Antibodies targeting PDL1 and methods of using same

Abstract

To provide an isolated antibody which specifically binds human PDL1, to provide a pharmaceutical composition thereof, and to provide a method of use thereof.SOLUTION: Provided are an isolated antibody having binding specificity for human PDL1, the isolated antibody that specifically binds human PDL1 comprising: HCDR1, HCDR2 and HCDR3 sequences having specific sequences respectively; and LCDR1, LCDR2 and LCDR3 sequences having different specific sequences respectively, as well as pharmaceutical compositions thereof, and methods of use thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to isolated antibodies that specifically bind to human PDL1, as well as pharmaceutical compositions and methods of use thereof. The present invention further relates to nucleic acids encoding said antibodies, vectors comprising said nucleic acids, host cells comprising said nucleic acids or said vectors, and methods of producing said antibodies. [Background technology]

[0002] PDL1 (CD274, B7-H1) is a 40 kDa type I transmembrane protein. PDL1 is a membrane surface glycoprotein ligand for PD-1, a major immune checkpoint receptor expressed by activated T and B cells, and mediates immunosuppression. PDL1 is involved in suppressing immune system responses during chronic infections, pregnancy, tissue allogeneic transplantation, autoimmune diseases, and cancer. PDL1 is known to be expressed in both antigen-presenting cells and cancer cells in human head and neck squamous cell carcinoma, melanoma, and brain tumors, thyroid, thymus, esophagus, lung, breast, gastrointestinal tract, colorectum, liver, pancreas, kidney, adrenal cortex, bladder, urothelium, ovary, and skin (Non-Patent Documents 1 to 7). PDL1 is rarely expressed in normal tissues, but is inducibly expressed at tumor sites (Non-Patent Documents 8 and 9). PDL1 downregulates T cell activation and cytokine secretion by binding to PD-1 (Non-Patent Document 10). Activated PD-1 by PDL1 potentially provides an immune-permissive environment for tumor initiation and growth. PDL1 also exerts a negative control on T cell function through its interaction with another receptor, B7.1 (B7-1, CD80).

[0003] Inhibition of PDL1 / PD-1 interaction leads to potent antitumor activity. Various antibodies against PDL1 are already known (see Patent Documents 1 and 2), and many clinical antibodies that disrupt PD-1 signaling have been developed. These antibodies fall into two major categories: those targeting PD-1 (nivolumab, Bristol-Myers Squibb; pembrolizumab, Merck, Whitehouse Station, NJ; pidilizumab, CureTech, Yavne, Israel) and those targeting PDL1 (MPDL3280A (Genentech, South San Francisco, CA); MEDI4736 (MedImmune / AstraaZeneca); BMS-936559 (Bristol-Myers Squibb); MSB0010718C (EMD Serono, Rockland, MA)) (for review, see 11). Targeting PDL1 and PD-1 appear to result in different biological effects. PD-1 antibodies prevent the interaction of PD-1 with both its ligands PDL1 and PDL2. PDL1 antibodies do not prevent the interaction of PD-1 with PDL2, but the effect of this interaction is unknown. However, PDL1 antibodies interfere with the interaction of PDL1 with not only PD-1 but also B7-1 (Non-Patent Document 12), which is thought to result in negative signaling to T cells. Promising early data have been shown by blocking PDL1, and four anti-PDL1 mAbs, namely atezolizumab and MEDI4736 (both Fc null mutants of human IgG1), MSB001078C (IgG1), and BMS-936559 (IgG4), are currently in clinical trials (Non-Patent Document 13).

[0004] To date, no satisfactory methods have been disclosed for inducing a strong immune response in cancer patients. Thus, there is a need in the art to obtain therapeutic modulators that improve the PDL1 / PD-1 interaction and to obtain methods for overcoming the immunosuppressive mechanisms observed in cancer patients. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 079174 [Patent Document 2] International Publication No. 2017 / 118321 [Non-patent literature]

[0006] [Non-Patent Document 1] Katsuya Y, et al., Lung Cancer.88(2):154-159(2015) [Non-Patent Document 2] Nakanishi J, et al., Cancer Immunol Immunother.56(8):1173-1182(2007) [Non-Patent Document 3] Nomi T, et al., Clin Cancer Res.13(7):2151-2157(2007) [Non-Patent Document 4] Fay AP, et al., J Immunother Cancer.3:3(2015) [Non-Patent Document 5] Strome SE, et al., Cancer Res.63(19):6501-6505(2003) [Non-Patent Document 6] Jacobs JF,et al.Neuro Oncol.11(4):394-402(2009) [Non-Patent Document 7] Wilmotte R,et al.Neuroreport.16(10):1081-1085(2005) [Non-Patent Document 8] Dong H, et al., Nat Med.8(8):793-800(2002) [Non-Patent Document 9] Wang et al., Onco Targets Ther.9:5023-5039(2016) [Non-Patent Document 10] Freeman et al.,2000;Latchman et al.,2001 [Non-Patent Document 11] Postow MA et al.,J Clin Oncol.Jun 10;33(17):1974-82(2015) [Non-Patent Document 12] Butte MJ, et al., Immunity 27:111-122, (2007) [Non-Patent Document 13] Chester C.,et al.,Cancer Immunol Immunother Oct;65(10):1243-8(2016) Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide antibodies that specifically bind to human PDL1 protein and have advantageous properties in therapeutic applications, such as high affinity and improved efficacy, as well as improved biophysical properties, such as solubility, development and stability. [Means for solving the problem]

[0008] In one aspect, the invention relates to novel PDL1 antibodies.

[0009] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody of the invention and a pharma- ceutically acceptable carrier.

[0010] In another aspect, the present invention relates to the antibody of the invention or the composition of the invention for use as a medicament.

[0011] 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 of said treatment.

[0012] In one aspect, the invention relates to the use of an antibody of the invention or a composition of the invention in the manufacture of a medicament for the treatment of cancer in a subject in need of such treatment.

[0013] In another aspect, the invention relates to a method for treating cancer in a subject in need of such treatment, comprising administering to said subject a therapeutically effective amount of an antibody of the invention or a composition of the invention.

[0014] In yet another aspect, the invention relates to a nucleic acid encoding an antibody of the invention. In a further aspect, the invention relates to a vector comprising said nucleic acid. In a further aspect, the invention relates to a host cell comprising said nucleic acid or said vector.

[0015] In another aspect, the invention relates to a method for producing an antibody of the invention, the method comprising the step of culturing a host cell containing a nucleic acid or vector of the invention.

[0016] The following aspects, advantageous features and preferred embodiments of the invention, taken individually or in combination, further contribute to solving the object of the invention:

[0017] 1. An isolated antibody having binding specificity for 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 NO: 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 NO: 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 NO: 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 any 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 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 18, 21, 24, 49, 52 and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18; (f) a light chain variable region CDR3 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 19, 22, 25, 50, 53 and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19.

[0018] 2. The antibody of item 1, wherein the antibody comprises: (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; (b) 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; (c) 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; (d) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 8, 9 and 10, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; (e) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 11, 12 and 13, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 23, 24 and 25, respectively; (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; (g) 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; (h) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, respectively, and the LLCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (i) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 39, 40 and 41, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; (j) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 42, 43 and 44, respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 54, 55 and 56, respectively.

[0019] 3. The antibody of item 1, comprising: (a) an HCDR1 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:1; (b) an HCDR2 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:2; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:3; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 17; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 18; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 19.

[0020] 4. The antibody of item 1, comprising: (a) an HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) an HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:6; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 7; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 21; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 22.

[0021] 5. The antibody of item 1, comprising: (a) an HCDR1 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO: 32; (b) an HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 33; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 34; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 48; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 49; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 50.

[0022] 6. The antibody of item 1, comprising: (a) an HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36; (b) an HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 37; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 38; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:51; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 52; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:53.

[0023] 7. The antibody of any one of the preceding items, wherein the antibody comprises a heavy chain variable region (VH), wherein said VH is VH1, VH3 or VH4, preferably VH3 or VH4, more preferably VH3.

[0024] 8. The antibody comprises a light chain variable region (VL), wherein the VL is Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1; a framework FR4 selected from Vκ FR4, particularly Vκ1 FR4, Vκ3 FR4, and VλFR4, comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more identity with an amino acid sequence selected from any one of SEQ ID NO:64 to SEQ ID NO:70, preferably a Vλ FR4 set forth in any one of SEQ ID NO:64 to SEQ ID NO:70, preferably a Vλ FR4 set forth in SEQ ID NO:64 or 65, more preferably a Vλ FR4 set forth in SEQ ID NO:64; The antibody of any one of the preceding items.

[0025] 9.Antibodies A heavy chain variable region comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 14, 15, 16, 45, 46 and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16; A light chain variable region comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27; The antibody of any one of the preceding items.

[0026] 10.Antibody, (a) a heavy chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO:14 and a light chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO:26; (b) a heavy chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid sequence SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid sequence SEQ ID NO: 26; (c) a heavy chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid sequence SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid sequence SEQ ID NO: 27; (d) a heavy chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO: 45 and a light chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO: 57; (f) a heavy chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO: 46 and a light chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO: 58; or (g) a heavy chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO: 47 and a light chain variable region comprising an amino acid sequence that is 90% or more identical to amino acid SEQ ID NO: 57; The antibody of any one of the preceding items.

[0027] 11.Antibody, A heavy chain variable region comprising 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; a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 57 and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27; The antibody of any one of the preceding items.

[0028] 12.Antibody, (a) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26, (b) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 26; (c) the VH sequence of SEQ ID NO: 16 and the 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) the VH sequence of SEQ ID NO: 46 and the 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 The antibody of any one of the preceding items.

[0029] 13.Antibodies (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, particularly less than 100 pM, preferably less than 50 pM, more preferably less than 10 pM, as measured by surface plasmon resonance (SPR), and in particular the antibody is an scFv (monovalent affinity); (ii) 10 -3 s -1 Below, 10 -4 s -1 Less than or equal to 10 -5 s -1 The following K off and in particular, said antibody is an scFv. (iii) 10 3 M -1 s -1 That's it, 10 4 M -1 s -1 That's it, 10 5 M -1 s -1 More than or equal to 10 6 M -1 s -1 More than K on and in particular, said antibody is an scFv. (iv) has cross-reactivity with Cynomolgus PDL1, in particular binds with a KD of less than 5 nM, in particular less than 1 nM, in particular less than 500 pM, in particular less than 100 pM, preferably less than 10 pM, as measured by SPR, in particular wherein the antibody is an scFv; (v) has no cross-reactivity with mouse PDL1, particularly as measured by SPR; and / or (vi) does not bind to human PDL2, particularly as measured by SPR; An antibody according to any of the preceding items.

[0030] 14. The antibody has the following characteristics: (i) has the ability to neutralize PDL1 / PD-1 interaction with a potency (relative potency) of greater than 1.5, e.g., greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, as compared to avelumab, as measured by ELISA; The relative potency is the IC in ng / mL of avelumab as measured by ELISA. 50 Value of IC in ng / mL of said antibody as measured by ELISA 50 is the ratio of In particular, the antibody is an scFv and / or (ii) optionally, having the ability to neutralize PDL1 / PD-1 interaction with a potency (relative potency) of greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, relative to avelumab, as measured by an NFAT reporter gene assay; The relative potency is the IC in ng / mL of avelumab as measured in an NFAT reporter gene assay. 50 Values ​​of IC in ng / mL of said antibodies as measured in an NFAT reporter gene assay 50 is the ratio of In particular, the antibody is an scFv and / or (iii) has the ability to neutralize the PDL1 / B7-1 interaction with a potency (relative potency) of greater than 1.5, e.g., greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, as measured by ELISA, compared to avelumab; The relative potency is the IC in ng / mL of avelumab as measured by ELISA. 50 Value of IC in ng / mL of said antibody as measured by ELISA 50 is the ratio of In particular, the antibody is an scFv. The antibody of any one of the preceding items.

[0031] 15. The antibody, (i) when in scFv format, has a melting temperature (Tm) of 60°C or more, preferably 65°C or more, more preferably 70°C or more, as measured by differential scanning fluorimetry; In particular, the antibody is formulated in 50 mM phosphate-citrate buffer, pH 6.4, 150 mM NaCl. (ii) when in scFv format, the antibody of the invention exhibits less than 5% loss in monomer content after 5 successive freeze-thaw cycles at a starting concentration of 10 mg / ml, preferably less than 3%, more preferably less than 1%; In particular, the antibody is formulated in 50 mM phosphate-citrate buffer, 150 mM NaCl, pH 6.4, and / or (iii) when in scFv format, the antibody of the invention exhibits a loss of less than 15%, such as less than 12%, such as less than 10%, such as less than 7%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, preferably less than 1%, of monomer content after storage at 4°C for 2 weeks or more, in particular 4 weeks or more, when at a starting concentration of 10 mg / ml; In particular, the antibodies of the invention are formulated in 50 mM phosphate citrate buffer, 150 mM NaCl, pH 6.4. An antibody according to any of the preceding items.

[0032] 16. The antibody of 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 ankyrin-based domains, fynomers, avimers, anticalins, binding domains based on scaffold alternatives limited to fibronectin, and binding sites engineered into the antibody constant region (e.g. F-star's Modular Antibody Technology™).

[0033] 17. The antibody of any one of the preceding items, wherein the antibody is a single chain variable fragment (scFv) or Fv.

[0034] 18. The antibody of 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.

[0035] 19. The antibody of item 16, wherein the antibody is an IgG selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, preferably, the antibody is IgG1.

[0036] 20. The antibody of any preceding item, wherein the antibody is chimeric or humanized.

[0037] 21. An antibody that binds to essentially the same epitope as any one of the antibodies of items 1 to 20.

[0038] 22. The antibody of any one of the preceding items, which is a multispecific molecule, particularly a multispecific molecule having one or more second functional molecules.

[0039] 23. The antibody is a single chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a cyclic dimeric scDb (CD-scDb), a bispecific T cell binding domain (BiTE; tandem-di-scFv), a tandem-tri-scFv, a tribody (Fab-(scFv)2) or a bibody (Fab-(scFv)1), a Fab, a Fab-Fv2, a Morrison (IgG CH3-scFv fusion (Morrison L) or an IgG CL-scFv fusion (Morrison H)), triabody, scDb-scFv, bispecific Fab2, di-mini antibody, tetrabody, scFv-Fc-scFv fusion, scFv-HSA-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, 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), 23. The antibody of item 22, which is in a format selected from the group consisting of: Fv, scFv, scDb, tandem-di-scFv, tandem-tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD, MATCH and DuoBodie fused to the N- and / or C-terminus of either chain of one heterodimeric Fc domain or the other heterodimeric domain.

[0040] 24. A pharmaceutical composition comprising the antibody of any one of items 1 to 23 and a pharma- ceutically acceptable carrier.

[0041] 25. The antibody of any one of items 1 to 23 or the composition of item 24 for use as a medicament.

[0042] 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 of said treatment.

[0043] 27. Use of the antibody according to any one of items 1 to 23 or the composition according to item 24 for treating cancer in a subject in need of such treatment.

[0044] 28. Use of the antibody according to any one of items 1 to 23 or the composition according to item 24 for the manufacture of a medicament for treating cancer in a subject in need of said treatment.

[0045] 29. A method for treating cancer in a subject in need of such treatment, comprising administering to said subject a therapeutically effective amount of the antibody of any one of items 1 to 23 or the composition of item 24.

[0046] 30. A nucleic acid encoding the antibody of any one of items 1 to 23.

[0047] 31. A vector comprising the nucleic acid of item 31.

[0048] 32. A host cell comprising the nucleic acid of item 31 or the vector of item 32.

[0049] 33. A method for producing the antibody according to any one of items 1 to 23, comprising the step of culturing a host cell containing the nucleic acid according to item 31 or the vector according to item 31.

[0050] 34. A kit comprising the antibody of any one of items 1 to 23 or the composition of item 24. [Brief description of the drawings]

[0051] [Figure 1] Neutralization of the PDL1 / PD-1 interaction by rabbit IgG clones with the best affinity for PDL1. The absorbance measured by ELISA is expressed in ng / ml as a function of the molecular concentration of 33-03-G02 (A) or 37-20-B03 (B). Avelumab was used as a control. [Diagram 2]Neutralization of the PDL1 / B7-1 interaction by rabbit IgG clones 33-03-G02 (A) or 37-20-B03 (B) with the best affinity for PDL1. The absorbance measured by ELISA is expressed as a function of the molecule concentration in ng / ml. Avelumab was used as a control. [Diagram 3] Figure 1 shows the 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 percentage inhibition ratio, corresponding 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 control. [Figure 4] Figure 4A shows the effect of CDR set and framework selection on the neutralization of PDL1 / PD-1 interaction in NFAT-luciferase reporter gene assay. The percentage of inhibition corresponding to the luminescence signal obtained in the assay is expressed in ng / ml as a function of scFv concentration. Avelumab was used as a control. Figure 4B shows the effect of domain optimization on the neutralization potency of PDL1 / PD-1 interaction in NFAT-luciferase reporter gene assay. The percentage of inhibition corresponding to the luminescence signal obtained in the assay is expressed in ng / ml as a function of scDbs concentration. Avelumab was used as a control. [Diagram 5] Figure 1 shows the neutralization potency of PDL1 / PD-1 interaction by scDb-scFvs PRO963 and PRO1057 (A), PRO1186 and PRO1430 (B), PRO1431 and PRO1432 (C), PRO1473 (D), PRO1476 (E), PRO1479 (F) and PRO1482 (G) in the presence of recombinant human serum albumin in a reporter gene assay. The percentage inhibition ratio corresponding 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 control. [Figure 6]Figure 1 shows the effect of bivalent molecule potency and of LC or HC scFv fusions in Morrison format on the neutralization potency of PDL1 / PD-1 interaction in an NFAT-luciferase reporter gene assay. The percentage of inhibition corresponding 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 control. [Figure 7] PD-1 / PDL1 competition ELISA. All molecules potently blocked the interaction between PD-1 and PDL1 with IC50 values ​​similar or less than the control IgG avelumab. [Figure 8] B7-1 / PDL1 competition ELISA. All molecules potently blocked the interaction between B7-1 and PDL1, similar to avelumab. [Figure 9] Ex vivo T cell activation assay. PBMCs were stimulated with 10 ng / ml SEA and treated with serial dilutions of scFv PRO997 or scDb PRO885 for 96 h. T cell activation was assessed by IL-2 quantification by ELISA in harvested supernatants. Treatment with PRO885 and PRO997 resulted in significant IL-2 secretion. PRO997 showed higher potency than avelumab. Compared to avelumab, PRO885 showed significantly higher efficacy. Data were fitted using a sigmoidal 4PL fit (GraphPad Prism). [Figure 10]Antitumor activity of anti-PDL1 IgG1 (PRO1137) treatment in human HCC827 NSCLC xenografts using immunodeficient NOG mouse strain and allogeneic human peripheral blood mononuclear cells (hPBMCs). Mice were administered anti-PDL1 IgG1 (PRO1137) or vehicle ip on days 0, 3, 7, and 10. Tumor volumes were measured twice weekly and mice were euthanized on days 17 and 18. Tumor volumes were normalized as relative tumor volume to tumor volume at the start of treatment. (A) Mean relative tumor volume in mice reconstituted with PBMCs from two donors (n=8 mice per group). Dotted lines indicate number of treatments. (B) Mean relative tumor volume in mice reconstituted with PBMCs from donor B (n=4 mice per group). (C) Individual relative tumor volumes in mice reconstituted with PBMCs from two donors. Each symbol represents an individual animal within the same treatment group. (D) Individual relative tumor volumes in mice reconstituted with PBMCs from donor B. Each symbol represents an individual animal within the same treatment group. [Figure 11] Figure 1 shows HCC827 xenografts in NOG mice replaced with hPBMC. Figure 2 shows the body weight of NOG mice loaded with HCC827 after treatment with anti-PDL1 IgG1 (PRO1137) therapy. Body weight was measured twice weekly and mice were euthanized on days 17 and 18. Body weight was normalized relative to the body weight at the start of treatment. [Figure 12] Figure 1 shows the evaluation of anti-PDL1 IgG1 (PRO1196) antitumor efficacy in human HCC827 NSCLC xenografts in NOG mice engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC). Antitumor activity of PRO1196 (0.1 mg) was compared to avelumab (0.1 mg) or vehicle treatment (palivizumab, 0.1 mg). Mice were treated at days 0, 5, 10, 15 and 20 (dotted lines). Tumor growth and body weight were recorded twice weekly. Tumor volumes were normalized as relative tumor volume to tumor volume at the start of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present invention provides antibodies that specifically bind to human PDL1 protein, as well as pharmaceutical compositions, methods of making, and methods of using such antibodies and compositions.

[0053] Unless otherwise defined, 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 pertains.

[0054] The terms "comprise" and "comprise" are used herein in their open-ended, non-limiting sense unless otherwise specified. Thus, with respect to such latter embodiments, the term "comprise" encompasses its narrower concept of "consisting of."

[0055] The terms "a" and "an" and "the" and similar terms in the context of describing the present invention are intended to encompass the singular and plural, unless otherwise stated herein or clearly contradicted by the context (particularly in the context of the claims which follow). For example, the term "cell" includes a plurality of cells and also mixtures thereof. When the plural is used for compounds, salts, etc., this may also refer to a single compound, salt, etc.

[0056] In a first aspect, the present invention relates to an antibody that specifically binds to human PDL1.

[0057] As used herein, the term "antibody" includes the following: Whole antibodies or single chains thereof, and any antigen-binding fragments (i.e., "antigen-binding portions") thereof or single chains thereof, as well as molecules comprising antibody CDRs, VH regions or VL regions, including but not limited to multispecific antibodies. A naturally occurring "whole antibody" is a glycoprotein comprising two or more heavy (H) chains and two or more light (L) chains linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) and interspersed more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody is believed to mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0058] 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 whole antibody that retain 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 Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region, Fd fragments consisting of the VH and CH1 domains, Fv fragments consisting of the VL and VH domains of a single arm of an antibody, as well as binding domains based on ankyrin-based domains, fynomers, avimers, anticalins, scaffolding alternatives limited to fibronectin, and binding sites integrated into the constant region of an antibody (e.g., F-star's Modular Antibody Technology™).

[0059] The term "complementarity determining region" ("CDR") is used in accordance with the Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering system), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering system), ImMunoGenTics (IMGT) numbering system (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999), Lefranc, M.-P. et al. The amino acid sequence of the constant region is determined using any of a number of well-known systems, such as those described in, for example, by Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering). For example, in the Kabat classical format, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered as 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered as 24-34 (LCDR1), 50-56 (LCDR2) and 95-102 (HCDR3). In Chothia, the CDR amino acid residues of the VH are numbered as 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3) and the CDR amino acid residues of the VL are numbered as 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) of the human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) of the human VL.In IMGT, the CDR amino acid residues of the VH are numbered approximately as 26-35 (HCDR1), 51-57 (HCDR2) and 93-102 (HCDR3), and the CDR amino acid residues of the VL are numbered approximately as 27-32 (LCDR1), 50-52 (LCDR2) and 89-97 (LCDR3) ("Kabat" numbering). In IMGT, the CDRs of an antibody can be determined using the IMGT / DomainGap Align program. In the context of the present invention, the numbering system proposed by Honegger & Pluckthun ("AHo") (Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670) is used, unless otherwise stated.

[0060] In addition, the following residues are defined as CDRs according to the AHo numbering system: LCDR1 (also called CDR-L1): L24-L42; LCDR2 (also called CDR-L2): L58-L72; LCDR3 (also called CDR-L3): L107-L138; HCDR1 (also referred to as CDR-H1): H27-H42; HCDR2 (also referred to as CDR-H2): H57-H76; HCDR3 (also called CDR-H3): H108-H138.

[0061] As a reminder, the Honegger & Pluckthun numbering system is convenient for accounting for slight variations in length, as well as gaps in sequence, that are found in naturally occurring antibodies in different VH and VL subfamilies, particularly in the CDRs. Thus, in a given antibody variable domain, not all positions 1 to 149 are typically occupied by an amino acid residue.

[0062] Antigen-binding moieties may be incorporated into maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, scDb-scFv, v-NAR and bis-scFv (see Holliger and Hudson, 2005, Nature Biotechnology, 23, 1126-36). Antigen-binding moieties of antibodies may be fused to a polypeptide-based scaffold, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes a monobody of a fibronectin polypeptide). Antigen-binding moieties may be incorporated into a single chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), together with a complementary light chain polypeptide, to 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).

[0063] The term "binding specificity" as used herein refers to the ability of an individual antibody to react with one antigenic determinant and not with a different antigenic determinant. As used herein, the term "specifically binds" or "specific" refers to a significant and reproducible interaction, e.g., binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to the target more readily and / or to a greater extent, with a stronger affinity, avidity, than it binds to other targets. "Specific binding" in its broadest sense (and unless otherwise defined) refers to the ability of an antibody to discriminate between a target of interest and unrelated molecules, for example, as determined according to a specificity assay method known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) test, and peptide scan. For example, a standard ELISA assay can be performed. Scoring can be performed by standard colorimetric reactions (e.g., reaction of the secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). The reaction in a particular well is recorded by optical density (e.g., at 450 nm). A typical background (=negative reaction) can be about OD of 0.1, and a typical positive reaction can be about OD of 1. This means that the ratio between positive and negative scores can be 10-fold or more. As a further example, an SPR assay can be performed, in which specific binding is indicated by a difference between background and signal of 10-fold or more, preferably 100-fold or more. Typically, the determination of binding specificity is performed not by the use of a single control molecule, but by using about 3-5 unrelated molecules (e.g., skim milk, transferrin, etc.). The antibody of the present invention has binding specificity for human PDL1. In a particular embodiment, the antibody of the present invention has binding specificity for human PDL1 but not for human PDL2, especially in the measurement by SPR.

[0064] 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 antigen specificity (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 (such PDL1 molecules from other species). Thus, in one embodiment, the antibody of the present invention has binding specificity with human PDL1 and PDL1 of cynomolgus monkey (also known as Cynomolgus monkey or "Cynomolgus"). Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0065] Suitably, the antibody of the present invention is a monoclonal antibody. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to antibodies that are substantially identical to or derived from the same amino acid sequence from the same genetic source. A monoclonal antibody composition exhibits binding specificity and affinity for a particular epitope or binding specificity and affinity for a particular set of epitopes.

[0066] Antibodies of the present invention include, but are not limited to, chimeric and humanized.

[0067] The term "chimeric antibody" refers to (a) Chimeric antibodies in which the constant regions or portions thereof have been modified, substituted or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or modified class, effector function and / or species, or to an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) to confer novel properties; or (b) the variable region or a portion thereof has been modified, substituted, or exchanged for a variable region having a different or altered antigen specificity; For example, a mouse antibody can be modified by replacing its constant region with the constant region of a human immunoglobulin. By replacing the constant region with a human constant region, the chimeric antibody can maintain its specificity in recognizing an antigen while reducing the antigenicity in humans compared to the original mouse antibody.

[0068] As used herein, a "humanized" antibody is an antibody that is less immunogenic in humans while retaining the reactivity of non-human antibodies. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining parts of the antibody (i.e., the constant and variable region framework parts) with their human counterparts. Further framework region modifications can be made within the human framework sequences or within CDR sequences derived from the germline of other mammalian species. The humanized antibodies of the invention can include amino acid residues not encoded by human sequences (e.g., by random or site-specific mutagenesis in vitro, by somatic mutation in vivo, or by conservative substitutions that promote stability or high production). See, e.g., 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. Examples of other humanization techniques include, but are not limited to, the Xoma technology disclosed in U.S. Patent No. 5,766,886.

[0069] The term "recombinant humanized antibody" as used herein includes all human antibodies that are prepared, expressed, constructed or isolated by recombinant means, including, for example, antibodies isolated from a host cell that has been transformed to express the humanized antibody, e.g., from a transfectome, as well as antibodies prepared, expressed, constructed or isolated by any other means including splicing of all or part of a human immunoglobulin gene (sequence relative to other DNA sequences).

[0070] The term "PDL1" refers in particular to human PDL1 having UniProt ID number Q9NZQ7, represented herein as SEQ ID NO: 63. Suitably, the antibodies of the invention target PDL1, in particular human PDL1 having UniProt ID number Q9NZQ7, represented herein as SEQ ID NO: 63. Suitably, the antibodies of the invention target human and cynomolgus monkey (Macaca fascicularis) PDL1, in particular as measured by surface plasmon resonance (SPR), and preferably do not cross-react with mouse PDL1. 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, as measured by SPR.

[0071] The antibodies of the present invention are PDL1 inhibitors. The term "blocker" or "blocking antibody" or "inhibitor" or "inhibitory 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, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The antibodies of the present invention target, reduce or inhibit the ability of PDL1 to bind to its binding partners, thereby preventing 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.

[0072] The antibodies of the invention described herein include, but are not limited to, isolated humanized monoclonal antibodies as described in the Examples. Examples of such anti-human PDL1 antibodies are those having the sequences listed in Table 1. Further details regarding the generation and characterization of the antibodies described herein are provided in the Examples.

[0073] The isolated antibody of the present invention having binding specificity for human PDL1 comprises the following heavy chain variable region (VH) and light chain variable region (VL): (a) said VH comprises three complementarity determining regions, designated HCDR1, HCDR2 and HCDR3, in order; (b) said VL comprises three complementarity determining regions, designated LCDR1, LCDR2 and LCDR3, in that order;

[0074] The present invention provides antibodies that specifically bind to PDL1 protein, the antibodies comprising a VH CDR having the amino acid sequence of any one of the VH CDRs listed in Table 1. In particular, the present invention provides antibodies that specifically bind to PDL1 protein, the antibodies having a VH CDR of any one, two, three or more amino acid sequences of the VH CDRs listed in Table 1.

[0075] The present invention also provides an antibody that specifically binds to a PDL1 protein, the antibody comprising a VL CDR having the 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, the antibody having a VL CDR having the amino acid sequence of any one, two, three or more of the VL CDRs listed in Table 1.

[0076] Other antibodies of the invention include those that contain mutated amino acids but specifically bind to PDL1 protein and have CDR regions that are 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the CDR regions represented by the sequences in Table 1. In one aspect, other antibodies of the invention include those that have mutant amino acid sequences that specifically bind to PDL1 protein and have CDR regions in which only 1, 2, 3, 4, or 5 amino acids are mutated compared to the CDR regions represented by the sequences in Table 1.

[0077] The term "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences refers to the fact that two or more sequences or subsequences are identical. "Percent (%) sequence identity" and "homology" with respect to a nucleic acid, peptide, polypeptide, or antibody sequence 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 of a particular nucleic acid, peptide, or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways that are within the skill of the art, such as using publicly available computer software (e.g., BLAST, BLAST-2, or ALIGN software). Those skilled in the art can determine appropriate parameters for calculating the alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences to be compared.

[0078] For sequence comparison, typically one sequence is used as a control sequence to which a test sequence is to be compared. When using a sequence comparison algorithm, test and control sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and sequence algorithm program parameters are assigned. Default program parameters can be used, or alternative parameters can be assigned. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the program parameters.

[0079] Two examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity include 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 analyses 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 uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and is incorporated into the ALIGN program (version 2.0). In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J. Mol. Biol. 48:444-453, 1970), which is incorporated into the GAP program of the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0080] The term "amino acid" refers to naturally occurring 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 those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The terms "polypeptide" and "protein" are used interchangeably herein in reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence implicitly encompasses conservatively modified variants thereof.

[0081] The present invention provides an isolated antibody having binding specificity for human PDL1, the antibody comprising: (a) a heavy chain variable region CDR1 (HCDR1) 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 NO: 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 selected from any of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2; and (c) a heavy chain variable region CDR3 (HCDR3) 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 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 selected from any 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 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 of SEQ ID NOs: 19, 22, 25, 50, 53 and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19; Includes.

[0082] Suitably, the isolated antibody of the invention having binding specificity for human PDL1 comprises (a) a heavy chain variable region CDR1 (HCDR1) comprising, or preferably consisting of, a sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of 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; (b) a heavy chain variable region CDR2 (HCDR2) comprising, or preferably consisting of, a sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 2, 6, 9, 12, 33, 37, 40, or 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2; (c) a heavy chain variable region CDR3 (HCDR3) comprising, or preferably consisting of, a sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence 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, or preferably consisting of, a sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 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, or preferably consisting of, a sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 18, 21, 24, 49, 52, or 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18; (f) a light chain variable region CDR3 (LCDR3) comprising, or preferably consisting of, a sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 19, 22, 25, 50, 53, or 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19; Includes.

[0083] In one embodiment, the antibody of the invention having binding specificity for human PDL1 comprises (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; (b) 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; (c) 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; (d) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 8, 9 and 10, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; (e) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 11, 12 and 13, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 23, 24 and 25, respectively; (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; (g) 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; (h) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, respectively, and the LLCDR1, LCDR2 and CDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively; (i) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 39, 40 and 41, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; (j) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 42, 43 and 44, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 54, 55 and 56, respectively; Includes.

[0084] In one embodiment, an antibody of the invention which has binding specificity for 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. In another embodiment, an antibody of the invention which has binding specificity for human PDL1 comprises 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.

[0085] Suitably, the antibody of the invention having binding specificity for human PDL1 comprises (a) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 17, 18 and 19, respectively; (b) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 4, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 20, 21 and 22, respectively; (c) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 5, 6 and 7, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 20, 21 and 22, respectively; (d) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 8, 9 and 10, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 17, 18 and 19, respectively; (e) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 11, 12 and 13, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 23, 24 and 25, respectively; (f) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 48, 49 and 50, respectively; (g) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 35, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 51, 52 and 53, respectively; (h) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 36, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 51, 52 and 53, respectively; (i) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 39, 40 and 41, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 48, 49 and 50, respectively; (j) HCDR1, HCDR2 and HCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 42, 43 and 44, respectively, and LCDR1, LCDR2 and LCDR3 sequences that have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 54, 55 and 56, respectively; Includes.

[0086] In one embodiment, the antibody of the invention having binding specificity for human PDL1 comprises HCDR1, HCDR2 and HCDR3 sequences having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 1, 2 and 3, respectively; and LCDR1, LCDR2 and LCDR3 sequences having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 17, 18 and 19, respectively. In another embodiment, the antibody of the invention having binding specificity for human PDL1 comprises HCDR1, HCDR2 and HCDR3 sequences having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 32, 33 and 34, respectively; and LCDR1, LCDR2 and LCDR3 sequences having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identity to SEQ ID NOs: 48, 49 and 50, respectively.

[0087] Suitably, the antibody of the invention having binding specificity for human PDL1 comprises (a) an HCDR1 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:1; (b) an HCDR2 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:2; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:3; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 17; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 18; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 19; Suitably, the antibody of the invention having binding specificity for human PDL1 comprises (a) HCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO:1; (b) an HCDR2 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO:2; (c) an HCDR3 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO:3; (d) LCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 17; (e) an LCDR2 comprising, and preferably consisting of, an amino acid sequence having at least 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 18; (f) an LCDR3 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 19; Includes.

[0088] In a further embodiment, the antibody of the invention that has binding specificity for human PDL1 has (a) an HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) an HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:6; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 7; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 21; (f) an 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, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 4 or SEQ ID NO: 5; (b) an HCDR2 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO:6; (c) an HCDR3 comprising, or preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 7; (d) LCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 20; (e) an LCDR2 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 21; (f) an LCDR3 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 22; Includes.

[0089] Suitably, the antibody of the invention having binding specificity for human PDL1 comprises (a) an HCDR1 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO: 32; (b) an HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 33; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 34; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 48; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 49; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 50; Suitably, the antibody of the invention having binding specificity for human PDL1 comprises (a) HCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 32; (b) an HCDR2 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 33; (c) an HCDR3 comprising, or preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 34; (d) LCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 48; (e) an LCDR2 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 49; (f) an LCDR3 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO: 50; Includes.

[0090] In a further embodiment, the antibody of the invention that has binding specificity for human PDL1 has (a) an HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36; (b) an HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 37; (c) an HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 38; (d) an LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO:51; (e) an LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 52; (f) an LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 53; Suitably, the antibody of the invention having binding specificity for human PDL1 comprises (a) HCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 35 or SEQ ID NO: 36; (b) an HCDR2 comprising, or preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 37; (c) an HCDR3 comprising, or preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 38; (d) LCDR1 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 51; (e) an LCDR2 comprising, and preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 52; (f) an LCDR3 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO: 53; Includes.

[0091] In a further embodiment, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), the antibody comprising 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, classified according to sequence identity and homology. Methods for determining sequence homology, for example using homology search matrices such as BLOSUM (Henikoff, S. & Henikoff, JG, Proc. Natl. Acad. Sci. USA 89 (1992) 10915-10919), and for grouping sequences by homology, are well known to those skilled in the art. The various subfamilies of VH, Vκ and Vλ can be identified, for example, as shown in Knappik et al., J. Mol. Biol. 296 (2000) 57-86, where VH is classified as VH1A, VH1B and VH2 to VH6, Vκ is classified as Vκ1 to Vκ4, and Vλ is classified as Vλ1 to Vλ3. In vivo, the Vκ, Vλ and VH chains of an antibody are the result of random rearrangement of the V and J sites of germline κ chains, the V and J sites of germline λ chains, and the V, D and J sites of heavy chains, respectively. Which subfamily a given antibody variable chain belongs to is determined by the corresponding V sites, in particular by the framework regions FR1 to FR3. Thus, in the present invention, any VH sequence characterized only by a particular set of framework regions HFR1 to HFR3 may be combined with any HFR4 sequence, e.g., an HFR4 sequence derived from one of the heavy chain germline J sites, or an HFR4 sequence derived from a rearranged VH sequence.

[0092] Suitably, the invention provides an isolated antibody that specifically binds to PDL1 (eg, human PDL1 protein), said antibody comprising VH1A, VH1B, VH3 or VH4.

[0093] 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 derived from SEQ ID NO: 15 belong to the VH1 family (regions marked in non-bold in Table 1). Suitably, a VH belonging to the VH1 family as used herein is a VH comprising FR1 to FR4 that have 85% or more, preferably 90% or more, more preferably 95% or more sequence identity with FR1 to FR4 of SEQ ID NO: 15.

[0094] 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 derived from SEQ ID NO: 16 belong to the VH3 family (regions marked in non-bold in Table 1). Optimally, a VH belonging to the VH3 family as used herein is a VH comprising FR1 to FR4 that have 85% or more, preferably 90% or more, more preferably 95% or more sequence identity with FR1 to FR4 of SEQ ID NO: 16.

[0095] 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 derived from SEQ ID NO: 14 belong to the VH4 family (regions marked in non-bold in Table 1). Optimally, a VH belonging to the VH4 family as used herein is a VH comprising FR1 to FR4 that have 85% or more, preferably 90% or more, more preferably 95% or more sequence identity with FR1 to FR4 of SEQ ID NO: 14.

[0096] Examples of VH sequence alternatives are described in Knappik et al., J. Mol. Biol. 296 (2000) 57-86.

[0097] In one embodiment, the isolated antibody of the invention comprises a VH4 or VH3 domain.

[0098] Suitably, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), said antibody comprising Vκ framework FR1, FR2 and FR3, in particular Vκ1 or Vκ3 framework, preferably Vκ1 framework FR1 to 3, and VκFR4, in particular framework FR4 selected from Vκ1 FR4, Vκ3 FR4 and Vλ FR4. A suitable Vκ1 framework FR1 to 3 is set out in SEQ ID NO: 26 (FR regions are marked in Table 1 in non-bold). A suitable Vκ1 framework FR1 to 3 comprises an amino acid sequence corresponding to FR1 to 3 and having 60% or more, 70% or more, 80% or more, 90% or more identity with the amino acid sequence derived from SEQ ID NO: 26 (FR regions are marked in Table 1 in non-bold).

[0099] Examples of alternative Vκ1 sequences, as well as examples of Vκ2, Vκ3 and Vκ4 sequences, are described in Knappik et al., J. Mol. Biol. 296 (2000) 57-86.

[0100] Suitable Vλ FR4s are represented by SEQ ID NO: 64 to SEQ ID NO: 70. In a preferred embodiment, the Vλ FR4 is represented by SEQ ID NO: 64 or 65, more preferably, the Vλ FR4 is represented by SEQ ID NO: 64. In one embodiment, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), the antibody comprising a Vλ FR4 comprising an amino acid sequence selected from any of SEQ ID NO: 64 to SEQ ID NO: 70, preferably having 60% or more, 70% or more, 80% or more, 90% or more identity to the amino acid sequence of SEQ ID NO: 64 or 65, more preferably SEQ ID NO: 64.

[0101] Thus, in one embodiment, the invention provides an antibody comprising: (i) the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences: 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; b. 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; or 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; (ii) a VH3 or VH4 domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1; A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly a Vλ FR4 comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably a Vλ FR4 represented by any one of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64; A VL domain comprising a VL framework comprising:

[0102] In another embodiment, the invention provides an antibody having binding specificity to human PDL1, comprising: (i) 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; (ii) a VH1A, VH1B, VH3 or VH4 domain framework sequence, preferably a VH1A or VH1B domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1; A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly a Vλ FR4 comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably a Vλ FR4 comprising an amino acid sequence of any one of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64; A VL domain comprising a VL framework comprising:

[0103] In a specific embodiment, the invention provides an antibody comprising: (i) 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; (ii) a VH3 or VH4 domain framework sequence, preferably a VH4 domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1; A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly a Vλ FR4 comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably a Vλ FR4 represented by any one of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64; A VL domain comprising a VL framework comprising:

[0104] In a preferred specific embodiment, the present invention provides an antibody comprising: (i) 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; (ii) a VH3 or VH4 domain framework sequence, preferably a VH3 domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1; A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly a Vλ FR4 comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any one of SEQ ID NO: 64 to SEQ ID NO: 70, preferably a Vλ FR4 represented by any one of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64; A VL domain comprising a VL framework comprising:

[0105] In one embodiment, the present invention provides an antibody having binding specificity for human PDL1, (i) the CDR domains CDR1, CDR2 and CDR3, (ii) human Vκ framework regions FR1 to FR3, in particular human Vκ1 framework regions FR1 to FR3; (iii) (a) a human Vλ germline sequence for FR4, in particular a Vλ germline sequence selected from SEQ ID NOs: 64 to 70, preferably SEQ ID NO: 64; and (b) a Vλ-based sequence having one or two mutations, in particular one mutation, compared to the human Vλ germline sequence closest to FR4, comprising an amino acid sequence selected from any of SEQ ID NO: 64 to SEQ ID NO: 70, preferably SEQ ID NO: 64; FR4, selected from The present invention provides an antibody comprising a VL comprising:

[0106] The present invention provides isolated antibodies that specifically bind to PDL1 (eg, human PDL1 protein), the antibodies comprising a VH domain listed in Table 1.

[0107] The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising a VH amino acid sequence listed in Table 1, and wherein the mutations in the framework sequence (e.g., a non-CDR sequence) (various non-limiting examples of mutations include additions, substitutions, or deletions) are about 10 amino acids or less.

[0108] The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising a VH amino acid sequence listed in Table 1, and wherein the mutations in the framework sequence (e.g., a non-CDR sequence) (various non-limiting examples of mutations include additions, substitutions, or deletions) are about 20 amino acids or less.

[0109] Other antibodies of the present invention include those that contain mutated amino acids but specifically bind to PDL1 and have a VH region that has 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to a VH region represented by a sequence listed in Table 1.

[0110] The present invention provides isolated antibodies that specifically bind to a PDL1 protein, said antibodies comprising a VL domain listed in Table 1.

[0111] The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising a VL amino acid sequence listed in Table 1, and wherein the mutations in the framework sequence (e.g., a non-CDR sequence) (various non-limiting examples of mutations include additions, substitutions, or deletions) are about 10 amino acids or less.

[0112] The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising a VL amino acid sequence listed in Table 1, and wherein the mutations in the framework sequence (e.g., a non-CDR sequence) (various non-limiting examples of mutations include additions, substitutions, or deletions) are about 20 amino acids or less.

[0113] Other antibodies of the present invention include those that contain mutated amino acids but specifically bind to PDL1 and have a VL region that has 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to a VL region represented by a sequence listed in Table 1.

[0114] The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising: a heavy chain variable region comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more, 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; a light chain variable region comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more, identical to the amino acid sequence of SEQ ID NO: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27; Includes.

[0115] In one embodiment, the antibody of the invention has binding specificity for human PDL1, A heavy chain variable region comprising 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; a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 57 and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27; Includes.

[0116] In one embodiment, the antibody of the invention that has binding specificity for human PDL1 comprises: (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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 14; and A VL sequence comprising an amino acid sequence identical to SEQ ID NO: 26 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more; (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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 15; and A VL sequence comprising an amino acid sequence identical to SEQ ID NO: 26 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more; (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 which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 16, preferably comprising the G56A and Y105F mutations (AHo numbering), and a VL sequence comprising an amino acid sequence which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 27, and preferably comprising the S9A and A51P mutations (AHo numbering); (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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 45; and A VL sequence comprising an amino acid sequence identical to SEQ ID NO: 57 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more; (e) the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 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 identical to SEQ ID NO: 46 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more, and preferably comprising the V2S, V25A, I44V, G56A, V82K, F89V and Y105F mutations (AHo numbering); and a VL sequence comprising an amino acid sequence which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 58, and preferably comprising I2F, M4L and A51P mutations (AHo numbering); or (f) 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 that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to SEQ ID NO: 47, preferably comprising the V25A and I44V mutations (AHo numbering), and A VL sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to SEQ ID NO:57.

[0117] In one embodiment, the antibody of the invention that has binding specificity for human PDL1 comprises: (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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 14; and A VL sequence comprising an amino acid sequence identical to SEQ ID NO: 26 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more; (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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 15; and A VL sequence comprising an amino acid sequence identical to SEQ ID NO: 26 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more; (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 which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 16, preferably comprising the G56A and Y105F mutations (AHo numbering), and a VL sequence comprising an amino acid sequence which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 27, and preferably comprising the S9A and A51P mutations (AHo numbering); (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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 45; and A VL sequence comprising an amino acid sequence identical to SEQ ID NO: 57 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more; (e) 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 identical to SEQ ID NO: 46 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more, and preferably comprising the V2S, V25A, I44V, G56A, V82K, F89V and Y105F mutations (AHo numbering); and a VL sequence comprising an amino acid sequence which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 58, and preferably comprising I2F, M4L and A51P mutations (AHo numbering); or (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 that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to SEQ ID NO: 47, preferably comprising the V25A and I44V, G56A, V82K and F89V mutations (AHo numbering), and A VL sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to SEQ ID NO:57.

[0118] In a preferred embodiment, the antibody of the invention that has binding specificity for 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; A VH sequence comprising an amino acid sequence which is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more identical to SEQ ID NO: 16, preferably comprising the G56A and Y105F mutations (AHo numbering), and A VL sequence comprising an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, identical to SEQ ID NO: 27, and preferably the VL comprises S9A and A51P mutations (AHo numbering).

[0119] In a further embodiment, the isolated antibody of the invention that has 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) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 26; (c) the VH sequence of SEQ ID NO: 16 and the 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) the VH sequence of SEQ ID NO: 46 and the VL sequence of SEQ ID NO: 58, or (f) the VH sequence of SEQ ID NO: 47 and the VL sequence of SEQ ID NO: 57. In a preferred embodiment, an isolated antibody of the invention with binding specificity for human PDL1 comprises the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 26. In a more preferred embodiment, an isolated antibody of the invention with binding specificity for human PDL1 comprises the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 27.

[0120] 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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, preferably 90% or more 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 represented by 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 represented by 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.

[0121] Other antibodies of the invention with binding specificity for human PDL1 include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but have 60% or more, 70% or more, 80% or more, 90% or more or 95% or more identity with the sequences set forth in Table 1. In one embodiment, it comprises a mutated amino acid sequence in which only 1, 2, 3, 4 or 5 amino acids have been mutated in the variable region when compared to the sequence of the variable region set forth in Table 1, but substantially the same activity is maintained. As used herein, the term "substantially the same activity" refers to an activity that is substantially equal to or greater than 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%, or 110%, or 120%, or 130%, or 140%, or 150%, or 160%, or 170%, or 180%, or 190%, for example, at most 200%, as compared to the activity measured in a parent antibody, e.g., an antibody of the invention, particularly an antibody of the invention listed in Table 1.

[0122] As long as each of these antibodies can bind PDL1 and the antigen-binding specificity is provided primarily 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), provided that each antibody contains VH CDR1, 2 and 3 and VL CDR1, 2 and 3 and allows the formation of other PDL1-binding molecules of the invention. Such "mixed and matched" PDL1-binding antibodies can be tested using well-known binding assays, such as those described in the Examples (e.g., ELISA). When VH CDR sequences are mixed and matched, the CDR1, CDR2 and / or CDR3 sequences from a particular VH sequence should be replaced with a structurally similar CDR sequence(s). Likewise, when VL CDR sequences are mixed and matched, the CDR1, CDR2 and / or CDR3 sequence from a particular VL sequence should be replaced with a structurally similar CDR sequence(s). As will be apparent to one of ordinary skill in the art, novel VH and VL sequences can be generated from the CDR sequences presented herein for the monoclonal antibodies of the invention by mutating one or more VH and / or VL CDR region sequences to have a structurally similar sequence.

[0123] In another embodiment, the present invention provides antibodies comprising an amino acid sequence having homology to a sequence set forth in Table 1, which bind to PDL1 and maintain the desired functional properties of those antibodies set forth in Table 1.

[0124] For example, the invention provides an isolated monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16, selected from the group consisting of SEQ ID NO: 14, 15, 16, 45, 46, and 47; the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27, which is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: The antibody specifically binds to human PDL1 protein.

[0125] In one embodiment, the VH and / or VL amino acid sequence may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a sequence set forth in Table 1. In one embodiment, the VH and / or VL amino acid sequence may be identical except for amino acid substitutions of only 1, 2, 3, 4 or 5 amino acids.

[0126] In one embodiment, an antibody of the invention has a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, One or more of these CDR sequences have a particular amino acid sequence based on the antibodies described herein or conservative modifications thereof, which maintain the desired functional properties of the PDL1-binding antibodies of the invention.

[0127] The term "conservatively modified variants" or "conservative variants" applies to amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to encoding identical or essentially identical amino acid sequences, or, in the case of a nucleic acid that does not code for an amino acid sequence, essentially identical sequences. 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. Thus, at any position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide describes every possible silent variation of the nucleic acid. One of skill in the art will recognize that each codon in a nucleic acid, except AUG (usually the only codon for methionine) and TGG (usually the only codon for tryptophan), can be modified to produce a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0128] "Conservatively modified variants" or "conservative variants" with respect to a polypeptide sequence include individual 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 that result in functionally equivalent amino acids are known. Such conservatively modified variants may in addition be, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (see 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 change the binding characteristics of the antibody containing the amino acid sequence.

[0129] Accordingly, the present invention provides an isolated monoclonal antibody comprising or consisting of a heavy chain variable region comprising a CDR1, CDR2 and CDR3 sequence, and a light chain variable region comprising a CDR1, CDR2 and CDR3 sequence, the heavy chain variable region CDR1 comprises, preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 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; the heavy chain variable region CDR2 comprises, preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 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; the heavy chain variable region CDR3 comprises, preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 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; the light chain variable region CDR1 comprises, preferably consists of, an amino acid sequence selected from any 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; the light chain variable region CDR2 comprises, preferably consists of, an amino acid sequence selected from any 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; the light chain variable region CDR3 comprises, preferably consists of, an amino acid sequence selected from any 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; The antibody can specifically bind to PDL1 and block PD-1 / PDL1 interaction.

[0130] In one embodiment, the antibody of the invention has heavy and light chain variable regions optimized for expression in mammalian cells, one or more of which have specific amino acid sequences 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 invention. Thus, the invention provides an isolated monoclonal antibody optimized for expression in mammalian cells, comprising a heavy and light chain variable region, the heavy chain variable region comprises an amino acid sequence selected from any of SEQ ID NOs: 14, 15, 16, 45, 46, and 47, preferably SEQ ID NO: 14 or 16, preferably SEQ ID NO: 16, and conservatively modified sequences thereof; the light chain variable region comprises an amino acid sequence selected from any of SEQ ID NOs: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, preferably SEQ ID NO: 27, and conservatively modified sequences thereof; The antibody can specifically bind to PDL1 and block PD-1 / PDL1 interaction.

[0131] In one embodiment, an antibody of the invention has full-length heavy chain sequences and full-length light chain sequences optimized for expression in mammalian cells, one or more of which have specific amino acid sequences based on the antibodies described herein or conservative modifications thereof, wherein the antibody maintains the desired functional properties of the PDL1-binding antibodies of the invention.

[0132] As used herein, the term "optimized" means that the nucleotide sequence has been modified to encode an amino acid sequence using codons that are preferred in the producing cell or organism (typically, for example, a eukaryotic cell of the genus Pichia, a Chinese Hamster Ovary cell (CHO) or a human cell). An optimized nucleotide sequence is engineered to retain, completely or as far as possible, the amino acid sequence encoded by the original starting nucleotide sequence, known as the "parental" sequence. As used herein, optimized sequences are engineered to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also envisioned in the present invention. An amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0133] Another type of variable region modification is to improve one or more binding properties (e.g. affinity) of the antibody of interest by mutating amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL, known as "affinity maturation". The introduction of the mutation(s) can be performed by site-directed mutagenesis or PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties of interest can be assessed in in vitro or in vivo assays described herein and illustrated in the Examples. Conservative modifications (as described above) can also be introduced. Mutations may be amino acid substitutions, additions or deletions. Moreover, typically only one, two, three, four or five residues in the CDR regions are altered.

[0134] An "affinity matured" antibody is one that has one or more modifications in one or more variable domains that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not possess such modifications. In one embodiment, an affinity matured antibody may have nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies are produced by well-known procedures. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by shuffling VH- and VL-domains. Random mutagenesis of hypervariable region ("HVR") and / or framework residues is described, for example, 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).

[0135] In one embodiment, the present invention provides an isolated monoclonal antibody comprising a VH3 comprising G56A and Y105F mutations, and in particular an amino acid sequence represented by SEQ ID NO: 16, and a VL, preferably comprising S9A, A51P mutation, and in particular an amino acid sequence represented by SEQ ID NO: 27.

[0136] In one embodiment, an "affinity matured" antibody of the invention comprises a VH4 comprising V25A, I44V, G56A, V82K, F89V mutations, in particular the amino acid sequence set forth in SEQ ID NO: 47, and a VL, preferably comprising the amino acid sequence set forth in SEQ ID NO: 57. In yet another embodiment, an "affinity matured" antibody of the invention comprises a VH4 comprising V2S, V25A, I44V, G56A, V82K, F89V, Y105F mutations, in particular the amino acid sequence set forth in SEQ ID NO: 46, and a VL comprising I2F, M4L, A51P mutations, in particular the amino acid sequence set forth in SEQ ID NO: 58.

[0137] Antibodies of the invention can also be prepared by using as starting material an antibody having one or more of the VH and / or VL sequences set forth herein to engineer a modified antibody, which may have altered properties over the starting antibody. The antibody can be engineered by modifying one or more residues in one or both of the variable regions (i.e., VH and / or VL), for example, one or more CDR regions and / or one or more framework regions. Additionally, or alternatively, the antibody can be engineered by modifying residues in the constant region, for example to alter the effector function of the antibody.

[0138] One of the possible engineering of variable regions is CDR grafting. Antibodies interact with target antigens mainly through amino acid residues located in the six complementarity determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector in which the CDR sequences from a particular naturally occurring antibody are fused to framework sequences from a different antibody with different properties (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. Patent No. 5,225,539 (Winter); and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 (Queen et al.)).

[0139] Such framework sequences can be obtained from public DNA databases or from published publications describing germline antibody gene sequences and rearranged antibody sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Tomlinson, IM, et al., 1992 J.fol.Biol.227:776-798, and Cox, JP Let al., 1994 Eur.J Immunol.24:827-836, the disclosures of which are incorporated herein by reference. For example, germline DNA sequences for human heavy and light chain variable region genes and rearranged antibody sequences are present in the "IMGT" database (available on the Internet at www.imgt.org; Lefranc, MP et al., 1999 Nucleic Acids Res. 27:209-212, the disclosures of which are incorporated herein by reference).

[0140] Examples of framework sequences used in the antibodies of the present invention include those structurally similar to the framework sequences used by the selected antibodies of the present invention, such as the consensus sequences and / or framework sequences used by the monoclonal antibodies of the present invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be grafted onto framework regions having identical sequences present in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations compared to the germline sequences. For example, as a specific example, it is known to be useful to mutate residues in framework regions to maintain or enhance the antigen-binding ability of an antibody (see, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 (Queen et al.)).

[0141] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be used, including those that include the five major idiotypes of human immunoglobulins and their antigen-binding fragments, as well as those that include immunoglobulins of other animal species, preferably in their humanized form, so long as the resulting polypeptide contains one or more binding regions that specifically bind to PDL1.

[0142] In one aspect, the present invention relates to a method for producing non-immunoglobulin-based antibodies using a non-immunoglobulin scaffold onto which the CDRs of the present invention can be grafted. Known or future developed non-immunoglobulin frameworks and scaffolds can be used as long as they contain specific binding regions for the target PDL1 protein. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Waltham, Massachusetts), ankyrin (Molecular Partners AG, Zurich, Switzerland), lipocalin (Pieris Proteolab AG, Freising, Germany), small molecule immunopharmaceuticals (Trubion Pharmaceuticals, Seattle, Washington), maxybody (Avidia, Mountain View, California), protein A (Affibody AG, Sweden) and affilin (γ-crystallin or ubiquitin) (Scil Proteins, Halle, Germany).

[0143] Suitably, the antibodies of the invention specifically bind to PDL1 and are characterised by one or more of the following parameters: (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, particularly less than 100 pM, preferably less than 50 pM, more preferably less than 10 pM, more preferably less than 5 pM, particularly when measured by surface plasmon resonance (SPR), and particularly wherein the antibody is an scFv; (ii) 10 -3 s -1 Below, 10 -4 s -1 Less than or equal to 10 -5 s -1 The following K off and in particular, said antibody is an scFv. (iii) 10 3 M -1 s -1 That's it, 10 4 M-1 s -1 That's it, 10 5 M -1 s -1 More than or equal to 10 6 M -1 s -1 More than K on and in particular, said antibody is an scFv. (iv) has cross-reactivity with Cynomolgus PDL1, in particular binds with a KD of less than 5 nM, in particular less than 1 nM, in particular less than 500 pM, in particular less than 100 pM, preferably less than 10 pM, for Cynomolgus PDL1, as measured by surface plasmon resonance, in particular where the antibody is an scFv, has no cross-reactivity with mouse PDL1, in particular measured by surface plasmon resonance; and / or (v) In particular, it does not bind to human PDL2 as measured by SPR.

[0144] 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 regions of an antibody "arm" interact with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0145] "Binding affinity" broadly 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, "binding affinity," "binds to," "binds with," or "binding to" as used herein refer to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody fragment and an antigen). The affinity of a molecule X with its partner Y is usually measured by the dissociation constant (K D) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies usually bind to antigens slowly and tend to dissociate quickly, whereas high affinity antibodies usually bind to antigens quickly and tend to remain bound for a long time. Various methods for measuring binding affinity are known, any of which can be used to solve the problems of the present invention. Specific exemplary embodiments for measuring binding affinity (i.e., binding strength) are described below.

[0146] As used herein, the term "K assoc ", "Ka" or "K on " refers to the association rate of a particular antibody-antigen interaction, while the term "K dis ", "Kd" or "K off " refers to the dissociation rate of a particular antibody-antigen interaction. In one embodiment, the term "KD" as used herein refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The "KD" or "KD value" or "KD value" of the present invention refers to the dissociation constant of a particular antibody-antigen interaction. D " or "K DIn one embodiment, the "Affinity Value" is measured using a surface-plasmon resonance assay using a MASS-1 SPR instrument (Sierra Sensors). To measure affinity, an antibody specific for the Fc region is immobilized on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using a standard amine-coupling procedure with rabbit IgG (Bethyl Laboratories, Cat. No. A120-111A). Rabbit monoclonal antibodies in the B cell supernatant are captured by immobilized anti-rabbit IgG antibodies. A minimum IgG concentration in the B cell supernatant is required for adequate capture. After monoclonal antibody capture, human PDL1 (Peprotech) is injected at a concentration of 90 nM over the flow cell for 3 minutes, and protein dissociation from the IgG captured on the sensor chip is allowed for 5 minutes. After each injection cycle, the surface is regenerated with two injections of 10 mM glycine-HCl. The apparent dissociation (kd) and association (ka) constants, as well as the apparent dissociation equilibrium constants (KD) were calculated using the one-to-one Langmuir binding model with the MASS-1 analysis software (Analyzer, Sierra Sensors). 2 Relative values ​​of Chi normalized to the extrapolated maximum binding level of the analyte 2 ) to monitor the quality of the fit and use it as an indicator for maintaining the quality of the curve fitting. 2 A small value of r indicates high accuracy of fitting to the one-to-one Langmuir binding model. The results are considered valid if the response unit (RU) of ligand binding is 2% or more of the RU of antibody capture. Samples showing RU of ligand binding less than 2% of the RU of antibody capture are considered to indicate the absence of specific binding of PDL1 to the captured antibody. The equilibrium dissociation constant (K D ) is the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0147] Suitably, the affinity of the antibody of the present invention to PDL1 may be higher than the affinity of PDL1 to PD-1. It goes without saying that a higher affinity of the PDL1 antibody compared to the affinity of PDL1 to PD-1 may be particularly useful for dissociating or neutralizing a 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 to PDL1 may be equal to or higher than the affinity of avelumab to PD-1. In one embodiment, the PDL1 antibody of the present invention neutralizes the PD-1 / PDL1 interaction with equal or higher potency than avelumab. In yet another embodiment, the PDL1 antibody of the present invention neutralizes the B7-1 / PDL1 interaction with equal or higher potency than avelumab. The binding affinity of the antibody may be determined, for example, by the dissociation constant (KD). A strong affinity is represented by a low KD, whereas a weak affinity is represented by a high KD.

[0148] Thus, in a suitable embodiment, the antibody of the present invention may have a KD of 1-50,000 pM, 1-40,000 pM, 1-30,000 pM, 1-20,000 pM, 1-10,000 pM, 1-5,000 pM, 1-2,500 pM, 1-1,000 pM, 1-750 pM, 1-500 pM, 1-250 pM, 1-100 pM, 1-50 pM, or 1-10 pM. In suitable embodiments, the antibodies of the invention may have a KD of less than about 50nM, less than about 45nM, less than about 40nM, less than about 35nM, less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, less than about 10nM, less than about 9nM, less than about 8nM, less than about 7nM, less than about 6nM, less than about 5nM, less than about 4nM, less than about 3nM, less than 2nM, less than 1nM, less than 0.5nM, less than 0.25nM, less than 100pM, less than 10pM or less than 5pM, particularly when measured by SPR, and particularly when said antibodies are scFv. Suitably, the antibodies of the invention have a KD of less than 5nM, particularly when measured by SPR. Suitably, the antibodies of the invention have a KD of less than 1nM, particularly when measured by SPR. Suitably, the antibodies of the invention have a KD of less than 100pM, particularly when measured by SPR. Suitably, the antibody of the present invention has a KD of less than 50 pM, particularly when measured by SPR.Preferably, the PDL1-BD of the present invention binds to human PDL1 with a KD of less than 10 pM, particularly when measured by SPR.Preferably, the PDL1-BD of the present invention binds to human PDL1 with a KD of less than 5 pM, particularly when measured by SPR.

[0149] Suitably, the antibody of the invention has an affinity for human PDL1 of 10 3 M -1 s -1 That's it, 10 4 M -1 s -1 That's it, 5 x 10 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 5 x 10 5 M -1 s -1 That's it, 10 6 M-1 s -1 That's it, 5 x 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 That's it, 5 x 10 7 M -1 s -1 More than K on Preferably, the antibodies of the invention bind at a rate of 10 5 M -1 s -1 Above, especially 10 6 M -1 s -1 More than K on Has speed.

[0150] Suitably, the antibody of the invention has an affinity for human PDL1 of 10 -3 s -1 Below, 3 x 10 -3 s -1 Below, 5 x 10 -3 s -1 Below, 10 -4 s -1 Below, 5 x 10 -4 s -1 Below, 10 -5 s -1 Below, 5 x 10 -5 s -1 Below, 10 -6 s -1 Less than or equal to 10 -7 s -1 The following K off Preferably, the antibodies of the invention bind at a rate of 10 -3 s -1 Below, 10 -4 s -1 The following, especially 10 -5 s -1 The following K off Has speed.

[0151] Suitably, the antibodies of the invention specifically bind to PDL1 and are characterised by one or more of the following parameters: (i) has the ability to neutralize PDL1 / PD-1 interaction with a potency (relative potency) of greater than 1.5, e.g., greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, as compared to avelumab, as measured by ELISA; The relative potency is the IC in ng / mL of avelumab as measured by ELISA. 50 Value of IC in ng / mL of said antibody as measured by ELISA 50 is the ratio of In particular, the antibody is an scFv, and (ii) optionally, having the ability to neutralize PDL1 / PD-1 interaction with a potency (relative potency) of greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, relative to avelumab, as measured by an NFAT reporter gene assay; The relative potency is the IC in ng / mL of avelumab as measured in an NFAT reporter gene assay. 50 Values ​​of IC in ng / mL of said antibodies as measured in an NFAT reporter gene assay 50 is the ratio of In particular, the antibody is an scFv, and (iii) has the ability to neutralize PDL1 / B7.1 interaction with a potency (relative potency) of greater than 1.5, e.g., greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, as measured by ELISA, compared to avelumab; The relative potency is the IC in ng / mL of avelumab as measured by ELISA. 50 Value of IC in ng / mL of said antibody as measured by ELISA 50 is the ratio of In particular, said antibody is an scFv.

[0152] Suitably, the antibodies of the present invention have useful biophysical properties.

[0153] Suitably, the antibodies of the invention, when in scFv format, have a melting temperature (Tm) of 55°C or higher, such as 60°C or higher, preferably 65°C or higher, more preferably 70°C or higher, as measured by differential scanning fluorimetry, particularly when the antibodies are formulated in 50 mM phosphate-citrate buffer at pH 6.4, 150 mM NaCl. DSF has been previously reported (Egan, et al., MAbs, 9(1)(2017), 68-84; Niesen, et al., Nature Protocols, 2(9)(2007) 2212-2221). The transition midpoint of thermal denaturation of scFv constructs is measured by differential scanning fluorimetry using the fluorescent dye SYPRO® Orange (see Wong & Raleigh, Protein Science 25(2016) 1834-1840). Samples in pH 6.4 phosphate-citrate buffer are prepared in a total volume of 100 μl with a final protein concentration of 50 μg / mL and a final concentration of 5×SYPRO® orange. 25 μl of the prepared samples are added in triplicate to a white-walled AB gene PCR plate. The assay is performed in a qPCR machine used as a thermal cycler, and the fluorescence emission is detected using a custom dye calibration routine in the software. The PCR plate containing the test samples is heated in 1°C increments from 25°C to 96°C, with 30 seconds pause after each temperature increase. The total assay time is approximately 2 hours. The Tm is calculated by the software GraphPad Prism using the mathematical second derivative to calculate the inflection point of the curve. The Tm reported is the average of three measurements.

[0154] Suitably, the antibodies of the invention, when in scFv format, exhibit a loss of less than 5%, preferably less than 3%, more preferably less than 1%, of monomer content after being subjected to 5 successive freeze-thaw cycles at a starting concentration of 10 mg / ml of the antibody of the invention, particularly when said antibody is formulated in 50 mM phosphate citrate buffer, pH 6.4, 150 mM NaCl.

[0155] Suitably, the antibodies of the invention when in scFv format exhibit a loss of less than 15%, such as less than 12%, such as less than 10%, such as less than 7%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, preferably less than 1%, after storage for 2 weeks or more, particularly for 4 weeks or more, at 4°C at a starting concentration of 10 mg / ml of the antibody of the invention, particularly when the antibody of the invention is formulated in 50 mM phosphate citrate buffer at pH 6.4, 150 mM NaCl.

[0156] The loss of monomer content is measured 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 outlined in USP Chapter 621. The method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. Separation of molecules occurs between the void volume (V0) and the total permeate volume (VT) of a particular column. SE-HPLC measurements are performed on a Chromaster HPLC system (Hitachi High-Tech Corporation) equipped with an automatic sample injection device and a UV detector set to a detection wavelength of 280 nm. The instrument is controlled by the software EZChrom Elite (Agilent Technologies, Version 3.3.2 SP2), which also supports the analysis of the resulting chromatograms. Protein samples are clarified by centrifugation and maintained at a temperature of 4-6 °C in the autosampler before injection. For the analysis of scFv samples, a Shodex KW403-4F column (Showa Denko, #F6989202) was used with a standardized 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 loading per injection was 5 μg. Samples were detected by a UV detector at a wavelength of 280 nm and data were recorded using an appropriate software set. The resulting chromatograms were analyzed from V0 to VT, excluding matrix-related peaks with elution times longer than 10 min.

[0157] As used herein, the term "recognize" refers to an antibody finding and interacting with (eg, binding to) its structural epitope.

[0158] As used herein, the terms "compete" or "cross-compete" and related terms are used interchangeably herein and refer to the ability of an antibody to interfere with the binding of other antibodies or binding agents to PDL1 in a standard competitive binding assay.

[0159] The ability or extent to which an antibody according to the invention can prevent the binding of other antibodies or binding molecules to PDL1, i.e. whether it can cross-compete, can be determined using standard competitive binding assays. Particularly suitable quantitative cross-competition assays use FACS- or AlphaScreen-based methods to allow competition between a labeled (e.g. His-tagged, biotinylated or radiolabeled) antibody or fragment thereof and a non-labeled antibody or fragment thereof and measure their binding to the target. In general, a cross-competing antibody or fragment thereof is one that binds to a target in such a way that, for example in a cross-competition assay, in the presence of a second antibody or fragment thereof alone during the assay, the displacement recorded by an immunoglobulin single variable domain or polypeptide of the invention is up to 100% (e.g. in a FACS-based competitive assay) relative to the maximum theoretical degree of displacement by an antibody or fragment thereof with cross-blocking ability present in a given amount to be tested (e.g. displacement by an untreated (e.g. unlabeled) antibody or fragment thereof required for cross-blocking). Preferably, the cross-competing antibody or fragment thereof has a recorded displacement of 10% to 100%, preferably 50% to 100%.

[0160] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of molecules with chemically active surface groupings, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. "Conformational" and "linear" epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. As used herein, the term "conformational epitope" refers to amino acid residues in an antigen that are located on the surface when the polypeptide chain is folded to form the native protein, and that exhibit a significant decrease in the rate of HD displacement upon Fab binding. Conformational epitopes include, but are not limited to, functional epitopes. The term "linear epitope" refers to an epitope in which all of the positions of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the (continuous) protein.

[0161] The present invention also provides antibodies that bind to the same epitope as the PDL1-binding antibodies listed in Table 1. Accordingly, additional antibodies are identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies of the invention in PDL1 binding assays.

[0162] Suitably, the isolated antibody of the present invention is selected from the group consisting of: These include monoclonal antibodies, chimeric antibodies, IgG antibodies, Fab, Fv, scFv, dsFv, scAb, STAB, and binding domains based on alternative scaffolds, such as but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectin, and binding sites engineered into the constant region of antibodies (e.g. F-star's Modular Antibody Technology™).

[0163] Suitably, the isolated antibody of the invention is an Fv. Suitably, the isolated antibody of the invention is an scFv antibody fragment. A "single-chain Fv" or "scFv" or "sFv" antibody fragment comprises the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for target binding. A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. In general, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding (see, for example, Pluckthun, 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 scFv format with a linker as set forth in SEQ ID NO: 28. In yet another embodiment, the isolated antibody of the invention is a single chain variable fragment (scFv) as set forth 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 set forth in SEQ ID NO: 31.

[0164] Suitably, the isolated antibody of the invention is an IgG antibody fragment. The term "isotype" refers to an antibody class, such as IgM, IgE, IgG (e.g., IgG1 or IgG4), that arises due to differences in heavy chain constant region genes. Isotype also includes modifications to any one of these classes to alter Fc function, such as to enhance or reduce effector function or binding to Fc receptors. In one embodiment, the isolated antibody of the invention is an IgG selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, preferably IgG1.

[0165] Suitably, the isolated antibody of the present invention comprises 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; A VH sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 14; and and a VL sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 26. In a more specific embodiment, the antibody of the present invention is an IgG1 comprising 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; a heavy chain sequence comprising an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 90%, identical to SEQ ID NO: 93; and and a light chain sequence comprising an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, identical to SEQ ID NO: 92. Suitably, the isolated antibody of the invention is an IgG1 comprising 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; A VH sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 14; and and a VL sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 26. In a more specific embodiment, the antibody of the present invention is an IgG1 comprising 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; A VH sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 16; and The IgG1 comprises a VL sequence that contains an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO:27.

[0166] Suitably, the isolated antibody of the present invention comprises 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 comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 45; and and a VL sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 57. In a more specific embodiment, the antibody of the present invention is an IgG1 comprising 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 heavy chain sequence comprising an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 90%, identical to SEQ ID NO: 91; and a light chain sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 90.

[0167] Suitably, the isolated antibody of the present invention 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; A VH sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 45; and and a VL sequence comprising an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 57. In a more specific embodiment, the antibody of the present invention is an IgG1 comprising 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 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO: 47; and The IgG1 comprises a VL sequence that contains an amino acid sequence that is 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, identical to SEQ ID NO:57.

[0168] In other specific embodiments of the invention, the isolated antibody of the invention is a multispecific molecule, particularly a multispecific molecule having at least a second functional molecule, such as a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific molecule.

[0169] The term "multispecific molecule" or "multispecific antibody" as used herein refers to an antibody that binds to two or more different epitopes on at least two different targets (e.g., PDL1 and another target different from PDL1) or to two or more different epitopes on the same target. The term "multispecific molecule" includes bispecific, trispecific, tetraspecific, pentaspecific and hexaspecific antibodies. The term "bispecific antibody" as used herein refers to an antibody that binds to two different epitopes on two different targets or on the same target. The term "trispecific antibody" as used herein refers to an antibody that binds to three different epitopes on three different targets or on the same target.

[0170] The antibodies of the invention can also be derivatized or linked to other functional molecules, such as other peptides or proteins (e.g., other antibodies or ligands for a receptor), thereby resulting in a multispecific molecule that binds to two or more binding sites and / or different target molecules. The antibodies of the invention can in fact be derivatized or linked to multiple other functional molecules, thereby resulting in a multispecific molecule that binds to two or more different binding sites and / or target molecules. To obtain a multispecific molecule of the invention, the antibodies of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, etc.) to one or more other binding molecules (e.g., other antibodies, antibody fragments, peptides, or binding mimetics), thereby resulting in a multispecific molecule.

[0171] Thus, the present invention encompasses multispecific molecules comprising one or more first binding specificities 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 encompasses multispecific molecules comprising one or more first binding specificities for PDL1 and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of PDL1 different from the first target epitope. The multispecific molecule may further comprise a third binding specificity in addition to the first and second target epitopes.

[0172] In yet another embodiment, the invention encompasses multispecific molecules comprising monovalent, bivalent or polyvalent specificity for PDL1, preferably monovalent specificity.

[0173] In other specific embodiments of the invention, the isolated antibody of the invention is a monovalent or multivalent, eg, bivalent, trivalent, tetravalent, pentavalent, hexavalent, PDL1-specific molecule.

[0174] The term "monovalent molecule" or "monovalent antibody" as used herein refers to an antibody that binds to a single epitope on a target molecule (eg, PDL1).

[0175] The term "multivalent antibody" refers to a single binding molecule with multiple valencies, where "valency" refers to the number of antigen-binding moieties that bind to an epitope on the same target molecule. Thus, a single binding molecule can bind to multiple target molecules or to multiple binding sites on a target molecule that includes multiple copies of an epitope. Examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, etc. As used herein, the term "bivalent antibody" refers to an antibody with two antigen-binding moieties (each binding to the same epitope).

[0176] Suitably, the isolated antibody of the present invention is a multispecific molecule (e.g. a bispecific molecule) and / or a multivalent molecule (e.g. a monovalent specificity molecule for PDL1, a bivalent specificity molecule for PDL1), which may be selected from any suitable multispecific, e.g. bispecific, antibody format known in the art, including but not limited to single chain diabody (scDb), tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), bispecific T cell binding domain (BiTE; tandem-di-scFv), tandem-tri-scFv, tribody (Fab-(scFv)2) or bibody (Fab-(scFv)1), Fab, Fab-Fv2, Morrison (IgG CH3-scFv fusion (Morrison L) or IgG CL-scFv fusion (Morrison H)), triabody, scDb-scFv, bispecific Fab2, di-minibody, tetrabody, scFv-Fc-scFv fusion, scFv-HSA-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, 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) ), IgG-scFv fusions such as Ts2Ab (dsscFv linked to the C-terminus of the heavy chain), bispecific antibodies based on heterodimeric Fc domains such as Knob-into-Hole antibodies (KiHs) (bispecific IgG prepared by KiH technology), Fv, scFv, scDb, tandem-di-scFv, tandem-tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD fused to the N- and / or C-terminus of either chain of one heterodimeric Fc domain or the other heterodimeric domain, MATCH (WO 2016 / 0202457, Egan T., et al.,mAbs 9(2017)68-84) and formats based on DuoBodie (bispecific IgG prepared by Duobody technology) (MAbs.2017 Feb / Mar;9(2):182-212.doi:10.1080 / 19420862.2016.1268307). Single chain diabodies (scDb) or scDb-scFv are particularly suitable for use in the present invention.

[0177] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a VH linked 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 complementary domains on the other chain to form two antigen-binding sites. In a specific embodiment, the polypeptide linker is one or two units of four glycine and one serine amino acid residues (GGGGS). n , n=1 or 2, preferably 1. Diabodies may be bivalent or bispecific. Details of diabodies are described, for example, in 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).

[0178] A bispecific scDb (especially a 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 linked 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-VHB-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 the first antigen and the VLB and VHB together form an antigen-binding site for the second antigen.

[0179] The linker L1 is in particular a peptide of 2 to 10 amino acids, more particularly 3 to 7 amino acids, and even more particularly 5 amino acids, and the linker L3 is in particular a peptide of 1 to 10 amino acids, more particularly 2 to 7 amino acids, and even more particularly 5 amino acids. In a specific embodiment, the linker L1 and / or L3 is a peptide of 1 or 2 units consisting of 4 glycine amino acid residues and 1 serine amino acid residue ((GGGGS) n , n=1 or 2, preferably n=1).

[0180] The intermediate linker L2 is in particular a peptide of 10 to 40 amino acids, more particularly 15 to 30 amino acids, and more particularly 20 to 25 amino acids. In a specific embodiment, the linker L2 is one or more units consisting of four glycine amino acid residues and one serine amino acid residue ((GGGGS) n , n=1, 2, 3, 4, 5, 6, 7 or 8, preferably n=4).

[0181] In one embodiment of the invention, the isolated antibody is a multispecific and / or multivalent antibody in scDb-scFv format. 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, said 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, said linker is a peptide of 4 glycine amino acid residues and 1 serine amino acid residue ((GGGGS) n , n=1, 2, 3, 4, 5, 6, 7 or 8, preferably n=2).

[0182] In one embodiment of the invention, the isolated antibody is a multispecific and / or multivalent antibody in the MATCH format described in WO 2016 / 0202457, Egan T. et al., mAb 9 (2017) 68-84.

[0183] The multispecific and / or multivalent molecules of the invention can be produced using any useful antibody production method known in the art (see, for example, Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14 for the generation of bispecific constructs, Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 and WO 99 / 57150 for bispecific diabodies and tandem scFvs). Further examples of suitable methods for the preparation of bispecific constructs of the invention include the techniques of Genmab (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750). Methods for the production of bispecific antibodies comprising the Fc portion of a functional antibody 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).

[0184] Other antibodies that can be used in the multispecific and multivalent molecules of the invention are murine, chimeric, and humanized monoclonal antibodies.

[0185] Multispecific molecules of the invention can be prepared by conjugating several binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be prepared separately and then conjugated to one another. When the binding specificities are proteins or peptides, covalent conjugation can be achieved using a variety of coupling or cross-linking agents. Exemplary cross-linkers include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (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 Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132, Brennan et al., 1985 Science 229:81-83, and Glennie et al., 1987 J. Immunol. 139:00:002367-2375. Conjugating agents include SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0186] When the binding specificity is an antibody, conjugation can be performed in which the C-terminal hinge regions of the two heavy chains are linked by sulfhydryls. In a specific embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues (e.g., one) prior to conjugation.

[0187] Alternatively, two or more binding specificities can be encoded in the same vector, 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 molecules of the invention can be single-chain molecules comprising one single chain antibody and one binding determinant, or single-chain multispecific molecules comprising two binding determinants. A multispecific molecule may comprise two or more single-chain molecules. For example, methods for preparing multispecific molecules are described in U.S. Pat. Nos. 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498 and 5,482,858.

[0188] The binding of the bispecific molecules to their specific targets can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays specifically detects the presence of protein-antibody complexes, usually by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0189] In a further aspect, the present invention provides a nucleic acid encoding the 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 PDL1 protein. Such a nucleic acid sequence can be optimized for expression in a mammalian cell.

[0190] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to one or more deoxyribonucleotides or ribonucleotides, in single- or double-stranded form, and polymers thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, natural, and unnatural, that have similar binding properties as the reference nucleic acid, and that are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses the sequence specified, as well as conservatively modified variants thereof (e.g., degenerate codon substitutions) and their complementary sequences. Specifically, degenerate codon substitutions may be created by substituting a sequence with mixed bases and / or deoxyinosine residues at the third position of one or more selected (or all) codons, as described below (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).

[0191] The present invention provides substantially purified nucleic acid molecules encoding polypeptides comprising segments or domains of the PDL1-binding antibody chains described above, wherein, when expressed from a suitable expression vector, the polypeptides encoded by these nucleic acid molecules are capable of binding to the PDL1 antigen.

[0192] Also provided in the present invention are polynucleotides that encode one or more CDR regions, typically all three CDR regions, from the heavy or light chain of a PDL1-binding antibody listed in Table 1. Some other polynucleotides encode all or substantially all of the heavy and / or light chain variable region sequences of a PDL1-binding antibody listed in Table 1. Due to the degeneracy of the code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence.

[0193] Polynucleotide sequences can be prepared by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding PDL1-binding antibodies (e.g., sequences described in the Examples below). Direct chemical synthesis of nucleic acids can be carried out by methods known in the art, such as the phosphotriester method (Narang et al., 1979, Meth. Enzymol. 68:90), the phosphodiester method (Brown et al., Meth. Enzymol. 68:109, 1979), the diethyl phosphoramidite method (Beaucage et al., Tetra. Lett., 22:1859, 1981), and the solid-phase method (U.S. Pat. No. 4,458,066). For example, introduction of mutations into a polynucleotide sequence by PCR can be carried out as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. 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.

[0194] The present invention also provides expression vectors and host cells for producing the above-mentioned PDL1-binding antibodies.

[0195] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be introduced into a host cell and integrated into the genome of the host cell, thereby replicating along with the host genome.

[0196] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention also includes other forms of expression vectors, such as viral vectors, such as replication defective retroviruses, adenoviruses and adeno-associated viruses, which serve equivalent functions.

[0197] The term "operably linked" refers to the functional relationship of two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional control sequence to a transcribed sequence. For example, promoter or enhancer sequences are operably linked to a coding sequence if they stimulate or regulate the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional control sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they act in cis. However, some transcriptional control sequences (e.g., enhancers) need not be physically close to or adjacent to the coding sequence whose transcription they enhance.

[0198] A variety of expression vectors can be used to express polynucleotides encoding PDL1-binding antibody chains or binding fragments. Viral-based and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors that typically carry expression cassettes for protein or RNA expression, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). For example, non-viral vectors useful for the expression of PDL1-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invitrogen, San Diego, Calif.), MPS V vectors, and various other vectors for protein expression known in the art. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papilloma viruses, HBP Epstein-Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV)-based vectors. See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995 and Rosenfeld et al., Cell 68:143, 1992.

[0199] The choice of expression vector depends on the host cell in which the vector is to be expressed. Typically, an expression vector contains a promoter and other control sequences (e.g., enhancers) operably linked to the polynucleotide encoding the PDL1-binding antibody. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. Cultivation of the transformed organism under non-inducing conditions allows for unbiased growth of the population carrying the coding sequence, since expression of the product is tolerable to the host cell. In addition to the promoter, other control elements may be required or desirable for efficient expression of the PDL1-binding antibody. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression may be enhanced by enhancers appropriate for the cell system used (see, e.g., 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 CMV enhancer may be used to enhance expression in mammalian host cells.

[0200] The expression vector may be provided with a secretory signal sequence to allow the polypeptide encoded by the inserted PDL1-binding antibody sequence to be expressed as a fusion protein. In many cases, the inserted PDL1-binding antibody sequence is linked to a signal sequence prior to insertion into the vector. The vector used to receive the sequences encoding the light and heavy chain variable region domains of the PDL1-binding antibody also optionally encodes a constant region or a portion thereof. Such vectors allow expression of the variable region as a fusion protein with the constant region, thereby resulting in the production of complete antibodies and antigen-binding fragments thereof. Typically, such constant regions are of human origin.

[0201] 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 such terms refer not only to the particular cell but also to the progeny of such a cell. Such progeny may not, in fact, be identical to the parent cell, since certain modifications may occur over time, due to mutation or the influence of environmental conditions, but are still intended to be included within the scope of the term "host cell" as used herein.

[0202] Host cells for harboring and expressing PDL1-binding antibody chains may be prokaryotic or eukaryotic. Escherichia coli is one of the prokaryotic hosts useful for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use include Bacillus bacteria (e.g., Bacillus subtilis) and other enterobacteria (e.g., Salmonella, Serratia, and various Pseudomonas species). In these prokaryotic hosts, expression vectors can also be constructed, which typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. In addition, various known promoters can be included, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system derived from lambda phage. These promoters typically control expression, optionally with an operator sequence, and also contain sequences such as ribosome binding sites to initiate and terminate transcription and translation. Other microorganisms (e.g., 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.

[0203] In one embodiment, mammalian host cells are used to express and produce the PDL1-binding polypeptides of the invention. For example, they may be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These include any normal mortal or normal or abnormal immortalized animal or human cells. Many suitable host cell lines capable of secreting intact immunoglobulins have been developed, such as CHO cells, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture systems to express polypeptides is reviewed in, for example, Winnacker, FROM GENES TO CLONES, VCH Publishers, NYNY 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter and enhancer (Queen, et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters are constitutive, cell-specific, stage-specific, and / or regulatable or controllable promoters. 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 early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations, all of which are known in the art.

[0204] The method of introducing an expression vector containing a polynucleotide sequence of interest varies depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other host cells. (See Sambrook et al., supra, for an overview). Other methods include, for example, electroporation, calcium phosphate treatment, transformation with liposomes, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced DNA uptake, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression is usually required. For example, cell lines stably expressing PDL1-binding antibody chains or binding fragments can be prepared using the expression vectors of the invention, which contain a viral origin of replication or an endogenous expression element and a selectable marker gene. After introduction of the vector, the cells may be grown in rich medium for 1-2 days before switching them to selective medium. The purpose of using a selectable marker is to confer resistance to selection, the presence of which allows cells that preferentially express the introduced sequence to grow in selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type. Thus, the invention provides a method of producing an antibody of the invention, said method comprising the step of culturing, in particular expressing, a host cell containing a nucleic acid or vector encoding an antibody of the invention, whereby said antibody of the invention or a fragment thereof is expressed.

[0205] In a further aspect, the invention relates to a pharmaceutical composition comprising an antibody of the invention and a pharma- ceutically acceptable carrier, which enhances or stabilizes the composition or facilitates preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other media that are physiologically compatible.

[0206] The pharmaceutical compositions of the present invention can be administered by various well-known methods. The route or method of administration varies depending on the desired effect. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or in the vicinity of the target site. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., injection or irrigation). Depending on the route of administration, the active compounds (i.e., antibodies and multispecific molecules) may be coated with a material to protect the compound from the action of acids and other natural conditions which may result in the inactivation of the compound.

[0207] The pharmaceutical composition of the present invention can be prepared according to methods known and routinely practiced in the art. For example, see 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 amount or effective amount of PDL1-binding antibody is used in the pharmaceutical composition of the present invention. The PDL1-binding antibody is prepared in a pharma- ceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered at regular intervals, or the dose may be gradually decreased or increased depending on the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly useful to formulate the components into unit dosage forms. As used herein, "unit dosage form" refers to physically discrete units conveniently arranged as dosage units for the subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0208] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so that the amount of active ingredient, composition and method of administration is effective to achieve the desired therapeutic response in a particular patient without causing toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention or its esters, salts or amides used, the timing of administration, the route of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, symptoms, general health, past medical history of the patient being treated, and other factors.

[0209] The antibody is usually administered multiple times. The interval between single doses can be weekly, monthly or yearly. The administration intervals can be irregular depending on the measurement of the patient's blood concentration of the PDL1-binding antibody. Alternatively, the antibody can be administered as a sustained release formulation, in which case frequent administration is not required. The amount and frequency of administration varies depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies. The amount and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive administration, a relatively low dose is administered at relatively infrequent intervals for an extended period of time. Some patients may continue to receive treatment for the rest of their lives. In therapeutic administration, a relatively high dose may be required at relatively short intervals until the progression of the disease is slowed or terminated, preferably until the patient shows partial or complete improvement of the symptoms of the disease. The patient can then receive a preventive regimen.

[0210] The antibodies of the invention have in vitro and in vivo diagnostic and therapeutic utility: for example, these molecules can be administered to cells in culture in vitro or in vivo, or to a subject in vivo, for example, to treat, prevent, or diagnose a variety of disorders.

[0211] In one aspect, the invention relates to an antibody of the invention or a composition of the invention for use as a medicament.

[0212] 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 disorder, in particular cancer, in a subject in need thereof.

[0213] In another aspect, the invention relates to the use of an antibody of the invention or a composition of the invention for the treatment of a proliferative disorder, in particular cancer, in a subject in need thereof.

[0214] In a further aspect, the present invention relates to the use of an antibody of the invention or a composition of the invention in the manufacture of a medicament for the treatment of a proliferative disorder, in particular cancer, in a subject in need of such treatment.

[0215] In one aspect, the invention provides a method for treating a proliferative disorder, particularly cancer, in a subject in need of such treatment comprising administering to said subject a therapeutically effective amount of an antibody of the invention or a composition of the invention.

[0216] The term "subject" includes humans and non-human animals. Non-human animals include, for example, all vertebrate mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" and "subject" are used interchangeably herein.

[0217] 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 adverse effects caused by a disease, or may be therapeutic in the sense of slowing disease progression. As used herein, "treatment" includes any treatment of a mammalian (e.g., human) disease, including, for example: (a) inhibiting the disease, i.e., slowing its progression, and (b) relieving the disease, i.e., causing regression of the disease.

[0218] The term "therapeutically effective amount" or "effective amount" refers to the amount of a drug that, when administered to a mammal or other subject to be treated for a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" varies depending on the drug, the disease and its severity of the subject being treated, as well as the age, weight, etc.

[0219] In one embodiment, the proliferative disease is cancer. The term "cancer" refers to a disease characterized by rapid uncontrollable proliferation of stray cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, but are 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, e.g., both terms include solid tumors, and tumors that are, e.g., diffuse or circulating liquid tumors. As used herein, the term "cancer" or "tumor" includes pre-cancers, as well as malignant cancers and tumors. The term "cancer" is used herein to mean the widespread nature of tumors, and includes 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 gland cancer, bladder cancer, breast cancer, gastric 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. unremovable or metastatic melanoma), renal cell carcinoma, sarcoma, glioblastoma, multiple myeloma, or gastrointestinal cancer, in particular colon cancer or colorectal adenoma. Head and neck tumors, endometrial cancer, Cowden syndrome, Lhermitte-Duclos disease, Bannayan-Zonana syndrome, prostatic hyperplasia, neoplasia, especially epithelial, 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 combinations 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.

[0220] The antibody of the present invention or the composition of the present invention inhibits the growth of solid tumors, but also liquid tumors. In yet another embodiment, the proliferative disease is a solid tumor. The term "solid tumor" refers in particular to breast, ovarian, colon, rectal, prostate, gastrointestinal (particularly gastric), cervical, lung (e.g. non-small cell lung cancer and small cell lung cancer), and head and neck tumors. Furthermore, depending on the tumor type and the particular combination used, it is possible to reduce the tumor volume. The antibody of the present invention or the composition of the present invention is also suitable for preventing the metastasis of tumors, as well as the growth or development of micrometastases in subjects with cancer.

[0221] The term "prevent" or "prevention" refers to the complete inhibition of the progression of the disease or any secondary effects of the disease. As used herein, the term "prevent" or "prevention" encompasses preventing the development of a disease or condition in an individual who has been diagnosed as having a predisposition to the disease, but has not been diagnosed as having the disease.

[0222] In one aspect, 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 contents, such as: instructions, other reagents (e.g., a label, a therapeutic agent or an agent useful for chelating or otherwise coupling the antibody to a label or therapeutic agent, or a radioprotective composition), equipment or other materials for preparing the antibody molecule for administration, a pharma- ceutically acceptable carrier, and equipment or other materials for administration to a subject. In a particular embodiment, the kit comprises a pharma- ceutically effective amount of the antibody of the present invention. In yet another embodiment, the kit comprises a pharma- ceutical effective amount of a lyophilized antibody of the present invention, and a diluent, and optional instructions. The kit may further comprise a filter needle for reconstitution and a needle for injection.

[0223] [Table 1]

[0224] [Table 2]

[0225]

Table 3

[0226]

Table 4

[0227]

Table 5

[0228]

Table 6

[0229]

Table 7

[0230]

Table 8

[0231]

Table 9

[0232]

Table 10

[0233]

Table 11

[0234]

Table 12

[0235] [Table 13]

[0236] [Table 14]

[0237] [Table 15]

[0238] [Table 16]

[0239] Throughout the documents of this application, there may be discrepancies between the descriptions in the specification (e.g., Tables 1-3) and the sequence listing, but in such cases the descriptions in the specification shall prevail.

[0240] For clarity, certain features of the present invention are described in the context of separate embodiments, but it is clear that they may be combined in one embodiment. Conversely, various aspects of the present invention described in the description of one embodiment may be divided for brevity and applied in appropriate subcombinations. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and components thereof are specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0241] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0242] To the extent permitted by the patent laws of each country, all patents, patent applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

[0243] The following examples illustrate the invention described above, but are not intended to limit the scope of the invention in any manner. Other test models known to those skilled in the relevant art may also be used to confirm the utility of the claimed invention. EXAMPLES

[0244] Novel antibody against human PDL1: Example 1: Generation of rabbit antibodies against human PDL1:

[0245] Rabbits were immunized with recombinantly produced and purified human PDL1 extracellular domain. During the immunization procedure, the strength of the humoral immune response to the antigen in the serum of each rabbit that still gave rise to detectable binding of polyclonal serum antibodies was qualitatively evaluated by measuring the maximum dilution (titer) against the antigen. Serum antibody titers against the immobilized antigen (recombinant human PDL1 extracellular domain) were evaluated using ELISA. All immunized rabbits had a serum titer of at least 1:2.64 × 10 6 The serum from the same rabbit before the first antigen injection was used as a background control.

[0246] Example 2: Identification and selection by Hit: A flow cytometry-based sorting procedure was developed that allows specific detection and isolation of high affinity hPDL1-binding B cells during the hit identification procedure. To identify hPDL1-binding B cells, hPDL1 ECD was labeled with the fluorescent dye R-phycoerythrin (R-PE). Because the PD-1-binding site on labeled PDL1 as well as the binding site of anti-PDL1 neutralizing antibodies are potentially blocked by the bulky R-PE label, the accessibility of the epitope could be confirmed by flow cytometry. The Fc portion of human IgG1 or the PD-1 extracellular domain fused to avelumab was captured on protein G beads, and the binding of R-PE-labeled PDL1 was confirmed by flow cytometry. The fluorescence intensity showed a proportional relationship to the amount of labeled PDL1 that bound to the receptor immobilized on the beads. Binding of PDL1 to PD-1 and neutralizing antibodies was confirmed, whereas binding of irrelevant cytokines to anti-PDL1 antibodies was not detected.

[0247] screening: Due to the difficulty of purifying individual rabbit antibodies at a high-throughput culture scale, the results obtained during the screening phase were based on assays performed with unpurified antibodies from culture supernatants of antibody-secreting cells (ASCs). Such supernatants allow a large number of antibodies to be ranked relative to each other, but do not provide absolute values ​​other than binding ability. Supernatants from every individual cultured clone were collected over a period of 4 weeks. At the end of the culture period, rabbit monoclonal antibodies in each cell culture supernatant were characterized in a high-throughput ELISA for binding to recombinant human PDL1 extracellular domain. PDL1-bound supernatants were further characterized for their binding kinetics to human and cynomolgus PDL1. In addition, the neutralization potential of PDL1 / PD-1 interaction was measured by cell-based reporter gene assays as well as by competitive ELISA. Neutralization of PDL1 / B7-1 interaction was also evaluated by competitive ELISA. With the exception of binding kinetics, reported values ​​from the high-throughput screen were to be interpreted as "yes" or "no" results, which were based on single measurements (not dose-response). Mouse PDL1 binding potential of supernatants was analyzed by direct ELISA, and binding kinetics were determined for positive supernatants only.

[0248] Direct ELISA for hPDL1 binding: 50 μl of PBS containing 500 ng / ml PDL1 was added to the ELISA plate and coated overnight at 4° C. The next day, the plate was washed three times in overflow mode with 450 μl of washing buffer (PBS, 0.005% Tween 20) per well, and 300 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) per well was added and treated for 1 h at room temperature on a nutating mixer. The plate was then washed three times in overflow mode with 450 μl of washing buffer, 50 μl of each supernatant was added, and the plate was incubated for 1.5 h at room temperature with gentle shaking. After washing three times in overflow mode with 450 μl of washing buffer, 50 μl of HRP-conjugated goat and rabbit IgG antibodies were added to each well. After 1 hour incubation at room temperature on a rotating mixer, the plates were washed with 450 μl of wash buffer per well, followed by the addition of 50 μl of TMB (3,3',5,5'-tetramethylbenzidine, KPL, Cat. No. 53-00-00). After 5-10 minutes of color development, the enzymatic reaction was stopped by the addition of 50 μl of 1 M HCl per well, and the plates were read at 450 nm using 690 nm as the reference wavelength.

[0249] Affinity for hPDL1 by SPR: The binding affinity of antibodies to human PDL1 was measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). In the affinity screening, 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 procedure. Rabbit monoclonal antibodies in B cell supernatants were captured by immobilized anti-rabbit IgG antibodies. A minimum IgG concentration in the B cell supernatant was required for sufficient capture. After monoclonal antibody capture, human PDL1 (Peprotech) was injected at a concentration of 90 nM over the flow cell for 3 min, and protein dissociation from the IgG captured on the sensor chip was allowed to proceed for 5 min. After each injection cycle, the surface was regenerated with two injections of 10 mM glycine-HCl. The apparent dissociation (kd) and association (ka) constants, as well as the apparent dissociation equilibrium constant (K D ) was calculated using the one-to-one Langmuir binding model with MASS-1 analysis software (Analyzer, Sierra Sensors, Inc.), and Chi 2 Relative values ​​of Chi normalized to the extrapolated maximum binding level of the analyte 2 ) was used to monitor the quality of the fit and to serve as an index for maintaining the quality of the curve fitting. 2 When the value of Chi is small, it means that the fitting to the one-to-one Langmuir binding model is highly accurate. 2 The relative values ​​of were ≦10%. Results were considered valid if the response units (RU) for ligand binding were ≧2% of the RU for antibody capture. Samples showing RU for ligand binding less than 2% of the RU for antibody capture were considered to indicate no specific binding of PDL1 to the captured antibody.

[0250] PDL1 / PD-1 Blocking ELISA: 50 μl of PBS containing 2 μg / ml PD-1 was added and the ELISA plate was coated overnight at 4°C. The next day, the plate was washed 3 times in overflow mode with 450 μl of wash buffer per well, and 300 μl of blocking buffer per well was added and treated on a rotating mixer for 1 h at room temperature. PDL1 was then diluted in blocking buffer at a concentration 20-fold higher than the desired final concentration of 250 ng / ml. To further adjust the assay sensitivity, some clones were analyzed in the presence of 40 ng / ml PDL1. Next, 114 μl of each supernatant was diluted in blocking buffer containing 6 μl of PDL1 in a non-binding plate, and the plate was incubated on a rotating mixer for 1 h at room temperature. The ELISA plate was washed 3 times in overflow mode with 450 μl of wash buffer per well, and 50 μl of each dilution was added to the ELISA plate. The plate was incubated at room temperature for 1.5 h with gentle shaking. After washing three times with 450 μl of washing buffer per well, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After 1 h of incubation at room temperature, the plate was washed three times with 450 μl of washing buffer and 50 μl of TMB was added, followed by color development for 5-10 min. Finally, the enzyme reaction was stopped by adding 50 μl of 1 M HCl, and the plate was measured at 450 nm with 690 nm as the reference wavelength.

[0251] PDL1 / B7-1 Blocking ELISA: 50 μl of PBS containing 4 μg / ml of B7-1 was added and the ELISA plate was coated overnight at 4°C. The next day, the plate was washed 3 times in overflow mode with 450 μl of wash buffer per well, 300 μl of blocking buffer per well was added and treated for 1 h at room temperature on a rotating mixer. PDL1 was then diluted in blocking buffer at a concentration 20 times higher than the desired final concentration of 500 ng / ml. 114 μl of each supernatant was then diluted in blocking buffer containing 6 μl of PDL1 in a non-binding plate and the plate was incubated for 1 h at room temperature on a rotating mixer. The ELISA plate was washed 3 times in overflow mode with 450 μl of wash buffer per well and 50 μl of each dilution was added to the ELISA plate. The plate was incubated for 1.5 h at room temperature with gentle shaking. After washing three times with 450 μl of washing buffer per well, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After 1 h of incubation at room temperature, the plate was washed three times with 450 μl of washing buffer and 50 μl of TMB was added, followed by color development for 5-10 min. Finally, the enzyme reaction was stopped by adding 50 μl of 1 M HCl, and the plate was measured at 450 nm with 690 nm as the reference wavelength.

[0252] Blocking PDL1 / PD-1 in cell-based assays (reporter gene): To further analyze the Hits, their ability to neutralize the PDL1 / PD-1 interaction when both interacting molecules were expressed on the cell surface was tested using CHO / PDL1 / TCR activators and Jurkat / PD-1 cells. 35,000 CHO / PDL1 / TCR activators in 100 μl of cell culture medium (DMEM / F12, 10% FCS) were added to the inner wells of a white culture plate and incubated for 16-20 hours at 37°C and 5% CO2. The next day, 95 μl of cell culture medium was removed from each well and 50 μl of the supernatant of the screened B cells or the positive control avelumab was added at concentrations determined as giving 0%, 50% and 100% of the maximum signal, and the plate was incubated for 30 minutes at 37°C. Effector Jurkat cells diluted to 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS) were then added at 50 μl per well and the plates were incubated for 6 h at 37° C. and 5% CO2. Finally, luciferase substrate (BPS Bioscience) prepared according to the manufacturer's protocol was added at 50 μL per well, the plates were incubated for 30 min in the dark and luminescence was measured using a Topcount.

[0253] Species specificity by SPR: cyno and mouse We also measured the binding kinetics to cynomolgus and mouse PDL1 using a similar SPR setup as described for binding to human PDL1, but replacing human PDL1 with cynomolgus or mouse PDL1, respectively.

[0254] Screening Hits Selection: The pharmacological properties of the final clonal monoclonal antibodies in B cell supernatants are shown in Table 4.

[0255] Example 3: Hit confirmation: Cloning and production: Following identification of selected clones for hit confirmation, these rabbit antibodies were cloned, expressed and purified for further characterization. Cloning of the corresponding light and heavy chain variable domains was performed by in vitro ligation of DNA fragments into appropriate mammalian expression vectors. These expression vectors containing consensus sequences of the constant regions of the light and heavy chains of rabbit IgG were co-expressed, resulting in their assembly and secretion of fully functional rabbit monoclonal IgG. After vector construction, the sequences of the resulting constructs were confirmed again, and the plasmid DNA was amplified and purified and subjected to transfection into mammalian cells.

[0256] Expression vectors for rabbit antibody heavy and light chains were transfected into mammalian suspension cell lines for transient heterologous expression by lipid-based transfection reagents. Conditions, such as the ratio of heavy and light chain vectors, were optimized to increase the expression level of secreted monoclonal IgG. Expression cultures were cultured for 7 days in a shaking incubator. At the end of the heterologous expression period, cell culture supernatants were collected by centrifugation. Secreted rabbit IgG was then affinity purified by Protein A beads. The IgG-loaded beads were washed, and the purified antibody was eluted by pH shift. The identity, content, and purity of the eluted fractions were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV absorption at 280 nm, and size-exclusion high-performance liquid chromatography (SE-HPLC). Table 5 summarizes the production and characterization of rIgG.

[0257] [Table 17]

[0258] [Table 18]

[0259] Affinity for hPDL1 by SPR: The binding kinetics of purified monoclonal rabbit antibodies against human PDL1 were measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). As most of the antibodies showed very slow off-rates, experiments were performed at 37°C in a buffer containing high salt concentration to clarify the binding affinity of different antibodies. Antibodies specific for the Fc region of rabbit IgG (Bethyl Laboratories, Cat. No. A120-111A) were immobilized on a sensor chip (SPR-2 affinity sensor, High Capacity Amine, Sierra Sensors) using standard amine coupling procedures. Rabbit monoclonal antibodies were captured by immobilized anti-rabbit IgG antibodies. After capture of the monoclonal antibody, two-fold serial dilutions of PDL1 ranging from 90 to 0.35 nM were tested in HEPES buffer containing 150 mM NaCl and 150 mM MgCl2 for 5 min to test binding to IgG captured on the biosensor chip and dissociation of the protein from IgG captured on the sensor chip. After each injection cycle, the surface was regenerated with two injections of 10 mM glycine-HCl. The apparent dissociation (kd) and association (ka) constants, as well as the apparent dissociation equilibrium constant (K D ) was calculated using the one-to-one Langmuir binding model with MASS-1 analysis software (Analyzer, Sierra Sensors, Inc.), and Chi 2 Relative values ​​of Chi normalized to the extrapolated maximum binding level of the analyte 2 ) was used to monitor the quality of the fit and to serve as an index for maintaining the quality of the curve fitting. 2 When the value of Chi is small, it means that the fitting accuracy to the one-to-one Langmuir binding model is high. Most Hits are 2 The relative values ​​of were below 10%. Table 6 shows the rabbit IgG antibodies that were selected for further development.

[0260] [Table 19]

[0261] PDL1 / PD-1 Blocking ELISA Titer: The potency of neutralizing PDL1 binding to PD-1 was evaluated in a competitive ELISA. ELISA plates were coated overnight at 4°C with 50 μl of 4 μg / ml PD-1 in PBS. The next day, plates were washed three times in overflow mode with 450 μl washing buffer (PBS, 0.005% Tween 20) per well, and 300 μl blocking buffer (PBS, 1% BSA, 0.2% Tween 20) per well was added and incubated for 1 h at room temperature on a nutating mixer. PDL1 was then diluted in blocking buffer to a final concentration of 1 ng / ml. Next, in the non-binding plate, 120 μl of buffer containing serial dilutions of PDL1 ranging from 300 to 0.005 ng / ml of the rIgG to be tested was prepared per well, and the plate was incubated at room temperature for 30 min. The ELISA plate was washed three times in overflow mode with 450 μl of wash buffer, and 50 μl of each dilution was added to adjacent wells of the ELISA plate as replicate samples. The plate was incubated for 90 min at room temperature with gentle shaking. After washing three 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 1 h of incubation at room temperature, the plate was washed three times with 450 μl of wash buffer and 50 μl of TMB was added before color development for 5–10 min. Finally, 50 μl of 1 M HCl was added to stop the enzymatic reaction, and the plate was read at 450 nm with 690 nm as the reference wavelength.

[0262] Rabbit IgG from clones 33-03-G02 and 37-20-B03 showed high potency in neutralizing the PDL1 / PD-1 interaction. Clone 37-20-B03 had approximately 2-fold higher potency than avelumab (Table 7). Dose-response curves obtained for selected clones are shown in Figure 1.

[0263] [Table 20]

[0264] Titer in PDL1 / B7-1 Blocking ELISA: The potency of neutralizing the PDL1 / B7-1 interaction was evaluated in a competitive ELISA. ELISA plates were coated overnight at 4°C with 50 μl of PBS containing 4 μg / ml of B7-1. The next day, plates were washed three times in overflow mode with 450 μl of washing buffer (PBS, 0.005% Tween 20) per well and 300 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) per well were added and treated for 1 h at room temperature on a nutating mixer. PDL1 was then diluted to 40 ng / ml in blocking buffer. Next, in the non-binding plate, 120 μl of buffer containing serial dilutions of PDL1 ranging from 900 to 0.015 ng / ml of the rIgG to be tested was prepared per well and the plate was incubated at room temperature for 30 min. The ELISA plate was washed three times with 450 μl of wash buffer in overflow mode, and 50 μl of each dilution was added twice to adjacent wells of the ELISA plate as replicate samples. The plate was incubated for 90 min at room temperature with gentle shaking. 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 1 h of incubation at room temperature, the plate was washed three times with 450 μl of wash buffer and 50 μl of TMB was added before color development for 5–10 min. Finally, the enzyme reaction was stopped by adding 50 μl of 1 M HCl, and the plate was measured at 450 nm with 690 nm as the reference wavelength. The selected rabbit IgG was able to block PDL1 / B7-1 interaction with a potency comparable to that of avelumab, as shown in Table 8. The dose-response curves obtained for the selected clones are shown in Figure 2.

[0265] [Table 21]

[0266] Potency in cell-based PDL1 / PD-1 blocking assay (reporter gene): The potency of neutralizing PDL1 binding 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 a white cell culture plate and incubated for 16-20 h at 37 °C and 5% CO2. The next day, 95 μl of cell culture medium was removed from each well and 50 μl of a two-fold dilution series of the molecules to be tested (from 3,000 to 0.46 ng / ml) and the control avelumab solution were added. Then, 50 μl of effector Jurkat cells diluted to 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS) were added to each well and the plate was incubated for 6 h at 37 °C and 5% CO2. Finally, luciferase substrate (BPS Bioscience), prepared according to the manufacturer's protocol, was added at 50 μL per well, the plate was incubated in the dark for 30 min, and luminescence was measured using a Topcount. The selected clones were able to block PDL1 / PD-1 interaction in a cell-based reporter gene assay (see Table 9). The dose-response curves obtained for the selected clones are shown in Figure 3.

[0267] [Table 22]

[0268] Binding to PDL1 expressing cells by FACS: Binding titers to PDL1-expressing cells were measured for selected IgGs. 50,000 CHO-PDL1-expressing cells were distributed into round-bottom non-tissue culture treated 96-well plates. Cells were washed twice with 100 μl PBS by centrifugation at 400×g for 5 min. Cells were resuspended in 100 μl of serial dilutions of the test rIgGs and the control IgG avelumab from 2,000 to 0.128 ng / ml in staining buffer (PBS, heat inactivated BCS 2%, 2 mM EDTA). After 1 h incubation at 4°C on a rotating mixer, cells were washed three times with 100 μl staining buffer and 5 min centrifugation steps at 400×g. Next, the rabbit IgG-treated cells were resuspended in 100 μl of staining buffer containing 2 μg / ml of goat anti-rabbit IgG (APC-labeled), and the avelumab (human IgG1)-treated cells were resuspended in 100 μl of staining buffer containing 2 μg / ml of goat anti-human IgG (APC-labeled). The plates were incubated for 1 h at 4°C on a rotating mixer. The plates were washed three times with 100 μl of staining buffer and resuspended in staining buffer in a final volume of 50 μl. Finally, the APC signal of 20,000 events per well was analyzed by flow cytometry using a Novocyte flow cytometer system (ACEA Bioscience). Binding of PDL1-expressing cells was confirmed for all evaluated rabbit IgGs. The binding titers of selected rabbit IgGs to intracellular PDL1 are shown in Table 10.

[0269] [Table 23]

[0270] Species specificity by SPR:cyno: Using a similar setup as described for binding to human PDL1, binding kinetics to cynomolgus PDL1 was also measured, but in this case human PDL1 was replaced by cynomolgus PDL1. Binding to cynomolgus PDL1 was confirmed for all selected IgGs (Table 11).

[0271] [Table 24]

[0272] Species specificity by SPR: Mouse: Binding kinetics to cynomolgus monkey PDL1 was also measured using a similar setup as described for binding to human PDL1, but in this case human PDL1 was replaced by mouse PDL1. No binding to mouse PDL1 was detected for selected rabbit IgGs from clones 33-03-G02 and 37-20-B03.

[0273] Example 4: Selection of clones for humanization Based on the data obtained in the Hit validation, all clones were humanized by fusing the CDRs to VH3, VH4 or VH1A or VH1B based frameworks. To obtain the best affinity and potency, two clones that showed the best affinity to human PDL1 as rIgG, 33-03-G02 and 37-20-B03, were given different structures by grafting and further optimized. The following grafting variants were applied for clones 33-03-G02 and 37-20-B03: CDR grafting - grafting rabbit CDRs onto a human framework; IF graft - CDR graft + graft of all rabbit VL / VH interface residues; Full grafting - CDR grafting + framework residues following the AHo humanization protocol (antigen interface (AIF) residues (rabbit residues potentially in contact with the antigen (following AHo)) were limited to no more than 20% residue changes, taking into account solvent accessibility during interface formation, thereby reducing the number of mutations (rabbit framework residues)).

[0274] Proteins were heterologously expressed as insoluble inclusion bodies by overnight small-scale expression with induction in E. coli (except for PRO997, which was produced in mammalian CHO-S cells, similar to the rIgG expression described above). Inclusion bodies were isolated from homogenized cell pellets by a centrifugation protocol that included several washing steps to remove cell debris and other host cell-derived impurities. The purified inclusion bodies were solubilized in a denaturing buffer, and the scFvs were refolded according to a scalable refolding protocol that yields milligram quantities of natively folded monomeric scFvs. The scFvs were purified using a standardized protocol. The refolded product was captured by affinity chromatography to obtain purified scFvs. Only the main fraction with the desired purity was used, as no amount of the sample was available for SEC polishing. In addition, the melting temperatures of the scFvs were measured by differential scanning fluorimetry (DSF) (details below). Table 12 summarizes the preparation of VH4 CDR-grafted scFv molecules. Since two clones contained an unpaired cysteine ​​residue in their CDR-loops, a C57S mutation was introduced into clone 37-20-B03 as shown in Table 12.

[0275] Further graft mutants were engineered for several selected clones and their initial preparation and characterization are summarized in Tables 13 (AHo numbering) and 14.

[0276] Example 5: Pharmacokinetic analysis of humanized scFvs: Next, the main pharmacokinetic properties of the humanized scFvs were analyzed using an assay system similar to that described for the Hit confirmation phase, but with appropriate modifications according to the different formats of the scFv molecules.

[0277] 5.1 Affinity for human PDL1 The affinity of humanized scFvs for human PDL1 was measured by SPR analysis with a T200 instrument (Biacore, GE Healthcare). In this experiment, Fc-tagged human PDL1 was captured using the Human Antibody Capture kit from GE Healthcare. After each sample injection cycle, the CM5 sensor chip was regenerated and the antigen was newly captured. Using a dose-response multi-cycle kinetic assay with varying sample concentrations from 0.12 to 30 nM diluted in running buffer, scFvs were injected as samples for 5 min and protein dissociation was allowed to proceed for 12 min. The resulting sensograms were fitted using a 1:1 binding model. As shown in Table 15, binding to human PDL1 was confirmed for the humanized scFvs tested.

[0278] 5.2. Neutralization of PDL1 / PD-1 interaction by competitive ELISA: The potency of neutralizing PDL1 binding to PD-1 was evaluated by competitive ELISA following the same procedure as described above. 50 Values ​​are expressed as the IC of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC 50 :(I C 50アベルマブ / I C 50試験scFv )) As summarized in Table 16, potency was up to 5-fold higher than avelumab in this assay. 50 It is clear that all scFvs tested had potencies equal to or higher than avelumab.

[0279] 5.3. Neutralization of PDL1 / B7-1 interaction by competitive ELISA The potency of neutralizing PDL1 binding to B7-1 was evaluated by competitive ELISA following the same procedure as described above. Individual IC 50 Values ​​are expressed as the IC of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC 50 :(I C 50アベルマブ / I C 50試験scFv )) As summarized in Table 17, potency was up to 10-fold higher than avelumab in this assay. 50 It is clear that the calculated potency of the scFvs tested was higher or equal to that of avelumab.

[0280] Neutralization of PDL1 / PD-1 interaction in NFAT reporter gene assays The ability to neutralize PDL1 binding to PD-1 was assessed in a cell-based reporter gene assay as described above. Each molecule to be tested, as well as the control avelumab, were added to the plate as serial dilutions. The individual IC 50 Values ​​are expressed as the IC of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC 50 :(I C 50アベルマブ / I C 50試験scFv )) As summarized in Table 18, potency was up to 5-fold higher than avelumab in this assay. 50 It is clear that the titer can be calculated. The scFvs tested had higher or similar potencies.

[0281] [Table 25]

[0282] [Table 26]

[0283] [Table 27]

[0284] [Table 28]

[0285] [Table 29]

[0286] [Table 30]

[0287] [Table 31]

[0288] 5.5. Binding to hPDL1-expressing cells by flow cytometry: Binding titers to PDL1 expressing cells were measured for several molecules. During hit confirmation, the same cell lines were used (CHO-PDL1 and CHO-K1), but the scFvs were detected by APC-labeled protein L. Each molecule to be tested, as well as the control avelumab, were added to the plate in serial dilutions. The individual IC 50 Values ​​are expressed as the IC of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC 50 :(I C 50アベルマブ / I C 50試験scFv The potencies are summarized in Table 19.

[0289] [Table 32]

[0290] 5.6. Species cross-reactivity (binding to cynomolgus monkey and mouse PDL1 by SPR) Cross-reactivity to cynomolgus monkey PDL1 was measured in a similar assay to that used for human PDL1, using recombinant PDL1 from Shino Biological Co., Ltd. Table 20 summarizes the affinities obtained for all tested scFvs. All tested scFvs that showed binding to human PDL1 also showed binding to cynomolgus monkey PDL1.

[0291] [Table 33]

[0292] 5.7. PDL1 vs. PDL2 selectivity by SPR The binding of humanized scFv to PDL2 was tested by SPR analysis using a T200 instrument (Biacore, GE Healthcare). In this experiment, Fc-tagged human PDL2 was captured using the Human Antibody Capture kit from GE Healthcare. After each sample injection cycle, the CM5 sensor chip was regenerated and new antigen was captured. The scFv was injected as a sample diluted to a concentration of 180 nM in running buffer for 5 minutes, and protein separation was allowed to proceed for 12 minutes. All humanized scFvs tested, listed in Table 21, were not observed to bind to PDL2.

[0293] [Table 34]

[0294] Example 6: Biophysical characterization of humanized scFv: Selected domains with better affinity than avelumab were produced on a larger scale (0.2L to 1.2L expression volume). After purification, the protein samples were concentrated to 10mg / mL or more using a centrifugal concentration tube with a molecular weight cutoff of 5kD. The preparation of materials for stability evaluation is summarized in Table 22.

[0295] 6.1. Storage stability test: The humanized scFv were subjected to stability studies (e.g., 4-week stability studies) in which the scFv was formulated at 10 mg / ml in aqueous buffer (final buffer composition 50 mM NaCiP, 150 mM NaCl, pH 6.4) and stored at -80°C or below, 4°C and 40°C for 4 weeks. At a minimum, monomeric and oligomeric fractions in the formulation were assessed by integration of SE-HPLC peak areas after 1 week, 2 weeks and at the end of each study. Other time points were recorded for some molecules. Table 23 compares measurements obtained at the 7th day and 28th day endpoints of the study.

[0296] [Table 35]

[0297] [Table 36]

[0298] 6.2. Freeze-thaw stability test: In addition to the storage stability tests described above, the suitability of the scFv molecules with good performance was evaluated by freeze-thaw (F / T) cycling (colloidal stability). For F / T stability evaluation, the same analytical methods (SE-HPLC, SDS-PAGE) and parameters (% monomer content and % monomer loss) as those of the storage stability test were applied to monitor the quality of the molecules over 5 F / T cycles. Table 24 illustrates the course of % monomer content loss over 5 repeated F / T cycles. Since no specific freeze-thaw test was performed, the freeze-thaw data obtained for the −80° C. samples used in the storage stability test over 28 days are shown in the graph below. After repeated F / T cycles, no molecules showed a monomer content loss of more than 4%.

[0299] [Table 37]

[0300] 6.3.Heat Denaturation: Thermal denaturation data for selected scFv constructs was obtained from DSF measurements and is shown in Table 25. The resulting Tm values ​​were determined by fitting the data to the Boltzmann equation. Table 25 summarizes the melting temperatures calculated by DSF.

[0301] [Table 38]

[0302] Multispecific molecules comprising antibodies of the invention: Exemplary multispecific molecules comprising antibodies of the invention are shown in Table 3.

[0303] Example 7: Affinity for PDL1, CD137, HSA and MSA: method: The affinity of different species to PDL1 was measured by SPR measurements using a Biacore T200 instrument (GE Healthcare). Antibodies specific for the Fc region of human IgG were immobilized on a sensor chip (CM5 sensor chip, GE Healthcare) by amine coupling. In all formats, except the Morrison format that contains Fc, PDL1-Fc chimeric proteins from different species were captured by the immobilized antibodies. Three-fold serial dilutions (0.12-90 nM) of PDL1-specific molecules were injected into the flow cell for 3 min and separation was monitored for 10 min. After each injection cycle, the surface was regenerated with one injection of 3 M MgCl2 solution. The apparent separation (k d ) and meeting (k a The kinetic constants, as well as the apparent dissociation equilibrium constants (KD), were calculated using a one-to-one Langmuir binding model. The affinity of different species to CD137 was measured using the same setup as for PDL1, except that the CD137-Fc chimeric proteins from different species were captured by immobilized antibodies.

[0304] Fc-containing formats were directly captured by an antibody specific for the Fc region of human IgG. Two-fold serial dilutions ranging from 90 to 0.35 nM of PDL1 extracellular domain or CD137 extracellular domain were used to test for binding to IgG captured on the biosensor chip. After each injection cycle, the surface was regenerated with one injection of 3 M MgCl2 solution.

[0305] The affinity of the molecules for different species of serum albumin (SA) was measured by SPR measurements using a Biacore T200 instrument (GE Healthcare). SA was directly coupled to a CM5 sensor chip (GE Healthcare) using amine coupling chemistry. After regeneration counting and surface performance testing to determine the best assay conditions, dose-response measurements were performed and the resulting binding curves were double-referenced (empty reference channel and no sample injection) and fitted using a 1:1 Langmuir model to obtain kinetic parameters. The assay was performed in 1x PBS-Tween buffer at pH 5.5.

[0306] result: Measurement of the binding kinetics of the humanized constructs shows differences in affinity for PDL1 when comparing the CDRs of clone 33-03-G02 with the structural grafts (STRs), with the STR grafts showing a 20-fold increase in affinity compared to the CDR grafts of the same clone (PRO885 vs. PRO1126 in Table 26). The CDR grafts from clone 37-20-B03 (PRO997) show approximately 2-fold higher affinity when compared to the STR grafts of clone 33-03-G02. The binding affinity of the CDR grafts of 33-03-G02 is comparable to the binding capacity of the parental scFvs when they are combined in different multispecific formats (PRO830 compared to PRO885, PRO951, PRO1123, PRO1124, PRO963, PRO966, PRO1057, PRO1058, PRO1059 and PRO1060, Table 26). The scFv from both clones show almost identical affinity to human and cynomolgus PDL1 (see PRO977 and PRO830 in Table 26).

[0307] [Table 39]

[0308] [Table 40]

[0309] Example 8: Blocking PDL1 / PD-1 interaction in a cell-based reporter gene assay using CHO cells expressing PDL1 and TCR activator molecules, and Jurkat cells expressing PD-1 and containing a luciferase gene under an NFAT response element: method: Engineered Jurkat T cells stably expressing a NFAT (nuclear factor of activated T cells)-luciferase reporter and human PD-1 in a bioluminescent reporter gene assay serve as effector T cells. Cells stably expressing human PDL1 and T cell receptor (TCR) activators serve as antigen-presenting cells. Co-culturing the two cell lines induces activation of the Jurkat NFAT pathway via binding of the TCR activator / TCR complex. In response to binding of PDL1-expressing cells, PD-1 signaling in PD-1 effector T cells inhibits T cell function and the NFAT pathway is inhibited. Blocking the interaction of PD-1 and PDL1 receptors leads to reactivation of the NFAT pathway.

[0310] 35,000 CHO / PDL1 / TCR activator (BPS Bioscience) cells in 100 μl of cell culture medium (DMEM / F12, 10% FCS) were added to the inner wells of a white cell 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 each molecule to be tested was added in a two-fold dilution series from 3,000 to 0.46 ng / ml, along with the control avelumab. Effector Jurkat cells expressing PD-1 (BPS Bioscience) were then diluted at 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS) and 50 μl was added to each well and the plate was incubated at 37°C and 5% CO2 for 6 hours. Finally, luciferase substrate (BPS Bioscience), prepared according to the manufacturer's protocol, was added at 50 μL per well, the plates were incubated in the dark for 30 min and luminescence was measured using a Topcount.

[0311] result: To evaluate the effect of CDR set and framework selection on potency to neutralize PDL1 binding to PD-1, three anti-PDL1 scFvs were tested in an NFAT reporter gene cell-based assay. PRO830 contains the CDR set of clone 33-03-G02 grafted onto a VH4 framework, while 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 scFvs tested, with an IC of 42.88 ng / ml. 50 value of 34.09ng / ml 50 The potency of the same CDR set was approximately 2-fold higher when grafted onto the VH4 framework instead of the VH1 framework. 50 The values ​​were 11.12 ng / ml vs. 21.29 ng / ml (Figure 4A and Table 27).

[0312] The potency of neutralizing PDL1 binding to PD-1 was measured using bispecific molecules with the 33-03-G02 PDL1 domain before (CDR grafting) and after (structure grafting) domain optimization. CDR grafting (PRO885) was compared to structure grafting (PRO1126). Domain optimization was compared to IC 50 The neutralization titers were increased three-fold, with values ​​of 137.2 ng / ml for PRO885 and 48.15 ng / ml for PRO1126 (Figure 4B and Table 27).

[0313] Two trispecific molecules with a CDR-grafted anti-PDL1 domain of clone 33-03-G02 and two different human serum albumin binding domains were also evaluated for their potency in neutralizing the PDL1 / PD-1 interaction, particularly for their half-life extension. The HSA domain of PRO1057 also binds mouse serum albumin. Experiments were performed in the presence of 25 mg / ml HSA. Neutralization potency (IC 50 = 665.1 ng / ml) was lower than that of avelumab (Figure 5 and Table 27).

[0314] [Table 41]

[0315] In serum, the so-called Morrison format was tested in a cell-based titer reporter gene assay. In this format, one specificity is generated by the IgG part (bivalent) and two scFvs with specificity for a second target are linked to the heavy (HC) or light (LC) chain of the IgG via flexible peptide linkers. All Morrison molecules tested carried the CDR-grafted anti-PDL1 domain of clone 33-03-G02 on both arms of the IgG. The two constructs PRO1059 and PRO1060 differ in that two anti-CD137 scFvs are fused to either the heavy (HC) or light (LC) chain. PRO1062 has the same structure as PRO1060 but with a different CD137 domain. The neutralization titers of all molecules were comparable (Figure 6 and Table 27).

[0316] Example 9: Blocking PDL1 interaction with PD-1 and B7-1 using competitive ELISA These assays were performed to evaluate the ability of PDL1 inhibitors to block the interaction between PDL1 and PD-1 or between PDL1 and B.71. Different formats, including scFv, scDb, scDb-scFv and Morrison, were analyzed in a competitive ELISA and compared to the control IgG avelumab.

[0317] PDL1 / PD-1 Competitive ELISA: ELISA microplates were coated with 4 μg / ml human PD-1 overnight at 4°C and washed three times with 450 μl of wash buffer per well. To each well, 300 μl of PBS containing 1% BSA and 0.2% Tween (dilution buffer) was added, and the plates were blocked for 1 h at room temperature. Three-fold serial dilutions of inhibitors were prepared in dilution buffer containing 1 ng / ml of biotinylated human PDL1 to final concentrations ranging from 300 to 0.005 ng / ml. The mixtures were preincubated for 1 h at room temperature with gentle shaking on a rotary mixer (21 rpm) and added to the microplate after three washing cycles with 450 μl of wash buffer per well. The plates were incubated for 1.5 h at room temperature with gentle shaking and washed three times with 450 μl of wash buffer per well, after which 10 ng / ml of streptavidin-polyHRP40 was added to each microplate well. After 1 h incubation at room temperature, the plate was washed three times with 450 μl of wash buffer and TMB was added. After 6 min, the enzyme reaction was stopped by adding 1 M HCl and the absorbance was measured at 450 nm with 690 nm as the reference wavelength. IC 50 To calculate values, control subtracted values ​​were used to perform a 4-parameter logistic (4PL) curve fit in Graph Pad Prism.

[0318] As illustrated in Figure 7 and Table 28, all PDL1 inhibitors blocked the interaction of PDL1 with PD-1 when tested in a competitive ELISA. scFv PRO830 blocked the interaction with comparable potency, while PRO997 and PRO1013 had IC values ​​significantly lower than avelumab. 50 All molecules maintained their inhibitory properties when combined with a multispecific format (i.e., scDbs or Morrison). PRO885 was less potent than avelumab, whereas PRO1126, which contains an improved anti-PDL1 domain, had a lower IC 50 The Morrison format was slightly less potent when compared to avelumab. We also demonstrated the neutralizing effect of PRO1057 in the presence of human serum albumin, but with IC 50 The value was about twice as high.

[0319] PDL1 / B7-1 competitive ELISA: ELISA microplates were coated overnight at 4°C with 4 μg / ml human B7-1 and washed three times with 450 μl of wash buffer per well. To each well, 300 μl of PBS containing 1% BSA and 0.2% Tween (dilution buffer) was added and the plates were blocked for 1 h at room temperature. Three-fold serial dilutions of inhibitors were prepared in dilution buffer containing 40 ng / ml of biotinylated PDL1 to final concentrations ranging from 900 to 0.015 ng / ml. The mixtures were preincubated for 1 h at room temperature with gentle shaking on a rotary mixer (21 rpm) and added to the microplate after three washing cycles with 450 μl of wash buffer per well. The plates were incubated for 1.5 h at room temperature with gentle shaking and washed three times with 450 μl of wash buffer per well, after which 10 ng / ml of streptavidin-polyHRP40 was added to each microplate well. After 1 h incubation at room temperature, the plate was washed three times with 450 μl of wash buffer and TMB was added. After 6 min, the enzyme reaction was stopped by adding 1 M HCl and the absorbance was measured at 450 nm with 690 nm as the reference wavelength. IC 50To calculate values, control subtracted values ​​were used to perform a 4-parameter logistic (4PL) curve fit in Graph Pad Prism.

[0320] All PDL1 inhibitors, except PRO1126, were tested for their ability to block the interaction of B7-1 with PD-1. PRO830 showed comparable potency to avelumab, whereas PRO997 and PRO1013 showed lower IC 50 All scDbs and Morrison also inhibited the interaction between PDL1 and B.7-1. The scDb PRO885 showed similar potency to avelumab, but the IC of Morrison was lower. 50 The values ​​were approximately 2 to 3.4 times lower. The data are shown in Figure 8 and Table 28.

[0321] [Table 42]

[0322] Example 10: Evaluation of the stimulatory effect of PDL1 blockade and simultaneous CD137 stimulation in human PBMCs stimulated with the superantigen SEA using a cell-based assay: In this experiment, the synergistic effect of PD-1 / PDL1 blockade 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 CD137 on T cells, respectively. By applying anti-PDL1×CD137 molecules, two T cell regulatory signaling pathways were simultaneously targeted, i.e., inhibition of the inhibitory PD-1 / PDL1 pathway and activation of the CD137 pathway via immunological synapse formation mediated by the bispecific anti-PDL1×CD137 molecule (PRO885). T cell activation through the secretion of interleukin-2 (IL-2) was evaluated and compared to the effect mediated by PDL1 blockade mediated by the benchmarking control antibody avelumab. In addition, the anti-PDL1 scFv, PRO997, was tested and compared with avelumab in the same experimental setup.

[0323] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human whole blood by density gradient centrifugation. NK cells were then depleted from the PBMCs using anti-CD56 antibody and the MACS cell separation kit (Miltenyi Biotec). 100,000 PBMCs were then added per well to a 96-well plate, followed by the addition of serial dilutions of PRO885, PRO997 and avelumab in assay buffer containing SEA at a concentration of 10 ng / ml. After 96 hours of incubation at 37°C and 5% CO2, cell supernatants were harvested and human interleukin-2 (IL-2) levels in the culture supernatants were quantified using the BioLegend IL-2 human ELISA MAX assay according to the kit instructions. IL-2 concentrations were interpolated and back-calculated from the IL-2 standard curve, and EC 50 To calculate the values, they were plotted against the concentrations of avelumab and PRO885.

[0324] As shown in Figure 9, IL-2 was secreted by T cells following stimulation of CD137 by blocking PD-1 / PDL1 interaction and simultaneous addition of the bispecific molecule PRO885. When compared to avelumab, PRO885 demonstrated higher T cell activation and better potency (PRO885:EC 50 =39.92ng / ml, avelumab:EC 50 = 69.89 ng / ml, Table 29). This finding demonstrates that the bispecific anti-PDL1 x CD137 scDb PRO885 is able to induce T cell stimulation more potently than the simple blockade of PDL1 by avelumab. Furthermore, the high affinity anti-PDL1 scFv PRO997 was found to be more potent than avelumab in stimulating T cells (PRO997:EC 50 = 40.86ng / ml, avelumab:EC 50 =90.18ng / ml, Table 29).

[0325] [Table 43]

[0326] Example 11: Evaluation of anti-tumor efficacy of anti-PDL1 antibodies in a human cell line-derived lung cancer xenograft model HCC827: The anti-tumor activity of anti-PDL1 IgG1 antibody PRO1137 (SEQ ID NO: 90 and 91) was evaluated in human HCC827 NSCLC xenografts using Taconic's immunodeficient NOG mouse line and allogeneic human peripheral blood mononuclear cells. The transplanted human T lymphocytes show xenoreactivity against foreign major histocompatibility (MHC) class I and II and other antigens derived from the mouse cells. As a result, the T lymphocytes show inflammatory infiltration of various organs, leading to the death of the animals after several weeks, and this process is known as xenograft-versus-host disease (xGVHD). Treatment with immunomodulatory antibodies (e.g., anti-PDL1 and anti-CD137) has been shown to result in exacerbation of xGVHD (Sanmamed MF et al. Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2- / -IL2Rgnull immunodeficient mice. Cancer Res 2015;75(17):3466-3478).

[0327] Study set-up and treatment schedule: Female NOG mice were dosed with 5 × 10 6 HCC827 cells were injected into one limb. Cells were injected in 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 tumor engraftment (80–100 mm 3 After successfully growing 5×10 median tumor volume, mice were intravenously injected with 5×10 6 On the day of randomization, four mice in each group were reconstituted with PBMCs from donor A and an additional four mice were reconstituted with PBMCs from donor B. Treatments began 1–2 h after PBMC injection and were applied as follows:

[0328] [Table 44]

[0329] Based on the in vitro activity of the antibody blocking PD-1 / PDL1 interaction in the NF-AT reporter gene assay, the dose of PRO1137 was set at 0.2 mg to obtain the same relative activity as the dose of 0.1 mg avelumab (per mouse). Thus, a dose of 0.2 mg of PRO1137 is comparable to a dose of 0.1 mg avelumab and can be expressed as 1 relative unit (1r.U). Body weight and caliper measurements of tumor volume were performed twice weekly. Animals were sacrificed at predefined time points depending on the study results. All animals were sacrificed at the "same" time points (days 17 and 18). For throughput reasons, the first half of each group was sampled and processed on day 1, and the remaining second half of each group was sampled and processed the same the next day. Animals reconstituted with PBMCs from two different donors adequately represented the two sampling cohorts.

[0330] Results: Anti-PDL1 PRO1137 anti-tumor activity in human HCC827 NSCLC xenografts using immunodeficient NOG mouse line and allogeneic human peripheral blood mononuclear cells (hPBMC) was evaluated by measuring tumor volume (Figure 10). Tumor volume was measured twice a week, and mice were euthanized on days 17 and 18. Tumor volume was normalized as the relative tumor volume to the tumor volume at the start of treatment. As shown in Figure 10, 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 body weight (median value), which suggests that the molecule is well tolerated at the dose levels tested (Figure 11).

[0331] Example 12: Evaluation of the antitumor effect of human PRO1137 in NOG mice engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC): The anti-tumor activity of human PRO1196 (anti-PDL1 IgG1, SEQ ID NOs: 92 and 93) was compared to vehicle treatment or avelumab in human HCC827 NSCLC xenografts using NOG mice engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC).

[0332] Study set-up and treatment schedule: Female NOG mice engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC) were injected subcutaneously with HCC827 NSCLC cells. Mice were injected with 5×10 6 HCC827 cells were injected into the NOG mice. The cells were injected as a mixture of 50% cell suspension in PBS and 50% Matrigel in a total injection volume of 100 μl. After injection of tumor cells into NOG mice and successful tumor engraftment (80–100 mm per group), the tumor cells were cultured in 100% PBS and 50% Matrigel. 3 Median tumor volume (n=10) and mice (n=10) were randomized into treatment groups.

[0333] [Table 45]

[0334] Body weight and caliper tumor volume measurements were performed twice weekly. Tumors were harvested on days 25, 29 and 30 after treatment.

[0335] Results: The antitumor activity of PRO1196 (anti-PDL1 IgG1, SEQ ID NOs: 92 and 93) in human HCC827 NSCLC xenografts using immunodeficient NOG mouse strains engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC) was evaluated by measuring tumor volume (Figure 12). Tumor volumes were measured twice weekly and mice were euthanized on days 25, 29 and 30. Tumor volumes were normalized as relative tumor volume to tumor volume at the start of treatment. As shown in Figure 12, treatment with PRO1196 and avelumab resulted in mitigation of tumor growth compared to the control group.

[0336] Example 13: Evaluation of the antitumor efficacy of PDL1 blockade and combination with local stimulation of CD137 in a tumor-bearing MC38 colon cancer model: In addition, the antitumor activity of multispecific antibodies comprising the PDL1 domain of the invention may be tested in the MC38 colon cancer model in tumor-bearing C57BL / 6 mice with intact immune systems. This model has also been used by others to show enhanced antitumor activity upon combined treatment with a CD137 agonist and a PD-1 / PDL1 antagonist (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).

[0337] Since neither the anti-CD137 nor the anti-PDL1 domains of the tested multispecific antibodies cross-react with mouse PDL1 and mouse CD137, an engineered human CD137 knock-in model established by CrownBio may be used. In this model, the extracellular and transmembrane domains of mouse CD137 were replaced with the respective sequences of human CD137 in a C57BL / 6 mouse background using the CRISPR / Cas9 system. In addition, a modified MC38 tumor cell line expressing human PDL1 instead of mouse PDL1 under the control of the CMV promoter may be used. The effect of the multispecific antibody on tumor volume may be compared with combination therapy with a humanized IgG1 containing the same PDL1-specific variable domain as the multispecific antibody, and with a humanized IgG4 with the same CD137-specific variable domain. To obtain further evidence of a local anti-tumor immune response, the number of tumor-infiltrating lymphocytes (e.g. CD8+, CD4+ and regulatory T cells) may be analyzed by flow cytometry. To examine the immune system modulation following anti-CD137 / anti-PDL1 treatment systemically, the number of CD4+ and CD8+ T cells in the liver and spleen may be analyzed by flow cytometry and possibly immunohistochemistry. In addition, the systemic IFNγ level may be analyzed using a quantitative ELISA method. To further characterize the safety profile of anti-CD137 / anti-PDL1 combination therapy, clinical chemistry pathology parameters mainly related to liver toxicity (observed in clinical anti-CD137 therapy), such as high levels of alanine aminotransferase, glutathione dehydrogenase and aspartate aminotransferase, may be evaluated.

Claims

1. An isolated antibody having binding specificity to human PDL1, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively; LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively; Including, The antibody, (i) a heavy chain variable region comprising an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 58, or (ii) An antibody comprising a heavy chain variable region having an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 47, and a light chain variable region having an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO:

57. [0023] The antibody according to claim 2, comprising a heavy chain variable region comprising at least 2S, 25A, 44V, 56A, 82K, 89V, and 105F (AHo numbering), and a light chain variable region comprising at least 2F, 4L, and 51P (AHo numbering); The antibody of claim 1, wherein the antibody described in (ii) comprises a heavy chain variable region comprising at least 25A, 44V, 56A, 82K, and 89V (AHo numbering). (i) a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 58; or (ii) the VH sequence of SEQ ID NO: 47 and the VL sequence of SEQ ID NO: 57; The antibody of claim 2 .

4. The antibody, (i) binds to human PDL1 with a dissociation constant (KD) of less than 100 pM as measured by surface plasmon resonance (SPR); (ii) 10 to human PDL1 as measured by SPR －３ s －１ The following K ｏｆｆ and combine with (iii) 10 to human PDL1 as measured by SPR ５ M －１ s －１ Above K ｏｎ and combine with (iv) has cross-reactivity with Cynomolgus PDL1 and binds to Cynomolgus PDL1 with a KD of less than 100 pM as measured by SPR; and (v) has no cross-reactivity with mouse PDL1 as measured by SPR. (vi) does not bind to human PDL2 as measured by SPR; (vii) has the ability to neutralize the PDL1 / PD-1 interaction with a potency (relative potency) of greater than 2 compared to avelumab as measured by ELISA, wherein the relative potency is greater than or equal to the IC in ng / mL of avelumab as measured by ELISA. ５０ value and the IC in ng / mL of the antibody measured by ELISA. ５０ a ratio of the antibody to a value, (viii) has the ability to neutralize the PDL1 / PD-1 interaction with a potency (relative potency) of greater than 2 compared to avelumab as measured by an NFAT reporter gene assay, wherein the relative potency is greater than or equal to the IC in ng / mL of avelumab as measured in an NFAT reporter gene assay. ５０ values ​​and IC in ng / mL of the antibodies as measured in the NFAT reporter gene assay. ５０ a ratio of the antibody to a value, wherein the antibody is an scFv, and / or (ix) has the ability to neutralize the PDL1 / B7-1 interaction with a potency (relative potency) of greater than 2 compared to avelumab as measured by ELISA, wherein the relative potency is greater than or equal to the IC in ng / mL of avelumab as measured by ELISA. ５０ value and the IC in ng / mL of the antibody measured by ELISA. ５０ value, wherein the antibody is an scFv. An antibody described in any one of claims 1 to 3.

5. The antibody, (i) when in scFv format, the antibody has a melting temperature (Tm) of 65° C. or greater when formulated in 50 mM phosphate-citrate buffer, pH 6.4, 150 mM NaCl, as measured by differential scanning fluorimetry; (ii) when in scFv format, the antibody exhibits less than 5% loss of monomer content after being subjected to five successive freeze-thaw cycles at a starting concentration of 10 mg / ml when formulated in 50 mM phosphate citrate buffer, pH 6.4, 150 mM NaCl; and / or (iii) when in scFv format, the antibody exhibits less than 15% loss of monomer content after storage for 2 weeks or more at 4° C. at a starting concentration of 10 mg / ml when formulated in 50 mM phosphate citrate buffer, pH 6.4, 150 mM NaCl; An antibody described in any one of claims 1 to 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, an Fv, a scFv, a dsFv, and an scAb.

7. The antibody of claim 6, which is an scFv, and the scFv has an amino acid sequence selected from the group consisting of SEQ ID NO:61 and SEQ ID NO:

62.

8. The antibody of claim 1 , wherein the antibody is a multispecific molecule, the multispecific molecule having one or more second functional molecules.

9. 9. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

10. An antibody according to any one of claims 1 to 8 for use as a medicament.

11. 10. An antibody according to any one of claims 1 to 8 for use in the treatment of cancer.

12. A nucleic acid encoding an antibody according to any one of claims 1 to 8.

13. A method for producing an antibody according to any one of claims 1 to 8, comprising culturing a host cell containing the nucleic acid of claim 12.

Images