Antibody targeting PDL1 and method using the same

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, as well as a pharmaceutical composition and method of use thereof. 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 technology] 【0002】 PDL1 (CD274, B7-H1) is a 40 kDa type I transmembrane protein. PDL1 is a membrane-surface glycoprotein ligand for PD-1 and is a major immune checkpoint receptor expressed by activated T cells and B cells, mediating immunosuppression. PDL1 is involved in the suppression of immune system responses in chronic infections, pregnancy, allogeneic tissue transplantation, autoimmune diseases, and cancer. PDL1 is known to be expressed in both antigen-donor cells and cancer cells in human head and neck squamous cell carcinoma, melanoma, and brain tumors, as well as in the thyroid, thymus, esophagus, lung, chest, gastrointestinal tract, colon, liver, pancreas, kidney, adrenal cortex, bladder, urothelium, ovaries, and skin (Non-Patent Literature 1-7). PDL1 is rarely expressed in normal tissues and is inductively expressed in tumor sites (Non-Patent Literature 8 and 9). By binding to PD-1, PDL1 downregulates T cell activation and cytokine secretion (Non-Patent Literature 10). PD-1 activated by PDL1 potentially provides an immune-tolerant environment for tumor development and growth. PDL1 also negatively regulates T cell function through interactions with other receptors 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 main categories: those that target PD-1 (nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck, White House Station, NJ), pidilizumab (CureTech, Yavne, Israel)) and those that target PDL1 (MPDL3280A (Genentech, South San Francisco, CA), MEDI4736 (MedImmune / AstraaZeneca), BMS-936559 (Bristol-Myers Squibb), MSB0010718C (EMD Serono, Rockland, MA)) (see Non-Patent Literature 11 for review). The resulting biological effects appear to differ between targeting PDL1 and targeting PD-1. PD-1 antibodies prevent interaction between PD-1 and its ligands, PDL1 and PDL2. PDL1 antibodies do not prevent interaction between PD-1 and PDL2, although 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 Literature 12), which is thought to result in negative signaling to T cells. Promising early data have been shown by blocking PDL1, and currently four anti-PDL1 mAbs, namely atezolizumab and MEDI4736 (both Fc-null mutants of human IgG1), MSB001078C (IgG1), and BMS-936559 (IgG4), are in clinical trials (Non-Patent Literature 13). 【0004】 To date, no satisfactory method for inducing a robust immune response in cancer patients has been disclosed. Therefore, there is a need in this field to obtain therapeutic modulators that improve the PDL1 / PD-1 interaction and to obtain methods for overcoming the immunosuppressive mechanisms observed in cancer patients. [Prior art documents] [Patent Documents] 【0005】 [Patent Document 1] International Publication No. 2013 / 079174 [Patent Document 2] International Publication No. 2017 / 118321 [Non-patent literature] 【0006】 [Non-Patent Document 1] Katsuya Y, et al., Lung Cancer.88(2):154-159(2015) [Non-Patent Document 2] Nakanishi J, et al., Cancer Immunol Immunother.56(8):1173-1182(2007) [Non-Patent Document 3] Nomi T, et al., Clin Cancer Res.13(7):2151-2157(2007) [Non-Patent Document 4] Fay AP, et al., J Immunother Cancer.3:3(2015) [Non-Patent Document 5] Strome SE, et al., Cancer Res.63(19):6501-6505(2003) [Non-Patent Document 6] Jacobs JF,et al.Neuro Oncol.11(4):394-402(2009) [Non-Patent Document 7] Wilmotte R,et al.Neuroreport.16(10):1081-1085(2005) [Non-Patent Document 8] Dong H, et al., Nat Med.8(8):793-800(2002) [Non-Patent Document 9] Wang et al., Onco Targets Ther.9:5023-5039(2016) [Non-Patent Document 10] Freeman et al.,2000;Latchman et al.,2001 [Non-Patent Document 11] Postow MA et al.,J Clin Oncol.Jun 10;33(17):1974-82(2015) [Non-Patent Document 12] Butte MJ, et al., Immunity 27:111-122, (2007) [Non-Patent Document 13] Chester C.,et al.,Cancer Immunol Immunother Oct;65(10):1243-8(2016) [Overview of the Initiative] [Problems that the invention aims to solve] 【0007】 The object of the present invention is to provide an antibody that specifically binds to the human PDL1 protein and has beneficial properties for therapeutic applications, such as high affinity and improved efficacy, as well as improved biophysical properties, such as solubility, spreadability, and stability. [Means for solving the problem] 【0008】 In one embodiment, the present invention relates to a novel PDL1 antibody. 【0009】 In one embodiment, 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 an antibody or composition of the present invention for use as a pharmaceutical agent. 【0011】 In one embodiment, the present invention relates to an antibody or composition of the present invention for use in the treatment of a subject requiring treatment for cancer. 【0012】 In one embodiment, the present invention relates to the use of an antibody or composition of the present invention in the manufacture of a therapeutic agent for a subject requiring treatment for cancer. 【0013】 In another embodiment, the present invention relates to a method for treating cancer in a subject who requires treatment for cancer, comprising administering a therapeutically effective amount of the antibody or 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 yet another aspect, the present invention relates to a vector comprising the nucleic acid. In yet another aspect, the present invention relates to a host cell comprising the nucleic acid or the vector. 【0015】 In another embodiment, the present invention relates to a method for producing the antibody of the present invention, the method comprising the step of culturing a host cell containing 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 sections, each individually or in combination, further contribute to solving the problems of the present invention: 【0017】 1. Isolated antibodies having binding specificity to human PDL1, including the following: (a) A heavy chain variable region CDR1 comprising, preferably consisting of, an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, preferably SEQ ID NO: 1 or 32, more preferably SEQ ID NO: 1, (b) A heavy chain variable region CDR2 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2, (c) A heavy chain variable region CDR3 comprising, preferably comprising, 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, (d) A light chain variable region CDR1 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 17, 20, 23, 48, 51, and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17, (e) A light chain variable region CDR2 comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 18, 21, 24, 49, 52, and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18, (f) A light chain variable region CDR3 comprising, preferably comprising, an amino acid sequence selected from any of SEQ ID NOs: 19, 22, 25, 50, 53, and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19. 【0018】 2. The antibody from item 1, which includes the following: (a) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively, (b) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively, (c) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 5, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively, (d) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 8, 9, and 10, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively, (e) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 11, 12, and 13, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 23, 24, and 25, respectively, (f) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively, (g) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively, (h) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 36, 37, and 38, respectively, and the LLCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively, (i) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 39, 40, and 41, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively, (j) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 42, 43, and 44, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 54, 55, and 56, respectively. 【0019】 3. Antibodies from item 1, including the following: (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1, (b) HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 2, (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 3, (d) LCDR1, which preferably consists of the amino acid sequence of SEQ ID NO: 17, (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 18, (f) LCDR3 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 19. 【0020】 4. Antibodies from item 1, including the following: (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, (b) HCDR2 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 6, (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 7, (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20, (e) LCDR2, which contains, preferably consists of, the amino acid sequence of SEQ ID NO: 21, (f) LCDR3 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 22. 【0021】 5. Antibodies from item 1, including the following: (a) HCDR1 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 32, (b) HCDR2 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 33, (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 34, (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 48, (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 49, (f) LCDR3 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 50. 【0022】 6. Antibodies from item 1, including the following: (a) HCDR1 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, (b) HCDR2 comprising, preferably comprising, the amino acid sequence of Sequence ID No. 37, (c) HCDR3 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 38, (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 51, (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 52, (f) LCDR3 comprising, preferably comprising, the amino acid sequence of Sequence ID No. 53. 【0023】 7. An antibody comprising a heavy chain variable region (VH), wherein the VH is VH1, VH3, or VH4, preferably VH3 or VH4, more preferably VH3, according to any one of the above items. 【0024】 8. The antibody includes 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, Framework FR4 comprising an amino acid sequence having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from Vκ FR4, particularly Vκ1 FR4, Vκ3 FR4, and VλFR4, and particularly selected from any of SEQ ID NOs: 64 to 70, preferably Vλ FR4 described in any of SEQ ID NOs: 64 or 65, more preferably Vλ FR4 described in SEQ ID NOs: 64, and a framework FR4, An antibody for any one of the above items, including the above. 【0025】 9. Antibodies, A heavy chain variable region containing an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NOs: 14, 15, 16, 45, 46, and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16, selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46, and 47, A light chain variable region containing an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NOs. 26, 27, 57, and 58, preferably SEQ ID NOs. 26 or 27, more preferably SEQ ID NOs. 27, An antibody for any one of the above items, including the above. 【0026】 10. Antibodies, (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) Heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 15 and light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 26, (c) Heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 16 and light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 27, (d) Heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 45 and 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) Heavy chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 47 and light chain variable region containing an amino acid sequence that is 90% or more identical to amino acid sequence number 57, An antibody for any one of the above items, including the above. 【0027】 11. Antibodies, A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46, and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16, A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27, An antibody for any one of the above items, including the above. 【0028】 12. Antibodies, (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, comprising. 【0029】 13. The antibody is (i) When measured by surface plasmon resonance (SPR), it 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 particularly, the antibody is scFv (monovalent affinity), (ii) When measured by SPR, it binds to human PDL1 with a K of 10 6 s -1 or less, 10 -4 s -1 or less, or 10 -5 s -1 or less of K off and binds, and particularly, the antibody is scFv. (iii) When measured by SPR, it binds to human PDL1 with a K of 10 3 M [[ID=�9]] -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 binds, and particularly, the antibody is scFv. (iv) Having cross-reactivity with Cynomolgus PDL1, and in particular binding to Cynomolgus PDL1 with a KD of less than 5 nM, especially less than 1 nM, especially less than 500 pM, especially less than 100 pM, preferably less than 10 pM, as measured by SPR, and in particular the antibody is scFv, (v) In particular, when measured by SPR, there was no cross-reactivity with mouse PDL1. and / or (vi) In particular, when measured by SPR, it does not bind to human PDL2. An antibody for any of the items mentioned above. 【0030】 14. Antibodies have the following characteristics: (i) When measured by ELISA, it has the ability to neutralize PDL1 / PD-1 interaction with a titer (relative titer) greater than 1.5, for example greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The aforementioned relative titer is the IC50 ng / mL of avelumab measured by ELISA. 50 The IC value of the antibody measured by ELISA is ng / mL. 50 It is the ratio to the value, In particular, the antibody is scFv, and / or (ii) Optionally, when measured by an NFAT reporter gene assay, it has the ability to neutralize the PDL1 / PD-1 interaction at a titer (relative titer) greater than 1.5, for example greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The aforementioned relative titer is the IC50 ng / mL of avelumab measured in the NFAT reporter gene assay. 50 The IC50 value of the antibody measured in the NFAT reporter gene assay, in ng / mL, is the IC50 value. 50 It is the ratio to the value, In particular, the antibody is 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, for example greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The aforementioned relative titer is the IC50 ng / mL of avelumab measured by ELISA. 50 The IC value of the antibody measured by ELISA is ng / mL. 50 It is the ratio to the value, In particular, the antibody is scFv, An antibody for any one of the items mentioned above. 【0031】 15. The antibody said above, (i) When in scFv format, when measured by differential scanning fluorescence quantitative analysis, it has a melting temperature (Tm) of 60°C or higher, preferably 65°C or higher, more preferably 70°C or higher. In particular, the antibody is formulated in 50 mM phosphate-citrate buffer, pH 6.4, and 150 mM NaCl. (ii) When in scFv format, if the antibody of the present invention is at an initial concentration of 10 mg / ml, after 5 consecutive freeze-thaw cycles, it shows a monomer content loss of less than 5%, preferably less than 3%, more preferably less than 1%. In particular, the antibody is formulated in 50 mM phosphate-citrate buffer, 150 mM NaCl, pH 6.4, and / or (iii) When in scFv format, if the antibody of the present invention is at an initial concentration of 10 mg / ml, after storage at 4°C for 2 weeks or more, particularly 4 weeks or more, 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%. In particular, the antibody of the present invention is formulated in 50 mM phosphate citrate buffer, 150 mM NaCl, pH 6.4. An antibody for any of the items mentioned above. 【0032】 16. An antibody of any of the above items, selected from the group consisting of a monoclonal antibody, a chimeric antibody, Fab, Fv, scFv, dsFv, scAb, and a binding domain based on a scaffold substitute limited to an ankyrin-based domain, fynomer, avimer, anticalin, and fibronectin, and a binding site incorporated into the constant region of the antibody (e.g., F-star's Modular Antibody Technology®). 【0033】 17. An antibody for any one of the above items, wherein the antibody is a single-chain variable partial 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, more preferably having 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. An antibody of any of the above items, wherein the antibody is a chimeric or humanized. 【0037】 21. An antibody that binds to essentially the same epitope as one of the antibodies listed in items 1-20. 【0038】 22. An antibody of any one of the above items, which is a multispecific molecule, and in particular a multispecific molecule having one or more secondary functional molecules. 【0039】 23. The antibody may be a single-chain diabody (scDb), tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), bispecific T cell binding site (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-mini antibody, tetrabody, scFv-Fc-scFv fusion, scFv-HSA-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, bsAb (scFv linked to the C-terminus of the light chain), Bs1Ab (scFv linked to the N-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 (s Antibodies of item 22, in a format selected from the group consisting of IgG-scFv fusions such as cFv, Ts2Ab (dsscFv linked to the C-terminus of the heavy chain), bispecific antibodies based on heterodimeric Fc domains such as Knob-into-Hole antibodies (KiHs), Fv, scFv, scDb, tandem-di-scFv, tandem-tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD, MATCH, and DuoBodie fused to the N and / or C-terminus of either one heterodimeric Fc domain or the other heterodimeric domain. 【0040】 24. A pharmaceutical composition comprising one antibody from items 1 to 23 and a pharmaceutically acceptable carrier. 【0041】 25. A composition of one antibody from items 1 to 23 or item 24, for use as a pharmaceutical agent. 【0042】 26. A composition of one antibody from item 1 to 23 or item 24 for use in the treatment of a subject requiring treatment for cancer. 【0043】 27. Use of any one antibody from items 1 to 23 or the composition of item 24 for the treatment of cancer in a subject requiring treatment. 【0044】 28. Use of any one antibody from items 1 to 23 or a composition of item 24 for the manufacture of a therapeutic agent in a subject requiring treatment for cancer. 【0045】 29. A method for treating cancer in a subject who requires treatment for cancer, comprising administering a therapeutically effective amount of one antibody from item 1 to 23 or a composition of item 24 to the subject. 【0046】 30. A nucleic acid that codes for one of the antibodies listed in items 1-23. 【0047】 31. A vector containing the nucleic acid of item 31. 【0048】 32. Host cells containing the nucleic acid of item 31 or the vector of item 32. 【0049】 33. A method for producing one antibody from any one of items 1 to 23, comprising the step of culturing a host cell containing the nucleic acid of item 31 or the vector of item 31. 【0050】 34. A kit containing one antibody from items 1 to 23 or the composition of item 24. [Brief explanation of the drawing] 【0051】 [Figure 1] This study demonstrates the neutralization of the PDL1 / PD-1 interaction by a rabbit IgG clone with the best affinity for PDL1. Absorbance, measured by ELISA, is expressed in ng / ml as a function of the molecular concentration of 33-03-G02(A) or 37-20-B03(B). Avelumab was used as a control. [Figure 2]This study demonstrates the neutralization of the PDL1 / B7-1 interaction by rabbit IgG clones 33-03-G02(A) or 37-20-B03(B), which have the best affinity for PDL1. Absorbance measured by ELISA is expressed as a function of molecular concentration in ng / ml. Avelumab was used as a control. [Figure 3] This study demonstrates the neutralization of the PDL1 / PD-1 interaction by a selected rabbit IgG clone exhibiting the best affinity for PDL1 in a cell-based reporter gene assay. The percentage inhibition rate corresponding to the luminescence signal obtained in the assay is expressed as a function of molecular concentration in ng / ml. Avelumab was used as a control. [Figure 4] Figure 4A shows the effect of CDR set and framework selection on the neutralization of the PDL1 / PD-1 interaction in the NFAT-luciferase reporter gene assay. The percentage inhibition rate corresponding to the luminescence signal obtained in the assay is expressed in ng / ml as a function of scFv concentration. Avelumab was used as a control. Figure 4B shows the effect of domain optimization on the neutralization titer of the PDL1 / PD-1 interaction in the NFAT-luciferase reporter gene assay. The percentage inhibition rate corresponding to the luminescence signal obtained in the assay is expressed in ng / ml as a function of scDbs concentration. Avelumab was used as a control. [Figure 5] This report shows the neutralizing titers of the PDL1 / PD-1 interaction by scDb-scFvs PRO963 and PRO1057 (A), PRO1186 and PRO1430 (B), PRO1431 and PRO1432 (C), PRO1473 (D), PRO1476 (E), PRO1479 (F), and PRO1482 (G) in the presence of recombinant human serum albumin in reporter gene assays. The percentage inhibition rate corresponding to the luminescence signal obtained in the assay is expressed as a function of molecular concentration in ng / ml. Avelumab was used as a control. [Figure 6]This report describes the effect of the titer of the divalent molecule and the Morrison-format LC or HC scFv fusion on the neutralizing titer of the PDL1 / PD-1 interaction in the NFAT-luciferase reporter gene assay. The percentage of inhibition corresponding to the luminescence signal obtained in the assay is expressed as a function of molecular concentration in ng / ml. Avelumab was used as a control. [Figure 7] PD-1 / PDL1 competitive ELISA. All molecules potently blocked the interaction between PD-1 and PDL1 with IC50 values ​​comparable to or lower than those of the control IgG, avelumab. [Figure 8] B7-1 / PDL1 competitive ELISA. All molecules, like avelumab, potently blocked the interaction between B7-1 and PDL1. [Figure 9] Ex vivo T cell activation assay. PBMCs were stimulated with 10 ng / ml SEA and treated for 96 hours with serial dilutions of scFv PRO997 or scDb PRO885. T cell activation was evaluated by IL-2 quantification by ELISA of the recovered supernatant. Treatment with PRO885 and PRO997 resulted in significant IL-2 secretion. PRO997 showed a higher titer than avelumab. PRO885 showed a significantly higher efficacy compared to avelumab. Data were fitted using sigmoid4PL fit (GraphPad Prism). [Figure 10]This study demonstrates the antitumor activity of anti-PDL1 IgG1 (PRO1137) therapy in human HCC827 NSCLC xenograft using immunodeficient NOG mouse strains and allogeneic human peripheral blood mononuclear cells (hPBMCs). Mice were administered either anti-PDL1 IgG1 (PRO1137) or a vehicle via ip administration on days 0, 3, 7, and 10. Tumor volume was measured twice weekly, 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. (A) Mean relative tumor volume in mice reconstituted with PBMCs from two donors (n=8 mice per group). The dotted line indicates the number of treatments. (B) Mean relative tumor volume in mice reconstituted with PBMCs from donor B (n=4 mice per group). (C) Individual relative tumor volume in mice reconstituted with PBMCs from two donors. Each symbol represents an individual animal within the same treatment group. (D) Individual relative tumor volumes in mice reconstituted with PBMCs derived from donor B. Each symbol represents an individual animal within the same treatment group. [Figure 11] This shows xenotransplantation of HCC827 in NOG mice replaced with hPBMCs. It also shows the body weight of NOG mice loaded with HCC827 after treatment with anti-PDL1 IgG1 (PRO1137). Body weight was measured twice weekly, and mice were euthanized on days 17 and 18. Body weight was normalized as relative body weight to body weight at the start of treatment. [Figure 12] This study evaluates the antitumor effect of anti-PDL1 IgG1 (PRO1196) in human HCC827 NSCLC xenografting in NOG mice grafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs). The antitumor activity of PRO1196 (0.1 mg) was compared with avelumab (0.1 mg) or vehicle treatment (palivizumab, 0.1 mg). Mice were treated on days 0, 5, 10, 15, and 20 (dotted line). Tumor growth and body weight were recorded twice weekly. Tumor volume was normalized as relative tumor volume to the tumor volume at the start of treatment. [Modes for carrying out the invention] 【0052】 This invention provides an antibody that specifically binds to the human PDL1 protein, a pharmaceutical composition, a method for producing such an antibody and composition, and a method for using such an antibody and composition. 【0053】 Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those generally understood by those skilled in the art relating to the present invention. 【0054】 The terms “include” and “inclusive” are used in this specification in their open-ended, non-restrictive sense unless otherwise specified. Therefore, with respect to such latter embodiments, the term “include” encompasses its narrower sub-concept, “consisting of.” 【0055】 In the context describing this invention, the terms "a," "an," "the," and similar terms are to be singular and plural unless explicitly stated in this specification or clearly denied in the context (particularly in the context of the claims described later). For example, the term "cell" encompasses multiple cells and mixtures thereof. When the plural form is used for compounds, salts, etc., it may also mean a single compound, salt, etc. 【0056】 In a first embodiment, the present invention relates to an antibody that specifically binds to human PDL1. 【0057】 The terms "antibody" and other similar terms used herein include the following: All antibodies or their single chains, and any antigen-binding fragments thereof (i.e., “antigen-binding moieties”) or their single chains, and molecules containing antibody CDRs, VH regions, or VL regions (including, but not limited to, multispecific antibodies). Naturally occurring “all antibodies” are glycoproteins containing two or more heavy (H) chains and two or more light (L) chains linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this specification) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this specification) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further divided into a hypervariable region called the complementarity-determining region (CDR) and scattered, more conserved regions called the framework region (FR). Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody is thought to mediate the binding of immunoglobulins to host tissues or factors, 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,” “the antigen-binding fragment,” and “antigen-binding moiety” refer to one or more fragments of a complete antibody that possess the ability to specifically bind to a given antigen (e.g., PDL1). Examples of binding fragments encompassed by the term “antigen-binding moiety” of an antibody include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinking in a hinge region, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of VL and VH domains of a single arm of the antibody, and binding domains based on scaffold substitutes limited to ankyrin-based domains, fynomers, avimers, anticalin, and fibronectin, as well as binding sites incorporated into the constant region of the antibody (e.g., F-star's Modular Antibody Technology®). 【0059】 The term "complementarity-determining region" ("CDR") is used in 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), and ImMunoGenTics (IMGT) numbering system (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999), Lefranc, M.-P. et al. This refers to the amino acid sequence of a specific region determined using one of many well-known methods, such as those described in al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering system) and Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering system). For example, in Kabat's classical format, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered as 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered as 24-34 (LCDR1), 50-56 (LCDR2), and In Chothia, the CDR amino acid residues of VH are numbered as 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), while the CDR amino acid residues of VL are numbered as 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Combining the Kabat and Chothia CDR definitions, 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 CDR of an antibody can be determined using the IMGT / DomainGap Align program. In the context of this invention, unless otherwise specified, the numbering scheme proposed by Honegger & Pluckthun ("AHo") (Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670) is used. 【0060】 Furthermore, the following residues are defined as CDRs according to the AHo numbering scheme: LCDR1 (also referred to as CDR-L1): L24-L42; LCDR2 (also known as CDR-L2): L58-L72; LCDR3 (also known as CDR-L3): L107-L138; HCDR1 (also known as CDR-H1): H27-H42; HCDR2 (also known as CDR-H2): H57-H76; HCDR3 (also known as CDR-H3): H108-H138. 【0061】 For the sake of clarity, the numbering system by Honegger and Pluckthun is well-suited to explaining the slight variations in length and sequence gaps observed in naturally occurring antibodies across different VH and VL subfamilies, particularly in CDRs. Therefore, in a given antibody variable domain, not all positions from 1 to 149 are typically occupied by amino acid residues. 【0062】 The antigen-binding moiety may be incorporated into 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 moiety of an antibody may be fused to a polypeptide-based scaffold, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, describing the monobody of the fibronectin polypeptide). A pair of antigen-binding regions may be formed by incorporating the antigen-binding moiety, along with a complementary light chain polypeptide, into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1) (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” refers to the ability of an individual antibody to react with one antigenic determinant and not with a different antigenic determinant. As used in this specification, “specifically binding” or “specific” refers to a significant and reproducible interaction, such as 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) is an antibody that binds to that target more readily and / or more strongly, with stronger affinity and binding activity than it binds to other targets. “Specific binding” in its broadest sense (and unless otherwise defined) refers to the ability of an antibody to distinguish molecules unrelated to the target of interest, as determined, for example, by specificity assay methods of known art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) assays, and peptide scans. For example, a standard ELISA assay can be performed. Scoring can be performed by a standard colorimetric reaction (e.g., a reaction between a secondary antibody containing horseradish peroxide 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) may have an OD of approximately 0.1, and a typical positive reaction may have an OD of approximately 1. This means that the ratio between positive and negative scores can be 10-fold or more. As a further example, an SPR assay can also 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, the determination of binding specificity is performed not by using a single control molecule, but by using approximately 3-5 unrelated molecules (e.g., skim milk, transferrin, etc.). The antibody of the present invention has binding specificity to human PDL1. In certain embodiments, the antibody of the present invention has binding specificity to human PDL1, particularly in SPR measurements, but does not bind to human PDL2. 【0064】 Appropriately, the antibodies of the present invention are isolated antibodies. 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 antibodies that specifically bind to antigens other than PDL1). However, an isolated antibody that specifically binds to PDL1 may have cross-reactivity with other antigens (such PDL1 molecules from other species). Therefore, in one embodiment, the antibody of the present invention has binding specificity to human PDL1 and PDL1 of cynomolgus monkeys (also known as Cynomolgus monkeys or “Cynomolgus”). Furthermore, the isolated antibody may substantially not contain other cell-derived materials and / or chemicals. 【0065】 More appropriately, the antibodies of the present invention are monoclonal antibodies. As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” mean an antibody that is substantially identical to, or derived from, an amino acid sequence of the same gene source. Monoclonal antibody compositions exhibit binding specificity and affinity to a specific single epitope, or to a specific group of epitopes. 【0066】 The antibodies of the present invention include, but are not limited to, chimeric and humanized antibodies. 【0067】 The term "chimeric antibody" is, (a) A chimeric antibody in which the constant region or a portion thereof is modified, substituted, or exchanged so 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 (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.), thereby conferring novel properties, or (b) A variable region or a portion thereof that has been modified, replaced, or exchanged with a variable region having different or modified antigen specificity. It is one of the following antibody molecules. For example, a mouse antibody can be modified by substituting its constant region with the constant region of a human immunoglobulin. This substitution with the human constant region can reduce the antigenicity of the chimeric antibody in humans compared to the original mouse antibody, while maintaining its specificity in recognizing the antigen. 【0068】 The “humanized” antibodies used herein are antibodies that exhibit low immunogenicity in humans while maintaining the reactivity of non-human antibodies. This can be achieved, for example, by maintaining the non-human CDR region and substituting the remaining portion of the antibody (i.e., the framework portion of the constant and variable regions) with their human counterparts. Further modifications of the framework region may be performed within the human framework sequence or within the CDR sequence derived from the germline of other mammalian species. The humanized antibodies of the present invention may include amino acid residues not encoded by the human sequence (for example, by random or site-directed mutagenesis in vitro, or by somatic mutation 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. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of humanization techniques include, but are not limited to, the Xoma technique 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, including, for example, antibodies isolated from a transfectome, from host cells transformed to express a humanized antibody, and antibodies prepared, expressed, constructed or isolated by any other means, including splicing of all or part of a human immunoglobulin gene (a sequence against the base sequence of other DNA). 【0070】 The term "PDL1" specifically refers to human PDL1 having UniProt ID number Q9NZQ7, which is represented in this specification as Sequence ID No. 63. Preferably, the antibody of the present invention targets PDL1, and in particular human PDL1 represented by UniProt ID number Q9NZQ7, which is represented in this specification as Sequence ID No. 63. Preferably, the antibody of the present invention targets human and cynomolgus monkey (Macaca fascicularis) PDL1 when measured by surface plasmon resonance (SPR), and preferably does not cross-react with mouse PDL1. Preferably, the antibody of the present invention has binding specificity to human PDL1. In particular, the antibody of the present invention does not bind to human PDL2 when measured by SPR. 【0071】 The antibody of the present invention is a PDL1 inhibitor. The terms “blocker,” “blocking antibody,” “inhibitor,” “inhibiting antibody,” “antagonist,” or “antagonist antibody” refer to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The antibody of the present invention targets, reduces, or inhibits the binding ability of PDL1 to its binding partner, thereby interfering with the function of PDL1. In particular, the antibody of the present invention blocks the interaction of PDL1 with PD-1. In some embodiments, the antibody of the present invention blocks the interaction of PDL1 with PD-1 and B7-1. 【0072】 The antibodies of the present invention described herein include, but are not limited to, isolated humanized monoclonal antibodies as described in the examples. Examples of such anti-human PDL1 antibodies are antibodies having the sequences listed in Table 1. Further details regarding the preparation and characterization of the antibodies described herein are provided in the examples. 【0073】 The isolated antibody of the present invention, which has binding specificity to human PDL1, comprises the following heavy chain variable region (VH) and light chain variable region (VL): (a) The VH includes, in order, three complementary determination regions: HCDR1, HCDR2, and HCDR3. (b) The VL includes three complementary decision regions, in order: LCDR1, LCDR2, and LCDR3. 【0074】 The present invention provides an antibody that specifically binds to the PDL1 protein, wherein the antibody comprises a VH CDR having one of the amino acid sequences of the VH CDRs listed in Table 1. In particular, the present invention provides an antibody that specifically binds to the PDL1 protein, wherein the antibody comprises a VH CDR having one, two, three or more amino acid sequences of the VH CDRs listed in Table 1. 【0075】 The present invention also provides an antibody that specifically binds to the PDL1 protein, wherein the antibody comprises a VL CDR having one of the amino acid sequences of the VL CDRs listed in Table 1. In particular, the present invention provides an antibody that specifically binds to the PDL1 protein, wherein the antibody comprises a VL CDR having one, two, three or more amino acid sequences of the VL CDRs listed in Table 1. 【0076】 Other antibodies of the present invention include those that contain mutated amino acids but specifically bind to the PDL1 protein and have a CDR region 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 the CDR region represented by the sequence listed in Table 1. In one embodiment, 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 compared to the CDR region represented by the sequence listed in Table 1. 【0077】 In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "identity" refer to the fact that two or more sequences or subsequences are identical. "Percent (%) sequence identity" and "homology" in relation to nucleic acid, peptide, polypeptide, or antibody sequences are defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to a particular nucleic acid, peptide, or polypeptide sequence, provided that a sequence alignment is created, gaps are introduced as necessary to achieve the maximum possible sequence identity %, and no conservative substitutions are considered as part of the sequence identity. Alignment aimed at determining amino acid sequence identity % can be performed in various ways, which are within the scope of the art in the prior art, for example, by using publicly available computer software (e.g., BLAST, BLAST-2, or ALIGN software). Those skilled in the art can determine appropriate parameters for alignment calculation, including any algorithms necessary to achieve the maximum possible alignment over the full length of the sequences to be compared. 【0078】 For sequence comparison, typically, one sequence is used as a control sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and control sequences are input into a computer, sub-sequence coordinates are assigned as needed, and parameters for the sequence algorithm program are assigned. Default program parameters can be used, or alternative parameters can be assigned. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence in comparison with the control sequence, based on the program parameters. 【0079】 Two examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, 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 the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties, 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 PAM250 matrix, with 16, 14, 12, 10, 8, 6, or 4 gap weights and 1, 2, 3, 4, 5, or 6 length weights. 【0080】 The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to natural amino acids. Natural amino acids refer to those encoded by the genetic code, as well as retrospectively modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. The terms "polypeptide" and "protein" are used interchangeably in this specification when relating to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics 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 includes its conservatively modified variants. 【0081】 The present invention provides an isolated antibody having binding specificity to human PDL1, and the antibody is (a) A heavy chain variable region CDR1 (HCDR1) preferably comprising, and more preferably consisting of, an amino acid sequence of SEQ ID NO: 1 or 32, more preferably SEQ ID NO: 1, selected from any one of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39 and 42, (b) A heavy chain variable region CDR2 (HCDR2) comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40 and 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2, (c) A heavy chain variable region CDR3 (HCDR3) comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 3, 7, 10, 13, 34, 38, 41 and 44, preferably SEQ ID NO: 3 or 34, more preferably SEQ ID NO: 3, (d) A light chain variable region CDR1 (LCDR1) comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 17, 20, 23, 48, 51, and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17, (e) A light chain variable region CDR2 (LCDR2) comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 18, 21, 24, 49, 52, and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18, (f) A light chain variable region CDR3 (LCDR3) comprising, preferably consisting of, an amino acid sequence selected from any of SEQ ID NOs: 19, 22, 25, 50, 53, and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19, Includes. 【0082】 Appropriately, the isolated antibody of the present invention having binding specificity to human PDL1 is, (a) A heavy chain variable region CDR1 (HCDR1) preferably comprising a 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, which 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 amino acid sequence of SEQ ID NO: 1, (b) A heavy chain variable region CDR2 (HCDR2) preferably comprising a 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, which 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 amino acid sequence of SEQ ID NO: 2, (c) A heavy chain variable region CDR3 (HCDR3) preferably comprising a 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, which 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 amino acid sequence of SEQ ID NO: 3, (d) A light chain variable region CDR1 (LCDR1) preferably comprising a 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, which 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 amino acid sequence of SEQ ID NO: 17, (e) A light chain variable region CDR2 (LCDR2) preferably comprising a 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, which 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 amino acid sequence of SEQ ID NO: 18, (f) A light chain variable region CDR3 (LCDR3) preferably comprising a 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, which 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 amino acid sequence of SEQ ID NO: 19, Includes. 【0083】 In one embodiment, the antibody of the present invention having binding specificity to human PDL1 is, (a) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively, (b) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively. , and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively, (c) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 5, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively, (d) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 8, 9, and 10, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively, (e) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 11, 12, and 13, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 23, 24, and 25, respectively, (f) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively, (g) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively, (h) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 36, 37, and 38, respectively, and the LLCDR1, LCDR2, and CDR3 sequences of sequence numbers 51, 52, and 53, respectively, (i) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 39, 40, and 41, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively, (j) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 42, 43, and 44, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 54, 55, and 56, respectively, Includes. 【0084】 In one embodiment, the antibody of the present invention having binding specificity to human PDL1 comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs. 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs. 17, 18, and 19, respectively. In another embodiment, the antibody of the present invention having binding specificity to human PDL1 comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs. 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs. 48, 49, and 50, respectively. 【0085】 Appropriately, the antibody of the present invention having binding specificity to human PDL1 is, (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 with SEQ ID NOs. 17, 18, and 19, respectively. (b) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 4, 6, and 7 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 20, 21, and 22 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, 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 sequence numbers 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 sequence numbers 20, 21, and 22 respectively. (d) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 8, 9, and 10 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 17, 18, and 19 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. (e) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 11, 12, and 13 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 23, 24, and 25 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. (f) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 32, 33, and 34 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 48, 49, and 50 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. (g) HCDR1, HCDR2, and HCDR3 sequences that are identical to SEQ ID NOs. 35, 37, and 38 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to SEQ ID NOs. 51, 52, and 53 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. (h) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 36, 37, and 38 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 51, 52, and 53 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. (i) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 39, 40, and 41 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 48, 49, and 50 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, (j) HCDR1, HCDR2, and HCDR3 sequences that are identical to sequence numbers 42, 43, and 44 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively, and LCDR1, LCDR2, and LCDR3 sequences that are identical to sequence numbers 54, 55, and 56 by 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. Includes. 【0086】 In one embodiment, the antibody of the present invention having binding specificity to human PDL1 is, Each of the following HCDR1, HCDR2, and HCDR3 sequences have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with sequence numbers 1, 2, and 3, respectively. Each comprises 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. In other embodiments, the antibody of the present invention having binding specificity to human PDL1 is: Each of the following HCDR1, HCDR2, and HCDR3 sequences have 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with sequence numbers 32, 33, and 34, respectively. Each of these sequences includes LCDR1, LCDR2, and LCDR3 sequences that have identity with sequence numbers 48, 49, and 50 by 60 or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. 【0087】 Appropriately, the antibody of the present invention having binding specificity to human PDL1 is, (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1, (b) HCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 2, (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 3, (d) LCDR1, which preferably consists of the amino acid sequence of SEQ ID NO: 17, (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 18, (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 19, Includes. More appropriately, the antibody of the present invention having binding specificity to human PDL1 is, (a) An HCDR1 comprising, preferably consisting thereof, 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 with SEQ ID NO: 1, (b) An HCDR2 comprising, preferably 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 with SEQ ID NO: 2, (c) An HCDR3 comprising, preferably 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 with SEQ ID NO: 3, (d) LCDR1 comprising, preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 17, (e) LCDR2 comprising, preferably comprising, an amino acid sequence having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18, (f) LCDR3 comprising, preferably, 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 with SEQ ID NO: 19, Includes. 【0088】 In further embodiments, the antibody of the present invention having binding specificity to human PDL1 is, (a) HCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, (b) HCDR2 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 6, (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 7, (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20, (e) LCDR2, which contains, preferably consists of, the amino acid sequence of SEQ ID NO: 21, (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 22, Includes. More 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: 4 or SEQ ID NO: 5, preferably comprising the same. (b) An HCDR2 comprising, preferably 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 with SEQ ID NO: 6, (c) An HCDR3 comprising, preferably, 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 with SEQ ID NO: 7, (d) LCDR1 comprising, preferably consisting of, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 20, (e) LCDR2 comprising, preferably consisting thereof, 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 with SEQ ID NO: 21, (f) LCDR3 comprising, preferably, 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 with SEQ ID NO: 22, Includes. 【0089】 Appropriately, the antibody of the present invention having binding specificity to human PDL1 is, (a) HCDR1 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 32, (b) HCDR2 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 33, (c) HCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 34, (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 48, (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 49, (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 50, Includes. More appropriately, the antibody of the present invention having binding specificity to human PDL1 is, (a) an HCDR1 comprising, preferably 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 with SEQ ID NO: 32, (b) An HCDR2 comprising, preferably, an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 33, (c) An HCDR3 comprising, preferably 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 with SEQ ID NO: 34, (d) LCDR1 comprising, preferably, 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 with SEQ ID NO: 48, (e) LCDR2 comprising, preferably consisting thereof, 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 with SEQ ID NO: 49, (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 50, preferably consisting thereof, Includes. 【0090】 In further embodiments, the antibody of the present invention having binding specificity to human PDL1 is, (a) HCDR1 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, (b) HCDR2 comprising, preferably comprising, the amino acid sequence of Sequence ID No. 37, (c) HCDR3 comprising, preferably comprising, the amino acid sequence of SEQ ID NO: 38, (d) LCDR1 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 51, (e) LCDR2 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 52, (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 53, Includes. More appropriately, the antibody of the present invention having binding specificity to human PDL1 is, (a) an HCDR1 comprising, preferably 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 with SEQ ID NO: 35 or SEQ ID NO: 36, (b) An HCDR2 comprising, preferably 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 with SEQ ID NO: 37, (c) An HCDR3 comprising, preferably, 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 with SEQ ID NO: 38, (d) LCDR1 comprising, preferably, 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 with SEQ ID NO: 51, (e) LCDR2 comprising, preferably consisting thereof, 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 with SEQ ID NO: 52, (f) LCDR3 comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 53, Includes. 【0091】 In further embodiments, the present invention provides isolated antibodies that specifically bind to PDL1 (e.g., human PDL1 protein), wherein the antibodies comprise 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 heavy and light chain sequences of antibodies, classified according to sequence identity and homology. Methods for determining sequence homology using homology search matrices such as BLOSUM (Henikoff, S. & Henikoff, JG, 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, for example, as shown in Knappik et al., J.Mol.Biol.296(2000)57-86, which classifies VH into VH1A, VH1B, and VH2 through VH6, Vκ into Vκ1 through Vκ4, and Vλ into Vλ1 through Vλ3. In vivo, the Vκ, Vλ, and VH chains of an antibody are the result of random rearrangement of the V and J sites of the germline κ chain, the V and J sites of the germline λ chain, and the V, D, and J sites of the heavy chain, respectively. The subfamily to which a given antibody variable chain belongs is determined by the corresponding V site, particularly by the framework regions FR1 through FR3. Therefore, in the present invention, any VH sequence characterized by only a specific 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 sites of the heavy chain germline, or an HFR4 sequence derived from a rearranged VH sequence. 【0092】 More specifically, the present invention provides isolated antibodies that specifically bind to PDL1 (e.g., human PDL1 protein), wherein the antibodies include VH1A, VH1B, VH3, or VH4. 【0093】 A specific example of a VH belonging to the VH1 family is represented by Sequence ID No. 15. In particular, the framework regions FR1 to FR4 derived from Sequence ID No. 15 belong to the VH1 family (regions marked in Table 1, not in bold). Preferably, a VH belonging to the VH1 family as used herein is a VH containing FR1 to FR4 that has 85% or more, preferably 90% or more, and more preferably 95% or more sequence identity with FR1 to FR4 of Sequence ID No. 15. 【0094】 A specific example of a VH belonging to the VH3 family is represented by Sequence ID No. 16. In particular, the framework regions FR1 to FR4 derived from Sequence ID No. 16 belong to the VH3 family (regions marked in Table 1, not in bold). Optimally, the VH belonging to the VH3 family used herein is a VH containing FR1 to FR4 that has 85% or more, preferably 90% or more, and more preferably 95% or more sequence identity with FR1 to FR4 of Sequence ID No. 16. 【0095】 A specific example of a VH belonging to the VH4 family is represented by Sequence ID No. 14. In particular, the framework regions FR1 to FR4 derived from Sequence ID No. 14 belong to the VH4 family (regions marked in Table 1, not in bold). Optimally, the VH belonging to the VH4 family used herein is a VH containing FR1 to FR4 that has 85% or more, preferably 90% or more, and more preferably 95% or more sequence identity with FR1 to FR4 of Sequence ID No. 14. 【0096】 Examples of VH sequence substitutes are described in Knappik et al., J.Mol.Biol.296(2000)57-86. 【0097】 In one embodiment, the isolated antibody of the present invention contains a VH4 or VH3 domain. 【0098】 Preferably, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), wherein the antibody comprises Vκ frameworks FR1, FR2, and FR3, particularly Vκ1 or Vκ3 frameworks, preferably Vκ1 frameworks FR1 to FR3, and VκFR4, particularly framework FR4 selected from Vκ1 FR4, Vκ3 FR4, and Vλ FR4. Suitable Vκ1 frameworks FR1 to FR3 are described in SEQ ID NO: 26 (in Table 1, the FR region is marked in a non-bold font). Suitable Vκ1 frameworks FR1 to FR3 include amino acid sequences corresponding to FR1 to FR3 that have 60% or more, 70% or more, 80% or more, and 90% or more identity with the amino acid sequence derived from SEQ ID NO: 26 (in Table 1, the FR region is marked in a non-bold font). 【0099】 Examples of alternative Vκ1 sequences, as well as examples of Vκ2, Vκ3, and Vκ4 sequences, are described in Knappik et al., J.Mol.Biol.296(2000)57-86. 【0100】 Suitable Vλ FR4s are represented by SEQ ID NOs: 64 to 70. In preferred embodiments, Vλ FR4 is represented by SEQ ID NOs: 64 or 65, and more preferably by SEQ ID NOs: 64. In one embodiment, the present invention provides an isolated antibody that specifically binds to PDL1 (e.g., human PDL1 protein), wherein the antibody comprises a Vλ FR4 having an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NOs: 64 or 65, preferably SEQ ID NOs: 64, selected from any of SEQ ID NOs: 64 to 70, preferably SEQ ID NOs: 64 or 65, more preferably SEQ ID NOs: 64. 【0101】 Therefore, in one embodiment, the present invention provides an antibody comprising the following: (i) The following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences: a. The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively. b. The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively, or c. The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 36, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively. (ii) VH3 or VH4 domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1, A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ Vλ FR4 having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any of SEQ ID NOs. 64 to 70, preferably Vλ FR4 represented by any of SEQ ID NOs. 64, more preferably Vλ FR4 represented by SEQ ID NO. 64, and the Vλ FR4 represented by the Vκ1 FR4, Vκ3 FR4 and Vλ FR4, respectively, and a framework FR4, A VL domain that includes the VL framework. 【0102】 In other embodiments, the present invention provides an antibody having binding specificity to human PDL1, which includes: (i) the HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 5, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively. (ii) VH1A, VH1B, VH3 or VH4 domain framework sequence, preferably VH1A or VH1B domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1, A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ FR4, Vλ FR4 having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any of SEQ ID NOs. 64 to 70, preferably Vλ FR4 having an amino acid sequence of any of SEQ ID NOs. 70, more preferably Vλ FR4 represented by SEQ ID NO. 64, and A VL domain that includes the VL framework. 【0103】 In specific embodiments, the present invention provides an antibody comprising the following: (i) the HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively. (ii) VH3 or VH4 domain framework sequence, preferably VH4 domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1, A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ Vλ FR4 having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any of SEQ ID NOs. 64 to 70, preferably Vλ FR4 represented by any of SEQ ID NOs. 64, more preferably Vλ FR4 represented by SEQ ID NO. 64, and the Vλ FR4 represented by the Vκ1 FR4, Vκ3 FR4 and Vλ FR4, respectively, and a framework FR4, A VL domain that includes the VL framework. 【0104】 In a preferred specific embodiment, the present invention provides an antibody comprising: (i) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. (ii) VH3 or VH4 domain framework sequence, preferably VH3 domain framework sequence, and (iii) Vκ framework FR1, FR2 and FR3, particularly FR1 to FR3 of Vκ1 or Vκ3, preferably FR1 to FR3 of Vκ1, A framework FR4 selected from VκFR4, particularly Vκ1 FR4, Vκ3 FR4 and Vλ Vλ FR4 having 60% or more, 70% or more, 80% or more, or 90% or more identity with an amino acid sequence selected from any of SEQ ID NOs. 64 to 70, preferably Vλ FR4 represented by any of SEQ ID NOs. 64, more preferably Vλ FR4 represented by SEQ ID NO. 64, and the Vλ FR4 represented by the Vκ1 FR4, Vκ3 FR4 and Vλ FR4, respectively, and a framework FR4, A VL domain that includes the VL framework. 【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, especially human Vκ1 framework regions FR1 to FR3, (iii) (a) A sequence of human Vλ germline for FR4, particularly selected from SEQ ID NOs. 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 a human Vλ germline sequence having the amino acid sequence most closely related to FR4, selected from any of SEQ ID NOs. 64 to SEQ ID NOs. 70, preferably SEQ ID NO. 64, FR4 selected from The present invention provides an antibody containing VL (vein ligation) which includes the present antibody. 【0106】 The present invention provides isolated antibodies that specifically bind to PDL1 (e.g., human PDL1 protein), the antibodies comprising the VH domains listed in Table 1. 【0107】 The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising the VH amino acid sequence listed in Table 1, wherein the mutations in the framework sequence (e.g., a sequence other than CDR) (additions, substitutions, or deletions, as various non-limiting examples of mutations) are approximately 10 amino acids or less. 【0108】 The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising the VH amino acid sequence listed in Table 1, wherein the mutations in the framework sequence (e.g., a sequence other than CDR) (additions, substitutions, or deletions, as various non-limiting examples of mutations) 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 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 the VH region represented by the sequence listed in Table 1. 【0110】 The present invention provides an isolated antibody that specifically binds to the PDL1 protein, the antibody comprising the VL domains listed in Table 1. 【0111】 The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising the VL amino acid sequence listed in Table 1, wherein the mutations in the framework sequence (e.g., a sequence other than CDR) (additions, substitutions, or deletions, as various non-limiting examples of mutations) are approximately 10 amino acids or less. 【0112】 The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody comprising the VL amino acid sequence listed in Table 1, wherein the mutations in the framework sequence (e.g., a sequence other than CDR) (additions, substitutions, or deletions, as various non-limiting examples of mutations) are approximately 20 amino acids or less. 【0113】 Other antibodies of the present invention include those that contain mutated amino acids but specifically bind to PDL1 and have a VL region that 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 the VL region represented by the sequence listed in Table 1. 【0114】 The present invention also provides an isolated antibody that specifically binds to PDL1, the antibody being, 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 an amino acid sequence that is identical to 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, A light chain variable region comprising an amino acid sequence that is identical to, by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, 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 an amino acid sequence that is identical to, Includes. 【0115】 In one embodiment, the antibody of the present invention has binding specificity to human PDL1, A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 45, 46, and 47, preferably SEQ ID NO: 14 or 16, more preferably SEQ ID NO: 16, A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 57, and 58, preferably SEQ ID NO: 26 or 27, more preferably SEQ ID NO: 27, Includes. 【0116】 In one embodiment, the antibody of the present invention having binding specificity to human PDL1 includes the following: (a) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 14 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and VL sequences containing the same amino acid sequence as SEQ ID NO. 26 in proportion to 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 90% or more, (b) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 5, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 15 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and VL sequences containing the same amino acid sequence as SEQ ID NO. 26 in proportion to 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 90% or more, (c) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively. A VH sequence containing the same amino acid sequence as SEQ ID NO: 16 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably the VH containing the G56A and Y105F mutations (AHo numbering), and A VL sequence containing the same amino acid sequence as SEQ ID NO: 27 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably containing the S9A and A51P mutations (AHo numbering), (d) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 45 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and VL sequences containing the same amino acid sequence as SEQ ID NO: 57 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, (e) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 36, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively. A VH sequence containing the same amino acid sequence as SEQ ID NO: 46 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably containing V2S, V25A, I44V, G56A, V82K, F89V and Y105F mutations (AHo numbering), and A VL sequence containing the same amino acid sequence as SEQ ID NO: 58 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably the VL containing the I2F, M4L and A51P mutations (AHo numbering), or (f) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively. VH sequences that are identical to sequence number 47 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably containing V25A and I44V mutations (AHo numbering), and A VL sequence containing the same amino acid sequence as SEQ ID NO: 57 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. 【0117】 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 sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 14 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and VL sequences containing the same amino acid sequence as SEQ ID NO. 26 in proportion to 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 90% or more, (b) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 15 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and VL sequences containing the same amino acid sequence as SEQ ID NO. 26 in proportion to 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 90% or more, (c) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. A VH sequence containing the same amino acid sequence as SEQ ID NO: 16 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably the VH containing the G56A and Y105F mutations (AHo numbering), and A VL sequence containing the same amino acid sequence as SEQ ID NO: 27 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably containing the S9A and A51P mutations (AHo numbering), (d) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 45 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and VL sequences containing the same amino acid sequence as SEQ ID NO: 57 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, (e) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively. A VH sequence containing the same amino acid sequence as SEQ ID NO: 46 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably containing V2S, V25A, I44V, G56A, V82K, F89V and Y105F mutations (AHo numbering), and A VL sequence containing the same amino acid sequence as SEQ ID NO: 58 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and preferably the VL containing the I2F, M4L and A51P mutations (AHo numbering), or (f) The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively. A VH sequence that is identical to sequence number 47 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably the VH containing V25A and I44V, G56A, V82K and F89V mutations (AHo numbering), and A VL sequence containing the same amino acid sequence as SEQ ID NO: 57 by 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. 【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, and preferably comprising 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, and preferably comprising 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 amino acids or 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 sequences of the variable regions described in Table 1, while maintaining substantially the same activity. As used herein, the term "substantially the same activity" refers to the activity measured in the parental antibody, for example, compared with the antibodies of the present invention, particularly the antibodies of the present invention described in Table 1, and is 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】 The sequences of VH CDR1, 2, and 3, and VL CDR1, 2, and 3, can be “mixed and matched” (i.e., CDRs from different antibodies can be mixed and matched) insofar as each of these antibodies can bind to PDL1 and its antigen-binding specificity is mainly provided by the CDR1, 2, and 3 regions, provided that each antibody contains VH CDR1, 2, and 3, as well as VL CDR1, 2, and 3, and that it forms other PDL1-binding molecules of the present invention. Such “mixed and matched” PDL1-binding antibodies can be tested using well-known binding experiments, as 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 substituted with structurally similar CDR sequences (including multiple sequences). Similarly, when VL CDR sequences are mixed and matched, CDR1, CDR2, and / or CDR3 sequences derived from a particular VL sequence should be replaced with structurally similar CDR sequences (including multiple sequences). As will be obvious to those skilled in the art, novel VH and VL sequences can be produced by mutating one or more VH and / or VL CDR region sequences from the CDR sequences shown in this specification with respect to the monoclonal antibodies of the present invention, so as to have structurally similar sequences. 【0123】 In another embodiment, the present invention provides antibodies comprising amino acid sequences homologous to those listed in Table 1, wherein the antibodies bind to PDL1 and maintain the desired functional properties of those antibodies as listed 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. The heavy chain variable region includes an amino acid sequence that is 80% or more, 90% or more, or 95% or more identical to the amino acid sequence 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 includes an amino acid sequence that is 80% or more, 90% or more, or 95% or more identical to the amino acid sequence of SEQ ID NOs. 26, 27, 57, and 58, preferably SEQ ID NOs. 26 or 27, more preferably SEQ ID NOs. 27. The aforementioned antibody specifically binds to the human PDL1 protein. 【0125】 In one embodiment, the VH and / or VL amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences listed in Table 1. In one embodiment, the VH and / or VL amino acid sequences 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 containing CDR1, CDR2 and CDR3 sequences, and a light chain variable region containing CDR1, CDR2 and CDR3 sequences, One or more of these CDR sequences have a specific amino acid sequence based on the antibody described in this specification or its conserved modification, 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 acids and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant means encoding the same or essentially the same amino acid sequence, or, in the case of nucleic acids that do not encode amino acid sequences, essentially the same sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position where alanine is identified by the codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid mutations are “silent mutations,” and they are a type of conservatively modified mutation. Every nucleic acid sequence in this specification encoding a polypeptide describes a silent mutation of any available nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG (usually the sole codon for methionine) and TGG (usually the sole codon for tryptophan)) can be modified for the production of a functionally identical molecule. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is implicitly present in each described sequence. 【0128】 A “conservatively modified variant” or “conservative variant” of a polypeptide sequence includes individual substitutions, deletions, or additions to the polypeptide sequence that result in amino acid substitutions by chemically similar amino acids. A table of conservative substitutions that result in functionally equivalent amino acids is publicly known. Such conservatively modified variants may also include, but are not excluded from, the polymorphic variants, interspecific homologs, and alleles of the present invention. The following eight groups include amino acids that are mutually conservative substitutions: 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 antibody binding properties, including the amino acid sequence. 【0129】 Therefore, 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, and preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 1, 4, 5, 8, 11, 32, 35, 36, 39, and 42, preferably SEQ ID NO: 1 or 32, more preferably SEQ ID NO: 1, or a conserved variant thereof. The heavy chain variable region CDR2 comprises, and preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 2, 6, 9, 12, 33, 37, 40, and 43, preferably SEQ ID NO: 2 or 33, more preferably SEQ ID NO: 2, or a conserved variant thereof. The heavy chain variable region CDR3 comprises, and preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 3, 7, 10, 13, 34, 38, 41, and 44, preferably SEQ ID NO: 3 or 34, more preferably SEQ ID NO: 3, or a conserved variant thereof. The light chain variable region CDR1 comprises, and preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 17, 20, 23, 48, 51, and 54, preferably SEQ ID NO: 17 or 48, more preferably SEQ ID NO: 17, or a conserved variant thereof. The light chain variable region CDR2 comprises, and preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 18, 21, 24, 49, 52, and 55, preferably SEQ ID NO: 18 or 49, more preferably SEQ ID NO: 18, or a conserved variant thereof. The light chain variable region CDR3 comprises, and preferably consists of, an amino acid sequence selected from any of SEQ ID NOs: 19, 22, 25, 50, 53, and 56, preferably SEQ ID NO: 19 or 50, more preferably SEQ ID NO: 19, or a conserved variant thereof. This 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 in this specification or its conserved modifications, 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, comprising a heavy chain variable region and a light chain variable region, optimized for expression in mammalian cells. The heavy chain variable region includes 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 conserved modification sequence. The light chain variable region includes 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 a conserved modification sequence thereof. This 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, one or more of these sequences having a specific amino acid sequence based on the antibody described herein or its conserved modifications, and the antibody maintains the desired functional properties of the PDL1-binding antibody of the present invention. 【0132】 In this specification, the term “optimized” means that a nucleotide sequence has been modified to encode an amino acid sequence using preferred codons in a producing cell or organism (generally, for example, cells of the genus Pichia, Chinese hamster ovary cells (CHO), or human cells). The optimized nucleotide sequence is processed to retain, to the greatest extent possible, the amino acid sequence encoded by the original start nucleotide sequence known as the “parent” sequence. In this specification, the optimized sequence is processed to have preferred codons in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also recalled in the present invention. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as the optimized sequence. 【0133】 Other types of variable region modification involve improving one or more binding properties (e.g., affinity) of an antibody of interest by mutating amino acid residues within the CDR1, CDR2, and / or CDR3 regions of the VH and / or VL, known as "affinity maturation." The introduction of mutations (including multiple mutations) can be performed by site-directed mutagenesis or PCR-mediated mutagenesis, and the resulting effect on antibody binding or other functional properties can be evaluated in in vitro or in vivo assays described in this specification and illustrated in the examples. Conservative modifications (such as those described above) can also be introduced. Mutations may be amino acid substitutions, additions, or deletions. Furthermore, 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 parental antibody that does not have such modifications. In one embodiment, an affinity matured antibody may have a nanomolar or picomolar affinity for the 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 ("HVR") and / or framework residues is described, for example, in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813(1994), Schier et al. Gene 169:147-155(1995), Jackson et al, J. Immunol. 154(7):3310-9(1995) and Hawkins et al, J. Mol. Biol. 226:889-896(1992). 【0135】 In one embodiment, the 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, and 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 an antibody having one or more VH and / or VL sequences as shown in this specification as an initiating material to process into a modified antibody, which may have modified properties compared to the initiating antibody. The antibody can be processed by modifying one or both of the variable regions (i.e., VH and / or VL), for example, one or more residues in one or more CDR regions and / or one or more framework regions. Alternatively, the antibody can be processed by modifying residues in the constant region to, for example, alter the effector function of the antibody. 【0138】 One possible modification of the variable region is the creation of a CDR graft. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). Therefore, the amino acid sequences within the CDRs exhibit greater diversity among individual antibodies than the sequences outside the CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the characteristics of specific naturally occurring antibodies by constructing expression vectors that fuse CDR sequences from specific naturally occurring antibodies to 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., USA 86:10029-10033; U.S. Patent No. 5,225,539 (Winter); and U.S. Patents No. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 (Queen et al.)). 【0139】 Such framework sequences can be obtained from public DNA databases or from publicly available literature describing germline antibody gene sequences or rearranged antibody sequences. For example, germline DNA sequences of human heavy chain and light chain variable region genes are available in the "VBase" human germline sequence database (available on the internet at www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, Tomlinson, IM, et al., 1992 J. fol. Biol. 227:776-798, and Cox, JP Let al., 1994 Eur. J Immunol. 24:827-836, the disclosures thereof incorporated herein by reference. For example, germline DNA sequences for human heavy and light chain variable region genes and rearranged antibody sequences are available in the "IMGT" database (available online at www.imgt.org, Lefranc, MP et al., 1999 Nucleic Acids Res. 27:209-212; the contents of these disclosures are incorporated herein by reference). 【0140】 Examples of framework sequences used in the antibodies of the present invention include those structurally similar to the framework sequences used by selected antibodies of the present invention, such as consensus sequences and / or framework sequences used by monoclonal antibodies of the present invention. The VH CDR1, 2, and 3 sequences, and the VL CDR1, 2, and 3 sequences, may be grafted onto framework regions having sequences identical to those present in germline immunoglobulin genes from which the framework sequences originate, or the CDR sequences may be grafted onto framework regions containing one or more mutations compared to the germline sequences. For example, it is known that mutating residues in the framework region is useful to maintain or enhance the antigen-binding ability of antibodies (see, for example, U.S. Patents 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, including the five main idiotypes of their antigen-binding fragments, or those containing immunoglobulins from other animal species, preferably in humanized forms. 【0142】 In one embodiment, the present invention relates to a method for producing a non-immunoglobulin-based antibody using a non-immunoglobulin scaffold capable of grafting the CDR of the present invention. Known or future-developed non-immunoglobulin frameworks and scaffolds can be used, as long as they contain a specific binding region to the target PDL1 protein. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Waltham, Massachusetts), Ankyrin (Molecular Partners AG, Zurich, Switzerland), Lipocalin (Pieris Proteolab AG, Freising, Germany), Small Molecule Immunotherapy (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】 Suitablely, the antibody of the present invention specifically binds to PDL1 and is characterized by one or more of the following parameters: (i) When measured particularly by surface plasmon resonance (SPR), the antibody 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, and more preferably less than 5 pM, and in particular the antibody is scFv, (ii) When measured by SPR, 10 -3 s -1 The following 10 -4 s -1 The following, or 10 -5 s -1 The following K off It binds, and in particular, the antibody is scFv, (iii) When measured by SPR, 10 3 M -1 s -1 The above 10 4 M-1 s -1 The above 10 5 M -1 s -1 Above, or 10 6 M -1 s -1 The above K on It binds, and in particular, the antibody is scFv, (iv) Having cross-reactivity with cynomolgus PDL1, and in particular binding to cynomolgus PDL1 at 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, in particular the antibody is scFv, and in particular does not have cross-reactivity with mouse PDL1 when measured by surface plasmon resonance, and / or (v) In particular, it does not bind to human PDL2 when measured by SPR. 【0144】 As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within the range of each antigenic site, the variable region of the antibody "arm" interacts with the antigen at numerous sites through weak non-covalent bonds, and the more interactions there are, the stronger the affinity. 【0145】 In a broad sense, "binding affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity," "to bind to," "to bind with," or "to bind to" as used in this specification refers to the intrinsic binding affinity that reflects the 1:1 interaction between the components of the binding pair (e.g., an antibody fragment and an antigen). The affinity of molecule X to its partner Y is usually expressed by the dissociation constant (K). DAffinity can be expressed as follows. Affinity can be measured by common methods of the public art, including the methods described in this specification. Antibodies with low affinity typically tend to bind slowly to antigens and dissociate quickly, while antibodies with high affinity typically tend to bind quickly to antigens and maintain binding for a long time. Various methods for measuring binding affinity are known, and any of them can be used to solve the problems of the present invention. Specific exemplary embodiments for measuring binding affinity (i.e., binding strength) are described below. 【0146】 The term "K" used in this specification assoc ", "Ka or "K on " refers to the association rate of a specific antibody-antigen interaction, while the term "K" used herein refers to the association rate of a specific antibody-antigen interaction. dis ", "Kd", or "K off "KD" refers to the separation rate of a specific antibody-antigen interaction. In one embodiment, the term "KD" as used herein refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as molar concentration (M). In the present invention, "KD" or "KD value" or "K D " or "K DIn one embodiment, the "affinity value" is measured using a surface-plasmon resonance assay with a MASS-1 SPR instrument (Sierra Sensors). When measuring affinity, an antibody specific to the Fc region is immobilized on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) with rabbit IgG (Bethyl Laboratories, catalog number A120-111A) using a standard amine-coupling procedure. Rabbit monoclonal antibodies in the B cell supernatant are captured by the immobilized anti-rabbit IgG antibody. A minimum IgG concentration in the B cell supernatant is necessary to ensure sufficient capture. After capturing the monoclonal antibodies, human PDL1 (Peprotech) is injected into the flow cell at a concentration of 90 nM for 3 minutes, and the protein is separated 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 separation (kd) and association (ka) constants, as well as the apparent separation equilibrium constant (KD), were calculated using a one-to-one Langmuir coupling model with MASS-1 analysis software (Analyzer, Sierra Sensors). 2 The relative value of (Chi normalized by the maximum binding level of the extrapolated sample) 2 Based on this, the quality of the fit is monitored and used as an indicator for maintaining the quality of curve fitting. 2 A small value of this indicates high accuracy in fitting to a one-to-one Langmuir binding model. The result is considered valid if the reaction units (RUs) in ligand binding are 2% or more of the RUs in antibody capture. Samples showing ligand binding RUs of less than 2% of the RUs in antibody capture are considered to indicate the absence of specific binding of PDL1 to the captured antibody. Equilibrium dissociation constant (K) D ) is the ratio k off / k on It is calculated as follows. See, for example, Chen et al, J.Mol.Biol.293:865-881(1999). 【0147】 Appropriately, 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 higher affinity of the PDL1 antibody 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 another embodiment, the PDL1 antibody of the present invention neutralizes the B7-1 / PDL1 interaction. Appropriately, 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 with 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 with a titer equal to or higher than that of avelumab. The binding affinity of the antibody may be determined, for example, by the dissociation constant (KD). Strong affinity is represented by a low KD (Key Degree), while weak affinity is represented by a high KD. 【0148】 Therefore, in appropriate embodiments, the antibodies of the present invention may have KDs of 1-50,000 pM, 1-40,000 pM, 1-30,000 pM, 1-20,000 pM, 1-10,000 pM, 1-5,000 pM, 1-2,500 pM, 1-1,000 pM, 1-750 pM, 1-500 pM, 1-250 pM, 1-100 pM, 1-50 pM, and 1-10 pM. In a suitable embodiment, the antibody of the present invention may 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 measured by SPR, and the antibody is scFv. Preferably, the antibody of the present invention has a KD of less than 5 nM, particularly when measured by SPR. Preferably, the antibody of the present invention has a KD of less than 1 nM, particularly when measured by SPR. Preferably, the antibody of the present invention has a KD of less than 100 pM, particularly when measured by SPR. Preferably, the antibody of the present invention has a KD of less than 50 pM, especially when measured by SPR. Preferably, the PDL1-BD of the present invention binds to human PDL1 with a KD of less than 10 pM, especially when measured by SPR. Preferably, the PDL1-BD of the present invention binds to human PDL1 with a KD of less than 5 pM, especially when measured by SPR. 【0149】 More precisely, the antibody of the present invention, when measured by surface plasmon resonance (SPR), has a ratio of 10 to human PDL1. 3 M -1 s -1 The above 10 4 M -1 s -1 The above 5 x 10 4 M -1 s -1 The above 10 5 M -1 s -1 The above 5 x 10 5 M -1 s -1 The above 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 Binds at the following rate. Preferably, the antibody of the present invention has a K of 10 5 M -1 s -1 Above, especially 10 6 M -1 s -1 Above K on rate. 【0150】 Suitably, the antibody of the present invention specifically 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 Binds at the following rate. Preferably, the antibody of the present invention has a K of 10 -3 s -1 Below, 10 -4 s -1 Below, especially 10 -5 s -1 Below K off rate. 【0151】 Suitably, the antibody of the present invention specifically binds to PDL1 and is characterized by one or more of the following parameters: (i) When measured by ELISA, it has the ability to neutralize PDL1 / PD-1 interaction with a titer (relative titer) greater than 1.5, for example greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The aforementioned relative titer is the IC50 ng / mL of avelumab measured by ELISA. 50 The IC value of the antibody measured by ELISA is ng / mL. 50 It is the ratio to the value, In particular, the antibody is scFv, and (ii) Optionally, when measured by an NFAT reporter gene assay, it has the ability to neutralize the PDL1 / PD-1 interaction at a titer (relative titer) greater than 1.5, for example greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The aforementioned relative titer is the IC50 ng / mL of avelumab measured in the NFAT reporter gene assay. 50 The IC50 value of the antibody measured in the NFAT reporter gene assay, in ng / mL, is the IC50 value. 50 It is the ratio to the value, In particular, the antibody is scFv, and (iii) When measured by ELISA, it has the ability to neutralize the PDL1 / B7.1 interaction with a titer greater than 1.5, for example greater than 2, greater than 2.5, preferably greater than 3, more preferably greater than 4, compared to avelumab. The aforementioned relative titer is the IC50 ng / mL of avelumab measured by ELISA. 50 The IC value of the antibody measured by ELISA is ng / mL. 50 It is the ratio to the value, In particular, the antibody in question is scFv. 【0152】 Ideally, the antibodies of the present invention possess useful biophysical properties. 【0153】 Appropriately, when the antibody of the present invention is in scFv format, and particularly when the antibody is prepared in 50 mM phosphate-citrate buffer in 150 mM NaCl at pH 6.4, it has a melting temperature (Tm) of 55°C or higher, for example 60°C or higher, preferably 65°C or higher, and more preferably 70°C or higher, as measured by differential scanning fluorescence (DSF). 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 fluorescence (DSF) using the fluorescent dye SYPRO® Orange (see Wong & Raleigh, Protein Science 25(2016) 1834-1840). A phosphate-citrate buffer sample at pH 6.4 is prepared in a total volume of 100 μl at a final protein concentration of 50 μg / mL and a final concentration of 5×SYPRO® orange. 25 μl of the prepared sample is added three times to a white-walled 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 in the software. The PCR plate containing the test sample is heated in 1°C increments from 25°C to 96°C, with a 30-second pause after each temperature increase. The total assay time is approximately 2 hours. Tm is calculated using the GraphPad Prism software with a mathematical second derivative, and the inflection point of the curve is determined. The output Tm is the average of the three measurements. 【0154】 Appropriately, when the antibody of the present invention is in scFv format, after being subjected to five 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 pH 6.4, 50 mM phosphate citrate buffer in 150 mM NaCl, it exhibits a monomer content loss of less than 5%, preferably less than 3%, more preferably less than 1%. 【0155】 Appropriately, when the antibody of the present invention is in scFv format, after storage at 4°C for at least two weeks, particularly at least four weeks, at an initial concentration of 10 mg / ml of the antibody of the present invention, it exhibits 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%, when the antibody of the present invention is prepared in pH 6.4, 50 mM phosphate citrate buffer in 150 mM NaCl. 【0156】 Monomer content loss is measured by calculating the area under the curve of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stable phase and a liquid mobile phase, as outlined in USP Chapter 621. This method utilizes a hydrophobic stable phase and an aqueous mobile phase to separate molecules based on their size and shape. Molecular separation occurs between the void volume (V0) and total osmotic volume (VT) of a specific column. SE-HPLC measurements are performed using a Chromaster HPLC system (Hitachi High-Tech Corporation) equipped with an automated sample injection device and an ultraviolet detector set to a detection wavelength of 280 nm. The system is controlled by the software EZChrom Elite (Agilent Technologies, Version 3.3.2 SP2), which also supports the analysis of the resulting chromatograms. Protein samples are clarified by centrifugation and maintained at a temperature of 4-6°C in the autosampler before injection. For the analysis of scFv samples, a Shodex KW403-4F column (Showa Denko, #F6989202) was used, with a standardized buffered saline mobile phase (50 mM sodium phosphate, pH 6.5, 300 mM sodium chloride) at the recommended flow rate of 0.35 mL / min. The target sample addition amount per injection was 5 μg. The sample was detected by a UV detector at a wavelength of 280 nm, and the data was recorded using an appropriate software set. The resulting chromatogram was analyzed in the range of V0 to VT, and matrix-related peaks with elution times longer than 10 minutes were excluded. 【0157】 As used in this specification, the term "recognize" refers to an antibody discovering its structural epitope and interacting with it (for example, by binding). 【0158】 The terms “competing” or “cross-competing” and related terms used in this specification are interchangeable and refer to the ability of an antibody to prevent the binding of other antibodies or binders to PDL1 in a standard competitive binding assay. 【0159】 The ability or extent to which the antibody according to the present invention can prevent other antibodies or binding molecules from binding to PDL1, i.e., whether it can cross-compete, can be determined using a standard competitive binding assay. In particular, a suitable quantitative cross-competition assay involves using a FACS- or AlphaScreen-based method to pit a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or fragment thereof against an unlabeled antibody or fragment thereof and measuring their binding to the target. Generally, a cross-competition antibody or fragment thereof binds to the target in such a manner that, for example in a cross-competition assay, the substitution recorded by the immunoglobulin single variable domain or polypeptide of the present invention, in the presence of the second antibody or fragment thereof alone during the assay, is up to 100% (e.g., in a FACS-based competitive assay) relative to the maximum theoretical degree of substitution by the antibody or fragment thereof having cross-blocking ability present in a predetermined amount to be tested (e.g., substitution by the untreated (e.g., unlabeled) antibody or fragment thereof required for cross-blocking). Preferably, the cross-competition antibody or fragment thereof has 10% to 100% recorded substitutions, preferably 50% to 100%. 【0160】 The term "epitope" refers to a determinant of a protein that can specifically bind to an antibody. Epitopes typically consist of molecules with chemically active surface groupings, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge properties. "Structural" and "linear" epitopes are distinguished by the fact that binding to the former, rather than the latter, is lost in the presence of a denaturing solvent. As used herein, "structural epitope" refers to an amino acid residue in an antigen that is located on the surface when a polypeptide chain folds to form a native protein, and whose HD substitution rate is significantly reduced by Fab binding. Structural epitopes include, but are not limited to, functional epitopes. The term "linear epitope" refers to an epitope where all interaction sites between the protein and the interacting molecule (e.g., antibody) are linearly aligned along the (continuous) primary amino acid sequence of the protein. 【0161】 The present invention also provides antibodies that bind to the same epitopes as the PDL1-binding antibodies listed in Table 1. Therefore, further antibodies are identified based on their ability to cross-compete with other antibodies of the present invention in PDL1 binding assays (e.g., statistically significantly inhibit binding competitively). 【0162】 Appropriately, the isolated antibody of the present invention is selected from the group consisting of: Examples include monoclonal antibodies, chimeric antibodies, IgG antibodies, Fab, Fv, scFv, dsFv, scAb, STAB, and binding domains based on alternative scaffolds, such as, but not limited to, ankyrin-based domains, finomers, avimers, anticarin, fibronectin, and binding sites incorporated into the constant region of antibodies (e.g., F-star's Modular Antibody Technology®). 【0163】 Suitablely, the isolated antibody of the present invention is Fv. Suitablely, the isolated antibody of the present invention is an scFv antibody fragment. A "single-chain Fv" or "scFv" or "sFv" antibody fragment comprises the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. Generally, the Fv polypeptide further comprises 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 comprises the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. Generally, the scFv polypeptide further comprises 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, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315). In a specific embodiment, the functional fragment is in scFv format including a 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】 More appropriately, the isolated antibody of the present invention is an IgG antibody fragment. The term “isotype” refers to an antibody class, such as IgM, IgE, or IgG (e.g., IgG1 or IgG4), resulting from differences in heavy chain constant region genes. Isotypes also include modifications to alter Fc function, such as modifications to enhance or reduce effector function or binding to the Fc receptor, for example, modifications to any one of these classes. In one embodiment, the isolated antibody of the present invention is IgG, preferably IgG1, selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 【0165】 Appropriately, the isolated antibody of the present invention is The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively, VH sequences containing the same amino acid sequence as SEQ ID NO: 14 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 comprising a VL sequence containing 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, the same amino acid sequence as SEQ ID NO: 26. In a more specific embodiment, the antibody of the present invention is, The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 4, 6, and 7, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 20, 21, and 22, respectively, A heavy chain sequence containing the same amino acid sequence as SEQ ID NO: 93 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 containing a light chain sequence containing 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, the same amino acid sequence as SEQ ID NO: 92. Appropriately, the isolated antibody of the present invention is, The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 14 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 comprising a VL sequence containing 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, the same amino acid sequence as SEQ ID NO: 26. In a more specific embodiment, the antibody of the present invention is, The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 16 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 containing a VL sequence that contains the same amino acid sequence as SEQ ID NO: 27 in proportion to 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 90% or more. 【0166】 Appropriately, the isolated antibody of the present invention is The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 45 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 containing a VL sequence containing 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, the same amino acid sequence as SEQ ID NO: 57. In a more specific embodiment, the antibody of the present invention is, The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 35, 37, and 38, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 51, 52, and 53, respectively. A heavy chain sequence containing the same amino acid sequence as SEQ ID NO: 91 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 containing a light chain sequence that contains 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, the same amino acid sequence as SEQ ID NO: 90. 【0167】 Appropriately, the isolated antibody of the present invention is The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 45 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 containing a VL sequence containing 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, the same amino acid sequence as SEQ ID NO: 57. In a more specific embodiment, the antibody of the present invention is, The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 48, 49, and 50, respectively. VH sequences containing the same amino acid sequence as SEQ ID NO: 47 in 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, and IgG1 containing a VL sequence that contains 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, the same amino acid sequence as SEQ ID NO: 57. 【0168】 In another specific embodiment of the present invention, the isolated antibody of the present invention is a multispecific molecule, in particular a multispecific molecule having at least a second functional molecule, such as a bispecific, triplicate, quadruplicate, quinticate, or hexaspecific molecule. 【0169】 As used herein, the terms “multispecific molecule” or “multispecific antibody” refer to an antibody that binds to two or more different epitopes on at least two different targets (e.g., PDL1 and another target different from PDL1), or an antibody that binds to two or more different epitopes on the same target. The term “multispecific molecule” includes bispecific, triplicate, quadruplicate, quinticate, and sextipate antibodies. As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes on two different targets, or to two different epitopes on the same target. As used herein, the term “triplicate antibody” refers to an antibody that binds to three different targets, or to three different epitopes on the same target. 【0170】 The antibodies of the present invention can be derivatized or linked to other functional molecules such as other peptides or proteins (e.g., ligands for other antibodies or receptors), thereby creating multispecific molecules that bind to two or more binding sites and / or different target molecules. In fact, the antibodies of the present invention can 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 mimics) (e.g., by chemical bonding, gene fusion, non-covalent association, etc.), thereby creating multispecific molecules. 【0171】 Therefore, the present invention includes multispecific molecules comprising one or more first binding specificities for PDL1 and a second binding specificity for a second target epitope. For example, the second target epitope is located on a target molecule other than PDL1. Therefore, the present invention includes multispecific molecules comprising one or more first binding specificities for PDL1 and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of PDL1 that is different from the first target epitope. In addition to the first and second target epitopes, the multispecific molecules may further include a third binding specificity. 【0172】 In yet another embodiment, the present invention includes multispecific molecules that have monovalent, divalent, or polyvalent specificity with respect to PDL1, preferably monovalent specificity. 【0173】 In another specific embodiment of the present invention, the isolated antibody of the present invention is a monovalent, or for example, a bivalent, trivalent, tetravalent, pentavalent, or hexavalent PDL1-specific molecule. 【0174】 As used herein, the terms "monovalent molecule" or "monovalent antibody" refer 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 valencies, where "valency" refers to the number of antigen-binding sites that bind to the same epitope on the 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 the epitope. Examples of multivalent antibodies include, but are not limited to, divalent, trivalent, tetravalent, and pentavalent antibodies. As used herein, the term "divalent antibody" refers to an antibody having two antigen-binding sites (each binding to the same epitope). 【0176】 Preferably, the isolated antibody of the present invention is a multispecific molecule (e.g., a bispecific molecule) and / or a polyvalent molecule (e.g., a monovalent specific molecule for PDL1, a bivalent specific molecule for PDL1), which may be selected from any suitable multispecific, e.g., bispecific antibody format known in the art, 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 binding site (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-mini antibody, tetrabody, scFv-Fc-scFv fusion, scFv-HSA-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, bsAb (scFv linked to the C-terminus of the light chain), Bs1Ab (scFv linked to the N-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 both the heavy and light chains) ), IgG-scFv fusions such as Ts2Ab (dsscFv linked to the C-terminus of the heavy chain), bispecific antibodies based on heterodimer Fc domains such as Knob-into-Hole antibodies (KiHs) (bispecific IgG prepared by KiH technology), Fv, scFv, scDb, tandem-di-scFv, tandem-tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD fused to the N and / or C-terminus of either the heterodimer Fc domain or the other heterodimer domain, MATCH (International Publication No. 2016 / 0202457, Egan T., et al.)This format is 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 this invention. 【0177】 The term "diabody" refers to an antibody fragment having two antigen-binding sites, the fragment containing VH linked to VL within the same polypeptide chain (VH-VL). By using a linker that is too short to cause pairing between two domains on the same chain, these domains pair with complementary domains on other chains to form two antigen-binding sites. In a specific embodiment, the polypeptide linker is one or two units (GGGGS) consisting 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 divalent 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 monomer scDb) contains, in particular, two variable region heavy chain domains (VH) or fragments thereof and two variable region light chain domains (VL) or fragments thereof, linked by linkers L1, L2, and L3, in the order of VHA-L1-VLB-L2-VHB-L3-VLA, VHA-L1-VHB-L2-VLB-L3-VLA, VLA-L1-VLB-L2-VHB-L3-VHA, VLA The variants are -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, where the VLA and VHA domains together form an antigen-binding site for the first antigen, and the VLB and VHB domains together form an antigen-binding site for the second antigen. 【0179】 Linker L1 is a peptide consisting of 2 to 10 amino acids, more specifically 3 to 7 amino acids, and even more specifically 5 amino acids, and linker L3 is a peptide consisting of 1 to 10 amino acids, more specifically 2 to 7 amino acids, and even more specifically 5 amino acids. In specific embodiments, linker L1 and / or L3 are one or two units consisting of four glycine amino acid residues and one serine amino acid residue ((GGGGS) n Includes n=1 or n=2, preferably n=1. 【0180】 The intermediate linker L2 is a peptide of 10 to 40 amino acids, specifically 15 to 30 amino acids, and more specifically 20 to 25 amino acids. In a specific embodiment, the linker L2 is one or more units ((GGGGS)) consisting of four glycine amino acid residues and one serine amino acid residue. n , including 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 polyvalent 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 with 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, e.g., 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 is a peptide of four glycine amino acid residues and one serine amino acid residue ((GGGGS) n , including 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 as described in International Publication No. 2016 / 0202457, Egan T. et al., mAb 9(2017)68-84. 【0183】 The multispecific and / or polyvalent molecules of the present invention can be produced using any of the useful antibody production methods known in the prior art (for example, for the preparation of bispecific constructs, see Fischer, N. & Leger, O., Pathobiology 74(2007)3-14; for bispecific diabody and tandem scFvs, see Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907(2012)713-727 and International Publication No. 99 / 57150). Further examples of preferred methods for preparing 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 producing bispecific antibodies containing the Fc moiety of functional antibodies are also known techniques (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 include 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 together. When the binding specificity moieties are proteins or peptides, covalent conjugate formation can be achieved 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 sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., 1984 J.Exp.Med.160:1686 and Liu, MA et al., 1985 Proc.Natl.Acad.Sci.USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins.Mitt. No.78, 118-132, Brennan et al., 1985 Science 229:81-83, and Glennie et al., 1987 J.Immunol.139:00:002367-2375. Conjugates include SATA and sulfo-SMCC, both of which are available from Pierce Chemical (Rockford, Illinois). 【0186】 When the binding specificity moiety is an antibody, a conjugate can be performed by linking the C-terminal hinge regions of two heavy chains with sulfhydryl residues. In a specific embodiment, the hinge region is modified to include an odd number (e.g., one) sulfhydryl residues before the conjugate. 【0187】 Alternatively, two or more binding specificity moieties can be encoded in the same vector, expressed, and assembled in the same host cell. This method is particularly useful when the bispecific molecule is an mAb×mAb, mAb×Fab, Fab×F(ab')2, or ligand×Fab fusion protein. The multispecific molecule of the present invention may 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. Patents 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498, and 5,482,858. 【0188】 The binding of bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosolvent assays (ELISA), radioimmunoassays (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays typically detects the presence of a protein-antibody complex by using a labeling reagent (e.g., antibody) specific to the complex of interest. 【0189】 In a further embodiment, the present invention provides nucleic acids encoding the antibody of the present invention. The present invention also provides nucleic acid sequences encoding the CDR, VH, VL, full-length heavy chain and full-length light chain 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 this specification and refers to one or more deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, as well as polymers thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bindings, which may be synthetic, natural, or unnatural products, and which have similar binding properties as reference nucleic acids and are metabolized in a similar manner to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothionates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, a particular nucleic acid sequence also implicitly includes, in addition to the specified sequence, its conservatively modified variants (e.g., codon degenerate substitutions) and its complementary sequences. Specifically, as described later, degenerate codon substitutions may be induced by creating a sequence in which the third position of one or more (or all) selected codons is replaced with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). 【0191】 The present invention provides substantially purified nucleic acid molecules encoding polypeptides containing segments or domains of the PDL1-binding antibody chain described above. When expressed from a suitable expression vector, the polypeptides encoded by these nucleic acid molecules can exhibit binding ability to the PDL1 antigen. 【0192】 Furthermore, the present invention provides polynucleotides that encode one or more CDR regions, usually all three, derived from the heavy or light chain of the PDL1-binding antibody listed in Table 1. Several other polynucleotides encode all or substantially all of the heavy and / or light chain variable region sequences of the PDL1-binding antibody listed in Table 1. Due to coding degeneracy, various nucleic acid sequences encode each immunoglobulin amino acid sequence. 【0193】 Polynucleotide sequences can be prepared by de novo solid-phase DNA synthesis or by PCR mutation introduction of existing sequences encoding PDL1-binding antibodies (e.g., sequences described in the examples below). Direct chemical synthesis of nucleic acids can be carried out by known techniques (e.g., phosphotriester method (Narang et al., 1979, Meth. Enzymol. 68:90), phosphodiester method (Brown et al., Meth. Enzymol. 68:109, 1979), diethylphosphoramidite method (Beaucage et al., Tetra. Lett., 22:1859, 1981), and solid-phase method (U.S. Patent No. 4,458,066)). For example, the introduction of mutations into polynucleotide sequences by PCR can be carried out as described in, for example, PCR Technology: Principles and Applications for DNA Amplification, HAErlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991. 【0194】 The present invention also provides an expression vector and host cells for producing the above-mentioned PDL1-binding antibody. 【0195】 The term "vector" is intended to refer to a polynucleotide molecule that can transport other polynucleotides to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA loop that can ligate further DNA fragments. Another type of vector is a viral vector, which can ligate further DNA fragments into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be introduced into a host cell, integrated into the host cell's genome, and thereby replicated together 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”). Generally, expression vectors useful in recombinant DNA technology often take the form of plasmids. Hereinafter, “plasmid” and “vector” are used interchangeably, as plasmids are the most commonly used form of vector. However, in this invention, expression vectors include other forms (e.g., viral vectors (e.g., non-replicating retroviruses, adenoviruses, and adeno-associated viruses)) and perform equivalent functions. 【0197】 The term "mobilely ligated" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcription regulatory sequence and the sequence being transcribed. For example, promoter or enhancer sequences are mobilely ligated to a coding sequence if they stimulate or regulate the transcription of that coding sequence in a suitable host cell or other expression system. Generally, promoter transcription regulatory sequences that are mobilely ligated to a sequence being transcribed are physically adjacent to the sequence being transcribed; i.e., they act in cis. However, for some transcription regulatory sequences (e.g., enhancers), it is not necessary for them to be physically close to or adjacent to the coding sequence in which they intend to enhance transcription. 【0198】 Various expression vectors can be used to express polynucleotides encoding PDL1-binding antibody chains or binding fragments. Virus-based and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Examples of non-viral vectors and systems include plasmids, episomal vectors with expression cassettes typically for expressing proteins or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). Examples of non-viral vectors useful for expressing 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 vectors, and various other protein expression vectors known in the art. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40-based vectors, papillomaviruses, HBP Ibsteinbar 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) that are operably ligated to a polynucleotide encoding a PDL1-binding antibody. In one embodiment, an inducible promoter is used to prevent the expression of the inserted sequence under conditions other than induction conditions. Examples of inducible promoters include arabinose, lacZ, metallothionein promoters, or heat shock promoters. By culturing transformed organisms under non-inducible conditions, the expression of the product is made acceptable to the host cell, allowing for the growth of a population with the coding sequence without bias. In addition to the promoter, other regulatory elements may be required or desirable for the efficient expression of the PDL1-binding antibody. Typical examples of these elements include the ATG start codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency may be enhanced using an enhancer suitable for the cell line being 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, expression in mammalian host cells may be enhanced using an SV40 enhancer or a CMV enhancer. 【0200】 The expression vector may contain a secretion signal sequence to express a polypeptide encoded by the inserted PDL1-binding antibody sequence as a fusion protein. In many cases, the inserted PDL1-binding antibody sequence is ligated to the signal sequence before insertion into the vector. The vector used to receive the sequences encoding the light chain and heavy chain variable region domains of the PDL1-binding antibody may also encode the constant region or a portion thereof. Such a vector allows for the expression of the variable region as a fusion protein with the constant region, resulting in the production of a complete antibody and its antigen-binding fragment. Typically, such a constant region is of human origin. 【0201】 The terms “recombinant host cell” or simply “host cell” refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to a specific cell but also to its offspring. Such offspring may undergo certain modifications over time due to mutation or the influence of surrounding conditions, and therefore may not be identical to the parent cell in practice, but 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 suitable microbial hosts include Bacillus bacteria (e.g., Bacillus subtilis) and other Enterobacteria (e.g., Salmonella, Serratia, and various Pseudomonas species). Expression vectors can also be constructed in these prokaryotic hosts, which typically contain expression regulatory sequences (e.g., replication origins) compatible with the host cell. Furthermore, various known promoters are available, such as lactose promoters, tryptophan (trp) promoters, β-lactamase promoters, or λ-phage-derived promoters. These promoters typically control expression along with operator sequences as needed, and initiate and terminate transcription and translation by having ribosome binding site sequences, etc. The PDL1-binding polypeptide of the present invention can also be expressed using other microorganisms (e.g., yeast). Insect cells can also be used in combination with baculovirus vectors. 【0203】 In one embodiment, mammalian host cells are used to express and produce the PDL1-binding polypeptide of the present invention. For example, these may be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines carrying exogenous expression vectors. These also include any conventional lethal, conventional, or abnormal immortalized animal or human cells. 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 expressing polypeptides is outlined, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, NYNY 1987. Mammalian host cell-like expression vectors may include expression regulatory sequences, such as replication origins, promoters and enhancers (Queen, et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters are constitutive, cell-specific, stage-specific, and / or regulated or controllable promoters. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late-stage 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 for introducing an expression vector containing the target polynucleotide sequence 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. above for an overview). Other methods include, for example, electroporation, calcium phosphate treatment, liposome transformation, injection and microinjection, ballistic, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-induced DNA uptake enhancement, and ex vivotransduction. Stable expression is usually required for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express PDL1-binding antibody chains or binding fragments can be prepared using the expression vector of the present invention, which contains a viral replication origin or endogenous expression element and a selectable marker gene. After introducing the vector, the cells may be grown in a rich medium for 1-2 days before being switched to the selection medium. The purpose of using a selectable marker is to confer resistance to selection, and its presence allows cells that suitably express the introduced sequence to grow in the selection medium. Stably transfected cells with resistance can be grown using tissue culture techniques appropriate to the cell type. Accordingly, the present invention provides a method for producing the antibody of the present invention, the method comprising the step of culturing, particularly expressing, a host cell containing a nucleic acid or vector encoding the antibody of the present invention, thereby expressing the antibody or a fragment thereof of the present invention. 【0205】 In a further embodiment, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier enhances or stabilizes the composition or facilitates the preparation of the composition. Examples of pharmaceutically acceptable carriers include solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and other physiologically compatible media. 【0206】 The pharmaceutical compositions of the present invention can be administered by various well-known methods. The route or method of administration varies depending on the desired effect. Administration may be intravenous, intramuscular, intraperitoneal, subcutaneous, or near the target site. The pharmaceutically acceptable carrier must be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., infusion or irrigation). Depending on the route of administration, the active compounds (i.e., antibodies and multispecific molecules) may be coated with a material that protects the compounds from the action of acids and other natural conditions that may lead to the inactivation of the compounds. 【0207】 The pharmaceutical compositions of the present invention can be prepared according to known, routinely practiced methods 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, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective or effective amount of PDL1-conjugated antibody is used in the pharmaceutical composition of the present invention. The PDL1-conjugated antibody is prepared in a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage plan is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, it may be administered as a single bolus, divided into several doses administered at regular intervals, or the dose may be gradually reduced or increased depending on the urgency of the therapeutic situation. Composing the component into unit dosage forms is particularly useful for facilitating administration and ensuring dose uniformity. As used herein, "unit dosage form" refers to physically separate units that have been appropriately adjusted as dosage units for a subject to be treated, and each unit contains a predetermined amount of an active compound calculated to produce the desired therapeutic effect in conjunction with the necessary pharmaceutical carrier. 【0208】 The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to ensure that the amount of active ingredient, composition, and method of administration is effective in achieving the desired therapeutic response in a particular patient without causing toxicity to the patient. The selected dosage level depends on various pharmacokinetic factors, such as the activity of the particular composition of the present invention or its ester, salt, or amide used, the timing of administration, the route of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, age, sex, weight, symptoms, general health condition, the patient's medical history, and other factors. 【0209】 Antibodies are usually administered in multiple doses. Single doses may be administered weekly, monthly, or yearly. The administration interval may be irregular depending on the patient's blood concentration of PDL1-binding antibody. Alternatively, the antibody can be administered as a sustained-release formulation, in which case frequent administration is not necessary. The dosage and frequency of administration vary depending on the antibody's half-life in the patient. Generally, humanized antibodies have longer half-lives than chimeric antibodies and non-human antibodies. Dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. Prophylactic administration involves relatively low doses administered over a long period at relatively infrequent intervals. Some patients may continue treatment for the rest of their lives. Therapeutic administration may require relatively high doses at relatively short intervals until the progression of the disease slows or ends, preferably until the patient shows partial or complete improvement in the symptoms of the disease. Afterward, the patient may receive a prophylactic regimen. 【0210】 The antibodies of the present invention have utility for in vitro and in vivo diagnosis and treatment. For example, these molecules can be administered, for example, in vitro or in vivo into cultured cells, or in vivo to a subject, for the treatment, prevention, or diagnosis of various disorders. 【0211】 In one embodiment, the present invention relates to an antibody or composition of the present invention for use as a pharmaceutical agent. 【0212】 In one embodiment, the present invention relates to an antibody or composition of the present invention for use in the treatment of subjects requiring treatment for proliferative disorders, particularly cancer. 【0213】 In other embodiments, the present invention relates to the use of antibodies or compositions of the present invention for the treatment of subjects requiring treatment for proliferative disorders, particularly cancer. 【0214】 In a further embodiment, the present invention relates to the use of antibodies or compositions of the present invention in the manufacture of therapeutic agents for subjects requiring treatment of proliferative disorders, particularly cancer. 【0215】 In one embodiment, the present invention provides a method for treating a subject who requires treatment for a proliferative disorder, particularly cancer, comprising administering a therapeutically effective amount of the antibody or composition of the present invention to the subject. 【0216】 The term "subject" includes both humans and non-human animals. Non-human animals include, for example, all vertebrates, such as mammals and non-mammals (e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles). Unless otherwise specified, the terms "patient" and "subject" are interchangeable in this specification. 【0217】 As used herein, the terms “treatment,” “to treat,” “to treat,” and “treated” refer to obtaining a desired pharmacological and / or physiological effect. Such effect may be therapeutic in the sense of partially or completely curing a disease and / or adverse effects caused by the disease, or it may be therapeutic in the sense of delaying the progression of the disease. As used herein, “treatment” also encompasses all treatments of diseases in mammals (e.g., humans), including, for example: (a) inhibiting the disease, i.e., suppressing its progression, and (b) alleviating the disease, i.e., causing a regression of the disease. 【0218】 The terms "therapeutic effective dose" or "effective dose" refer to the amount of a drug that, when administered to a mammal or other being treated for a disease, is sufficient to carry out the treatment for that disease. The therapeutic effective dose varies depending on the drug, the disease and its severity in the subject being treated, as well as age, weight, and other factors. 【0219】 In one embodiment, the proliferative disorder is cancer. The term “cancer” means a disease characterized by the rapid and uncontrollable proliferation of vagrant cells. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers described in this specification, but not limited to, include: 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 in this specification, for example, both terms encompass solid tumors and fluid tumors that spread or circulate, for example. In this specification, the terms “cancer” or “tumor” encompass precancerous conditions as well as malignant cancers and tumors. In this specification, the term “cancer” is used to mean the widespread nature of a tumor and encompasses 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 tumors), esophageal cancer, ovarian cancer, cervical cancer, colorectal cancer, rectal cancer, prostate cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), vaginal cancer, thyroid cancer, melanoma (e.g., unremovable or metastatic melanoma), renal cell carcinoma, sarcoma, gliablastoma, multiple myeloma, or gastrointestinal cancer, particularly colorectal cancer or colorectal adenoma. Head and neck tumors, endometrial cancer, Cowden syndrome, Lhermitt-Dukuro disease, Bannayan-Zonana syndrome, prostatic hyperplasia, tumorigenesis, particularly epithelial, preferably breast cancer or squamous cell carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia (e.g., Philadelphia chromosome-positive chronic myeloid leukemia), acute lymphoblastic leukemia (e.g., Philadelphia chromosome-positive acute lymphoblastic leukemia), non-Hodgkin lymphoma, plasma cell myeloma, Hodgkin lymphoma, leukemia and combinations thereof. In a preferred embodiment, the cancer is lung cancer, preferably non-small cell lung cancer (NSCLC). In another embodiment, the cancer is colorectal cancer. 【0220】 The antibodies or compositions of the present invention inhibit the growth of solid tumors, but also inhibit fluid tumors. In yet another embodiment, the proliferative disease is a solid tumor. The term “solid tumor” means, in particular, breast cancer, ovarian cancer, colorectal cancer, rectal cancer, prostate cancer, gastrointestinal cancer (especially stomach cancer), cervical cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), and head and neck tumors. Furthermore, depending on the tumor type and the specific combination used, it is possible to reduce tumor volume. The antibodies or compositions of the present invention are also suitable for preventing tumor metastasis and preventing the proliferation or growth of micrometastatic lesions in subjects with cancer. 【0221】 The term “prevent” or “prevention” means the complete suppression of disease progression or any secondary effects of disease. As used herein, the term “prevent” or “prevention” includes preventing the onset of 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 embodiment, the present invention relates to a kit comprising the antibody of the present invention or a pharmaceutical composition of the present invention. The kit may include one or more other contents, such as: instructions for use; other reagents (e.g., labels, therapeutic agents or agents useful for chelating or otherwise coupling the antibody to the label or therapeutic agent, or a radioprotective composition); apparatus or other materials for preparing antibody molecules for administration; a pharmaceutically acceptable carrier; and apparatus or other materials for administration to a subject. In a particular embodiment, the kit comprises a pharmaceutically effective amount of the antibody of the present invention. In yet another embodiment, the kit comprises a pharmaceutically effective amount of lyophilized antibody of the present invention, a diluent, and optional instructions for use. The kit may further include a filter needle for re-preparation and a needle for injection. 【0223】 [Table 1] 【0224】 [Table 2] 【0225】 Table 3 【0226】 Table 4 【0227】 Table 5 【0228】 Table 6 【0229】 Table 7 【0230】 Table 8 【0231】 Table 9 【0232】 Table 10 【0233】 Table 11 【0234】 Table 12 【0235】 [Table 13] 【0236】 [Table 14] 【0237】 [Table 15] 【0238】 [Table 16] 【0239】 Throughout the documents of this application, there may be discrepancies between the description in the specification (e.g., Tables 1-3) and the sequence listing; however, in such cases, the description in the specification shall prevail. 【0240】 For clarity, certain features of the present invention are described in the context of separate embodiments, but it is obvious that they may be combined within a single embodiment. Conversely, for brevity, various forms of the present invention described in the description of a single embodiment may be divided and applied as appropriate subcombinations. All combinations of embodiments relating to the present invention are specifically encompassed in the present invention and are disclosed in this specification as if every combination were individually and explicitly disclosed. In addition, all subcombinations of various embodiments and their components are also specifically encompassed in the present invention and are disclosed in this specification as if every such subcombination were individually and explicitly disclosed. 【0241】 The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention other than those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are included in the appended claims. 【0242】 To the extent permitted by the patent laws of each country, all patents, patent applications, publications, test methods, documents, and other materials cited in this specification are incorporated by reference. 【0243】 The following examples illustrate the present invention as described above, but are not intended to limit the scope of the invention in any way. The usefulness of the claimed invention can also be confirmed using other test models known to those skilled in the art. [Examples] 【0244】 Novel antibodies against human PDL1: Example 1: Generation of rabbit antibodies against human PDL1: 【0245】 Rabbits were immunized with recombinantly produced and purified human PDL1 extracellular domains. During immunization, the intensity of the humoral immune response to the antigen was qualitatively assessed in the serum of each rabbit, where detectable binding of polyclonal serum antibodies still occurred, by measuring the maximum dilution (titer) against the antigen. Serum antibody titers against the immobilized antigen (recombinant human PDL1 extracellular domain) were evaluated using ELISA. All immunized rabbits had serum titers of at least 1:2.64 × 10⁶. 6 It showed very high titer in dilution. Serum obtained from the same rabbit before the initial antigen injection was used as a background control. 【0246】 Example 2: Identification and selection using Hit: We developed a flow cytometry-based sorting procedure that enables the specific detection and isolation of high-affinity hPDL1-binding B cells during the hit identification procedure. To identify hPDL1-binding B cells, hPDL1 ECDs were labeled with the fluorescent dye R-phycoerythrin (R-PE). The PD-1 binding site and the anti-PDL1 neutralizing antibody binding site on labeled PDL1 were potentially blocked by the bulky R-PE label, allowing us to confirm epitope accessibility by flow cytometry. The extracellular domain of PD-1 fused to the Fc portion of human IgG1 or avelumab was captured on protein G beads, and the binding of R-PE-labeled PDL1 was confirmed by flow cytometry. The fluorescence intensity showed a proportional relationship to the amount of labeled PDL1 bound to the receptor immobilized on the beads. Binding of PDL1 to PD-1 and the neutralizing antibody was confirmed, while binding of unrelated cytokines to the anti-PDL1 antibody was not detected. 【0247】 screening: Due to the difficulty of purifying individual rabbit antibodies at high-throughput culture scales, the results obtained during the screening phase were based on assays using unpurified antibodies from the culture supernatant of antibody-secreting cells (ASCs). While such supernatants allow for ranking of individual antibodies across a large number of cells, absolute values ​​other than binding ability cannot be obtained. Supernatants were collected from all individually cultured clones for more than four weeks. At the end of the culture period, rabbit monoclonal antibodies in each cell culture supernatant were characterized by high-throughput ELISA for binding to the recombinant human PDL1 extracellular domain. Further characterization of binding dynamics to human and cynomolgus monkey PDL1 was performed using PDL1-bound supernatants. In addition, the potential for neutralizing PDL1 / PD-1 interactions was measured by cell-based reporter gene assays and competitive ELISA. Neutralization of PDL1 / B7-1 interactions was also evaluated by competitive ELISA. Except for binding kinetics, the reported values ​​from high-throughput screening were interpreted as either "yes" or "no," and were based on single measurements (not dose-response). The mouse PDL1 binding potential of the supernatant was analyzed by direct ELISA, and binding kinetics were measured only for positive supernatants. 【0248】 Direct ELISA for hPDL1 binding: 50 μl of PBS containing 500 ng / ml PDL1 was added to an ELISA plate and coated overnight at 4°C. The following day, the plate was washed three times in overflow mode with 450 μl of wash buffer per well (PBS, 0.005% Tween 20), and 300 μl of blocking buffer per well (PBS, 1% BSA, 0.2% Tween 20) was added and the plates were treated on a nutating mixer at room temperature for 1 hour. 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 plates were incubated at room temperature for 1.5 hours with gentle vibration. 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 on a rotary mixer at room temperature for 1 hour, the plate was washed with 450 μl of washing buffer per well, and then 50 μl of TMB (3,3',5,5'-tetramethylbenzidine, KPL, catalog no. 53-00-00) was added. After allowing color development 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 according to SPR: The binding affinity of antibodies to human PDL1 was measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). For affinity screening, a standard amine coupling procedure was used to immobilize a sensor chip (SPR-2 affinity sensor, High Capacity Amine, Sierra Sensors) with an antibody specific to the Fc region of rabbit IgG (Bethyl Laboratories, catalog number A120-111A). Rabbit monoclonal antibodies in B cell supernatant were captured by the immobilized anti-rabbit IgG antibody. A minimum IgG concentration in the B cell supernatant was necessary for sufficient capture. After capturing the monoclonal antibody, human PDL1 (Peprotech) was injected into a flow cell at a concentration of 90 nM for 3 minutes, and protein separation from the captured IgG on the sensor chip was performed for 5 minutes. After each injection cycle, the surface was regenerated by injecting 10 mM glycine-HCl twice. Apparent separation (kd) and association (ka) constants, and apparent separation equilibrium constant (K D ) was calculated using a one-to-one Langmuir coupled model and MASS-1 analysis software (Analyzer, Sierra Sensors), and Chi 2 The relative value of (Chi normalized by the maximum binding level of the extrapolated sample) 2 Based on this, the quality of the fit was monitored and used as an indicator for maintaining the quality of curve fitting. 2 When this value is small, it means that the fitting accuracy to the one-to-one Langmuir couple model is high. In most hits, Chi 2 The relative value was 10% or less. The result was considered valid if the reaction unit (RU) in ligand binding was 2% or more of the RU in antibody capture. Samples showing ligand-bound RU of less than 2% of the RU in antibody capture were considered to indicate that there was no specific binding of PDL1 to the captured antibody. 【0250】 PDL1 / PD-1 blocking ELISA: 50 μl of PBS containing 2 μg / ml PD-1 was added, and the ELISA plate was coated overnight at 4°C. The following day, the plate was washed three 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 swirling mixer at room temperature for 1 hour. Next, PDL1 was diluted in blocking buffer to a concentration 20 times higher than the desired final concentration of 250 ng / ml. The assay sensitivity was further adjusted, and several clones were analyzed in the presence of 40 ng / ml PDL1. Next, 114 μl of each supernatant in an unbound plate was diluted in blocking buffer containing 6 μl of PDL1, and the plate was incubated on a swirling mixer at room temperature for 1 hour. The ELISA plate was washed three 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 each well three times with 450 μl of washing buffer, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed three times with 450 μl of washing buffer, 50 μl of TMB was added, and the plate was allowed to develop color 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 following day, the plate was washed three times in overflow mode with 450 μl of wash buffer per well, and 300 μl of blocking buffer was added per well. The plates were then treated on a swirling mixer at room temperature for 1 hour. Next, PDL1 was diluted in blocking buffer to a concentration 20 times higher than the desired final concentration of 500 ng / ml. Then, 114 μl of each supernatant in an unbound plate was diluted in blocking buffer containing 6 μl of PDL1, and the plates were incubated on a swirling mixer at room temperature for 1 hour. The ELISA plate was washed three 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 plates were incubated at room temperature for 1.5 hours with gentle shaking. After washing each well three times with 450 μl of washing buffer, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed three times with 450 μl of washing buffer, 50 μl of TMB was added, and the plate was allowed to develop color 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. 【0252】 Blocking of PDL1 / PD-1 in cell-based assays (reporter genes): To further analyze the hits, the ability of both interacting molecules to neutralize the PDL1 / PD-1 interaction when 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 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 following day, 95 μl of cell culture medium was removed from each well, and 50 μl of the supernatant of screened B cells, or avelumab at concentrations measured to produce 0%, 50%, and 100% of the maximum signal, was added. 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 a rate of 50 μl, and the plates were 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 a rate of 50 μL per well, and the plates were incubated in the dark for 30 minutes. Luminescence was measured using Topcount. 【0253】 Species specificity by SPR: cyno and mouse Furthermore, using the same SPR setup as described for binding to human PDL1, we measured the binding kinetics to PDL1 in cynomolgus monkeys and mice, but in each case, human PDL1 was replaced with cynomolgus monkey or mouse PDL1, respectively. 【0254】 Selection of screening hits: Table 4 shows the pharmacological properties of the monoclonal antibody of the final clone in the B cell supernatant. 【0255】 Example 3: Confirmation of Hit: Cloning and production: Following the identification of 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, DNA fragments were in vitro ligated into appropriate mammalian expression vectors. These expression vectors, containing consensus sequences of the invariant regions of the light and heavy chains of rabbit IgG, were co-expressed and associated to secrete fully functional rabbit monoclonal IgG. After vector construction, the sequences of the resulting constructs were re-verified, plasmid DNA was amplified and purified, and then transfected into mammalian cells. 【0256】 Expression vectors for rabbit antibody heavy and light chains were transfected into mammalian suspension cell lines for transient xenoexpression using lipid-based transfection reagents. Conditions such as the ratio of heavy and light chain vectors were optimized to enhance the expression level of secreted monoclonal IgG. Expression cells were cultured for 7 days in a shaking cell culture system. At the end of the xenoexpression period, the cell culture supernatant was collected by centrifugation. Next, the secreted rabbit IgG was affinity-purified using protein A beads. The IgG-loaded beads were washed, and the purified antibody was eluted by pH shift. The identity, content, and purity of the eluted fractions were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV absorption at 280 nm, and size exclusion high-performance liquid chromatography (SE-HPLC). Table 5 summarizes the production and characterization of rIgG. 【0257】 [Table 17] 【0258】 [Table 18] 【0259】 Affinity for hPDL1 according to SPR: The binding kinetics of purified monoclonal rabbit antibodies to human PDL1 were measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). Since most antibodies showed very slow off-rates, experiments were conducted at 37°C in a high-salt buffer to clearly distinguish between different antibodies and their corresponding binding affinities. Using a standard amine coupling procedure, antibodies specific to the Fc region of rabbit IgG (Bethyl Laboratories, catalog number A120-111A) were immobilized on a sensor tip (SPR-2 affinity sensor, High Capacity Amine, Sierra Sensors). The rabbit monoclonal antibodies were then captured by the immobilized anti-rabbit IgG antibodies. After capturing the monoclonal antibody, PDL1 was serially diluted 2-fold in HEPES buffer containing 150 mM NaCl and 150 mM MgCl2 in the range of 90–0.35 nM. The test involved binding to IgG captured on the biosensor chip and separation of proteins from the captured IgG, performed over 5 minutes. After each injection cycle, the surface was regenerated by injecting 10 mM glycine-HCl twice. The apparent separation (kd) and association (ka) constants, as well as the apparent separation equilibrium constant (K), were determined. D ) was calculated using a one-to-one Langmuir coupled model and MASS-1 analysis software (Analyzer, Sierra Sensors), and Chi 2 The relative value of (Chi normalized by the maximum binding level of the extrapolated sample) 2 Based on this, the quality of the fit was monitored and used as an indicator for maintaining the quality of curve fitting. 2 When this value is small, it means that the fitting accuracy to the one-to-one Langmuir couple model is high. Most hits are Chi 2 The relative value 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 of PDL1 in neutralizing the binding of PD-1 was evaluated in a competitive ELISA. 50 μl of PBS containing 4 μg / ml PD-1 was added to an ELISA plate and coated overnight at 4°C. The following day, the plate was washed three times in overflow mode with 450 μl of wash buffer per well (PBS, 0.005% Tween 20), and 300 μl of blocking buffer per well (PBS, 1% BSA, 0.2% Tween 20) was added and the plates were treated on a nutating mixer at room temperature for 1 hour. Next, PDL1 was diluted to a final concentration of 1 ng / ml with the blocking buffer. Then, in an unbound plate, 120 μl of buffer containing a series dilution of PDL1 ranging from 300 to 0.005 ng / ml for the rIgG to be tested was prepared in each well, and the plate was incubated at room temperature for 30 minutes. The ELISA plate was washed three times in overflow mode with 450 μl of wash buffer, and 50 μl of each dilution was added to adjacent wells of the ELISA plate to create repeat samples. The plate was incubated at room temperature for 90 minutes with gentle shaking. After washing three times with 450 μl of wash buffer, 50 μl of 10 ng / ml streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed three times with 450 μl of wash buffer, 50 μl of TMB was added, and the plate was allowed to develop color 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. 【0262】 Rabbit IgG derived from clones 33-03-G02 and 37-20-B03 showed high titers that neutralized the PDL1 / PD-1 interaction. Clone 37-20-B03 had approximately twice the titer of avelumab (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 that neutralizes the PDL1 / B7-1 interaction was evaluated in a competitive ELISA. 50 μl of PBS containing 4 μg / ml B7-1 was added to an ELISA plate and coated overnight at 4°C. The following day, the plate was washed three times in overflow mode with 450 μl of wash buffer per well (PBS, 0.005% Tween 20), and 300 μl of blocking buffer per well (PBS, 1% BSA, 0.2% Tween 20) was added and the plates were treated on a nutating mixer at room temperature for 1 hour. Next, PDL1 was diluted to 40 ng / ml with the blocking buffer. Then, in an unbound plate, 120 μl of buffer containing a series dilution of PDL1 ranging from 900 to 0.015 ng / ml for the rIgG to be tested was prepared in each well, and the plate was incubated at room temperature for 30 minutes. The ELISA plate was washed three times in overflow mode with 450 μl of wash buffer, and 50 μl of each dilution was added twice to adjacent wells of the ELISA plate to create repeat samples. The plate was incubated at room temperature for 90 minutes with gentle shaking. After washing three times with 450 μl of wash buffer, 50 μl of streptavidin-polyHRP40 was added to each well of the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed three times with 450 μl of wash buffer, 50 μl of TMB was added, and the plate was allowed to develop for 5–10 minutes. Finally, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl, 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 at a titer equivalent to that of avelumab, as shown in Table 8. The dose-response curves obtained for the selected clones are shown in Figure 2. 【0265】 [Table 21] 【0266】 Titer in cell-based PDL1 / PD-1 blocking assays (reporter gene): The titer of PD-1 in neutralizing PDL1 binding was evaluated in a cell-based reporter gene assay. 35,000 CHO / PDL1 / TCR activated cells in 100 μl of cell 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 following day, 95 μl of cell medium was removed from each well, and a 2x dilution series of the molecule to be tested (from 3,000 to 0.46 ng / ml) and 50 μl of control avelumab solution were added. 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 a rate of 50 μL per well, the plates were 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 for the selected clones are shown in Figure 3. 【0267】 [Table 22] 【0268】 FACS binding to PDL1-expressing cells: The binding titer to PDL1-expressing cells was measured for selected IgGs. 50,000 CHO-PDL1-expressing cells were distributed into round-bottom, untreated 96-well plates. The cells were washed twice with 100 μl of PBS by centrifugation at 400 × g for 5 minutes. The cells were resuspended in 100 μl of serial dilution preparations of the rIgGs to be tested and the control IgG avelumab, prepared to 2,000–0.128 ng / ml in staining buffer (PBS, heat-inactivated BCS 2%, 2 mM EDTA). After incubation on a rotary mixer at 4°C for 1 hour, the cells were washed three times by centrifugation at 400 × g for 5 minutes with 100 μl of staining buffer. Next, cells treated with rabbit IgG were resuspended in 100 μl of staining buffer containing 2 μg / ml goat anti-rabbit IgG (APC labeled), and cells treated with avelumab (human IgG1) were also resuspended in 100 μl of staining buffer containing 2 μg / ml goat anti-human IgG (APC labeled). The plates were incubated on a rotary mixer at 4°C for 1 hour. The plates were washed three times with 100 μl of staining buffer and resuspended in a final volume of 50 μl of staining buffer. Finally, the APC signal for 20,000 events per well was analyzed by flow cytometry using a Novocyte flow cytometry system (ACEA Bioscience). Binding of PDL1-expressing cells was confirmed for all evaluated rabbit IgGs. The binding titers of selected rabbit IgGs to intracellular PDL1 are shown in Table 10. 【0269】 [Table 23] 【0270】 Species specificity by SPR: cyno: Using the same setup as described for binding to human PDL1, we also measured the binding kinetics to cynomolgus monkey PDL1, but in this case, human PDL1 was replaced with cynomolgus monkey PDL1. Binding to cynomolgus monkey PDL1 was confirmed for all selected IgGs (Table 11). 【0271】 [Table 24] 【0272】 Species specificity by SPR: Mouse: Using the same setup as described for binding to human PDL1, the binding kinetics to cynomolgus monkey PDL1 were also measured, but in this case, human PDL1 was replaced with mouse PDL1. No binding to mouse PDL1 was detected for selected rabbit IgG derived from clones 33-03-G02 and 37-20-B03. 【0273】 Example 4: Selection of clones for humanization Based on the data obtained during hit confirmation, all clones were humanized by fusing the CDR to a VH3, VH4, or VH1A or VH1B-based framework. To obtain the best affinity and titer, two clones that showed the best affinity to human PDL1 as rIgG, 33-03-G02 and 37-20-B03, were further optimized by grafting to confer different structures. The following graft variants were applied to clones 33-03-G02 and 37-20-B03: CDR graft - Grafting rabbit CDR onto a human framework. IF graft - CDR graft + graft of all rabbit VL / VH interface residues. Complete graft - CDR graft + framework residues following the AHo humanization protocol (antigen interface (AIF) residues (rabbit residues that potentially come into contact with the antigen (AHo)) are limited to more than 20% residue changes, taking into account the ease of solvent access during interface formation, thereby reducing the number of mutations (rabbit framework residues)). 【0274】 Proteins were heterologously expressed as insoluble inclusion bodies by small-scale overnight expression with induction in E. coli (however, PRO997 was produced in mammalian CHO-S cells, similar to the rIgG expression described above). Inclusion bodies were isolated from the homogenized cell pellet by a centrifugation protocol that removed cell residue and impurities from other host cells, including several washing steps. The purified inclusion bodies were solubilized in denaturation buffer, and the scFvs were refolded according to a scalable refolding protocol that produced milligrams of naturally folded monomer scFv. During this process, the scFvs were purified using a standardized protocol. The refolded product was captured by affinity chromatography to obtain purified scFvs. Since an amount of sample suitable for SEC polishing was not available, only the main fraction with the desired purity was used. In addition, the melting temperature of scFvs was measured by differential scanning fluorescence (DSF) (details are described later). Table 12 summarizes the preparation of VH4 CDR grafted scFv molecules. Since the two clones contained unpaired cysteine ​​residues in their CDR loops, the C57S mutation was introduced into clone 37-20-B03, as shown in Table 12. 【0275】 Further graft variants were designed for several selected clones, and their initial preparation and characterization are summarized in Tables 13 (AHo numbering) and 14. 【0276】 Example 5: Pharmacokinetic analysis of humanized scFvs: Next, using the same assay system as described for the hit confirmation phase, we analyzed the major pharmacokinetic properties of humanized scFvs, but with appropriate modifications depending on the different formats of the scFv molecule. 【0277】 5.1 Affinity for human PDL1 The affinity of humanized scFvs for human PDL1 was measured by SPR analysis using a T200 instrument (Biacore, GE Healthcare). In this experiment, Fc-tagged human PDL1 was captured using a Human Antibody Capture kit from GE Healthcare. After each sample injection cycle, the CM5 sensor chip was regenerated and new antigen was captured. Using a dose-response multicycle reaction rate assay with varying sample concentrations of 0.12–30 nM diluted in running buffer, scFvs was injected as a sample for 5 minutes, and protein dissociation was allowed to proceed for 12 minutes. The resulting samplegram was fitted using a 1:1 binding model. As shown in Table 15, binding to human PDL1 was confirmed with 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 that neutralizes the binding of PDL1 to PD-1 was evaluated by competitive ELISA. Individual ICs in each plate 50 The IC values ​​were compared to the IC values ​​of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC) 50 :(I C 50アベルマブ / I C 50試験scFv Table 16 summarizes the titers, and in this assay, a maximum IC ratio of 5-fold was observed for avelumab. 50 It is clear that this can be calculated. All scFvs tested had a titer equivalent to or higher than avelumab. 【0279】 5.3. Neutralization of PDL1 / B7-1 interaction by competitive ELISA Following the same procedure as described above, the titer that neutralizes the binding of PDL1 to B7-1 was evaluated by competitive ELISA. Individual ICs in each plate 50 The IC values ​​were compared to the IC values ​​of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC) 50 :(I C 50アベルマブ / I C 50試験scFv Table 17 summarizes the titers, and in this assay, up to 10-fold IC2 was observed for avelumab. 50 It is clear that this can be calculated. The tested scFvs had a higher or equivalent potency to avelumab. 【0280】 5.4. Neutralization of PDL1 / PD-1 interaction in NFAT reporter gene assays The ability to neutralize the binding of PDL1 to PD-1 was evaluated using the cell-based reporter gene assay described above. Similar to the control avelumab, each molecule under test was added to the plate as a serial dilution. Individual ICs in each plate were evaluated. 50 The IC values ​​were compared to the IC values ​​of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC) 50 :(I C 50アベルマブ / I C 50試験scFv Table 18 summarizes the titers, and in this assay, a maximum IC of 5 times was observed for avelumab. 50 It is clear that this can be calculated. The tested scFvs had a higher or equivalent titer. 【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 titer to PDL1-expressing cells was measured for several molecules. The same cell lines (CHO-PDL1 and CHO-K1) were used during hit confirmation, but scFv was detected by APC-labeled protein L. Similar to the control avelumab, each molecule tested was added to plates in serial dilutions. Individual ICs in each plate were measured. 50 The IC values ​​were compared to the IC values ​​of avelumab, a control molecule added to each plate. 50 Calibrated against (relative IC) 50 :(I C 50アベルマブ / I C 50試験scFv The titers are summarized in Table 19. 【0289】 [Table 32] 【0290】 5.6. Cross-reactivity between species (binding of PDL1 to cynomolgus monkeys and mice by SPR) Cross-reactivity with cynomolgus monkey PDL1 was measured using the same assay as that used for human PDL1, with recombinant PDL1 from Shino Biological being used. Table 20 summarizes the affinity obtained for all tested scFvs. All tested scFvs that showed binding to human PDL1 also showed binding to cynomolgus monkey PDL1. 【0291】 [Table 33] 【0292】 5.7. Selectivity between PDL1 and PDL2 by SPR The binding of humanized scFv to PDL2 was tested using SPR analysis with a T200 instrument (Biacore, GE Healthcare). In this experiment, Fc-tagged human PDL2 was captured using a Human Antibody Capture kit from GE Healthcare. After each sample infusion cycle, the CM5 sensor chip was regenerated and new antigens were captured. scFv was injected as a sample diluted to a concentration of 180 nM in running buffer for 5 minutes, and protein separation was allowed to proceed for 12 minutes. None of the humanized scFv tested, as listed in Table 21, showed binding to PDL2. 【0293】 [Table 34] 【0294】 Example 6: Biophysical characterization of humanized scFv: Selected domains exhibiting better affinity than avelumab were prepared on a larger scale (expression volume of 0.2 L to 1.2 L). Furthermore, after purification, the protein samples were concentrated to over 10 mg / mL using 5 kD molecular weight cut centrifugation tubes. The preparation of materials for stability evaluation is summarized in Table 22. 【0295】 6.1. Storage stability test: Humanized scFv was subjected to stability testing (e.g., a 4-week stability test). For this test, scFv was prepared at a concentration of 10 mg / ml in a buffered aqueous solution (final buffer composition: 50 mM NaCiP, 150 mM NaCl, pH 6.4) and stored for 4 weeks at -80°C, 4°C, and 40°C. At a minimum, monomer and oligomer fractions in the formulation were evaluated by integrating SE-HPLC peak areas at 1 week, 2 weeks, and after the completion of each test. Records were also made for several molecules at other time points. Table 23 compares the measured values ​​obtained at the endpoints on day 7 and day 28 of the test. 【0296】 [Table 35] 【0297】 [Table 36] 【0298】 6.2. Freeze-thaw stability test: In addition to the aforementioned storage stability tests, the suitability of high-performing scFv molecules was evaluated by freeze-thaw (F / T) cycles (colloidal stability). To evaluate F / T stability, the same analytical methods (SE-HPLC, SDS-PAGE) and parameters (% monomer content and % monomer loss) as in the storage stability tests were applied, and the quality of the molecules was monitored over 5 F / T cycles. Table 24 illustrates the process of % monomer content loss over 5 repeated F / T cycles. Since no special freeze-thaw tests were performed, the freeze-thaw data obtained over 28 days from the -80°C samples used in the storage stability tests is shown in the graph below. After repeated F / T cycles, no molecules showed monomer content loss greater than 4%. 【0299】 [Table 37] 【0300】 6.3. Thermal denaturation: Thermal denaturation data for selected scFv structures were obtained from DSF measurements and are shown in Table 25. The obtained Tm values ​​were measured by fitting the data to Boltzmann's equation. Table 25 summarizes the melting temperatures calculated by DSF. 【0301】 [Table 38] 【0302】 Multispecific molecules containing antibodies of the present invention: Table 3 shows exemplary multispecific molecules containing the antibody of the present invention. 【0303】 Example 7: Affinity for PDL1, CD137, HSA, and MSA: method: Affinity for PDL1 from different species was measured using SPR measurement with a Biacore T200 instrument (GE Healthcare). Antibodies specific to the Fc region of human IgG were immobilized on a sensor chip (CM5 sensor chip, GE Healthcare) by amine coupling. In all formats, except for the Morrison format which included Fc, PDL1-Fc chimeric proteins from different species were captured by the immobilized antibody. A 3-fold serial dilution (0.12-90 nM) of PDL1-specific molecules was injected into a flow cell over 3 minutes, and separation was monitored for 10 minutes. After each injection cycle, the surface was regenerated by injecting a 3 M MgCl2 solution once. Apparent separation (k d ) and meetings (k a The rate constant and apparent separation equilibrium constant (KD) were calculated using a one-to-one Langmuir binding model. The affinity for CD137 from different species was measured using the same setup as for PDL1, except that CD137-Fc chimeric proteins from different species were captured by immobilized antibodies. 【0304】 The Fc-containing format was directly captured by antibodies specific to the Fc region of human IgG. Binding to IgG captured on the biosensor chip was tested using 2-fold serial dilutions of the PDL1 extracellular domain or CD137 extracellular domain in the range of 90–0.35 nM. After each injection cycle, the surface was regenerated by a single injection of 3 M MgCl2 solution. 【0305】 The affinity of molecules to different species of serum albumin (SA) was measured using a Biacore T200 instrument (GE Healthcare) by SPR measurement. SA was directly coupled to a CM5 sensor chip (GE Healthcare) using an amine coupling chemical method. After determining the best assay conditions by recalculating counting and surface performance testing, the dose response was measured, and the resulting coupling curve was double-referenced (empty reference channel and no sample injection) and fitted using a 1:1 Langmuir model to obtain kinetic parameters. The assay was performed in 1× PBS-Tween buffer at pH 5.5. 【0306】 result: Measurements of the binding kinetics of humanized constructs revealed differences in affinity for PDL1 when comparing the CDR and structural graft (STR) of clone 33-03-G02. The STR graft showed a 20-fold improvement in affinity compared to the CDR graft of the same clone (Table 26, PRO885 vs. PRO1126). Compared to the STR graft of clone 33-03-G02, the CDR graft derived from clone 37-20-B03 (PRO997) showed approximately twice the affinity. The binding affinity of the 33-03-G02 CDR graft is equivalent to the binding ability of the parent scFv when combined in different multispecificity formats (Table 26, when comparing PRO830 with PRO885, PRO951, PRO1123, PRO1124, PRO963, PRO966, PRO1057, PRO1058, PRO1059, and PRO1060). The scFv derived from both clones exhibits nearly identical affinity to human and cynomolgus monkey 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 activator molecules, and Jurkat cells expressing PD-1 and containing a luciferase gene under the NFAT response element: method: In a bioluminescent reporter gene assay, a modified Jurkat T cell stably expressing human PD-1 and an NFAT (nuclear factor of activated T cells)-luciferase reporter are used as effector T cells. Cells stably expressing human PDL1 and T cell receptor (TCR) activators are used as antigen-presenting cells. By co-culturing the two cell lines, activation of the Jurkat NFAT pathway is induced via the binding of the TCR activator / TCR complex. In response to the binding of PDL1-expressing cells, PD-1 signaling in PD-1 effector T cells inhibits T cell function and thus inhibits the NFAT pathway. Blocking the interaction between PD-1 and the PDL1 receptor leads to reactivation of the NFAT pathway. 【0310】 35,000 CHO / PDL1 / TCR activator (BPS Bioscience) cells in 100 μl of cell 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 following day, 95 μl of cell medium was removed from each well, and 50 μl of each molecule to be tested was added in a 2-fold dilution series of 3,000–0.46 ng / ml, along with control avelumab. Next, PD-1-expressing effector Jurkat cells (BPS Bioscience) 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, 50 μL of luciferase substrate (BPS Bioscience), prepared according to the manufacturer's protocol, was added to each well, the plates were incubated in the dark for 30 minutes, and luminescence was measured using Topcount. 【0311】 result: To evaluate the effect of CDR set and framework selection on the titer that neutralizes the binding of PDL1 to PD-1, three anti-PDL1 scFvs were tested in an NFAT reporter gene cell-based assay. PRO830 contained a CDR set of clone 33-03-G02 grafted onto a VH4 framework, while PRO997 and PRO1013 contained CDR sets of clone 37-20-B03 grafted onto a VH4 or VH1 framework, respectively. PRO830 had the lowest titer among the three scFvs tested, with an IC50 of 42.88 ng / ml. 50 The value is 34.09 ng / ml IC2. 50 It has a titer equivalent to avelumab, which has a value. PRO997 is the most potent molecule. When grafted onto the VH4 framework instead of the VH1 framework, the titer of the same CDR set was approximately twice as high. 50 The values ​​were 11.12 ng / ml versus 21.29 ng / ml. (Figure 4A and Table 27) 【0312】 The titer for neutralizing the binding of PDL1 to PD-1 was measured using a bispecific molecule containing the 33-03-G02 PDL1 domain, both before (CDR graft) and after (structural graft) domain optimization. The CDR graft (PRO885) was compared with the structural graft (PRO1126). Domain optimization was performed using IC. 50 The values ​​were 137.2 ng / ml for PRO885 and 48.15 ng / ml for PRO1126, indicating a threefold improvement in neutralizing potency (Figure 4B and Table 27). 【0313】 Furthermore, we evaluated the titer of two triplicate molecules possessing anti-PDL1 domains grafted from clone 33-03-G02, and two different human serum albumin-binding domains, focusing on their ability to neutralize PDL1 / PD-1 interactions, particularly their prolonged half-lives. The HSA domain of PRO1057 also binds to mouse serum albumin. Experiments were conducted in the presence of 25 mg / ml HSA. Neutralization titer (IC) was evaluated. 50 The concentration (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 arises from the IgG moiety (bivalent), and two scFvs with specificity for a second target are linked to the heavy chain (HC) or light chain (LC) of IgG via a flexible peptide linker. All Morrison molecules tested carried anti-PDL1 domains via CDR grafts of clone 33-03-G02 on both arms of IgG. The 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 structure 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 PDL1 interaction with PD-1 and B7-1 using competitive ELISA. These assays were performed to evaluate the ability of PDL1 inhibitors to block interactions between PDL1 and PD-1, or between PDL1 and B.71. Different formats, including scFv, scDb, scDb-scFv, and Morrison, were analyzed in competitive ELISA and compared to the control IgG avelumab. 【0317】 PDL1 / PD-1 competitive ELISA: ELISA microplates were coated overnight at 4°C with 4 μg / ml 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 plates were blocked at room temperature for 1 hour. A 3-fold serial dilution series of the inhibitor was prepared in dilution buffer containing 1 ng / ml biotinylated human PD-1 to final concentrations ranging from 300 to 0.005 ng / ml. The mixtures were pre-incubated at room temperature for 1 hour with gentle shaking using a rotary mixer (21 rpm), and after three washing cycles with 450 μl of wash buffer per well, they were added to the microplates. The plates were incubated at room temperature for 1.5 hours with gentle shaking, washed three times with 450 μl of wash buffer per well, and then 10 ng / ml streptavidin-polyHRP40 was added to the wells of each microplate. After incubation at room temperature for 1 hour, the plate was washed three times with 450 μl of washing buffer, and TMB was added. After 6 minutes, 1 M HCl was added to stop the enzymatic reaction, and the absorbance was measured at 450 nm using 690 nm as the reference wavelength. IC 50 To calculate the values, we used the values ​​obtained by subtracting the control and performed a 4-parameter logistic (4PL) curve fit in Graph Pad Prism. 【0318】 As illustrated in Figure 7 and Table 28, all PDL1 inhibitors blocked the interaction between PDL1 and PD-1 when tested in competitive ELISA. scFv PRO830 blocked the interaction at a comparable titer, while PRO997 and PRO1013 showed significantly lower IC50s than avelumab. 50 The values ​​indicated that they were potent inhibitors. When combined with multispecific formats (i.e., scDbs or Morrison), all molecules maintained their inhibitory properties. PRO885 was not more potent than avelumab, while PRO1126, which contained an improved anti-PDL1 domain, had a lower IC50. 50 Values ​​were measured. Compared to avelumab, Morrison format had a slightly lower titer. It also showed a neutralizing effect on PRO1057 in the presence of human serum albumin, but IC 50 The value was about twice as high. 【0319】 PDL1 / B7-1 competitive ELISA: Using 4 μg / ml human B7-1, ELISA microplates were coated overnight at 4°C 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 at room temperature for 1 hour. A 3-fold serial dilution series of the inhibitor was prepared in dilution buffer containing 40 ng / ml biotinylated PDL1 to achieve final concentrations ranging from 900 to 0.015 ng / ml. The mixtures were pre-incubated at room temperature for 1 hour with gentle shaking using a rotary mixer (21 rpm), and after three washing cycles with 450 μl of wash buffer per well, they were added to the microplates. The plates were incubated at room temperature for 1.5 hours with gentle shaking, washed three times with 450 μl of wash buffer per well, and then 10 ng / ml streptavidin-polyHRP40 was added to each well of the microplate. After incubation at room temperature for 1 hour, the plate was washed three times with 450 μl of washing buffer, and TMB was added. After 6 minutes, 1 M HCl was added to stop the enzymatic reaction, and the absorbance was measured at 450 nm using 690 nm as the reference wavelength. IC 50To calculate the values, we used the values ​​obtained by subtracting the control and performed a 4-parameter logistic (4PL) curve fit in Graph Pad Prism. 【0320】 For all PDL1 inhibitors except PRO1126, their ability to block the interaction between B7-1 and PD-1 was tested. PRO830 showed comparable potency to avelumab, but PRO997 and PRO1013 had lower IC50s. 50 These were measured values. All scDb and Morrison inhibited the interaction between PDL1 and B.7-1. scDb PRO885 showed comparable titer to avelumab, but Morrison's IC50 was lower. 50 The values ​​were approximately 2 to 3.4 times lower. The data is shown in Figure 8 and Table 28. 【0321】 [Table 42] 【0322】 Example 10: Evaluation of the stimulating effects of PDL1 blockade and simultaneous CD137 stimulation in human PBMCs stimulated with the superantigen SEA using a cell-based assay: This experiment evaluated the synergistic effects of PD-1 / PDL1 inhibition and CD137 agonism. In the assay, peripheral blood mononuclear cells (PBMCs) stimulated with the superantigen staphylococcal enterotoxin A (SEA) were used to induce PDL1 expression on antigen-donor cells (APCs) and T cells, respectively, and CD137 expression on T cells. By applying the anti-PDL1×CD137 molecule, two T cell regulatory signaling pathways were simultaneously targeted: inhibitory inhibition of the PD-1 / PDL1 pathway and activation of the CD137 pathway via immunological synapse formation mediated by the bispecific anti-PDL1×CD137 molecule (PRO885). T cell activation mediated by interleukin-2 (IL-2) secretion was evaluated and compared to the effect mediated by PDL1 inhibition mediated by the benchmarking control antibody avelumab. In addition, PRO997, an anti-PDL1 scFv agent, was tested and compared to avelumab 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 reduced from the PBMCs using an anti-CD56 antibody and a MACS cell isolation kit (Miltenyi Biotec). Then, 100,000 PBMCs were added per well to a 96-well plate, followed by the addition of a dilution series of PRO885, PRO997, and avelumab in an assay buffer containing SEA at a concentration of 10 ng / ml. After 96 hours of incubation at 37°C and 5% CO2, the cell supernatant was collected, and the human interleukin-2 (IL-2) level in the culture supernatant was quantified using the BioLegend IL-2 Human ELISA MAX assay according to the kit's instructions. The IL-2 concentration was interpolated from the IL-2 standard curve and calculated in reverse, and the EC (Emission Control Value) was used. 50 To calculate the values, the concentrations of avelumab and PRO885 were plotted against each other. 【0324】 As shown in Figure 9, IL-2 was secreted by T cells following the blocking of the PD-1 / PDL-1 interaction and simultaneous stimulation of CD137 by the addition of the bispecific molecule PRO885. Compared to avelumab, PRO885 showed higher T cell activation and better titer (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 simple PDL1 blocking by avelumab. Furthermore, it was revealed that the high-affinity anti-PDL1 scFv PRO997 is more potent than avelumab in stimulating T cells (PRO997:EC). 50 =40.86 ng / ml, avelumab: EC 50 =90.18ng / ml, Table 29). 【0325】 [Table 43] 【0326】 Example 11: Evaluation of the antitumor efficacy of anti-PDL1 antibody 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 human HCC827 NSCLC xenograft using Taconic's immunodeficient NOG mouse strain and allogeneic human peripheral blood mononuclear cells. Transplanted human T lymphocytes exhibit xenobiotic reactivity to exogenous major histocompatibility (MHC) classes I and II and other antigens derived from mouse cells. As a result, T lymphocytes show inflammatory infiltration into various organs, leading to animal death after several weeks, and this process is known as xenograft-versus-host disease (xGVHD). Treatment with immunomodulatory antibodies (e.g., anti-PDL1 and anti-CD137) has been shown to 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 processing schedule: Female NOG mice were given 5 × 10 6 One HCC827 cell was injected into one limb. The cells were injected in a mixture of 50% PBS cell suspension and 50% Matrigel, with a total injection volume of 100 μl. Tumor cells were injected into NOG mice, and tumor transplantation (80-100 mm) was performed. 3 After successfully measuring the median tumor volume, intravenous injection was administered to mice in 5 × 10 6 Human PBMCs were used for replacement. On the day of random assignment, four mice in each group were reconstituted with PBMCs from donor A, and four more mice were reconstituted with PBMCs from donor B. Treatment was started 1-2 hours after PBMC injection and applied as follows. 【0328】 [Table 44] 【0329】 Based on the in vitro activity of antibodies blocking PD-1 / PDL-1 interaction in NF-AT reporter gene assays, the dose of PRO1137 to obtain the same relative activity as a model dose of 0.1 mg of avelumab (per mouse) was set to 0.2 mg. Therefore, a dose of 0.2 mg of PRO1137 is equivalent to a dose of 0.1 mg of avelumab and can be expressed as 1 relative unit (1 r.U). Body weight and calipas splint measurement of tumor volume were performed twice weekly. Animals were sacrificed at predetermined time points according to the test results. All animals were sacrificed at the "same" time point (days 17 and 18). For processing capacity reasons, samples were collected and processed from the first half of each group on day 1, and the same sample collection and processing was performed from the remaining second half of each group on the following day. Animals reconstituted with PBMCs from two different donors adequately reflected the two sampling cohorts. 【0330】 Results: The antitumor activity of anti-PDL1 PRO1137 in human HCC827 NSCLC xenografts using immunodeficient NOG mouse strains and allogeneic human peripheral blood mononuclear cells (hPBMCs) was evaluated by measuring tumor volume (Figure 10). Tumor volume was measured twice weekly, 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 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), which suggests that the molecule is well tolerable at test dose levels (Figure 11). 【0331】 Example 12: Evaluation of the antitumor effect of human PRO1137 in NOG mice grafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs): The antitumor activity of human PRO1196 (anti-PDL1 IgG1, SEQ ID NOs. 92 and 93) was compared with vehicle therapy or avelumab in human HCC827 NSCLC xenografts using NOG mice grafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs). 【0332】 Test setup and processing schedule: Female NOG mice grafted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs) were subcutaneously injected with HCC827 NSCLC cells. 5 × 10⁶ cells were injected into one limb of each mouse. 6 Individual HCC827 cells were injected. The cells were injected as a mixture of 50% PBS cell suspension and 50% Matrigel, with a total injection volume of 100 μl. After successfully injecting tumor cells into NOG mice and transplanting the tumors (80-100 mm per group), the tumor cells were examined. 3 Based on the median tumor volume, mice (n=10) were randomized to the treatment group. 【0333】 [Table 45] 【0334】 Weight measurements and tumor volume measurements using a caliper were performed twice a week. Tumors were collected on days 25, 29, and 30 after treatment. 【0335】 Results: The antitumor activity of PRO1196 (anti-PDL1 IgG1, SEQ ID NOs. 92 and 93) in human HCC827 NSCLC xenografts using immunodeficient NOG mouse lines transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (UCB HSCs) was evaluated by measuring tumor volume (Figure 12). Tumor volume was measured twice weekly, and 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 avelumab resulted in suppression of tumor growth compared to the control group. 【0336】 Example 13: Evaluation of the antitumor efficacy of PDL1 block and its combination with local stimulation of CD137 in a cancer-bearing MC38 colorectal cancer model: 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 tumor-bearing C57BL / 6 mice with 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 multispecific antibody under test cross-reacts with mouse PDL1 and mouse CD137, a modified 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 using the CRISPR / Cas9 system in a C57BL / 6 mouse background. In addition, a modified MC38 tumor cell line expressing human PDL1 instead of mouse PDL1 under the control of a CMV promoter can be used. The effect of the multispecific antibody on tumor volume can be compared with combination therapy with humanized IgG1 containing the same PDL1-specific variable domain as the multispecific antibody, and with humanized IgG4 containing the same CD137-specific variable domain. To obtain further evidence of a local anti-tumor immune response, the number of tumor-infiltrating lymphocytes (e.g., CD8+, CD4+, and regulatory T cells) can be analyzed by flow cytometry. To systemically examine the modulation of the immune system following anti-CD137 / anti-PDL1 treatment, the number of CD4+ and CD8+ T cells in the liver and spleen can be analyzed by flow cytometry and possibly immunohistochemistry. Furthermore, systemic IFNγ levels can be analyzed using quantitative ELISA. In addition, to characterize the safety profile of anti-CD137 / anti-PDL1 combination therapy, clinicochemopathological parameters mainly related to hepatotoxicity (observed in anti-CD137 therapy in clinical practice), such as high levels of alanine aminotransferase, glutenate dehydrogenase, and aspartate aminotransferase, can be evaluated.

Claims

[Claim 1] An isolated antibody having binding specificity to human PDL1, The HCDR1, HCDR2, and HCDR3 sequences of sequence numbers 1, 2, and 3, respectively, Each includes the LCDR1, LCDR2, and LCDR3 sequences of sequence numbers 17, 18, and 19, respectively. The aforementioned antibody (i) A heavy chain variable region containing an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 14, and a light chain variable region containing an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 26, (ii) A heavy chain variable region containing an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 15, and a light chain variable region containing an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 26, Or, (iii) A heavy chain variable region containing an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 16, and a light chain variable region containing an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 27, antibody. [Claim 2] The aforementioned antibody (i) When measured by surface plasmon resonance (SPR), it binds to human PDL1 with a dissociation constant (KD) of less than 10 pM, (ii) When measured by SPR, 10 －３ s －１ The following KK ｏｆｆ Combined, (iii) When measured by SPR, 10 ３ M －１ s －１ The above K ｏｎ Combined, (iv) It exhibits cross-reactivity with Cynomorgus PDL1, and when measured by SPR, it binds to Cynomorgus PDL1 at a KD of less than 5 nM. (v) When measured by SPR, there was no cross-reactivity with mouse PDL1. (vi) When measured by SPR, it does not bind to human PDL2. (vii)When measured by ELISA, it has the ability to neutralize the PDL1 / PD-1 interaction with a titer (relative titer) of more than 3 times compared to abelumab, and the relative titer is the ng / mL IC ５０ value of abelumab measured by ELISA divided by the ng / mL IC ５０ value of the antibody (where the antibody is scFv) measured by ELISA, and (viiii) When measured by the NFAT reporter gene assay, it has the ability to neutralize the PDL1 / PD-1 interaction at a titer greater than 4 (relative titer) compared to avelumab, and the said relative titer is equal to the IC50 of avelumab ng / mL measured by the NFAT reporter gene assay. ５０ The IC50 value of the antibody (where the antibody is scFv) measured in the NFAT reporter gene assay, ng / mL IC50. ５０ It is the ratio to the value, and / or, (ix) When measured by ELISA, it has the ability to neutralize the PDL1 / B7-1 interaction with a titer greater than 4 (relative titer) compared to avelumab, and the said relative titer is equal to the IC50 of avelumab measured by ELISA at ng / mL. ５０ The IC50 value of the antibody (where the antibody is scFv) measured by ELISA is ng / mL. ５０ It is the ratio to the value. The antibody according to claim 1. [Claim 3] The aforementioned antibody (x) When the format is scFv, and the antibody is prepared in pH 6.4, 150 mM NaCl, 50 mM phosphate-citrate buffer, it has a melting temperature (Tm) of 60°C or higher when measured by differential scanning fluorescence measurement. When the (xi)scFv format is used, and the antibody is prepared in 50 mM phosphate citrate buffer in 150 mM NaCl at pH 6.4, the antibody is subjected to five consecutive freeze-thaw cycles at an initial concentration of 10 mg / ml, and shows a monomer content loss of less than 5%. and / or, (xi) When the format is scFv, and the antibody is prepared in 50 mM phosphate citrate buffer in 150 mM NaCl at pH 6.4, the antibody exhibits a monomer content loss of less than 5% after storage at 4°C for more than two weeks at an initial concentration of 10 mg / ml. The antibody according to claim 1 or 2. [Claim 4] The antibody according to any one of claims 1 to 3, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, Fab, Fv, scFv (excluding scFv containing a heavy chain variable region containing an amino acid sequence identical to or more than the amino acid sequence of SEQ ID NO: 16 and a light chain variable region containing an amino acid sequence identical to or more than the amino acid sequence of SEQ ID NO: 27), dsFv, and scAb. [Claim 5] The antibody according to claim 4, wherein the scFv has an amino acid sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO:

30. [Claim 6] The antibody according to any one of claims 1 to 5, wherein the antibody is a multispecific molecule, and the multispecific molecule has one or more second functional molecules. [Claim 7] A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. [Claim 8] An antibody according to any one of claims 1 to 6, for use as a pharmaceutical agent. [Claim 9] An antibody according to any one of claims 1 to 6, for use in the treatment of cancer. [Claim 10] A nucleic acid encoding an antibody according to any one of claims 1 to 6. [Claim 11] A method for producing an antibody according to any one of claims 1 to 6, comprising the step of culturing a host cell containing the nucleic acid of claim 10.

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