Antibodies targeting PDL1 and methods of use thereof

Abstract

To provide an isolated antibody which specifically binds to human PDL1, and pharmaceutical compositions and methods of use thereof.SOLUTION: The present invention provides an isolated antibody having binding specificity for human PDL1, the antibody including HCDR1, HCDR2, and HCDR3 sequences respectively having specific sequences, and LCDR1, LCDR2, and LCDR3 sequences respectively having different specific sequences, the isolated antibody specifically binding to human PDL1, and pharmaceutical compositions and methods of use thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an isolated antibody that specifically binds to human PDL1, and its pharmaceutical composition and method of use. The present invention further relates to a nucleic acid encoding the antibody, a vector containing the nucleic acid, a host cell containing the nucleic acid or the vector, and a method for producing the antibody.

Background Art

[0002] PDL1 (CD274, B7-H1) is a 40 kDa type I transmembrane protein. PDL1 is a membrane surface glycoprotein ligand for PD-1, a major immune checkpoint receptor expressed by activated T cells and B cells, and mediates immunosuppression. PDL1 is involved in the suppression of immune system responses among chronic infections, pregnancy, tissue allotransplantation, 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, chest, digestive tract, colorectal, liver, pancreas, kidney, adrenal cortex, bladder, urothelium, ovary, and skin, etc. (Non-Patent Documents 1 to 7). PDL1 is hardly expressed in normal tissues and 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). PD-1 activated by PDL1 potentially provides an immune-tolerant environment for tumorigenesis and growth. PDL1 also negatively regulates T cell function through interaction with another receptor B7.1 (B7-1, CD80).

[0003] Inhibition of the PDL1 / PD-1 interaction results in 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 belong to 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 / AstraZeneca), BMS-936559 (Bristol-Myers Squibb), MSB0010718C (EMD Serono, Rockland, MA)) (see Non-Patent Document 11 for a review). The resulting biological effects appear to be different between targeting PDL1 and targeting PD-1. 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, and the effect of this interaction is not understood. However, PDL1 antibodies also interfere with the interaction of PDL1 not only with PD-1 but also with B7-1 (Non-Patent Document 12), which is thought to result in negative signaling to T cells. Promising initial data have been shown by blocking PDL1, and currently four anti-PDL1 mAbs, namely atezolizumab and MEDI4736 (both human IgG1 Fc null mutants), MSB001078C (IgG1), and BMS-936559 (IgG4), are being subjected to clinical trials (Non-Patent Document 13).

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

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide an antibody that specifically binds to human PDL1 protein and has beneficial properties for therapeutic use, such as high affinity and improved efficacy, and improved biophysical properties, such as solubility, developability, and stability.

Means for Solving the Problems

[0008] In one aspect, the present invention relates to a novel PDL1 antibody.

[0009] In one aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

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

[0011] In one aspect, the present invention relates to the antibody of the present invention or the composition of the present invention for use in the treatment of a subject in need of treatment for cancer.

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

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

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

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

[0016] The aspects, advantageous features and preferred embodiments of the present invention summarized in the following items further contribute to the solution of the problems of the present invention, either alone or in combination:

[0017] 1. An isolated antibody having binding specificity for human PDL1, comprising: (a) A heavy chain variable region CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, preferably SEQ ID NO: 1 or 32, more preferably SEQ ID NO: 1, preferably consisting of or consisting essentially of, and (b) A heavy-chain variable region CDR2 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NO: 2, 6, 9, 12, 33, 37, 40, and 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2, and (c) A heavy-chain variable region CDR3 comprising, preferably consisting of, an amino acid sequence selected from any 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, and (d) A light-chain variable region CDR1 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NO: 17, 20, 23, 48, 51, and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17, and (e) A light-chain variable region CDR2 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NO: 18, 21, 24, 49, 52, and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18, and (f) A light-chain variable region CDR3 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NO: 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 NO: 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 17, 18, and 19, respectively, and (b) The HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 20, 21, and 22, respectively, and (c) The HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 5, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 20, 21, and 22, respectively, and (d) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 8, 9, and 10, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, and (e) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 11, 12, and 13, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 23, 24, and 25, and (f) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 32, 33, and 34, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 48, 49, and 50, and (g) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 35, 37, and 38, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 51, 52, and 53, and (h) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 36, 37, and 38, and respectively, the LLCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 51, 52, and 53, and (i) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 39, 40, and 41, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 48, 49, and 50, and (j) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 42, 43, and 44, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 54, 55, and 56.

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

[0020] 4. An 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, and (b) An HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 6, and (c) An HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 7, and (d) An LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20, and (e) An LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 21, and (f) An LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 22.

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

[0022] 6. An 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, and (b) An HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 37, and (c) An HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 38, and (d) An LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 51, and (e) An LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 52, and (f) An LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 53.

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

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

[0025] 9. The antibody is a heavy chain variable region comprising an amino acid sequence that is 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, and a light chain variable region comprising an amino acid sequence that is 90% or more identical to 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. An antibody against any one of the above items, including

[0026] 10. The antibody is (a) A heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 14 and a light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 26, (b) A heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 15 and a light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 26, (c) A heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 16 and a light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 27, (d) A heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 45 and a light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 57, (f) A heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 46 and a light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 58, or (g) A heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 47 and a light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 57, An antibody against any one of the above items, including

[0027] 11. The antibody is Any one 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 heavy chain variable region containing the amino acid sequence, and Any one 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, a light chain variable region containing the amino acid sequence, and An antibody against any one of the above items, including

[0028] 12. The antibody is (a) VH sequence of SEQ ID NO: 14 and VL sequence of SEQ ID NO: 26, (b) VH sequence of SEQ ID NO: 15 and VL sequence of SEQ ID NO: 26, (c) VH sequence of SEQ ID NO: 16 and VL sequence of SEQ ID NO: 27, (d) VH sequence of SEQ ID NO: 45 and VL sequence of SEQ ID NO: 57, (f) VH sequence of SEQ ID NO: 46 and VL sequence of SEQ ID NO: 58, or (g) VH sequence of SEQ ID NO: 47 and VL sequence of SEQ ID NO: 57 An antibody of any one of the above items.

[0029] 13. The antibody is (i) When measured by surface plasmon resonance (SPR), 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, and in particular, the antibody is scFv (monovalent affinity), (ii) When measured by SPR, binds to human PDL1 with a K of 10 -3 s -1 or less, 10 -4 s -1 or less, or 10 -5 s -1 or less of K off and in particular, the antibody is scFv, (iii) When measured by SPR, binds to human PDL1 with a K of 10 3 M -1 s -1 or more, 10 4 M -1 s -1 or more, 10 5 M -1 s -1 or more, or 10 6 M -1 s -1 or more of K on and in particular, the antibody is scFv, (iv) having cross-reactivity with cynomolgus PDL1, and in particular, when measured by SPR, binding with a KD of less than 5 nM, particularly less than 1 nM, particularly less than 500 pM, particularly less than 100 pM, preferably less than 10 pM to cynomolgus PDL1, and in particular, the antibody being a scFv, (v) in particular, when measured by SPR, having no cross-reactivity with mouse PDL1, and / or (vi) in particular, when measured by SPR, not binding to human PDL2, an antibody of any of the above items.

[0030] 14. The antibody has the following characteristics: (i) When measured by ELISA, having the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) of more than 1.5, for example more than 2, more than 2.5, preferably more than 3, more preferably more than 4, compared to avelumab, The relative titer is the ratio of the ng / mL IC 50 value of the antibody measured by ELISA to the ng / mL IC 50 value of avelumab measured by ELISA, and in particular, the antibody being a scFv, and / or, (ii) Optionally, when measured by the NFAT reporter gene assay, having the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) of more than 1.5, for example more than 2, more than 2.5, preferably more than 3, more preferably more than 4, compared to avelumab, The relative titer is the ratio of the ng / mL IC 50 value of the antibody measured in the NFAT reporter gene assay to the ng / mL IC 50 value of avelumab measured in the NFAT reporter gene assay, and in particular, the antibody being a scFv, and / or, (iii) When measured by ELISA, it has the ability to neutralize the PDL1 / B7-1 interaction with a titer (relative titer) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The relative titer is the ng / mL IC 50 value of avelumab measured by ELISA, divided by the ng / mL IC 50 value of the antibody measured by ELISA, In particular, when the antibody is a scFv, The antibody of any one of the above items.

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

[0032] 16. The antibody according to any one of the above items, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, Fab, Fv, scFv, dsFv, scAb, and ankyrin-based domains, fynomer, avimer, anticalin, a binding domain based on a scaffold substitute limited to fibronectin, and a binding site incorporated into the constant region of an antibody (e.g., F-star’s Modular Antibody Technology (trademark)).

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

[0034] 18. The antibody of item 17, wherein the scFv is 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, and more preferably has the amino acid sequence of 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, and preferably, the antibody is IgG1.

[0036] 20. The antibody according to any one of the above items, wherein the antibody is chimeric or humanized.

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

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

[0039] 23. The antibody is selected from the group consisting of single-chain diabody (scDb), tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), bispecific T cell engaging (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-terminal group of the light chain), Bs2Ab (scFv linked to the N-terminal group of the heavy chain), Bs3Ab (scFv linked to the C-terminus of the heavy chain), Ts1Ab (scFv linked to the N-terminal groups of the heavy and light chains), Ts2Ab (dsscFv linked to the C-terminus of the heavy chain), and other IgG-scFv fusions, bispecific antibodies based on heterodimeric Fc domains such as Knob-into-Hole antibodies (KiHs), 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 termini of either chain of one heterodimeric Fc domain or the other heterodimeric domain, the antibody of item 22 in a format selected from the group.

[0040] 24. A pharmaceutical composition comprising any one of the antibodies of items 1 to 23 and a pharmaceutically acceptable carrier.

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

[0042] 26. An antibody of any one of Items 1 to 23 or the composition of Item 24 for use in such treatment in a subject in need of cancer treatment.

[0043] 27. Use of an antibody of any one of Items 1 to 23 or the composition of Item 24 in the treatment of a subject in need of cancer treatment.

[0044] 28. Use of an antibody of any one of Items 1 to 23 or the composition of Item 24 for the manufacture of a medicament for the treatment of a subject in need of cancer treatment.

[0045] 29. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of any one of Items 1 to 23 or the composition of Item 24.

[0046] 30. A nucleic acid encoding an 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 an antibody of any one of Items 1 to 23, the method comprising culturing a host cell comprising the nucleic acid of Item 31 or the vector of Item 31.

[0050] 34. A kit comprising an antibody of any one of Items 1 to 23 or the composition of Item 24.

Brief Description of the Drawings

[0051]

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Mode for Carrying Out the Invention

[0052] The present invention provides an antibody that specifically binds to human PDL1 protein, as well as a pharmaceutical composition, a manufacturing method, and a method of using such an antibody and composition.

[0053] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art related to the present invention.

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

[0055] The terms "a", "an", "the" and similar terms in the context of describing the present invention are to be construed to include the singular and plural forms (especially in the context of the claims hereinafter) unless specified herein or clearly contradicted by the context. For example, the term "cell" includes a plurality of cells and mixtures thereof. When the plural form is used for compounds, salts, etc., this may also mean 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, terms such as "antibody" include the following: All antibodies or their single chains, as well as any antigen-binding fragments thereof (i.e., "antigen-binding portions") or their single chains, and molecules containing antibody CDRs, VH regions or VL regions (including, but not limited to, bispecific antibodies). Naturally occurring "all antibodies" are glycoproteins containing two or more heavy (H) chains and two or more light (L) chains that are linked to each other 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 intervening more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies are thought to mediate binding to host tissues or factors of immunoglobulins, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0058] As used herein, terms such as "antigen-binding fragment", "its antigen-binding fragment", "antigen-binding portion", etc. refer to one or more fragments of a complete whole antibody that retain the ability to specifically bind to a given antigen (e.g., PDL1). Examples of binding fragments included in the term "antigen-binding portion" of an antibody include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains, F(ab)2 fragments, divalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of VL and VH domains of a single arm of an antibody, and binding domains based on scaffold alternatives limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectin, and binding sites incorporated into the constant region of an antibody (e.g., F-star’s Modular Antibody Technology (trademark)).

[0059] The term "complementary determining region" ("CDR") refers to the amino acid sequences of certain regions determined using any of a number of well-known methods described in, for example, 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), the ImMunoGenTics (IMGT) numbering system (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999), Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering system), and Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering). For example, in the classical Kabat 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 89-97 (LCDR3). In Chothia, the CDR amino acid residues of VH are numbered as 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of VL are numbered as 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Combining the CDR definitions of Kabat and Chothia, the CDR consists of the amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH, and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of human VL.In IMGT, the CDR amino acid residues of VH are numbered approximately 26 - 35 (HCDR1), 51 - 57 (HCDR2), and 93 - 102 (HCDR3), and the CDR amino acid residues of VL are numbered approximately 27 - 32 (LCDR1), 50 - 52 (LCDR2), and 89 - 97 (LCDR3) (numbering by "Kabat"). In IMGT, the CDRs of an antibody can be determined using the program IMGT / DomainGap Align. In the context of the present invention, unless otherwise specified, the numbering system proposed by Honegger & Plückthun ("AHo") (Honegger & Plückthun, J. Mol. Biol. 309 (2001) 657 - 670) is used.

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

[0061] For the sake of clarity, the numbering system by Honegger & Plückthun is convenient for explaining the slight variations in length, as well as the gaps in the sequence, which are found in different VH and VL subfamilies, particularly in the CDRs, in naturally occurring antibodies. Thus, in a given antibody variable domain, not all positions from 1 to 149 are usually occupied by amino acid residues.

[0062] The antigen-binding portion may be incorporated into a maxibody, minibody, intrabody, diabody, triabody, tetrabody, scDb-scFv, v-NAR, and bis-scFv (see Holliger and Hudson, 2005, Nature Biotechnology, 23, 1126-36). For example, the antigen-binding portion of an antibody may be fused to a scaffold based on a polypeptide such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes a monobody of a fibronectin polypeptide). An antigen-binding portion may be incorporated into a single-chain molecule containing 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. Patent No. 5,641,870).

[0063] As used herein, the term "binding specificity" means the ability of an individual antibody to react with one antigenic determinant and not with a different antigenic determinant. As used herein, the terms "specifically bind" or "specific" refer to a significant and reproducible interaction, e.g., binding between a target and an antibody, which 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) binds to that target more readily and / or more strongly than to other targets, with a greater affinity and binding activity. "Specific binding" in its broadest sense (and when not otherwise defined) refers to the ability of an antibody to distinguish a target molecule from molecules unrelated to the target, as determined, for example, according to known art specificity assay methods. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) assays, and peptide scanning. For example, a standard ELISA assay can be performed. Scoring can be performed by a standard colorimetric reaction (e.g., reaction of a secondary antibody with horseradish peroxidase and tetramethylbenzidine using 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 an OD of about 0.1, and a typical positive reaction can be an OD of about 1. This means that the ratio between the 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 of 10-fold or more, preferably 100-fold or more, between the background and the signal. Typically, determination of binding specificity is performed by using about 3 to 5 unrelated molecules (e.g., skim milk, transferrin, etc.) in combination, rather than using a single control molecule. The antibodies of the present invention have binding specificity for human PDL1. In certain embodiments, the antibodies of the present invention have binding specificity for human PDL1, but do not bind to human PDL2, particularly as measured by SPR.

[0064] Preferably, the antibody of the present invention is an isolated antibody. As used herein, the term "isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to PDL1 substantially does not contain an antibody that specifically binds to an antigen 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 cynomolgus monkey (also known as cynomolgus macaque or "cynomolgus") PDL1. Further, the isolated antibody may substantially not contain materials and / or chemicals derived from other cells.

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

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

[0067] The term "chimeric antibody" refers to (a) an antibody in which the constant region or a portion thereof is 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 a completely different molecule (such as an enzyme, toxin, hormone, growth factor, drug, etc.) to confer new properties, or (b) an antibody in which the variable region or a portion thereof is modified, substituted, or exchanged with a variable region having a different or modified antigen specificity. It is any of the antibody molecules. For example, a mouse antibody can be modified by replacing its constant region with the constant region of a human immunoglobulin. By replacement with the human constant region, a chimeric antibody can have reduced antigenicity in humans compared to the original mouse antibody while maintaining its specificity in recognizing an antigen.

[0068] As used herein, a "humanized" antibody is an antibody that has low immunogenicity in humans while maintaining the reactivity of an antibody from a non-human source. This can be obtained, for example, by maintaining the non-human CDR regions and replacing the remaining portions of the antibody (i.e., the constant region and the framework portions of the variable regions) with their human counterparts. Further modifications of the framework regions may be made within the human framework sequences or within the CDR sequences derived from the germline of other mammalian species. The humanized antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., by random or site-specific mutagenesis in vitro, or by somatic mutations in vivo, or by conservative substitutions that promote stability or high production). See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984, Morrison and Oi, Adv. Immunol., 44:65-92, 1988, Verhoeyen et al., Science, 239:1534-1536, 1988, Padlan, Molec. Immunol., 28:489-498, 1991, and Padlan, Molec. Immunol., 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] As used herein, the term "recombinant humanized antibody" encompasses all human antibodies prepared, expressed, constructed, or isolated by recombinant means, such as antibodies isolated from host cells transformed to express a humanized antibody, for example, from a transfectome, as well as any other means including splicing of all or part of a human immunoglobulin gene (sequence relative to the base sequence of other DNA) that are prepared, expressed, constructed, or isolated.

[0070] The term "PDL1" specifically refers to human PDL1 having UniProt ID number Q9NZQ7 and is represented herein as SEQ ID NO: 63. Appropriately, the antibodies of the present invention target PDL1, particularly human PDL1 as shown by UniProt ID number Q9NZQ7 and represented as SEQ ID NO: 63 herein. Appropriately, the antibodies of the present invention target human and cynomolgus monkey (Macaca fascicularis) PDL1, preferably do not cross-react with mouse PDL1, particularly when measured by surface plasmon resonance (SPR). Appropriately, the antibodies of the present invention have binding specificity for human PDL1. In particular, the antibodies of the present invention do not bind to human PDL2 when measured by SPR.

[0071] The antibodies of the present invention are PDL1 inhibitors. The terms "blocker" or "blocking antibody" or "inhibitor" or "inhibiting antibody" or "antagonist" or "antagonist antibody" mean an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The antibodies of the present invention target, reduce, and inhibit the binding ability of PDL1 to its binding partner, thereby interfering with the function of PDL1. In particular, the antibodies of the present invention block the interaction of PDL1 with PD-1. In some embodiments, the antibodies of the present invention block the interaction of PDL1 with PD-1 and B7-1.

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

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

[0074] The present invention provides an antibody that specifically binds to the PDL1 protein, and the antibody comprises a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 1. In particular, the present invention provides an antibody that specifically binds to the PDL1 protein, and the antibody has a VH CDR having an amino acid sequence of any one, two, three, or more of the VH CDRs listed in Table 1.

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

[0076] Other antibodies of the present invention include those containing mutated amino acids, which specifically bind to the PDL1 protein and have a CDR region 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 with the CDR region represented by the sequences listed in Table 1. In one aspect, other antibodies of the present invention include mutant amino acid sequences that specifically bind to the PDL1 protein and have a CDR region in which only 1, 2, 3, 4, or 5 amino acids are mutated as compared to the CDR region represented by the sequences listed in Table 1.

[0077] The terms "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences refer to the fact that the two or more sequences or subsequences are the same. "Percent (%) sequence identity" and "homology" with respect to a nucleic acid, peptide, polypeptide or antibody sequence are defined as the percentage of nucleotide or amino acid residues in a candidate sequence that have identity with the nucleotide or amino acid residues of a particular nucleic acid, peptide or polypeptide sequence, where an alignment of the sequences is made and gaps are introduced as necessary to achieve the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. Alignments for the purpose of determining amino acid sequence identity % can be made in a variety of ways, which are within the skill of the art in the prior art, for example using commonly available computer software (such as BLAST, BLAST-2 or ALIGN software). One of ordinary skill in the art can determine appropriate parameters for alignment calculations, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences to be compared.

[0078] For array comparison, typically one array is used as the control array against which the test array is to be compared. When using an array comparison algorithm, the test and control arrays are input into a computer, sub-array coordinates are assigned as necessary, and parameters for the array algorithm program are assigned. It is also possible to use default program parameters or to assign alternative parameters. The array comparison algorithm then calculates the percentage of array identity of the test array in comparison to the control array based on the program parameters.

[0079] Two examples of algorithms suitable for determining the percentage of array identity and array similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The percentage of 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 percentage of 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) and uses a Blossom 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0080] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and mimetics that function in a manner similar to natural amino acids. Natural amino acids refer to those encoded by the genetic code, as well as amino acids that are modified post - factually, such as hydroxyproline, γ - carboxyglutamic acid, and O - phosphoserine. The terms "polypeptide" and "protein" are used interchangeably herein when referring to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics that mimic the corresponding natural amino acids, as well as to natural and non - natural 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 to human PDL1, and the antibody (a) a heavy - chain variable - region CDR1 (HCDR1) comprising, preferably consisting of, more preferably consisting of the 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, more preferably consisting of the 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; (c) a heavy - chain variable - region CDR3 (HCDR3) comprising, preferably consisting of, more preferably consisting of the 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; (d) a light - chain variable - region CDR1 (LCDR1) comprising, preferably consisting of, more preferably consisting of the 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 one of SEQ ID NOs: 18, 21, 24, 49, 52 and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18, and (f) A light chain variable region CDR3 (LCDR3) comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 19, 22, 25, 50, 53 and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19, and comprising.

[0082] Suitably, the isolated antibody of the invention having binding specificity to human PDL1 (a) A heavy chain variable region CDR1 (HCDR1) comprising, 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 with 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, and (b) A heavy chain variable region CDR2 (HCDR2) comprising, 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 with 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, 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 with 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, and (d) 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, and 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 with it, preferably consisting of it, a light chain variable region CDR1 (LCDR1), (e) 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 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 with it, preferably consisting of it, a light chain variable region CDR2 (LCDR2), (f) 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, and 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 with it, preferably consisting of it, a light chain variable region CDR3 (LCDR3), comprising.

[0083] In one embodiment, the antibody of the present invention having binding specificity to human PDL1 (a) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, (b) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 4, 6, and 7 and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 20, 21, and 22, (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, and (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, and (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, and (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, and (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, and (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, and (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, and (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, and comprising.

[0084] In one embodiment, the antibodies of the invention having binding specificity to human PDL1 each comprise HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, respectively. In other embodiments, the antibodies of the invention having binding specificity to human PDL1 each comprise 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.

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

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

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

[0088] In a further embodiment, the antibody of the present invention having binding specificity to human PDL1 is (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, preferably consisting of the same, (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, preferably consisting of the same, (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, preferably consisting of the same, (d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, preferably consisting of the same, (e) LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, preferably consisting of the same, (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 22, preferably consisting of the same, comprises. Suitably, the antibody of the present invention having binding specificity to human PDL1, (a) 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, preferably consisting of, and HCDR1, (b) 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, preferably consisting of, and HCDR2, (c) 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, preferably consisting of, and HCDR3, (d) 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, preferably consisting of, and LCDR1, (e) 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, preferably consisting of, and LCDR2, (f) 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, preferably consisting of, and LCDR3, comprises.

[0089] Suitably, the antibody of the present invention having binding specificity to human PDL1, (a) An HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 32, and (b) An HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 33, and (c) An HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 34, and (d) An LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 48, and (e) An LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 49, and (f) An LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 50, and including. Appropriately, the antibody of the present invention having binding specificity to human PDL1 is (a) An HCDR1 comprising 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, preferably consisting of the same, and (b) An HCDR2 comprising 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, preferably consisting of the same, and (c) An HCDR3 comprising 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, preferably consisting of the same, and (d) An LCDR1 comprising 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, preferably consisting of the same, and (e) 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, preferably consisting of the same, and LCDR2, (f) an amino acid sequence containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 50, and LCDR3, comprising.

[0090] In a further embodiment, the antibody of the invention having binding specificity to human PDL1 is (a) HCDR1 containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, (b) HCDR2 containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 37, (c) HCDR3 containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 38, (d) LCDR1 containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 51, (e) LCDR2 containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 52, (f) LCDR3 containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 53, comprising. Suitably, the antibody of the invention having binding specificity to human PDL1 is (a) HCDR1 containing 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, preferably consisting of the same, (b) HCDR2 containing 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, preferably consisting of the same, (c) 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, preferably consisting of the HCDR3, (d) 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, preferably consisting of the LCDR1, (e) 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, preferably consisting of the LCDR2, (f) an amino acid sequence comprising, preferably consisting of, the LCDR3 of SEQ ID NO: 53, and comprising.

[0091] In a further embodiment, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), said 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 families of antibody heavy and light chain sequences that are classified according to sequence identity and homology. Methods for determining sequence homology using homology search matrices such as BLOSUM (Henikoff, S. & Henikoff, J.G., Proc. Natl. Acad. Sci. USA 89 (1992) 10915-10919), and methods 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 as shown, for example, in Knappik et al., J. Mol. Biol. 296 (2000) 57-86, according to which VH is classified into VH1A, VH1B and VH2 to VH6, Vκ is classified into Vκ1 to Vκ4, and Vλ is classified into Vλ1 to Vλ3. In vivo, the Vκ chain, Vλ chain and VH chain of an antibody are each the result of random rearrangement of the V and J segments of the germline κ chain, the V and J segments of the germline λ chain, and the V, D and J segments of the heavy chain, respectively. To which subfamily a given antibody variable chain belongs is determined by the corresponding V segment, particularly 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, such as an HFR4 sequence derived from one of the J segments of the heavy chain germline, or an HFR4 sequence derived from a rearranged VH sequence.

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

[0093] Specific examples of VH belonging to the VH1 family are 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 (the regions marked other than in bold in Table 1). Suitably, the VH belonging to the VH1 family used herein is a VH comprising FR1 to FR4 having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more with FR1 to FR4 of SEQ ID NO: 15.

[0094] Specific examples of VH belonging to the VH3 family are 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 (the regions marked other than in bold in Table 1). Optimally, the VH belonging to the VH3 family used herein is a VH comprising FR1 to FR4 having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more with FR1 to FR4 of SEQ ID NO: 16.

[0095] Specific examples of VH belonging to the VH4 family are 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 (the regions marked other than in bold in Table 1). Optimally, the VH belonging to the VH4 family used herein is a VH comprising FR1 to FR4 having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more with FR1 to FR4 of SEQ ID NO: 14.

[0096] Examples of alternatives to the VH sequence 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 a Vκ framework FR1, FR2, and FR3, particularly a Vκ1 or Vκ3 framework, preferably Vκ1 framework FR1 to 3, and a framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4, and Vλ FR4. Suitable Vκ1 framework FR1 to 3 is set forth in SEQ ID NO: 26 (in Table 1, the FR regions are marked other than in bold). Suitable Vκ1 framework FR1 to 3 corresponds to FR1 to 3 and comprises an amino acid sequence 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 (in Table 1, the FR regions are marked other than in 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 FR4 is represented by SEQ ID NO: 64 to SEQ ID NO: 70. In a preferred embodiment, Vλ FR4 is represented by SEQ ID NO: 64 or 65, and more preferably, 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), said antibody comprising a Vλ FR4 comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more identity with the amino acid sequence selected from any of SEQ ID NO: 64 to SEQ ID NO: 70, preferably SEQ ID NO: 64 or 65, more preferably SEQ ID NO: 64.

[0101] Thus, in one embodiment, the present 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 NO: 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 20, 21, and 22, respectively, b. respectively, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 35, 37 and 38, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 51, 52 and 53, respectively, or c. respectively, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36, 37 and 38, and the 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, and a Vλ FR4 having an amino acid sequence selected from Vκ FR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more identity with any of SEQ ID NOs: 64 to 70, preferably a Vλ FR4 represented by any of SEQ ID NOs: 64 to 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64, and a framework FR4 selected from a VL domain comprising a VL framework comprising.

[0102] In other embodiments, the present invention provides an antibody having binding specificity to human PDL1, which comprises: (i) respectively, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 5, 6 and 7, 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, and VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly an amino acid sequence selected from any of SEQ ID NO: 64 to SEQ ID NO: 70 and having 60% or more, 70% or more, 80% or more, 90% or more identity, preferably a Vλ FR4 containing an amino acid sequence of any of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64, a framework FR4 selected from, A VL domain comprising a VL framework containing

[0103] In a specific embodiment, the present invention provides an antibody comprising: (i) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 32, 33 and 34 respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 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, and VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly an amino acid sequence selected from any of SEQ ID NO: 64 to SEQ ID NO: 70 and having 60% or more, 70% or more, 80% or more, 90% or more identity, preferably a Vλ FR4 represented by any of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64, a framework FR4 selected from, A VL domain comprising a VL framework containing

[0104] In a preferred specific embodiment, the present invention provides an antibody comprising: (i) HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1, 2 and 3 respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 17, 18 and 19 respectively, (ii) A VH3 or VH4 domain framework sequence, preferably a VH3 domain framework sequence, and (iii) The Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1, and Vκ FR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly a Vλ FR4 having an amino acid sequence selected from any of SEQ ID NO: 64 to SEQ ID NO: 70 and having an identity of 60% or more, 70% or more, 80% or more, 90% or more with the amino acid sequence, preferably a Vλ FR4 represented by any of SEQ ID NO: 64 to SEQ ID NO: 70, more preferably a Vλ FR4 represented by SEQ ID NO: 64, and a framework FR4 selected from A VL domain comprising a VL framework comprising

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

[0106] The present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), and the antibody comprises a VH domain listed in Table 1.

[0107] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises the VH amino acid sequence listed in Table 1, and the mutations in the framework sequence (e.g., the sequence that is not a CDR), as various non-limiting examples of mutations, such as addition, substitution, or deletion, are about 10 amino acids or less.

[0108] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises the VH amino acid sequence listed in Table 1, and the mutations in the framework sequence (e.g., the sequence that is not a CDR), as various non-limiting examples of mutations, such as addition, substitution, or deletion, 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 with the VH region represented by the sequence described in Table 1.

[0110] The present invention provides an isolated antibody that specifically binds to the PDL1 protein, wherein the antibody comprises the VL domain listed in Table 1.

[0111] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises the VL amino acid sequence listed in Table 1, and the mutations in the framework sequence (e.g., the sequence that is not a CDR), as various non-limiting examples of mutations, such as addition, substitution, or deletion, are about 10 amino acids or less.

[0112] The present invention also provides an isolated antibody that specifically binds to PDL1, wherein the antibody comprises the VL amino acid sequence listed in Table 1, and the mutations in the framework sequence (e.g., the sequence that is not a CDR), as various non-limiting examples of mutations, such as addition, substitution, or deletion, are about 20 amino acids or less.

[0113] Other antibodies of the present invention include mutated amino acids, but specifically bind to PDL1 and have a VL region represented by the sequence described in Table 1 and 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 with the VL region.

[0114] The present invention also provides an isolated antibody that specifically binds to PDL1, and the antibody has 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, and 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 amino acid sequence, and 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, and 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 amino acid sequence. and comprises

[0115] In one embodiment, the antibody of the present invention has binding specificity to human PDL1, and has a heavy chain variable region comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46, and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16, and a light chain variable region comprising any one of the amino acid sequences 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. and comprises

[0116] In one embodiment, the antibody of the present invention having binding specificity to human PDL1 includes the following: (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 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, (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 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, (c) The HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 20, 21, and 22, respectively, A VH sequence comprising an amino acid sequence that is 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: 16, preferably 90% or more identical, and preferably comprising the G56A and Y105F mutations (AHo numbering), said VH, 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: 27, preferably 90% or more identical, and preferably comprising the S9A and A51P mutations (AHo numbering), said VL, (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 identical to SEQ ID NO: 45, preferably 90% or more identical, 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, preferably 90% or more identical, (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 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: 46, preferably 90% or more identical, and preferably comprising the V2S, V25A, I44V, G56A, V82K, F89V, and Y105F mutations (AHo numbering), said VH, 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: 58, preferably 90% or more identical, and preferably comprising the I2F, M4L, and A51P mutations (AHo numbering), said VL, or (f) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 35, 37, and 38, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 51, 52, and 53, 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 identical to SEQ ID NO: 47, preferably 90% or more identical, and preferably comprising the V25A and I44V mutations (AHo numbering), said VH, 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 having binding specificity for human PDL1 comprises the following: (a) respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, and respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, 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 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, (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 identical to SEQ ID NO: 15, preferably 90% or more identical, 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: 26, preferably 90% or more identical, (c) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, respectively, a VH sequence comprising an amino acid sequence that is 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: 16, preferably 90% or more identical, and preferably comprising the G56A and Y105F mutations (AHo numbering), said VH, 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: 27, preferably 90% or more identical, and preferably comprising the S9A and A51P mutations (AHo numbering), said VL, (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 identical to SEQ ID NO: 45, preferably 90% or more identical, 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, preferably 90% or more identical. (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 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: 46, preferably 90% or more identical, preferably comprising the V2S, V25A, I44V, G56A, V82K, F89V, and Y105F 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: 58, preferably 90% or more identical, preferably comprising the 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, 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 present invention having binding specificity to human PDL1 comprises the following: 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: 16, preferably comprising the G56A and Y105F mutations (AHo numbering), said VH, 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: 27, preferably comprising the S9A and A51P mutations (AHo numbering), said VL.

[0119] In a further embodiment, the isolated antibody of the present invention having binding specificity to human PDL1 comprises the following: (a) The VH sequence of SEQ ID NO: 14 and the 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) The VH sequence of SEQ ID NO: 45 and the 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, the isolated antibody of the present invention having binding specificity to 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, the isolated antibody of the present invention having 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 the antibody described in Table 1. In one embodiment, the antibody that specifically binds to PDL1 comprises 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 and 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, preferably 90% or more identity. 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 present invention having binding specificity to human PDL1 include those in which the amino acids or the nucleic acids encoding the amino acids are mutated, but have 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more identity with the sequences described in Table 1. In one embodiment, it comprises a mutant amino acid sequence in which only 1, 2, 3, 4, or 5 amino acids are mutated in the variable region when compared with the sequence of the variable region described in Table 1, but substantially the same activity is maintained. As used herein, the term "substantially the same activity" refers to the activity measured in the parental antibody, for example, compared with the antibody of the present invention, particularly the antibody of the present invention described in Table 1, and substantially 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 100% or more, or 110% or more, or 120% or more, or 130% or more, or 140% or more, or 150% or more, or 160% or more, or 170% or more, or 180% or more, or 190% or more, for example, activity shown by activity equivalent to at most 200%.

[0122] These antibodies can each bind to PDL1, and as long as their antigen-binding specificities are provided mainly by the regions of CDR1, 2, and 3, the sequences of VH CDR1, 2, and 3, as well as the sequences of VL CDR1, 2, and 3, can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched), provided that each antibody must contain VH CDR1, 2, and 3, as well as VL CDR1, 2, and 3, and must also form other PDL1-binding molecules of the present invention. Such "mixed and matched" PDL1-binding antibodies can be tested using well-known binding experiments, 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 derived from a particular VH sequence should be replaced with structurally similar CDR sequences (including plural). Similarly, when VL CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequences derived from a particular VL sequence should be replaced with structurally similar CDR sequences (including plural). As will be apparent to those skilled in the art, novel VH and VL sequences can be created by mutating one or more VH and / or VL CDR region sequences to have structurally similar sequences to the CDR sequences shown in this specification for the monoclonal antibodies of the present invention.

[0123] In another embodiment, the present invention provides an antibody comprising an amino acid sequence having homology with the sequences described in Table 1, wherein the antibody binds to PDL1 and maintains the desired functional characteristics of those antibodies described in Table 1.

[0124] For example, the present invention provides an isolated monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is 80% or more, 90% or more, or 95% or more identical to the 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, The light 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 selected from the group consisting of SEQ ID NO: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27. The antibody specifically binds to human PDL1 as a 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 the sequences listed 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, the antibody of the present 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 specific amino acid sequence based on the antibodies described herein or conservative modifications thereof, and the antibody maintains the desired functional properties of the PDL1-binding antibody of the present invention.

[0127] The terms "conservatively modified variant" or "conservative variant" apply to amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a conservatively modified variant refers to one that encodes the same or essentially the same amino acid sequence, or, in the case of a nucleic acid that does not encode an amino acid sequence, refers to an essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid mutations are "silent mutations" and are one type of conservatively modified mutation. Every nucleic acid sequence herein that encodes a polypeptide describes every possible silent mutation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, the usual codon for methionine, and TGG, the usual codon for tryptophan) can be modified for the production of a functionally identical molecule. Thus, each silent mutation of a nucleic acid encoding a polypeptide is implicitly present in each described sequence.

[0128] "Conservatively modified variant" or "conservative variant" with respect to a polypeptide sequence includes individual substitutions, deletions, or additions to the polypeptide sequence that result in amino acid substitutions by chemically similar amino acids. Tables of conservative substitutions that result in functionally equivalent amino acids are known. Such conservatively modified variants may additionally be polymorphic variants, interspecies homologs, and alleles of the invention and are not excluded therefrom. 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 modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence.

[0129] Accordingly, the present invention provides an isolated monoclonal antibody comprising or consisting of a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, The heavy chain variable region CDR1 comprises 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, preferably consisting of it, The heavy chain variable region CDR2 comprises 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, preferably consisting of it, The heavy chain variable region CDR3 comprises 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, preferably consisting of it, The light chain variable region CDR1 comprises 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, preferably consisting of it, The light chain variable region CDR2 comprises 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, preferably consisting of the same, The light chain variable region CDR3 comprises 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, preferably consisting of the same, The antibody can specifically bind to PDL1 and block the PD-1 / PDL1 interaction.

[0130] In one embodiment, the antibody of the present invention has a heavy chain variable region and a light chain variable region optimized for expression in mammalian cells, and one or more of these sequences have a specific amino acid sequence based on the antibody described herein or its conservative modification, and the antibody retains the desired functional properties of the PDL1-binding antibody of the present invention. Accordingly, the present invention provides an isolated monoclonal antibody optimized for expression in mammalian cells, comprising a heavy chain variable region and a light chain variable region, 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 its conservative modified sequence, 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 its conservative modified sequence, The antibody can specifically bind to PDL1 and block the PD-1 / PDL1 interaction.

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

[0132] As used herein, the term "optimized" means that the nucleotide sequence has been modified to encode an amino acid sequence using codons preferred in the production cell or organism (generally, for example, cells of the eukaryotic genus Pichia, Chinese hamster ovary cells (CHO) or human cells). The optimized nucleotide sequence is engineered to retain, completely or to the extent possible, the amino acid sequence encoded by the original starting nucleotide sequence known as the "parent" sequence. As used herein, the optimized sequences have been engineered to have codons preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated in the present invention. The amino acid sequence encoded by the 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 within the CDR1, CDR2, and / or CDR3 regions of VH and / or VL, known as "affinity maturation". The introduction of the mutation(s) can be carried out by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding, or other desired functional properties, can be evaluated in in vitro or in vivo assays as described herein and exemplified in the examples. Conservative modifications (such as those described above) can also be introduced. The mutation can be an amino acid substitution, addition or deletion. Further, typically, only one, two, three, four or five residues within the CDR region are modified.

[0134] An "affinity matured" antibody 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 as compared to the parent antibody that does not have such modifications. In one embodiment, an affinity matured antibody may have nanomolar or picomolar affinity for a target antigen. Affinity matured antibodies are made by well-known procedures. For example, Marks et al, Bio / Technology 10:779-783 (1992) describes affinity maturation by shuffling of VH- and VL-domains. Random mutagenesis of hypervariable regions ("HVRs") 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 invention provides an isolated monoclonal antibody comprising VH3 containing the G56A and Y105F mutations, particularly the amino acid sequence represented by SEQ ID NO: 16, and preferably VL containing S9A, containing the A51P mutation, particularly the amino acid sequence represented by SEQ ID NO: 27.

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

[0137] The antibodies of the present invention can further be prepared by using, as starting materials, antibodies having one or more VH and / or VL sequences shown herein in the preparation of modified antibodies, which may have modified properties compared to the starting antibodies. The processing of the antibodies can be carried out by modifying one or both of the variable regions (i.e., VH and / or VL), for example, one or more residues within one or more CDR regions and / or one or more framework regions. Further, or alternatively, the antibodies can be processed by modifying residues within the constant region, for example, to modify the effector function of the antibody.

[0138] One of the possible variable region processes is CDR grafting. Antibodies interact with the target antigen mainly through the amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). Therefore, the amino acid sequences within the CDRs have more diversity among 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 that fuses the CDR sequences from a particular naturally occurring antibody to the framework sequences from different antibodies 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., U.S.A. 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 a framework array can be obtained from a public DNA database or a known document that describes the gene sequences of germline antibodies or rearranged antibody sequences. For example, the germline DNA sequences of the genes of the human heavy and light chain variable regions are present in the "VBase" human germline sequence database (available on the Internet as www.mrc-cpe.cam.ac.uk / vbase), and Kabat, E.A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Tomlinson, I.M., et al., 1992 J. fol. Biol. 227:776-798, and Cox, J.P.L. et al., 1994 Eur. J Immunol. 24:827-836, the disclosures of which are incorporated herein by reference. For example, the germline DNA sequences for the genes of the human heavy and light chain variable regions and rearranged antibody sequences are present in the "IMGT" database (available on the Internet as www.imgt.org, Lefranc, M.P. 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 that are structurally similar to the framework sequences used by the selected antibodies of the present invention, such as consensus sequences and / or framework sequences used by the monoclonal antibodies of the present invention. The VH CDR1, 2, and 3 sequences, as well as the VL CDR1, 2, and 3 sequences, can also be grafted into framework regions having the same sequences as those present in the germline immunoglobulin genes from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions that contain one or more mutations as compared to the germline sequences. For example, as a specific example, it is known that it is useful to mutate residues in the framework region in order to maintain or enhance the antigen-binding ability of the 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 as long as the resulting polypeptide contains one or more binding regions that specifically bind to PDL1. Such frameworks or scaffolds include those containing human immunoglobulins, those containing the five main idiotypes of their antigen-binding fragments, and those containing immunoglobulins of other animal species, preferably in their humanized forms.

[0142] In one aspect, the present invention relates to a method for producing a non-immunoglobulin-based antibody using a non-immunoglobulin scaffold onto which a CDR of the present invention can be grafted. As long as it contains a specific binding region for the target PDL1 protein, known or future-developed non-immunoglobulin frameworks and scaffolds can be used. Examples of 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 present invention specifically bind to PDL1 and are characterized by one or more of the following parameters: (i) When measured particularly by surface plasmon resonance (SPR), it 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 the antibody is a scFv, (ii) When measured by SPR, it binds to human PDL1 with a K of 10 -3 s -1 or less, 10 -4 s -1 or less, or 10 -5 s -1 or less of K, particularly when the antibody is a scFv, off wherein the antibody binds to human PDL1 with a K of 10 (iii) When measured by SPR, it binds to human PDL1 with a K of 10 3 M -1 s -1 or more, 10 4 M-1 s -1 Above, 10 5 M -1 s -1 Above, or 10 6 M -1 s -1 Above of K on Linked by, in particular, when the antibody is scFv, (iv) Having cross-reactivity with cynomolgus PDL1, in particular, when measured by surface plasmon resonance, binding with a KD of less than 5 nM, particularly less than 1 nM, particularly less than 500 pM, particularly less than 100 pM, preferably less than 10 pM to cynomolgus PDL1, in particular, when the antibody is scFv, in particular, when measured by surface plasmon resonance, having no cross-reactivity with mouse PDL1, and / or (v) In particular, when measured by SPR, not binding to human PDL2.

[0144] As used herein, the term "affinity" means the strength of the interaction between an antibody and an antigen at a single antigenic site. Within the scope of each antigenic site, the variable regions of the antibody "arms" interact with the antigen by weak non-covalent forces at multiple sites, and the more interactions there are, the stronger the affinity.

[0145] "Binding affinity" generally refers to the strength of the non-covalent interaction between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, "binding affinity", "binds to", "binds with", or "binding to" as used in the present specification refers to the specific binding affinity reflecting the 1:1 interaction between the components of the binding pair (such as an antibody fragment and an antigen). The affinity of molecule X for its partner Y is usually the dissociation constant (K D) can be represented by. Affinity can be measured by general methods of the prior art, including the methods described in the present specification. Antibodies with low affinity usually tend to have slow binding to antigens and rapid dissociation, while antibodies with high affinity usually tend to have rapid binding to antigens and maintain the binding for a long time. Various methods for measuring binding affinity are known, and any of them can be used for solving the problems of the present invention. Specific exemplary embodiments for measuring binding affinity (i.e., the strength of binding) are described below.

[0146] As used herein, the term "K" assoc ", "Ka" or "K" on " shall refer to the association rate of a specific antibody-antigen interaction, while the term "K" dis ", "Kd" or "K" off " shall refer to the dissociation rate of a specific antibody-antigen interaction. In one embodiment, as used herein, the term "KD" shall refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as molar concentration (M). The "KD" or "KD value" or "K" D " or "K" DThe "value" is measured using a surface plasmon resonance assay with a MASS-1 SPR instrument (Sierra Sensors) in one embodiment. When measuring affinity, an antibody specific for the Fc region is immobilized on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using standard amine-coupling procedures with rabbit IgG (Bethyl Laboratories, catalog number A120-111A). Rabbit monoclonal antibodies in the B cell supernatant are captured by the immobilized anti-rabbit IgG antibody. The minimum IgG concentration in the B cell supernatant is necessary to effect sufficient capture. After capturing the monoclonal antibody, human PDL1 (Peprotech) is injected into the flow cell at a concentration of 90 nM for 3 minutes to effect separation of the protein from the IgG captured on the sensor chip for 5 minutes. After each injection cycle, the surface is regenerated by injecting 10 mM glycine-HCl twice. The apparent dissociation (kd) and association (ka) constants, and the apparent dissociation equilibrium constant (KD) are calculated using a 1:1 Langmuir binding model with MASS-1 analysis software (Analyzer, Sierra Sensors), and Chi 2 's relative value (Chi 2 normalized by the maximum binding level of the extrapolated analyte) is used to monitor the quality of the fit and is used as an indicator for maintaining the quality of curve fitting. When the value of Chi 2 is small, it means that the accuracy of the fit to the 1:1 Langmuir binding model is high. If the reaction units (RU) in ligand binding are 2% or more of the RU of antibody capture, the results are judged to be valid. Samples showing an RU of ligand binding less than 2% of the RU of antibody capture are considered to indicate no specific binding of PDL1 to the captured antibody. The equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on . See, for example, Chen et al, J. Mol. Biol. 293:865-881 (1999).

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

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

[0149] Suitably, the antibodies of the invention, when measured by surface plasmon resonance (SPR), are specific for human PDL1 at 10 3 M -1 s -1 or more, 10 4 M -1 s -1 or more, 5 × 10 4 M -1 s -1 or more, 10 5 M -1 s -1 or more, 5 × 10 5 M -1 s -1 or more, 10 6 M-1 s -1 Above, 5 × 10 6 M -1 s -1 Above, 10 7 M -1 s -1 Above, 5 × 10 7 M -1 s -1 Above K on Combine at the speed of. Preferably, the antibody of the present invention, when measured by SPR, has a K of 10 5 M -1 s -1 Above, especially 10 6 M -1 s -1 Above K on Have a speed of.

[0150] Suitably, the antibody of the present invention, when measured by surface plasmon resonance (SPR), binds to human PDL1 and is characterized by one or more of the following parameters: -3 s -1 Below, 3 × 10 -3 s -1 Below, 5 × 10 -3 s -1 Below, 10 -4 s -1 Below, 5 × 10 -4 s -1 Below, 10 -5 s -1 Below, 5 × 10 -5 s -1 Below, 10 -6 s -1 Below or 10 -7 s -1 Below K off Combine at the speed of. Preferably, the antibody of the present invention, when measured by SPR, has a K of 10 -3 s -1 Below, 10 -4 s -1 Below, especially 10 -5 s -1 Below K off Have a speed of.

[0151] Suitably, the antibody of the present invention specifically binds to PDL1 and is characterized by one or more of the following: (i) When measured by ELISA, it has the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The relative titer is the ratio of the ng / mL IC 50 value of the antibody measured by ELISA to the ng / mL IC 50 value of avelumab measured by ELISA. In particular, the antibody is an scFv, and (ii) Optionally, when measured by the NFAT reporter gene assay, it has the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The relative titer is the ratio of the ng / mL IC 50 value of the antibody measured in the NFAT reporter gene assay to the ng / mL IC 50 value of avelumab measured in the NFAT reporter gene assay. In particular, the antibody is an scFv, and (iii) When measured by ELISA, it has the ability to neutralize the PDL1 / B7.1 interaction with a titer (relative titer) greater than 1.5, such as greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The relative titer is the ratio of the ng / mL IC 50 value of the antibody measured by ELISA to the ng / mL IC 50 value of avelumab measured by ELISA. In particular, the antibody is an scFv.

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

[0153] Suitably, when the antibody of the present invention is in the scFv format, particularly when the antibody is prepared in 50 mM phosphate-citrate buffer with pH 6.4 and 150 mM NaCl, it has 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. DSF has already been 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 the scFv construct is measured by differential scanning fluorimetry using the fluorescent dye SYPRO® Orange (see Wong & Raleigh, Protein Science 25(2016)1834-1840). Samples in 50 mM phosphate-citrate buffer at pH 6.4 are prepared at a final protein concentration of 50 μg / mL and a final concentration of 5×SYPRO® Orange in a total volume of 100 μl. The prepared 25 μl samples are added three times to a white-wall AB gene PCR plate. The assay is performed in a qPCR instrument used as a thermal cycler, and fluorescence emission is detected using a custom dye calibration routine of the software. The PCR plate containing the test samples is heated from 25 °C to 96 °C at 1 °C increments and stopped for 30 seconds after each temperature increase. The total assay time is about 2 hours. Tm is calculated by the software GraphPad Prism using a mathematical second derivative, and the inflection point of the curve is calculated. The output Tm is the average of three measurements.

[0154] Suitably, when the antibody of the present invention is in the scFv format, after being subjected to 5 consecutive freeze-thaw cycles at an initial concentration of 10 mg / ml of the antibody of the present invention, particularly when the antibody is prepared in 50 mM phosphate-citrate buffer with pH 6.4 and 150 mM NaCl, it shows a loss of monomer content of less than 5%, preferably less than 3%, more preferably less than 1%.

[0155] Preferably, when the antibody of the present invention is in the scFv format, after storage at an initial concentration of 10 mg / ml of the antibody of the present invention at 4 °C for 2 weeks or more, particularly 4 weeks or more, especially when the antibody of the present invention is prepared in a 50 mM phosphate citrate buffer of pH 6.4 and 150 mM NaCl, it shows a loss of monomer content of less than 15%, for example less than 12%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1%.

[0156] The loss of monomer content is measured by calculating the area under the curve of the SEC-HPLC chromatogram. SEC-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, which is outlined in Chapter 621 of the USP. This method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. The separation of molecules occurs between the void volume (V0) and the total permeation volume (VT) of a specific column. The measurement by SEC-HPLC is performed in a Chromaster HPLC system (Hitachi High-Technologies Corporation) equipped with an autosampler injector and an ultraviolet detector set at a detection wavelength of 280 nm. The apparatus is controlled by software EZChrom Elite (Agilent Technologies, Version 3.3.2 SP2), and the analysis of the chromatogram obtained thereby is supported. The protein sample is clarified by centrifugation and maintained at a temperature of 4 - 6 °C in the autosampler before injection. When analyzing the scFv sample, a column Shodex KW403-4F (Showa Denko K.K., #F6989202) is used, and at that time, a standardized buffered saline mobile phase (50 mM sodium phosphate, pH 6.5, 300 mM sodium chloride) is used at a recommended flow rate of 0.35 mL / min. The target sample addition amount per injection was 5 μg. The sample is detected by an ultraviolet detector at a wavelength of 280 nm, and the data is recorded by an appropriate software set. The resulting chromatogram is analyzed in the range from V0 to VT, and at that time, matrix-related peaks with an elution time longer than 10 minutes are excluded.

[0157] As used herein, the term "recognize" refers to an antibody discovering its structural epitope and interacting (e.g., binding) therewith.

[0158] As used herein, the terms "compete" or "cross-compete" and related terms are used interchangeably herein and mean the ability of an antibody to prevent 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 a standard competitive binding assay. In a particularly suitable quantitative cross-competition assay, a FACS- or AlphaScreen-based method is used to compete between a labeled (e.g., His-tagged, biotinylated or radiolabeled) antibody or fragment thereof and an unlabeled antibody or fragment thereof and measure their binding to the target. Generally, a cross-competing antibody or fragment thereof binds to the target in such a manner that, for example, in a cross-competition assay, during the assay, in the presence of a secondary antibody or fragment thereof alone, the displacement recorded by an immunoglobulin single variable domain or polypeptide of the invention is up to 100% relative to the maximum theoretical degree of displacement by an antibody or fragment thereof having the cross-blocking ability present at the predetermined amount to be tested (e.g., in a FACS-based competition assay). 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 the determinant of a protein to which an antibody can specifically bind. Epitopes usually consist of molecules having 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 binding to the former rather than the latter is lost in the presence of a denaturing solvent. 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 the rate of HD substitution is significantly reduced by Fab binding. Conformational epitopes include, but are not limited to, functional epitopes. The term "linear epitope" means an epitope in which all positions of the interaction between a protein and an interacting molecule (e.g., an antibody) are linearly present along the (continuous) primary amino acid sequence of the protein.

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

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

[0163] Suitably, the isolated antibody of the present invention is an Fv. Suitably, the isolated antibody of the present invention is a scFv antibody fragment. A "single-chain Fv" or "scFv" or "sFv" antibody fragment contains the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains that enables the sFv to form a desirable structure for target binding. A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains that enables the scFv to form a desirable structure for antigen binding (see, for example, Pluckthun, The pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315). In a specific embodiment, the functional fragment is in scFv format containing the linker represented by SEQ ID NO: 28. In yet another embodiment, the isolated antibody of the present invention is a single-chain variable region fragment (scFv) represented by 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 present invention is a single-chain variable region fragment (scFv) represented by SEQ ID NO: 31.

[0164] Suitably, the isolated antibody of the present invention is an IgG antibody fragment. The term "isotype" refers to antibody classes such as IgM, IgE, IgG (such as IgG1 or IgG4) that result from differences in heavy chain constant region genes. Isotypes also include modifications for modifying Fc functions, such as modifications to any one of these classes made to enhance or reduce effector function or binding to Fc receptors. In one embodiment, the isolated antibody of the present 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 is respectively, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, 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 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, and is an IgG1. In a more specific embodiment, the antibody of the present invention is respectively, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 4, 6 and 7, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 21 and 22, a heavy 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: 93, 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: 92, and is an IgG1. Suitably, the isolated antibody of the present invention is respectively, the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 17, 18 and 19, 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 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, and is an IgG1. In a more specific embodiment, the antibody of the present invention 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: 16, 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: 27, and is an IgG1.

[0166] Suitably, the isolated antibody of the present invention 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 An IgG1 comprising a VL sequence having 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 the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 51, 52, and 53, respectively, a heavy chain sequence having 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: 91, and a light chain sequence having 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, and is an IgG1.

[0167] Suitably, the isolated antibody of the present invention 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 having 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 having 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, and is an IgG1. In a more specific embodiment, the antibody of the present invention 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: 47, 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, and an IgG1 comprising the same.

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

[0169] As used herein, the terms "multispecific molecule" or "multispecific antibody" refer to an antibody that binds to two or more different epitopes of at least two different targets (e.g., PDL1 and another target different from PDL1), or an antibody that binds to two or more different epitopes of the same target. The term "multispecific molecule" includes bispecific, trispecific, tetravalent specific, pentavalent specific, and hexavalent specific antibodies. As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes on two different targets or on the same target. As used herein, the term "trispecific antibody" refers to an antibody that binds to three different epitopes on three different targets or on the same target.

[0170] The antibodies of the present invention can also be derivatized or linked to other functional molecules such as other peptides or proteins (e.g., ligands for other antibodies or receptors), thereby generating multispecific molecules that bind to two or more binding sites and / or different target molecules. The antibodies of the present invention can actually be derivatized or linked to multiple other functional molecules, thereby becoming multispecific molecules that bind to two or more different binding sites and / or target molecules. To obtain the multispecific molecules of the present invention, the antibodies of the present invention can be functionally linked to one or more other binding molecules (e.g., other antibodies, antibody fragments, peptides or binding mimetics) (e.g., by chemical bonds, gene fusions, non-covalent associations, etc.), thereby becoming multispecific molecules.

[0171] Accordingly, the present invention encompasses multispecific molecules that include 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 a target molecule different from PDL1. Accordingly, the present invention encompasses multispecific molecules that include 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 that is different from the first target epitope. The multispecific molecule can further include a third binding specificity in addition to the first and second target epitopes.

[0172] In yet another embodiment, the present invention encompasses multispecific molecules that include monovalent, bivalent or multivalent specificities for PDL1, preferably monovalent specificities.

[0173] In other specific embodiments of the present invention, the isolated antibodies of the present invention are monovalent or multivalent PDL1-specific molecules such as bivalent, trivalent, tetravalent, pentavalent, hexavalent.

[0174] As used herein, the term "monovalent molecule" or "monovalent antibody" refers to an antibody that binds to a single epitope on a target molecule (e.g., PDL1).

[0175] The term "multivalent antibody" refers to a single binding molecule having multiple valences, where the "valence" is the number of antigen-binding portions that bind to epitopes 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 containing 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 having two antigen-binding portions (each binding to the same epitope).

[0176] Suitably, the isolated antibodies of the present invention are multispecific molecules (e.g., bispecific molecules) and / or multivalent molecules (e.g., monovalent specific molecules for PDL1, bivalent specific molecules for PDL1), which may be selected from any suitable multispecific, e.g., bispecific, antibody format known in the art, and such formats include, but are not limited to, single-chain diabody (scDb), tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), bispecific T cell engager (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-terminal group of the light chain), Bs2Ab (scFv linked to the N-terminal group of the heavy chain), Bs3Ab (scFv linked to the C-terminus of the heavy chain), Ts1Ab (scFv linked to the N-terminal groups of the heavy and light chains), Ts2Ab (dsscFv linked to the C-terminus of the heavy chain), IgG-scFv fusions such as Knob-into-Hole antibodies (KiHs) (bispecific IgG prepared by the KiH technology), bispecific antibodies based on heterodimeric Fc domains, 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., a format based on mAbs 9(2017)68 - 84 and DuoBodie (bispecific IgG prepared by Duobody technology) (MAbs. 2017 Feb / Mar;9(2):182 - 212.doi:10.1080 / 19420862.2016.1268307). Single - chain diabody (scDb) or scDb - scFv is particularly suitable for use in the present invention.

[0177] The term "diabody" refers to an antibody fragment having two antigen - binding sites, and the fragment contains VH linked to VL (VH - VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, those domains pair with the complementary domains of the other chain to form two antigen - binding sites. In a specific embodiment, the polypeptide linker consists of one or two units (GGGGS) composed of four glycine amino acid residues and one serine amino acid residue n , including n = 1 or 2, preferably including 1. The diabody may be bivalent or bispecific. Details of the diabody are described, for example, in European Patent No. 404097, International Publication No. 93 / 01161, Hudson et al., Nat. Med. 9:129 - 134(2003), and Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444 - 6448(1993). Triabody and tetrabody are also described in Hudson et al., Nat. Med. 9:129 - 134(2003).

[0178] A bispecific scDb (especially a bispecific monomeric scDb) particularly comprises two variable heavy chain domains (VH) or fragments thereof and two variable light chain domains (VL) or fragments thereof, which are linked by linkers L1, L2, and L3. Their order is 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. The VLA and VHA domains together form an antigen-binding site for the first antigen, and VLB and VHB together form an antigen-binding site for the second antigen.

[0179] Linker L1 is particularly a peptide of 2 to 10 amino acids, more specifically 3 to 7 amino acids, and even more specifically 5 amino acids. Linker L3 is particularly a peptide of 1 to 10 amino acids, more specifically 2 to 7 amino acids, and even more specifically 5 amino acids. In a specific embodiment, linker L1 and / or L3 comprises one or two units ((GGGGS) n , where n = 1 or 2, preferably n = 1).

[0180] The intermediate linker L2 is particularly a peptide of 10 to 40 amino acids, specifically 15 to 30 amino acids, and even more specifically 20 to 25 amino acids. In a specific embodiment, the linker L2 comprises one or more units ((GGGGS) n , where n = 1, 2, 3, 4, 5, 6, 7, or 8, preferably n = 4).

[0181] In one embodiment of the present invention, the isolated antibody is a multispecific and / or multivalent antibody in the 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) by a flexible Gly-Ser linker. In one embodiment, the flexible Gly-Ser linker is a peptide of 2 to 40 amino acids, such as 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10 amino acids, particularly 10 amino acids. In a specific embodiment, the linker comprises 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 = 2).

[0182] In one embodiment of the present invention, the isolated antibody is a multispecific and / or multivalent antibody in the MATCH format described in International Publication No. WO 2016 / 0202457, Egan T. et al., mAb 9 (2017) 68-84.

[0183] The multispecific and / or multivalent molecules of the present invention can be produced using any of the useful antibody production methods well-known in the prior art (for example, with regard to the preparation of bispecific constructs, see Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14; with regard to bispecific diabodies and tandem scFvs, see Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 and WO 99 / 57150). Further specific examples of suitable methods for the preparation of the bispecific constructs of the present 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 preparing bispecific antibodies containing 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 present invention are mouse, chimeric, and humanized monoclonal antibodies.

[0185] The multispecific molecules of the present invention can be prepared by conjugating several binding specificity moieties using methods known in the prior art. For example, each binding specificity moiety of a bispecific molecule can be prepared separately and then conjugated to each other. When the binding specificity moiety is a protein or peptide, covalent conjugation can be performed using various coupling or crosslinking agents. Examples of crosslinking agents 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 sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, M A 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. Examples of conjugating agents include SATA and sulfo-SMCC, both of which are available from Pierce Chemical Company (Rockford, Illinois).

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

[0187] Alternatively, two or more binding specificity moieties can be encoded, expressed, and assembled in the same host cell in the same vector. This method is particularly useful when the bispecific molecule is an mAb×mAb, mAb×Fab, Fab×F(ab’)2, or ligand×Fab fusion protein. The multispecific molecule of the present invention can be a single-chain molecule containing one single-chain antibody and one binding determinant, or a single-chain multispecific molecule containing two binding determinants. The multispecific molecule may contain two or more single-chain molecules. For example, methods for preparing multispecific molecules are described in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498, and U.S. Patent No. 5,482,858.

[0188] The binding of the bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays typically specifically detects the presence of a protein-antibody complex by using a labeling reagent (e.g., an antibody) specific for the complex of interest.

[0189] In a further aspect, the present invention provides a nucleic acid encoding an antibody of the present invention. The present invention also provides nucleic acid sequences encoding CDRs, VH, VL, full-length heavy chains, and full-length light chains of an antibody that specifically binds to the PDL1 protein. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0190] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in the present specification, and refers to one or more deoxyribonucleotides or ribonucleotides, and polymers thereof, in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, natural, and non-natural, which have similar binding properties as reference nucleic acids, and which are metabolized in a similar manner as reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., codon degeneracy substitutions) and complementary sequences in addition to the explicitly recited sequence. Specifically, as described below, degenerate codon substitutions may be achieved by substituting the third position of one or more selected (or all) codons with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0191] The present invention provides a substantially purified nucleic acid molecule encoding a polypeptide comprising a segment or domain of the above-described PDL1-binding antibody chain. When expressed from a suitable expression vector, the polypeptide encoded by these nucleic acid molecules can exhibit the ability to bind to the PDL1 antigen.

[0192] In addition, the present invention provides a polynucleotide encoding one or more CDR regions, usually all three CDR regions, derived from the heavy or light chain of the PDL1-binding antibody described in Table 1. Some other polynucleotides encode all or substantially all of the heavy and / or light chain variable region sequences of the PDL1-binding antibody described in Table 1. Due to the degeneracy of coding, various nucleic acid sequences encode each immunoglobulin amino acid sequence.

[0193] The polynucleotide sequence can be prepared by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding a PDL1-binding antibody (e.g., the sequences described in the following examples). Direct chemical synthesis of nucleic acids can be carried out by methods of known techniques (e.g., the phosphotriester method (Narang et al., 1979, Meth. Enzymol. 68:90), the phosphodiester method (Brown et al., Meth. Enzymol. 68:109, 1979), the diethylphosphoramidite method (Beaucage et al., Tetra. Lett., 22:1859, 1981), and the solid-phase method (U.S. Patent No. 4,458,066)). For example, mutagenesis of a polynucleotide sequence by PCR can be carried out as described in, for example, PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, N.Y., 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 an expression vector and a host cell for producing the above PDL1-binding antibody.

[0195] The term "vector" is intended to refer to a polynucleotide molecule that can transport other polynucleotides to which it is ligated. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can ligate additional DNA fragments. Other types of vectors are viral vectors, which can ligate additional DNA fragments to the viral genome. Certain vectors are capable of autonomous replication in the host cells 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 host cells and integrated into the host cell genome, thereby replicating with the host genome.

[0196] Furthermore, certain vectors can induce the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology often take the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because the plasmid is the form of the vector most commonly used. However, in the present invention, expression vectors include other forms (e.g., viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses)), and they perform equivalent functions.

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

[0198] To express a polynucleotide encoding a PDL1-binding antibody chain or binding fragment, various expression vectors can be used. 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 typically having an expression cassette for expressing protein or RNA, 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, California), MPS V vector, and various other vectors for protein expression known in the art. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40-based vectors, papillomaviruses, 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 selection of an expression vector depends on the host cell in which the vector is to be expressed. Typically, an expression vector includes a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding a PDL1-binding antibody. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequence under conditions other than the inducing conditions. Examples of inducible promoters include, for example, arabinose, lacZ, metallothionein promoter or heat shock promoter. By culturing the organism transformed under non-inducing conditions, the expression of the product is made acceptable to the host cell, so that a population having the coding sequence can be grown without bias. In addition to the promoter, other control elements are required or may be desirable for efficient expression of the PDL1-binding antibody. These elements typically include an ATG start codon and adjacent ribosome binding site or other sequences. In addition, the expression efficiency may be enhanced by an enhancer suitable for the cell line used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994 and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to enhance expression in mammalian host cells.

[0200] A secretion signal sequence may be placed in the expression vector to express the polypeptide encoded by the inserted PDL1-binding antibody sequence as a fusion protein. Often, the inserted PDL1-binding antibody sequence is ligated to the signal sequence before insertion into the vector. Also, the vector used to receive the sequences encoding the light and heavy chain variable region domains of the PDL1-binding antibody may also encode a constant region or a portion thereof in some cases. Such a vector results in the expression of the variable region as a fusion protein with the constant region, thereby leading to the production of a complete antibody and its antigen-binding fragments. Typically, such a constant region is of human origin.

[0201] The term "recombinant host cell" or simply "host cell" means a cell into which a recombinant expression vector has been introduced. It should be understood that such term refers not only to a particular cell but also to the progeny of such cell. Such progeny may not actually be identical to the parental cell due to mutations or effects of surrounding conditions, which may gradually introduce certain modifications, provided that they are included within the scope of the term "host cell" as used herein.

[0202] The host cell for accommodating and expressing the PDL1-binding antibody chain may be a prokaryote or a eukaryote. Escherichia coli is one of the prokaryotic hosts useful for cloning and expressing the polynucleotide of the present invention. Other microbial hosts suitable for use include bacteria of the genus Bacillus (e.g., Bacillus subtilis) and other enterobacteria (e.g., bacteria of the genus Salmonella, Serratia, and various species of the genus Pseudomonas). In these prokaryotic hosts, an expression vector can also be constructed, which typically includes an expression regulatory sequence (e.g., an origin of replication) compatible with the host cell. Further, various known promoters are included, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system derived from λ phage. These promoters typically control expression, optionally together with an operator sequence, and initiate and terminate transcription and translation by having a ribosome-binding site sequence and the like. Also, the PDL1-binding polypeptide of the present invention can be expressed using other microorganisms (e.g., yeast). Insect cells can also be used in combination with a baculovirus vector.

[0203] In one embodiment, mammalian host cells are used to express and produce the PDL1-binding polypeptide of the present invention. For example, they may be hybridoma cell lines that express endogenous immunoglobulin genes, or mammalian cell lines that carry exogenous expression vectors. These include any normal lethal or normal or abnormally immortalized animal or human cells. For example, many suitable host cell lines capable of secreting complete 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 for expressing polypeptides is outlined, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y. N.Y. 1987. Expression vectors for mammalian host cells can include expression control sequences, such as origins of replication, promoters, and enhancers (Queen, et al., Immunol. Rev. 89:49-68, 1986), and the 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, the metallothionein promoter known in the prior art, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (e.g., the human early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations.

[0204] The method of introducing an expression vector containing the polynucleotide sequence of interest varies depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other host cells. (See Sambrook et al. supra for an overview). Other methods include, for example, electroporation, calcium phosphate treatment, transformation by liposomes, injection and microinjection, biolistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O’Hare, Cell 88:223, 1997), enhancement of DNA uptake by drugs, and ex vivo transduction. Stable expression is usually required for the long-term, high-yield production of recombinant proteins. For example, a cell line stably expressing a PDL1-binding antibody chain or binding fragment can be prepared using the expression vector of the present invention containing a viral replication origin 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 the selection medium. The purpose of using a selectable marker is to confer resistance to selection, and the presence thereof allows cells that preferably express the introduced sequence to grow in the selection medium. Stably transfected cells having resistance can be grown using tissue culture techniques suitable for the cell type. Accordingly, the present invention provides a method for producing the antibody of the present invention, said method comprising culturing, in particular expressing, a host cell containing a nucleic acid or vector encoding the antibody of the present invention, whereby said antibody or fragment thereof of the present invention is expressed.

[0205] In a further aspect, the present invention relates to a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier enhances or stabilizes the composition, or facilitates the preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other media having physiological compatibility.

[0206] The pharmaceutical compositions of the invention can be administered by a variety of well-known methods. The route or method of administration will vary depending on the desired effect. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered near the target site. The pharmaceutically acceptable carrier must be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., injection or infusion). Depending on the route of administration, the active compounds (i.e., antibodies and multispecific molecules) may be coated with a material that protects the compound from the action of acids and other natural conditions that may cause inactivation of the compound.

[0207] The pharmaceutical composition of the present invention can be prepared according to known methods routinely practiced in the prior art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000 and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition is preferably manufactured under GMP conditions. Typically, a therapeutically effective amount or an effective amount of the PDL1-binding antibody is used in the pharmaceutical composition of the present invention. The PDL1-binding antibody is prepared in a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosing schedule is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, it may be administered in several divided doses at regular intervals, or the dose may be tapered or increased depending on the urgency of the therapeutic situation. In facilitating administration and equalizing the dose, it is particularly useful to formulate the ingredients in unit dosage form. As used herein, "unit dosage form" refers to physically discrete units suitable as unit dosages for the subjects to be treated, each unit containing a predetermined quantity of the 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 as to be an amount of the active ingredient, composition and method of administration that is effective to achieve the desired therapeutic response in a particular patient without causing toxicity to the patient. The dosage level selected will depend upon a variety of pharmacokinetic factors, for example, the activity of the particular composition of the present invention or its ester, salt or amide used, the time of administration, the route of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other agents, compounds and / or materials used in combination with the particular composition being used, age, sex, weight, symptoms, general health, past medical history of the patient being treated, and other factors.

[0209] Antibodies are usually administered multiple times. The intervals between single administrations can be weekly, monthly, or yearly. Depending on the measurement results of the blood concentration of the patient's PDL1-binding antibody, the administration intervals may be irregular. Alternatively, the antibody can be administered as a sustained-release formulation, in which case it is not necessary to administer it frequently. The dosage and frequency of administration vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit a longer half-life than chimeric antibodies and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic administration, a relatively low dosage is administered at relatively infrequent intervals over a long period. Some patients may continue treatment for the rest of their lives. In therapeutic administration, relatively high dosages may be required at relatively short intervals until the progression of the disease slows down or ends, preferably until the patient shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient can receive administration in a prophylactic regimen.

[0210] The antibodies of the present invention have utility for diagnosis and treatment in vitro and in vivo. For example, these molecules can be administered for the treatment, prevention, or diagnosis of various disorders, for example, into cultured cells in vitro or in vivo, or into a subject in vivo.

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

[0212] In one aspect, the present invention relates to the antibody or composition of the present invention for use in the treatment of a subject in need of treatment for a proliferative disorder, particularly cancer.

[0213] In other aspects, the present invention relates to the use of the antibody or composition of the present invention for the treatment of a subject in need of treatment for a proliferative disorder, particularly cancer.

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

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

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

[0217] As used herein, the terms "treatment", "treating", "treat", "treated", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in the sense of partially or completely curing the disease and / or adverse effects caused by the disease, or may be therapeutic in the sense of delaying disease progression. As used herein, "treatment" includes any treatment of a disease in a mammal (e.g., a human), for example: (a) inhibiting the disease, i.e., suppressing its progression, and (b) alleviating the disease, i.e., causing regression of the disease.

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

[0219] In one embodiment, the proliferative disease is cancer. The term "cancer" means a disease characterized by the rapid, uncontrollable growth of errant cells. Cancer cells may spread locally or throughout the body to other parts via the bloodstream and lymphatic system. 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, for example, both terms include solid tumors and, for example, disseminated or circulating liquid tumors. As used herein, the term "cancer" or "tumor" includes precancer, as well as malignant cancers and tumors. The term "cancer" is used herein to mean the broadness of a tumor 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 cancer, bladder cancer, breast cancer, gastric cancer (e.g., gastric tumor), 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., inoperable or metastatic melanoma), renal cell carcinoma, sarcoma, glioblastoma, multiple myeloma, or gastrointestinal cancer, particularly colon cancer or colorectal adenoma, head and neck tumors, endometrial cancer, Cowden syndrome, Lhermitte-Duclos disease, Bannayan-Zonana syndrome, prostate hyperplasia, tumor formation, particularly epithelial, preferably breast cancer or squamous cell carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia (e.g., Philadelphia chromosome-positive chronic myelogenous leukemia), acute lymphocytic leukemia (e.g., Philadelphia chromosome-positive acute lymphocytic leukemia), non-Hodgkin lymphoma, plasmacytic myeloma, Hodgkin lymphoma, leukemia, and combinations thereof. In a preferred embodiment, the cancer is lung cancer, preferably non-small cell lung cancer (NSCLC). In other embodiments, 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 inhibits liquid tumors. In yet another embodiment, the proliferative disease is a solid tumor. The term "solid tumor" particularly means breast cancer, ovarian cancer, colorectal cancer, rectal cancer, prostate cancer, gastrointestinal cancer (particularly gastric cancer), cervical cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), and tumors of the head and neck. Furthermore, depending on the tumor type and the specific 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 tumor metastasis and preventing the growth or development of micrometastases in a subject having cancer.

[0221] The term "prevent" or "prevention" refers to the complete suppression of the progression of a disease or any secondary effects of the disease. As used herein, the term "prevent" or "prevention" includes preventing the onset of a disease or symptoms 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 the antibody of the present invention or the pharmaceutical composition of the present invention. The kit may contain one or more other contents as follows: instructions for use, other reagents (e.g., labels, therapeutic agents or agents useful for chelating or otherwise coupling an antibody to a label or therapeutic agent, or a radiation protection composition), devices or other materials for preparing antibody molecules for administration, a pharmaceutically acceptable carrier, and devices or other materials for administration to a subject. In a particular embodiment, the kit contains a pharmaceutically effective amount of the antibody of the present invention. In yet another embodiment, the kit contains a pharmaceutically effective amount of the lyophilized antibody of the present invention, a diluent, and any instructions for use. The kit may further contain a filter needle for reconstitution and an injection needle.

[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 differences between the descriptions in the specification (e.g., Tables 1 to 3) and the sequence listing. In such cases, the descriptions in the specification shall be regarded as the main text.

[0240] For clarity, specific features of the present invention are described in the context of separate embodiments, but it is obvious that they may be combined within one embodiment. Conversely, for the sake of brevity, the various forms of the present invention described in the description of one embodiment may be divided and applied as appropriate sub - combinations. All combinations of embodiments related to the present invention are specifically included in the present invention and shall be regarded as disclosed in this specification as if each combination was individually and clearly disclosed. In addition, all sub - combinations of various embodiments and their components are also specifically included in the present invention and shall be regarded as disclosed in this specification as if each such sub - combination was individually and clearly disclosed in this specification.

[0241] The present invention is not limited in scope by the specific embodiments described in this specification. In fact, various modifications of the invention other than those described in this specification 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 in this specification are hereby incorporated by reference into this specification.

[0243] The following examples illustrate the invention described above, but in no way are intended to limit the scope of the invention. Other test models known to those skilled in the art of this related technology can also be used to confirm the utility of the claimed invention.

Example

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

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

[0246] Example 2: Identification and selection by Hit: During the Hit identification procedure, a flow cytometry-based sorting procedure was developed that enabled the specific detection and isolation of high-affinity hPDL1-binding B cells. To identify hPDL1-binding B cells, hPDL1 ECD was labeled with the fluorescent dye R-phycoerythrin (R-PE). Since the PD-1 binding site on labeled PDL1 and the binding site of the anti-PDL1 neutralizing antibody are potentially blocked by the bulky R-PE label, the epitope accessibility could be confirmed by flow cytometry. The extracellular domain of PD-1 fused to the Fc portion of human IgG1 or abelumab 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 binding to the receptor immobilized on the beads. While the binding of PDL1 to PD-1 and the neutralizing antibody was confirmed, the binding of irrelevant cytokines to the anti-PDL1 antibody was not detected.

[0247] Screening: At high-throughput culture scales, it is difficult to purify individual rabbit antibodies, so the results obtained during the screening phase were based on assays performed with unpurified antibodies from the culture supernatants of antibody-secreting cells (ASCs). Such supernatants allow ranking of each antibody in large numbers, but absolute values other than binding ability cannot be obtained. Supernatants were collected from every individually cultured clone over a period of more than 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 the recombinant human PDL1 extracellular domain. Using the PDL1-binding supernatants, further characterization was performed for the binding kinetics to human and cynomolgus monkey PDL1. In addition, the neutralization potential of the PDL1 / PD-1 interaction was measured by cell-based reporter gene assays and competitive ELISA. Also, the neutralization of the PDL1 / B7-1 interaction was evaluated by competitive ELISA. Except for the binding kinetics, the reported values of the high-throughput screening were interpreted as "positive" or "negative" results, which were based on single measurements (not dose responses). The mouse PDL1 binding potential of the supernatants was analyzed by direct ELISA, and the binding kinetics were measured only for the positive supernatants.

[0248] Direct ELISA for hPDL1 binding: 50 μl of PBS containing 500 ng / ml of 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 wash buffer (PBS, 0.005% Tween 20) per well, and 300 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added per well and treated on a nutating mixer for 1 hour at room temperature. Next, the plate was washed three times in overflow mode with 450 μl of wash buffer, 50 μl of each supernatant was added, and the plate was incubated for 1.5 hours at room temperature with gentle shaking. After washing three times in overflow mode with 450 μl of wash buffer, 50 μl of HRP-conjugated goat and rabbit IgG antibodies were added to each well. After incubation for 1 hour at room temperature on a rotating mixer, the plate was washed with 450 μl of wash buffer per well, and then 50 μl of TMB (3,3’,5,5’-tetramethylbenzidine, KPL, catalog number 53-00-00) was added. After developing color for 5 - 10 minutes, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl per well, and the plate was measured at 450 nm using 690 nm as the reference wavelength.

[0249] Affinity for hPDL1 by SPR: Using a MASS-1 SPR instrument (Sierra Sensors), the binding affinity of antibodies against human PDL1 was measured by surface plasmon resonance (SPR). In affinity screening, an antibody specific to the Fc region of rabbit IgG (Bethyl Laboratories, catalog number 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 the B cell supernatant were captured by the immobilized anti-rabbit IgG antibody. The minimum IgG concentration in the B cell supernatant is necessary to allow sufficient capture. After capturing the monoclonal antibody, human PDL1 (Peprotech) was injected into the flow cell at a concentration of 90 nM for 3 minutes, and separation of the protein from the IgG captured on the sensor chip was allowed for 5 minutes. After each injection cycle, the surface was regenerated by injecting 10 mM glycine-HCl twice. The apparent dissociation (kd) and association (ka) constants, and the apparent dissociation equilibrium constant (K D ) were calculated using a 1:1 Langmuir binding model with MASS-1 analysis software (Analyzer, Sierra Sensors), and the quality of the fit was monitored based on the relative value of Chi 2 (normalized by the maximum binding level of the extrapolated analyte of Chi 2 ), and this was used as an indicator for maintaining the quality of curve fitting. When the value of Chi 2 is small, it means that the accuracy of the fit to the 1:1 Langmuir binding model is high. For most Hits, the relative value of Chi 2 was 10% or less. When the reaction unit (RU) in ligand binding was 2% or more of the RU of antibody capture, the result was judged to be valid. Samples showing an RU of ligand binding less than 2% of the RU of 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 of 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, 300 μl of blocking buffer was added per well, and the plate was processed on a rotary mixer at room temperature for 1 hour. Next, PDL1 was diluted in blocking buffer at a concentration 20-fold higher than the desired final concentration of 250 ng / ml. The assay sensitivity was further adjusted, and several clones were analyzed in the presence of 40 ng / ml of PDL1. Next, 114 μl of each supernatant in the unbound plate was diluted with blocking buffer containing 6 μl of PDL1, and the plate was incubated on a rotary mixer at room temperature for 1 hour. 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 hours with gentle shaking. After washing 3 times with 450 μl of wash buffer per well, 50 μl of 10 ng / ml of streptavidin-poly HRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of wash buffer, 50 μl of TMB was added, and the color was developed for 5 - 10 minutes. Finally, 50 μl of 1 M HCl was added to stop the enzymatic reaction, and the plate was measured at 450 nm using 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 was added per well, and the plate was treated on a rotary mixer at room temperature for 1 hour. Next, PDL1 was diluted in blocking buffer at a concentration 20-fold higher than the desired final concentration of 500 ng / ml. Next, 114 μl of each supernatant in the unbound plate was diluted with blocking buffer containing 6 μl of PDL1, and the plate was incubated on a rotary mixer at room temperature for 1 hour. 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 hours with gentle shaking. After washing 3 times with 450 μl of wash buffer per well, 50 μl of 10 ng / ml streptavidin-poly HRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of wash buffer, 50 μl of TMB was added, and the color was developed for 5 - 10 minutes. Finally, 50 μl of 1 M HCl was added to stop the enzyme reaction, and the plate was measured at 450 nm using 690 nm as the reference wavelength.

[0252] Blocking of 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 activator and Jurkat / PD-1 cells. 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 culture plate and incubated at 37 °C and 5% CO2 for 16 - 20 hours. The next day, 95 μl of cell culture medium was removed from each well, and 50 μl of the supernatant of the screened B cells, or the positive control abelumab at concentrations measured to yield 0%, 50%, and 100% of the maximal signal, was added, and the plate was incubated at 37 °C for 30 minutes. Next, effector Jurkat cells diluted to 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS) were added to each well at 50 μl, 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 plate was incubated in the dark for 30 minutes, and luminescence was measured using Topcount.

[0253] Species specificity by SPR: cyno and mouse Also, using the same SPR setup as described for binding to human PDL1, the binding kinetics to cynomolgus and mouse PDL1 were measured, replacing human PDL1 with cynomolgus or mouse PDL1, respectively.

[0254] Selection of screening Hits: The pharmacological properties of the final clones of monoclonal antibodies in the B cell supernatant are shown in Table 4.

[0255] Example 3: Confirmation of Hit: Cloning and production: Following the identification of the selected clones for Hit confirmation, these rabbit antibodies were cloned, expressed, and purified for further characterization. For the cloning of the corresponding light and heavy chain variable domains, in vitro ligation of the DNA fragments was performed into appropriate mammalian expression vectors. These expression vectors containing the consensus sequences of the constant regions of the light and heavy chains of rabbit IgG were co-expressed, allowed to associate, and secreted 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 for transfection into mammalian cells.

[0256] Expression vectors for rabbit heavy and light chains were transfected into a mammalian suspension cell line for transient heterologous expression using a lipid-based transfection reagent. To enhance the expression level of the secreted monoclonal IgG, conditions such as the ratio of heavy and light chain vectors were optimized. The cultured cells for expression were cultured for 7 days in a shaking culture apparatus. At the end of the heterologous expression period, the cell culture supernatant was collected by centrifugation. Next, the secreted rabbit IgG was affinity purified using Protein A beads. The beads loaded with IgG were washed, and the purified antibody was eluted by pH shift. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet absorption at 280 nm, and size exclusion high performance liquid chromatography (SE-HPLC) were used to analyze the identity, content, and purity of the eluted fractions. Table 5 summarizes the production and characterization of rIgG.

[0257]

Table 17

[0258]

Table 18

[0259] Affinity for hPDL1 by SPR: Using a MASS-1 SPR instrument (Sierra Sensors), the binding kinetics of a purified monoclonal rabbit antibody to human PDL1 was measured by surface plasmon resonance (SPR). Since most of the antibodies showed a very slow off-rate, the experiments were carried out at 37 °C in a buffer containing high salt concentration to clarify the differences in binding affinity due to antibody differences. Using a standard amine coupling procedure, an antibody specific for the Fc region of rabbit IgG (Bethyl Laboratories, catalog number A120-111A) was immobilized on a sensor chip (SPR-2 affinity sensor, High Capacity Amine, Sierra Sensors). The rabbit monoclonal antibody was captured by the immobilized anti-rabbit IgG antibody. After capturing the monoclonal antibody, PDL1 serially diluted two-fold in the range of 90 - 0.35 nM in HEPES buffer containing 150 mM NaCl and 150 mM MgCl2 was used for the test, and the binding to IgG captured on the biosensor chip and the separation of the protein from IgG captured on the sensor chip were tested for 5 minutes. After each injection cycle, 10 mM glycine-HCl was injected twice to regenerate the surface. The apparent dissociation (kd) and association (ka) constants, and the apparent dissociation equilibrium constant (K D ) were calculated using a 1:1 Langmuir binding model with MASS-1 analysis software (Analyzer, Sierra Sensors), and the quality of the fit was monitored based on the relative value of Chi 2 (Chi 2 normalized by the maximum binding level of the extrapolated analyte), and this was used as an indicator for maintaining the quality of curve fitting. When the value of Chi 2 is small, it means that the accuracy of the fit to the 1:1 Langmuir binding model is high. For most Hits, the relative value of Chi 2 was 10% or less. Table 6 shows the rabbit IgG antibodies selected for further development.

[0260]

Table 19

[0261] Titer in PDL1 / PD-1 blocking ELISA: The titer neutralizing the binding of PDL1 to PD-1 was evaluated in a competitive ELISA. 50 μl of PBS containing 4 μg / ml of PD-1 was added to the ELISA plate and coated overnight at 4°C. The next day, the plate was washed 3 times in overflow mode with 450 μl of wash buffer (PBS, 0.005% Tween 20) per well, 300 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added per well, and the plate was treated on a nutating mixer at room temperature for 1 hour. Next, PDL1 was diluted with the blocking buffer to a final concentration of 1 ng / ml. Next, in the unbound plate, 120 μl of buffer containing serial dilutions of PDL1 in the range of 300 to 0.005 ng / ml of the test rIgG was prepared per well, and the plate was incubated at room temperature for 30 minutes. The ELISA plate was washed 3 times in overflow mode with 450 μl of wash buffer, 50 μl of each dilution was added to adjacent wells of the ELISA plate as replicate samples. The plate was incubated at room temperature for 90 minutes while gently shaking. After washing 3 times with 450 μl of wash buffer, 50 μl of 10 ng / ml streptavidin-poly HRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of wash buffer, 50 μl of TMB was added, and the color was developed for 5 to 10 minutes. Finally, 50 μl of 1 M HCl was added to stop the enzyme reaction, and the plate was measured at 450 nm using 690 nm as the reference wavelength.

[0262] Rabbit IgG derived from clones 33-03-G02 and 37-20-B03 showed high titers neutralizing the PDL1 / PD-1 interaction. Clone 37-20-B03 had a titer approximately 2-fold higher than that of abelumab (Table 7). The dose-response curves obtained for the selected clones are shown in Figure 1.

[0263]

Table 20

[0264] Titer in PDL1 / B7-1 blocking ELISA: The titer neutralizing the PDL1 / B7-1 interaction was evaluated in a competitive ELISA. 50 μl of PBS containing 4 μg / ml of B7-1 was added to the ELISA plate and coated overnight at 4°C. The next day, the plate was washed 3 times in overflow mode with 450 μl of wash buffer (PBS, 0.005% Tween 20) per well, 300 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added per well, and the plate was treated on a nutating mixer at room temperature for 1 hour. Next, PDL1 was diluted to 40 ng / ml with the blocking buffer. Next, in the unbound plate, 120 μl of buffer containing serial dilutions of PDL1 in the range of 900 - 0.015 ng / ml of the rIgG to be tested was prepared per well, and the plate was incubated at room temperature for 30 minutes. The ELISA plate was washed 3 times in overflow mode with 450 μl of wash buffer, 50 μl of each dilution was added twice to adjacent wells of the ELISA plate as replicate samples. The plate was incubated at room temperature for 90 minutes with gentle shaking. After washing 3 times with 450 μl of wash buffer, 50 μl of streptavidin-poly HRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed 3 times with 450 μl of wash buffer, 50 μl of TMB was added, and the color was developed for 5 - 10 minutes. Finally, 50 μl of 1 M HCl was added to stop the enzymatic reaction, and the plate was measured at 450 nm using 690 nm as the reference wavelength. The selected rabbit IgG was able to block the PDL1 / B7-1 interaction with a titer equivalent to that of abelumab, as shown in Table 8. The dose-response curve obtained for the selected clone is shown in Figure 2.

[0265]

Table 21

[0266] Potency (reporter gene) in cell-based PDL1 / PD-1 blocking assay: The potency to neutralize 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 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 a two-fold dilution series of the molecule to be tested (from 3,000 to 0.46 ng / ml) and a control abelumab solution were added at 50 μl. Next, 50 μl of effector Jurkat cells diluted to 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS) 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, and the plate was incubated in the dark for 30 minutes, and luminescence was measured using Topcount. The selected clones were able to block the PDL1 / PD-1 interaction in a cell-based reporter gene assay (see Table 9). The dose-response curves obtained in the selected clones are shown in Figure 3.

[0267]

Table 22

[0268] Binding to PDL1-expressing cells by FACS: The binding titer to PDL1-expressing cells was measured for the selected IgGs. 50,000 CHO-PDL1-expressing cells were dispensed into round-bottom non-tissue culture-treated 96-well plates. The cells were washed twice with 100 μl of PBS by centrifugation at 400 × g for 5 minutes. The rIgGs to be tested and the control IgG abelumab were prepared as serial dilutions in 100 μl of staining buffer (PBS, 2% BCS inactivated by heating, 2 mM EDTA) at 2,000 to 0.128 ng / ml, and the cells were resuspended therein. After incubation at 4°C for 1 hour on a rotary mixer, the cells were washed three times by 100 μl of staining buffer and a centrifugation step at 400 × g for 5 minutes. Next, the cells treated with rabbit IgG were resuspended in 100 μl of staining buffer containing 2 μg / ml of goat anti-rabbit IgG (APC-labeled), and the cells treated with abelumab (human IgG1) were resuspended in 100 μl of staining buffer containing 2 μg / ml of goat anti-human IgG (APC-labeled). The plates were incubated at 4°C for 1 hour on a rotary mixer. The plates were washed three times with 100 μl of staining buffer and resuspended in staining buffer at 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 cytometry system (ACEA Bioscience). Binding to PDL1-expressing cells was confirmed for all evaluated rabbit IgGs. The binding titer of the selected rabbit IgGs to intracellular PDL1 is shown in Table 10.

[0269]

Table 23

[0270] Species specificity by SPR: cyno: Using the same settings as described for the binding to human PDL1, the binding kinetics to cynomolgus PDL1 were also measured, in which case human PDL1 was replaced with cynomolgus PDL1. Binding to cynomolgus PDL1 was confirmed for all selected IgGs (Table 11).

[0271]

Table 24

[0272] Species specificity by SPR: Mouse: Using the same settings as described for the binding to human PDL1, the binding kinetics to cynomolgus PDL1 were also measured, in this case replacing human PDL1 with mouse PDL1. For the selected rabbit IgG derived from clone 33-03-G02 and 37-20-B03, no binding to mouse PDL1 was detected.

[0273] Example 4: Selection of clones for humanization Based on the data obtained in the Hit confirmation, all clones were humanized by fusing the CDRs to VH3, VH4 or VH1A or VH1B-based frameworks. To obtain the best affinity and titer, two clones showing 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 graft mutants were applied for clones 33-03-G02 and 37-20-B03: CDR grafting - grafting of rabbit CDRs onto the human framework, IF grafting - CDR grafting + grafting of residues at all rabbit VL / VH interfaces, Full grafting - CDR grafting + framework residues (antigen interface (AIF) residues (rabbit residues potentially contacting the antigen (following AHo)) following the AHo humanization protocol, limiting the residue changes to 20% or more considering the solvent accessibility during interface formation, thereby reducing the number of mutations (rabbit framework residues)).

[0274] Heterologous expression of the protein as insoluble inclusion bodies was carried out by overnight small-scale expression with induction in Escherichia coli (however, for PRO997, it was produced in mammalian CHO-S cells as in the above rIgG expression). Inclusion bodies were isolated from the homogenized cell pellet by a centrifugation protocol that removes cell debris and other host cell-derived impurities, including several washing steps. The purified inclusion bodies were solubilized in denaturing buffer and the scFvs were refolded according to a scalable refolding protocol that yields milligram amounts of the naturally folded monomeric scFv. At this time, 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 because an amount sufficient for SEC polishing of the sample could not be secured. In addition, the melting temperature of the scFvs was measured by differential scanning fluorimetry (DSF) (details will be described later). Table 12 summarizes the production of VH4 CDR-grafted scFv molecules. Since two clones contained cysteine residues with mismatches in their CDR-loops, the C57S mutation was introduced into clone 37-20-B03 as shown in Table 12.

[0275] Additional graft variants were designed for several selected clones and are summarized in Table 13 (AHo numbering) and Table 14 for their initial preparation and characterization.

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

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

[0278] 5.2. Neutralization of PDL1 / PD-1 interaction by competitive ELISA: Following the same procedure as described above, the titer for neutralizing the binding of PDL1 to PD-1 was evaluated by competitive ELISA. The individual IC 50 values in each plate were calibrated against the IC 50 of the control molecule abelumab added to each plate (relative IC 50 : (IC 50アベルマブ / IC 50試験scFv )). As summarized for the titers in Table 16, it is clear that in this assay, an IC 50 up to 5-fold that of abelumab can be calculated. All of the tested scFvs had titers equal to or higher than that of abelumab.

[0279] 5.3. Neutralization of PDL1 / B7-1 interaction by competitive ELISA Following the same procedure as described above, the titer for neutralizing the binding of PDL1 to B7-1 was evaluated by competitive ELISA. The individual IC 50 values in each plate were calibrated against the IC 50 of the control molecule abelumab added to each plate (relative IC 50 : (IC 50アベルマブ / IC 50試験scFv )) As summarized for the titer in Table 17, in this assay, up to 10-fold the IC for abelumab can be calculated in this assay. 50 It is clear that the scFvs tested had titers higher than or equivalent to abelumab.

[0280] 5.4 Neutralization of PDL1 / PD-1 interaction in NFAT reporter gene assay The ability to neutralize the binding of PDL1 to PD-1 was evaluated in the cell-based reporter gene assay as described above. Similar to the control abelumab, each molecule to be tested was added to the plate as a serial dilution. The individual IC values in each plate were calibrated against the IC of abelumab, the control molecule added to each plate (relative IC: (IC / IC)). 50 value was calibrated against the IC of abelumab, the control molecule added to each plate 50 (relative IC 50 : (IC 50アベルマブ / IC 50試験scFv )). As summarized for the titer in Table 18, in this assay, up to 5-fold the IC for abelumab can be calculated. 50 It is clear that the scFvs tested had titers higher than or equivalent to abelumab.

[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: The binding titers to PDL1-expressing cells were measured for several molecules. During hit confirmation, the same cell lines (CHO-PDL1 and CHO-K1) were used, and the scFv was detected by APC-labeled protein L. Similar to the control abelumab, each molecule to be tested was added to the plate at serial dilutions. The individual IC 50 values in each plate were calibrated against the IC 50 of the control molecule abelumab added to each plate (relative IC 50 : (IC 50アベルマブ / IC 50試験scFv ). The titers are summarized in Table 19.

[0289]

Table 32

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

[0291]

Table 33

[0292] 5.7. Selectivity of PDL1 to PDL2 by SPR The binding of the humanized scFv to PDL2 was tested by SPR analysis using a T200 instrument (Biacore, GE Healthcare). In this experiment, human PDL2 with an Fc tag was captured using a Human Antibody Capture kit from GE Healthcare. After each sample injection cycle, the CM5 sensor chip was regenerated and a new antigen was captured. The scFv was injected as a sample diluted to a concentration of 180 nM in the running buffer for 5 minutes, and the separation of the protein was allowed to proceed for 12 minutes. No binding to PDL2 was observed for any of the humanized scFvs tested, which are listed in Table 21.

[0293]

Table 34

[0294] Example 6: Biophysical Characterization of Humanized scFv: Selected domains with better affinity than abelumab were produced on a larger scale (expression volume of 0.2 L to 1.2 L). Further, after purification, the protein sample was concentrated to 10 mg / mL or more using a centrifugal concentration tube with a molecular weight cut-off of 5 kD. The production of materials for stability evaluation is summarized in Table 22.

[0295] 6.1. Storage Stability Test: The humanized scFv was subjected to a stability test (e.g., a 4-week stability test). At that time, the scFv was prepared at 10 mg / ml in a buffered aqueous solution (the final buffer composition was 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, using the fractions of monomer and oligomer in the formulation, it was evaluated by integrating the SE-HPLC peak area after 1 week, 2 weeks, and after the end of each test. For other time points, it was recorded for several molecules. Table 23 compares the measured values obtained at the endpoints on the 7th day and 28th day of the test.

[0296]

Table 35

[0297]

Table 36

[0298] 6.2. Freeze-thaw stability test: In addition to the above-mentioned storage stability test, the compatibility of scFv molecules with excellent performance was evaluated by freeze-thaw (F / T) cycles (colloidal stability). For F / T stability evaluation, the same analytical methods (SE-HPLC, SDS-PAGE) and parameters (% monomer content and % monomer loss) as those in the storage stability test were applied to monitor the quality of the molecules in 5 F / T cycles. Table 24 illustrates the process of % monomer content loss in 5 repeated F / T cycles. Since no special freeze-thaw test was conducted, the freeze-thaw data obtained from the -80°C samples used in the storage stability test over 28 days are shown in the following graph. After repeated F / T cycles, there were no molecules with a monomer content loss greater than 4%.

[0299]

Table 37

[0300] 6.3. Thermal denaturation: Thermal denaturation data of the selected scFv constructs were obtained from DSF measurements and are shown in Table 25. The obtained Tm values were measured by fitting the data to the Boltzmann equation. Table 25 summarizes the melting temperatures calculated by DSF.

[0301]

Table 38

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

[0303] Example 7: Affinity for PDL1, CD137, HSA and MSA Method: The affinity for different species of PDL1 was measured by SPR measurement using a Biacore T200 instrument (GE Healthcare). An antibody specific for the Fc region of human IgG was immobilized on a sensor chip (CM5 sensor chip, GE Healthcare) by amine coupling. In all formats, except for the Morrison format containing Fc, PDL1-Fc chimeric proteins from different species were captured by the immobilized antibody. Serial three-fold dilutions (0.12 - 90 nM) of PDL1-specific molecules were injected into the flow cell for 3 minutes and dissociation was monitored for 10 minutes. After each injection cycle, a 3M MgCl2 solution was injected once to regenerate the surface. The apparent dissociation (k d ) and association (k a ) rate constants, and the apparent dissociation equilibrium constant (KD) were calculated using a 1:1 Langmuir binding model. The affinity for different species of CD137 was measured using the same settings as for PDL1, except that CD137-Fc chimeric proteins from different species were captured by the immobilized antibody.

[0304] The format containing Fc was directly captured by an antibody specific for the Fc region of human IgG. Using a two-fold serial dilution in the range of 90 to 0.35 nM of the PDL1 extracellular domain or the CD137 extracellular domain, the binding to IgG captured on the biosensor chip was tested. After each injection cycle, a 3M MgCl2 solution was injected once to regenerate the surface.

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

[0306] Results: As a result of measuring the binding kinetics of the humanized constructs, when comparing the CDR of clone 33-03-G02 with the structure graft (STR), a difference in affinity for PDL1 was shown, and the STR graft showed a 20-fold improvement in affinity compared to the CDR graft of the same clone (PRO885 vs PRO1126 in Table 26). The CDR graft from clone 37-20-B03 (PRO997) showed approximately twice the affinity when compared to the STR graft of clone 33-03-G02. The binding affinity of the CDR grafts of 33-03-G02 became equivalent to the binding ability of the parental scFv when they were combined into different multispecific formats (when comparing PRO830 with PRO885, PRO951, PRO1123, PRO1124, PRO963, PRO966, PRO1057, PRO1058, PRO1059, and PRO1060, Table 26). The scFvs from both clones showed almost equivalent affinity for human and cynomolgus PDL1 (see PRO977 and PRO830 in Table 26).

[0307]

Table 39

[0308]

Table 40

[0309] Example 8: Blocking of PDL1 / PD-1 interaction in a cell-based reporter gene assay using CHO cells expressing PDL1 and TCR activating factor molecules and Jurkat cells expressing PD-1 and containing a luciferase gene under the NFAT response element Method: In a bioluminescent reporter gene assay, an NFAT (nuclear factor of activated T cells)-luciferase reporter and engineered Jurkat T cells stably expressing human PD-1 are made to function as effector T cells. Cells stably expressing human PDL1 and T cell receptor (TCR) activating factor are made to function as antigen-presenting cells. By co-culturing the two cell lines, activation of the Jurkat NFAT pathway is induced via binding of the TCR activating factor / TCR complex. In response to binding of PDL1-expressing cells, PD-1 signaling in the PD-1 effector T cells inhibits T cell function and the NFAT pathway is inhibited. Blocking of the interaction between the 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, along with control abelumab, was added in a 2-fold dilution series from 3,000 to 0.46 ng / ml. Next, effector Jurkat cells (BPS Bioscience) expressing PD-1 were diluted to 400,000 cells / ml in assay buffer (RPMI1640 with 10% FCS), 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 plate was incubated in the dark for 30 minutes, and luminescence was measured using Topcount.

[0311] Results: To evaluate the effect of CDR set and framework selection on the titer neutralizing the binding of PDL1 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 the VH4 framework, and PRO997 and PRO1013 contain the CDR set of clone 37-20-B03 grafted onto the VH4 or VH1 framework, respectively. PRO830 had the lowest titer among the three scFvs tested, with an IC 50 value of 42.88 ng / ml and a titer equivalent to that of abelumab with an IC 50 value of 34.09 ng / ml. PRO997 was the most potent molecule. When grafted onto the VH4 framework instead of the VH1 framework, the titer of the same CDR set was approximately 2-fold higher. The IC 50 values were 11.12 ng / ml vs. 21.29 ng / ml, respectively. (Figure 4A and Table 27)

[0312] The titer neutralizing the binding of PDL1 to PD-1 was measured before (CDR grafting) and after (structural grafting) domain optimization using bispecific molecules having the 33-03-G02 PDL1 domain. The CDR graft (PRO885) was compared to the structural graft (PRO1126). Domain optimization improved the neutralizing titer three-fold with an IC 50 value of 137.2 ng / ml for PRO885 and 48.15 ng / ml for PRO1126. (Figure 4B and Table 27).

[0313] Also, two trispecific molecules having an anti-PDL1 domain with CDR grafting of clone 33-03-G02 and two different human serum albumin binding domains were used to evaluate the titer neutralizing the PDL1 / PD-1 interaction, particularly for the extension of the half-life. The HSA domain of PRO1057 was also bound to mouse serum albumin. The experiments were conducted in the presence of 25 mg / ml of HSA. The neutralizing titer (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 provided by the IgG portion (bivalent), and two scFvs having specificity for a second target are linked via a flexible peptide linker to the heavy chain (HC) or light chain (LC) of the IgG. All Morrison molecules tested were assumed to carry the anti-PDL1 domain by CDR grafting of clone 33-03-G02 on both arms of the IgG. Two constructs, PRO1059 and PRO1060, differ in that two anti-CD137 scFvs are fused to either the heavy chain (HC) or the light chain (LC). PRO1062 has the same configuration as PRO1060 but has a different CD137 domain. The neutralizing titers of all molecules were equivalent. (Figure 6 and Table 27).

[0316] Example 9: Blocking of the interaction of PDL1 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 B7-1. Different formats including scFv, scDb, scDb-scFv and moripan were analyzed in competitive ELISA and compared with the control IgG abelumab.

[0317] PDL1 / PD-1 competitive ELISA: ELISA microplates were coated overnight at 4°C with 4 μg / ml of human PD-1 and washed three times with 450 μl of wash buffer per well. 300 μl of PBS (dilution buffer) containing 1% BSA and 0.2% Tween was added to each well and the plate was blocked for 1 hour at room temperature. A three-fold serial dilution series of the inhibitor was prepared in dilution buffer containing 1 ng / ml of biotinylated human PDL1 to a final concentration in the range of 300 to 0.005 ng / ml. The mixtures were pre-incubated for 1 hour at room temperature with gentle shaking on a rotary mixer (21 rpm) and then added to the microplates after three wash cycles with 450 μl of wash buffer per well. The plates were incubated for 1.5 hours at room temperature with gentle shaking, washed three times with 450 μl of wash buffer per well, and then 10 ng / ml of streptavidin-poly HRP40 was added to the wells of each microplate. After incubation for 1 hour at room temperature, the plates were washed three times with 450 μl of wash buffer, and TMB was added. After 6 minutes, 1 M HCl was added to stop the enzyme reaction and the absorbance was measured at 450 nm using 690 nm as the reference wavelength. IC 50 For calculation of the IC value, the values subtracted by the control were used to perform a four-parameter logistic (4PL) curve fitting in Graph Pad Prism.

[0318] As illustrated in Figure 7 and Table 28, when tested in competitive ELISA, all PDL1 inhibitors blocked the interaction between PDL1 and PD-1. scFv PRO830 blocked the interaction with comparable potency, while PRO997 and PRO1013 showed significantly lower IC 50 values, i.e., they were potent inhibitors. When combined with the multispecific formats (i.e., scDbs or Morisons), all molecules maintained their inhibitory properties. PRO885 was not more potent than avelumab, while PRO1126, which contains an improved anti-PDL1 domain, showed low IC 50 values. The Morrison format had slightly lower potency when compared to avelumab. The neutralizing effect of PRO1057 in the presence of human serum albumin was also shown, but the IC 50 values were approximately two-fold higher.

[0319] PDL1 / B7-1 Competitive ELISA: ELISA microplates were coated overnight at 4 °C with 4 μg / ml of human B7-1 and washed three times with 450 μl of wash buffer per well. 300 μl of PBS (dilution buffer) containing 1% BSA and 0.2% Tween was added to each well and the plates were blocked for 1 h at room temperature. Serial three-fold dilutions of the inhibitors were prepared in dilution buffer containing 40 ng / ml of biotinylated PDL1 to give 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 microplates after three wash cycles with 450 μl of wash buffer per well. The plates were incubated for 1.5 h at room temperature with gentle shaking, washed three times with 450 μl of wash buffer per well, and then 10 ng / ml of streptavidin-poly HRP40 was added to the wells of each microplate. After incubation for 1 h at room temperature, the plates were washed three times with 450 μl of wash buffer, and TMB was added. After 6 min, 1 M HCl was added to stop the enzyme reaction and the absorbance was measured at 450 nm using 690 nm as the reference wavelength. IC 50For value calculation, a 4-parameter logistic (4PL) curve fit was performed in Graph Pad Prism using the value obtained by subtracting the control.

[0320] For all PDL1 inhibitors except PRO1126, their ability to block the interaction between B7-1 and PD-1 was tested. PRO830 showed a potency equivalent to that of avelumab, while PRO997 and PRO1013 had low IC 50 values. All scDb and moricizine also inhibited the interaction between PDL1 and B.7-1. scDb PRO885 showed a potency equivalent to that of avelumab, while the IC 50 value of moricizine was approximately 2 - 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 by PDL1 block and simultaneous CD137 stimulation in human PBMCs stimulated with superantigen SEA using a cell-based assay: In this experiment, the synergistic effect of PD-1 / PDL1 inhibition and CD137 agonism was evaluated. In the assay, peripheral blood mononuclear cells (PBMCs) stimulated with the superantigen Staphylococcal enterotoxin A (SEA) were used to induce the expression of PDL1 on antigen-presenting cells (APCs) and T cells, and CD137 on T cells, respectively. By applying the anti-PDL1×CD137 molecule, two T cell regulatory signaling pathways were simultaneously targeted, namely, 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). The activation of T cells by the secretion of interleukin-2 (IL-2) was evaluated and compared with the effect mediated by PDL1 inhibition mediated by the benchmark control antibody, abelumab. In addition, PRO997, an anti-PDL1 scFv, was tested and compared with abelumab in the same experimental setup.

[0323] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human whole blood by density gradient centrifugation. Next, NK cells were depleted from the PBMCs using an anti-CD56 antibody and a MACS cell separation kit (Miltenyi Biotec). Next, 100,000 PBMCs per well were added to 96-well plates, followed by the preparation and addition of dilution series of PRO885, PRO997, and abelumab in assay buffer containing SEA at a concentration of 10 ng / ml. After culturing for 96 hours at 37°C and 5% CO2, the cell supernatants were collected, and the human interleukin-2 (IL-2) levels in the culture supernatants were quantified using the IL-2 Human ELISA MAX assay from BioLegend according to the kit's instructions. The IL-2 concentration was interpolated from the IL-2 standard curve and back-calculated and plotted against the concentrations of abelumab and PRO885 for EC 50 value calculation.

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

[0325]

Table 43

[0326] Example 11: Evaluation of the antitumor efficacy of anti-PDL1 antibodies in the human cell line-derived lung cancer xenograft model HCC827: The antitumor activity of the anti-PDL1 IgG1 antibody PRO1137 (SEQ ID NOs: 90 and 91) was evaluated in a human HCC827 NSCLC xenograft using the immunodeficient NOG mouse strain from Taconic and allogeneic human peripheral blood mononuclear cells. The transplanted human T lymphocytes exhibit foreign reactivity against foreign major histocompatibility (MHC) class I and II and other antigens derived from mouse cells. As a result, the T lymphocytes show inflammatory infiltration into various organs, leading to the death of the animals after a few weeks, and this process is known as xenogeneic graft-versus-host disease (xGVHD). Treatment with immunomodulatory antibodies (e.g., anti-PDL1 and anti-CD137) has been shown to exacerbate 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] Test setup and treatment schedule: Female NOG mice were injected with 5×10 6 HCC827 cells per limb. The cells were injected in a total volume of 100 μl in a mixture of 50% cell suspension in PBS and 50% Matrigel. After injecting the tumor cells into the NOG mice and achieving successful tumor engraftment (median group tumor volume of 80 - 100 mm 3 ), intravenous injection into the mice was replaced with 5×10 6 human PBMC. On the day of random assignment, four mice in each group were reconstituted with donor A's PBMC, and another four mice were reconstituted with donor B's PBMC. Treatment was initiated 1 - 2 hours after PBMC injection and applied as follows.

[0328]

Table 44

[0329] Based on the in vitro activity of an antibody that blocks the PD-1 / PDL1 interaction in an NF-AT reporter gene assay, the dose of PRO1137 to obtain the same relative activity was set at 0.2 mg using the dose of 0.1 mg of abelumab (per mouse) as a model. Therefore, the dose of 0.2 mg of PRO1137 is comparable to the dose of 0.1 mg of abelumab and can be expressed as 1 relative unit (1 r.U). Body weight measurements and caliper measurements of tumor volume were performed twice a week. Animals were sacrificed at predetermined time points according to the test results. All animals were sacrificed at the "same" time points (days 17 and 18). Due to processing capacity reasons, sample collection and processing were performed on the first half of each group on day 1, and the same sample collection and processing were performed on the remaining second half of each group the next day. Animals reconstituted with PBMC from two different donors appropriately reflected the two sampling cohorts.

[0330] Results: The antitumor activity of anti-PDL1 PRO1137 in human HCC827 NSCLC xenografts using immunodeficient NOG mouse strain 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 relative tumor volume to the tumor volume at the start of treatment. As shown in Figure 10, treatment with the PRO1137 monoclonal antibody showed a reduction in tumor growth compared to the vehicle control group. In particular, treatment with PRO1137 did not result in a decrease in body weight (median value), suggesting that the molecule is well tolerated at the test dose levels (Figure 11).

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

[0332] Test setup and treatment schedule: HCC827 NSCLC cells were injected subcutaneously into female NOG mice engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC). Five × 10 6 cells were injected into one limb of the mice. The cells were injected as a mixture of a 50% cell suspension in PBS and 50% Matrigel in a total injection volume of 100 μl. After injecting the tumor cells into the NOG mice and achieving successful tumor engraftment (median tumor volume of 80 - 100 mm 3 per group), the mice (n = 10) were randomized to treatment groups.

[0333]

Table 45

[0334] Body weight measurements and tumor volume measurements by caliper were performed twice a week. 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 an immunodeficient NOG mouse strain engrafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSC) was evaluated by measuring tumor volume (Figure 12). Tumor volume was measured twice a week and the mice were euthanized on days 25, 29, and 30. Tumor volume was normalized as relative tumor volume to the tumor volume at the start of treatment. As shown in Figure 12, treatment with PRO1196 and atezolizumab resulted in suppression 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 MC38 colorectal cancer model bearing cancer In addition, the antitumor activity of the multispecific antibody containing the PDL1 domain of the present invention can be tested in the MC38 colorectal cancer model of cancer-bearing C57BL / 6 mice having a complete immune system. This model has also been used elsewhere, and enhanced antitumor activity has been shown by 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 domain nor the anti-PDL1 domain of the multi-specific antibody to be tested cross-reacts with mouse PDL1 and mouse CD137, the engineered human CD137 knock-in model established by CrownBio can be used. In this model, the extracellular and transmembrane domains of mouse CD137 were replaced with the respective sequences of human CD137 in the C57BL / 6 mouse background using the CRISPR / Cas9 system. Additionally, a modified MC38 tumor cell line expressing human PDL1 instead of mouse PDL1 under the control of the CMV promoter can be used. The effect of the multi-specific antibody on tumor volume can be compared with combination therapies with humanized IgG1 containing the same PDL1-specific variable domain as the multi-specific antibody and humanized IgG4 having the same CD137-specific variable domain. To obtain further evidence of a local anti-tumor immune response, the numbers of tumor-infiltrating lymphocytes (e.g., CD8+, CD4+ and regulatory T cells) can be analyzed by flow cytometry. To globally test the modulation of the immune system following anti-CD137 / anti-PDL1 treatment, the numbers of CD4+ and CD8+ T cells in the liver and spleen can be analyzed by flow cytometry and perhaps immunohistochemistry. Furthermore, systemic IFNγ levels can be analyzed using a quantitative ELISA method. Additionally, to characterize the safety profile of the anti-CD137 / anti-PDL1 combination therapy, clinical chemical pathology parameters associated with high levels of liver toxicity (e.g., alanine aminotransferase, glutamate dehydrogenase and aspartate aminotransferase), mainly observed in anti-CD137 therapy during clinical practice, can be evaluated.

Claims

1. An isolated antibody having binding specificity to human PDL1, comprising, respectively, the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, and, respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 17, 18, and 19, and an antibody containing the same.

2. The antibody according to claim 1, wherein the antibody comprises a heavy chain variable region (VH), and the VH is VH3 or VH4, preferably VH3.

3. The antibody comprises a light chain variable region (VL), and the VL is Vκ framework FR1, FR2, and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1, Vκ FR4, particularly Vκ1 FR4, Vκ3 FR4, and an amino acid sequence selected from VλFR4, particularly having 60% or more, 70% or more, 80% or more, 90% or more identity with an amino acid sequence selected from any of SEQ ID NOs: 64 to 70, preferably Vλ FR4 as described in any of SEQ ID NOs: 64 to 70, preferably Vλ FR4 as described in SEQ ID NOs: 64 or 65, more preferably Vλ FR4 as described in SEQ ID NO: 64, and a framework FR4 containing the same, and the antibody according to claim 1 or 2.

4. The antibody comprises a heavy chain variable region containing an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 14, 15, and 16, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16, and has 90% or more identity with the amino acid sequence, a light chain variable region containing an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 26 and 27, preferably SEQ ID NO: 27, and has 90% or more identity with the amino acid sequence, and the antibody according to any one of claims 1 to 3.

5. (a) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 26, or (b) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 26, or (c) the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 27, and the antibody according to any one of claims 1 to 4.

6. The antibody when measured by surface plasmon resonance (SPR), 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, (ii) When measured by SPR, binds to human PDL1 with a K of 10 －３ s －１ or less, 10 －４ s －１ or less, or 10 －５ s －１ or less, and ｏｆｆ is combined with (iii) When measured by SPR, for human PDL1, 10 ３ M －１ s －１ or more, 10 ４ M －１ s －１ or more, 10 ５ M －１ s －１ or more, or 10 ６ M －１ s －１ or more of K ｏｎ binds, (iv) It has cross-reactivity with cynomolgus PDL1, and in particular, when measured by SPR, it binds to cynomolgus PDL1 with a 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. (v) In particular, when measured by SPR, it does not have cross-reactivity with mouse PDL1. (vi) In particular, when measured by SPR, it does not bind to human PDL2. (vii) When measured by ELISA method, it has the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) more than 2, preferably more than 3, more preferably more than 4, compared to avelumab. The relative titer is the ratio of the ng / mL IC ５０ value of abciximab measured by the ELISA method to the ng / mL IC ５０ value of the antibody measured by the ELISA method, and In particular, the antibody is a scFv. (viii) When measured by NFAT reporter gene assay, it has the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) more than 2, preferably more than 3, more preferably more than 4, compared to avelumab. The relative titer is the ratio of the ng / mL IC ５０ value of abelumab measured in the NFAT reporter gene assay to the ng / mL IC ５０ value of the antibody measured in the NFAT reporter gene assay, and In particular, the antibody is a scFv, and / or (ix) When measured by ELISA method, it has the ability to neutralize the PDL1 / B7-1 interaction with a titer (relative titer) more than 2, preferably more than 3, more preferably more than 4, compared to avelumab. The relative titer is the ratio of the ng / mL IC ５０ value of abciximab measured by the ELISA method to the ng / mL IC ５０ value of the antibody measured by the ELISA method, and In particular, the antibody is a scFv. The antibody according to any one of claims 1 to 5.

7. The antibody is (i) When in scFv format, in particular when the antibody is prepared in 50 mM phosphate-citrate buffer with pH 6.4 and 150 mM NaCl, it has a melting temperature (Tm) of 60 °C or higher, preferably 65 °C or higher, more preferably 70 °C or higher when measured by differential scanning fluorimetry. (ii) When in scFv format, after subjecting the antibody of the present invention to 5 consecutive freeze-thaw cycles at an initial concentration of 10 mg / ml, in particular when the antibody of the present invention is prepared in 50 mM phosphate citrate buffer with pH 6.4 and 150 mM NaCl, it shows a loss of monomer content of less than 5%, preferably less than 3%, more preferably less than 1%, and / or (iii)When in scFv format, the antibody of the present invention, after storage at an initial concentration of 10 mg / ml at 4 °C for 2 weeks or more, particularly 4 weeks or more, particularly when the antibody is prepared in 50 mM phosphate-citrate buffer at pH 6.4, 150 mM NaCl, shows a loss of monomer content of less than 15%, for example less than 12%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1%. The antibody according to any one of claims 1 to 6.

8. The antibody according to any one of claims 1 to 7, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, Fab, Fv, scFv, dsFv, scAb, STAB, and ankyrin-based domains, fynomer, avimer, anticalin, a binding domain based on a scaffold substitute limited to fibronectin, and a binding site incorporated into the constant region of the antibody (e.g., F-star's Modular Antibody Technology (trademark)), preferably scFv.

9. The antibody according to claim 8, which is scFv and 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, preferably SEQ ID NO:

31.

10. The antibody according to any one of claims 1 to 7, wherein the antibody is a multispecific molecule, particularly the multispecific molecule has one or more second functional molecules.

11. A pharmaceutical composition comprising the isolated antibody according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. The antibody according to any one of claims 1 to 10 or the composition according to claim 11 for use as a medicament.

13. The antibody according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 for use in the treatment of cancer.

14. A nucleic acid encoding the antibody according to any one of claims 1 to 10.

15. A method for producing the antibody according to any one of claims 1 to 10, the method comprising culturing a host cell comprising the nucleic acid of claim 14.

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