 15.

[0111] A specific example of a VH belonging to the VH3 family is represented by SEQ ID NO: 16. In particular, the framework regions FR1 to FR4 taken from SEQ ID NO: 16 belong to the VH3 family (Table 1, non-bold marked regions). Suitably, the VH belonging to the VH3 family used herein comprises FR1 to FR4 having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with FR1 to FR4 of SEQ ID NO: 16.

[0112] A specific example of a VH belonging to the VH4 family is represented by SEQ ID NO: 14. In particular, the framework regions FR1 to FR4 taken from SEQ ID NO: 14 belong to the VH4 family (Table 1, non-bold marked regions). Suitably, the VH belonging to the VH4 family used herein comprises FR1 to FR4 having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with FR1 to FR4 of SEQ ID NO: 14.

[0113] Further examples of VH sequences can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86.

[0114] In one embodiment, the isolated antibody of the invention comprises a VH4 or VH3 domain.

[0115] Suitably, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), wherein the antibody comprises Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 framework, preferably Vκ1 framework FR1 to 3, and framework FR4, the framework FR4 is selected from Vκ FR4, in particular Vκ1 FR4, Vκ3 FR4 and Vλ FR4. Suitable Vκ1 framework FR1 to 3 are listed in SEQ ID NO: 26 (Table 1, FR region is marked in non-bold). Suitable Vκ1 framework FR1 to 3 comprises an amino acid sequence having at least 60, 70, 80, 90% identity with an amino acid sequence corresponding to FR1 to 3 and selected from SEQ ID NO: 26 (Table 1, FR region is marked in non-bold).

[0116] Further examples of Vκ1 sequences, as well as further examples of Vκ2, Vκ3 or Vκ4 sequences can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86.

[0117] Suitable Vλ FR4 is shown in SEQ ID NO: 64 to SEQ ID NO: 70. In a preferred embodiment, Vλ FR4 is shown in SEQ ID NO: 64 or 65, more preferably, Vλ FR4 is shown in SEQ ID NO: 64. In one embodiment, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), wherein the antibody comprises Vλ FR4, and the Vλ FR4 comprises an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably SEQ ID NO: 64 or 65, more preferably SEQ ID NO: 64 having at least 60, 70, 80, 90% identity.

[0118] Therefore, in one embodiment, the present invention thus provides an antibody comprising:

[0119] (i) the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences:

[0120] a. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively;

[0121] b. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; or

[0122] c. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively;

[0123] (ii) a VH3 or VH4 domain framework sequence; and

[0124] (iii) a VL domain comprising a VL framework comprising Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and a framework FR4, wherein the framework FR4 is selected from Vκ FR4, in particular Vκ1FR4, Vκ3 FR4 and Vλ FR4, in particular Vλ FR4 comprising an amino acid sequence that is at least 60, 70, 80, 90% identical to any one of the amino acid sequences selected from SEQ ID NO: 64 to SEQ ID NO: 70, preferably, Vλ FR4 is as shown in SEQ ID NO: 64 to SEQ ID NO: 70, more preferably, Vλ FR4 is as shown in SEQ ID NO: 64.

[0125] Therefore, in another embodiment, the present invention provides an antibody having binding specificity to human PDL1, comprising:

[0126] (i) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 5, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively;

[0127] (ii) a VH1A, VH1B, VH3 or VH4 domain framework sequence, preferably a VH1A or VH1B domain framework sequence; and

[0128] (iii) a VL domain comprising a VL framework comprising Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and a framework FR4, wherein the framework FR4 is selected from Vκ FR4, in particular Vκ1FR4, Vκ3 FR4 and Vλ FR4, in particular Vλ FR4 comprising an amino acid sequence that is at least 60, 70, 80, 90% identical to an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably Vλ FR4 comprising an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably, Vλ FR4 is as shown in SEQ ID NO: 64.

[0129] Therefore, in a specific embodiment, the present invention provides an antibody comprising:

[0130] (i) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively;

[0131] (ii) a VH3 or VH4 domain framework sequence, preferably a VH4 domain framework sequence; and

[0132] (iii) a VL domain comprising a VL framework comprising Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and a framework FR4, wherein the framework FR4 is selected from Vκ FR4, in particular Vκ1FR4, Vκ3 FR4 and Vλ FR4, in particular Vλ FR4 comprising an amino acid sequence that is at least 60, 70, 80, 90% identical to any one of the amino acid sequences selected from SEQ ID NO: 64 to SEQ ID NO: 70, preferably, Vλ FR4 is as shown in SEQ ID NO: 64 to SEQ ID NO: 70, more preferably, Vλ FR4 is as shown in SEQ ID NO: 64.

[0133] Therefore, in a preferred embodiment, the present invention provides an antibody comprising:

[0134] (i) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively,

[0135] and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively;

[0136] (ii) a VH3 or VH4 domain framework sequence, preferably a VH3 domain framework sequence; and

[0137] (iii) a VL domain comprising a VL framework comprising Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and a framework FR4, wherein the framework FR4 is selected from Vκ FR4, in particular Vκ1FR4, Vκ3 FR4 and Vλ FR4, in particular Vλ FR4 comprising an amino acid sequence that is at least 60, 70, 80, 90% identical to any one of the amino acid sequences selected from SEQ ID NO: 64 to SEQ ID NO: 70, preferably, Vλ FR4 is as shown in SEQ ID NO: 64 to SEQ ID NO: 70, more preferably, Vλ FR4 is as shown in SEQ ID NO: 64.

[0138] Thus, in one embodiment, the invention provides an antibody having binding specificity to human PDL1 and comprising a VL comprising:

[0139] (i) CDR domains CDR1, CDR2 and CDR3;

[0140] (ii) human Vκ framework region FR1 to FR3, in particular human Vκ1 framework region FR1 to FR3;

[0141] (iii) FR4, which is selected from (a) a human Vλ germline sequence of FR4, in particular a Vλ germline sequence selected from the following group: SEQ ID NO: 64 to 70, preferably SEQ ID NO: 64; and (b) a Vλ-based sequence, which has one or two mutations, in particular one mutation, compared to the closest human Vλ germline sequence of FR4, and the closest human Vλ germline sequence of FR4 comprises an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably SEQ ID NO: 64.

[0142] The present invention provides an isolated antibody that specifically binds to PDL1 (eg, human PDL1 protein), wherein the antibody comprises a VH domain listed in Table 1.

[0143] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises a VH amino acid sequence listed in Table 1, wherein no more than about 10 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein, as various non-limiting examples, the mutation is an addition, substitution or deletion).

[0144] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises a VH amino acid sequence listed in Table 1, wherein no more than about 20 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein, as various non-limiting examples, the mutation is an addition, substitution or deletion).

[0145] Other antibodies of the invention include amino acid sequences that have been mutated but still specifically bind to PDL1 and have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity in the VH region to the VH region described in the sequence described in Table 1.

[0146] The present invention provides an isolated antibody that specifically binds to a PDL1 protein, wherein the antibody comprises a VL domain listed in Table 1.

[0147] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises a VL amino acid sequence listed in Table 1, wherein no more than about 10 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein, as various non-limiting examples, the mutation is an addition, substitution or deletion).

[0148] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises a VL amino acid sequence listed in Table 1, wherein no more than about 20 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein, as various non-limiting examples, the mutation is an addition, substitution or deletion).

[0149] Other antibodies of the invention include amino acid sequences that have been mutated but still specifically bind to PDL1 and have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity in the VL region to the VL region described in the sequence described in Table 1.

[0150] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, preferably at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16; and a light chain variable region comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, preferably at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 126, 27, 57 and 58, preferably SEQ ID NOs: 26 or 27, more preferably SEQ ID NO: 27.

[0151] In one embodiment, an antibody of the present invention having binding specificity to human PDL1 comprises: a heavy chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16; and a light chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 57 and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27.

[0152] In one embodiment, an antibody of the invention having binding specificity for human PDL1 comprises:

[0153] (a) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 14; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26;

[0154] (b) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 5, 6 and 7, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively, a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 15; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26;

[0155] (c) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 16; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26, preferably, wherein the VH comprises G56A and Y105F mutations (AHo numbering), and the VL comprises S9A and A51P mutations (AHo numbering);

[0156] (d) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 45; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 57;

[0157] (e) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 46; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 46. NO:58 is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to an amino acid sequence; preferably, wherein the VH comprises V2S, V25A, I44V, G56A, V82K, F89V and Y105F mutations (AHo numbering), and the VL comprises I2F, M4L and A51P mutations (AHo numbering); or

[0158] (f) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; a VH sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 47; and a VL sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 57, preferably, wherein the VH comprises V25A, I44V, G56A, V82K and F89V mutations (AHo numbering).

[0159] In one embodiment, an antibody of the invention having binding specificity for human PDL1 comprises:

[0160] (a) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 14; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26;

[0161] (b) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively, a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 15; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26;

[0162] (c) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 16; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 27; preferably, wherein the VH comprises G56A and Y105F mutations (AHo numbering), and the VL comprises S9A and A51P mutations (AHo numbering);

[0163] (d) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs:32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs:48, 49 and 50, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO:45; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO:57;

[0164] (e) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identical, preferably at least 90 percent identical, to SEQ ID NO: 46; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identical, preferably at least 90 percent identical, to SEQ ID NO: NO:58 is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to an amino acid sequence; preferably, wherein the VH comprises V2S, V25A, I44V, G56A, V82K, F89V and Y105F mutations (AHo numbering), and the VL comprises I2F, M4L and A51P mutations (AHo numbering); or

[0165] (f) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; a VH sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 47; and a VL sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 57, preferably, wherein the VH comprises V25A, I44V, G56A, V82K and F89V mutations (AHo numbering).

[0166] In a preferred embodiment, an antibody of the invention having binding specificity to human PDL1 comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively, a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 16; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26, preferably, wherein the VH comprises G56A and Y105F mutations (AHo numbering), and the VL comprises S9A and A51P mutations (AHo numbering).

[0167] In another embodiment, the isolated antibody of the present invention having binding specificity to human PDL1 comprises: (a) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26; (b) a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 26; (c) a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 27; (d) a VH sequence of SEQ ID NO: 45 and a VL sequence of SEQ ID NO: 57; (e) a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 58; or (f) a VH sequence of SEQ ID NO: 47 and a VL sequence of SEQ ID NO: 57. In a preferred embodiment, the isolated antibody of the present invention having binding specificity to human PDL1 comprises a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26. In a more preferred embodiment, the isolated antibody of the present invention having binding specificity to human PDL1 comprises the VH sequence of SEQ ID NO:16 and the VL sequence of SEQ ID NO:27.

[0168] In one embodiment, the antibody that specifically binds to PDL1 is an antibody described in Table 1. In one embodiment, the antibody that specifically binds to PDL1 comprises an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, preferably at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62. In one embodiment, the antibody that specifically binds to PDL1 is as shown in SEQ ID NO: 29 or SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 29, more preferably SEQ ID NO: 31. In one embodiment, the antibody that specifically binds to PDL1 is as shown in SEQ ID NO: 60 or SEQ ID NO: 61 or SEQ ID NO: 62, preferably SEQ ID NO: 60, more preferably SEQ ID NO: 62.

[0169] Other antibodies of the invention having binding specificity to human PDL1 include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but have at least 60, 70, 80, 90 or 95% identity with the sequences described in Table 1. In one embodiment, the amino acid sequences are included in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable region when compared to the variable region described in the sequence described in Table 1, while retaining substantially the same activity. As used herein, the term "substantially the same activity" means that the activity represented by substantially the same activity is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% or at least 110%, or at least 120%, or at least 130%, or at least 140%, or at least 150%, or at least 160%, or at least 170%, or at least 180%, or at least 190%, for example up to 200% of the activity determined for the parent antibody (e.g., an antibody of the invention, particularly an antibody of the invention described in Table 1).

[0170] Given that each of these antibodies can bind to PDL1 and that the antigen binding specificity is primarily provided by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain VH CDR1, 2, and 3 and VL CDR1, 2, and 3) to produce other PDL1-binding binding molecules of the present invention. Such "mixed and matched" PDL1-binding antibodies can be tested using binding assays known in the art and binding assays described in the Examples (e.g., ELISA). When mixing and matching VH CDR sequences, the CDR1, CDR2, and / or CDR3 sequences of a specific VH sequence should be replaced with structurally similar CDR sequences. Similarly, when mixing and matching VL CDR sequences, the CDR1, CDR2, and / or CDR3 sequences of a specific VL sequence should be replaced with structurally similar CDR sequences. It will be apparent to one of ordinary skill in the art that new VH and VL sequences can be generated by mutating one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences of the monoclonal antibodies of the invention set forth herein.

[0171] In yet another embodiment, the present invention provides an antibody comprising an amino acid sequence homologous to the sequence described in Table 1, and said antibody binds to PDL1 and retains the desired functional properties of those antibodies described in Table 1.

[0172] For example, the present invention provides an isolated monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 90% or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NOs: 14 or 16, more preferably SEQ ID NO: 16; the light chain variable region comprises an amino acid sequence that is at least 80%, at least 90% or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 126, 27, 57 and 58, preferably SEQ ID NOs: 26 or 27, more preferably SEQ ID NO: 27; wherein the antibody specifically binds to the PDL1 protein.

[0173] In one embodiment, the VH and / or VL amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in Table 1. In one embodiment, the VH and / or VL amino acid sequences may be identical except for amino acid substitutions at no more than 1, 2, 3, 4 or 5 amino acid positions.

[0174] In one embodiment, an antibody of the present invention has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences have a specified amino acid sequence based on the antibodies described herein or conservative modifications thereof, wherein the antibody retains the desired functional properties of the PDL1-binding antibodies of the present invention.

[0175] The term "conservatively modified variant" or "conservative variant" applies to amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or when the nucleic acid does not encode an amino acid sequence, it refers to substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is clearly specified by a codon, the codon can be changed to any one of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG and TGG, AUG is usually the only codon for methionine, and TGG is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each of the described sequences.

[0176] For polypeptide sequences, "conservatively modified variants" or "conservative variants" include single substitutions, deletions or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. These conservatively modified variants are in addition to and do not exclude the following: polymorphic variants, interspecies homologs and alleles of the present invention. The following eight groups contain amino acids that are conservatively substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In one embodiment, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing the amino acid sequence.

[0177] Thus, the present invention provides an isolated monoclonal antibody comprising or consisting of a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein:

[0178] The heavy chain variable region CDR1 comprises any one selected from SEQ ID NO: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, preferably SEQ ID NO: 1 or 32, more preferably SEQ ID NO: 1, or a conservative variant thereof, and preferably consists of the same; the heavy chain variable region CDR2 comprises any one selected from SEQ ID NO: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2, or a conservative variant thereof, and preferably consists of the same; the heavy chain variable region CDR3 comprises any one selected from SEQ ID NO: 3, 7, 10, 13, 34, 38, 41 and 44, preferably SEQ ID NO: 3 or 34, more preferably SEQ ID NO: 3, or a conservative variant thereof, and preferably consists of the same;

[0179] The light chain variable region CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 17, 20, 23, 48, 51 and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17, or a conservative variant thereof, and preferably consists of the same; the light chain variable region CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 18, 21, 24, 49, 52 and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18, or a conservative variant thereof, and preferably consists of the same; and the light chain variable region CDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 19, 22, 25, 50, 53 and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19, or a conservative variant thereof, and preferably consists of the same;

[0180] The antibody specifically binds to PDL1 and is capable of blocking the PD-1 / PDL1 interaction.

[0181] In one embodiment, the antibody of the present invention is optimized for expression in mammalian cells, the antibody having a heavy chain variable region and a light chain variable region, wherein one or more of these sequences have a specified amino acid sequence based on the antibodies described herein or conservative modifications thereof, wherein the antibody retains the desired functional properties of the PDL1 binding antibody of the present invention. Therefore, the present invention provides an isolated monoclonal antibody optimized for expression in mammalian cells, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NOs: 14 or 16, more preferably SEQ ID NO: 16, and conservative variants thereof; and the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 57 and 58, preferably SEQ ID NOs: 26 or 27, more preferably SEQ ID NO: 27, and conservative variants thereof; wherein the antibody specifically binds to PDL1 and is capable of blocking PD-1 / PDL1 interaction.

[0182] In one embodiment, an antibody of the invention is optimized for expression in a mammalian cell and has a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of these sequences has a specified amino acid sequence based on an antibody described herein or a conservative modification thereof, wherein the antibody retains the desired functional properties of the PDL1 binding antibody of the invention.

[0183] As used herein, the term "optimized" refers to changing a nucleotide sequence to use codons that are preferred in a production cell or organism (typically a eukaryotic cell, such as a Pichia cell, a Chinese hamster ovary cell (CHO) or a human cell) to encode an amino acid. The optimized nucleotide sequence is engineered to retain the amino acid sequence originally encoded by the starting nucleotide sequence (also referred to as the "parent" sequence) completely or as much as possible. The optimized sequence herein has been engineered to have codons that are preferred in mammalian cells. However, it is also contemplated herein that these sequences are optimally expressed in other eukaryotic cells or prokaryotic cells. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as optimized.

[0184] Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR1, CDR2 and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the target antibody, referred to as "affinity maturation". Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and the effects on antibody binding or other intended functions can be assessed in in vitro or in vivo assays as described herein and provided in the Examples. Conservative modifications (as described above) can be introduced. Mutations can be amino acid substitutions, additions or deletions. In addition, typically no more than 1, 2, 3, 4 or 5 residues are changed in the CDR regions.

[0185] An "affinity matured" antibody is an antibody having one or more changes in one or more of its variable domains that result in improved affinity of the antibody for the antigen compared to a parent antibody that does not have those changes. In one embodiment, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by protocols known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of hypervariable region ("HVR") and / or framework residues is described, e.g., in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0186] In one embodiment, the present invention provides an isolated monoclonal antibody comprising a VH3 comprising G56A and Y105F mutations, in particular comprising the amino acid sequence according to SEQ ID NO: 16; and preferably comprising a VL comprising S9A; A51P mutations, in particular comprising the amino acid sequence according to SEQ ID NO: 27.

[0187] In one embodiment, the "affinity matured" antibody of the invention comprises: a VH4 comprising V25A; I44V; G56A; V82K; F89V mutations, in particular comprising the amino acid sequence according to SEQ ID NO: 47; and preferably comprises a VL comprising the amino acid sequence according to SEQ ID NO: 57. In another embodiment, the "affinity matured" antibody of the invention comprises: a VH4 comprising V2S; V25A; I44V; G56A; V82K; F89V; Y105F mutations, in particular comprising the amino acid sequence according to SEQ ID NO: 46; and preferably comprises a VL comprising I2F; M4L; A51P mutations, in particular comprising the amino acid sequence according to SEQ ID NO: 58.

[0188] The antibodies of the invention can be further prepared by the following method: using antibodies having one or more VH and / or VL sequences as shown herein as starting materials to engineer modified antibodies, which modified antibodies may have different properties from the starting antibodies. Antibodies can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), such as within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues within the constant region, for example to alter the effector function of the antibody.

[0189] One type of variable region engineering that can be performed is CDR transplantation. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). Therefore, the amino acid sequences in the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific naturally occurring antibody can be expressed by constructing an expression vector that contains the CDR sequences from that specific naturally occurring antibody and grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad., USA 86:10029-10033; U.S. Pat. No. 5,225,539 (Winter), and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (Queen et al.)).

[0190] Such framework sequences can be obtained from public DNA databases or public references including germline antibody gene sequences or rearranged antibody sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can also be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase) and Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM et al., 1992 J. fol. Biol. 227: 776-798; and Cox, JPL et al., 1994 Eur. J Immunol. 24: 827-836; the contents of each are expressly incorporated herein by reference. For example, germline DNA sequences of human heavy and light chain variable region genes and rearranged antibody sequences can be found in the "IMGT" database (available online at www.imgt.org; see Lefranc, MP et al., 1999 Nucleic Acids Res. 27:209-212; the contents of each of which are expressly incorporated herein by reference).

[0191] Examples of framework sequences for antibodies of the invention are those that are structurally similar to the framework sequences used by the selected antibodies of the invention, e.g., the consensus sequences and / or framework sequences used by the monoclonal antibodies of the invention. The VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be transplanted onto a framework region having the same sequence as that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be transplanted onto a framework region comprising one or more mutations compared to the germline sequence. For example, it has been found that, in some cases, it is beneficial to mutate residues within the framework region to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (Queen et al.)).

[0192] A variety of antibody / immunoglobulin frameworks or scaffolds can be used, as long as the resulting polypeptide includes at least one binding region that specifically binds to PDL1. Such frameworks or scaffolds include the five major idiotypes of human immunoglobulins, antigen-binding fragments thereof, and immunoglobulins from other animal species, preferably with humanized features.

[0193] On the one hand, the present invention relates to a method for producing a non-immunoglobulin-based antibody using a non-immunoglobulin scaffold, and the CDR of the present invention can be transplanted onto the non-immunoglobulin scaffold. Known or future non-immunoglobulin frameworks and scaffolds can be used as long as they contain binding regions that are specific to the target PDL1 protein. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immune drugs (Trubion Pharmaceuticals Inc., Seattle, Wash.), large antibodies (Avidia, Inc., Mountain View, Calif), protein A (Affibody AG, Sweden) and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0194] Suitably, the antibodies of the invention specifically bind to PDL1 and are characterized by one or more of the following parameters:

[0195] (i) binds to human PDL1 with a dissociation constant (KD) of less than 10 nM, particularly less than 5 nM, particularly less than 1 nM, particularly less than 500 pM, more particularly less than 100 pM, preferably less than 50 pM, more preferably less than 10 pM, more preferably less than 5 pM, particularly as measured by surface plasmon resonance (SPR), particularly wherein the antibody is a scFv;

[0196] (ii) K of binding to human PDL1 off Rate is 10 -3 s -1 or lower, or 10 -4 s -1 or lower, or 10 -5 s -1 or lower, as measured by SPR, in particular, wherein the antibody is a scFv;

[0197] (iii) K of binding to human PDL1 on The rate is at least 10 3 M -1 s -1 or higher, at least 10 4 M -1 s-1 or higher, at least 10 5 M -1 s -1 or higher, at least 10 6 M -1 s -1 or higher, as measured by SPR, in particular, wherein the antibody is a scFv;

[0198] (iv) cross-reacts with cynomolgus monkey (Macaca fascicularis) PDL1, in particular binds to Macaca fascicularis PDL1 with a KD of less than 5 nM, in particular less than 1 nM, in particular less than 500 pM, more in particular less than 100 pM, preferably less than 10 pM, as measured by surface plasmon resonance, in particular wherein the antibody is a scFv; does not cross-react with Mus musculus PDL1, in particular as measured by SPR; and / or

[0199] (v) does not bind to human PDL2, particularly as measured by SPR.

[0200] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at many sites through weak non-covalent forces; the greater the interaction, the stronger the affinity.

[0201] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" or "binding to..." refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody fragment and an antigen). The affinity of a molecule X and its partner Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including the methods described herein. Low-affinity antibodies will generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies will generally bind to antigens faster and tend to maintain longer binding times. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity, i.e., binding strength, are described below.

[0202] The term "K assoc ”, “Ka” or “K on " refers to the association rate of a specific antibody-antigen interaction, and the term "K" as used herein dis ”, “Kd” or “K off" refers to the dissociation rate of a specific antibody-antigen interaction. In one embodiment, the term "KD" as used herein refers to the dissociation constant, which is obtained by the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). In one embodiment, the "KD" or "KD value" or "KD value" according to the present invention is D ” or “K D The "value" was measured by surface plasmon resonance assay using a MASS-1 SPR instrument (Sierra Sensors). To measure affinity, an antibody specific for the Fc region of rabbit IgG (Bethyl Laboratories, Cat. No. A120-111A) was immobilized on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using a standard amine coupling protocol. Sensors). Rabbit monoclonal antibodies in the B-cell supernatant were captured by immobilized anti-rabbit IgG antibodies. The minimum concentration of IgG in the B-cell supernatant was required to be sufficient for capture. After capture of the monoclonal antibody, human PDL1 (Peprotech) was injected into the flow cell at a concentration of 90nM for 3 minutes, and then the protein was allowed to dissociate from the IgG captured on the sensor chip for 5 minutes. After each injection cycle, the surface was regenerated by two injections of 10mM glycine-HCl. The apparent dissociation rate constant (kd) and association rate constant (ka) as well as the apparent dissociation equilibrium constant (KD) were calculated using the one-to-one Langmuir binding model using MASS-1 analysis software (Analyzer, SierraSensors) and were calculated based on the relative Chi 2 (Will Chi 2 The quality of the fit was monitored by normalizing to the extrapolated maximum binding level of the analyte, where the relative Chi 2 It is a measure of the quality of the curve fit. 2 The smaller the value of , the more accurate the fit to the one-to-one Langmuir binding model. Results were considered valid if the ligand-bound response units (RU) were at least 2% of the antibody-captured RU. Samples with ligand-bound RUs less than 2% of the antibody-captured RUs were considered to have no specific binding of PDL1 to the captured antibody. The equilibrium dissociation constant (K D ) is calculated as the ratio K off / K on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0203] Suitably, the affinity of the antibodies of the present invention for PDL1 may be higher than the affinity of PDL1 for PD-1. It should be understood that the higher affinity of the PDL1 antibody compared to the affinity of PDL1 for PD-1 may be particularly useful for dissociating or neutralizing the preformed PD-1 / PDL1 complex. In one embodiment, the PDL1 antibody of the present invention neutralizes the PD-1 / PDL1 interaction. In another embodiment, the PDL1 antibody of the present invention neutralizes the B7-1 / PDL1 interaction. Suitably, the affinity of the PDL1 antibody of the present invention for PDL1 may be comparable to or higher than the affinity of Avelumab for PD-1. In one embodiment, the PDL1 antibody of the present invention neutralizes the PD-1 / PDL1 interaction with a potency equal to or higher than that of Avelumab. In another embodiment, the PDL1 antibody of the present invention neutralizes the B7-1 / PDL1 interaction with a potency equal to or higher than that of Avelumab. The binding affinity of the antibody can be determined, for example, by the dissociation constant (KD). A lower KD indicates a stronger affinity, while a higher KD indicates a weaker affinity.

[0204] Thus, in suitable embodiments, the KD of an antibody of the invention may be 1 to 50,000 pM, 1 to 40,000 pM, 1 to 30,000 pM, 1 to 20,000 pM, 1 to 10,000 pM, 1 to 5,000 pM, 1 to 2,500 pM, 1 to 1,000 pM, 1 to 750 pM, 1 to 500 pM, 1 to 250 pM, 1 to 100 pM, 1 to 50 pM, 1 to 10 pM. In suitable embodiments, the KD of an antibody of the invention may be less than about 50 nM, less than about 45 nM, less than about 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, less than 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.25 nM, less than 100 pM, less than 10 pM, or less than 5 pM, particularly as measured by SPR, particularly wherein the antibody is a scFv. Suitably, an antibody of the invention has a KD of less than 5 nM, particularly as measured by SPR. Suitably, an antibody of the invention has a KD of less than 1 nM, particularly as measured by SPR. Suitably, an antibody of the invention has a KD of less than 100 pM, particularly as measured by SPR. Suitably, the antibodies of the invention have a KD of less than 50 pM, in particular as measured by SPR. Preferably, the PDL1-BD of the invention binds to human PDL1 with a KD of less than 10 pM, in particular as measured by SPR. More preferably, the PDL1-BD of the invention binds to human PDL1 with a KD of less than 5 pM, in particular as measured by SPR.

[0205] Suitably, the K of the antibody of the present invention binding to human PDL1 on The rate is at least 10 3 M -1 s -1 or higher, at least 10 4 M -1 s -1 or higher, at least 5x10 4 M -1 s -1 or higher, at least 10 5 M -1 s -1 or higher, at least 5x10 5 M -1 s -1 or higher, at least 10 6 M - 1 s -1 or higher, at least 5x10 6M -1 s -1 or higher, at least 10 7 M -1 s -1 or higher, at least 5x10 7 M -1 s -1 or higher, as measured by surface plasmon resonance (SPR). Preferably, the K of the antibodies of the invention is on The rate is at least 10 5 M -1 s -1 or higher, especially at least 10 6 M -1 s -1 or higher, as measured by SPR.

[0206] Suitably, the K of the antibody of the present invention binding to human PDL1 off Rate is 10 -3 s -1 or lower, 3x10 -3 s -1 or lower, 5x10 -3 s -1 or less, 10 -4 s -1 or lower, 5x10 -4 s -1 or less, 10 -5 s -1 or lower, 5x10 -5 s -1 or less, 10 -6 s -1 or lower or 10 -7 s -1 or lower, as measured by surface plasmon resonance (SPR). Preferably, the K of the antibodies of the invention is off Rate is 10 -3 s -1 or less, 10 -4 s -1 or lower, especially 10 -5 s -1 or lower, as measured by SPR.

[0207] Suitably, the antibodies of the invention specifically bind to PDL1 and are characterized by one or more of the following parameters:

[0208] (i) having the ability to neutralize the PDL1 / PD-1 interaction, with a potency relative to the potency of avelumab (relative potency) (measured in an ELISA assay) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4; and wherein the relative potency is the IC of avelumab 50 The values ​​(in ng / mL, as measured in an ELISA assay) are related to the IC 50 values ​​(in ng / mL as measured in an ELISA assay), particularly wherein the antibody is a scFv; and

[0209] (ii) optionally, having an ability to neutralize the PDL1 / PD-1 interaction, with a potency relative to the potency of avelumab (relative potency) (measured in a NFAT reporter gene assay) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4; and wherein the relative potency is the IC of avelumab 50 The values ​​(in ng / mL, as measured in the NFAT reporter gene assay) were compared with the IC 50 values ​​(in ng / mL as measured in a NFAT reporter gene assay), particularly wherein the antibody is a scFv; and

[0210] (iii) having an ability to neutralize the PDL1 / B7.1 interaction with a potency relative to the potency of avelumab (relative potency) (measured in an ELISA assay) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4; and wherein the relative potency is the IC of avelumab 50 The values ​​(in ng / mL, as measured in an ELISA assay) are related to the IC 50 The ratio of values ​​(in ng / mL as measured in an ELISA assay), in particular, wherein the antibody is a scFv. Suitably, the antibodies of the invention have advantageous biophysical characteristics.

[0211] Suitably, when in scFv form, the melting temperature (Tm) of the antibody of the invention (determined by differential scanning fluorimetry) is at least 55°C, for example at least 60°C, preferably at least 65°C, more preferably at least 70°C, in particular, wherein the antibody is formulated in 50 mM phosphate citrate buffer at pH 6.4, 150 mM NaCl. DSF has been described previously (Egan et al., MAbs, 9(1)(2017), 68-84; Niesen et al., Nature Protocols, 2(9)(2007) 2212-2221). The transition midpoint of the thermal unfolding of the scFv construct is determined by differential scanning fluorimetry using a fluorescent dye Orange determined (see Wong & Raleigh, Protein Science 25 (2016) 1834-1840). Samples were prepared at a final protein concentration of 50 μg / mL in citrate phosphate buffer at pH 6.4 and contained 5x The final concentration of Orange was 100 μl in a total volume of 100 μl. 25 microliters of the prepared sample were added in triplicate to a white-walled AB gene PCR plate. The analysis was performed in a qPCR machine used as a thermal cycler, and the fluorescence emission was detected using the custom dye calibration program of the software. The PCR plate containing the test samples was subjected to a temperature change from 25°C to 96°C in increments of 1°C, with a pause of 30s after each temperature increase. The total assay time was approximately two hours. Tm was calculated by calculating the inflection point of the curve using the mathematical second derivative method using GraphPad Prism software. The reported Tm is the average of three measurements.

[0212] Suitably, when in scFv format, when the starting concentration of the antibody of the invention is 10 mg / ml, and in particular wherein the antibody is formulated in 50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.4, the antibody of the invention loses less than 5%, preferably less than 3%, more preferably less than 1% of its monomer content after 5 consecutive freeze-thaw cycles.

[0213] Suitably, when in scFv format, the antibody of the invention loses less than 15%, such as less than 12%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1% of its monomer content after storage at 4°C for at least 2 weeks, in particular at least 4 weeks, when the starting concentration of the antibody of the invention is 10 mg / ml, and in particular wherein the antibody is formulated in 50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.4.

[0214] The loss of monomer content was determined by calculating the area under the curve of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, as described in USP Chapter 621. The method uses a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. The separation of molecules occurs between the void volume (V0) and the total permeation volume (VT) of a specific column. SE-HPLC measurements were performed on a Chromaster HPLC system (Hitachi High-Technologies Corporation), which was equipped with automatic sample injection and a UV detector with a detection wavelength set to 280 nm. The equipment was controlled by the software EZChrom Elite (Agilent Technologies, Version 3.3.2SP2), which also supports the analysis of the resulting chromatograms. The protein sample was cleared by centrifugation before injection, and the temperature of the autosampler was maintained at 4-6°C. For analysis of scFv samples, a Shodex KW403-4F column (Showa Denko Co., Ltd., #F6989202) was used, with a standard buffered saline mobile phase (50 mM sodium phosphate pH 6.5, 300 mM sodium chloride) at a recommended flow rate of 0.35 mL / min. The target sample load for each injection was 5 μg. Samples were detected by a UV detector at a wavelength of 280 nm and data were recorded by a suitable software suite. The resulting chromatogram was analyzed in the range of V0 to VT, thereby excluding matrix-related peaks with elution times>10 minutes.

[0215] As used herein, the term "recognizes" refers to an antibody that finds and interacts with (eg, binds to) its conformational epitope.

[0216] The terms "competition" or "cross-competition" and related terms are used interchangeably herein and refer to the ability of one antibody to interfere with the binding of another antibody or binding agent to PDL1 in a standard competitive binding assay.

[0217] Standard competitive binding assays can be used to determine the ability or degree to which an antibody can interfere with the binding of another antibody or binding molecule to PDL1, and therefore determine whether it can be said to be cross-competing according to the present invention. A particularly suitable quantitative cross-competition assay uses a FACS-based or AlphaScreen-based method to measure the competition of a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or fragment thereof with another antibody or fragment thereof in terms of their binding to the target. Typically, a cross-competing antibody or fragment thereof, for example, in a cross-competition assay, will bind to the target so that during the assay and in the presence of a second antibody or fragment thereof, the recorded displacement of the immunoglobulin single variable domain or polypeptide of the present invention is up to 100% (e.g., in a FACS-based competition assay) of the maximum theoretical displacement of a potential cross-blocking antibody or fragment thereof to be tested (e.g., a cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked is replaced). Preferably, the recorded displacement of a cross-competing antibody or fragment thereof is between 10% and 100%, more preferably between 50% and 100%.

[0218] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains), usually with specific three-dimensional structural characteristics and specific charge characteristics. The difference between a "conformational" epitope and a "linear" epitope is that in the presence of a denaturing solvent, the binding to the former is lost, while the binding to the latter is not lost. As used herein, the term "conformational epitope" refers to antigenic amino acid residues that are aggregated together on the surface when the polypeptide chain is folded to form a native protein, and they show a significantly reduced HD exchange rate due to Fab binding. Conformational epitopes include but are not limited to functional epitopes. The term "linear epitope" refers to an epitope in which all interaction points between a protein and an interacting molecule (such as an antibody) occur linearly (continuously) along the primary amino acid sequence of the protein.

[0219] The present invention also provides antibodies that bind to the same epitope as the PDL1 binding antibodies listed in Table 1. Thus, other antibodies can be identified based on their ability to cross-compete with (e.g., competitively inhibit binding of) other antibodies of the invention in a PDL1 binding assay.

[0220] Suitably, the isolated antibody of the present invention is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, IgG antibodies, Fab, Fv, scFv, dsFv, scAb, STAB and binding domains based on other scaffolds, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectin and binding sites constructed in antibody constant regions (e.g., Modular Antibody Technology of F-star). TM ).

[0221] Suitably, the antibody of the present invention is Fv. Suitably, the antibody of the present invention is scFv antibody fragment. "Single-chain Fv" or "scFv" or "sFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form a desired target binding structure. "Single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form a desired antigen binding structure (see, e.g., Plückthun, The pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315). In a specific embodiment, the functional fragment is in the form of a scFv comprising a linker according to SEQ ID NO: 28. In another embodiment, the isolated antibody of the invention is a single chain variable fragment (scFv) as shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 60, SEQ ID NO: 61 or SEQ ID NO: 62. In a preferred embodiment, the isolated antibody of the invention is a single chain variable fragment (scFv) as shown in SEQ ID NO: 31.

[0222] Suitably, the antibody of separation of the present invention is an IgG antibody fragment. The term "isotype" refers to the antibody class (e.g., IgM, IgE, IgG such as IgG1 or IgG4) provided by the heavy chain constant region gene. Isotype also includes a modified form of one of these classes, wherein modified after the Fc function, for example to enhance or reduce effector function or with the combination of Fc receptors. In one embodiment, the antibody of separation of the present invention is an IgG, preferably IgG1, selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0223] Suitably, the isolated antibody of the present invention is an IgG1 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 14; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26. In a more specific embodiment, the antibody of the present invention is an IgG1, which comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively; a heavy chain sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 93; and a light chain sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 92. Suitably, the isolated antibody of the present invention is an IgG1 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 14; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 26.In a more specific embodiment, the antibody of the present invention is an IgG1, which comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 16; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 27.

[0224] Suitably, the isolated antibody of the present invention is an IgG1 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 45; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 57. In a more specific embodiment, the antibody of the invention is an IgG1 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; a heavy chain sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 91; and a light chain sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 90.

[0225] Suitably, the isolated antibody of the present invention is an IgG1 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 45; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 57. In a more specific embodiment, the antibody of the invention is an IgG1 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 47; and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent identical to SEQ ID NO: 57.

[0226] In another specific embodiment of the present invention, the isolated antibody of the present invention is a multispecific molecule, in particular a multispecific molecule having at least a second functional molecule, such as a bispecific molecule, a trispecific molecule, a tetraspecific molecule, a pentaspecific molecule, a hexaspecific molecule.

[0227] As used herein, the term "multispecific molecule" or "multispecific antibody" refers to an antibody that binds to two or more different epitopes on at least two or more different targets (e.g., PDL1 and another target different from PDL1), or an antibody that binds to two or more different epitopes on the same target. The term "multispecific molecule" includes bispecific, trispecific, tetraspecific, pentaspecific, and hexaspecific antibodies. As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes on two different targets or the same target. As used herein, the term "trispecific antibody" refers to an antibody that binds to three different targets or three different epitopes on the same target.

[0228] The antibodies of the present invention can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand of a receptor) to produce multispecific molecules that bind to at least two binding sites and / or different target molecules. In fact, the antibodies of the present invention can be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and / or target molecules. In order to produce multispecific molecules of the present invention, the antibodies of the present invention can be functionally linked to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to produce multispecific molecules.

[0229] Thus, the present invention includes multispecific molecules comprising at least one first binding specificity for PDL1 and a second binding specificity for a second target epitope. For example, the second target epitope is present on another target molecule different from PDL1. Thus, the present invention includes multispecific molecules comprising at least one first binding specificity for PDL1 and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of PDL1 that is different from the first target epitope. In addition to the first and second target epitopes, the multispecific molecule may also include a third binding specificity.

[0230] In another embodiment, the invention comprises a monovalent, bivalent or multivalent multispecific molecule specific for PDL1, preferably monovalent.

[0231] In another specific embodiment of the present invention, the isolated antibody of the present invention is a monovalent or multivalent PDL1-specific molecule, such as a bivalent, trivalent, tetravalent, pentavalent, or hexavalent one.

[0232] As used herein, the term "monovalent molecule" or "monovalent antibody" refers to an antibody that binds to a single epitope on a target molecule (eg, PDL1).

[0233] The term "multivalent antibody" refers to a single binding molecule with more than one valency, where "valency" is described as the number of antigen binding moieties that bind to an epitope on the same target molecule. In this way, a single binding molecule can bind to more than one target molecule, or to more than one binding site on a target molecule (which contains multiple copies of an epitope). Examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, and the like. As used herein, the term "bivalent antibody" refers to an antibody with two antigen binding moieties, each of which binds to the same epitope.

[0234] Suitably, the isolated antibody of the present invention is a multispecific molecule, such as a bispecific molecule, and / or a multivalent molecule, such as a monovalent molecule for a PDL1-specific molecule, a divalent molecule for a PDL1-specific molecule, which is an antibody format selected from any suitable multispecific (e.g., bispecific) format known in the art, including but not limited to formats based on: single-chain diabody (scDb), tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), bispecific T cell engager (BiTE; tandem bi-scFv), tandem tri-scFv, triabody (Fab-(scFv)2) or diabody (Fab-(scFv)1), Fab, Fab-Fv2, Morrison (IgG CH3-scFv fusion (Morrison L) or IgG CL-scFv fusion (Morrison H)), triabodies, scDb-scFv, bispecific Fab2, diabodies, tetrabodies, scFv-Fc-scFv fusions, scFv-HSA-scFv fusions, diabodies, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions such as bsAb (scFv linked to the C-terminus of the light chain), Bs1Ab (scFv linked to the N-terminus of the light chain), Bs2Ab (scFv linked to the N-terminus of the heavy chain), Bs3Ab (scFv linked to the C-terminus of the heavy chain), Ts1Ab (linked to the N-terminus of both the heavy and light chains). The invention relates to bispecific antibodies based on heterodimeric Fc domains, such as Knob-into-Hole antibodies (KiH) (bispecific IgG prepared by KiH technology); Fv, scFv, scDb, tandem bi-scFv, tandem tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD, MATCH fused to the N- and / or C-terminus of any chain of a heterodimeric Fc domain or any other heterodimeric domain (described in WO2016 / 0202457; Egan et al., 2016:1151-1163); and Fv, scFv, scDb, tandem bi-scFv, tandem tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD, MATCH fused to the N- and / or C-terminus of any chain of a heterodimeric Fc domain or any other heterodimeric domain. T. et al., mAbs 9 (2017) 68-84) and Duobody (bispecific IgG prepared by Duobody technology) (MAbs. 2017 Feb / Mar; 9 (2): 182-212. doi: 10.1080 / 19420862.2016.1268307). Particularly suitable for use herein are single-chain diabodies (scDb) or scDb-scFv.

[0235] The term "diabodies" refers to antibody fragments with two antigen binding sites that comprise a VH connected to a VL in the same polypeptide chain (VH-VL). 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 to create two antigen binding sites. In certain embodiments, the polypeptide linker comprises one or two units of four (4) glycine amino acid residues and one (1) serine amino acid residue (GGGGS) n , wherein n=1 or 2, preferably 1. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP 404097, WO 93 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003) and Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0236] The bispecific scDb, in particular the bispecific monomeric scDb, in particular comprises two variable heavy chain domains (VH) or fragments thereof and two variable light chain domains (VL) or fragments thereof, which are connected by linkers L1, L2 and L3 in the following order: VHA-L1-VLB-L2-VHB-L3-VLA, VHA-L1-VHB-L2-VLB-L3-VLA, VLA-L1-VLB-L2-VHB-L3-VHA, VLA-L1-V HB-L2-VLB-L3-VHA, VHB-L1-VLA-L2-VHA-L3-VLB, VHB-L1-VHA-L2-VLA-L3-VLB, VLB-L1-VLA-L2-VHA-L3-VHB or VLB-L1-VHA-L2-VLA-L3-VHB, wherein the VLA and VHA domains together form an antigen binding site for a first antigen, and VLB and VHB together form an antigen binding site for a second antigen.

[0237] The linker L1 is in particular a peptide of 2-10 amino acids, more in particular 3-7 amino acids, most in particular 5 amino acids, and the linker L3 is in particular a peptide of 1-10 amino acids, more in particular 2-7 amino acids, most in particular 5 amino acids. In a specific embodiment, the linker L1 and / or L3 comprises one or two units of four (4) glycine amino acid residues and one (1) serine amino acid residue (GGGGS) n , wherein n=1 or 2, preferably n=1.

[0238] The intermediate linker L2 is particularly a peptide of 10-40 amino acids, more particularly 15-30 amino acids, most particularly 20-25 amino acids. In a specific embodiment, the linker L2 comprises one or more units of four (4) glycine amino acid residues and one (1) serine amino acid residue (GGGGS) n , wherein n=1, 2, 3, 4, 5, 6, 7 or 8, preferably n=4.

[0239] In one embodiment of the invention, the isolated antibody is a multispecific and / or multivalent antibody in the form of scDb-scFv. The term "scDb-scFv" refers to an antibody format in which a single-chain Fv (scFv) fragment is fused to a single-chain diabody (scDb) via a flexible Gly-Ser linker. In one embodiment, the flexible Gly-Ser linker is a peptide of 2-40 amino acids, such as 2-35, 2-30, 2-25, 2-20, 2-15, 2-10 amino acids, in particular 10 amino acids. In a specific embodiment, the linker comprises four (4) glycine amino acid residues and one (1) serine amino acid residue (GGGGS) n , wherein n=1, 2, 3, 4, 5, 6, 7 or 8, preferably n=2.

[0240] In one embodiment of the invention, the isolated antibody is a multispecific and / or multivalent antibody in the MATCH format as described in WO 2016 / 0202457; Egan T. et al., mAbs 9 (2017) 68-84.

[0241] The multispecific and / or multivalent molecules of the invention can be prepared using any convenient antibody preparation method known in the art (for production of bispecific constructs, see, e.g., Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14; for production of bispecific diabodies and tandem scFvs, see, e.g., Hornig, N. & -Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 and WO 99 / 57150). Specific examples of suitable methods for preparing the bispecific constructs of the present invention also include Genmab technology (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus technology (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750) technology. Methods for producing bispecific antibodies comprising a functional antibody Fc portion are also known in the art (see, for example, Zhu et al., Cancer Lett. 86 (1994) 127-134); and Suresh et al., Methods Enzymol. 121 (1986) 210-228).

[0242] Other antibodies that can be used in the multispecific and multivalent molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

[0243] The multispecific molecules of the invention can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent bonding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phthalimide (oPDM), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229: 81-83 and Glennie et al., 1987 J. Immunol. 139: 2367-2375. Conjugating agents were SATA and sulfo-SMCC, both available from Pierce Chemical Co., Ltd. (Rockford, Ill.).

[0244] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, e.g., one, prior to conjugation.

[0245] Alternatively, two or more binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. The multispecific molecule of the present invention can be a single chain molecule comprising a single chain antibody and a binding determinant, or can be a single chain multispecific molecule comprising two binding determinants. The multispecific molecule can comprise at least two single chain molecules. Methods for preparing multispecific molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0246] Binding of the bispecific molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays typically detects the presence of a specific target protein-antibody complex by employing a labeled reagent (e.g., antibody) specific for the target complex.

[0247] In another aspect, the present invention provides a nucleic acid encoding an antibody of the present invention. The present invention also provides a nucleic acid sequence encoding the CDR, VH, VL, full-length heavy chain and full-length light chain of an antibody that specifically binds to a PDL1 protein. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0248] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein, and refers to one or more deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, specific nucleic acid sequences also implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, as described below, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0249] The present invention provides substantially purified nucleic acid molecules encoding polypeptides comprising the above-mentioned segments or domains of PDL1 binding antibody chains. When expressed from a suitable expression vector, the polypeptides encoded by these nucleic acid molecules can exhibit PDL1 antigen binding ability.

[0250] The present invention also provides polynucleotides that encode at least one CDR region of the heavy chain or light chain of the PDL1 binding antibodies listed in Table 1 and generally encode all three CDR regions. Some other polynucleotides encode all or substantially all variable region sequences of the heavy chain and / or light chain of the PDL1 binding antibodies listed in Table 1. Due to the degeneracy of codons, there will be multiple nucleic acid sequences encoding each immunoglobulin amino acid sequence.

[0251] The polynucleotide sequence can be generated de novo by solid phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding a PDL1 binding antibody (e.g., a sequence described in the Examples below). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations into a polynucleotide sequence by PCR can be performed by methods described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0252] The present invention also provides an expression vector and a host cell for producing the above-mentioned PDL1 binding antibody.

[0253] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell they import (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). After importing the host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell, thereby replicating together with the host genome.

[0254] In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA techniques are typically in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector form. However, the present invention is intended to include such other forms of expression vectors with equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0255] The term "operably linked" refers to the functional relationship between two or more polynucleotide (e.g., DNA) fragments. Typically, it refers to the functional relationship between a transcription regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, it is operably linked to the coding sequence. Typically, a promoter transcription regulatory sequence operably linked to a transcribed sequence is physically continuous with the transcribed sequence, i.e., they are cis. However, some transcription regulatory sequences, such as enhancers, do not have to be physically continuous or located near the coding sequence whose transcription is enhanced.

[0256] Various expression vectors can be used to express polynucleotides encoding PDL1 binding antibody chains or binding fragments. Both viral-based expression vectors and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (usually with expression cassettes for expressing proteins or RNA) and artificial chromosomes (see, for example, Harrington et al., Nat Genet. 15: 345, 1997). For example, non-viral vectors that can be used to express PDL1 binding polynucleotides and polypeptides in mammalian (eg, human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invitrogen, San Diego, Calif.), MPS V vectors and many other vectors known in the art for expressing other proteins. Useful viral vectors include retrovirus-based, adenovirus-based, adeno-associated virus-based, herpes virus-based, SV40-based, papillomavirus-based, HBP Epstein Barr virus-based, vaccinia virus-based, and Semliki Forest virus-based (SFV) vectors. See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0257] The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Typically, the expression vector comprises a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding a PDL1 binding antibody. In one embodiment, an inducible promoter is used to prevent the expression of the inserted sequence under non-inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. The culture of the transformed organism can be amplified under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by host cells. In addition to the promoter, other regulatory elements may be required or desired for effective expression of the PDL1 binding antibody. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by including an enhancer suitable for the cell system in use (see, for example, Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153: 516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[0258] The expression vector can also provide a secretion signal sequence position to form a fusion protein with the polypeptide encoded by the inserted PDL1 binding antibody sequence. Typically, the inserted PDL1 binding antibody sequence is connected to the signal sequence before being included in the vector. The vector used to receive the sequence encoding the PDL1 binding antibody light chain and heavy chain variable domains sometimes also encodes the constant region or part thereof. This vector allows the variable region to be expressed as a fusion protein with the constant region, resulting in the production of a complete antibody and its antigen-binding fragment. Typically, such a constant region is human.

[0259] The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended to refer not only to the specific subject cell, but also to the progeny of that cell. Because certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0260] The host cell containing and expressing the PDL1 binding antibody chain can be prokaryotic or eukaryotic. Escherichia coli is a prokaryotic host that can be used to clone and express the polynucleotides of the present invention. Other applicable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia and various Pseudomonas. In these prokaryotic hosts, expression vectors can also be prepared, which generally contain expression control sequences (e.g., replication origins) compatible with the host cells. In addition, there will be any number of various well-known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or promoter systems of bacteriophage λ. Promoters generally control expression (optionally together with operator sequences) and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express the PDL1 binding polypeptides of the present invention. Insect cells can also be used in combination with baculovirus vectors.

[0261] In one embodiment, mammalian host cells are used to express and produce the PDL1 binding polypeptides of the present invention. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes, or they can be mammalian cell lines with exogenous expression vectors. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally described in Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences, such as replication origins, promoters and enhancers (see, e.g., Queen et al., Immunol. Rev. 89: 49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific and / or adjustable or regulatable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPS V promoters, tetracycline-inducible CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0262] The method of introducing an expression vector comprising a polynucleotide sequence of interest depends on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts. (See generally Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic method, virions, immunoliposomes, polycations: nucleic acid conjugates, naked DNA, artificial virions, fusions with herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), reagent-enhanced DNA absorption and in vitro transduction. In order to produce recombinant proteins for a long time and in high yield, stable expression is generally required. For example, a cell line stably expressing a PDL1 binding antibody chain or binding fragment can be prepared using the expression vector of the present invention, the expression vector comprising a viral replication origin or an endogenous expression element and a selectable marker gene. After the vector is introduced, the cells can be grown in a rich culture medium for 1-2 days and then converted to a selective culture medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows cells that successfully express the introduced sequence to grow in a selective medium. Resistant, stably transfected cells can be propagated using tissue culture techniques appropriate to the cell type. Therefore, the invention provides a method for producing an antibody of the invention, wherein the method comprises the following steps: culturing a host cell comprising (particularly expressing) a nucleic acid or vector encoding an antibody of the invention, thereby expressing the antibody of the invention or its fragment.

[0263] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers enhance or stabilize the composition, or facilitate the preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc.

[0264] The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. The route and / or mode of administration depends on the desired result. Administration can be intravenous, intramuscular, intraperitoneal or subcutaneous, or administered near the target site. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody and multispecific molecule, can be coated with a material to protect the compound from acid and other natural conditions that may inactivate the compound.

[0265] The pharmaceutical compositions of the present invention can be prepared according to methods well known in the art and commonly practiced. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition is preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or an effective dose of PDL1 binding antibody is used in the pharmaceutical composition of the present invention. The PDL1 binding antibody is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniform dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0266] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and the like.

[0267] Antibodies are usually administered multiple times. The intervals between single doses can be weekly, monthly or annual. Intervals can also be irregular, as indicated by measuring the blood levels of PDL1 binding antibodies in patients. Alternatively, antibodies can be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency depend on the half-life of the antibody in the patient. Typically, the half-life of humanized antibodies is longer than that of chimeric antibodies and non-human antibodies. The dose and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment throughout their lives. In therapeutic applications, relatively high doses are sometimes required to be administered at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete relief of symptoms of the disease. Thereafter, a preventive regimen can be administered to the patient.

[0268] The antibodies of the invention have in vitro and in vivo diagnostic and therapeutic uses. For example, these molecules can be administered to cells in culture (e.g., in vitro or in vivo) or in a subject (e.g., in vivo) to treat, prevent or diagnose a variety of diseases.

[0269] In one aspect, the invention relates to an antibody of the invention or a composition of the invention for use as a medicament.

[0270] In one aspect, the invention relates to an antibody of the invention or a composition of the invention for use in the treatment of a proliferative disease, particularly cancer, in a subject in need thereof.

[0271] In another aspect, the present invention relates to the use of an antibody of the present invention or a composition of the present invention for treating a proliferative disease, in particular cancer, in a subject in need thereof.

[0272] In another aspect, the present invention relates to the use of an antibody of the present invention or a composition of the present invention for the preparation of a medicament for treating a proliferative disease, in particular cancer, in a subject in need thereof.

[0273] In one aspect, the invention provides a method of treating a proliferative disease, particularly cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of the invention or a composition of the invention.

[0274] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0275] As used herein, the terms "treatment", "treating", "treat", "treated" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in the sense of partially or completely curing a disease and / or attributing adverse effects of the disease or delaying the progression of the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, such as a human, and includes: (a) inhibiting the disease, i.e., preventing its development; and (b) relieving the disease, i.e., causing regression of the disease.

[0276] The term "therapeutically effective amount" or "effective amount" refers to the amount of an agent that, when administered to a mammal or other subject to treat a disease, is sufficient to cause such treatment of the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0277] In one embodiment, the proliferative disease is cancer. The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or spread to other parts of the body through the blood and lymphatic system. Examples of various cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms cover solid and liquid (e.g., diffuse or circulating) tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors. The term "cancer" used herein refers to a wide range of tumors, including all solid and hematological malignancies. Examples of such tumors include, but are not limited to, benign or particularly malignant tumors, solid tumors, brain cancer, kidney cancer, liver cancer, adrenal cancer, bladder cancer, breast cancer, abdominal cancer (e.g., gastric tumors), esophageal cancer, ovarian cancer, cervical cancer, colon cancer, rectal cancer, prostate cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), vaginal cancer, thyroid cancer, melanoma (e.g., unresectable or metastatic melanoma), renal cell carcinoma, sarcoma, glioblastoma, multiple myeloma or gastrointestinal cancer, in particular colon cancer or colorectal adenoma, head and neck tumors, ovarian cancer ...ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ov Endometrial cancer, Cowden syndrome, Lhermitte-Duclos disease, Bannayan-Zonana syndrome, prostatic hyperplasia, neoplasms, especially neoplasms of epithelial characteristics, preferably breast cancer or squamous cell carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia (e.g. Philadelphia chromosome positive chronic myeloid leukemia), acute lymphocytic leukemia (e.g. Philadelphia chromosome positive acute lymphocytic leukemia), non-Hodgkin's lymphoma, plasma cell myeloma, Hodgkin's lymphoma, leukemia and any combination thereof. In a preferred embodiment, the cancer is lung cancer, preferably non-small cell lung cancer (NSCLC). In another embodiment, the cancer is colorectal cancer.

[0278] The antibodies of the present invention or the compositions of the present invention inhibit the growth of solid tumors, but also inhibit the growth of liquid tumors. In another embodiment, the proliferative disease is a solid tumor. The term "solid tumor" refers in particular to breast cancer, ovarian cancer, colon cancer, rectal cancer, prostate cancer, abdominal cancer (especially gastric cancer), cervical cancer, lung cancer (such as non-small cell lung cancer and small cell lung cancer) and head and neck tumors. In addition, depending on the tumor type and the specific combination used, a reduction in tumor volume can be obtained. The antibodies of the present invention or the compositions of the present invention are also suitable for preventing the metastatic spread of tumors in subjects with cancer and the growth or development of micrometastases (micrometastases).

[0279] The term "preven" or "prevention" refers to the complete inhibition of the development of a disease or any secondary effects of a disease. As used herein, the term "prevent" includes preventing a disease or condition from occurring in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0280] On the one hand, the present invention relates to a kit comprising an antibody of the present invention or a pharmaceutical composition of the present invention. The kit may include one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or reagents that can be used to chelate or otherwise couple antibodies to labels or therapeutic agents, or radioprotective compositions; devices or other materials for preparing antibody molecules for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject. In a specific embodiment, the kit comprises a pharmaceutically effective amount of an antibody of the present invention. In another embodiment, the kit comprises a pharmaceutically effective amount of a lyophilized form of the antibody of the present invention and a diluent, and optionally includes instructions for use. The kit may also include a filter needle for reconstitution and a needle for injection.

[0281] Table 1: Examples of PDL1 antibodies of the invention (CDR residues are shown in bold and italic letters).

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290] Table 2. Other sequences relevant to the present invention.

[0291]

[0292]

[0293] Table 3. Examples of multispecific molecules comprising antibodies of the invention.

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] Throughout this application, if there is a discrepancy between the text of the specification (eg, Tables 1 to 3) and the sequence listing, the text of the specification shall prevail.

[0313] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and are disclosed herein as if each combination was explicitly disclosed individually. In addition, all subcombinations of the various embodiments and elements thereof are also specifically encompassed by the present invention and are disclosed herein as if each such subcombination was explicitly disclosed individually herein.

[0314] The present invention is not limited to the scope of the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0315] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference to the extent permitted by the respective patent laws.

[0316] The following examples illustrate the above invention, but are not intended to limit the scope of the invention in any way.Other test models known to those skilled in the relevant art can also determine the beneficial effects of the claimed invention.

[0317] Example

[0318] Novel antibodies against human PDL1.

[0319] Example 1: Generation of rabbit antibodies against human PDL1

[0320] Rabbits have been immunized with recombinantly produced and purified human PDL1 extracellular domain. During the immunization, the strength of the humoral immune response to the antigen was qualitatively assessed by determining the maximum dilution (titer) of each rabbit's serum at which the polyclonal serum antibodies were still able to produce detectable binding to the antigen. Serum antibody titers against the immobilized antigen (recombinant human PDL1 extracellular domain) were assessed using an enzyme-linked immunosorbent assay (ELISA). All immunized rabbits showed very high titers of at least 1:2.64 x 10 6 Diluted serum. Serum from the same rabbit before the first antigen injection was used as background control.

[0321] Example 2: Hit identification and selection.

[0322] In the hit identification program, a flow cytometry-based sorting protocol was developed that specifically detects high-affinity hPDL1-binding B cells and isolates them. To identify hPDL1-binding B cells, the hPDL1 ECD was labeled with the fluorescent dye R-phycoerythrin (R-PE). Since the PD-1 binding sites on the labeled PDL1 and the binding sites of the anti-PDL1 neutralizing antibodies may be blocked by the bulky R-PE label, the accessibility of the antigenic determinant was confirmed by flow cytometry. The PD-1 extracellular domain fused to the Fc portion of human IgG1 or avelumab was captured on protein G beads, and the binding of R-PE-labeled PDL1 was confirmed by flow cytometry. The fluorescence intensity is proportional to the amount of labeled PDL1 bound to the receptor immobilized on the beads. The binding of PDL1 to PD-1 and neutralizing antibodies has been confirmed, while the binding of irrelevant cytokines to the anti-PDL1 antibodies has not been detected.

[0323] filter:

[0324] The results obtained in the screening phase are based on assays with unpurified antibodies from culture supernatants of antibody-secreting cells (ASCs), because the scale of high-throughput culture does not allow purification of single rabbit antibodies. These supernatants allow a large number of antibodies to be ranked relative to each other, but do not provide absolute values ​​except for binding affinity. Supernatants are collected from each individually cultured clone over the course of at least four weeks. At the end of the culture period, the binding of rabbit monoclonal antibodies in each cell culture supernatant to the recombinant human PDL1 extracellular domain was characterized in a high-throughput ELISA. The binding kinetics of the supernatant binding to PDL1 to human and cynomolgus monkey PDL1 were further characterized. In addition, the neutralization potential of the PDL1 / PD-1 interaction was determined by competitive ELISA as well as a cell-based reporter gene assay. The neutralization of the PDL1 / B7-1 interaction was also determined by competitive ELISA. In addition to binding kinetics, the reported values ​​of high-throughput screening should be interpreted as answers "yes" or "no", which are based on single-point measurements (no dose response). Supernatants were analyzed for mouse PDL1 binding potential by direct ELISA and only positive supernatants were determined for binding kinetics.

[0325] Direct ELISA for hPDL1 binding

[0326] The ELISA plate was coated overnight at 4°C by adding 50 μl of PBS containing 500 ng / ml PDL1. The next day, the plate was washed 3 times in overflow mode with 450 μl wash buffer (PBS, 0.005% Tween 20) per well, and 300 μl blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added to each well and placed on a nutating mixer for 1 hour at room temperature. Then, the plate was washed 3 times in overflow mode with 450 μl wash buffer, 50 μl of each supernatant was added, and the plate was incubated with gentle stirring at room temperature for 1.5 hours. After washing 3 times with 450 μl wash buffer in overflow mode, 50 μl of HRP-conjugated goat and rabbit IgG antibodies were added to each well. After incubation for 1 h at room temperature on a nutating mixer, the plate was washed with 450 μl wash buffer per well, and then 50 μl TMB (3,3',5,5'-tetramethylbenzidine, KPL, Cat. No. 53-00-00) was added. After 5 to 10 minutes of development, the enzymatic reaction was stopped by adding 50 μl 1M HCl per well and the plate was read at 450 nm with a reference wavelength of 690 nm.

[0327] Affinity for hPDL1 determined by SPR

[0328] The binding affinity of antibodies to human PDL1 was measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). For affinity screening, antibodies specific to the rabbit IgG Fc region (Bethyl Laboratories, Cat. No. A120-111A) were fixed on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using a standard amine coupling protocol. Rabbit monoclonal antibodies in the supernatant of B-cells were captured by fixed anti-rabbit IgG antibodies. The minimum concentration of IgG in the supernatant of B cells is required to be sufficient for capture. After capturing the monoclonal antibody, human PDL1 (Peprotech) was injected into the flow cell at a concentration of 90nM for 3 minutes, and then the protein was dissociated from the IgG captured on the sensor chip for 5 minutes. After each injection cycle, 10mM glycine-HCl was injected twice to regenerate the surface. The apparent dissociation rate constant (k) was calculated using a one-to-one Langmuir binding model using MASS-1 analysis software (Analyzer, Sierra Sensors). d ) and the association rate constant (k a ) and the apparent dissociation equilibrium constant (K D ), and according to the relative Chi 2 (Will Chi 2The quality of the fit was monitored by normalizing to the extrapolated maximum binding level of the analyte, where the relative Chi 2 It is a measure of the quality of the curve fit. 2 The smaller the value of , the more accurate the fit to the one-to-one Langmuir binding model. For most hits, relative to Chi 2 The value was less than 10%. The results were considered valid if the ligand-bound response units (RU) were at least 2% of the antibody-captured RU. Samples with ligand-bound RU less than 2% of the antibody-captured RU were considered to have no specific binding of PDL1 to the captured antibody.

[0329] PDL1 / PD-1 blocking ELISA

[0330] ELISA plates were coated overnight at 4°C by adding 50 μl of PBS containing 2 μg / ml PD-1. The next day, the plates were washed 3 times in overflow mode with 450 μl wash buffer per well, and 300 μl blocking buffer was added to each well and placed on a nutating mixer for 1 hour at room temperature. PDL1 was then diluted in blocking buffer at a concentration 20 times higher than the desired final concentration of 250 ng / ml. The assay sensitivity was further adjusted and several clones were analyzed in the presence of 40 ng / ml PDL1. Next, in a non-binding plate, 114 μl of each supernatant was diluted with 6 μl of blocking buffer containing PDL1 and the plates were incubated on a nutating mixer for 1 hour at room temperature. The ELISA plate was washed 3 times in overflow mode with 450 μl wash buffer per well, and 50 μl of each dilution was then added to the ELISA plate. The plate was incubated for 1.5 hours at room temperature with gentle agitation. After washing each well with 450 μl of washing buffer 3 times, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of washing buffer and developed for 5 to 10 minutes after adding 50 μl of TMB. Finally, the enzymatic reaction was stopped by adding 50 μl of 1M HCl and the plate was read at 450 nm with 690 nm as the reference wavelength.

[0331] PDL1 / B7-1 blocking ELISA

[0332] ELISA plates were coated overnight at 4°C by adding 50 μl of PBS containing 4 μg / ml B7-1. The next day, the plates were washed 3 times in overflow mode with 450 μl wash buffer per well, and 300 μl blocking buffer was added to each well and placed on a nutating mixer for 1 hour at room temperature. PDL1 was then diluted in blocking buffer at a concentration 20 times higher than the desired final concentration of 500 ng / ml. Next, in a non-binding plate, 114 μl of each supernatant was diluted with 6 μl of blocking buffer containing PDL1, and the plates were incubated on a nutating mixer for 1 hour at room temperature. The ELISA plate was washed 3 times in overflow mode with 450 μl wash buffer per well, and 50 μl of each dilution was then added to the ELISA plate. The plate was incubated for 1.5 hours at room temperature with gentle agitation. After washing each well with 450 μl of washing buffer 3 times, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of washing buffer and developed for 5 to 10 minutes after adding 50 μl of TMB. Finally, the enzymatic reaction was stopped by adding 50 μl of 1M HCl and the plate was read at 450 nm with 690 nm as the reference wavelength.

[0333] PDL1 / PD-1 blockade in cell-based assays (reporter gene)

[0334] To further characterize the hits, CHO / PDL1 / TCR activated cells and Jurkat / PD-1 cells were used to test their ability to neutralize the PDL1 / PD-1 interaction when both interacting molecules were expressed simultaneously on the cell surface. 35,000 CHO / PDL1 / TCR activated cells in 100 μl of cell culture medium (DMEM / F12, 10% FCS) were added to the inner wells of the leukocyte culture plate and incubated at 37°C and 5% CO2 for 16-20 hours. The next day, 95 μl of cell culture medium was removed from each well and 50 μl of the screened B cell supernatant or positive control was added, where the concentration of avelumab was determined to produce 0%, 50% and 100% of the maximum signal, and the plate was incubated at 37°C for 30 minutes. Then, 50 μl of effector Jurkat cells diluted at 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS) were added to each well, and the plates were incubated for 6 hours at 37°C and 5% CO2. Finally, 50 μl of luciferase substrate (BPS Bioscience) prepared according to the manufacturer's protocol was added to each well, and the plates were incubated in the dark for 30 minutes, and luminescence was measured using a Topcount.

[0335] Species specificity determined by SPR: cynomolgus monkey and mouse

[0336] The binding kinetics to cynomolgus monkey and mouse PDL1 were also determined using the same SPR setup as described for binding to human PDL1, but replacing human PDL1 with cynomolgus monkey or mouse PDL1, respectively.

[0337] Filter hit selection

[0338] Table 4 lists the pharmacological properties of the final cloned monoclonal antibodies in the B cell supernatants.

[0339] Example 3: Hit confirmation.

[0340] Cloning and production

[0341] After identifying the clones selected for hit confirmation, these rabbit antibody clones are expressed and purified for further characterization. The cloning of the corresponding light chain and heavy chain variable domains requires the DNA fragments to be connected in vitro to suitable mammalian expression vectors. These expression vectors contain the consensus sequence of rabbit IgG light chain and heavy chain constant domains to allow the assembly and secretion of fully functional rabbit monoclonal IgG when co-expressed. After vector construction, the sequence of the resulting construct is confirmed again, and the plasmid DNA is amplified and purified for mammalian cell transfection.

[0342] The expression vectors of rabbit antibody heavy chain and light chain were transfected into mammalian suspension cell lines by lipid-based transfection reagents to perform transient heterologous expression. Conditions such as the ratio of heavy chain to light chain vectors were optimized to obtain a stable expression level of secreted monoclonal IgG. The expression culture was cultured in an oscillating incubator for 7 days. At the end of the heterologous expression phase, the cell culture supernatant was collected by centrifugation. Subsequently, the secreted rabbit IgG was affinity purified by protein A beads. The beads of the loaded IgG were washed, and the purified antibodies were eluted by pH changes. The eluted fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV absorbance at 280nm and size exclusion high performance liquid chromatography (SE-HPLC) to verify identity, content and purity. Table 5 summarizes the preparation and characterization of rIgG.

[0343]

[0344] Table 5. Summary of analytical data for rabbit monoclonal antibody preparation.

[0345]

[0346] Affinity for hPDL1 determined by SPR

[0347] The binding kinetics of purified monoclonal rabbit antibodies to human PDL1 were determined by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). Since most antibodies show very slow off rates, experiments were performed in a high salt concentration buffer at 37 ° C to distinguish the binding affinities of different antibodies. Antibodies specific to the rabbit IgG Fc region (Bethyl Laboratories, Cat. No. A120-111A) were fixed on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using a standard amine coupling protocol. Rabbit monoclonal antibodies were captured by fixed anti-rabbit IgG antibodies. After capturing the monoclonal antibody, the binding of PDL1 (ranging from 90 to 0.35 nM) to the IgG captured on the biosensor chip was tested in two-fold serial dilutions in HEPES buffer containing 150 mM NaCl and 150 mM MgCl2, and the protein was dissociated from the IgG captured on the sensor chip for 5 minutes. After each injection cycle, the surface was regenerated by two injections of 10 mM glycine-HCl. The apparent dissociation rate constant (k) was calculated using the one-to-one Langmuir binding model using MASS-1 analysis software (Analyzer, Sierra Sensors). d ) and the association rate constant (k a ) and the apparent dissociation equilibrium constant (K D ), and according to the relative Chi 2 (Will Chi 2 The quality of the fit was monitored by normalizing to the extrapolated maximum binding level of the analyte, where the relative Chi 2 It is a measure of the quality of the curve fit. 2 The smaller the value of , the more accurate the fit to the one-to-one Langmuir binding model. For most hits, relative to Chi 2 Values ​​were below 10%. Table 6 shows the rabbit IgG antibodies selected for further development.

[0348] Table 6. Summary of affinity measurements of rabbit IgG 33-03-G02 and 37-20-B03 for hPDL1.

[0349]

[0350] Efficacy of PDL1 / PD-1 Blocking ELISA

[0351] The ability to neutralize the binding of PDL1 to PD-1 was assessed in a competitive ELISA. The ELISA plate was coated overnight at 4°C by adding 50 μl of PBS containing 4 μg / ml PD-1. The next day, the plate was washed 3 times in overflow mode with 450 μl wash buffer (PBS, 0.005% Tween 20) per well, and 300 μl blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added to each well and placed on a nutating mixer at room temperature for 1 hour. PDL1 was then diluted to a final concentration of 1 ng / ml in blocking buffer. Next, in a non-binding plate, 120 μl of a serial dilution of the test rIgG in a buffer containing PDL1 (ranging from 300 to 0.005 ng / ml) was prepared per well, and the plate was incubated at room temperature for 30 minutes. The ELISA plate was washed 3 times with 450 μl of wash buffer in overflow mode, and 50 μl of each dilution was added to the adjacent wells of the ELISA plate to obtain duplicate samples. The plate was incubated at room temperature for 90 minutes under gentle agitation. After washing 3 times with 450 μl of wash buffer, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of wash buffer and developed for 5 to 10 minutes after adding 50 μl of TMB. Finally, the enzymatic reaction was stopped by adding 50 μl of 1M HCl and the plate was read at 450 nm with 690 nm as the reference wavelength.

[0352] Rabbit IgG from clones 33-03-G02 and 37-20-B03 had high potency in neutralizing PDL1 / PD-1 interactions. Clone 37-20-B03 was almost twice as potent as avelumab (Table 7). The dose response curves obtained for the selected clones are shown in Figure 1 middle.

[0353] Table 7. Summary of the neutralization potency of rabbit IgG 33-03-G02 and 37-20-B03 in PDL1 / PD-1 competition ELISA.

[0354]

[0355] Efficacy of PDL1 / B7-1 Blocking ELISA

[0356] The efficacy of neutralizing the PDL1 / B7-1 interaction was evaluated in a competitive ELISA. The ELISA plate was coated overnight at 4°C by adding 50 μl of PBS containing 4 μg / ml B7-1. The next day, the plate was washed 3 times in overflow mode with 450 μl wash buffer (PBS, 0.005% Tween 20) per well, and 300 μl blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added to each well and placed on a nutating mixer at room temperature for 1 hour. PDL1 was then diluted to 40 ng / ml in blocking buffer. Next, in a non-binding plate, 120 μl of a serial dilution of the test rIgG in a buffer containing PDL1 (ranging from 900 to 0.015 ng / ml) was prepared per well, and the plate was incubated at room temperature for 30 minutes. The ELISA plate was washed three times with 450 μl of wash buffer in overflow mode, and 50 μl of each dilution was added to adjacent wells of the ELISA plate to obtain duplicate samples. The plate was incubated at room temperature for 90 minutes under gentle agitation. After washing three times with 450 μl of wash buffer, 50 μl of streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation for 1 hour at room temperature, the plate was washed three times with 450 μl of wash buffer and developed for 5 to 10 minutes after adding 50 μl of TMB. Finally, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl and the plate was read at 450 nm with a reference wavelength of 690 nm. The selected rabbit IgG was able to block the PDL1 / B7-1 interaction with a potency similar to that of avelumab, as shown in Table 8. The dose response curves obtained for the selected clones are shown in Figure 2 middle.

[0357] Table 8. Neutralization potency of selected rIgGs in PDL1 / B7-1 competition ELISA.

[0358]

[0359] Efficacy in cell-based PDL1 / PD-1 blockade assay (reporter gene)

[0360] The efficacy of neutralizing the binding of PDL1 to PD-1 was evaluated in a cell-based reporter gene assay. 35,000 CHO / PDL1 / TCR activated cells in 100 μl of cell culture medium (DMEM / F12, 10% FCS) were added to the inner wells of the leukocyte culture plate and incubated at 37°C and 5% CO2 for 16-20 hours. The next day, 95 μl of cell culture medium was removed from each well and 50 μl of 2-fold concentrated serial dilutions (3,000 to 0.46 ng / ml) of each molecule to be tested (including the reference avelumab) were added. Then, 50 μl of effector Jurkat cells diluted in assay buffer (RPMI1640 with 10% FCS) at 400,000 cells / ml were added to each well, and the plate was incubated at 37°C and 5% CO2 for 6 hours. Finally, 50 μl of luciferase substrate (BPS Bioscience) prepared according to the manufacturer's protocol was added to each well, and the plates were incubated in the dark for 30 minutes and luminescence was measured using a Topcount. The selected clones were able to block the PDL1 / PD-1 interaction in a cell-based reporter gene assay (Table 9). The dose response curves obtained for the selected clones are shown in Figure 3 middle.

[0361] Table 9. Summary of the neutralization potency of selected rIgGs for PDL1 / PD-1 interaction in reporter gene assays.

[0362]

[0363] Binding to PDL1-expressing cells determined by FACS

[0364] The binding potency of the selected IgG to PDL1 expressing cells was also determined. 50,000 CHO-PDL1 expressing cells were distributed into round bottom non-tissue culture treated 96-well plates. The cells were washed twice with 100 μl PBS by centrifugation at 400 x g for 5 minutes. The cells were resuspended in 100 μl of test rIgG and a serial dilution of the control IgG avelumab, which was prepared in staining buffer (PBS, 2% BCS heat inactivated, 2mM EDTA) with a concentration range of 2,000 to 0.128 ng / ml. After incubation at 4 ° C for 1 hour on a nutating mixer, the cells were washed 3 times with 100 μl staining buffer and centrifuged at 400 x g for 5 minutes. Then, the cells treated with rabbit IgG were resuspended in 100 μl of staining buffer containing 2 μg / ml APC-labeled goat anti-rabbit IgG, and the cells treated with avelumab (human IgG1) were resuspended in 100 μl of staining buffer containing 2 μg / ml APC-labeled goat anti-human IgG. The plate was incubated at 4 ° C for 1 hour on a nutating mixer. The plate was washed 3 times with 100 μl of staining buffer and then resuspended in a final volume of 50 μl of staining buffer. Finally, the APC signal of 20,000 events per well was analyzed by flow cytometry using a Novocyte flow cytometer system (ACEA Bioscience). For all evaluated rabbit IgGs, the binding of PDL1-expressing cells can be confirmed. Table 10 shows the binding efficacy of cell PDL1 with selected rabbit IgGs.

[0365] Table 10. Binding potency of selected rabbit IgGs to cellular PDL1.

[0366]

[0367] Species specificity determined by SPR: cynomolgus monkey

[0368] The binding kinetics to cynomolgus monkey PDL1 were also determined, using the same setup as described for binding to human PDL1, but in this case replacing human PDL1 with cynomolgus monkey PDL1. For all selected IgGs, binding to cynomolgus monkey PDL1 was demonstrated (Table 11).

[0369] Table 11. Summary of affinity measurements of selected rabbit IgGs to cynomolgus monkey PDL1.

[0370]

[0371] Species specificity determined by SPR: mouse

[0372] The binding kinetics to cynomolgus monkey PDL1 were also determined, using the same setup as described for binding to human PDL1, but in this case replacing human PDL1 with mouse PDL1. For selected rabbit IgGs from clones 33-03-G02 and 37-20-B03, no binding to mouse PDL1 was detected.

[0373] Example 4: Selection of clones for humanization

[0374] Based on the data obtained during hit validation, all clones were humanized by grafting CDRs onto frameworks based on VH3, VH4 or VH1A or VH1B. To obtain the best affinity and potency, two clones that showed the best affinity for human PDL1 as rIgG, 33-03-G02 and 37-20-B03, were further optimized with different structural grafts. The following grafting variants were applied to clones 33-03-G02 and 37-20-B03: CDR grafting – grafting of rabbit CDRs onto human framework; IF grafting – CDR grafting plus grafting of all rabbit VL / VH interface residues; Full grafting – CDR grafting plus framework residues following the AHo humanization protocol (antigen interface (AIF) residues (rabbit residues that may contact the antigen according to AHo) are limited to residues with a >20% change in solvent accessibility after interface formation to reduce the total number of mutations (rabbit framework disability)).

[0375] Proteins were heterologously expressed as insoluble inclusion bodies in E. coli (except PRO997, which was produced in mammalian CHO-S cells, similar to the above-mentioned rIgG expression) by small-scale induced overnight expression. The inclusion bodies were separated from the homogenized cell pellet by a centrifugation scheme that included several washing steps to remove cell debris and other host cell impurities. The purified inclusion bodies were dissolved in a denaturing buffer and the scFv was refolded by a scalable refolding scheme to produce milligram quantities of natively folded monomeric scFv. At this point, the scFv was purified using a standardized scheme. The refolded product was captured by affinity chromatography to obtain a purified scFv. Only the main fractions with the required purity were used because the available amount did not allow SEC polishing of the sample. In addition, the melting temperature of the scFv was determined by differential scanning fluorimetry (DSF) measurement (described in more detail later). Table 12 summarizes the preparation of VH4CDR graft scFv molecules. Since two of the clones contained unpaired cysteine ​​residues in the CDR loops, a C57S mutation was introduced in clone 37-20-B03, as shown in Table 12.

[0376] Additional transplant variants were designed for some selected clones and are described in Table 13 (AHo numbering) and Table 14 outlining their initial production and characterization.

[0377] Example 5: Pharmacodynamic characterization of humanized scFvs

[0378] In the following, the humanized scFvs were characterized for their main pharmacodynamic properties using the same assay system as described for the hit validation stage, but with some modifications adapted to the different format of the scFv molecule.

[0379] 5.1 Affinity for human PDL1

[0380] The affinity of humanized scFv for human PDL1 was determined by SPR analysis on a T200 device (Biacore, GE Healthcare). In this experiment, Fc-tagged human PDL1 was captured using the Human Antibody Capture kit from GE Healthcare. After each analyte injection cycle, the CM5 sensor chip was regenerated and the neoantigen was captured. The scFv was injected as an analyte for 5 minutes using a dose-response multi-cycle kinetic assay with concentrations ranging from 0.12 to 30 nM diluted in running buffer, and the protein was then allowed to dissociate for 12 minutes. The sensorgrams obtained were fitted using a 1:1 binding model. As shown in Table 15, binding to human PDL1 was confirmed for the humanized scFvs tested.

[0381] 5.2 Neutralization of PDL1 / PD-1 interaction by competitive ELISA

[0382] The potency of neutralizing the binding of PDL1 to PD-1 was assessed by competition ELISA using the same method as above. 50 The IC values ​​of the reference molecule Avelumab were collected on each plate. 50 Calibrate (relative to IC 50 :IC 50，阿维鲁单抗 / IC 50，测试scFv The potency is summarized in Table 16, which shows that up to 5-fold the IC of avelumab could be resolved in this assay. 50 All scFvs tested had similar or better potency than avelumab.

[0383] 5.3 Neutralization of PDL1 / B7-1 interaction by competition ELISA

[0384] The potency of neutralizing the binding of PDL1 to B7-1 was evaluated by competition ELISA using the same method as above. 50 The IC values ​​of the reference molecule Avelumab were collected on each plate. 50Calibrate (relative to IC 50 :IC 50，阿维鲁单抗 / IC 50，测试scFv The potency is summarized in Table 17, which shows that up to 10-fold the IC of avelumab can be resolved in this assay. 50 All scFvs tested had similar or better potency than avelumab.

[0385] 5.4 Neutralization of PDL1 / PD-1 interaction in NFAT reporter gene assay

[0386] The potency to neutralize the binding of PDL1 to PD-1 was assessed in a cell-based reporter gene assay as described above. Serial dilutions of each molecule to be tested, as well as the reference avelumab, were added to the plate. The single IC 50 The IC values ​​of the reference molecule Avelumab were collected on each plate. 50 Calibrate (relative to IC 50 :IC 50，阿维鲁单抗 / IC 50，测试scFv The potency is summarized in Table 18, which shows that up to 5-fold the IC of avelumab could be resolved in this assay. 50 The scFvs tested had better or similar potency.

[0387] Table 12. Summary of scFv preparation and initial stability data.

[0388]

[0389] Table 13. List of humanized scFv variants.

[0390]

[0391] **Unpaired Cys at position 57 in CDRH2 (AHo numbering)

[0392] Table 14. Production summary table.

[0393]

[0394] Table 15. Affinity of scFvs for human PDL1.

[0395]

[0396]

[0397] Table 16. Potency of scFvs in inhibiting the interaction between PDL1 and PD-1.

[0398]

[0399] Table 17. Potency of scFvs in inhibiting the interaction between PDL1 and B7-1.

[0400]

[0401] Table 18. Potency of scFvs in neutralizing PDL1 / PD-1 interaction in reporter gene assay.

[0402]

[0403] 5.5 Binding to hPDL1-Expressing Cells Determined by Flow Cytometry

[0404] For some molecules, the binding potency to PDL1 expressing cells was determined. The same cell lines as during hit validation were used (CHO-PDL1 and CHO-K1), but the scFvs were detected by APC-labeled protein L. Serial dilutions of each molecule to be tested, as well as the reference avelumab, were added to the plate. The single IC 50 The IC values ​​of the reference molecule Avelumab were collected on each plate. 50 Calibrate (relative to IC 50 :IC 50，阿维鲁单抗 / IC 50，测试scFv ). The efficacy is summarized in Table 19.

[0405] Table 19. Summary of the binding potency of the tested scFvs to cellular PDL1.

[0406]

[0407] 5.6 Species Cross-Reactivity (Binding to Cynomolgus Monkey and Mouse PDL1 as Determined by SPR)

[0408] Cross-reactivity with cynomolgus monkey PDL1 was determined using recombinant PDL1 produced by Sino Biological using a method similar to that used to measure binding to human PDL1. The affinities of all tested scFvs are summarized in Table 20. All tested scFvs that showed binding to human PDL1 also showed binding to cynomolgus monkey PDL1.

[0409] Table 20. Affinity of scFv for cynomolgus monkey PDL1

[0410]

[0411] 5.7 Selectivity for PDL1 and PDL2 determined by SPR

[0412] The binding of humanized scFv to PDL2 was tested by SPR analysis on a T200 device (Biacore, GE Healthcare). In this experiment, Fc-tagged human PDL2 was captured using the Human Antibody Capture kit from GE Healthcare. After each analyte injection cycle, the CM5 sensor chip was regenerated and the new antigen was captured. The scFv was injected as an analyte for 5 minutes at a concentration of 180nM diluted in running buffer, and the protein was then allowed to dissociate for 12 minutes. No binding to PDL2 was observed for all humanized scFvs tested listed in Table 21.

[0413] Table 21. Binding of ScFvs to mouse PDL1 and PDL2 as determined by SPR.

[0414]

[0415] Example 6: Biophysical Characterization of Humanized scFv

[0416] Selected domains with better affinity than Avelumab were produced on a larger scale (0.2L-1.2L expression). In addition, after purification, protein samples were concentrated to >10 mg / mL using centrifugal concentrators with a molecular weight cutoff of 5 kD. Table 22 summarizes the material preparation for stability assessment.

[0417] 6.1 Storage stability study

[0418] The humanized scFv was subjected to stability studies, such as a four-week stability study, in which the scFv was formulated in an aqueous buffer (final buffer, 50 mM NaCiP, 150 mM NaCl, pH 6.4) at 10 mg / ml and stored for four weeks at <-80°C, 4°C, and 40°C. The proportion of monomers and oligomers in the formulation was assessed by integration of SE-HPLC peak areas at least one week, two weeks, and after the end of each study. For some molecules, other time points were recorded. Table 23 compares the endpoint measurements obtained on day 7 and day 28 of the study.

[0419]

[0420]

[0421] 6.2 Freeze-thaw stability study

[0422] In addition to the above-mentioned storage stability study, the compatibility (colloid stability) of the highest-performance scFv molecules was also evaluated for freeze-thaw (F / T) cycles. In order to perform F / T stability evaluation, the same analytical method and parameters (monomer content % and monomer loss %) as the storage stability study (SE-HPLC, SDS-PAGE) were used to monitor the quality of the molecules along with five F / T cycles. Table 24 illustrates the monomer content loss % along with five repeated F / T cycles. Since no special freeze-thaw study was performed, the freeze-thaw data obtained from the -80°C samples obtained in the storage stability study within 28 days are shown in the figure below. After repeated F / T cycles, no molecule had a monomer content loss of>4%.

[0423] Table 24. F / T stability - % monomer loss after repeated freeze-thaw cycles.

[0424]

[0425] *% monomer content loss after F / T cycle X

[0426] NA: Not applicable

[0427] 6.3. Thermal unfolding

[0428] Table 25 shows the thermal unfolding data obtained from DSF measurements of selected scFv constructs. By fitting the data to the Boltzmann equation, the resulting Tm values ​​can be determined. Table 25 summarizes the melting temperatures measured by DSF.

[0429] Table 25. DSFs of domains meeting success criteria.

[0430]

[0431] NA: Not Applicable, ND: Not Determined

[0432] Multispecific molecules comprising antibodies of the invention

[0433] Table 3 includes exemplary multispecific molecules comprising antibodies of the invention.

[0434] Example 7: Affinity for PDL1, CD137, HSA and MSA.

[0435] method:

[0436] The affinity of PDL1 to different species was determined by SPR measurement using Biacore T200 equipment (GE Healthcare). Antibodies specific to the Fc region of human IgG were fixed on a sensor chip (CM5 sensor chip, GE Healthcare) by amine coupling. For all forms, except the Morrison form containing Fc, the fixed antibody captured the PDL1-Fc chimeric protein of different species. Three times serial dilutions (0.12-90nM) of molecules specific to PDL1 were injected into the flow cell for three minutes, and the dissociation was monitored for 10 minutes. After each injection cycle, the surface was regenerated with a single injection of 3M MgCl2 solution. The apparent dissociation constant (kd) and association rate constant (ka) and apparent dissociation equilibrium constant (KD) were calculated using a one-to-one Langmuir binding model. The affinity of CD137 to different species was determined using the same settings as PDL1 (except that the CD137-Fc chimeric protein of different species was captured by fixed antibodies).

[0437] The Fc-containing format was captured directly by an antibody specific for the Fc region of human IgG. Two-fold serial dilutions of the PDL1 extracellular domain or the CD137 extracellular domain (ranging from 90 to 0.35 nM) were tested for binding to the IgG captured on the biosensor chip. After each injection cycle, the surface was regenerated with one injection of 3M MgCl2 solution.

[0438] The affinity of the molecules to serum albumin (SA) of different species was determined by SPR measurements using a Biacore T200 device (GE Healthcare). SA was directly coupled to a CM5 sensor chip (GE Healthcare) using amine coupling chemistry. After performing a regeneration search and surface performance tests to find the optimal assay conditions, the dose response was measured and the obtained binding curves were double referenced (empty reference channel and zero analyte injection) and fitted using a 1:1 Langmuir model to obtain the kinetic parameters. The assay was performed in 1X PBS-Tween buffer at pH 5.5.

[0439] result:

[0440] Binding kinetic measurements of the humanized constructs showed differences in binding affinity to PDL1, with the STR graft having a 20-fold improvement in affinity over the CDR graft of the same clone when comparing the CDR and structural grafts (STRs) of clone 33-03-G02 (PRO885 vs. PRO1126 in Table 26). The CDR graft derived from clone 37-20-B03 (PRO997) showed approximately twice the affinity compared to the STR graft of clone 33-03-G02. When the CDR graft of 33-03-G02 was combined into different multispecific formats, its binding affinity was similar to that of the parental scFv (compare PRO830 vs. PRO885, PRO951, PRO1123, PRO1124, PRO963, PRO966, PRO1057, PRO1058, PRO1059, and PRO1060, Table 26). The affinities of scFvs derived from both clones to human and cynomolgus monkey PDL1 were almost identical (see PRO977 and PRO830 in Table 26).

[0441]

[0442]

[0443] Example 8: Blockade of PDL1 / PD-1 interaction measured in a cell-based reporter gene assay using CHO cells expressing PDL1 and TCR activating molecules and Jurkat cells expressing PD-1 and containing the luciferase gene under a NFAT response element.

[0444] method

[0445] In the bioluminescent reporter gene analysis, Jurkat T cells stably expressing NFAT (nuclear factor of activated T cells)-luciferase reporter gene and human PD-1 act as effector T cells. Cells stably expressing human PDL1 and T cell receptor (TCR) activators act as antigen presenting cells. Co-culture of two cell lines induces the activation of Jurkat NFAT pathways by cross-linking of TCR activators / TCR complexes. After the participation of PDL1-expressing cells, PD-1 signaling in PD-1 effector T cells inhibits T cell function and leads to NFAT pathway inhibition. Blocking of PD-1 and PDL1 receptor interactions leads to the reactivation of NFAT pathways.

[0446] 35,000 CHO / PDL1 / TCR activation (BPSBioscience) cells in 100 μl of cell culture medium (DMEM / F12, 10% FCS) were added to the inner wells of the leukocyte culture plate and incubated at 37°C and 5% CO2 for 16-20 hours. The next day, 95 μl of cell culture medium was removed from each well and 50 μl of 2-fold concentrated serial dilutions (3,000 to 0.46 ng / ml) of each molecule to be tested (including reference avelumab) were added. Then, 50 μl of PD-1-expressing effector Jurkat cells (BPSBioscience) diluted at 400,000 cells / ml in assay buffer (RPMI1640 containing 10% FCS) were added to each well, and the plate was incubated at 37°C and 5% CO2 for 6 hours. Finally, 50 μl of luciferase substrate (BPS Bioscience) prepared according to the manufacturer's protocol was added per well, and the plate was incubated in the dark for 30 min and luminescence was measured using a Topcount.

[0447] result

[0448] To evaluate the impact of CDR set and framework selection on potency in neutralizing PDL1 binding to PD-1, three anti-PDL1 scFvs were tested in an NFAT reporter cell-based assay. PRO830 contains the CDR set of clone 33-03-G02 grafted onto a VH4 framework, and PRO997 and PRO1013 contain the CDR set of clone 37-20-B03 grafted onto a VH4 or VH1 framework, respectively. PRO830 had the lowest potency of the three tested scFvs, with an IC 50 The value was 42.88 ng / ml and had a similar potency to avelumab. The IC 50 The value was 34.09 ng / ml. PRO997 was the most potent molecule. When grafted onto the VH4 framework, the same CDR set was about 2-fold more potent than when grafted onto the VH1 framework. IC 50 The values ​​were 11.12ng / ml and 21.29ng / ml respectively. Figure 4 A and Table 27)

[0449] For bispecific molecules with the 33-03-G02 PDL1 domain, the neutralization potency of PDL1 binding to PD-1 was determined before (CDR graft) and after (structural graft) domain optimization. The CDR graft (PRO885) was compared to the structural graft (PRO1126). Domain optimization increased the neutralization potency threefold, with an IC of PRO885 of 50 The value is 137.2ng / ml, and the IC of PRO1126 50The value is 48.15ng / ml. Figure 4 B and Table 27).

[0450] Two trispecific molecules with an anti-PDL1 domain of the CDR graft of clone 33-03-G02 and two different human serum albumin binding domains (for half-life extension) were also evaluated for their potency to neutralize the PDL1 / PD-1 interaction. The HSA domain of PRO1057 also binds to mouse serum albumin. Experiments were performed in the presence of 25 mg / ml HSA. Neutralization potency (IC 50 =665.1ng / ml) is lower than Avelumab. ( Figure 5 and Table 27).

[0451] Table 27. Neutralization of PDL1 PD-1 interaction in NFAT reporter assay.

[0452]

[0453] NA: Not applicable

[0454] * :IC 50，阿维鲁单抗 (ng / ml) / IC 50，测试分子 (ng / ml)

[0455] In serum, the so-called Morrison format was tested in a cell-based potency reporter assay. In this format, the IgG part carries one specificity (bivalent) and two scFvs specific for the second target are linked to the heavy chain (HC) or light chain (LC) of the IgG via a flexible peptide linker. All Morrison molecules tested carried the anti-PDL1 domain of the CDR graft of clone 33-03-G02 on both IgG arms. The two constructs PRO1059 and PRO1060 differed in the fusion of the two anti-CD137 scFvs on the heavy chain (HC) or on the (LC). PRO1062 has the same architecture as PRO1060 but has a different CD137 domain. The neutralization potency of all molecules was similar ( Figure 6 and Table 27).

[0456] Example 9: Blocking the interaction of PDL1 with PD-1 and B7-1 using competition ELISA.

[0457] These assays were performed to evaluate the ability of PDL1 inhibitors to block the interaction between PDL1 and PD-1 or PDL1 and B.71. Different formats, including scFvs, scDbs, scDb-scFv, and Morrison, were analyzed in competition ELISAs and compared to the reference IgG avelumab.

[0458] PDL1 / PD-1 competition ELISA

[0459] The ELISA microplate coated with 4 μg / ml human PD-1 at 4 ° C overnight was washed 3 times with 450 μl wash buffer per well. Block the plate for 1 hour at room temperature by adding 300 μl PBS (dilution buffer) containing 1% BSA and 0.2% Tween to each well. In a dilution buffer containing 1 ng / ml biotinylated human PDL1, the inhibitor was serially diluted in 3-fold steps to a final concentration of 300 to 0.005 ng / ml. The mixture was pre-incubated at room temperature for 1 hour with slow stirring on a rotary agitator (21 rpm) and added to the microplate after washing 3 cycles with 450 μl wash buffer per well. The plate was incubated at room temperature for 1.5 hours with slow stirring, and then 10 ng / ml streptavidin-polyHRP40 was added to the wells of each microplate after washing 3 times with 450 μl wash buffer per well. After incubation for 1 h at room temperature, the plates were washed 3 times with 450 μl of wash buffer and TMB substrate solution was added. After 6 min, the enzymatic reaction was stopped by adding 1 M HCl and the absorbance was measured at 450 nm using 690 nm as the reference wavelength. To calculate the IC 50 Values ​​were used and four-parameter logistic (4PL) curve fitting was performed in Graph Pad Prism using reference-subtracted values.

[0460] like Figure 7 As shown in Table 28, all PDL1 inhibitors blocked the interaction of PD-1 with PDL1 when tested in a competition ELISA. scFv PRO830 blocked the interaction with similar potency to avelumab, while the IC values ​​of PRO997 and PRO1013 were 50 The values ​​were significantly lower than those of avelumab and therefore more potent inhibitors. When combined in multispecific formats, i.e., scDbs or Morrisons, all molecules retained their inhibitory properties. PRO885 was less potent than avelumab, while for PRO1126, which contains a modified anti-PDL1 domain, the IC 50 The Morrison format was slightly less potent than avelumab. Neutralization of PRO1057 was also demonstrated in the presence of human serum albumin, with IC 50 The value is approximately twice as high.

[0461] PDL1 / B7-1 competition ELISA

[0462] ELISA microplates coated with 4 μg / ml human B7-1 overnight at 4°C were washed three times with 450 μl wash buffer per well. The plates were blocked for 1 hour at room temperature by adding 300 μl of PBS (dilution buffer) containing 1% BSA and 0.2% Tween to each well. The inhibitors were serially diluted in 3-fold steps to a final concentration of 900 to 0.015 ng / ml in dilution buffer containing 40 ng / ml biotinylated PDL1. The mixture was pre-incubated at room temperature for 1 hour with slow stirring on a rotary agitator (21 rpm) and added to the microplate after washing for 3 cycles with 450 μl wash buffer per well. The plate was incubated at room temperature for 1.5 hours with slow stirring, and then 10 ng / ml streptavidin-polyHRP40 was added to each well of the microplate after washing 3 times with 450 μl wash buffer per well. After incubation for 1 h at room temperature, the plates were washed 3 times with 450 μl of wash buffer and TMB substrate solution was added. After 6 min, the enzymatic reaction was stopped by adding 1 M HCl and the absorbance was measured at 450 nm using 690 nm as the reference wavelength. To calculate the IC 50 Values ​​were used to perform a four-parameter logistic (4PL) curve fit in Graph Pad Prism using the reference-subtracted values.

[0463] All PDL1 inhibitors except PRO1126 were tested for their ability to block the interaction of PD-1 with B7-1. PRO830 showed similar potency to avelumab, while the IC 50 All scDbs and Morrisons also inhibited the interaction between PDL1 and B.7-1. scDb PRO885 had a potency similar to that of avelumab, while the IC values ​​of Morrisons were 50 The value was reduced by about 2-3.4 times. Figure 8 and Table 28.

[0464] Table 28. Blocking of PDL1 interaction with PD-1 and B7-1 using competitive ELISA

[0465]

[0466] NA: Not applicable

[0467] *:IC 50，阿维鲁单抗 (ng / ml) / IC 50，测试分子 (ng / ml)

[0468] Example 10: Evaluation of the stimulatory effects of simultaneous PDL1 blockade and CD137 stimulation in a cell-based assay using human PBMCs stimulated with the superantigen SEA.

[0469] In this experiment, the synergistic effect of PD-1 / PDL1 inhibition and CD137 agonism was evaluated. The assay used peripheral blood mononuclear cells (PBMCs) stimulated with the superantigen Staphylococcal Enterotoxin A (SEA) to induce the expression of PDL1 on antigen presenting cells (APCs) and T cells and the expression of CD137 on T cells, respectively. By applying anti-PDL1xCD137 molecules, two T cell regulatory signaling pathways were simultaneously targeted: inhibition of the inhibitory PD-1 / PDL1 pathway and the activating CD137 pathway through the formation of immune synapses mediated by the bispecific anti-PDL1xCD137 molecule (PRO885). T cell activation was assessed by the secretion of interleukin 2 (IL-2) and compared with the effect mediated by PDL1 inhibition mediated by the benchmark reference antibody avelumab. In addition, the anti-PDL1 scFv PRO997 was tested in the same experimental setting and compared with avelumab.

[0470] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human whole blood by density gradient centrifugation. NK cells were then removed from the PBMCs using an anti-CD56 antibody and a MACS cell separation kit (Miltenyi Biotec). Next, 100,000 PBMCs were added to each well of a 96-well plate, followed by serial dilutions of PRO885, PRO997, and avelumab in assay buffer (containing SEA at a concentration of 10 ng / ml). After incubation for 96 hours at 37°C and 5% CO2, cell supernatants were collected, and human interleukin 2 (IL-2) levels in the culture supernatants were quantified using BioLegend's IL-2 Human ELISA MAX assay according to the kit instructions. IL-2 concentrations were interpolated from the IL-2 standard curve, back-calculated, and plotted against avelumab and PRO885 concentrations to calculate EC 50 value.

[0471] like Fig. 9 As shown in Figure 2, after simultaneous blockade of PD-1 / PDL1 interaction and stimulation of CD137 by the addition of the bispecific molecule PRO885, T cells secreted IL-2. PRO885 showed higher T cell activation and better efficacy than avelumab (PRO885, EC 50=39.92 ng / ml; avelumab, EC50 = 69.89 ng / ml, Table 29). This finding suggests that the bispecific anti-PDL1xCD137 scDb PRO885 is able to induce stronger T cell stimulation compared to blocking PDL1 with avelumab alone. In addition, the high-affinity anti-PDL1 scFv PRO997 was found to be more effective than avelumab in stimulating T cells (PRO997, EC50 = 69.89 ng / ml, Table 29). 50 =40.86ng / ml; Avelumab, EC 50 =90.18 ng / ml, Table 29).

[0472] Table 29. EC values ​​of PRO885 and PRO997 determined in PBMC assay using SEA stimulation 50 value.

[0473]

[0474] Example 11: Evaluation of the anti-tumor efficacy of anti-PDL1 antibodies in the human cell line-derived lung cancer xenograft model HCC827.

[0475] The anti-tumor activity of the anti-PDL1 IgG1 antibody PRO1137 (SEQ ID Nos: 90 and 91) was evaluated in human HCC827 NSCLC xenografts using the immunodeficient NOG mouse strain from Taconic and allogeneic human peripheral blood mononuclear cells. The implanted human T lymphocytes showed xeno-reactivity to foreign major histocompatibility (MHC) class I and II and other antigens from mouse cells. As a result, the T lymphocytes caused inflammatory infiltration in different organs, leading to the death of the animals after a few weeks, a process known as xenograft-versus-host disease (xGVHD). Treatment with immunomodulatory antibodies such as anti-PDL1 and anti-CD137 has been shown to exacerbate xGVHD (Sanmamed MF et al. Nivolumab andurelumab enhance antitumor activity of human T lymphocytes engrafted inRag2- / -IL2Rgnull immunodeficient mice. Cancer Res 2015; 75(17): 3466-3478).

[0476] Study Setting and Treatment Protocol: Female NOG mice were injected unilaterally with 5x10 6HCC827 cells. The cells were injected as a mixture of 50% cell suspension in PBS and 50% matrigel, with a total injection volume of 100 μl. After the tumor cells were injected into NOG mice and successfully implanted tumors (median tumor volume of 80-100 mm 3 ), 5x10 6 On the day of randomization, four mice per group were reconstituted with PBMCs from donor A and four mice were reconstituted with PBMCs from donor B. Treatments were initiated 1-2 hours after PBMC injection and proceeded as follows.

[0477]

[0478] Based on the in vitro activity of the antibody blocking the PD-1 / PDL1 interaction in the NF-AT reporter gene, a 0.2 mg dose of PRO1137 was set to achieve the same relative activity simulated by a 0.1 mg dose of avelumab (per mouse). Therefore, a 0.2 mg dose of PRO1137 can be expressed as one relative unit (1r.U) relative to a 0.1 mg dose of avelumab. Body weight and tumor volume were measured by caliper twice a week. Animals were killed at the specified time points based on the results of the study. All animals were executed at the "same" time points (on days 17 and 18). Due to production capacity reasons, sample collection and processing of the first half of each group was performed on the first day, and sample collection and processing of the second half of each group was performed on the second day. Animals reconstituted with PBMCs from two different donors had equal representation in both sampling groups.

[0479] result: The antitumor activity of anti-PDL1 PRO1137 in human HCC827 NSCLC xenografts was evaluated by measuring tumor volume using the immunodeficient NOG mouse strain and allogeneic human peripheral blood mononuclear cells (hPBMCs). Fig.10 Tumor volumes were measured twice a week until mice were sacrificed on day 17 or 18. Tumor volumes were normalized to the tumor volume at the start of treatment (relative tumor volume). Fig.10 As shown, treatment with PRO1137 monoclonal antibody showed reduced tumor growth compared to the vehicle control group. Notably, treatment with PRO1137 did not result in a decrease in median body weight, suggesting that the molecule was well tolerated at the dose levels tested ( Fig.11 ).

[0480] Example 12: Evaluation of the anti-tumor efficacy of PRO1137 in NOG mice transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC)

[0481] The antitumor activity of PRO1196 (anti-PDL1 IgG1; SEQ ID NOs: 92 and 93) was compared with vehicle treatment or with avelumab in human HCC827 NSCLC xenografts using the NOG mouse strain transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs).

[0482] Study Setting and Treatment Protocol: Female NOG mice transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs) were subcutaneously injected with HCC827 NSCLC cells. Mice received unilateral injection of 5x10 6 HCC827 cells were injected with a mixture of 50% cell suspension in PBS and 50% Matrigel (), with a total injection volume of 100 μl. Tumor cells were injected into NOG mice and successfully implanted into tumors (median tumor volume of 80-100 mm 3 ), mice (n=10) were randomly divided into treatment groups:

[0483]

[0484] Body weight and tumor volume were measured twice a week by caliper. Tumors were harvested on days 25, 29, and 30 after treatment.

[0485] result: The antitumor activity of PRO1196 (anti-PDL1 IgG1; SEQ ID NOs: 92 and 93) in human HCC827 NSCLC xenografts was evaluated by measuring tumor volume using immunodeficient NOG mice transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs). Fig.12 Tumor volumes were measured twice a week until mice were sacrificed on day 25, 29, or 30. Tumor volumes were normalized to the tumor volume at the start of treatment (relative tumor volume). Fig.12 In the study, treatment with PRO1196 and avelumab resulted in stabilization of tumor growth compared with the control group.

[0486] Example 13: Evaluation of the anti-tumor efficacy of PDL1 blockade and simultaneous local stimulation of CD137 in a syngeneic MC38 colon cancer model.

[0487] In addition, the anti-tumor activity of the multispecific antibodies comprising the PDL1 domain of the present invention will be tested in the MC38 colon cancer model of syngeneic C57BL / 6 mice with a complete immune system. Others have used this model to show enhanced anti-tumor activity by combining treatment with CD137 agonists and PD-1 / PDL1 antagonists (Chen S et al. Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector / memory CD8 T cells in a poorly immunogenic tumor model. Cancer Immunol Res 2014; 3(2): 149-160 and Rodriguez-Ruiz ME et al. Abscopal effects of radiotherapy are enhanced by combined immunostimulatory mAbs and are dependent on CD8 T cells and crosspriming. Cancer Res 2016; 76(20): 5994-6005).

[0488] Since the anti-CD137 domain and anti-PDL1 domain of the multispecific antibody to be tested do not cross-react with mouse PDL1 and mouse CD137, the engineered human CD137 knock-in model established by CrownBio will be used. In this model, the extracellular and transmembrane domains of mouse CD137 are replaced with the corresponding sequences of human CD137 in the C57BL / 6 mouse background using the CRISPR / Cas9 system. In addition, a modified MC38 tumor cell line will be used, which expresses human PDL1 under the control of the CMV promoter rather than mouse PDL1. The effect of the multispecific antibody on tumor volume is compared with the combined treatment with humanized IgG1 and humanized IgG4, and the humanized IgG1 contains the same PDL1-specific variable domains as the multispecific antibody, and the humanized IgG4 has the same CD137-specific variable domains. To provide further evidence of a local anti-tumor immune response, the occurrence of tumor-infiltrating lymphocytes (e.g., CD8+, CD4+, and regulatory T cells) will be analyzed by flow cytometry. To explore the modulation of the systemic immune system after anti-CD137 / anti-PDL1 treatment, the occurrence of CD4+ and CD8+ T cells in the liver and spleen will be analyzed by flow cytometry and possible immunohistochemistry. In addition, systemic IFN levels can be analyzed using a quantitative ELISA method. To further characterize the safety of anti-CD137 / anti-PDL1 combination therapy, clinical chemopathological parameters primarily associated with liver toxicity (observed clinically for anti-CD137 treatment), such as elevated levels of alanine aminotransferase, glutamate dehydrogenase, and aspartate aminotransferase, can be evaluated.

Claims

1. An antibody having binding specificity to human PDL1, comprising HCDR1, HCDR2, HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; Wherein the antibody comprises: (a) a heavy chain variable region consisting of an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 14; and a light chain variable region consisting of an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 26; (b) a heavy chain variable region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of the group consisting of SEQ ID NO: 15; and a light chain variable region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 26; or (c) a heavy chain variable region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of the group consisting of SEQ ID NO: 16; and a light chain variable region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

27.

2. The antibody of claim 1, wherein the antibody comprises: a heavy chain variable region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and a light chain variable region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

27.

3. The antibody according to claim 1, comprising: (a) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26; or (b) a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 26; or (c) a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO:

27.

4. The antibody according to claim 1, wherein the antibody: (i) the dissociation constant for binding to human PDL1 is less than 100 pM as measured by surface plasmon resonance; (ii) K of binding to human PDL1 off The rate was measured by surface plasmon resonance to be 10 -4 s -1 or lower; (iii) K of binding to human PDL1 on The rate was measured by surface plasmon resonance to be at least 10 6 M -1 s -1 or higher; (iv) cross-reacts with cynomolgus monkey PDL1 and has a dissociation constant for binding to cynomolgus monkey PDL1 of less than 50 pM as measured by surface plasmon resonance; (v) does not cross-react with Mus musculus PDL1, as measured by surface plasmon resonance; (vi) no binding to human PDL2, as measured by surface plasmon resonance; (vii) having an ability to neutralize the PDL1 / PD-1 interaction with a potency relative to the potency of avelumab measured in an ELISA assay of greater than 2; and wherein the potency is the IC of avelumab in ng / mL measured in an ELISA assay 50 The values ​​are compared with the IC values ​​of the antibodies in ng / mL measured in the ELISA assay. 50 (viii) having an ability to neutralize the PDL1 / PD-1 interaction with a potency greater than 2 relative to the potency of avelumab as measured in a NFAT reporter gene assay; and wherein the potency is an IC value of avelumab in ng / mL as measured in a NFAT reporter gene assay. 50 Values ​​are compared to the IC values ​​of the antibodies in ng / mL measured in the NFAT reporter gene assay. 50 The ratio of the values ​​of , wherein the antibody is a scFv; and (ix) having an ability to neutralize the PDL1 / B7-1 interaction with a potency relative to the potency of avelumab measured in an ELISA assay of greater than 2; and wherein the potency is the IC of avelumab in ng / mL measured in an ELISA assay 50 The values ​​are compared with the IC values ​​of the antibodies in ng / mL measured in the ELISA assay. 50 The ratio of the values ​​is as follows, wherein the antibody is a scFv.

5. The antibody according to claim 1, wherein the antibody: (i) when in scFv format, has a melting temperature of at least 60°C as determined by differential scanning fluorimetry, wherein the antibody is formulated in 50 mM phosphate citrate buffer with 150 mM NaCl at pH 6.4; (ii) when in scFv format, the antibody loses less than 3% of its monomer content after 5 consecutive freeze-thaw cycles when the starting concentration of the antibody of the invention is 10 mg / ml, wherein the antibody of the invention is formulated in 50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.4; and (iii) when in scFv format, the antibody of the invention loses less than 5% of its monomer content after storage at 4°C for at least 2 weeks when the starting concentration is 10 mg / ml, wherein the antibody is formulated in 50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.

4.

6. The antibody of claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a Fab, a Fv, a scFv, a dsFv, and a scAb.

7. The antibody of claim 6, wherein the antibody is a scFv.

8. The antibody according to claim 7, wherein the scFv consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:

31.

9. The antibody according to claim 8, wherein the scFv consists of the amino acid sequence of SEQ ID NO:

31. 10 . A pharmaceutical composition comprising the antibody according to claim 1 , and a pharmaceutically acceptable carrier.

11. Use of the antibody according to any one of claims 1 to 9 in the preparation of a medicament for treating lung cancer.

12. Use of the pharmaceutical composition according to claim 10 in preparing a medicament for treating lung cancer.

13. A nucleic acid encoding the antibody according to any one of claims 1 to 9.

14. A method for producing an antibody according to any one of claims 1 to 9 in vitro, the method comprising the following steps: Cultivate a host cell comprising the nucleic acid of claim 13.

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