WO2019114235A1 - Pdl1 monoclonal antibody and application thereof - Google Patents

Abstract

The present invention provides an isolated human PDL1-specific binding molecule, being an isolated antibody or antigen-binding fragment. The human PDL1-specific binding molecule provided by the present invention can block PD1/PDL1 signaling and upregulate secretion of IFN-γ.

Description

 Technical field

 [0001] The present invention is in the field of tumor therapy and molecular immunology and relates to various anti-PDL1 antibodies, pharmaceutical compositions thereof and uses thereof. In particular, the invention relates to a plurality of monoclonal antibodies against PDL1.

 Background technique

 [0002] T cell-mediated cellular immunity plays an important role in the recognition and killing of tumor cells. T cells pass through T cell receptor (TCR) and major tissues with specific antigens on the surface of tumor cells. The major histocompatibility complex (MHC) binds to recognize tumor cells. The interaction between TCR and MHC molecules is controlled by a series of immune checkpoints, including co-stimulatory signals and co-suppression signals that activate or inhibit T cells. Among them, PD1 and its ligand PDL1 pathway are inhibitory immunological checkpoints, which combine to transmit co-suppressive signals, can inhibit the immune activity of T cells, play an important role in immune tolerance, and are also important for tumor cell immune escape. the reason.

 [0003] Programmed death receptor 1 (PD1) is an important immunosuppressive molecule.

 A type I transmembrane protein that is a member of the CD28 superfamily, originally cloned from apoptotic mouse T cell hybridoma 2B4.11. Immunomodulation targeting PD1 has important implications for anti-tumor, anti-infective, anti-autoimmune diseases, and survival of organ transplants. PD1 has two binding ligands, PDL1(B7-Hl)fPPDL2(B7-DC), which have different expressions. PDL2 expression is limited, mainly expressed in activated macrophages, dendritic cells and a few tumors. PDL1 is widely expressed in activated T cells, B cells, macrophages, dendritic cells, and tumor cells, and is expressed in tissues such as the placenta, eyes, and epithelium, muscle, liver, and vascular endothelium. Therefore, the role of PDL1 in the body far exceeds that of PDL2.

 [0004] Programmed cell death 1 ligand 1, PDL1 is also known as a cluster of differentiation 274 (CD274) or a B7 homolog 1 (B7-H1). It is a protein in human body, encoded by the CD274 gene.

[0005] PDL1 has IgV and IgC-like regions, a transmembrane region, and a cytoplasmic tail, wherein the cytoplasmic tail region is associated with intracellular signal transduction, and IgV and IgC are involved in intercellular signal transduction. Studies have shown that TNF, IFN-Y, I L-4, granulocyte stimulating factor and IL-10 and other cytokines can up-regulate the expression of PDL1 in different cells. \¥0 2019/114235 卩(:17(:\2018/092330 达.

The role of this molecule has a wide range of tissue expression profiles, high expression in some tumor cell lines, and many studies have shown that it is related to the immune escape mechanism of tumors. The microenvironment of the tumor part can induce tumor

 Anti-tumor! Cell apoptosis.

 [0007] After binding, transmitting an inhibitory signal, inhibiting lymphocyte proliferation, activity, and inhibition

0041 cells differentiated into 11 ₁₁ and 71 ₁₁₇ cells and inhibited the release of inflammatory cytokines.

Peripheral lymphocytes are immune to their own antigens to prevent autoimmune diseases. but

 The inhibitory effect, thereby promoting the immune escape of the tumor.

_[0008] The mechanism of tumor immune escape includes: down-regulation of tumor cell membrane surface complex _(MHC) -like molecules, lack of immunostimulatory molecules, secretion of immunosuppressive cytokines, expression of death ligands or expression

occur. In addition, the tumor microenvironment consists of tumor cells, blood vessels, tissue fluids, mesenchymal cells, and a small amount of infiltrating lymphocytes. There are also some inflammatory cytokines in the tumor microenvironment, such as positive ₀ and positive sputum.

₁ will also be an effective target for anti-tumor therapy.

 Summary of invention

 technical problem

 Problem solution

Technical solution _[0010] The present invention utilizes a mammalian cell expression system to express recombinant _PDL1 as an antigen to immunize mice, and fuses mouse spleen cells with myeloma cells to obtain hybridoma cells. The present invention obtains a plurality of hybridoma cell lines by screening a large number of samples, and is capable of secreting a specific monoclonal antibody which specifically binds to _PDL1 , and the monoclonal antibody further obtains a chimeric antibody by molecular cloning or the like. The prepared chimeric antibody was able to bind to _{PDL1 on the} surface of human _T cells (Fig. ₁ , Table _III ) and had high affinity (Table _II ). A series of characterization and functional experiments were performed to show that these chimeric antibodies bind to activated _T cells and _DC cells (Figures ₂ and ₃ ) and are very effective at blocking the binding between _PDL1 and _PD1 or _{CD 80} . (Figure ₄ , Table _IV ). Species cross-reactivity experiments showed that chimeric antibodies had no or little cross-reactivity with cynomolgus monkeys and mice (Figure 5, Table _V ). In vitro functional assays have shown that these chimeric antibodies can promote the immune function of _T cell lines or primary _T cells (Fig. ₆ , ₇ ). Animal experiments show that these drug-forming antibodies can effectively inhibit tumors in humanized _PDL1 knock-in Growth in the mouse (Figure ₈ ). Finally, these antibodies were humanized to reduce immunogenicity and to obtain therapeutic antibodies. The following invention is thus provided:

_[0011] The invention provides antibodies that specifically bind to human _PDLl , which are produced from hybridoma cell lines. The hybridoma cell line provided by the present invention is derived from mouse immunization, cell fusion, and a series of operations such as screening can produce a monoclonal antibody which specifically blocks the binding of _PDL1 and _PD1/CD80 . (See Example ₁ ) _[0012] The present invention also provides a chimeric antibody that specifically binds to human _PDL1 , wherein a chimeric antibody is constructed by _ligating the variable region of a murine _PDL1 antibody to human _IgG1 and _K constant regions Heavy and light chains. (See Example ₂ )

_[0013] The chimeric antibodies provided by the present invention have high affinity in kinetic detection, wherein the equilibrium binding constant _K

_3⁄4 £ _3.89xl0

The kinetic parameters provided by one aspect of the invention were determined using the biomolecular interaction system _Octet-96 ( _{Pall Life Sciences, S-000959} ). (See Example ₃ )

_[0014] A chimeric antibody provided by one aspect of the invention binds to a cell surface antigen with an _EC50 of _62.6

In the range of n _g/mL and _{437.7 ng/mL} ; wherein the term _EC50 refers to the _{concentration for 50% of maximal effect} . (See Example ₄ )

_[0015] One aspect of the invention provides a chimeric antibody that binds to activated _T cells and _DC cells. (See example

5) \¥02019/114235 卩(:17(:\2018/092330

是指被测量的拮抗剂的半抑制浓度[0016] A chimeric antibody provided by another aspect of the invention blocks ?00 and ? 01, or binding 0080, 1050, respectively, the range in the range of 119.8 _{¾ / 11} and 178.5 _{¾ / 11} and 608.5 _{¾ / 11} and 1867 _{¾ / 11.} Of which, 1050

Refers to the semi-inhibitory concentration of the antagonist being measured

. (See Example 6)

公开的序列。 例如， 67-1(00 80,

[0017] In the present invention, unless otherwise specified, the 0080 is: 87-1; the specific protein sequence thereof is a sequence known in the prior art, and may refer to the existing literature or

The sequence disclosed. For example, 67-1 (00 80,

 [0018] A chimeric antibody provided by one aspect of the invention cross-reacts with a mouse without a species and cross-reacts with a species of cynomolgus monkey. (See Example 7)

细胞的免疫功能。 (参见实施例 8) The chimeric antibody provided by the invention can be enhanced

The immune function of the cells. (See Example 8)

， 本发明抗体可以增强正1^-丫的分泌 (参 见实施例 9) 。 The chimeric antibodies provided by the present invention have strong in vitro biological activities. Mixed lymphocyte reaction in allogeneic

The antibody of the present invention can enhance the secretion of positive 1^-丫 (see Example 9).

 [0021] The drug-forming antibody provided by the present invention has strong in vivo pharmacological activity. In tumor animal models, the antibodies of the invention are effective in inhibiting tumor growth and reducing tumor volume. (See Example 10)

[0022] One aspect of the invention provides an isolated human-specific binding molecule comprising and 1 ₅₎ , _&) three light chains 0

 50, $£010X0:56, $£(30)X0:62, $£(310X0:68, $£010X0:74, 8£〇10 NO:80, or $£(30)X0:86;

[0027] ( ₁₁₁₎ Heavy chain 00113 is selected from: $£(310X0:39, $£(310X0:45, $£(30)X0:

51, $£(310X0:57, $£(30)X0:63, $£(310X0:69, $£(310X0:75, 8£〇10 N0:81, or $£(30)X0:87; \¥02019/114235 卩(:17(:\2018/092330

40所示的轻链

41所示的轻 链 00112、 以及如

42所示的轻链 00113; 或者 The chain variable region contains

Light chain shown in 40

The light chain 00112 shown in 41, and as

Light chain 00113 shown in 42; or

(9) The heavy chain variable region comprises as

45所示的重链 00113; 和/或轻 链可变区包含如

46所示的轻链

47所示的轻 链 00112、 以及如

48所示的轻链 00113; 或者 N0: 44 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown in 45 contains

Light chain shown in 46

Light chain 00112 shown in 47, and as

Light chain 00113 shown in 48; or

[0034] (111) the heavy chain variable region comprises

51所示的重链 00113; 和/或轻 链可变区包含如

52所示的轻链

53所示的轻 链 00112、 以及如

54所示的轻链 00113; 或者 N0: 50 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown at 51 comprises

Light chain 52

Light chain 00112 shown in Figure 53, and as

Light chain 00113 shown in 54; or

(IV) the heavy chain variable region comprises as

57所示的重链 00113; 和/或轻 链可变区包含如

58所示的轻链

593所示的 轻链 00112、 以及如

60所示的轻链 00113; 或者 N0: 56 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown in 57 contains

Light chain shown in 58

Light chain 00112 shown in 593, and as

Light chain 00113 shown in 60; or

\¥0 2019/114235 卩（：17（：\2018/092330 (V) the heavy chain variable region comprises a heavy chain as shown at 61

\¥0 2019/114235 卩(:17(:\2018/092330

64所示的轻链

65所示的轻 链 00112、 以及如

66所示的轻链 00113; 或者 a heavy chain 00112 represented by NO: 62, and a heavy chain 00113 as indicated by 63; and/or a light chain variable region comprising

Light chain shown in 64

Light chain 00112 shown in 65, and as

Light chain 00113 as shown in 66; or

[0037] ( _{VI) the} heavy chain variable region comprises

69所示的重链 00113; 和/或轻 链可变区包含如

70所示的轻链

71所示的轻 链 00112、 以及如

72所示的轻链 00113; 或者 N0: 68 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown in 69 contains

Light chain shown at 70

Light chain 00112 shown in 71, and as

Light chain 00113 as shown in 72; or

73所示的重链

[0038] The heavy chain variable region comprises

Heavy chain shown in 73

75所示的重链 00113; 和/或轻 链可变区包含如

76所示的轻链

77所示的轻 链 00112、 以及如

78所示的轻链 00113; 或者 N0: 74 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown at 75 comprises

Light chain shown in 76

Light chain 00112 shown in 77, and as

Light chain 00113 shown in 78; or

[0039] a heavy chain variable region comprising

81所示的重链 00113; 和/或轻 链可变区包含如

82所示的轻链

83所示的轻 链 00112、 以及如

84所示的轻链 00113; 或者 N0: 80 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown at 81 comprises

Light chain shown in 82

Light chain 00112 shown in 83, and as

Light chain 00113 as shown in 84; or

( _{IX) the} heavy chain variable region comprises as

87所示的重链 00113; 和/或轻 链可变区包含如

88所示的轻链

89所示的轻 链 00112、 以及如

90所示的轻链匚0尺3。 N0: 86 shows the heavy chain 00112, and as

The heavy chain 00113; and/or the light chain variable region shown at 87 comprises

Light chain shown in 88

Light chain 00112 shown in 89, and as

The light chain shown at 90 is 0 feet 3 .

 [0041] In some embodiments of the invention, the antibody is a full length antibody.

或?(& )' ₂或₈〇 。 [0042] In some embodiments of the invention, the antigen-binding fragment is

Or ?(&)' ₂ or ₈ 〇.

In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein:

( _{1) The} heavy chain variable region selection

 NO: 33, and $£(30) X0: 35;

[0045] (9) the light chain variable region is selected from the group consisting of:

24, $£(310 X0: 26; $£(30) X0: 28, $£(310 X0: 30, $£(310 X0: 32, 8£〇10 \¥02019/114235 卩(:17(:\2018/092330

NO: 34, and $£(3 0) X0: 36;

( ₁₁₁₎ a sequence having at least 80% homology to the sequence of ₍₁₎ , ₍₃₎ .

 In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein:

[0058] ( ₁₎ Blocking 0!^ and ? a combination of 01 or 0080;

 In some embodiments, the monoclonal antibody or antigen binding portion thereof is used in the manufacture of a medicament for inhibiting tumor cell growth in a patient.

 In some embodiments, the monoclonal antibody or antigen binding portion thereof is used in the manufacture of a medicament for the treatment of an infectious disease.

 One aspect of the invention provides an isolated nucleic acid encoding one or both of an antibody light chain variable region and an antibody heavy chain variable region, wherein

(!) A fragment encoding the heavy chain variable region of the antibody is selected from the group consisting of: SEQ ID NO: 1 _^ 8 £ 10 10 N0: \¥02019/114235 卩(:17(:\2018/092330

3, $£010X0:5, SEQIDNO:7, $£(310X0:9, $£010X0: 11, 8£〇10 N0: 13, $£(310X0: 15, and $£(30)X0: 17;

(9) a fragment encoding the variable region of the light chain of the antibody is selected from the group consisting of:

 4, $£(310X0:6, SEQIDN0:8, $£(310X0: 10, $£(310X0: 12, 8£〇10 N0: 14, $£(30)X0: 16, and $£(310X0: 18;

 (111) a sequence having at least 80% homology to the sequence of (1), (3).

 The invention also provides a vector comprising the nucleic acid molecule.

 The invention also provides a host cell comprising the nucleic acid molecule or vector.

单克隆抗体； 该抗人

单克隆抗体为所述的分离的人

特异性结合分子。 The present invention also provides a conjugate comprising an anti-human covalently linked to an isotope, an immunotoxin, and/or a chemical

Monoclonal antibody;

Monoclonal antibody is the isolated person

Specific binding molecules.

特异性结合分子和 /或 所述的结合物与固体介质或半固体介质偶联形成。 [0071] The present invention also provides a conjugate comprising the isolated human

The specific binding molecule and/or the conjugate is formed by coupling with a solid medium or a semi-solid medium.

单克隆抗体和 /或结合物和 /或偶联物在制备治疗疾 病的药物中的应用； The present invention also relates to the said anti-antibody

Use of monoclonal antibodies and/or conjugates and/or conjugates for the preparation of a medicament for the treatment of a disease;

 [0073] The disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma or melanoma.

人源化单克隆抗体或其抗原结合部 分、 核酸分子、 载体、 宿主细胞、 结合物、 或者偶联物， 以及任选的药学上可 接受的载体或赋形剂， 以及任选的其它生物活性物质。 The present invention provides a composition containing anti-human

A humanized monoclonal antibody or antigen binding portion thereof, nucleic acid molecule, vector, host cell, conjugate, or conjugate, and optionally a pharmaceutically acceptable carrier or excipient, and optionally other biological activity substance.

的所述抗体或所述抗原结合片段的复合物的试剂。 The invention also relates to a kit comprising the isolated antibody or antigen-binding fragment thereof and a set for detecting binding to a human

An agent of the antibody or a complex of the antigen-binding fragment.

 The invention is further described below: In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. Also, the proteins and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunologically relevant terms, and laboratory procedures used herein are terms and routine steps that are widely used in the corresponding art. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

[0077] The term "antibody" as referred to herein includes intact antibodies and any antigen-binding fragments thereof (ie, "antigen-binding portions" \¥0 2019/114235 卩(:17(:\2018/092330

""" or "single chain.""Antibody" refers to at least two heavy ( _3⁄4 ) chains and two light ( _3⁄4 chain glycoproteins, or antigen-binding portions thereof) that are linked together by a disulfide bond. Each heavy chain Heavy chain variable region (abbreviated herein as _V)

Composition, they are arranged from the amino terminus to the carboxy terminus in the following order: ₁₁₁ , ₀₀₁₁₁ , ? _{112, 00112,} foot-3, _{00113,? 114} . The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system ((:.

_[0078] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to retention with an antigen (

 The function can be performed by a fragment of a full length antibody. The term "binding layer" included in the "antigen-binding portion" of an antibody

The synthetic linkers are joined together, which allows them to make a protein chain, and!!

 The original binding moiety is included. These antibody fragments are obtained by conventional techniques well known to those skilled in the art, and the utility of these fragments is screened in the same manner as intact antibodies.

_{[0079] As} used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (:

Other antigens such as 1 molecule may have cross-reactivity. Moreover, the isolated antibody can be substantially free of other cellular materials and _/ or chemicals. \¥0 2019/114235 卩(:17(:\2018/092330

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single molecule composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope

序列已被移植到人构架序列上的抗体。 The term "human antibody" as used herein includes an antibody having a variable region in which both the framework regions and regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. Human antibodies of the invention may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein does not include a germline derived from another mammalian species such as a mouse.

The sequence has been grafted to an antibody on a human framework sequence.

 The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity, which has a variable region in which both the framework region and the sputum region are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising: fused to an immortalized cell: 8 cells, the 8 cell is transgenic from a genome having a human heavy chain transgene and a light chain transgene Obtained in human animals (eg, transgenic mice).

的任何其他方法 制备、 表达、 产生或分离的抗体。 这些重组人抗体具有其中构架区和

区均 源自人种系免疫球蛋白序列的可变区。 但是在某些实施方案中， 这些重组人抗 体可以经历体外诱变（或者， 当使用人 ¾序列的转基因动物时， 经历体内体细胞 诱变）， 因此重组抗体

区的氨基酸序列尽管是源自人种系

序列 并与之相关的序列， 但可能不是在体内天然存在于人抗体种系的所有组成成分〇·

The term "recombinant human antibody" as used herein includes all human antibodies produced, expressed, produced or isolated by recombinant methods, eg: (from transgenic or transchromosomal animals (eg, mice) for human immunoglobulin genes or An antibody isolated from a hybridoma (described further below) prepared therefrom, (3⁄4) an antibody isolated from a host cell transformed with a human antibody, such as a transfectoma, (〇) an antibody isolated from a recombinant combinatorial human antibody library

Any other method of preparing, expressing, producing or isolating antibodies. These recombinant human antibodies have a framework region and

The regions are all derived from the variable regions of human germline immunoglobulin sequences. However, in certain embodiments, these recombinant human antibodies can undergo in vitro mutagenesis (or, when using a human sequence of transgenic animals, undergo in vivo somatic mutagenesis), thus recombinant antibodies

The amino acid sequence of the region, although derived from the human germline

Sequences and sequences associated with them, but may not be all components of the human antibody germline that are naturally present in the body〇

The column has been grafted to the antibody on the human framework sequence. Other framework region modifications can also be made within the human framework sequence. \¥0 2019/114235 卩(:17(:\2018/092330

_{[0085] The} term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, eg, wherein the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human. Antibody to antibodies.

_[0086] As used herein, the term ": ^ or _{^" "refers} to a particular antibody - antigen interaction rate of binding, and herein, the term": ^ _{"or" "refers} to a particular antibody - antigen interaction dissociation rate The term " ₁ ^" as used herein refers to the dissociation constant, which is obtained from the ratio of ₁ ^ to ₁ ^ (ie ₁ ^ _/1 ^) and is expressed as the molar concentration (: _M ). The value of the antibody may be As determined by methods established in the art. A preferred method of determining antibodies: using surface plasmon resonance, preferably using a biosensor system.

_[0087] "Homology" refers to a bad sequence between two polynucleotide sequences when aligned or most preferably two polypeptide sequences Xiang similarity.

 See or link to a polynucleotide that is not linked in nature.

 Advantageous effects of the invention

 Beneficial effect

Clonal antibodies combined with tumor vaccine for tumor immunotherapy can effectively enhance the immune activation of tumor vaccines. Head

 Two years of popular targets in the field of lung cancer treatment.

 Brief description of the drawing

 DRAWINGS

[0094] 图 4b_{: ELISA}检测 PDL1嵌合抗体阻断 CD80与 PDL1之间的相互作用；

Figure 4b _{: ELISA} detection of PDL1 chimeric antibodies blocking the interaction between CD80 and PDL1;

Figure 5a _{: ELISA} detects cross-reactivity of PDL1 chimeric antibodies with cynomolgus PDL1 species;

Figure 5b _{: FACS} detection of PDL1 chimeric antibodies cross-reactive with mouse PDL1 species;

[0097] FIG. _6: Luciferase assay kit PDL1 chimeric antibody on the immune response of Jurkat cells; [0098] FIG. _{7: ELISA} detection experiment PDL1 MLR secreted chimeric antibody enhancement of IFN- _Y;

Figure 8 _{: PDL1} drug-forming antibodies inhibit tumor growth in PDL1 humanized transgenic mice.

9a-9c _: Table 1, a sequence number table of the anti-PDL1 antibody of the present invention;

Figure 10 _: Table 2, Human Mouse PDL1 Chimeric Antibody Kinetic Analysis Table;

Figure 11 _: Table 3, cell binding half effective concentration table of human murine PDL1 chimeric antibody;

Figure 12 _: Table IV, ligand blocking test table for human murine PDL1 chimeric antibody;

Figure 13 _: Table 5. Cross-reactivity table of human murine PDL1 chimeric antibodies.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention discloses monoclonal antibodies and their applications, and those skilled in the art can learn from the contents of the present invention and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described in the preferred embodiments, and it is obvious that those skilled in the art can modify and adapt the methods and applications described herein to implement and apply the present invention without departing from the scope of the present invention. Invention technology.

 The monoclonal antibodies provided by the present invention and the materials and reagents used in the application thereof are commercially available.

 [0107] The present invention will be further illustrated below in conjunction with the embodiments:

Example 1 _: Screening of animal-free sore and anti-PDL1 murine antibodies

[0109] Balb/c mice of appropriate age were selected for immunoinjection. After the hPDL1-mFc fusion protein was used as an antigen and mixed with complete Freund's adjuvant (Sigma-Aldrich), the mice were immunized by subcutaneous injection to stimulate the corresponding B lymphocyte clones. Subsequently, immunized mice were boosted by intraperitoneal injection of 100 1:1 emulsified hPDL1-mFc in incomplete Freund's adjuvant (Sigma-Aldrich) approximately every two to three weeks. The mouse spleen lymphocytes were removed by aseptic operation, mixed with the prepared SP2/0 myeloma cells in a certain ratio (spleen cells 1×10 ⁸ , myeloma cells 2×10 ⁷ ), and polyethylene glycol (Sigma, P7181) was added. ) Perform cell fusion.

 [0110] After fusion, the fused cells were added to a 96-well plate, 0.1 mL of HAT medium was added to each well, and placed in a carbon dioxide incubator, and cultured at 37 ° C; on the fourth day, 0.1 mL of HT medium was added to each well; On day 7, the medium was completely replaced with HT medium. Screening was performed on days 8-12, and positive cells were subcloned by limiting dilution, expanded, and tested by ELISA and FACS. The specific methods are as follows:

2/mL of hPDL1-His was coated on a 96-well plate (Coming, catalog #9018), 4. C overnight incubation. Plates were washed three times with PBST (PBS containing 0.05% Tween-20) and blocked with 5% skim milk powder (Oxoid, catalog #LP0031B). After the plate was washed three times with PBST, hybridoma supernatant was added, 37 ° C for 2 hours. ^"After the plate was washed, added 1/5000 diluted HRP labeled goat anti-mouse IgG secondary antibody (BioLegend, catalog # 405306), 37. C was incubated for 1 hour. After washing the plate, TMB (Tiangen, catalog # PA107-02) was added for color development.

Binding of antibodies to B16-hPDL1 cells (endogenous mouse PDL1 gene knockout) was detected by FACS. Trypsin-digested 2.5x10 ⁵ B 16-hPDL1 cells were added to each well in a 96-well plate (Coming, catalog #3799) and incubated with the hybridoma supernatant for 30 minutes at 4 °C. After washing the cells twice with buffer (PBS containing 1% BSA and 5 mM EDTA), APC-labeled goat anti-mouse IgG secondary antibody (BD, catalog #550826) was added and incubated at 4 ° C for 30 minutes. After the cells are washed with buffer, in Attune

 Analysis on a Nxt flow cytometer (Thermo Fisher).

 The PDL1 antibody is capable of blocking the binding of PDL1 to PD1, and thus the hybridoma supernatant is further screened using a ligand blocking assay. lJ ^ig/mL of hPDl-hFc was coated in 96-well plates at 4 ° C overnight. After washing the plate for 3 times, it was blocked with 5% skim milk powder at 37 ° C for 2 hours. After washing the plate three times, pre-mixed hybridoma supernatant and 200 ng/mL biotinylated hPDL1-hFc were added and incubated for 1 hour at room temperature. After washing the plate, HRP-labeled streptavidin was added and incubated for 1 hour at room temperature. After washing the plate, add TMB color.

 The PDL1 antibody is capable of blocking the binding of PDL1 to CD80, and thus the hybridoma supernatant is further screened using a ligand blocking assay. 2 / 11 of hCD80-hFc was coated in 96-well plates at 4 ° C overnight. After washing the plate for 3 times, it was blocked with 5% skim milk powder at 37 ° C for 2 hours. After washing the plate three times, pre-mixed hybridoma supernatant and 1000 ng/mL biotinylated hPDL1-hFc were added and incubated for 1 hour at room temperature. After washing the plate, HRP-labeled streptavidin was added and incubated for 1 hour at room temperature. After washing the plate, add TMB color.

The cross-reactivity of PDL1 antibody with cynomolgus PDL1 was examined using an ELISA method. Will 2 _[i g/mL of anti-mouse IgG Fc antibody (Sigma, catalog #M428〇) was coated onto a 96-well plate at 4 ° C overnight. Block with 5% skim milk powder for 2 hours at 37 °C. After washing the plate with PBST, the hybridoma supernatant was added and incubated for 30 minutes at room temperature. After washing the plate three times with PBST, the biotin-labeled cynomolgus monkey PDL1 (SinoBiologi cal, catalog

 #90251-C08H) Add to the plate and incubate for 1 hour at room temperature. The plate was washed three times with PBST, and 1/1000 diluted HRP-labeled streptavidin was added and incubated for 1 hour at room temperature. After washing the plate, add TMB color.

 Example 2: Cloning of murine antibody cDNA and construction of chimeric antibody

 The variable region gene sequences of the hybridoma antibody heavy and light chains were obtained using the degenerate primer PCR method. Hybridoma Monoclonal cells were isolated using Trizol (invitrogen, catalog #15596-018) and total RNA was isolated using Superscript III First-Strand Synthesis System (invitrogen, catalog

 #18080-051) , Reverse transcription using RNA as a template to obtain a cDNA library. Using the obtained cDNA library as a template, PCR was performed using degenerate primers (Zhou H, et al., Nucleic Acids Research 22: 888-889 (1994), Chardes T et al „ FEBS Letters 452: 386-394 (1999)) The PCR product was detected by agarose gel electrophoresis, and the PCR amplification products of the heavy and light chain variable regions were expected to be 400 base pairs. The PCR product was cloned into pClone007 vector (Tsingke, catalog #TSV-007BS) and transformed. Trans 5ot (Transgen, catalog#)

 CD201-02), and selected 3-6 E. coli monoclonals on agar plates for gene sequencing. In some cases, PCR products can also be used directly for gene sequencing. By the above method, the variable region full-length gene sequences of the heavy and light chains of the antibody are finally obtained. By NCBI

 Ig-BLAST (https://www.ncbi.nlm.nih.gov/projects/igblast/) Further analysis yielded complementarity determining regions for anti-weight and light chains (Complementarity Determining)

 Region) and the framework region's gene and amino acid sequences (Kabat E.A., et al., 1991, Sequences of proteins of immunological interest, in: NIH Publication No.

 91-3242, US Department of Health and Human Services, Bethesda, MD). See the antibody sequence listing and Figure 9a-Figure 9c (Table I) for specific information.

The heavy and light chains of the chimeric antibodies were constructed by ligating the heavy and light chain variable regions of the murine PDL1 antibody to human IgG1 (or IgG1 mutant N297A) and the K constant region, respectively. A suitable restriction site is introduced into the variable region of the heavy chain and the light chain by PCR, and cloned into the corresponding chimeric antibody expression vector, respectively. The chimeric antibody heavy and light chain plasmids were introduced into 293F cells by lipofection and continued for 6 days. The antibody in the cell culture supernatant was purified using a Protein A column (GE Healthcare, catalog #17549112). The Protein A column was washed with 1 mM PBS, and then the antibody was removed from 50 mM PBS (pH 3. 〇).

 filter. After the antibody solution was concentrated by Ultra-15 centrifugal concentrators (Millipore, catalog #ACS 500024), the quantitative antibody concentration was measured using a Nanodrop spectrophotometer. The endotoxin content in the purified antibody solution was determined using a Gel Clot TAL kit (Xiamen Bioendo Technology, catalog #010250) with a standard content of less than 1 EU/mL.

Example 3 _: Human mouse PDTJ chimeric antibody kinetics

The kinetic constant ( _{ka J3⁄4kn} ) of binding between the antibody and the antigen was measured using the biomolecular interaction system Octet-96 (Pall Life Sciences, S- ₀₀₀₉₅₉ ), and the equilibrium binding constant _K was further calculated. The hPDL1-mFc antigen protein was coupled to the surface of an AMC sensor (Pall Life Sciences, PN18-5099) and antibodies of different concentrations were added to measure the binding and dissociation between the PDL1 chimeric antibody and the sensor surface PDL1-mFc protein. Specifically, the AMC sensor was pre-wet for 10 minutes in buffer (PBS containing 0.02% Tween-20 and 0.1% BSA) and then equilibrated in the sample buffer of hPDL1-mFc for 5 minutes to couple the PDL1-mFc protein to Sensor surface. The PDL1-mFc-coupled AMC sensor was first equilibrated in buffer for 2 minutes and then placed in antibodies containing different concentrations (3-200).

nM) was incubated for 5 minutes in buffer to measure binding of the antibody to the PDL1-mFc protein; finally, the antigen- and antibody-conjugated sensor was repositioned in the sample buffer and waited for ₁₀ minutes to measure the antibody and hPDL1-mFc Dissociation of the protein. The measured data were fitted by a 1 _:1 model to obtain the binding and dissociation kinetic constants, and on this basis, the equilibrium binding constant _Kfl was further obtained. The experimental results are shown in Figure 10 (Table II).

Example 4 _: Binding of human ilPDL1 chimeric antibody to B16-hPDL1 cells

 The human and mouse chimeric antibodies were further tested for binding to cell surface hPDL1 using the FACS method. B16-hPD L1 cells (endogenous mouse PDL1 gene knockout) with FACS buffer (1% in PBS)

BSA) was resuspended to 4x10 ⁶ cells/mL, and 50 μL/well was added to a 96-well round bottom plate (Coming, catalog #3795). The purified antibody to be tested was added to the well (50 ^ ₁₁₇ well) at a certain concentration, and incubated at 4 ° C for 1 hour. After the cells were washed three times with buffer, a PE-labeled fluorescent secondary antibody was added, 4. C was incubated for 0.5 hours. Cell with FAC The buffer was washed three times, resuspended to 200 _[iL/ well, and the fluorescence signal was detected using an Attune Nxt flow cytometer (Thermo Fisher). The results are shown in Figure 1. The combined effective half concentration is shown in Figure 11 (Table III).

Example 5 _: Binding of human ilPDL1 chimeric antibodies to activated T cells and DC cells

The human and mouse chimeric antibodies were further tested for binding to activated T cells and dendritic cell surface hPD L1 using the FACS method. Peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood using density gradient centrifugation (Ficoll-Paque Premium, GE Healthcare, catalog #17-5442-02). Human CD3 ⁺ T cells were isolated from PBMC using the Pan T Cell Isolation Kit (Miltenyi, catalog #130-096-535) and activated with an equal amount of CD3/CD28 Dynabeads (Invitrogen 11132D) for 48 hours. Activated T cells were resuspended in FACS buffer (PBS containing 1% BSA) to 4x10 ⁶ cells/mL, 50

 ^L/hole is added to the 96-well round bottom plate (Coming, catalog #3795). The purified antibody to be tested was added to the well at a certain concentration (50 ^/3⁄4), and incubated at 4 ° C for 1 hour. After the cells were washed three times with buffer, a fluorescent secondary antibody labeled with PE was added, 4. C was incubated for 0.5 hours. The cells were washed three times with FACS buffer, resuspended to 200 pL/well, and the calender signal was detected using an Attune Nxt flow cytometer (Thermo Fisher). The result is shown in Figure 2.

Mononuclear cells (monocytes) were further isolated and purified from PBMCs using a CD14 Cell Isolation Kit (Miltenyi, catalog #130-050-201). Induction of monocyte differentiation into dendritic cells by adding cytokine 1000 U/mL GM-CSF (Prospec, catalog #CYT-22l) and 1000 U/mL IL-4 (Prospec, catalog #CYT21l) to the culture medium (Dendritic

Cells). Cytokines are replenished every 2-3 days and immature dendritic cells are harvested after 5-6 days. Dendritic cells were resuspended in FACS buffer (PBS containing 1% BSA) to 4x10 ⁶ cells/mL, 50 μL/well was added to 96-well round bottom plate (Coming, catalog

 #3795) . The purified antibody to be tested was added to the well (50 μL/well) at a certain concentration, and incubated at 4 ° C for 1 hour. After the cells were washed three times with buffer, a PE-labeled fluorescent secondary antibody was added, 4. C was incubated for 0.5 hours. The cells were washed three times with FACS buffer, resuspended to 200 pL/well, and the fluorescence signal was detected using an Attune Nxt flow cytometer (Thermo Fisher). The result is shown in Figure 3.

Example 6 _: Human mouse PDL1 chimeric antibody positron PD1 or interaction between CDS0 and PDL1

Whether the antibody blocked the binding between hPD1 or CD80 and hPDL1 was detected by an ELISA method. The hPDL1-hFc protein was biotinylated using the Biot in Labeling Kit-NH ₂ (Dojindo, catalog #LK03) kit. hPDl-hFc or hCD80-hFc was coated onto 96-well plates at 4 °C overnight. Use 5% skim milk powder Block at 2 °C for 2 hours. After washing the plate three times, pre-mixed biotinylated hPDL1-hFc and PDL1 chimeric antibodies were added and incubated for 1 hour at room temperature. The plate was washed with PBST, HRP-labeled streptavidin was added, and incubated for 1 hour at room temperature. After washing the plate, add TMB to develop color. The results are shown in Figures 4a and 4b, and the results are summarized in Figure 12 (Table IV).

Example 7: Human mouse PDTJ chimeric antibody species

 The human mouse PDL1 chimeric antibody was cross-reactive with cynomolgus species using an ELISA method. Quantitatively purified antibodies were coated onto 96-well plates at 4 °C overnight. It was blocked with 5% skim milk powder at 37 ° C for 2 hours. Biotinylated cynomolgus PDL1 (SinoBiological, catalog # 90251-C08H-200) was added to the plate and incubated for 1 hour at room temperature. The plate was washed three times with PBST, and 1/1000 diluted HRP-labeled streptavidin was added and incubated for 1 hour at room temperature. After washing the plate, add TMB color. The results are shown in Figure 5a and Figure 13 (Table V).

The FACS method was used to detect cross-reactivity of human murine PDL1 chimeric antibodies with mouse species. The wild-type B 16 cells were trypsinized with FACS buffer (PBS containing 1% BSA) resuspended to 1.25x10 ⁶ cells / mL. 80 pL of the cell suspension was mixed with 80 pL of the gradient diluted antibody and allowed to stand at room temperature for 30 minutes. After washing three times with PBST, a fluorescently labeled secondary antibody (BioLegend, catalog #409304), 4 was added. C was incubated for 1 hour. PBST wash? After the article, use the Attune Nxt flow cytometer (Thermo

 Fisher) Detects fluorescent signals. The results are shown in Figure 5b and Figure 13 (Table V).

 Example 8: Human ilPDL1 chimeric antibody enhances the sore-free function of Jurkat cells

 The Jurkat-PD 1 cells containing the luciferase reporter gene were used to detect the cellular biological activity of the human murine PDL 1 chimeric antibody. In this cell experiment, Raji/hPDL1 cells were used as antigen presenting cells, and Jurkat/hPD1/N FKB-luciferase cells were used as effector cells. Addition of human and murine PDL1 chimeric antibodies blocked the interaction of intercellular PD1/PD L1 and activated the expression of the Luciferase reporter gene in Jurkat/hPD1/NFKB-luciferase cells. Specifically, exogenously expressed hPDL1 was introduced into Raji cells by introducing exogenously expressed hPD1 and Luciferase reporter gene containing NFKB promoter into Jurkat cells. After mixing Raji/hPDL1 cells with Jurkat/hPD 1/NFKB-luciferase cells, add T cell receptor activator and gradient dilution of the antibody to be tested, and detect the luciferase assay kit (Promega, G7940) after 6-16 hours. . The results showed that the human and mouse PDL1 chimeric antibodies enhanced the fluorescence signal and showed a dose-dependent effect. The results are shown in Fig. 6.

Example 9: Allogeneic mixed R钿朐 assay of human murine PDTJ chimeric antibody (Mixed T.vmphocvte) Reaction. MLR)

 The in vitro biological activity of the human murine PDL1 chimeric antibody was detected using a mixed lymphocyte reaction. Peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood using density gradient centrifugation (Ficoll-Paque Premium, GE Healthcare, catalog #17-5442-02). Use CD 14 Cell Isolation

 Kit (Miltenyi, catalog

 #130-050-201) Monocytes were further isolated and purified from PBMC. By adding cytokine 1000 U/mL GM-CSF to the medium (Prospec, catalog

 #CYT-221) and 1000 U/mL IL-4 (Prospec, catalog #CYT211) to induce monocyte differentiation into dendritic cells. Cytokines are replenished every 2-3 days, and dendritic cells are harvested after 5-6 days for subsequent experiments.

Human CD3 ⁺ T cells using Pan T Cell Isolation Kit (Miltenyi, catalog

#130-096-535) Isolated from PBMC. CD3 ⁺ T cells and immature dendritic cells, as well as different concentrations of PDL1 antibody, were added to a 96-well round bottom plate (Coming, catalog #3799). After 5-6 days of cell culture, the supernatant was harvested and the cytokine content was quantified by ELISA. As a result, as shown in Fig. 7, the human murine P DL1 chimeric antibody was able to enhance the secretion of IFN-γ in the MLR.

 Example 10: Human ilPDL1 chimeric antibody inhibits tumor length

The tumor animal model described in the present invention is constructed by inoculating a genetically modified mouse colon cancer cell line MC38/mPDL1 ^K0 /hPDL1 in a PDL1 humanized transgenic mouse for use in a mouse. A special tumor animal model for testing the in vivo efficacy of a drug-derived PDL 1 antibody.

The PDL1 humanized transgenic mice (7-11 weeks old) were injected subcutaneously with a 0.1 mL volume of 5× ^{10 5} MC38/mPDL1 ^K °/h PDL1 tumor cells. Seven days after the tumor cells were inoculated, the PDL1 antibody, the reference antibody or the control PBS was injected twice a week at a dose of 10 mg/kg (dose volume of 10 mL/kg) for a total of 6 administrations. The subcutaneous tumor volume was measured twice a week using a vernier caliper during the experiment. As shown in Fig. 8, after 6 administrations, the antibody to be tested significantly inhibited tumor growth, and some tumors almost disappeared after drug treatment.

 The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present invention. And retouching should also be regarded as the scope of protection of the present invention.

Claims

 \¥0 2019/114235 卩(:17(:\2018/092330 Claims [claim 1] Separated person

Specific binding molecules, including &) and 15),

[Claim 2]

Characterized in that: the specific binding molecule comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises

: heavy chain shown in 38

Heavy chain 00113 shown in 39; and / \¥0 2019/114235 卩(:17(:\2018/092330 or light chain variable region containing light chain as shown in ₄₀ )

NO:

Light chain 00113 shown in 42; or The heavy chain variable region contains

Heavy chain shown in 43

with/

; or The heavy chain variable region contains

Heavy chain shown in 49

: heavy chain shown in 50

The heavy chain 00113; and/or the light chain variable region shown at 51 contains

with/

3 or The heavy chain variable region contains

Heavy chain shown in 61

with/

; or The heavy chain variable region contains

Heavy chain shown in 67

: Heavy chain shown in 68

The heavy chain 00113; and / or the light chain variable region shown in 69 contains

N0:

Light chain 00113 shown in ₇₂ ; or \¥02019/114235 卩(:17(:\2018/092330 heavy chain variable zone contains as

: Heavy chain shown in 74

Heavy chain 00113 shown in 75; and /

; or The heavy chain variable region contains

with/

; or The heavy chain variable region contains

with/

. [Claim 3] The separated person according to claim 2

A specific binding molecule characterized in that the antibody is a full length antibody. [Claim 4] The separated person according to claim 2

A specific binding molecule characterized in that the antigen-binding fragment is Fab or ?(&15)' ₂ or 8〇. [Claim 5] The separated person according to claim 1.

A specific binding molecule, characterized in that: the specific binding molecule comprises a light chain variable region and a heavy chain variable region, wherein: the heavy chain variable region is selected from the group consisting of: $£(30)X0: 19. $£(^10X0:21, $£(30)X0:

[Claim 6] The separated person according to claim 1

a specific binding molecule, characterized in that: the specific binding molecule comprises a light chain variable region and a heavy chain variable region, wherein the light chain is variable \¥02019/114235 卩(:17(:\2018/092330 region sequence has at least 80% homology with sequence (X); heavy chain variable region sequence has at least 80% homology with sequence (丫) The sequence (丫) is selected from: $£(30)X0:

[claim 7] The separated person according to claim 1

a specific binding molecule, characterized in that: the specific binding molecule comprises a light chain variable region and a heavy chain variable region, wherein: the heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

 ; or The heavy chain variable region is

And the light chain variable region is

[Claim 8] The separated person according to claim 1

a specific binding molecule characterized by: \¥0 2019/114235 卩(:17(:\2018/092330) The specific binding molecule exhibits at least one of the following properties: blocking the binding of ?00 to ?01 or 0080;

Combine Increased positive sputum production in mixed lymphocyte reaction _(MLR) assays. [Claim 9]

 Use in a medicament for inhibiting the growth of tumor cells in a patient. [Claim 10]

 Use in the treatment of infectious disease drugs. [Claim 11] An isolated nucleic acid encoding one or both of an antibody light chain variable region and an antibody heavy chain variable region, characterized in that: A fragment encoding the heavy chain variable region of the antibody is selected from the group consisting of: SEQ ID NO: 1 _^ 8 £© NO: 3

[Claim 12] An isolated nucleic acid encoding one or both of an antibody light chain variable region and an antibody heavy chain variable region, wherein: the nucleic acid is associated with a variable region encoding the heavy chain of the antibody The sequence shown by the fragment ( _{X) and the} sequence represented by the fragment (V) encoding the heavy chain variable region of the antibody have at least

[Claim 13] A vector comprising: the nucleic acid molecule according to any one of claims 11 or 12. \¥0 2019/114235 卩(:17(:\2018/092330 [Claim 14] A host cell, comprising: the nucleic acid molecule of any one of claims ₁₁ or ₁₂ , or Containing the vector of claim ₁₃ . _[ Claim _15] A conjugate characterized by: the conjugate comprising an isotope, an immunotoxin, and _/or

One kind of conjugate _{[Claim 16],} wherein: one consisting of anti-human according to any one of claims ₁ to _7,

 The medium is coupled to form. [Claim 17]

 Cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma or melanoma [Claim 18] The use of the conjugate according to claim ₁₅ in the preparation of a medicament for treating a disease; the disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, Bladder cancer, pancreatic cancer, glioma or melanoma. [Claim 19] The use of the conjugate according to claim ₁₆ for the preparation of a medicament for treating a disease; the disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer , bladder cancer, pancreatic cancer, glioma or melanoma. [Claim 20] A composition, characterized in that the composition contains a main substance ( _M ) and an auxiliary substance (; the main substance ( _M ) is selected from the separation according to any one of claims _{1 to 7} . of

Vector of claim _{13. The} host cell of claim _14, or a conjugate as claimed in claim in claim _15, wherein said one or more conjugate of claim _16; the auxiliary material (N) is selected from From a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances. [Claim 21] A kit, comprising: the kit comprising any one of claims _1-7 \¥0 2019/114235 An isolated human-specific binding molecule of 17(:17(:\2018/092330) and an agent for detecting an immune complex formed after specifically binding to the molecule.