CN105777906B - Anti- PD-L1 human antibody and its application - Google Patents
Anti- PD-L1 human antibody and its application Download PDFInfo
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Abstract
The present invention relates to anti-PD-L1 human antibody and its applications, and specifically, the present invention screens the human antibody that can specifically bind PD-L1 by yeast display, and further improves the affinity of antibody by affinity maturation.Anti- PD-L1 human antibody specificity of the invention is good, and affinity is high, and stability is good, the activation of enhancing T cell can be combined by the T cell with activation, and have significant inhibiting effect to tumour growth.The invention further relates to the purposes that the anti-PD-L1 human antibody is used to diagnose, treat tumour relevant to PD-L1.
Description
Technical field
The invention belongs to field of pharmaceutical biology, are related to anti-PD-L1 human antibody and its medical usage.
Background technique
T cell needs antigen presenting cell APC to provide two to static T lymphocyte when responding to exogenous antigen
A signal: first signal is: T cell identifies the Antigenic Peptide in conjunction with MHC molecule by TCR, is passed by TCR/CD3 complex
Pass antigen recognizing signal;Second signal is: being provided by a series of costimulatory molecules;T cell could be activated normally in this way,
To generate normal immune response.Effect difference is generated according to second signal, costimulatory molecules can be divided into positivity and pierced altogether
Swash molecule and negativity costimulatory molecules, the adjusting of positivity and negativity costimulatory signal and balance between the two are in immunity of organism
Important adjustment effect is played during the entire process of response.
PD-1 is a member of CD28 receptor family, which further includes CTLA4, CD28, ICOS and BTLA.The family is most
First member CD28 and ICOS are the (Hutloff found by that can increase the function of T cell proliferation after addition monoclonal antibody
Deng (1999) Nature 397:263-266;Hansen etc. (1980) Immunogenics 10:247-260).The ligand of PD-1
Including PD-L1 and PD-L2, existing result of study shows receptor and activation that T cell can be lowered after ligand binding and related thin
Secretion (Freeman etc. (2000) J Exp Med 192:1027-34 of intracellular cytokine;Latchman etc. (2001) Nat Immunol
2:261-8;Carter etc. (2002) Eur J Immunol 32:634-43;Ohigashi etc. (2005) Clin cancer Res
11:2947-53)。
PD-L1 (B7-H1) is cell surface glycoprotein, belongs to B7 family, has IgV and IgC sample area, transmembrane region and endochylema
Area tail portion.The gene was found for the first time in 1999 and clones (Dong H etc. (1999) Nat Med 5:1365-1369), it with
Receptor PD1 interaction in T cell, plays an important role in terms of the negativity regulation of immune response.PD-L1 in addition to and T
The PD-1 effect expressed on cell is outer, and the PD-L1 expressed in T cell can interact with the CD80 on APC conducts negative sense
Signal inhibits the function of T cell.PD-L1 on the cell in macrophage lineage other than expressing, in the mankind normally organize
Expression quantity is lower, but having fastened but in some tumour cells has higher expression, such as: lung cancer, oophoroma, colon cancer and
Melanoma (Iwai etc. (2002) PNAS 99:12293-7;Ohigashi etc. (2005) Clin Cancer Res 11:2947-
53).It is existing the results show that the highly expressed PD-L1 of tumour cell is by increasing the apoptosis of T cell to escaping in the immune of tumour
It plays an important role in ease.The P815 tumor cell line of researcher's discovery, transfection PD-L1 gene can resist specificity in vitro
The cracking of CTL has stronger oncogenicity and invasion for rear in its Mice Inoculated body.These biological characteristics can pass through resistance
Disconnected PD-L1 and reverse.The mouse of PD1 gene is knocked out, blocks PD-L1/PD-1 access, then inoculated tumour cell cannot form tumour
(Dong H etc. (2002) Nat Med 8:793-800).
There is still a need in conjunction with PD-L1 high-affinity and PD-1 can be blocked anti-in conjunction with PD-L1 for this field
PD-L1 antibody.
Summary of the invention
The present inventor utilizes yeast display, has obtained having by screening and affinity maturation good special
The anti-PD-L1 human antibody of property, higher compatibility and stability, has thus completed the present invention.
First aspect present invention is related to anti-PD-L1 antibody or its antigen-binding portion thereof comprising selected from such as next group
CDR region:
(1) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:1-3, light chain CDR1, CDR2, CDR3
Sequence respectively as shown in SEQ ID NO:4-6, or be greater than with the identity of above-mentioned sequence 70% respectively, 80%, 85%,
90%, 95% sequence;
(2) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:7-9, light chain CDR1, CDR2, CDR3
Sequence respectively as shown in SEQ ID NO:10-12, or be greater than with the identity of above-mentioned sequence 70% respectively, 80%, 85%,
90%, 95% sequence;
(3) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:13-15, light chain CDR1, CDR2,
The sequence of CDR3 respectively as shown in SEQ ID NO:16-18, or be greater than with the identity of above-mentioned sequence 70% respectively, 80%,
85%, 90%, 95% sequence;
(4) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:1,2,19, light chain CDR1, CDR2,
The sequence of CDR3 respectively as shown in SEQ ID NO:4-6, or be greater than with the identity of above-mentioned sequence 70% respectively, 80%, 85%,
90%, 95% sequence;
(5) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:7,20,9, light chain CDR1, CDR2,
The sequence of CDR3 respectively as shown in SEQ ID NO:10-12, or be greater than with the identity of above-mentioned sequence 70% respectively, 80%,
85%, 90%, 95% sequence;
(6) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:13-15, light chain CDR1, CDR2,
The sequence of CDR3 respectively as shown in SEQ ID NO:21,17,18, or be greater than with the identity of above-mentioned sequence 70% respectively, 80%,
85%, 90%, 95% sequence.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention, further includes being selected from
Heavy chain variable region framework region such as next group:
1) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:22-25, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence;
2) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:30-33, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence;
3) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:38-41, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence;
4) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:30-33, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention, further includes being selected from
Light chain variable region framework region such as next group:
1) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:26-29, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence;
2) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:30-33, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence;
3) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:38-41, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence;
4) sequence of FR1, FR2, FR3, FR4 are same with above-mentioned sequence as shown in SEQ ID NO:30-33, or respectively respectively
One property is greater than 70%, 80%, 85%, 90%, 95%, 99% sequence.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention comprising selected from such as
Next group of heavy chain variable region:
The identity with above-mentioned sequence is greater than its sequence as shown in SEQ ID NO:47,49,51,53,54, or respectively
70%, 80%, 85%, 90%, 95%, 99% sequence.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention comprising selected from such as
Next group of light chain variable region:
The identity with above-mentioned sequence is greater than its sequence as shown in SEQ ID NO:48,50,52,55,56, or respectively
70%, 80%, 85%, 90%, 95%, 99% sequence.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention is whole antibody, Shuan Te
Heterogenetic antibody, scFv, Fab, Fab', F (ab')2Or Fv.
In one embodiment of the invention, when it is scFv, the anti-PD-L1 antibody or its antigen-binding portion thereof
Heavy chain and light chain variable region between also contain link peptide.
In specific embodiments of the present invention, the sequence of the link peptide is as shown in SEQ ID NO:67.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention, is whole antibody.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention, heavy chain constant region choosing
From IgG, IgM, IgE, IgD and IgA.
In embodiments of the invention, heavy chain constant region is selected from IgG1, IgG2, IgG3 and IgG4.
In specific embodiments of the present invention, heavy chain constant region IgG1.
In specific embodiments of the present invention, the amino acid sequence of IgG1 is as shown in SEQ ID NO:68.
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one according to a first aspect of the present invention, constant region of light chain κ
Type or λ type.
In specific embodiments of the present invention, the amino acid sequence of κ type constant region of light chain is as shown in SEQ ID NO:70.
In specific embodiments of the present invention, the amino acid sequence of λ type constant region of light chain is as shown in SEQ ID NO:72.
Second aspect of the present invention is related to nucleic acid molecules, and it includes the nucleic acid sequence for capableing of encoding antibody heavy variable region, institutes
The antibody heavy chain variable region stated includes selected from the amino acid sequence such as next group:
(i) SEQ ID NO:1-3;
(ii) SEQ ID NO:7-9;
(iii) SEQ ID NO:13-15;
(iv) SEQ ID NO:1,2,19;
(v) SEQ ID NO:7,20,9.
The nucleic acid molecules of any one according to a second aspect of the present invention, the antibody heavy chain variable region include selected from such as next
The amino acid sequence of group:
SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54 or on
State the replacement for arriving several amino acid in sequence framework region containing one.
In embodiments of the invention, the nucleic acid molecules include to be selected from the sequence as shown in SEQ ID NO:57-61.
In embodiments of the invention, the nucleic acid molecules also include the nucleic acid sequence for capableing of encoding antibody heavy constant region
Column;The heavy chain constant region is selected from IgG, IgM, IgE, IgD and IgA.
In embodiments of the invention, heavy chain constant region is selected from IgG1, IgG2, IgG3 and IgG4.
In specific embodiments of the present invention, heavy chain constant region IgG1.
In specific embodiments of the present invention, the nucleic acid sequence of IgG1 is as shown in SEQ ID NO:69.
Third aspect present invention is related to nucleic acid molecules, and it includes the nucleic acid sequence for capableing of encoding antibody light variable region, institutes
The antibody's light chain variable region stated includes selected from the amino acid sequence such as next group:
(i) SEQ ID NO:4-6;
(ii) SEQ ID NO:10-12;
(iii) SEQ ID NO:16-18;
(iv) SEQ ID NO:21,17,18.
The nucleic acid molecules of any one according to a third aspect of the present invention, the antibody's light chain variable region include selected from such as next
The amino acid sequence of group:
SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:56 or on
State the replacement for arriving several amino acid in sequence framework region containing one.
In embodiments of the invention, the nucleic acid molecules include to be selected from the sequence as shown in SEQ ID NO:62-66.
In embodiments of the invention, the nucleic acid molecules also include the nucleic acid sequence for capableing of encoding antibody light constant region
Column;The constant region of light chain is κ type or λ type.
In specific embodiments of the present invention, the nucleic acid sequence of κ type constant region of light chain is as shown in SEQ ID NO:70.
In specific embodiments of the present invention, the amino acid sequence of λ type constant region of light chain is as shown in SEQ ID NO:72.
Fourth aspect present invention is related to carrier, the nucleic acid molecules containing any one of second or third aspect of the present invention.
Carrier any one of according to a fourth aspect of the present invention, the nucleic acid molecules containing any one of second aspect of the present invention and
The nucleic acid molecules of any one of the third aspect.
Fifth aspect present invention is related to host cell, the nucleic acid molecules containing any one of second or third aspect of the present invention
Or the carrier of any one of fourth aspect present invention.
Sixth aspect present invention is related to conjugate, the anti-PD-L1 antibody containing any one of first aspect present invention or its
Antigen-binding portion thereof and other bioactive substances, the anti-PD-L1 antibody or its antigen-binding portion thereof are directly or by even
Tab segments and other bioactive substances are coupled.
In embodiments of the invention, other bioactive substances, which are selected from, directly or indirectly cell to be inhibited to grow
Or kill cell or by activation organism immune response to inhibit or kill cell, to reach the chemicals for the treatment of tumour
Matter, toxin, polypeptide, enzyme, isotope, cell factor or other biologically active single substances or compounding substances, such as
Auristatin MMAE, Auristatin MMAF, Maytansine DM1, Maytansine DM4, calicheamicin
(calicheamicin), the toxin such as duocarmycin MGBA, adriamycin (doxorubicin), ricin (WA), diphtheria toxin,
I131, interleukin class, tumor necrosis factor, chemotactic factor (CF), nano particle etc..
Seventh aspect present invention is related to composition (such as pharmaceutical composition), contains any one of first aspect present invention
Any one of the nucleic acid molecules of any one of anti-PD-L1 antibody or its antigen-binding portion thereof, second aspect or the third aspect, fourth aspect
Carrier, the 5th aspect any one of host cell or any one of sixth aspect present invention conjugate and optional medicine
Acceptable carrier or excipient on, and optional other bioactive substances.
The composition (such as pharmaceutical composition) of any one according to a seventh aspect of the present invention, other bioactive substances
Including but not limited to other antibody, fusion protein or drug (such as anti-tumor drug, such as Radiotherapy chemotherapy drug).
The invention further relates to reagent or kits, the anti-PD-L1 antibody containing any one of first aspect present invention or its
Antigen-binding portion thereof, the detection reagent or kit whether there is for detecting PD-L1 albumen or derivatives thereof.
The invention further relates to diagnostic reagent or kits, the anti-PD-L1 antibody containing any one of first aspect present invention
Or its antigen-binding portion thereof, the diagnostic reagent or kit (such as the people for (such as cell or tissue) in vitro or in vivo
Or animal model) diagnosis disease relevant to PD-L1 (such as tumour or virus infection, such as the highly expressed virus infection of PD-L1
Or the highly expressed tumour of PD-L1).
In embodiments of the invention, the anti-PD-L1 antibody or its antigen-binding portion thereof, which are also coupled to have, can be used for examining
It surveys or can be by fluorescent dye, chemical substance, polypeptide, enzyme, isotope, label etc. that other reagents detect.
In embodiments of the invention, the tumour include but is not limited to lung cancer, it is oophoroma, colon and rectum carcinoma, black
Melanoma, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, larynx
Cancer, cervical carcinoma, carcinoma of uterine body, osteosarcoma, thyroid cancer, prostate cancer.
In embodiments of the invention, the virus infection is including but not limited to acute, subacute or Chronic HBV,
HCV, HIV infection.
The invention further relates to the anti-PD-L1 antibody or its antigen-binding portion thereof, second party of any one of first aspect present invention
The host cell of any one of the carrier of any one of the nucleic acid molecules of any one of face or the third aspect, fourth aspect, the 5th aspect, the
Any one of the conjugate of any one of six aspects or the 7th aspect composition are used to prepare prevention or treatment disease relevant to PD-L1
The use of the drug of (such as tumour or virus infection, such as the highly expressed tumour of PD-L1 or the highly expressed virus infection of PD-L1)
On the way.
In embodiments of the invention, the tumour refers to tumour relevant to PD-L1, such as refers to high expression PD-
The tumour of L1.
In specific embodiments of the present invention, wherein the tumour include but is not limited to lung cancer, oophoroma, colon cancer,
The carcinoma of the rectum, melanoma, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer,
Nasopharyngeal carcinoma, laryngocarcinoma, cervical carcinoma, carcinoma of uterine body, osteosarcoma, thyroid cancer, prostate cancer.
In embodiments of the invention, the virus infection is including but not limited to acute, subacute or Chronic HBV,
HCV, HIV infection.
The invention further relates to the anti-PD-L1 antibody of any one of first aspect present invention or its antigen-binding portion thereof to be used to prepare
Diagnose relevant to PD-L1 disease (such as tumour or virus infection, for example, the highly expressed tumour of PD-L1 or PD-L1 it is highly expressed
Virus infection) reagent or kit in purposes.
In embodiments of the invention, the tumour refers to tumour relevant to PD-L1, such as refers to high expression PD-
The tumour of L1.
In specific embodiments of the present invention, wherein the tumour include but is not limited to lung cancer, oophoroma, colon cancer,
The carcinoma of the rectum, melanoma, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer,
Nasopharyngeal carcinoma, laryngocarcinoma, cervical carcinoma, carcinoma of uterine body, osteosarcoma, thyroid cancer, prostate cancer.
In embodiments of the invention, the virus infection is including but not limited to acute, subacute or Chronic HBV,
HCV, HIV infection.
In embodiments of the invention, wherein the anti-PD-L1 antibody or its antigen-binding portion thereof are also coupled have it is available
In detection or can be by fluorescent dye, chemical substance, polypeptide, enzyme, isotope, label etc. that other reagents detect.
The invention further relates to the anti-PD-L1 antibody of any one of first aspect present invention or its antigen-binding portion thereof to be used to prepare
The purposes of the drug of prevention or treatment disease relevant to CD80.
In the present invention, the relevant disease of the CD80 is, for example, disease relevant to CD80 high expression.
The invention further relates to prevention or relevant to the PD-L1 disease for the treatment of (such as tumour or virus infections, such as PD-L1
Highly expressed tumour or the highly expressed virus infection of PD-L1) method, the method includes to subject in need prevention or
The anti-PD-L1 antibody or its antigen-binding portion thereof, second aspect or third of any one of the first aspect present invention of therapeutically effective amount
The host cell of any one of the carrier of any one of the nucleic acid molecules of any one of aspect, fourth aspect, the 5th aspect, the 6th aspect are appointed
Any one of one conjugate or the 7th aspect composition, and optional be combined with radiation therapy (such as roentgen radiation x) method.
In embodiments of the invention, the tumour refers to tumour relevant to PD-L1, such as refers to high expression PD-
The tumour of L1.
In specific embodiments of the present invention, wherein the tumour include but is not limited to lung cancer, oophoroma, colon cancer,
The carcinoma of the rectum, melanoma, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer,
Nasopharyngeal carcinoma, laryngocarcinoma, cervical carcinoma, carcinoma of uterine body, osteosarcoma, thyroid cancer, prostate cancer.
In embodiments of the invention, the virus infection is including but not limited to acute, subacute or Chronic HBV,
HCV, HIV infection.
The invention further relates to prevention or the method for relevant to the CD80 disease for the treatment of, the method includes give it is in need by
The anti-PD-L1 antibody or its antigen-binding portion thereof of any one of the first aspect present invention of examination person's prevention or therapeutically effective amount.
In the present invention, the disease relevant to CD80 is, for example, to express relevant disease with CD80 high.
The present invention is described further below:
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The normally understood meaning of personnel institute.Also, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue
Culture, microbiology, immunology relational language and laboratory operation step be in corresponding field widely used term and often
Advise step.Meanwhile for a better understanding of the present invention, the definition and explanation of relational language is provided below.
In the present invention, term " antibody " refers to usually (each pair of to have " light " (L) chain by two pairs of identical polypeptide chains
With " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can be classified as μ,
δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.It, can in light chain and heavy chain
Become area to connect with constant region by area " J " of about 12 or more amino acid, heavy chain also includes about 3 or more amino
Area " D " of acid.Each heavy chain is by heavy chain variable region (VH) and heavy chain constant region (CH) composition.Heavy chain constant region is by 3 structural domains
(CH1、CH2 and CH3) it forms.Each light chain is by light chain variable region (VL) and constant region of light chain (CL) composition.Constant region of light chain is by one
Domain CLComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including the various of immune system
The combination of the first component (C1q) of cell (for example, effector cell) and classical complement system.VHAnd VLArea can also be subdivided into tool
There is denatured region (referred to as complementary determining region (CDR)), is interspersed with the more conservative region for being known as framework region (FR).Respectively
VHAnd VLBy in the following order: arranged from amino terminal to carboxyl terminal 3 of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4
CDR and 4 FR composition.Variable region (the V of each heavy chain/light chain pairHAnd VL) it is respectively formed paratope.Amino acid is to each area
The distribution of domain or structural domain follows Kabat Sequences of Proteins of Immunological Interest
(National Institutes of Health, Bethesda, Md. (1987and 1991)) or Chothia&Lesk
(1987)J.Mol.Biol.196:901-917;The definition of Chothia et al. (1989) Nature 342:878-883.Term
" antibody " is not limited by any specific method for generating antibody.Such as comprising, particularly, recombinant antibodies, monoclonal antibody
And polyclonal antibody.Antibody can be the antibody of different isotypes, for example, IgG is (for example, IgG1, IgG2, IgG3 or IgG4 are sub-
Type), IgA1, IgA2, IgD, IgE or IgM antibody.
In the present invention, " antigen-binding portion thereof " of term antibody refers to one or more parts of full length antibody, described
The ability for the same antigen (for example, PD-L1) that part keeps binding antibody to be combined is competed with complete antibody to the special of antigen
Property combine.Usually referring to, Fundamental Immunology, Ch.7 (Paul, W., ed., second edition, Raven Press,
N.Y. (1989), are incorporation by reference in its entirety, for all purposes.By recombinant DNA technology or it can pass through
The enzymatic or chemical disruption of complete antibody generate antigen-binding portion thereof.In some cases, antigen-binding portion thereof include Fab,
Fab'、F(ab')2、It is Fd, Fv, dAb and complementary determining region (CDR) segment, single-chain antibody (for example, scFv), chimeric antibody, dual anti-
Body (diabody) and such polypeptide, it includes at least one for being enough to assign the antibody of polypeptid specificity antigen binding capacity
Point.
In the present invention, term " Fd segment " means by VHAnd CHThe antibody fragment of 1 structural domain composition;Term " Fv segment "
Mean by the V of the single armed of antibodyLAnd VHThe antibody fragment of structural domain composition;Term " dAb segment " means by VHStructural domain composition
Antibody fragment (Ward et al., Nature 341:544-546 (1989));Term " Fab segment " means by VL、VH、CLAnd CH1 knot
The antibody fragment of structure domain composition;Term " F (ab')2Segment " means two Fab comprising connecting by the disulphide bridges on hinge area
The antibody fragment of segment.
In some cases, the antigen-binding portion thereof of antibody is single-chain antibody (for example, scFv), wherein VLAnd VHStructural domain
Connector by the way that single polypeptide chain can be produced as match to be formed monovalent molecule (see, e.g., Bird et al.,
Science 242:423-426 (1988) and Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883
(1988)).Such scFv molecule can have general structure: NH2-VLConnector-VH- COOH or NH2-VHConnector-VL-COOH.It closes
Suitable prior art connector (link peptide) is made of duplicate GGGGS amino acid sequence or its variant.For example, can be used has ammonia
Base acid sequence (GGGGS)4Connector, but can also be used its variant (Holliger et al. (1993),
Proc.Natl.Acad.Sci.USA 90:6444-6448).Other connectors for use in the present invention are by Alfthan et al.
(1995), Protein Eng.8:725-731, Choi et al. (2001), Eur.J.Immunol.31:94-106, Hu et al.
(1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J.Mol.Biol.293:41-56 and
Roovers et al. (2001), Cancer Immunol. description.In embodiments of the invention, the sequence of the link peptide is
(GGGGS)3。
In some cases, antibody is bispecific antibody, can be combined respectively with two kinds of antigens or epitope,
Light chain, heavy chain or its antigen-binding portion thereof including specifically binding the antibody of the first antigen, and specific binding second are anti-
Light chain, heavy chain or its antigen-binding portion thereof of former antibody.In embodiments of the invention, it is tied in the bispecific antibody
The light chain, heavy chain or its antigen-binding portion thereof for closing the antibody of the first antigen can be the antibody or its antigen knot of any one of the present invention
Part is closed, light chain, heavy chain or its antigen-binding portion thereof of the antibody of the second antigen of the specific binding can be other anti-PD-
L1 antibody or its antigen-binding portion thereof or antibody or its antigen-binding portion thereof for other antigens.
In some cases, antibody is double antibody, that is, bivalent antibody, wherein VHAnd VLStructural domain table in single polypeptide chain
Reach, but using too short connector so that do not allow to match between two structural domains of same chain, thus force structural domain with
The complementary domain of another chain match and generate two antigen-binding sites (see, e.g., Holliger P. et al.,
Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993) and Poljak R.J. et al., Structure 2:1121-
1123(1994))。
It can be used routine techniques well known by persons skilled in the art (for example, recombinant DNA technology or enzymatic or chemical disruption
Method) antigen-binding portion thereof (for example, above-mentioned antibody fragment) of antibody is obtained from given antibody (such as monoclonal antibody 2E12),
And by with for complete antibody in a manner of identical mode with regard to specificity screening antibody antigen-binding portion thereof.
In the present invention, the antigen-binding portion thereof includes single-chain antibody (scFv), chimeric antibody, double antibody, scFv-Fc
Bivalent molecule, dAb and complementary determining region (CDR) segment, Fab segment, Fd segment, Fab' segment, Fv and F (ab')2Segment.
In the present invention, the IgG1 heavy chain constant region includes various allografts, as G1m (f), G1m (z), G1m (z,
Or G1m (z, a, x) a).In embodiments of the invention, the IgG1 heavy chain constant region is G1m (f) type.
In the present invention, the κ constant region of light chain includes various allografts, such as Km1, Km1,2 or Km3.In the present invention
Embodiment in, the κ constant region of light chain be Km3 type.
In the present invention, the lambda light chain constant region includes various allografts, such as λ I, λ II, λ III, λ VI.In the present invention
Embodiment in, the lambda light chain constant region be II type of λ.
Antibody nucleic acids molecule of the present invention also can use traditional genetic engineering recombinant technique or chemical synthesis side
Method obtains.On the one hand, the sequence of antibody nucleic acids molecule of the present invention contains the heavy chain variable region or anti-of anti-PD-L1 antibody
The partial nucleic acid sequence of body molecule.On the other hand, the sequence of antibody nucleic acids molecule of the present invention also includes anti-PD-L1 antibody
Light chain variable region or antibody molecule partial nucleic acid sequence.On the other hand, the sequence of antibody nucleic acids molecule of the present invention
It further include the CDR sequence of heavy chain or light chain variable region.Complementary determining region (complementary determinant region,
CDR be) position in conjunction with epitope, the CDR sequence in the present invention by IMGT/V-QUEST (http: //
Imgt.cines.fr/textes/vquest/ it) is determined.But it is different CDR sequence that division methods obtain slightly not
Together.
An aspect of of the present present invention be related to II 61-62, B50-6, B60, B II 61 of encoding antibody B60-55, B and B50 heavy chain and
The nucleic acid molecules of light-chain variable sequence.II 61-62, B50-6, B60, B II 61 of antibody B60-55, B and B50 heavy chain variable region sequence
The nucleic acid molecules of column correspond respectively to SEQ ID N0:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61 and SEQ ID N0:59.II 61-62, B50-6, B60, B II 61 of antibody B60-55, B and B50 light-chain variable sequence
Nucleic acid molecules correspond respectively to SEQ ID N0:62, SEQ ID NO:63, SEQ ID N0:64, SEQ ID NO:62, SEQ
ID NO:65 and SEQ ID NO:66.The invention further relates to contain II 61-62, B50-6, B60, B II 61 of antibody B60-55, B
With B50 heavy chain and the nucleic acid molecules variant of light-chain variable sequence or similar body.
On the other hand, the invention further relates to the variants of various isolated nucleic acid molecules, specifically, the sequence of nucleic acid variants
Column and following nucleic acid sequence SEQ ID N0:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID
NO:61、SEQ ID N0:59、SEQ ID N0:62、SEQ ID NO:63、SEQ ID N0:64、SEQ ID NO:62、SEQ ID
The phase same sex of NO:65 and SEQ ID NO:66 is at least up to 70%, preferably at least up to 75%, more preferably at least up to 80%, more preferably
At least up to 85%, more preferably at least up to 90%, most preferably at least up to 95%.
The present invention further further relates to II 61-62, B50-6, B60, B II 61 of encoding antibody B60-55, B and B50 heavy chain
Variable region is the corresponding by isolated nucleic acid molecules of amino acid sequence SEQ ID NO:47,49,51,53,54,51.The present invention
It further relates to II 61-62, B50-6, B60, B II 61 of encoding antibody B60-55, B and B50 chain variable region amino acid sequence is SEQ
The corresponding nucleic acid molecules of ID NO:48,50,52,48,55,56.
The present invention relates to the recombinant expression carrier for containing the nucleic acid molecules, the host for being also related to having converted these molecules is thin
Born of the same parents.Moreover, the invention further relates to cultivate and separate under given conditions using the host cell for containing the nucleic acid molecules
To the method for inventing the antibody.
Antibody amino acids sequence
II 61-62, B50-6, B60, B II 61 of monoclonal antibody B60-55, B and B50 heavy chain and chain variable region amino acid sequence can
To be derived by from corresponding nucleic acid sequence.II 61-62, B50-6, B60, B II 61 of antibody B60-55, B and B50 weight chain variable
Region amino acid sequence is SEQ ID NO:47,49,51,53,54,51 respectively.Antibody B60-55, B II 61-62, B50-6, B60, B
II 61 and B50 chain variable region amino acid sequence is SEQ ID NO:48,50,52,48,55,56 respectively.
On the other hand, the amino acid sequence of the heavy chain variable region of antibody provided by the invention and SEQ ID NO:47,49,
51,53,54 or 51 sequence similarity is at least up to 70%, and preferably at least 75%, preferably at least 80%, preferably 85%,
It is further preferably at least 90%, most preferably at least 95%.
On the other hand, the amino acid sequence of the light chain variable region of antibody provided by the invention and SEQ ID NO:48,50,
52,48,55 or 56 sequence similarity is at least up to 70%, and preferably at least 75%, preferably at least 80%, preferably 85%,
It is further preferably at least 90%, most preferably at least 95%.
The amino of the CDR of the heavy chain and light chain variable region of II 61-62, B50-6, B60, B II 61 and B50 of antibody B60-55, B
Acid sequence determination is as follows:
The amino acid sequence of CDR1, CDR2 and CDR3 of antibody B60-55 heavy chain are respectively SEQ ID NO:1-3.Antibody
The amino acid sequence of CDR1, CDR2 and CDR3 of B60-55 light chain distinguish SEQ ID NO:4-6.
The amino acid sequence of CDR1, CDR2 and CDR3 of II 61-62 heavy chain of antibody B are respectively SEQ ID NO:7-9.Antibody
The amino acid sequence of CDR1, CDR2 and CDR3 of II 61-62 light chain of B are respectively SEQ ID NO:10-12.
The amino acid sequence of CDR1, CDR2 and CDR3 of antibody B50-6 heavy chain are respectively SEQ ID NO:13-15.Antibody
The amino acid sequence of CDR1, CDR2 and CDR3 of B50-6 light chain are respectively SEQ ID NO:16-18.
On the other hand, the amino acid sequence that the heavy chain of anti-PD-L1 antibody or the CDR of segment contain may be in SEQ ID
Occur the mutation of one or more amino acid on NO:1-3,7-9,13-15,19,20 or increases or lack.Preferably, it is mutated
Or the amino acid no more than 3 amino acid for adding or lacking.It is further preferred that the amino acid of mutation or addition or missing is no more than
2 amino acid.Most preferably, it is mutated or adds or the amino acid of missing is no more than 1 amino acid.
On the other hand, the amino acid sequence that the light chain of anti-PD-L1 antibody or the CDR of segment contain may be in SEQ ID
NO:4-6,10-12,16-18, the mutation for occurring one or more amino acid on 21, increase or lack.Preferably, it is mutated, adds
The amino acid no more than 3 amino acid for adding or lacking.It is further preferred that the amino acid of mutation, addition or missing is no more than 2 ammonia
Base acid.Most preferably, the amino acid for being mutated, adding or lacking is no more than 1 amino acid.
The amino of the FR of the heavy chain and light chain variable region of II 61-62, B50-6, B60, B II 61 and B50 of antibody B60-55, B
Acid sequence determination is as follows:
The sequence of antibody B60-55, B60 heavy chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:22-25.Gently
The sequence of chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:26-29.
The sequence of antibody B II 61-62 heavy chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:30-33.Light chain
The sequence of variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:34-37.
The sequence of antibody B50-6, B50 heavy chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:38-41.Gently
The sequence of chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:42-45.
The sequence of antibody B II 61 heavy chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:30-33.Light chain can
The sequence for becoming area FR1, FR2, FR3, FR4 is respectively SEQ ID NO:34,46,36,37.
On the other hand, the heavy chain of anti-PD-L1 antibody or the amino acid sequence of light chain variable region FR may be in SEQ ID
Occur the mutation of one or more amino acid on NO:22-46, increase or lack.Preferably, the ammonia for being mutated, adding or lacking
Base acid no more than 3 amino acid.It is further preferred that the amino acid of mutation, addition or missing is no more than 2 amino acid.Most preferably
, the amino acid for being mutated, adding or lacking is no more than 1 amino acid.
The amino acid of above-mentioned antibody or CDR region or framework region mutates, the variant after addition or missing is still protected
Stay the ability of specific binding human PD-L 1.The present invention also includes the variant of such antigen-binding portion thereof.
Monoclonal antibody variant of the invention can be obtained by traditional gene engineering method.Those skilled in the art is complete
Know the method using nucleic acid mutation transformation DNA molecular.In addition, the nucleic acid molecules of encoding heavy chain and light chain variant can also lead to
Cross chemical synthesis acquisition.
In the present invention, for determine the algorithm of sequence identity and sequence similarity percentage be such as BLAST and
2.0 algorithm of BLAST, they describe respectively in (1977) Nucl.Acid.Res.25:3389-3402 such as Altschul and
Altschul etc. (1990) J.Mol.Biol.215:403-410.Using for example described in document or default parameters, BLAST and
BLAST 2.0 is determined for amino acid sequence identity percentage of the invention.The software for executing BLAST analysis can lead to
It is for the public to obtain to cross National Biotechnology Information Center.
In the present invention, the amino acid sequence with amino acid sequence at least 70% sequence identity include with
The substantially same polypeptide sequence of the amino acid sequence, such as when use methods described herein are (for example, by using standard parameter
BLAST analysis) when, with polypeptide sequence of the present invention compared with contain at least 70% sequence identity, preferably at least 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
Those of higher sequence identity sequence.
In the present invention, term " carrier ", which refers to, can be inserted the polynucleotide for encoding certain albumen and make egg
A kind of white nucleic acid delivery vehicle for obtaining expression.Carrier can make the heredity of its carrying by conversion, transduction or transfection host cell
Substance element is expressed in host cell inner expression.For example, carrier includes: plasmid;Phasmid;Coemid;Manually
The artificial chromosome (PAC) of chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the source P1;Phagocytosis
Body such as λ bacteriophage or M13 bacteriophage and animal virus etc..Animal virus type as carrier have retrovirus (including
Slow virus), adenovirus, adeno-associated virus, herpesviral (such as herpes simplex virus), poxvirus, baculoviral, papillomatosis
Poison, papova viruses (such as SV40).A kind of element that carrier may be expressed containing various control, including promoter sequence
Column, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.In addition, carrier can also contain replication origin.
Carrier is it is also possible to include the ingredient for assisting it to enter cell, such as virion, liposome or protein coat, but not only only
There are these substances.
In the present invention, term " host cell " refers to importing the cell of carrier, including following many cell types, such as
The prokaryotic cells such as Escherichia coli or withered grass bacterium, such as yeast cells or Aspergillus fungal cell, such as S2 drosophila cell or Sf9 elder brother
Worm cell, or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, 293 cell of HEK
Or the zooblast of people's cell.
Antibody fragment of the invention can use the complete antibody molecule of hydrolysis obtain (referring to Morimoto et al.,
J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81
(1985)).In addition, these antibody fragments can also directly by recombinant host cell generate (reviewed in Hudson,
Curr.Opin.Immunol.11:548-557(1999);Little et al.,Immunol.Today,21:364-370
(2000)).For example, Fab' segment can directly be obtained from E.coli cell or chemical lotus root joins to form 2 segment of F (ab')
(Carter et al.,Bio/Technology,10:163-167(1992)).For another example, F (ab')2Segment can use leucine
Zipper GCN4 connection obtains.In addition, Fv, Fab or F (ab')2Segment can also be directly from recombinant host cell culture solution directly
It is isolated.Those skilled in the art have full knowledge that the other technologies for preparing antibody fragment.
In the present invention, " specific binding " refers to two intermolecular nonrandom association reactions, such as antibody and generation
Reaction between the antigen of the antibody.It herein, is detection in conjunction with binding affinity of the antibody of the first antigen to second of antigen
Less than or it is very weak.In some embodiments, certain antigen-specific antibodies refer to affinity (KD)≤10-5M (such as 10- 6M、10-7M、10-8M、10-9M、10-10M etc.) antigen is combined, wherein KD refers to the ratio (koff/kon) of dissociation yield and Percentage bound,
It can be measured using method familiar to those skilled in the art.
In embodiments of the invention, anti-PD-L1 antibody of the invention can specifically with human PD-L 1 or simultaneously and
Mouse PD-L1 is combined, without in conjunction with PD-L2 and B7H3.
In embodiments of the invention, anti-PD-L1 antibody of the invention can be with hPD-1 competitive binding hPD-L1.
In the present invention, disease relevant to PD-L1 is for example including tumour relevant with PD-L1 or virus infection, especially
It is tumour relevant to PD-L1 high expression or virus infection.
In embodiments of the invention, the tumour include but is not limited to lung cancer, it is oophoroma, colon and rectum carcinoma, black
Melanoma, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, larynx
Cancer, cervical carcinoma, carcinoma of uterine body, osteosarcoma, thyroid cancer, prostate cancer.
In embodiments of the invention, the virus infection is including but not limited to acute, subacute or Chronic HBV,
HCV, HIV infection.
In the present invention, common usage is deferred in 20 kinds of conventional amino acids and its abbreviation.Referring to Immunology-A
Synthesis (second edition, E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass.
(1991)) it, is incorporation by reference.
Advantageous effect of the invention
The present invention utilizes yeast display, has obtained having by screening and further affinity maturation good special
The anti-PD-L1 human antibody of property, higher compatibility and stability, the antibody can specifically with human PD-L 1 or simultaneously and
Mouse PD-L1 is combined, and not in conjunction with B7H3 and PD-L2, can enhance T cell and then the T cell with activation combines
Activation, to tumour growth have significant inhibiting effect.
Detailed description of the invention
Fig. 1: the inhibition that the anti-hPD-L1scFv of purifying combines hPD-L1/hPD-1 ligand-receptor.
X- axis indicates the fluorescence intensity of EGFP, and Y- axis indicates the fluorescence intensity of SA-PE.A is blank control, and B is negative right
According to C B50scFv, D B60scFv, E are II 61scFv of B.
The yeast improved with hPD-L1 affinity is obtained after the screening of Fig. 2 affinity maturation
Wherein X- axis indicates the fluorescence intensity (yeast that the myc positive indicates expression complete antibody segment) of myc, and Y- axis indicates
In conjunction with the fluorescence intensity of the SA-APC of antigenic capacity.
The antibody obtained after Fig. 3 affinity maturation is compared with the ability of hPD-1 competitive binding hPD-L1
Wherein abscissa is antibody concentration (unit: ng/ml), and ordinate is OD value.
Wherein A is B II 61 compared with II 61-62 of B, and B is the comparison of B50 and B50-6, and C is the ratio of B60 and B60-55
Compared with.
Fig. 4 ELISA method detection anti-hPD-L1 antibody and hPD-L1 binding ability
Wherein abscissa is antibody concentration (unit: ng/ml), and ordinate is OD value.
The ability of Fig. 5 competitive ELISA detection anti-hPD-L1 antibody and hPD-1 competitive binding hPD-L1
Wherein abscissa is antibody concentration (unit: ng/ml), and ordinate is OD value.
Wherein Graph#5 is II 61-62mAb of B, Graph#2 B50-6mAb, Graph#3 B60-55mAb.
Fig. 6 is detected by competitive ELISA methodThe ability of anti-hPD-L1 antibody and CD80 competitive binding hPD-L1
The detection of Fig. 7 anti-hPD-L1 antibody specificity
Wherein X- axis is EGFP fluorescence intensity, and Y- axis is the fluorescence intensity that corresponding antibody combines, and A is blank control, B
For negative control, C is II 61-62mAb of B, D B60-55mAb, E B50-6mAb;
It (1) is hPD-L1-EGFP albumen, (2) are hB7H3-EGFP, and (3) are hPD-L2-EGFP albumen.
Ability of Fig. 8 anti-hPD-L1 antibody in conjunction with mPD-L1
Wherein X- axis is EGFP fluorescence intensity, and Y- axis is the fluorescence intensity that corresponding antibody combines, and A is blank control, B
For negative control, C B60-55mAb, D are II 61-62mAb of B, E B50-6mAb;
It (1) is hPD-L1-EGFP albumen, (2) are mPD-L1-EGFP albumen.
Ability of Fig. 9 anti-hPD-L1 antibody in conjunction with machin PD-L1
Figure 10 anti-hPD-L1 antibody is to CD4+The activation of T cell
Inhibitory activity of Figure 11 anti-hPD-L1 antibody B50-6 to tumour growth
Inhibitory activity of II 61-62 of Figure 12 anti-hPD-L1 antibody B60-55, B to tumour growth
Wherein A is dosage when being 3mg/kg, inhibiting effect of B II 61-62mAb and B60-55 to tumour growth;B is
Inhibiting effect of II 61-62mAb of B of different dosages to tumour growth.
Figure 13 B60-55 is compared with the stability of antibody 2.41H90P
Wherein A is the IC50 value that B60-55 and antibody 2.41H90P are changed over time;
B is the ratio that antibody dimer changes over time;
C is the competitive ELISA result of B60-55 acceleration for stabilization experiment.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
The expression of 1 recombined human PD-L1, PD-1 of embodiment and the preparation of correlation EGFP cell
According to the amino acid sequence (Q9NZQ7) of human PD-L 1 on albumen database Uniprot, the extracellular knot of human PD-L 1 is obtained
The amino acid sequence (i.e. in Q9NZQ7 the 1st residue to 238 residues) in structure domain;Exempted from according to people on albumen database Uniprot
The amino acid constant region sequence (P01857) of epidemic disease globulin gamma1 (IgG1), obtains the domain amino acid sequence of human IgG1-Fc
Column (i.e. in P01857 the 104th residue to 330 residues);According to human immunoglobulin(HIg) on albumen database Uniprot
The amino acid constant region sequence (P01868) of gamma1 (IgG1), obtains the domain amino acid sequence of human IgG1-Fc (i.e.
The 98th residue is to 324 residues in P01868).Utilize DNAworks online tool (http://helixweb.nih.gov/
Dnaworks/ it) designs corresponding DNA sequences encoding and obtains hPD-L1-Fc, the gene of hPD-L1-muFc fusion protein, according to same
The method of sample obtains the gene of hPD-1-Fc.Enhanced green fluorescence protein is obtained according to information on albumen database Uniprot
EGFP amino acid sequence (C5MKY7), the amino acid sequence (Q9NZQ7) of human PD-L 1, the amino acid sequence of mouse PD-L1
(Q9EP73), the amino acid sequence (Q15116) of people PD1.Using DNAworks online tool (http: //
Helixweb.nih.gov/dnaworks/ it) designs corresponding DNA sequences encoding and obtains the gene of PD-L1-EGFP fusion protein,
The gene of hPD-1-EGFP and mPD-L1-EGFP are obtained after the same method.Its DNA piece is obtained by artificial synthesized mode
Section.HindIII of the synthetic gene order respectively through Fermentas company and EcoRI double digestion are subcloned into commercialization and carry
In body pcDNA4/myc-HisA (Invitrogen, V863-20), sequence verification constructs the accuracy of plasmid, obtains recombinant plasmid
DNA is i.e.: pcDNA4-hPD-L1-Fc, pcDNA4-hPD-L1-muFc, pcDNA4-hPD1-Fc, pcDNA4-hPD-L1-EGFP,
pcDNA4-hPD1-EGFP、pcDNA4-mPD-L1-EGFP。
Using reverse transcription-polymerase chain reaction RT-PCR technology from the Dendritic Cells (DC cell) of laboratory cultures
PD-L2, B7H3 gene of amplification people, expands in (monocyte that the DC cell separates in PBMC passes through from TNF-α maturation)
It is as follows to increase primer: PDL2-FHindIII:
GCGCAAGCTTGCCACCATGATCTTCCTCCTGCTAATG (SEQ ID NO:74), PDL2-R EcoI:
GCCGAATTCGATAGCACTGTTCACTTCCCTC (SEQ ID NO:75);HB7H3-F HindIII:
GCGCAAGCTTGCCACCATGCTGCGTCGGCGGGGCAGC (SEQ ID NO:76), hB7H3-R BamHI:
GCGCGAATTCGGCTATTTCTTGTCCATCATCTTC (SEQ ID NO:77);Obtained PCR product warp
HindIII and the EcoRI double digestion of Fermentas company are subcloned into the pcDNA4-hPD-L1-EGFP built, are surveyed
The accuracy of sequence verifying building plasmid, obtains recombinant plasmid dna i.e.: pcDNA4-hPD-L2-EGFP, pcDNA4-hB7H3-
EGFP。
By relevant EGFP Transfected Recombinant Plasmid to HEK293 (ATCC, CRL-1573TM) in cell, pass through after transfecting 48h
The expression of fluorescence-activation signal sorting (FACS) confirmation hPD1, hPD-L1, mPD-L1, hPD-L2, hB7H3.
PcDNA4-hPD-L1-Fc, pcDNA4-hPD-L1-muFc, pcDNA4-hPD1-Fc are transiently transfected to HEK293
Middle cell is used for protein production.PEI needed for recombinant expression plasmid is diluted with Freestyle293 culture medium and conversion is added
(Polyethylenimine) every group of plasmid/PEI mixture is separately added into cell suspension by solution, is placed on 37 DEG C, and 10%
CO2, cultivate in 90rpm;50 μ g/L insulin-like growth factor-1 (insulin-like growth factor- are added simultaneously
1, IGF-1).EX293 culture medium, 2mM Glutamine and 50 μ g/L IGF-1,135rpm cultures are added after four hours again.Two
Add 3.8mM sodium vedproate (VPA) after 14 hours.After culture 5~6 days, transient expression culture supernatant is collected, is passed through
Protein A affinity chromatography, preliminary purification obtain hPD-L1-Fc, hPD-L1-muFc and hPD-1-Fc protein sample, are used for
Following embodiment.Obtained protein sample carries out preliminary detection using SDS-PAGE, is clear that purpose band.
Embodiment 2: anti-hPD-L1 antibody, clonal expression and identification are screened from yeast display library
The human antibody of PD-L1 is directed to using yeast display screening.By cloning the spleen from 21 Healthy Peoples
With lymph node IgM, VH and VL gene in IgG cDNA to specific support constructs scFV yeast display library (among VH and VL
Catenation sequence be GGGGSGGGGSGGGGS (SEQ ID NO:67) link peptide), storage capacity be 5 × 108.By the ferment of 10 times of storage capacity
Female library recovery, induction yeast surface expresses antibody, rich in the way of magnetic bead sorting with the biotinylated hPD-L1 antigen of 100nM
Collect secondary, then does airflow classification with anti-myc antibody and biotinylated hPD-L1 and be enriched with again twice.Obtained yeast applies
Plate, picking monoclonal.Monoclonal yeast expanded and inducing expression after with anti-myc antibody and biotinylated hPD-L1 or
Antigen hPD-1 staining analysis is compareed, antigen positive/control yeast-negative yeast is positive yeast.
Yeast colony PCR and sequencing will be carried out by the yeast clone of FACS confirmation, PCR primer are as follows: pNL6-F:
GTACGAGCTAAAAGTACAGTG (SEQ ID NO:78);PNL6-R:TAGATACCCATACGACGTTC (SEQ ID NO:
79);The primer of sequencing is pNL6-R.Sequence is compared with BioEdit software after obtaining sequencing result.
It is passed through after the human IgG1's-Fc Gene Fusion mentioned by single-chain antibody scFv gene obtained above and before
HindIII and the EcoRI double digestion of Fermentas company are cloned into commercial carrier pEE6.4 (Lonza), according to molecule gram
Grand standard operation carries out clone and plasmid is small mentions.Plasmid after the extraction transient expression in HEK293 cell, and pass through
Protein A column purification.
HPD-L1-EGFP cell is taken, is resuspended in 0.5%PBS-BSA Buffer, above-mentioned anti-after purification is added
HPD-L1scFv antibody, while related control is set, negative control is the hIgG1 albumen of 2 μ g, and hPD-1- is added in positive control
Fc.Secondary antibody is the anti-hIg-PE of eBioscience.Flow cytometer is detected after dyeing.Energy is identified in this approach
The antibody of combination cell surface PD-L1 antigen.
HPD-L1-EGFP cell is taken, is resuspended in 0.5%PBS-BSA Buffer, above-mentioned anti-hPD- after purification is added
L1scFv antibody, while negative control is set, negative control is the hIgG1 albumen of 2 μ g, and 0.3 μ g hPD-1- is added in all samples
Fc-biotin, secondary antibody are the SA-PE of eBioscience, and flow cytometer is detected after dyeing, the result is shown in Figure 1.With
The antibody that the method identification can block cell surface PD-L1 antigen and PD-1 to combine.
It is B50, B60, B II 61 respectively that three plants of preferable antibody of characteristic are obtained after screening and identification.It can by result
To find out, the antibody of three plants of anti-hPD-L1 can block the combination of itself and receptor hPD-1.
Between above-mentioned heavy chain of antibody and light chain variable region containing connection peptide sequence GGGGSGGGGSGGGGS (SEQ ID NO:
67)。
The wherein heavy chain variable amino acid sequence of B50 are as follows:
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTKAAWYWIRQSPSRGLEWLGRTYFRSKWYNDYADSVK
SRLTINPDTSKNQFSLQLKSVSPEDTAVYYCARGQYTAFDIWGQGTMVTVSS (SEQ ID NO:51);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:13-15;Horizontal line portion is not drawn
It is respectively FR1,2,3,4, sequence number is respectively SEQ ID NO:38-41;
Its corresponding DNA sequence dna are as follows:
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGC
CATCTCCGGGGACAGTGTCTCTAGCACCAAGGCTGCTTGGTACTGGATCAGGCAGTCCCCTTCGAGAGGCCTTGAG
TGGCTGGGAAGGACATACTTCCGGTCCAAGTGGTATAATGACTATGCCGACTCTGTGAAAAGTCGATTAACCATCA
ACCCAGACACATCCAAGAACCAGTTCTCCCTGCAACTTAAGTCTGTGAGTCCCGAGGACACGGCTGTGTATTACTG
TGCAAGAGGGCAATACACTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCG TCTCTTCA (SEQ ID NO:
59);
Its chain variable region amino acid sequence are as follows:
QSALIQPASVSGSPGQSITISCTGTSSDVGGYDLVSWYQQYPGQAPRLIIYEVIKRPSGISDRFSGSK
SGNTASLTISGLQAEDEADYYCSSYAGRRLHGVFGGGTQLTVL (SEQ ID NO:56);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:21,17,18;Cross is not drawn
Line part is respectively FR1,2,3,4, and sequence number is respectively SEQ ID NO:42-45;
Its corresponding DNA sequence dna are as follows:
CAGTCTGCTCTGATTCAGCCTGCCTCCGTGTCTGGGTCCCCTGGACAGTCGATCACTATCTCCTGTAC
TGGCACCAGTAGTGATGTTGGAGGTTATGACCTTGTCTCCTGGTACCAACAGTACCCGGGCCAAGCCCCCAGACTC
ATCATTTATGAGGTCATTAAGCGGCCCTCAGGGATTTCTGATCGCTTCTCTGGTTCCAAGTCTGGCAACACGGCCT
CCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATGCAGGTAGACGTCTTCA
TGGTGTGTTCGGAGGAGGCACCCAGCTGACCGTCCTC (SEQ ID NO:66).
The heavy chain variable amino acid sequence of B60 are as follows:
QVQLVQSGAEVKKPASSVKVSCTASGGSFSTYAISWVRQAPGQGLEWMGGIIPIFGTTKYAQRFQGRV
TITADESTTTAYMELSSLISDDTALYYCTTSRGFSYGWFDYWGQGTLVTVSS (SEQ ID NO:53);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:1,2,19;Horizontal line is not drawn
Part is respectively FR1,2,3,4, and sequence number is respectively SEQ ID NO:22-25;
Its corresponding DNA sequence dna are as follows:
CAGGTCCAGCTTGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGCGTCCTCGGTCAAAGTCTCCTGCAC
GGCTTCTGGCGGCTCCTTCAGCACCTATGCTATCAGTTGGGTGCGACAGGCTCCTGGACAAGGGCTTGAATGGATG
GGCGGGATCATCCCCATCTTTGGTACAACTAAGTACGCACAGAGGTTCCAGGGCAGGGTCACGATTACCGCGGACG
AATCGACGACCACAGCCTACATGGAGCTGAGCAGCCTGATATCTGACGACACGGCCCTGTATTATTGTACGACGTC
TCGTGGATTCAGCTATGGCTGGTTTGACTACTGGGGCCAGGGTACCCTGGTCACCG TCTCCTCA (SEQ ID NO:
60);
Its chain variable region amino acid sequence are as follows:
EIVMTQSPATLSLSPGERATLSCRASQSVGIHLAWYQQKLGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGSLPRTFGQGTKVEIK (SEQ ID NO:48);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:4-6;Horizontal line part is not drawn
Respectively FR1,2,3,4, sequence number are respectively SEQ ID NO:26-29;
Its corresponding DNA sequence dna are as follows:
GAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG
TAGGGCCAGTCAGAGTGTTGGCATACACTTAGCCTGGTACCAACAGAAACTTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGTAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCA
CCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTTCTTTACCTCGGACGTTCGG
CCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:62).
The heavy chain variable amino acid sequence of B II 61 are as follows:
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSASWNWIRQSPSRGLEWLGRTYYRSKWYDDYAVSVK SRISINPDTSKNQFSLQLNSVTPEDTAVYYCARSQGRYFVNYGMDVWGQGTTVTVSS (SEQ ID NO:54);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:7,20,9;Horizontal line is not drawn
Part is respectively FR1,2,3,4, and sequence number is respectively SEQ ID NO:30-33;
Its corresponding DNA sequence dna are as follows:
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGC
CATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTTCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAG
TGGCTGGGAAGGACATATTACAGGTCCAAATGGTATGATGATTATGCAGTATCTGTGAAAAGTCGAATCAGCATCA
ACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTG
TGCAAGAAGCCAGGGACGATATTTTGTCAACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC
TCA (SEQ ID NO:61);
Its chain variable region amino acid sequence are as follows:
DIRLTQSPSSLSASVGDRITITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSG
TDFTLTISSLQPEDVATYYCQQSYFTPRGITFGPGTKVDIK (SEQ ID NO:55);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:10-12;Horizontal line portion is not drawn
It is respectively FR1,2,3,4, sequence number is respectively SEQ ID NO:34,46,36,37;
Its corresponding DNA sequence dna are as follows:
GACATCCGGTTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAATCACCATCACTTG
CCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC
TATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA
CCATCAGCAGTCTGCAACCTGAAGATGTTGCAACTTACTACTGTCAACAGAGTTACTTTACCCCCCGCGGGATCAC
TTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO:65).
The Yeast libraries building that embodiment 3:anti-hPD-L1scFv affinity improves
Respectively with pEE6.4-B50-Fc, II 61-Fc plasmid of pEE6.4-B60-Fc, pEE6.4-B is template, pEE6.4-F:
TCTGGTGGTGGTGGTTCTGCTAGC (SEQ ID NO:80) and cMyc-BBXhoI:GCCAGATCTCGAGCTATTACAAGT
CTTCTTCAGAAATAAGCTTTTGTTCTAGAATTCCG (SEQ ID NO:81) is that primer carries out standard PCR reaction.It obtains
NheI and BglII of the PCR product through Fermentas company are cloned into commercial carrier pCT302 (addgene:#41845),
Obtain pCT302-B50, pCT302-B60, pCT302-BII61 recombinant plasmid.With reference next to document Ginger etc. (2006)
Nat Protoc 1 (2): 755-68 method is produced using the PCR that the method for error prone PCR obtains scFv random mutation
Object.Primer used is T7proshort:TAATACGACTCACTATAGGG (SEQ ID NO:82) and Splice4/L:
GGCAGCCCCATAAACACACAGTAT (SEQ ID NO:83).Obtained PCR product is through Fermentas company GeneJET
Ethanol precipitation is concentrated into concentration greater than 1 μ g/ μ l to DNA purification Kit again after purification.Utilize Fermentas company
NheI and BamHI double digestion commercial carrier pCT302, at the same with Fermentas company FastAP dephosphorylation enzyme to digestion after
Carrier dephosphorylation, then after purification with Fermentas company GeneJET DNA purification Kit, ethanol precipitation concentration
It is greater than 1 μ g/ μ l to concentration.The method of bibliography Ginger etc. (2006) Nat Protoc 1 (2): 755-68, utilizes yeast
Electrotransformation and the method for In vivo recombination obtain the yeast library of affinity maturation.
Embodiment 4: the yeast anti-hPD-L1scFv screening that affinity improves is generated
Yeast library after the affinity maturation obtained above hPD-L1-Fc albumen of 10nM and 1nM is passed through two in turn
Formula sorting, the yeast product coated plate sorted, the identification of picking monoclonal.The method dyed using low concentration antigen, with before
Obtained wild-type yeast dyes the yeast monoclonal for determining that affinity improves as control, streaming.
Yeast colony PCR will be carried out by the yeast clone of FACS confirmation and sequencing, method are same as above.After affinity maturation
ScFv gene and human IgG1's-Fc Gene Fusion before after HindIII and EcoRI double digestion gram through Fermentas company
It is grand into commercial carrier pEE6.4, carry out clone according to the standard operation of molecular cloning and plasmid be small mentions.Plasmid after extraction
The transient expression in HEK293 cell, and pass through protein A column purification.
(binding) ability is combined to antibody according to the method in embodiment 2 and blocks (blocking) ability
Detection.
Binding ability experimental result is shown in Fig. 2, it can be seen that three strain antibodies obtained after affinity maturation it is affine
Power significantly improves.
Blocking ability experimental result is shown in Fig. 3, it can be seen that the competition of three strain antibodies that are obtained after affinity maturation
It is 0.837 μ g/ml (B II 61 is 0.884 μ g/ml), B50-6 4.56 that the IC50 of PD-1 combination PD-L1, which is respectively as follows: II 61-62 of B,
μ g/ml (B50 is 5.63 μ g/ml), B60-55 is 1.14 μ g/ml (B60 is 16.8 μ g/ml)
B50-6, the anti-hPD- that II tri- plants of affinity of 61-62 of B60-55, B improves are obtained after affinity maturation
L1scFv antibody sequence.Compared with B50, the mutation of amino acid D to N is had occurred in the area CDR1 of VL in B50-6;Compared with B60,
The mutation of amino acid S to N has occurred in the area CDR3 of VH in B60-55;Compared with BII61, BII61-62 occurs in the area CDR2 of VH
The mutation of amino acid S to G, in the area VL FR2 has occurred the mutation of amino acid I to V.Above-mentioned heavy chain of antibody and light chain variable region
Between containing connection peptide sequence GGGGSGGGGSGGGGS (SEQ ID NO:67).
The wherein heavy chain variable amino acid sequence of B50-6 are as follows:
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTKAAWYWIRQSPSRGLEWLGRTYFRSKWYNDYADSVK
SRLTINPDTSKNQFSLQLKSVSPEDTAVYYCARGQYTAFDIWGQGTMVTVSS (SEQ ID NO:51);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:13-15;Horizontal line portion is not drawn
It is respectively FR1,2,3,4, sequence number is respectively SEQ ID NO:38-41;
Its corresponding DNA sequence dna are as follows:
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGC
CATCTCCGGGGACAGTGTCTCTAGCACCAAGGCTGCTTGGTACTGGATCAGGCAGTCCCCTTCGAGAGGCCTTGAG
TGGCTGGGAAGGACATACTTCCGGTCCAAGTGGTATAATGACTATGCCGACTCTGTGAAAAGTCGATTAACCATCA
ACCCAGACACATCCAAGAACCAGTTCTCCCTGCAACTTAAGTCTGTGAGTCCCGAGGACACGGCTGTGTATTACTG
TGCAAGAGGGCAATACACTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCG TCTCTTCA (SEQ ID NO:
59);
Its chain variable region amino acid sequence are as follows:
QSALIQPASVSGSPGQSITISCTGTSSNVGGYDLVSWYQQYPGQAPRLIIYEVIKRPSGISDRFSGSK
SGNTASLTISGLQAEDEADYYCSSYAGRRLHGVFGGGTQLTVL (SEQ ID NO:52);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:16-18;Horizontal line portion is not drawn
It is respectively FR1,2,3,4, sequence number is respectively SEQ ID NO:42-45;
Its corresponding DNA sequence dna are as follows:
CAGTCTGCTCTGATTCAGCCTGCCTCCGTGTCTGGGTCCCCTGGACAGTCGATCACTATCTCCTGTAC
TGGCACCAGTAGTAATGTTGGAGGTTATGACCTTGTCTCCTGGTACCAACAGTACCCGGGCCAAGCCCCCAGACTC
ATCATTTATGAGGTCATTAAGCGGCCCTCAGGGATTTCTGATCGCTTCTCTGGTTCCAAGTCTGGCAACACGGCCT
CCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATGCAGGTAGACGTCTTCA
TGGTGTGTTCGGAGGAGGCACCCAGCTGACCGTCCTC (SEQ ID NO:64);
The heavy chain variable amino acid sequence of B60-55 are as follows:
QVQLVQSGAEVKKPASSVKVSCTASGGSFSTYAISWVRQAPGQGLEWMGGIIPIFGTTKYAQRFQGRV
TITADESTTTAYMELSSLISDDTALYYCTTSRGFNYGWFDYWGQGTLVTVSS (SEQ ID NO:47);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:1-3;Horizontal line part is not drawn
Respectively FR1,2,3,4, sequence number are respectively SEQ ID NO:22-25;
Its corresponding DNA sequence dna are as follows:
CAGGTCCAGCTTGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGCGTCCTCGGTCAAAGTCTCCTGCAC
GGCTTCTGGCGGCTCCTTCAGCACCTATGCTATCAGTTGGGTGCGACAGGCTCCTGGACAAGGGCTTGAATGGATG
GGCGGGATCATCCCCATCTTTGGTACAACTAAGTACGCACAGAGGTTCCAGGGCAGGGTCACGATTACCGCGGACG
AATCGACGACCACAGCCTACATGGAGCTGAGCAGCCTGATATCTGACGACACGGCCCTGTATTATTGTACGACGTC
TCGTGGATTCAACTATGGCTGGTTTGACTACTGGGGCCAGGGTACCCTGGTCACCG TCTCCTCA (SEQ ID NO:
57);
Its chain variable region amino acid sequence are as follows:
EIVMTQSPATLSLSPGERATLSCRASQSVGIHLAWYQQKLGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGSLPRTFGQGTKVEIK (SEQ ID NO:48);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:4-6;Horizontal line part is not drawn
Respectively FR1,2,3,4, sequence number are respectively SEQ ID NO:26-29;
Its corresponding DNA sequence dna are as follows:
GAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG
TAGGGCCAGTCAGAGTGTTGGCATACACTTAGCCTGGTACCAACAGAAACTTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGTAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCA
CCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTTCTTTACCTCGGACGTTCGG
CCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:62);
The heavy chain variable amino acid sequence of II 61-62 of B are as follows:
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSASWNWIRQSPSRGLEWLGRTYYRSKWYDDYAVSVK GRISINPDTSKNQFSLQLNSVTPEDTAVYYCARSQGRYFVNYGMDVWGQGTTVTVSS (SEQID NO:49);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:7-9;Horizontal line part is not drawn
Respectively FR1,2,3,4, sequence number are respectively SEQ ID NO:30-33;
Its corresponding DNA sequence dna are as follows:
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGC
CATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTTCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAG
TGGCTGGGAAGGACATATTACAGGTCCAAATGGTATGATGATTATGCAGTATCTGTGAAAGGTCGAATCAGCATCA
ACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTG
TGCAAGAAGCCAGGGACGATATTTTGTCAACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC
TCA (SEQ ID NO:58);
Its chain variable region amino acid sequence are as follows:
DIRLTQSPSSLSASVGDRITITCRASQSISSYLNWYQQKPGKAPKLLVYGASSLQSGVPSRFSGSGSG
TDFTLTISSLQPEDVATYYCQQSYFTPRGITFGPGTKVDIK (SEQ ID NO:50);
Wherein horizontal line part is respectively CDR1,2,3, and sequence number is respectively SEQ ID NO:10-12;Horizontal line portion is not drawn
It is respectively FR1,2,3,4, sequence number is respectively SEQ ID NO:34-37;
Its corresponding DNA sequence dna are as follows:
GACATCCGGTTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAATCACCATCACTTG
CCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTC
TATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA
CCATCAGCAGTCTGCAACCTGAAGATGTTGCAACTTACTACTGTCAACAGAGTTACTTTACCCCCCGCGGGATCAC
TTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO:63).
Embodiment 5:scFv type antibody is formatted as IgG type antibody
According to the amino acid constant region sequence of human immunoglobulin(HIg) gamma1 (IgG1) on albumen database Uniprot
(P01857), human IgG1's amino acid constant region sequence is obtained.Using DNAworks online tool (http: //
Helixweb.nih.gov/dnaworks/ it) designs corresponding DNA sequences encoding and obtains human IgG1's constant region gene, will screen
The B50-6 arrived, B60-55, heavy chain variable region VH sequence and the human IgG1's constant region gene sequences of BII61-62 are stitched together,
Simultaneously in 5 ' the end addition signal peptide sequences of VH: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCC
AGGTTCCACCGGT (SEQ ID NO:84);The gene chemical synthesis that will splice, through Fermentas company HindIII and EcoRI
Double digestion, which is subcloned, obtains pEE6.4-B50-6HC into carrier pEE6.4;pEE6.4-B60-55HC;pEE6.4-BⅡ61-
62HC.According to the amino acid constant region sequence (P01834) of human immunoglobulin(HIg) Kappa on albumen database Uniprot, obtain
People's Kappa chain constant region amino acid sequence.Utilize DNAworks online tool (http://helixweb.nih.gov/
Dnaworks/ it) designs corresponding DNA sequences encoding and obtains people's Kappa light chain constant region gene, the B60-55, B that screening is obtained
The light chain variable region VL sequence of II 61-62 is together with people's Kappa light chain constant region gene sequence assembly, while 5 ' in VL are held
Add signal peptide sequence: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCAC CGGT
(SEQ ID NO:84);Utilize DNAworks online tool (http://helixweb.nih.gov/dnaworks/) design pair
The DNA sequences encoding answered obtains people lambda (λ) light chain constant region gene, the light chain variable region VL for the B50-6 that screening is obtained
Sequence adds signal peptide sequences: ATGG together with people's lambda light chain constant region gene sequence assembly, while at the 5 ' ends of VL
AGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT (SEQ ID NO:84);It will splicing
Good gene chemical synthesis, is subcloned through Fermentas company HindIII and EcoRI double digestion into carrier pEE12.4 (Lonza)
Obtain pEE12.4-B50-6LC;pEE12.4-B60-55LC;pEE12.4-BⅡ61-62LC.
The big extraction reagent kit of plasmid (PL14) provided using AidLab company to heavy chain obtained above and light chain plasmids into
Row plasmid mentions greatly.The light chain of recombination to construct and heavy chain plasmid cotransfection HEK293 cell are subjected to antibody expression.It will recombinant expression
PEI (Polyethylenimine) solution needed for plasmid is diluted with Freestyle293 culture medium and conversion is added, by every group of matter
Grain/PEI mixture is separately added into cell suspension, is placed on 37 DEG C, 10%CO2, cultivate in 90rpm;50 μ g/L are added simultaneously
IGF-1.EX293 culture medium, 2mM Glutamine and 50ug/L IGF-1,135rpm culture are added after four hours again.24
Add 3.8mM VPA after hour.After culture 5~6 days, transient expression culture supernatant is collected, Protein A affinity chromatography is passed through
Method, purifying obtain II 61-62mAb antibody of anti-hPD-L1B50-6, B60-55, B.
Wherein IgG1 chain amino acid constant region sequence are as follows:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:68);
IgG1 chain constant region nucleic acid sequence are as follows:
GCCAGCACTAAGGGGCCCTCTGTGTTTCCACTCGCCCCTTCTAGCAAAAGCACTTCCGGAGGCACTGC
AGCACTCGGGTGTCTGGTCAAAGATTATTTCCCTGAGCCAGTCACCGTGAGCTGGAACTCTGGCGCCCTCACCTCC
GGGGTTCACACCTTTCCAGCCGTCCTGCAGTCCTCCGGCCTGTACTCCCTGAGCAGCGTCGTTACCGTGCCATCCT
CTTCTCTGGGGACCCAGACATACATCTGCAATGTCAACCATAAGCCTAGCAACACCAAGGTGGACAAAAAGGTCGA
GCCAAAGAGCTGCGATAAGACACACACCTGCCCTCCATGCCCCGCACCTGAACTCCTGGGCGGGCCTTCCGTTTTC
CTGTTTCCTCCCAAGCCCAAGGATACACTGATGATTAGCCGCACCCCCGAAGTCACTTGCGTGGTGGTGGATGTGA
GCCATGAAGATCCAGAAGTTAAGTTTAACTGGTATGTGGACGGGGTCGAGGTGCACAATGCTAAAACAAAGCCCAG
GGAGGAGCAATATAACTCCACATACAGAGTGGTGTCCGTTCTGACAGTCCTGCACCAGGACTGGCTGAACGGGAAG
GAATACAAGTGCAAGGTGTCTAATAAGGCACTGCCAGCCCCCATAGAGAAGACAATCTCTAAAGCTAAAGGCCAAC
CACGCGAGCCTCAGGTCTACACACTGCCACCATCCAGGGACGAACTGACCAAGAATCAGGTGAGCCTGACTTGTCT
CGTCAAAGGATTCTACCCAAGCGACATCGCCGTGGAGTGGGAATCCAACGGCCAACCAGAGAACAACTACAAGACC
ACCCCACCAGTCCTGGACTCTGATGGGAGCTTTTTCCTGTATTCCAAGCTGACAGTGGACAAGTCTCGGTGGCAAC
AGGGCAACGTGTTCAGCTGCTCCGTGATGCATGAAGCCCTGCATAACCACTATACCCAGAAAAGCCTCAGCCTGTC
CCCCGGGAAATAATGA (SEQ ID NO:69);
Kappa chain amino acid constant region sequence are as follows:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:70);
Kappa chain constant region nucleic acid sequence are as follows:
CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGTACCGC
TAGCGTTGTGTGCCTGCTGAATAACTTTTATCCACGGGAGGCTAAGGTGCAGTGGAAAGTGGACAATGCCCTCCAG
AGCGGAAATAGCCAAGAGTCCGTTACCGAACAGGACTCTAAAGACTCTACATACTCCCTGTCCTCCACACTGACCC
TCTCCAAGGCCGACTATGAGAAACACAAGGTTTACGCATGCGAGGTCACACACCAGGGACTCTCCTCTCCCGTGAC
CAAGAGCTTCAACCGGGGAGAATGC (SEQ ID NO:71);
B50-6 light chain (lambda) amino acid constant region sequence are as follows:
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAA
SSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS (SEQ ID NO:72);
B50-6 light chain (lambda) constant region nucleic acid sequence are as follows:
GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAA
GGCCACACTGGTGTGTCTCGTAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCC
GTCAAGGTGGGAGTGGAGACCACCAAACCCTCCAAACAAAGCAACAACAAGTATGCGGCCAGCAGCTACCTGAGCC
TGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGTCACGCATGAAGGGAGCACCGTGGAGAAGAC
AGTGGCCCCTGCAGAATGCTCT (SEQ ID NO:73).
Embodiment 6:anti-hPD-L1mAb CHARACTERISTICS IDENTIFICATION
Anti-hPD-L1 antibody and hPD-L1 the binding ability detection (ELISA method) of purifying:
To be coated with buffer (50mM Na2CO3, NaHCO3PH9.6 hPD-L1-muFc to 2 μ g/ml, 100 μ L/) are diluted
Well, 4 DEG C overnight.Get rid of liquid in plate, after PBST (pH7.4,0.05%Tween-20, V/V) board-washing, 3%BSA-PBS closing
1h.Antibody B50-6mAb, B60-55mAb and B μ 61-62mAb is subjected to 2 times of gradient dilutions since 2000ng/ml respectively, altogether
11 concentration, dilution (1%BSA-PBS) compare, 37 DEG C of incubation 2h.Goat anti-human igg-HRP (Goat anti-is added
Human IgG-HRP conjugated), it is incubated for 1h.Add soluble one pack system tmb substrate developing solution, room temperature is protected from light colour developing 5-
10min。2N H2SO450 μ L/well, color development stopping reaction.It sets and reads OD450nm- in MD SpectraMax Plus384 microplate reader
650nm value, application software SotfMax Pro v5.4 carry out data processing and mapping analysis, as a result see Fig. 4.
Determine that the combination antigen EC50 of three strain antibodies is respectively 40 μ g/ml (B60-55mAb), 18.3 μ g/ by the method
Ml (II 61-62mAb of B), and 28.1 μ g/ml (B50-6mAb).
Anti-hPD-L1 antibody and hPD-L1 the binding kinetics detection (SPR) of purifying:
The combination of anti-PD-L1 antibody B50-6mAb, BII61-62mAb and B60-55mAb for the human PD-L 1 of recombination
Dynamics uses BIAcore by surface plasmon resonance (surface plasmon resonance, SRP) method
X100 apparatus measures.The hPD-L1-Fc direct coated of recombination on CM5 biologic sensor chip to obtain about 1000 response lists
Position (response units, RU).It is for kinetic measurement, antibody is slow with HBS-EP+1 × (GE, cat#BR-1006-69)
Fliud flushing three times serial dilution (1.37nm to 1000nm), in 25 DEG C of sample introduction 120s, Dissociation time 30min, 10mM glycine-
HCl (pH2.0) regenerates 120s.Use simple one-to-one Languir binding model (3.2 editions (BIAcore of BIAcore evaluation software
Evaluation Software version 3.2)) calculations incorporated rate (kon) and dissociation rate (koff).Balance dissociation is normal
Number (kD) is with ratio koff/kon calculating.
The binding affinity of the anti-PD-L1 antibody of measurement is shown in Table 1.
Table 1anti-hPD-L1 antibody and hPD-L1 binding kinetics detect
Title | Kon(1/Ms) | Koff(1/s) | KD(M) |
B50-6mAb | 1.672E+5 | 1.370E-2 | 8.193E-8 |
B60-55mAb | 1.295E+6 | 2.222E-4 | 1.716E-10 |
BⅡ61-62mAb | 9.795E+4 | 4.264E-4 | 4.353E-9 |
The anti-hPD-L1 antibody and hPD-1 competitive binding hPD-L1 ability of purifying detect:
To be coated with buffer (50mM Na2CO3, NaHCO3PH9.6) dilution hPD-L1-hIgG is to 5 μ g/ml, and 4 DEG C overnight.
3%BSA-PBS closes 1h after PBST (pH7.4,0.05%Tween-20, V/V) board-washing.By the dense of anti-hPD-L1mAb to be measured
Degree is diluted to 100 μ g/ml, 1%BSA-PBST-0.05%Tween-20 (hPD-1-hIgG-biotin containing 10 μ g/ml) 1:
6 gradient dilutions, totally 9 dilutions, 37 DEG C of 2h.Streptavidin (the SA-HRP of horseradish peroxidase-labeled is added after board-washing
Conjugated) it is incubated at room temperature 1.5h.Soluble one pack system tmb substrate developing solution room temperature is added to be protected from light colour developing 5-10min, 2N
H2SO4Color development stopping reaction.It sets and reads OD in MD SpectraMax Plus384 microplate reader450nm-650nmValue, application software
SotfMax Pro v5.4 carries out data processing and mapping analysis, strong according to detection data and IC50 value analysis antibody competition ability
It is weak, as a result see Fig. 5.
It is respectively 0.255 μ g/ml 1.7nM by the IC50 that the method determines that three strain antibodies compete PD-1 combination PD-L1
(B60-55), 0.24 μ g/ml 1.6nM (II 61-62 of B), and 1.76 μ g/ml11.7nM (B50-6).
The anti-hPD-L1 antibody and CD80 competitive binding hPD-L1 ability of purifying detect:
Take that screening obtains three strain antibody B60-55, B II 61-62 and B50-6 it is evaluated by the method for competitive ELISA
Whether the combination of PD-L1 and CD80 can be blocked.The specific method is as follows: to be coated with buffer (50mM Na2CO3,
NaHCO3PH9.6) dilution hPD-L1-hFc is to 5 μ g/ml, and 4 DEG C overnight.PBST (pH7.4,0.05%Tween-20, V/V) board-washing
3%BSA-PBS closes 1h afterwards.It is 100 μ g/ml, 1%BSA-PBST- by the concentration dilution of anti-hPD-L1mAb to be measured
0.05%Tween-20 (hCD80-hFc-biotin, R&D:140-B1-100 containing 100 μ g/ml) 1:6 gradient dilution, totally 9
A dilution, 37 DEG C of 2h.Room Streptavidin (SA-HRPconjugated) of horseradish peroxidase-labeled is added after board-washing
Temperature is incubated for 1.5h.Soluble one pack system tmb substrate developing solution room temperature is added to be protected from light colour developing 5-10min, 2N H2SO4Color development stopping is anti-
It answers.Set reading OD450nm-650nm value, application software SotfMax Pro v5.4 in MD SpectraMax Plus384 microplate reader
Data processing and mapping analysis are carried out, it is strong and weak according to detection data and IC50 value analysis antibody competition ability, as a result see Fig. 6.
It is respectively 0.543 μ g/ml (B60- by the IC50 that the method determines that three strain antibodies compete CD80 combination PD-L1
55), 0.709 μ g/ml (II 61-62 of B), and 0.553 μ g/ml (B50-6).
Antibody whether the identification of specific recognition PD-L1: the anti-hPD-L1 of purifying and hPD-L1, hPD-L2, hB7H3
Combination
The HEK293 cell containing hPD-L1-EGFP, hB7H3-EGFP, hPD-L2-EGFP that Example 1 constructs is resuspended
In 0.5%PBS-BSA Buffer, anti-hPD-L1mAb albumen is added, negative control is hIgG Fc albumen, is incubated on ice
20min.EBioscience secondary antibody anti-hIg-PE is added after washing, on ice 20min.Cell is resuspended in 500 μ l after washing
In 0.5%PBS-BSA Buffer, flow cytometer is detected.As a result as shown in Figure 6.It can be seen from the results that we three
Strain antibody can cannot be shown very with hPD-L1-EGFP cell combination with hB7H3-EGFP, hPD-L2-EGFP cell combination
Good specificity.
The combination of the anti-hPD-L1 and mouse PD-L1 (mPD-L1) of purifying:
The HEK293 cell containing hPD-L1-EGFP, mPD-L1-EGFP that Example 1 constructs, is resuspended in 0.5%PBS-
In BSA Buffer, anti-hPD-L1mAb to be detected is added, negative control is hIgG Fc albumen, it is incubated for 20min on ice,
EBioscience secondary antibody anti-hIg-PE is added after washing, is incubated for 20min on ice.Cell is resuspended in 0.5% after washing
In PBS-BSABuffer, flow cytometer is detected.As a result as shown in Figure 7.By result it can be seen that B50-6mAb can be with
The PD-L1 (mPD-L1) of mouse is combined, and B60-55 and BII61-62 cannot be in conjunction with mPD-L1.
The combination of the anti-hPD-L1 and machin PD-L1 of purifying:
Using human lymphocyte separating liquid (ocean Tianjin Hao) separation machin peripheral blood PBMC, it is complete in RPMI that cell is resuspended
In culture medium, adjustment cell concentration is 1,000,000/milliliter, and 2,000,000 cell machin PBMC cells are then added in 24 orifice plates,
It is added phytohemagglutin phytolectin (phytohaemagglutinin, PHA) simultaneously, final concentration of 2 μ g/ml stimulation cell 48 hours, then
Cell is collected, antibody dyeing is added after cell is washed in FACS buffer solution.Using Isotype ctrl (anti-KLH) as yin
Property control, commercialization PE label anti human PD-L 1 antibody (Biolegend:329705) be used as positive control.Our antibody is made
For primary antibody, anti-hIg-PE is added after washed and is dyed as secondary antibody.Each step dyes 4 DEG C and is incubated for 30 minutes, dyeing
After with FACS buffer solution centrifuge washing cell twice, add secondary antibody or directly fixed with 2% paraformaldehyde after use Guava
Analysis.As a result as shown in Figure 8.The result shows that machin T cell stimulates expression PD-L1 through PHA, our three strain antibodies can be with
The machin T cell of activation combines.
Embodiment 7: measurement PD-L1 antibody is to CD4 in dendritic cells-T cell mixing lymph reaction+The activation of T cell
Effect
It is white thin from healthy donor periphery pyknohemia using human lymphocyte separating liquid (ocean Tianjin Hao) density gradient centrifugation
Separating peripheral blood mononuclear cells PBMC in born of the same parents.Then it is resuspended in the RPMI1640 of serum-free, in 10cm culture dish
Culture 1-2 hours removes not adherent cell, and by cell culture in the RPMI containing 10%FBS.With 250ng/ml GM-
CSF (Shanghai is general glad: 102-03) and 100ng/ml IL-4 (Shanghai is general glad: final concentration 101-04) adds cell factor, every 2-
The fresh culture of 3 days addition factor-containings.At the 6th day of culture, with the TNF-alpha of 50ng/ml (Shanghai is general glad:
103-01) make cell maturation and is incubated for it 24 hours.Harvest mature dendritic cells, with the dyeing of HLA-DR antibody determine its at
It is ripe.It is resuspended in RPMI complete medium, then every hole in 96 hole U-shaped base plates (Costar:3799) adds 200,000/ml.
Enter 50 μ l, puts in incubator and cultivate.
Using magnetic bead separation kit (Miltenyi Biotec:130-096-533) to specifications method from another
CD4 is separated in donor PBMC+T cell.Counting is resuspended in RPMI complete medium, and concentration is 2,000,000/ml, is then added to
50 μ l are added in the 96 hole U-shaped base plates containing dendritic cells, every hole.Gradient dilution is added in RPMI complete medium in every hole
100 μ l of PD-L1 antibody, antibody final concentration are respectively 100,10,1,0.1,0.01,0.001,0 μ g/ml.Culture takes after five days
Clearly, the level of IFN-r in IFN-r ELISA detection kit (ebioscience) detection supernatant is utilized.As a result see Fig. 9.It can be seen that
CD4 in mixed lymphocyte reaction (MLP) can be enhanced in PD-L1 antibody+T cell secretes gamma interferon, that is to say, that PD-L1 antibody
Enhance the activation of T cell.The EC50 value of II 61-62 of B is 0.078 μ g/ml (being equivalent to 0.5nM), and the EC50 value of B60-55 is
0.189 μ g/ml (is equivalent to 1.2nM.
The inhibitory activity of embodiment 7:anti-hPD-L1 antibodies on tumor growth
It has become clear that many tumours are using expression PD-1 ligand as the method for weakening Anti-tumor t cell response.?
It finds characteristically to express raised levels of PD-L1 in the tumour of several people and tumor-infiltrating leukocyte, and this raising
PD-L1 expression it is often associated with worse prognosis.Mouse tumor model shows that PD-L1 expression has similar increasing in tumour
Add, and shows that PD-1/PD-L1 approach is inhibiting the effect in tumour immunity.
We provide experiment and show to block PD-L1 to MC38 cell (mouse Colon and rectum in syngeneic C57B6 mouse herein
Cancer cell) tumour growth influence.
C57B6 mouse was inoculated with 1,000,000 MC38 cells (Chicago University professor Fu Yangxin gives) at the 0th day,
0th, 3,7,10 day with 10mg/kg anti-PD-L1 (B50-6) or PBS intraperitoneal injection of mice.Every three days measurement tumour major diameter and
Minor axis calculates gross tumor volume, draws tumor growth curve figure (referring to Figure 10), it can be seen that anti-PD-L1 (B50-6) can be with
Significantly inhibit tumour growth.
For that cannot identify the antibody B60-55 and BII61-62 of mouse PD-L1, using immune deficiency NOD/SCID (non-fertilizer
Fat diabetes/severe combined immunodeficiency) mice study its activity in vivo.By expressing NOD/SCID mouse subcutaneous transplanting
The K-1735 of human PD-L 1A375(ATCC, CRL-1619TM) and people peripheral blood mononuclear cells PBMC experiment come
Realize this research purpose.A375 and PBMC is mixed before the injection in the ratio of 5:1, and 100 μ l of total volume subcutaneous injection (contains 5,000,000
A375,1,000,000 PBMC), in the 0th, 7,14,21,28 day intraperitoneal injection of tumor inoculation, (Figure 11 A antibody dosage is antibody
3mg/kg;Figure 11 B antibody dosage is shown in Figure 11), PBS is as negative control.4-6 mouse of each experimental group.It observes twice a week
The formation of tumour, and with vernier caliper measurement tumour major diameter and minor axis, gross tumor volume is calculated, tumor growth curve figure (ginseng is drawn
See Figure 11), it can be seen that antibody B60-55 and BII-61-62 can significantly inhibit tumour growth.
Embodiment 8B60-55 is compared with antibody 2.41H90P (Medimmune LLC company) stability
The antibody 2.41H90P of confrontation PD-L1 antibody B60-55 and MedImmune LLC has carried out 45 DEG C of accelerated stabilities
Experiment, specific experiment method are as follows: by the anti-PD-L1 antibody of anti-PD-L1 antibody B60-55 and MedImmune LLC company
2.41H90P (according in patent US20130034559 2.14H9 method prepare, and renamed for 2.41H90P) it is dense
It is reduced to 10mg/ml, 100 μ g antibody is taken to be put into 200 μ lPCR pipes, 45 DEG C of water-baths, in the 0th day, the 10th day, the 20th day, the 30th day
It receives sample to be at war with ELISA and SEC-HPLC analysis experiment, competitive ELISA method acquires IC50 with described in previous embodiment 6.
SEC-HPLC carries out analysis experiment using Shimadzu LC20AT HPLC liquid chromatograph, and by sample concentration to 1mg/ml, flow velocity is
0.5ml/min loading, total applied sample amount are 50ug, carry out isocratic elution 30min, the result is shown in Figure 12 after loading.
Wherein A is B60-55 figure compared with the IC50 value that antibody 2.41H90P is changed over time, it can be seen that different time
The simple competitive power checked and accepted does not have significant changes;B is the ratio that antibody dimer changes over time, it can be seen that with the time
Increase, the case where B60-55 and 2.41H90P can show the decline of dimer ratio, but the speed of 2.41H90P decline
It is faster than B60-55, show that the stability of B60-55 is more preferable;C is the competitive ELISA curve of B60-55 acceleration for stabilization experiment, can be seen
B60-55 is able to maintain preferable activity and stability out.
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention
Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (19)
1. anti-PD-L1 antibody or its antigen-binding portion thereof comprising the CDR region selected from such as next group:
(1) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:1-3, the sequence of light chain CDR1, CDR2, CDR3
Column are respectively as shown in SEQ ID NO:4-6;
(2) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQ ID NO:1,2,19, light chain CDR1, CDR2, CDR3
Sequence respectively as shown in SEQ ID NO:4-6.
2. the anti-PD-L1 antibody of claim 1 or its antigen-binding portion thereof comprising sequence is as shown in SEQ ID NO:47 or 53
Heavy chain variable region.
3. the anti-PD-L1 antibody of claim 1 or its antigen-binding portion thereof comprising the light chain as shown in SEQ ID NO:48 can
Become area.
4. the anti-PD-L1 antibody of claim 1 or its antigen-binding portion thereof, be whole antibody, bispecific antibody, scFv,
Fab、Fab'、F(ab')2Or Fv.
5. the anti-PD-L1 antibody of claim 4 or its antigen-binding portion thereof contain between the heavy chain and light chain variable region of the scFv
There is link peptide.
6. the anti-PD-L1 antibody of claim 5 or its antigen-binding portion thereof, the sequence of the link peptide such as SEQ ID NO:67 institute
Show.
7. the anti-PD-L1 antibody of any one of claim 1-6 or its antigen-binding portion thereof, heavy chain constant region be selected from IgG,
IgM。
8. the anti-PD-L1 antibody of claim 7 or its antigen-binding portion thereof, heavy chain constant region be selected from IgG1, IgG2, IgG3 and
IgG4。
9. the anti-PD-L1 antibody of any one of claim 1-6 or its antigen-binding portion thereof, constant region of light chain is κ or λ.
10. nucleic acid molecules, it includes can encode antibody of any of claims 1-9 or its antigen-binding portion thereof
Nucleic acid sequence.
11. carrier contains nucleic acid molecules described in any one of claim 10.
12. host cell contains carrier described in nucleic acid molecules described in any one of claim 10 or claim 11.
13. conjugate, anti-PD-L1 antibody or its antigen-binding portion thereof containing any one of claim 1-9 also include choosing
From the part of the following group: toxin, enzyme, isotope and cell factor.
14. conjugate, anti-PD-L1 antibody or its antigen-binding portion thereof containing any one of claim 1-9 also include choosing
From the part of the following group: Auristatin MMAE, Auristatin MMAF, Maytansine DM1, Maytansine DM4, blocking
Miramycin, adriamycin, ricin (WA), diphtheria toxin, I131, interleukin, tumor necrosis factor, becomes at duocarmycin MGBA
Change the factor and nano particle.
15. pharmaceutical composition, anti-PD-L1 antibody or its antigen-binding portion thereof or power containing any one of claim 1-9
Benefit requires the conjugate of any one of 13-14, and optionally pharmaceutically acceptable carrier or excipient.
16. detection reagent or kit, anti-PD-L1 antibody or its antigen-binding portion thereof containing any one of claim 1-9,
The detection reagent or kit whether there is for detecting PD-L1 albumen.
17. the anti-PD-L1 antibody or any one of its antigen-binding portion thereof or claim 13-14 of any one of claim 1-9
Conjugate be used to prepare the purposes of drug, the drug is for treating tumour or Chronic HBV, HCV and HIV infection.
18. the purposes of claim 17, the tumour is the highly expressed tumour of PD-L1.
19. the purposes of claim 17 or 18, wherein the tumour is selected from: lung cancer, oophoroma, colon and rectum carcinoma, melanin
Tumor, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngocarcinoma,
Cervical carcinoma, carcinoma of uterine body and osteosarcoma.
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