CN105622753B - A kind of PD-1 monoclonal antibody and its application - Google Patents
A kind of PD-1 monoclonal antibody and its application Download PDFInfo
- Publication number
- CN105622753B CN105622753B CN201410613502.1A CN201410613502A CN105622753B CN 105622753 B CN105622753 B CN 105622753B CN 201410613502 A CN201410613502 A CN 201410613502A CN 105622753 B CN105622753 B CN 105622753B
- Authority
- CN
- China
- Prior art keywords
- antibody
- cell
- seq
- variable region
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention provides a kind of PD-1 monoclonal antibody and its application, monoclonal antibody of the invention, specificity is high, and affinity is strong, can significantly stimulate the secretion of IFN-γ, and can effectively activate CD8+And CD4+T cell.
Description
Technical field
The invention belongs to biomedicine fields, specifically, the present invention relates to a kind of PD-1 monoclonal antibody and its applications.
Background technique
The specific immune response of T cell be by T cell receptor in conjunction with Antigenic Peptide MHC compound after formed first thorn
What energizing signal was excited；Simultaneously T cell mediate immune response intensity and amplitude be then by immunity inspection point albumen (including altogether
Inhibiting factor and costimulatory molecules) it is adjusted.By 20 years of researches, it is thin that the molecule of immunity inspection point is found in adjusting T
Key effect is played in born of the same parents' immune response, these molecules include: 4 (Cytotoxic of cytotoxic lymphocite antigen
Lymphocyte antigen-4, CTLA-4), death protein 1 (Programmed death-1, PD-1), T cell is exempted from
Epidemic disease globulin and mucoprotein structure -3 (T cell immunoglobul in and mucin domain 3, TIM-3), T cell
Immunoglobulin and ITIM structure (T cell Ig and ITIM domain, TIGIT) etc..
Under normal physiological condition, the major function of immunity inspection point is to maintain self tolerance and protect normal tissue from exempting from
Epidemic disease system injury.In tumor microenvironment, since long-time contacts tumour antigen, lymphocyte can the certain co-suppressions of unconventionality expression
The factor, to cause tumor specific T cells that dysfunction occurs.A large amount of clinical data is shown for the anti-of coinhibitory signals
Body can be improved the response of tumor specific T cells.Therefore, these immunity inspection point albumen become anti-tumor immunotherapy
Potential target spot.
However, the performance of current antibody is still unsatisfactory.Therefore this field need to develop it is new, have excellent performance needle
To the antibody drug of above-mentioned target spot, the needs of to meet clinically.
Summary of the invention
The purpose of the present invention is to provide a kind of new PD-1 monoclonal antibodies and its application.
The first aspect of the present invention, provides a kind of heavy chain variable region of antibody, and the heavy chain variable region includes following
Three complementary determining region CDR:
CDR1 shown in SEQ ID NO:4,
CDR2 shown in SEQ ID NO:6, and
CDR3 shown in SEQ ID NO:8.
In another preferred example, the heavy chain variable region has amino acid sequence shown in SEQ ID NO:10.
The second aspect of the present invention, provides a kind of heavy chain of antibody, and the heavy chain has first aspect present invention institute
The heavy chain variable region and heavy chain constant region stated.
In another preferred example, the heavy chain constant region is source of people or source of mouse.
The third aspect of the present invention provides a kind of light chain variable region of antibody, and the light chain variable region, which has, to be selected from down
The complementary determining region CDR of group:
CDR1 ' shown in SEQ ID NO:14,
CDR2 ' shown in SEQ ID NO:16, and
CDR3 ' shown in SEQ ID NO:18.
In another preferred example, the light chain variable region has amino acid sequence shown in SEQ ID NO:20.
The fourth aspect of the present invention, provides a kind of light chain of antibody, and the light chain has third aspect present invention institute
The light chain variable region and constant region of light chain stated.
In another preferred example, the constant region of the light chain is source of people or source of mouse.
The fifth aspect of the present invention, provides a kind of antibody, and the antibody includes
(1) heavy chain variable region as described in the first aspect of the invention；And/or
(2) light chain variable region as described in the third aspect of the present invention.
In another preferred example, the antibody includes heavy chain as described in respect of the second aspect of the invention；And/or such as the present invention
Light chain described in fourth aspect.
In another preferred example, the antibody is the antibody of the anti-PD-1 albumen of specificity.
In another preferred example, the antibody includes: single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody
(such as human mouse chimeric antibody), source of mouse antibody or humanized antibody.
The sixth aspect of the present invention, provides a kind of recombinant protein, and the recombinant protein includes
(i) sequence of heavy chain variable region as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention
Sequence, the sequence of light chain variable region as described in the third aspect of the present invention, the sequence of light chain as described in the fourth aspect of the present invention
The sequence of column or the antibody as described in fifth aspect present invention；And
(ii) sequence label of optional assistance expression and/or purifying.
In another preferred example, the sequence label includes 6His label.
In another preferred example, the anti-PD-1 albumen of recombinant protein specificity.
The seventh aspect of the present invention provides a kind of polynucleotides, it encodes polypeptide selected from the group below:
(1) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair
Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention
Antibody；Or
(2) recombinant protein as described in sixth aspect present invention.
In another preferred example, the polynucleotides have shown in SEQ ID NO:3,5,7,9,13,15,17 or 19
Sequence.
The eighth aspect of the present invention provides a kind of carrier, it contains polynucleotides described in seventh aspect present invention.
In another preferred example, the carrier include: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus,
Mammalian cell virus such as adenovirus, retrovirus or other carriers.
The ninth aspect of the present invention provides a kind of genetically engineered host cell, it contains eighth aspect present invention
Polynucleotides described in seventh aspect present invention are integrated in the carrier or genome.
The tenth aspect of the present invention provides a kind of immune conjugate, which contains:
(a) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair
Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention, as described in fifth aspect present invention
Antibody or the recombinant protein as described in sixth aspect present invention；With
(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or
Enzyme.
In another preferred example, the conjugate is selected from: (magnetic is total by fluorescence or luminous marker, radioactively labelled substance, MRI
Vibration imaging) CT (x-ray tomography of electronic computer) contrast agent or can generate detectable product enzyme, radiation
Property nucleic, biotoxin, cell factor (such as IL-2), antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/receive
Rice stick, virion, liposome, magnetic nanosphere, pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or biphenyl base hydrolase-
Sample protein (BPHL)), chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
The eleventh aspect of the present invention, provides a kind of pharmaceutical composition, it contains:
(i) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair
Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention, as described in fifth aspect present invention
Antibody, the recombinant protein as described in sixth aspect present invention or the immune conjugate as described in tenth aspect present invention；And
(ii) pharmaceutically acceptable carrier.
In another preferred example, the pharmaceutical composition is injection type.
In another preferred example, the pharmaceutical composition is used to prepare the drug for the treatment of tumour, and the tumour is selected from
The following group: gastric cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, large intestine
Cancer, prostate cancer, cervical carcinoma, adrenal tumor or tumor of bladder.
The twelveth aspect of the present invention provides heavy chain variable region as described in the first aspect of the invention, such as present invention the
Two aspect described in heavy chain, light chain variable region as described in the third aspect of the present invention, light chain as described in the fourth aspect of the present invention,
Antibody as described in fifth aspect present invention, the recombinant protein as described in sixth aspect present invention or such as tenth aspect present invention
The purposes of the immune conjugate is used to prepare medicament, reagent, detection plate or kit；
The reagent, detection plate or kit are used for: PD-1 albumen in test sample；
The medicament is used to treat or prevent the tumour of expression PD-1 albumen.
In another preferred example, the tumour include: gastric cancer, it is lymthoma, liver cancer, leukaemia, tumor of kidney, lung cancer, small
Intestinal cancer, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer or adrenal tumor.
In another preferred example, the tumour is selected from: gastric cancer and follicular lymphoma.
In another preferred example, the reagent includes the immune particle of chip, coated antibody.
The thirteenth aspect of the present invention provides a kind of method of PD-1 albumen in test sample, and the method includes steps
It is rapid:
(1) sample is contacted with antibody described in fifth aspect present invention；
(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are PD-1 eggs in sample
It is white.
The fourteenth aspect of the present invention provides a kind of preparation method of recombinant polypeptide, and this method includes:
(a) under conditions suitable for the expression, host cell described in ninth aspect present invention is cultivated；
(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in fifth aspect present invention
Or recombinant protein described in sixth aspect present invention.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows PD1-1375 to the affinity analysis result of soluble human PD-1.
Fig. 2A and 2B shows the sequential structure of monoclonal antibody PD1-1375 of the present invention.
Fig. 3 shows PD1-1375 and PD-1 competitive ELISA testing result.
Fig. 4 shows PD1-1375 and PDL1 competitive ELISA testing result.
Fig. 5 shows that PD1-1375 activates PBMC experimental result.
Fig. 6 shows the thermal stability of DSC detection PD1-1375.
Fig. 7 shows the proliferation for blocking PD-1 that can increase the antitumor T cell of PD-1+.
Fig. 8 shows that PD1 antibody can significantly increase cell factor (the IFN-γ and TNF-α) generation of tumour patient TIL.
Fig. 9 shows that PD1 antibody can significantly inhibit FL tumour growth.
Figure 10 A and 10B show PD1 antibody, in conjunction with Rituximab drug, can further suppress RL tumour growth.
Specific embodiment
The present inventor is by extensive and in-depth research, and by largely screening, unexpectedly a kind of anti-PD-1 monoclonal is anti-
Body PD1-1375, the experimental results showed that, not only specificity is high for the monoclonal antibody, and affinity is strong, can significantly stimulate IFN-γ
Secretion, and can effectively activate CD8+And CD4+T cell.The present invention is completed on this basis.
PD-1 signal path
PD-1 immunity inspection point receptor is capable of the activity of restricted T cells, and limit in periphery when tissue inflammation response
Autoimmunity processed.This is that the immune main mechanism resisted is generated in tumor microenvironment.T cell induces the table of PD-1 after activation
It reaches.After with PD-1 ligand binding, the phosphorylase SHP2 for participating in t cell activation is suppressed.Meanwhile because PD-1 is tied
Conjunction inhibits TCR stop signal, when this signal paths can change the connection between T cell-APC or T cell-target cell
Between.PD-1 high is expressed in Treg cell, and Treg cell can enhance the proliferation of itself in the presence of PD-1 ligand.Because being permitted
The Treg cell of more tumour high-expression infiltrations, may further suppress the immune response of effector cell, so blocking PD-1 letter
Number access can be by reducing and/or inhibiting the activity of Treg cell in tumor to enhance anti-tumor immune response.
There are two ligand PD-L1 (i.e. B7-H1/CD274) and PD-L2 (i.e. B7-DC/CD273) by PD-1.
PD-1 is expressed in most of tumor infiltrating lymphocyte (TILs) of source kinds of tumors type.Height expression PD-1
CD4+TIL is concentrated mainly on CD4 in tumor tissues+Treg cell.The CD8 of height expression PD-1+TILs shows incompetent or tired
Exhausted state, by comparing PD-1 in melanoma-TILs, PD-1+TILs secretes less cell factor.Just as PD-1 high table
Up in the TILs of many tumours, the PD-1 ligand also high tumor cell surface for being expressed in kinds of tumors.On solid tumor cell surface
The ligand PD-L1 of main expression PD-1 promotes mouse tumor cell height expression PD-L1 to be able to suppress the antitumor of T cell mediation
Immune response.Really, these are found to be the function for blocking PD-1 signal path enhancing anti-tumor effect cell in tumor microenvironment
It can provide the foundation.Analysis based on immunohistochemistry (IHC) and flow cytometer obtains a variety of human tumors in certain level group
The high expression PD-1 ligand of molding.The expression pattern of PD-1 ligand by the main Immunotherapy Strategy for determining to block this access whether
It is feasible, because main function of the PD-1 ligand in cancer is considered as playing immunosupress in tumor microenvironment, and PD-
1 with its ligand, PD-L1 and PD-L2, the function of lymphocyte can only be inhibited in conjunction with after.
In a preferred embodiment of the invention, the amino acid sequence of the PD-1 are as follows:
DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC
RFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTL
VVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQ
TEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO.:1)
As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical structure feature
Different four glycan albumen is made of two identical light chains (L) and two identical heavy chains (H).Every light chain is total by one
Valence disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Each heavy chain and
The intrachain disulfide bond at light chain also regular interval.One end of each heavy chain has variable region (VH), is followed by multiple constant regions.Every
One end of light chain has variable region (VL), and the other end has constant region；The constant region of light chain is opposite with first constant region of heavy chain, gently
The variable region of chain and the variable region of heavy chain are opposite.Special amino acid residue forms boundary between the variable regions of the light chain and the heavy chain
Face.
As used herein, " variable " the certain parts for indicating variable region in antibody of term are different in sequence, its shape
Combination and specificity at various specific antibodies to its specific antigen.However, changeability and being unevenly distributed over entire anti-
In body variable region.It concentrates in light chain and heavy chain variable region three segments being known as in complementary determining region (CDR) or hypervariable region
In.More conservative part is known as framework region (FR) in variable region.Four FR are respectively contained in the variable region of native heavy and light chain
Area, they are generally in beta sheet configuration, are connected by three CDR of formation connection ring, can form part β folding in some cases
Stack structure.CDR in every chain is by the area FR firmly against the antigen for together forming antibody together and with the CDR of another chain
Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly
With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody
Cellular toxicity.
" light chain " of vertebrate antibodies (immunoglobulin) can be classified as obviously not according to the amino acid sequence of its constant region
One kind in same two classes (referred to as κ and λ).According to the amino acid sequence of its heavy chain constant region, immunoglobulin can be divided into not
Same type.Mainly there are 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them can also be further separated into subclass
(isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Light chain constant corresponding to different immunoglobulin like protein is distinguished
It is also known as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of different immunoglobulin like protein are known to those skilled in the art
's.
As used herein, term " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from a kind of substantially uniform group, i.e.,
The single antibody for including in the group be it is identical, in addition to a small number of mutation that may be present naturally occurred.Monoclonal antibody is high
Specifically it is directed to single antigen site.Moreover, (usually having for different determinants from conventional polyclonal antibody preparation
Different antibodies) it is different, each monoclonal antibody is for the single determinant on antigen.Other than their specificity, monoclonal
The benefit of antibody, which also resides in them, to be synthesized by hybridoma culture, will not be polluted by other immunoglobulins.Modifier
" monoclonal " illustrates the characteristic of antibody, is obtained from substantially uniform antibody population, this is not construed as needing to use appointing
What specific process produces antibody.
It is anti-that the invention also includes the monoclonals of the corresponding amino acid sequence with the anti-PD-1 protein monoclonal antibody
Body, the monoclonal antibody with the anti-PD-1 protein monoclonal antibody variable region chain, and other eggs with these chains
White matter or protein conjugate and fusion expressed product.Specifically, the present invention include have containing hypervariable region (complementary determining region,
CDR any protein or protein conjugate and fusion expressed product (the i.e. immune conjugate and fusion table of light chain and heavy chain)
Up to product), as long as the hypervariable region is identical as the hypervariable region of light chain of the invention and heavy chain or at least 90% homology, preferably extremely
Few 95% homology.
As it is known by the man skilled in the art, immune conjugate and fusion expressed product include: drug, toxin, cell factor
(cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and the anti-PD-1 protein monoclonal antibody or its
Segment in conjunction with and the conjugate of formation.The invention also includes in conjunction with the anti-PD-1 protein monoclonal antibody or its segment
Cell surface marker object or antigen.
The present invention not only includes complete monoclonal antibody, further includes having immunocompetent antibody fragment, such as Fab or
(Fab’)2Segment；Heavy chain of antibody；Antibody light chain.
As used herein, term " heavy chain variable region " and " VH" be used interchangeably.
As used herein, term " variable region " and " complementary determining region (complementarity determining
Region, CDR) " it is used interchangeably.
In a preferred embodiment of the invention, the heavy chain variable region of the antibody includes that following three complementations are determined
Determine area CDR:
CDR1, amino acid sequence are GWSLTGPG (SEQ ID NO:4), and coding nucleotide sequence is,
gggtggtcattaaccggccccggt(SEQ ID NO.:3)；
CDR2, amino acid sequence are IYGDGST (SEQ ID NO.:6), and coding nucleotide sequence is,
atatatggtgatggaagcaca(SEQ ID NO.:5)；
CDR3, amino acid sequence are AYEYAMDW (SEQ ID NO.:8), and coding nucleotide sequence is,
gcctatgagtatgctatggactgc(SEQ ID NO.:7)。
In another preferred example, the amino acid sequence of the heavy chain variable region are as follows:
DVQLQESGPGLVAPSQSLSITCTVSGWSLTGPGVNWVRQPPGKGLEWLGMIYGDGSTDYNSALKSRLS
ISKDNSKSQVFLKINSLQTDDTARYYCAYEYAMDWWGQGTSVTVSS(SEQ ID NO.:10)；
Its coding nucleotide sequence are as follows:
gatgtgcagcttcaggagtcgggacctggcctggtggcgccctcacagagcctgtccatcacatgcac
cgtctcagggtggtcattaaccggccccggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctg
ggaatgatatatggtgatggaagcacagactataattcagctctcaaatccagactgagcatcagcaaggacaact
ccaagagccaagttttcttaaaaataaacagtctgcaaactgatgacacagccaggtactactgtgcctatgagta
tgctatggactgctggggtcaaggaacctcagtcaccgtctcctcaa(SEQ ID NO.:9)。
In a preferred embodiment of the invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain
Constant region, the heavy chain constant region can be source of mouse or source of people.
As used herein, term " light chain variable region " and " VL" be used interchangeably.
In a preferred embodiment of the invention, the light chain variable region of antibody according to the present invention has and is selected from
The complementary determining region CDR of the following group:
CDR1 ', amino acid sequence are WSVSTSGKSY (SEQ ID NO:14), and coding nucleotide sequence is,
tggagtgtcagtacatctggcaagagttat(SEQ ID NO.:13)；
CDR2 ', amino acid sequence are LLS (SEQ ID NO:16), and coding nucleotide sequence is cttctgtcc
(SEQ ID NO.:15)
CDR3 ', amino acid sequence are YHIRDLT (SEQ ID NO:18), and coding nucleotide sequence is,
taccacattagggaccttacacg(SEQ ID NO.:17)
In another preferred example, the amino acid sequence of the light chain variable region are as follows:
DIVMTQSPASLAVSLGQRATISYRASWSVSTSGKSYMHWNQQKPGQPPRLLIYLLSNLESGVPARFSG
SGSGTDFTLNIHPVEEEDAATYYCYHIRDLT (SEQ ID NO.:20),
Its coding nucleotide sequence are as follows:
gacattgtgatgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcata
cagggccagctggagtgtcagtacatctggcaagagttatatgcactggaaccaacagaaaccaggacagccaccc
agactcctcatctatcttctgtccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacag
acttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgttaccacattagggaccttac
acg(SEQ ID NO.:19)。
In a preferred embodiment of the invention, the light chain of the antibody includes above-mentioned light chain variable region and light chain
Constant region, the constant region of light chain can be source of mouse or source of people.
In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all
Refer to specific binding PD-1 albumen antibody, such as with heavy chain variable region (such as amino acid sequence of SEQ ID NO.:10) and/
Or the albumen or polypeptide of light chain variable region (such as amino acid sequence of SEQ ID NO.:20).They can be with or without starting first
Methyllanthionine.
In another preferred example, the antibody is mouse or the people's mouse chimeric mAb of anti-PD-1 albumen, its weight
Chain constant region and/or constant region of light chain can be the heavy chain constant region or constant region of light chain of humanization.It is highly preferred that the people
The heavy chain constant region or constant region of light chain in source are the heavy chain constant region or constant region of light chain of human IgG1, IgG2 etc..
The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention
It is (i.e. immune even including any protein or protein conjugate and fusion expressed product with heavy chain and light chain containing variable region
Join object and fusion expressed product), as long as the variable region is identical as the variable region of the heavy chain of antibody of the present invention and light chain or at least
90% homology, preferably at least 95% homology.
Generally, the antigenic binding property of antibody can be described by being located at 3 specific regions of heavy chain and light chain variable region,
The section is partitioned into 4 frame areas (FR) by referred to as Variable Area (CDR), and the amino acid sequence of 4 FR is relatively conservative,
Association reaction is not participated in directly.These CDR form cyclic structure, the β-pleated sheet formed by FR therebetween phase on space structure
Mutually close, the CDR on CDR and corresponding light chain on heavy chain constitutes the antigen binding site of antibody.It can be by comparing similar
The amino acid sequence of the antibody of type determines be which Amino acid profile FR or CDR region domain.
The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because at least partly relating in them
And combine antigen.Therefore, the present invention includes those molecules with the monoclonal antibody light chain with CDR and heavy chain variable region, only
Want its CDR and CDR that identifies herein have 90% or more (preferably 95% or more, most preferably 98% or more) homology.
The present invention not only includes complete monoclonal antibody, further include have immunocompetent antibody segment or antibody with
The fusion protein that other sequences are formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.
As used herein, term " segment ", " derivative " refer to that be kept substantially antibody of the present invention identical with " analog "
Biological function or active polypeptide.Polypeptide fragment of the invention, derivative or the like, which can be (i), one or more
Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino
Sour residue, which can be, may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues
The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second
Glycol) fusion is formed by polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (as before
Lead sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or merge egg with what 6His label was formed
It is white).According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.
Antibody of the present invention refers to polypeptide with PD-1 protein binding activity, including above-mentioned CDR region.The term further includes tool
There is the variant form of polypeptide with antibody identical function of the present invention, comprising above-mentioned CDR region.These variant forms include (but simultaneously
It is not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid
Missing, insertion and/or substitution, and it is one or several (usually within 20, preferably in C-terminal and/or N-terminal addition
Ground is within 10, is more preferably within 5) amino acid.For example, in the art, with amino acid similar in performance
When being replaced, the function of protein is not usually changed.For another example, one or several ammonia are added in C-terminal and/or N-terminal
Base acid will not generally also change the function of protein.The term further includes the active fragment and reactive derivative of antibody of the present invention.
The variant form of the polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induction
Mutant, the encoded albumen of DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency, with
And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.
The present invention also provides other polypeptides, such as the fusion protein comprising human antibody or its segment.In addition to almost overall length
Outside polypeptide, the invention also includes the segments of antibody of the present invention.In general, the segment has at least about 50 companies of antibody of the present invention
Continue amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about
100 continuous amino acids.
In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention,
There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties
Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and generate.
Table 1
The present invention also provides encoding such antibodies or the polynucleotide molecules of its segment or its fusion protein.Of the invention
Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can
To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with
Coding region sequence shown in SEQ ID NO.:3,5,7,9,13,15,17,19 is identical or the variant of degeneracy.Such as this paper institute
With " variant of degeneracy " refers to that coding has amino acid sequence identical with polypeptide of the invention, but and SEQ in the present invention
The differentiated nucleic acid sequence of coding region sequence shown in ID NO.:3,5,7,9,13,15,17,19.
The polynucleotides for encoding mature polypeptide of the invention include: the coded sequence of an encoding mature polypeptide；Mature polypeptide
Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume
Code sequence.
The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include
The polynucleotides of additional code and/or non-coding sequence.
The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least
70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more
The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature
Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization
Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90% with
On, more preferably 95% or more when, just hybridizes.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:10
And/or mature polypeptide shown in SEQ ID NO.:20 has identical biological function and activity.
The nucleotide full length sequence of antibody of the invention or its segment can usually use PCR amplification method, recombination method or artificial
Synthetic method obtains.A kind of feasible method is that manually synthetic method synthesizes related sequence, especially fragment length
When shorter.In general, being then attached the very long segment of available sequence again by first synthesizing multiple small fragments.In addition, may be used also
The coded sequence of heavy chain and expression label (such as 6Hi s) are fused together, fusion protein is formed.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
Biomolecule (nucleic acid, albumen etc.) according to the present invention includes existing biomolecule in a separate form.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely
Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and
In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This
A little carriers can be used for converting host cell appropriate, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, streptomyces；The bacterium of salmonella typhimurium
Cell；Fungal cell's such as yeast；The insect cell of drosophila S2 or Sf9；CHO, COS7, zooblast of 293 cells etc..
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original
When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is using MgCl2.If desired, conversion can also use the side of electroporation
Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used
Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth
It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction)
Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as
Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used
Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
Antibody of the invention can be used alone, can also be with detectable marker (for diagnostic purpose), therapeutic agent, PK (egg
White kinases) combination of modified part or any the above substance combines or coupling.
Detectable marker for diagnostic purposes includes but is not limited to: fluorescence or luminous marker, radioactively labelled substance,
MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product
Enzyme.
Can in conjunction with antibody of the present invention or coupling therapeutic agent include but is not limited to: 1. radionuclides (Koppe etc.,
2005, (Cancer metastasis reviews) 24,539 is commented in metastasis of cancer)；2. biology poison (Chaudhary etc., 1989,
Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and
Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding
(PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and
Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. gold nano
(Lapotko etc., 2005, cancer communicates (Cancer letters) 239,36 to particle/nanometer rods；Huang etc., 2006, it is Americanized
Association magazine (Journal of the American Chemical Society) 128,2115)；5. virion (Peng
Deng 2004, gene therapy (Gene therapy) 11,1234)；6. liposome (Mamot etc., 2005, cancer research (Cancer
Research) 65,11631)；7. magnetic nanosphere；8. pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or xenyl hydrolysis
Enzyme-sample protein (BPHL))；10. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
The present invention also provides a kind of compositions.In preference, the composition is pharmaceutical composition, it contains
The antibody stated or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, these substances can be prepared
In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is usually about 5-8, and preferably pH is about
6-8, although pH value can be varied with the property and illness to be treated for being formulated substance.Prepared pharmaceutical composition
It can be administered by conventional route, including (but being not limited to): tumor is interior, peritonaeum is interior, intravenous or local administration.
Pharmaceutical composition of the invention can be directly used for combining PD-1 protein molecular, thus can be used for preventing and treating swollen
Tumor.In addition, also can be used simultaneously other therapeutic agents.
Pharmaceutical composition of the invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more
Good ground 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) and pharmaceutically acceptable carrier or tax of the present invention
Shape agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Drug system
Agent should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain
There are glucose and the aqueous solution of other adjuvants to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile
Under the conditions of manufacture.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg of about 1 microgram/kg body weight-daily
Weight.In addition, polypeptide of the invention can be also used together with other therapeutic agents.
It is that the immune conjugate of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety
Effective quantity typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg weight, preferably
The ground dosage is about 1 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration route, disease
The factors such as people's health status, within the scope of these are all skilled practitioners technical ability.
Hybridoma cell strain
The present invention also provides can produce the present invention for the hybridoma cell strain of PD-1 protein monoclonal antibody；It is preferred that
, the present invention provides the hybridoma cell strains for PD-1 protein monoclonal antibody of high-titer.
After obtaining the hybridoma for producing PD-1 protein monoclonal antibody of the invention, those skilled in the art can be square
Just antibody is prepared using the hybridoma cell strain.In addition, those skilled in the art can also easily know of the invention resist
The structure (such as heavy chain variable region and light chain variable region of antibody) of body, then can prepare list of the invention by recombination method
Clonal antibody.
The preparation of monoclonal antibody
Antibody of the invention can be prepared by various technologies known to those skilled in the art.For example, this hair
Bright antigen can be applied to animal to induce the generation of monoclonal antibody.For monoclonal antibody, can using hybridoma technology come
Preparation is (see Kohler et al., Nature 256；495,1975；Kohler et al., Eur.J.Immunol.6:511,1976；
Kohler et al., Eur.J.Immunol.6:292,1976；Hammerling et al., In Monoclonal Antibodies
And T Cell Hybridomas, Elsevier, N.Y., 1981) or recombinant DNA method (U.S. Patent number 4,816,567) can be used
Preparation.
Representative myeloma cell is effective integration, the stabilization Gao Shui for supporting by the antibody produced cell of selection antibody
It shows no increases in output and gives birth to and to those of culture medium (HAT medium matrix) sensitivity myeloma cell, including myeloma cell strain, such as mouse
The myeloma cell strain of class (is purchased from Salk including the myeloma cell strain derived from MOPC-21 and MPC-11 mouse tumor
Institute Cell Distribution Center, Santiago, California, the U.S.) and SP-2, NZ0 or
X63-Ag8-653 cell (is purchased from American Type Culture Collection, Luo Keweier, Maryland, the U.S.).
Human myeloma and mouse-human heteromyeloma's cell strain be also described for generate human monoclonal antibodies [Kozbor,
J.Immunol., (1984) 133:3001；Brodeur etc., the production technology and application (Monoclonal of monoclonal antibody
Antibodies Production Techniques and Applications), 51-63 pages (Marcel Dekker,
Inc., New York, 1987)].
Growth of Hybridoma Cell is analyzed in culture medium therein to detect and have the monoclonal of required specificity anti-
The generation of body, e.g., by external binding analysis for example, Enzyme Linked Immunoadsorbent Assay (ELISA) or radiommunoassay (RIA).
The position for expressing the cell of antibody can be detected with FACS.Then, hybridoma clone can be formed by limiting dilution procedures
It is subcloned (subcloned), and grows (Goding, monoclonal antibody (Monoclonal by standard method
Antibodies): principle and practicing (Principles and Practice), Academic Press (1986) 59-103
Page).The suitable culture medium used to reach this purpose includes, for example, DMEM or RPMI-1640 culture medium.In addition,
Hybridoma can be grown as ascites tumor in animal body.
Pass through conventional immunoglobulin purification from culture medium, ascites or serum by the monoclonal antibody of subclone secretion
Technique is suitably separated, these purifying process are for example, Protein A-agarose method (protein A-Sepharose), hydroxyl
Base apatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The present invention provides a kind of monoclonal antibodies for PD-1 albumen, anti-especially for the monoclonal of PD-1 albumen
Body.In of the invention one preferred scheme, monoclonal antibody is using culture hybridoma method preparation.Take hybridoma thin
The supernatant of born of the same parents' culture, slightly proposes IgG through saturated ammonium sulphate method, then by the antibody slightly mentioned through affinity column (Protein
G-Sephrose it) purifies.
In the preferred scheme of of the invention one, monoclonal antibody is using Balb/C mouse ascites production monoclonal antibody
Method preparation.About hybridoma is inoculated into the mouse peritoneal of sensitization, visible abdomen obviously swells within 10 days or so.Extract abdomen
Water is pure through affinity column (Protein G-Sephrose) after saturated ammonium sulphate method slightly mentions, then by the antibody slightly mentioned
Change.
The immunoglobulin of label
In a preference of the invention, the immunoglobulin has detectable marker.More preferably, the mark
Note object is selected from the group: colloid gold label object, colored labels or fluorescent marker.
Method known to those skilled in the art progress can be used in colloid gold label.In a preferred scheme of the invention
In, the monoclonal antibody colloid gold label of PD-1 albumen obtains the monoclonal antibody of colloid gold label.
PD-1 protein monoclonal antibody of the invention has specificity well, very high potency.
Detection plate and its material
Detection plate material commonly used in the art can be used in detection plate of the invention, using conventional detection plate preparation method system
At.
The present invention detects the plate for detecting immunity of PD-1 albumen, and the support plate including test-strips and support test-strips can such as adopt
With PVC polyester offset plate etc.；The test-strips are by filter sample paper, chromatographic material, nitrocellulose filter and blotting paper successively overlap joint group
At overlapping part can be fixedly connected using conventional method, such as adhesive tape；Wherein: the pre-coated colloid gold label of chromatographic material
Or the PD-1 protein monoclonal antibody or polyclonal antibody of coloured label, preferably resisted by the PD-1 protein monoclonal of colloid gold label
Body adsorbs detection line and nature controlling line on nitrocellulose filter；
In a preferred scheme: the PD-1 protein monoclonal antibody of pre-coated colloid gold label is to adopt on chromatographic material
Pre-coated, package amount 50 is carried out with the PD-1 protein monoclonal antibody solution that concentration is 0.5-1.5mg/ml colloid gold label
μl/cm2；Preferred concentration is 0.5 or 1.5mg/ml, 50 μ l/cm2；
Detection method and result judgement
Detection plate is laid flat, by sample drop on filter sample paper, tomographic results are observed in sample about 120 μ l, 3~5min.According to
The fringe position of appearance carrys out judging result.
Negative: there is apparent colour band in quality control region, detection zone, are shown as negative；
It is positive: only obvious colour band occur in quality control region, and be shown as positive without colour band in detection zone；
Invalid: quality control region, detection zone are without any colour band or colour band do not occur in quality control region and colour band occur in detection zone, table
Bright detection method mistake or detection plate go bad or failure, and Ying Chongxin exchanges detection plate detection for.
Method and sample
The present invention relates in the method for cell and/or the pattern detection tumour of histolysis.This method step is big
It causes as follows: obtaining cell and/or tissue samples；In the medium by sample dissolution；Detection PD-1 egg in the sample of the dissolution
White level.Sample used in the method for the present invention can be any sample including cell being present in cell-preservation liquid,
As used in liquid basal cell detection method.
Kit
The present invention also provides a kind of reagents for referring to and containing antibody (or its segment) or detection plate of the invention of the invention
Box, in a preference of the invention, the kit further includes container, operation instructions, buffer etc..
The present invention is further designed for the detection kit of detection PD-1 level, which includes identification PD-1 albumen
Antibody detect required common reagent and buffer for dissolving the cracking medium of sample, such as various buffers, detection mark
Note, detection substrate etc..The detection kit can be in-vitro diagnosis device.
Main advantages of the present invention are:
(1) antibody of the anti-PD-1 albumen of the present invention, specificity is high, and affinity is strong, has good thermal stability, and can
Largely to prepare, monoclonal antibody quality is easy to control.
(2) antibody of the anti-PD-1 albumen of the present invention is capable of the secretion of effective stimulus T cell IFN-γ.
(3) antibody of the anti-PD-1 albumen of the present invention is capable of the secretion of effective stimulus T cell TNF-α.
(4) antibody of the anti-PD-1 albumen of the present invention can activate CD8+And CD4+T cell and other expression PD-1 albumen
Cell.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is calculated by weight.Experimental material used in the embodiment of the present invention can obtain unless otherwise specified from commercially available channel.
The preparation of 1 monoclonal antibody of embodiment
1.1 animal immune
6-8 weeks female BAl BIc/C mice is taken, immunogene is DNA recombinant expression PD-1 protein 29 3T cell, amino acid sequence
It is as follows:
DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC
RFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTL
VVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQ
TEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO.:1), immune programme is shown in Table 2.Every time
Mouse docking blood sampling is carried out before immune, it is indirect as detection antigen coat using DNA recombinant expression PD-1 protein 29 3T cell
ELISA method detection mice serum potency.When immune serum potency titre reaches maximum and no longer raising extracting spleen cell into
Row fusion.
2 mouse immune program of table
| Immunization time | Immunizing dose (a cell) | Immunization route |
| Head exempts from (the 1st day) | 1×106/0.1mL | Tail vein injection |
| Two exempt from (the 14th day) | 1×106/0.1mL | Tail vein injection |
| Three exempt from (the 28th day) | 1×106/0.1ml | Tail vein injection |
| Four exempt from (the 42nd day) | 1×106/0.1ml | Tail vein injection |
1.2 cell fusions and culture
The splenocyte for collecting immune mouse according to a conventional method is merged with myeloma cell SP2/0, and fusion ratio spleen is thin
Born of the same parents: SP2/0=5:1, fused cell are sub-packed in 96 well culture plates, are placed in 37 DEG C, 5%CO2It is selectively cultivated in constant incubator.
It is changed full liquid 3 times after fusion with HAT culture medium；When hybridoma covers with a field of microscope, ELISA detection is carried out.
The screening of 1.3 positive hybridomas
When detection, screened using indirect ELISA: antigen selects PD-1-Fc fusion protein, and 2 μ g/ml wrapper sheets, 4 DEG C are overnight,
After PBST washing pats dry, the closing of 5% skimmed milk power is added, 37 DEG C of 2h or 4 DEG C of effects overnight, after PBST washing pats dry, are added miscellaneous
It hands over tumor supernatant and positive (P), negative (N) and blank control (blank is directly added into ELIAS secondary antibody), 37 DEG C of reactions 1h, PBST is set
Washing adds 37 DEG C of sheep anti mouse-HRP secondary antibody (Sigma A2554) (1:10000) reaction, 45~60min after patting dry, PBST is washed
It washs and pats dry rear TMB colour developing and 2M H2SO4It terminates.
It is positive hole, and rechecked that ELISA the selection result, which is greater than 0.6 with OD value,.By being continuously detected as twice
After the positive, by the positive hole, cell is expanded, and is frozen and is subcloned in time.
The foundation of 1.4 hybridoma cell lines
All it is positive hole for 2 screenings, is subcloned using limiting dilution assay, takes one piece of 96 hole cell culture
150 μ l HAT culture solutions are added in every hole in plate；Suspension is gently blown and beaten to the positive hole that need to be subcloned, draws 100 μ l cell suspensions
It is added in 96 porocyte plates, the doubling dilution since the first hole.There are about the hole of 100 cells and additions in cell count selection hole
Into the loading slot containing 6ml HAT culture solution, it is added to by 100 holes μ l/ in the cell plates of paving feeder layer, adds first three columns；It mends again
Add HAT culture medium 3ml, 100 holes μ l/ are added in the cell plates of paving feeder layer, add in three column；Add HAT culture medium 5ml again, 100
The hole μ l/ is added in the cell plates of paving feeder layer, six column after adding.When calculating the positive rate of every block of plate and reaching 100%, obtain stablizing thin
Born of the same parents' strain PD1-1375.It is transferred in tissue culture plate, expands and cultivate and largely freeze.
The mass production (ascites preparation) of 1.5 monoclonal antibodies
The BALB/C female mice of mass propgation hybridoma (PD1-1375), 8-10 week old is caused with atoleine in advance
It is quick, 1x10 is then injected intraperitoneally6Hybridoma/only, it after mouse web portion obviously expands to a certain extent, uses within about 10 days or so
No. 9 syringe needles extract ascites.The ascites of harvest PD-1-Fc fusion protein, which is coated with, reaches 1:10 by ELISA detection titer of ascites,
000 or more.Ascites fluid is after removal fibrin and processing of saltouing, with the purifying of Prote in G affinity column chromatography method.Collect egg
White peak efflux, with ultraviolet specrophotometer OD260 is used after phosphate buffer (PBS) dialysis, 280 measure antibody protein concentration
For 0.7-1.5mg/ml, indirect ELISA testing result shows: the potency of the monoclonal antibody of purifying is in 1:10000 or more.
It is sequenced using monoclonal antibody of the conventional method to acquisition, sequencing result is as shown in Figure 2.
Affinity analysis of 2 PD1-1375 of embodiment to soluble human PD-1
Soluble antigen albumen PD1-Fc is dissolved in PBS with the concentration of 5 μ g/ml and carries out antigen coat；
The PD1-1375 of various concentration gradient carries out antibody incubation；
Sheep anti-mouse igg-HRP (ProSci) is dissolved in dilution (1%BSA+0.05%PBST20) with 1:5000, is carried out
Secondary antibody is incubated for, colour developing；Experimental result is as shown in Figure 1, obtain the EC50=29.4ng/mL of PD1-1375 antibody.
PD1-Fc is made of the extracellular domain of the PD1 molecule of people plus the Fc segment of human IgG1, and amino acid sequence is such as
Under:
DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC
RFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTL
VVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQ
TEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.:2)
The detection of 3 affinity of embodiment
Chip coating: recombinant antigen PD-1-Fc to 20 μ g/ml is diluted with pH5.5,20mM NaAc buffer.According to amino
Sour coupling reagent kit operation instruction is coupled, and first activates CM5 chip surface with EDC-NHS, then coupling recombination PD-1 to CM5
Chip, coupling level-preset value are 2000RU, and final coupling amount is 2639.3RU.
KD test: PD1-1375 (anti-PD-1ab) sample is diluted with HBS-EP buffer, difference dilute sample concentration is extremely
500nM,250nM,125nM,62.5nM,31.25nM,15.625nM.Setting sample injection time be 3min, Dissociation time 30min, then
Raw buffer is pH2.0Gly-HCl, the reproduction time 30s of 20mM, and repeating sample introduction sample concentration is 100mM.
Examination with computer is carried out according to the protocol of BiaCoreX100control soft, testing result shows the present invention
The K of monoclonal antibodyD=0.34nM.
The compatibility of table 3 and known PD-1 antibody compares
By comparing with known PD-1 antibody, antibody compatibility of the present invention is significantly better than existing PD-1 antibody.Display
Monoclonal antibody of the invention has great exploitation patent medicine potentiality.
4 PD1-1375 of embodiment and PD-1 competitive ELISA
Coating: PD-L1-Fc is dissolved in PBS with the concentration of 5 μ g/ml and carries out antigen coat；Plate hole is added in every 100 μ L of hole
In, coating is overnight.
Closing: coated plate is washed 2 times with cleaning solution, and 120 μ L confining liquids are added in every hole, closing a period of time, is taken
Out, it dries spare.
Competitive reaction: 1 μ of PD1-1375 solution and 50 μ L concentration of 50 μ L series of concentrations is added in every hole in the plate prepared
The PD-1-biotin solution of g/ml is incubated for a period of time.
Board-washing: it is washed 5 times with cleaning solution.
Chromogenic reaction: 0.05%Streptavidin-HRP (Sigma) 100 μ L is added in every hole, is incubated for a period of time.
Terminate: terminate liquid is added in every hole, and the light absorption value in each hole is measured in microplate reader.
Experimental result is as shown in figure 3, measure EC50=2850ng/mL.
5 PD1-1375 of embodiment and PDL1 competitive ELISA
(1) be coated with: PD-1-Fc is dissolved in PBS with the concentration of 5 μ g/ml and carries out antigen coat；Plate is added in every 100 μ L of hole
Kong Zhong, coating is overnight.
(2) it closes: coated plate is washed 2 times with cleaning solution, 120 μ L confining liquids are added in every hole, a period of time is closed,
It takes out, dries spare.
(3) competitive reaction: the PD1-1375 solution and 50 μ L of 50 μ L series of concentrations of every hole addition are dense in the plate prepared
The PD-L1-biotin solution of 1 μ g/ml is spent, a period of time is incubated for.
(4) it board-washing: is washed 5 times with cleaning solution.
(5) chromogenic reaction: 0.05%Streptavidin-HRP (Sigma) 100 μ L is added in every hole, is incubated for a period of time.
(6) terminate: terminate liquid is added in every hole, and the light absorption value in each hole is measured in microplate reader.
Experimental result is as shown in Figure 4: EC50=1020ng/mL
6 PD1-1375 of embodiment activates PBMC experiment
CD3 antibody (being purchased from eBioc ience company) is dissolved in PBS with the concentration of 3 μ g/ml is coated with 96 orifice plates；Every hole
It is added 5 × 105PBMC is incubated for；The PD1-1375 that 100 μ L, 20 μ g/ml is added in every hole is incubated for 4 days；Supernatant is collected to try by ELISA
Agent box detects IFN-γ secretion.
Negative control: incoherent IgG.
Positive control: the blocking antibody (MedImmune) of PDL1.
Experimental result is as shown in Figure 5: the IFN-γ of PD1-1375 group secretion is significantly higher than control group.
7 DSC of embodiment detects PD1-1375 thermal stability
PD1-1375 is dissolved in PBS with the concentration of 2mg/mL；105 DEG C are warming up to from the speed of 1 DEG C/min from 15 DEG C.
Two peaks should be CH2, the Tm value of Fab, CH3 respectively.From meeting from height, the Tm value and CH2 of Fab is substantially overlapping.
As a result as shown in Figure 6, it can be seen that the antibody is with good stability.
ELISA detection activity, discovery variation are no more than +/- 5% to 4 DEG C of storage PD1-1375 after two months, therefore at this
Under part, which kept stablizing in 2 months.
Embodiment 8 blocks the proliferation of the PD-1 increase antitumor T cell of PD-1+
Experimental method:
(1) recovery freezes the single cell suspension of follicular lymphoma patient, removes dead cell, uses the micro- magnetic bead of CD20/19
(Miltenyi biotech firm, Germany), is enriched with the B cell of the bis- positives of CD20/19, i.e. tumour cell
(2) method T cell enrichment is selected with negative simultaneously, T cell is marked into CFSE (Invitrogen/Life Technologies
Company)
(3) by autologous tumor cells and after self T-cell mixed culture (1:1) five days, supernatant is collected, cell is cleaned
Afterwards, the antibody for putting on anti-human CD4/8/PD1, using flow cytometer observation CFSE in CD4/CD8/PD1+/- T cell
Dilute situation.
Experimental result is as shown in fig. 7, anti-PD1 antibody of the invention can dramatically increase CD4+PD1+And CD8+PD1+Double sun
The proliferation of the TIL (tumor infiltrating lymphocyte) of property.
The cytokine activation of 9 TIL of embodiment
Experimental method: being shown in the experimental method of embodiment 8, is tried using Bio-Plex Pro Human Th1/Th2 cell factor
After agent box (Bio Rad), the supernatant that above-described embodiment 8 is collected and the mixing of fluorescent magnetic bead, Bio-Plex array is used
Reader reads fluorescence data.
Experimental results are shown in figure 8, and PD1 antibody of the invention can significantly increase the cell factor of tumour patient TIL
(IFN-γ and TNF-α) generates, and activates T cell function.
10 lymthoma transplantation model of embodiment detects antibody activity
RL lymthoma, a kind of lymphoma cell line being transformed from people, purchased from purchased from ATCC.Subcutaneous transplantation arrives
SCID mice.
Ramos lymthoma, a kind of Burkitt lymphoma cell line from people, purchased from purchased from ATCC.Subcutaneous transplantation arrives
Nude mice.
Lymthoma transplantation model detects PD-1 antibody activity:
(1)1×106Lymphoma cell in logarithmic growth phase is subcutaneous through being injected into mouse.
After (2) 7 days, from 10 μ g α PD-1 antibody of tail vein injection.It is then primary every injection in 6 days, it injects 3 times.Control makes
With the PBS of same volume.
(3) it calculates tumor size daily since the 8th day, observes the death condition of mouse.
Experimental result is as shown in figure 9, RL-SCID model is indicated by the solid line；Ramos- nude mice model is indicated with perforated line, is infused
The tumour growth for penetrating two groups of mouse of α PD-1 antibody is substantially less than injection PBS and organizes mouse together.Therefore, PD1 antibody of the invention can
Significantly to inhibit FL tumour growth (P < 0.01), extend the life cycle (P < 0.01) of animal model.
Lymthoma transplantation model detects PD-1 antibody+rituximab combination medication activity:
Each tumor model is divided into three groups: PBS group, Rituximab group (200 μ g/mouse are injected intraperitoneally in RTX), benefit
Appropriate former times monoclonal antibody+PD1-1375 group (10 μ g/mouse of intravenous injection).Rituximab is injected in Day1 inoculated tumour, Day10,27,34,41
PD1-1375 is injected in monoclonal antibody (Rituximab), Day13,20,27,34,41.
Experimental result is as shown in Figure 10, injects the survival rate of the RL-SCID mouse of α PD-1+RTX antibody better than exclusive use
RTX, the two are better than control group.The survival rate for injecting the Ramos- nude mice of α PD-1 antibody is better than using RTX, and the two is better than
Control group.Therefore, according to the two animal experimental models, PD1 antibody of the invention, in conjunction with Rituximab drug, Ke Yijin
One step inhibits RL tumour growth, extends the life cycle of animal model.
It discusses:
In follicular lymphoma TIL, PD1 expression is dramatically increased；In gastric cancer TIL, PD1 and Tim3 expression are dramatically increased.
The PD1 antibody that the present invention develops can be stimulated significantly in the tumor-infiltrated T lymphocyte of follicular lymphoma patient (TIL), PD1
The proliferation of positive CD4+TIL；The PD1 antibody can significantly increase FL TIL cell factor generate, including INF- γ and
TNF-α.Other cell factors also have the phenomenon that expression increases, comprising: IL-2, GM-CSF, IL-10, IL-5；It can also significantly pierce
Swash the generation of CD4+/CD8+T cell cytokine in gastric cancer TIL；In the test of lymthoma Transplanted tumor model, PD1 antibody can be with
It significantly inhibits the growth of tumour, improve survival time of mice.It is used in combination with Rituximab, the improvement to animal model life cycle
It is more significant；In vitro experiment, combined occlusion PD1 and Tim3 shows the significant killing ability to stomach cancer cell line；It is above-mentioned
Test result explanation, the PD1 antibody are one plant with the superior antibody for blocking performance.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (20)
1. a kind of antibody, which is characterized in that the antibody is the antibody of anti-PD-1 albumen, and the antibody includes
(1) heavy chain variable region, the heavy chain variable region include following three complementary determining region CDR:
CDR1 shown in SEQ ID NO:4,
CDR2 shown in SEQ ID NO:6, and
CDR3 shown in SEQ ID NO:8；With
(2) light chain variable region, the light chain variable region include following three complementary determining region CDR:
CDR1 ' shown in SEQ ID NO:14,
CDR2 ' shown in SEQ ID NO:16, and
CDR3 ' shown in SEQ ID NO:18.
2. antibody as described in claim 1, which is characterized in that the heavy chain variable region has ammonia shown in SEQ ID NO:10
Base acid sequence and the light chain variable region have amino acid sequence shown in SEQ ID NO:20.
3. antibody as described in claim 1, which is characterized in that the antibody includes: single-chain antibody or double-chain antibody.
4. antibody as described in claim 1, which is characterized in that the antibody is monoclonal antibody.
5. antibody as described in claim 1, which is characterized in that the antibody includes: chimeric antibody, source of mouse antibody or people
Source antibody.
6. a kind of recombinant protein, which is characterized in that the recombinant protein includes
(i) sequence of antibody as described in claim 1；And
(ii) sequence label of optional assistance expression and/or purifying.
7. a kind of polynucleotides, which is characterized in that it encodes polypeptide selected from the group below:
(1) antibody as described in claim 1；Or
(2) recombinant protein as claimed in claim 6.
8. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 7.
9. carrier as claimed in claim 8, which is characterized in that the carrier includes: bacterial plasmid, bacteriophage, zymogen
Grain, plant cell virus or mammalian cell virus.
10. carrier as claimed in claim 8, which is characterized in that the carrier is adenovirus or retrovirus.
11. a kind of genetically engineered host cell, which is characterized in that it contains carrier or genome according to any one of claims 8
In be integrated with polynucleotides as claimed in claim 7.
12. a kind of immune conjugate, which is characterized in that the immune conjugate contains:
(a) antibody as described in claim 1 or recombinant protein as claimed in claim 6；With
(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor or radionuclide.
13. immune conjugate as claimed in claim 12, which is characterized in that the detectable marker is enzyme.
14. a kind of pharmaceutical composition, which is characterized in that it contains:
(i) as described in claim 1 antibody, recombinant protein as claimed in claim 6 or exempt from as claimed in claim 12
Epidemic disease conjugate；And
(ii) pharmaceutically acceptable carrier.
15. pharmaceutical composition as claimed in claim 14, which is characterized in that the pharmaceutical composition is injection type.
16. antibody as described in claim 1, recombinant protein as claimed in claim 6 are exempted from as claimed in claim 12
The purposes of epidemic disease conjugate is used to prepare medicament, reagent, detection plate or kit；
The reagent, detection plate or kit are used for: PD-1 albumen in test sample；
The medicament is used to treat or prevent the tumour of expression PD-1 albumen.
17. purposes as claimed in claim 16, which is characterized in that the tumour includes: gastric cancer, lymthoma, liver cancer, white blood
On disease, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer or kidney
Adenoncus tumor.
18. purposes as claimed in claim 17, which is characterized in that the tumour is selected from: gastric cancer and follicular lymphoma.
19. purposes as claimed in claim 16, which is characterized in that the reagent include chip, coated antibody be immunized it is micro-
Grain.
20. a kind of preparation method of recombinant polypeptide, which is characterized in that this method includes:
(a) under conditions suitable for the expression, host cell described in claim 11 is cultivated；
(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in claim 1 or claim
Recombinant protein described in 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410613502.1A CN105622753B (en) | 2014-11-04 | 2014-11-04 | A kind of PD-1 monoclonal antibody and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410613502.1A CN105622753B (en) | 2014-11-04 | 2014-11-04 | A kind of PD-1 monoclonal antibody and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105622753A CN105622753A (en) | 2016-06-01 |
| CN105622753B true CN105622753B (en) | 2019-04-09 |
Family
ID=56038133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410613502.1A Active CN105622753B (en) | 2014-11-04 | 2014-11-04 | A kind of PD-1 monoclonal antibody and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105622753B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3322732A2 (en) | 2015-07-13 | 2018-05-23 | Cytomx Therapeutics Inc. | Anti-pd-1 antibodies, activatable anti-pd-1 antibodies, and methods of use thereof |
| EP3569616B1 (en) * | 2017-01-13 | 2021-09-29 | Taizhou Hanzhong Biopharmaceutics, Inc. | Monoclonal antibody against pd-1 and applications thereof |
| CN108341871A (en) * | 2017-01-24 | 2018-07-31 | 三生国健药业（上海）股份有限公司 | Anti- PD-1 monoclonal antibodies and its preparation method and application |
| BR112019018630A2 (en) * | 2017-03-09 | 2020-04-28 | Xiamen University | recombinant hsv virus, viral vector, host cell, method for obtaining the recombinant hsv virus, pharmaceutical composition, method for treating a tumor and use of the recombinant hsv virus |
| CN108948193B (en) * | 2017-05-18 | 2022-12-09 | 上海健信生物医药科技有限公司 | Antibody molecules directed against TIM-3, antigen binding fragments and medical uses thereof |
| CN110404066B (en) * | 2018-04-28 | 2022-06-17 | 齐鲁制药有限公司 | Monoclonal antibody preparation for resisting human PD-1, combined medicament and application thereof |
| CN108948203B (en) * | 2018-08-09 | 2019-06-04 | 南京鼓楼医院 | Anti- PD-1 monoclonal antibody and its preparation method and application |
| CN110885376B (en) * | 2018-09-11 | 2020-10-09 | 上海洛启生物医药技术有限公司 | anti-CD 47/CD20 bispecific antibodies and uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009024531A1 (en) * | 2007-08-17 | 2009-02-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating and diagnosing hematologic malignancies |
| EP2172219A1 (en) * | 2008-10-02 | 2010-04-07 | SNU R&DB Foundation | Anti-cancer agent comprising an iNKT ligand and anti-PD-1 antibody or anti-PD-L1 antibody |
| CN103242448A (en) * | 2013-05-27 | 2013-08-14 | 郑州大学 | Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof |
-
2014
- 2014-11-04 CN CN201410613502.1A patent/CN105622753B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009024531A1 (en) * | 2007-08-17 | 2009-02-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating and diagnosing hematologic malignancies |
| EP2172219A1 (en) * | 2008-10-02 | 2010-04-07 | SNU R&DB Foundation | Anti-cancer agent comprising an iNKT ligand and anti-PD-1 antibody or anti-PD-L1 antibody |
| CN103242448A (en) * | 2013-05-27 | 2013-08-14 | 郑州大学 | Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105622753A (en) | 2016-06-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105622753B (en) | A kind of PD-1 monoclonal antibody and its application | |
| US11155632B2 (en) | Anti-CD47 monoclonal antibody and use thereof | |
| US10556954B2 (en) | Anti-PD-L1 nanobody, coding sequence and use thereof | |
| JP6753946B2 (en) | Anti-human PD-1 humanized monoclonal antibody and application | |
| CN105461808B (en) | Monoclonal antibody and its application | |
| CN107840891A (en) | The anti-MSLN antibody of high-affinity and its application | |
| CN105837689B (en) | anti-CD19 monoclonal antibody and preparation method thereof | |
| CN109942712B (en) | anti-PD-L1/VEGF bifunctional antibody and application thereof | |
| WO2018050039A1 (en) | Novel anti-pd-1 nano-antibody and application thereof | |
| EP3858856A1 (en) | Anti-b7-h3 monoclonal antibody and use thereof in cell therapy | |
| CN107686520A (en) | Anti- PD L1 nano antibodies and its application | |
| CN109563165A (en) | Anti- CTLA-4 antibody | |
| CN109096396A (en) | A kind of anti-PD-L1 humanization nano antibody and its application | |
| JP2022548066A (en) | Anti-PD-L1 Single Domain Antibodies and Derivatives and Uses Thereof | |
| CN106188281B (en) | The preparation and application of anti-norovirus GII.4 type source of mouse monoclonal antibody | |
| CN102321173B (en) | Humanized macrophage inhibitory factor 1 monoclonal antibody and application thereof | |
| EP3882276A1 (en) | Bispecific antibody, preparation method therefor and application thereof | |
| CN109096401A (en) | Anti- Her2 nano antibody and its coded sequence and purposes | |
| CN109776678A (en) | A kind of humanization PD-L1 monoclonal antibody, preparation method and application | |
| CN107840889A (en) | The anti-CD123 antibody of high-affinity and its application | |
| CN110396129A (en) | Humanization CD19 antigen binding single-chain antibody and its Chimeric antigen receptor, immunocyte and application | |
| CN109265547A (en) | A kind of anti-cd 47 antibody and its application | |
| CN109929037A (en) | Conjugate and its application for programmed death ligand | |
| CN106188282B (en) | The preparation and application of anti-norovirus GI.1 type source of mouse monoclonal antibody | |
| CN104098698B (en) | A kind of anti-cd 3 antibodies and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |