CN109096396A - A kind of anti-PD-L1 humanization nano antibody and its application - Google Patents

Abstract

The present invention provides a kind of anti-PD-L1 humanization nano antibody and its applications.Specifically, the present invention provides a kind of nano antibodies of anti-albumen programmed death ligand 1 (PD-L1).Anti- PD-L1 nano antibody provided by the invention can effectively block the combination of PD-L1 and Programmed death 1 (PD-1), therefore can successfully block PD-L1 to the inhibiting effect of the T cell of expression PD-1.PD-L1 nano antibody of the present invention can be applied to the prevention, diagnosis, treatment of PD-L1 related disease.The present invention also provides the PD-L1 nano antibody sequence of humanization, the nano antibody after humanization still has the function of blocking PD-L1 in conjunction with PD-1, and has high-affinity and high specific, can be used as tumour immunity target spot antibody drug.

Description

A kind of anti-PD-L1 humanization nano antibody and its application

Technical field

The present invention relates to biomedical or biopharmaceutical technology, relate more specifically to anti-PD-L1 nano antibody and its Using.

Background technique

Cell-mediated cellullar immunologic response is the basis of antineoplastic immune, and the activation of T cell and proliferation need dual letter Number.I.e. in addition to needing T cell receptor (TCR) and expression in the MHC- antigenic peptide complexes knot on the surface antigen presenting cell (APCs) It closes outside the first signal generated；A variety of costimulatory molecules are also needed to participate in the second signal (costimulatory signal) provided.

The immune response initial stage, T cell under the collective effect of TCR the first signal and costimulatory signal provided, Reach its threshold of activation and be activated, generate effect and memory cells, plays immune defense function, and the activation causes A series of up-regulated expression of collaboration stimulation inhibition molecules, to maintain it to be in lasting state of activation or inhibit its overactivity Proliferation.If lacking the second signal of costimulatory molecules offer, it will cause the reactionless or specific immunity of T cell resistance to By in addition inducing cell enter apoptosis.On the contrary, if costimulatory signal reflection excessively, it is extremely sharp to may cause immunocyte It is living, to cause various autoimmune diseases.Therefore, immunocyte positivity obtained and negativity costimulatory signal need Relative equilibrium is reached so that immune response is opened and can appropriately be terminated in due course, resists exotic antigen in body and invade and prevent itself It plays an important role in the generation of immunity disease.

With extensive and in-depth research, costimulatory molecules have become new one of the hot spot of immunological investigation.Mediate association Molecule with stimulus signal mainly includes tumor necrosis factor/Tumor Necrosis Factor Receptors (TNF/TNFR) superfamily and immune ball Superfamily protein, such as the big superfamily of CD28/B7 two.These costimulatory molecules conduct letter in such a way that portable ligand interacts Number.

The 1/ programmed death factor of the programmed death factor, 1 ligand 1 (PD-1/PD-L1) is as CD28/B7 collaboration stimulation point The newcomer of sub- superfamily can mediate negativity costimulatory signal, can effectively inhibit T, B cell function and proliferation, subtract simultaneously The secretion of few cell factor IL-2, IL-10 and IFN-γ, the immunological regulation which participates in are exempted from tumour immunity, transplanting It is all of great significance in the research of the diseases such as epidemic disease, virus infection, autoimmunity.

PD-1 is one of immunoglobulin B7-CD28 family member, is made of extracellular fragment, hydrophobicity transmembrane region, intracellular section, Its intracellular section contains immunity receptor Tyrosine Inhibitory Motifs (immunoreceptor tyrosine-based inhibitory Motif, ITIM), immunity receptor tyrosine convert motif (immunoreceptor tyrosine-based switch motif,ITSM).Wherein, the activation of ITSM and T effector cell response activity are closely related.PD-1 can be expressed in activation CD4⁺T cell, CD8⁺In T cell, B cell, natural killer T cells, monocyte and Dendritic Cells.In addition, PD-1 is also expressed In regulatory T cells (regulatory T cell, Treg), and the proliferation of Treg cell can be promoted, inhibit immune response.

1 ligand 1 of the programmed death factor (programmed death 1ligand 1, PD-L1) is also known as CD274, is B7 Family member is the ligand of PD-1.PD-L1 belongs to I type transmembrane protein, totally 290 amino acid, and molecular weight is about 30-35KD.People PD-L1 molecule constitutes property and is expressed in the non-lymphoid tissues such as placenta, the heart, liver, lung, kidney, skeletal muscle and part hematopoietic cell, in chest Also there is appropriate expression in the lymphoid tissues such as gland, lymph node and spleen.In addition, PD-L1 is in lung cancer, liver cancer, breast cancer, oophoroma etc. Also there is expression, and cancerous tissue can also raise its expression by induction on cancerous tissue cell.In the body of health, PD-1/PD- The activation of L1 signal path can utmostly reduce the damage being immunoreacted to surrounding tissue, avoid that autoimmune disease occurs. However, PD-1/PD-L1 signal path altered activation tumor infiltative lymphocyte makes the reduction of T cell immunological effect, to mediate swollen Tumor immunologic escape promotes tumour growth.Expression of the PD-L1 in tumour and the cancer of the esophagus, cancer of pancreas and other types of cancer Survival rate decline is related, and highlighting the access can be used as new promising immunotherapy of tumors target spot, and many to obtain Experiment confirms.

Nano antibody is as a kind of novel small antibody fragments, by the natural heavy chain antibody heavy chain variable region of hunchbacked class (VHH) clone obtains.Nano antibody have excellent biological characteristics, molecular weight 12-15KD, be complete antibody ten/ One, possess complete antigen binding site again while overcoming natural antibody mole-cules amount big drawback, there is good group Penetrability is knitted, specificity is high, good water solubility.Because of its special structural property, the excellent of conventional antibodies and small-molecule drug has been had both It the defects of gesture, the almost ideal development cycle for overcoming conventional antibodies is long, and stability is lower, and preservation condition is harsh, is increasingly becoming Newly emerging force in Antybody therapy of new generation shows wide application prospect in immunodiagnosis and treatment

This research is by focus development in conjunction with PD-L1 high-affinity, and the nanometer that PD-L1/PD-1 can be blocked to combine is anti- PD-L1 nano antibody after body, especially humanization still maintains higher blocking activity.

Summary of the invention

The present invention provides the nano antibodies that a species specificity is directed to PD-L1, and can effectively block PD-L1's and PD-1 In conjunction with.

In the first aspect of the present invention, a kind of VHH chain of anti-PD-L1 nano antibody is provided, the VHH chain includes complementation Determine that area CDR, the complementary determining region CDR include CDR1 shown in SEQ ID NO.:5, shown in SEQ ID NO.:6 CDR3 shown in CDR2 and SEQ ID NO.:7 (or being made of described CDR1, CDR2 and CDR3).

In another preferred example, the PD-L1 is human PD-L 1.

In another preferred example, any one amino acid sequence further includes optionally past adding in above-mentioned amino acid sequence Add, lack, modify and/or replace at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain and PD-L1 high-affinity combines, blocks derived sequence of the PD-L1 in conjunction with PD-1.

In another preferred example, the VHH chain further includes framework region FR, and described CDR1, CDR2 and CDR3 are by VHH chain Framework region FR1, FR2, FR3 and FR4 are separated.

In another preferred example, the framework region FR by

(a) FR3 shown in FR2 shown in FR1 shown in SEQ ID NO.:1, SEQ ID NO.:2, SEQ ID NO.:3, The composition of FR4 shown in SEQ ID NO.:4；Or

(b) shown in FR2 shown in FR1 shown in SEQ ID NO.:10, SEQ ID NO.:11, SEQ ID NO.:12 The composition of FR4 shown in FR3, SEQ ID NO.:13.

In another preferred example, the framework region FR contains FR1, FR2, FR3 and FR4 (SEQ ID NO.:1- 4 or SEQ ID NO.:10-13).

In another preferred example, the amino acid sequence such as SEQ ID NO.:8 of the VHH chain of the anti-PD-L1 nano antibody Or shown in 14.

In addition, also providing a kind of heavy chain variable region of anti human PD-L 1 antibody, the heavy chain variable region includes three complementations Area CDR1, CDR2 and CDR3 are determined, and 3 CDR include CDR1 shown in SEQ ID NO.:5, shown in SEQ ID NO.:6 CDR2, CDR3 shown in SEQ ID NO.:7.

Second aspect of the present invention provides a kind of anti-PD-L1 nano antibody, it is the nano antibody for PD-L1 epitope, And the VHH chain with the amino acid sequence as shown in SEQ ID NO.:8 or SEQ ID NO.:14.

In another preferred example, the anti-PD-L1 nano antibody is fixed (or being carried on) and is carried in solid carrier or semisolid Body.

Third aspect present invention provides a kind of polynucleotides, the polynucleotide encoding protein selected from the group below: this Resist for anti-PD-L1 nanometers described in the VHH chain of anti-PD-L1 nano antibody or second aspect of the present invention described in invention first aspect Body.

In another preferred example, the polynucleotides have the nucleotide sequence as shown in SEQ ID NO.:9 or 15.

In another preferred example, the polynucleotides include DNA or RNA.

Fourth aspect present invention, provides a kind of expression vector, and the expression vector contains described in third aspect present invention Polynucleotides.

Fifth aspect present invention, provides a kind of host cell, and the host cell contains described in fourth aspect present invention Expression vector or its genome in be integrated with polynucleotides described in third aspect present invention.

In another preferred example, the host cell includes prokaryotic cell or eukaryocyte.

In another preferred example, the host cell is selected from the group: Escherichia coli, yeast cells.

Sixth aspect present invention provides a kind of method for generating anti-PD-L1 nano antibody, comprising steps of

(a) under conditions of being suitble to generate nano antibody, host cell described in fifth aspect present invention is cultivated, to obtain The culture of the anti-PD-L1 nano antibody must be contained；And

(b) the anti-PD-L1 nano antibody is separated or recycled from the culture.

In another preferred example, the method also includes steps: (c) further purifies and/or modifies and to obtain step (b) PD-L1 nano antibody.

In another preferred example, the anti-PD-L1 nano antibody has the amino as shown in SEQ ID NO.:8 or 14 Acid sequence.

Seventh aspect present invention provides a kind of polypeptide (preferably recombinant polypeptide), and the polypeptide contains:

(a) the VHH chain of anti-PD-L1 nano antibody as described in the first aspect of the invention or such as second aspect of the present invention institute The anti-PD-L1 nano antibody stated；

(b) optional, with the polypeptide in conjunction with or the modification marker selected from the group below that is coupled: chemical markers or biology Marker.

In another preferred example, the polypeptide is fused polypeptide.

In another preferred example, the chemical labeling is isotope, immunotoxin and/or chemicals.

In another preferred example, the biomarker is biotin, Avidin or enzyme label.

In another preferred example, the fusions fix (or being carried on) in solid carrier or semi-solid carrier.

Eighth aspect present invention provides a kind of immune conjugate, which contains:

(a) the VHH chain of anti-PD-L1 nano antibody as described in the first aspect of the invention or such as second aspect of the present invention institute The anti-PD-L1 nano antibody stated；With

(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or Enzyme.

In another preferred example, the coupling moiety is drug or toxin.

In another preferred example, the coupling moiety is detectable marker.

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..

In another preferred example, the immune conjugate contains: multivalence (such as divalent) is as described in the first aspect of the invention Anti- PD-L1 nano antibody VHH chain, anti-PD-L1 nano antibody as described in respect of the second aspect of the invention.

In another preferred example, the multivalence refers to, comprising multiple heavy in the amino acid sequence of the immune conjugate Anti- PD-L1 described in the VHH chain of multiple anti-PD-L1 nano antibody as described in the first aspect of the invention, second aspect of the present invention Nano antibody.

In another preferred example, the immune conjugate fixes (or being carried on) in solid carrier or semi-solid carrier.

Ninth aspect present invention provides the purposes of anti-PD-L1 nano antibody described in second aspect of the present invention, for making Standby (a) is used to detect the reagent of PD-L1 molecule；(b) for blocking preparation of the PD-L1 in conjunction with PD-1；(c) for treating tumour Drug.

In another preferred example, the detection includes flow cytometer detection, cellular immunofluorescence detection.

Tenth aspect present invention provides a kind of pharmaceutical composition, contains:

(i) the VHH chain of the anti-PD-L1 nano antibody of first aspect present invention or anti-PD- as described in respect of the second aspect of the invention Immune conjugate described in L1 nano antibody or eighth 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, lymph cancer, adrenal tumor or tumor of bladder.

On the one hand the present invention the tenth, provides the one or more of anti-PD-L1 nano antibody described in second aspect of the present invention Purposes:

(i) for detecting human PD-L 1 molecule；

(ii) it is used for flow cytometer detection；

(iii) it is detected for cellular immunofluorescence；

(iv) for treating tumour；

(v) it is used for diagnosing tumor.

In another preferred example, the purposes is non-diagnostic and non-treatment.

The twelfth aspect of the present invention, provides a kind of recombinant protein, and the recombinant protein includes

(i) sequence of heavy chain variable region VHH as described in the first aspect of the invention or as described in respect of the second aspect of the invention The sequence of nano antibody；And

(ii) sequence label of optional assistance expression and/or purifying.

In another preferred example, the sequence label includes 6His label and HA label.

In another preferred example, the recombinant protein specifically binds to PD-L1 albumen.

The 13rd aspect of the present invention, provides VHH chain as described in the first aspect of the invention, such as second aspect of the present invention institute The purposes of immune conjugate described in the nano antibody or eighth aspect present invention stated, they be used to prepare medicament, reagent, Detection plate or kit；

Wherein, the reagent, detection plate or kit are used for: PD-L1 albumen in test sample；

Wherein, the medicament is used to treat or prevent the tumour of expression PD-L1 (i.e. PD-L1 is positive).

In another preferred example, the tumour includes: that melanoma, gastric cancer, lymthoma, liver cancer, leukaemia, kidney are swollen Tumor, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer or adrenal tumor.

Fourteenth aspect of the present invention provides a kind of method of PD-L1 albumen in test sample, and the method includes steps It is rapid:

(1) sample is contacted with nano antibody described in second aspect of the present invention；

(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are PD-L1 eggs in sample It is white.

The fifteenth aspect of the present invention provides a kind of method for treating disease, and the method includes applying to the object of needs Immune conjugate described in the nano antibody described in second aspect of the present invention or eighth aspect present invention.

In another preferred example, the object includes mammal, such as people.

The 16th aspect of the present invention, provides a kind of framework region FR of anti-PD-L1 nano antibody VHH chain, the VHH chain Framework region the FR FR1 as shown in SEQ ID NO.:1, SEQ ID NO.:2 shown in FR2, shown in SEQ ID NO.:3 The composition of FR4 shown in FR3, SEQ ID NO.:4.

The 17th aspect of the present invention, provides a kind of kit, includes: (a) such as second party of the present invention in the kit Nano antibody described in face, immune conjugate described in fusions, eighth aspect present invention described in seventh aspect present invention, sheet Invent recombinant protein described in the 12nd aspect；(b) container.

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 the 9 kinds of PD-L1 nano antibody positive colony light absorption values filtered out using display technique of bacteriophage.

Fig. 2 is the barrier effect of FACS Preliminary Identification PD-L1 nano antibody.The result shows that specificity of the invention is directed to The nano antibody Nb43 of PD-L1 has good barrier effect to the combination of PD-L1 and PD-1.

Fig. 3 is FACS detection humanization nano antibody (Nb43) and humanization PD-L1 nano antibody Fc fusion protein (MY1909) combination of PD-L1/PD-1 is blocked.

Fig. 4 shows that humanization PD-L1 nano antibody Fc fusion protein (MY1909) can stimulate the activation of T cell.

Fig. 5 shows that humanization PD-L1 nano antibody Fc fusion protein (MY1909) examines the inhibitory activity of tumour growth It surveys.

Fig. 6 shows that MY1909 detects the inhibitory activity of tumour growth.

Specific embodiment

The present inventor is successfully obtained a kind of anti-PD-L1 nanometers and resisted by extensive and in-depth research by largely screening Body.The experimental results showed that one plant of PD-L1 nano antibody that the present invention obtains can effectively block it is mutual between PD-L1 and PD-1 Effect, and the PD-L1 nano antibody after the present inventor's humanization also effectively can block PD-L1 in conjunction with PD-1.It is basic herein On complete the present invention.

Specifically, camel is immunized using the PD-L1 extracellular fragment antigen protein of source of people in the present invention, and the nanometer for obtaining high quality is anti- Body phage display library.Then PD-L1 protein molecular is coupled on ELISA Plate, shows the correct space knot of PD-L1 albumen Structure, antigen in this format screen immune nano Antibody geometric mean titer (camel heavy chain antibody bacteriophage using display technique of bacteriophage Show gene pool), to obtain the nano antibody gene of PD-L1 specificity.This gene is gone in Escherichia coli again, thus Obtaining can be in E. coli and specific high nano antibody strain.

As used herein, term " nano antibody of the present invention ", " anti-PD-L1 nano antibody of the invention ", " PD- of the present invention L1 nano antibody " is used interchangeably, and refers both to specific recognition and the nano antibody for being incorporated into PD-L1 (including human PD-L 1).Especially The preferably amino acid sequence of VHH chain nano antibody as shown in SEQ ID NO.:8 or 14.

QVQLQESGGGSVQAGGSLRLSCAASRFTASMGWFRQAPGKEREGIATVSGAASTNYADSVRGRFTISKDNAKNTLYL QINSLKPEDTAVYYCAADDDYYAFLSRGARDFRYWGQGTQVTVSS(SEQ ID NO:8)

QVQLQESGGGLVQPGGSLRLSCAASRFTASMGWFRQAPGKGLEGIATVSGAASTNYADSVKGRFTISKDNSKNTLYL QINSLRDEDTAVYYCAADDDYYAFLSRGARDFRYWGQGTLVTVSS(SEQ ID NO:14)

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, term " single domain antibody ", " nano antibody " have the same meaning, and refer to monoclonal antibody heavy chain can Become area, constructs the single domain antibody being only made of a heavy chain variable region, it is the smallest antigen binding fragment with complete function Section.After the antibody for usually first obtaining natural deletions light chain and heavy chain constant region 1 (CH1), then the variable region of monoclonal antibody heavy chain, structure Build the single domain antibody being only made of a heavy chain variable region.

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.

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 are formed in conjunction with antibody of the invention or its segment Conjugate.The invention also includes the cell surface marker object in conjunction with the anti-PD-L1 protein antibodies or its segment or resist It is former.

As used herein, term " heavy chain variable region " and " V_H" 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 three complementary determining regions CDR1, CDR2 and CDR3.

In another preferred example, the complementary determining region CDR includes CDR1 shown in SEQ ID NO.:5, SEQ ID CDR3 shown in CDR2 and SEQ ID NO.:7 shown in NO.:6 (or being made of described CDR1, CDR2 and CDR3).

RFTASMG(SEQ ID NO:5)

VSGAAST(SEQ ID NO:6)

AADDDYYAFLSRGARDFRY(SEQ ID NO:7)

In another preferred example, the heavy chain variable region of the antibody further includes framework region FR, described CDR1, CDR2 and CDR3 is separated by framework region FR1, FR2, FR3 and FR4 of VHH chain.

In another preferred example, the framework region FR by

(a) FR3 shown in FR2 shown in FR1 shown in SEQ ID NO.:1, SEQ ID NO.:2, SEQ ID NO.:3, The composition of FR4 shown in SEQ ID NO.:4；Or

(b) shown in FR2 shown in FR1 shown in SEQ ID NO.:10, SEQ ID NO.:11, SEQ ID NO.:12 The composition of FR4 shown in FR3, SEQ ID NO.:13.

QVQLQESGGGSVQAGGSLRLSCAAS(SEQ ID NO:1)

WFRQAPGKEREGIAT(SEQ ID NO:2)

NYADSVRGRFTISKDNAKNTLYLQINSLKPEDTAVYYC(SEQ ID NO:3)

WGQGTQVTVSS(SEQ ID NO:4)

QVQLQESGGGLVQPGGSLRLSCAAS(SEQ ID NO:10)

WFRQAPGKGLEGIAT(SEQ ID NO:11)

NYADSVKGRFTISKDNSKNTLYLQINSLRDEDTAVYYC(SEQ ID NO:12)

WGQGTLVTVSS(SEQ ID NO:13)

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.

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 the polypeptide of specific binding PD-L1 albumen, such as albumen or polypeptide with heavy chain variable region.They can with or without rise Beginning methionine.

The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention Including with the heavy chain containing variable region any protein or protein conjugate and fusion expressed product (i.e. immune conjugate and Fusion expressed product), as long as the variable region is identical as the heavy chain variable region of antibody of the present invention 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 variable region, referred to as variable The section is partitioned into 4 frame areas (FR) by region (CDR), and the amino acid sequence of 4 FR is relatively conservative, does not join directly With association reaction.These CDR form cyclic structure, and the β-pleated sheet formed by FR therebetween is close to each other on space structure, weight The CDR on CDR and corresponding light chain on chain constitutes the antigen binding site of antibody.It can be by comparing the antibody of same type Amino acid sequence determines be which Amino acid profile FR or CDR region domain.

The variable region of the heavy chain of antibody of the present invention is particularly interesting, because being at least partly related to combining in them anti- It is former.Therefore, the present invention includes those molecules with antibody heavy chain variable region with CDR, as long as its CDR and identify herein CDR has the homology of 90% or more (preferably 95% or more, most preferably 98% or more).

The present invention not only includes complete antibody, further includes the segment or antibody and other sequences with immunocompetent antibody Arrange the fusion protein 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-L1 protein binding activity, including above-mentioned CDR region.The term further includes With with antibody identical function of the present invention, polypeptide comprising above-mentioned CDR region variant form.These variant forms include (but It is not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino Missing, insertion and/or the substitution of acid, and C-terminal and/or N-terminal addition it is one or several (usually within 20, compared with Being goodly is more preferably within 5 within 10) amino acid.For example, in the art, with amino similar in performance When acid is replaced, the function of protein is not usually changed.For another example, one or several in C-terminal and/or N-terminal addition Amino acid will not generally also change the function of protein.The term further includes that the active fragment of antibody of the present invention and activity derive Object.

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 nano antibody or its segment.In addition to almost overall length Polypeptide outside, the invention also includes the segments of nano antibody of the present invention.In general, the segment has antibody of the present invention at least about 50 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably extremely Few 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

Initial residue	Representative substitution	It is preferred to replace
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

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 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 has identical with mature polypeptide 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 6His) 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 includes but is not limited to for diagnostic purposes: 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；2. biological poison；3. Cell factor such as IL-2 etc.；4. gold nano grain/nanometer rods；5. virion；6. liposome；7. magnetic nanosphere；8. medicine activates Enzyme (for example, DT- diaphorase (DTD) or biphenyl base hydrolase-sample protein (BPHL))；9. treating agent (for example, cis-platinum) or appointing The nano particle etc. of what form.

Pharmaceutical composition

The present invention also provides a kind of compositions.Preferably, the composition is pharmaceutical composition, it contains above-mentioned Antibody or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, nothing can be formulated in these substances In poison, 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 can be with It is 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-L1 protein molecular, thus can be used for treating tumour.This Outside, other therapeutic agents also be can be used simultaneously.

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 nano antibody (or its conjugate) and pharmaceutically acceptable carrier or figuration of the present invention Agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Pharmaceutical preparation It should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain The aqueous solution of glucose and other adjuvants is prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably in sterile item It is manufactured under part.The dosage of active constituent is therapeutically effective amount, such as about 50 mg/kg of about 10 micrograms/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 50 mg/kg weight, compared with Good ground dosage is about 10 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration way The factors such as diameter, patient health situation, within the scope of these are all skilled practitioners technical ability.

The nano antibody of label

In a preference of the invention, the nano antibody has detectable marker.More preferably, the label Object is selected from the group: isotope, 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 nano antibody colloid gold label of anti-PD-L1 obtains the nano antibody of colloid gold label.

Anti- PD-L1 nano antibody of the invention has specificity well, very high potency.

Detection method

The invention further relates to the methods of detection PD-L1 albumen.This method step approximately as: obtain cell and/or tissue Sample；In the medium by sample dissolution；Detect the level of the PD-L1 albumen in the sample of the dissolution.

In detection method of the invention, used sample is not particularly limited, and representative example is to be present in carefully Born of the same parents save the celliferous sample in liquid.

Kit

The present invention also provides a kind of kits containing antibody (or its segment) or detection plate of the invention, in the present invention A preference in, the kit further includes container, operation instructions, buffer etc..

The present invention also provides the detection kit for detecting PD-L1 level, which includes identification PD-L1 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.

Using

As described above, nano antibody of the invention has, extensive biologic applications are worth and clinical value, application are related to The multiple fields such as diagnosing and treating, basic medical research, biological study to disease relevant to PD-L1.One preferred Using be for for PD-L1 clinical diagnosis and targeted therapy.

Main advantages of the present invention include:

(a) nano antibody high specific of the present invention is directed to the PD-L1 albumen with correct space structure of people.

(b) affinity of nano antibody of the present invention is strong.

(c) production of nano antibody of the present invention is easy.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, 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, no Then percentage and number are weight percent and parts by weight.

Embodiment 1: the expression and purification of human PD-L 1 albumen

(1) nucleotide sequence of human PD-L 1 is synthesized on pCDNA3.1 (-) carrier, it is then that its extracellular fragment sequence is sub- It is cloned on pF Μ SE-IgG1 carrier；

(2) pF Μ SE-IgG1-hPD-L1 (ECD) plasmid of building is extracted with the big extraction reagent kit of Omega plasmid；

(3) culture HEK293F cell to OD be 2.0 × 10⁶A/mL；

(4) plasmid and transfection reagent PEI 1:3 are stood into 10min after mixing, are then added to HEK293F cell In, 37 DEG C, 6%CO₂It is cultivated 5-6 days in shaking table culture case；

(5) cell conditioned medium is collected, is combined 1h at room temperature with Protein A pearl；

(6) with phosphate buffer pH 7.0 wash pearl after, then with 0.1M pH 3.0Glycine elution albumen；

(7) by the albumen ultrafiltration of elution into PBS, sampling progress SDS-PAGE examines antigen purity after measuring yield It surveys, remaining albumen is stored in -80 DEG C of refrigerators；

(8) it chooses antigen of the purity greater than 90% and carries out the immune of subsequent camel.

The building in embodiment 2:PD-L1 nano antibody library

(1) 1mg hPD-L1 (ECD)-Fc antigen is mixed in equal volume with Freund's adjuvant, an Xinjiang two-humped camel is immunized, often Zhou Yici is immunized 3 times altogether, and stimulation B cell expresses the nano antibody of antigentic specificity；

After (2) 3 times immune, extract 100mL camel peripheral blood lymphocytes and extract total serum IgE；

(3) it synthesizes cDNA and expands VHH using sleeve type PCR；

(4) 20 μ g pMECS Vector for Phage Display (Biovector of restriction enzyme Pst I and Not I digestion is utilized Supply) and two segments of 10 μ g VHH and connection；

(5) connection product is converted to electricity and is turned in competent cell TG1, construct PD-L1 nano antibody library and measure library Hold, storage capacity size is 1.0 × 10⁹CFU；

At the same time, 24 clones of random picking carry out bacterium colony PCR detections, the results showed that the insertion rate in built library for up to 95.8%.The above results explanation obtains the qualified PD-L1 nano antibody phage display library of storage capacity and insertion rate.

The screening and identification of embodiment 3:PD-L1 nano antibody

Antibody screening:

(1) it is dissolved in 100mM NaHCO₃, 10 μ g hPD-L1 (ECD)-Fc antigen (10 μ g Fc in pH 8.2 NaHCO₃As control) it is coupled on NUNC ELISA Plate, 4 DEG C stand overnight；

(2) second days 100 μ L 0.01%BSA of addition, room temperature close 0.5h；

(3) after 2h, 100 μ L bacteriophages (2 × 10 are added¹¹Camel nano antibody phage display gene pool is immunized in CFU), room temperature Act on 0.5h；

(4) it is washed 5 times with 0.05%PBS+Tween-20, to wash off non-specific bacteriophage；

(5) it under being dissociated the bacteriophage specifically bound with PD-L1 with 100mM triethanolamine, and infects and is in logarithmic phase The e. coli tg1 cell of growth, 37 DEG C of culture 1h generate the screening that simultaneously purified phage is used for next round, identical to screen Cheng Chongfu 3 takes turns, and enrichment times are respectively 11.4 times and 270 times without enrichment.

The single positive colony of specificity is screened with the enzyme-linked immunoassay method (ELISA) of bacteriophage:

(1) after above-mentioned 3 wheel screening in the Tissue Culture Dish containing bacteriophage, 600 single bacterium colonies is selected and are inoculated in The TB culture medium of ampicillin containing 100 μ g/mL (contains 2.3g KH in 1L TB culture medium₂PO₄, 12.52g K₂HPO₄, 12g peptone, 24g yeast extract, 4mL glycerol) in, after growing to logarithmic phase, add the IPTG of final concentration 1mM, 28 DEG C of cultures Overnight；

(2) antibody slightly is mentioned using osmosis acquisition, and antibody is transferred in the elisa plate through antigen coat, in room temperature Lower placement 1h；

(3) unbonded antibody is washed away with PBST, the anti-HA antibody of mouse is added, and is that century biotechnology is limited purchased from Beijing health Company), 1h is placed at room temperature；

(4) unbonded antibody is washed away with PBST, and goat-anti-mouse alkaline phosphatase enzyme mark antibody is added, puts at room temperature Set 1h；

(5) it washes away unbonded antibody with PBST, alkaline phosphatase developing solution is added, on ELISA instrument, in 405nm wave It is long, read absorption value；

(6) when sample well OD value is greater than 3 times of control wells OD value or more (Ratio+/- > 3), it is judged to positive colony hole.Knot Fruit shows that 95 positive colonies occur altogether in 600 clones；

(7) bacterium in 95 positive colony holes is turned to shake the LB liquid being 100 μ g/mL containing 2mL ampicillin concentration In to extract plasmid and to be sequenced；

(8) 95 strain clone of comparative analysis series, finally obtains 9 kinds of CDR3 region sequence notable difference nano antibodies, and number is Nb1, Nb4, Nb16, Nb23, Nb36, Nb43, Nb96, Nb102, Nb156 (Fig. 1 shows its ELISA light absorption value).

Embodiment 4: the block function of flow cytometry Preliminary Identification nano antibody

(1) hPD-1 (ECD)-Biotin albumen (the preparation method is the same as that of Example 1, the verifying of SDS-PAGE purity), albumen are prepared Biotinylated method is referring to biotin reagent specification.

(2) stablize the building of the A375 transgenic cell of expression PD-L1.

(3) preparation PD-L1 nano antibody TG1 bacterial strain slightly mentions lysate, and preparation method is the same as embodiment 3.

(4) each sample takes 1 × 10⁶It is a surely to turn PD-L1 cell and be resuspended in 0.5%BSA-PBS buffer, in addition 50 μ L of crude extract is stated, while negative control (hIgG1) and blank group (PBS) are set, 5 μ g hPD-1 (ECD)-Fc- are added in every hole Biotin, 4 DEG C of incubation 20min.

(5) PBS washs 2 cells, and the SA-PE of eBioscience is added, and 4 DEG C of incubations 20min, PBS wash 2 cells It is as shown in Figure 2 with flow cytometer (BD FACS Calibur) testing result afterwards.

(6) from 9 plants of CDR3 region sequence notable difference nano antibodies that screening obtains, discovery wherein has for 1 plant (Nb 43) Apparent barrier effect.

Embodiment 5:PD-L1 blocking-up type nano antibody it is humanization modified

(1) homologous in structural database as template using PD-L1 nano antibody sequence shown in SEQ ID NO.:8 first The search of structure searches 1500 structures altogether, takes 40 knots of wherein E value=0.0 and sequence identity property >=70% Structure；

(2) structure alignment, and the chadogram according to crystal structure resolution sizes and building are carried out to this 40 structures, most 7 albumen including 3dwt are chosen eventually, carry out the multimode based on PD-L1 nano antibody sequence shown in SEQ ID NO.:8 Plate Blast search, 10 structures finally obtained, then according to the height sequence of scoring functions, the minimum structure of molpdf is chosen, Continue following work；

(3) optimum structure built to mould calculates the Solvent accessibility of residue, the i.e. folding of residue using ProtSA server Folded state is criterion relative to the ratio of the Solvent accessibilities area of unfolding state, and taking the residue greater than 40% is to be exposed to outside solvent Residue；

(4) optimum structure and DP-47 built to mould carry out sequence alignment, and replacement is exposed to the residue of solvent accordingly.Most A kind of humanization PD-L1 nano antibody is determined eventually, the coding of the amino acid sequence as shown in SEQ ID NO.14.Before and after humanization Antibody sequence is corresponding such as the following table 2:

Table 2

The tetraploid rice such as the following table 3 in antibody backbone area and DP-47 skeleton area before and after humanization:

Table 3

Embodiment 6: humanization PD-L1 blocking-up type nano antibody eukaryotic expression purifying

(1) by the PD-L1 nano antibody sequent synthesis after humanization to pF Μ SE-IgG1 carrier (be purchased from Invivogen), PF Μ SE-IgG1-Nb (humanized) plasmid is extracted with the big extraction reagent kit of Omega plasmid；

(2) culture HEK293F cell to OD be 2.0 × 10⁶A/mL；

(3) plasmid and transfection reagent PEI are stood into 10min according to 1:3 after mixing, it is thin to be then added to HEK293F In born of the same parents, 37 DEG C, 6%CO₂It is cultivated 5-6 days in shaking table culture case；

(4) cell conditioned medium is collected, is combined 1h at room temperature with Protein A pearl；

(5) with phosphate buffer pH 7.0 wash pearl after, then with 0.1M pH 3.0Glycine elution albumen；

(6) by the albumen ultrafiltration of elution into PBS, sampling carries out SDS-PAGE detection ability purity after measuring yield, remaining Albumen is stored in -80 DEG C of refrigerators；

(7) successful expression purifying obtains Nb43 high-purity humanization nano antibody (SEQ ID NO.:14) as the result is shown.

Embodiment 7: the block function of Flow cytometry humanization PD-L1 nano antibody

Method is with embodiment 4:

(1) each sample takes 1 × 10⁶The cell strain of a stable expression PD-L1 is resuspended in 0.5%BSA-PBS buffer In, be added the humanization PD-L1 nano antibody of 10 μ g purifying, while negative control (hIgG1) and blank group (PBS), institute are set There is sample that 5 μ g hPD-1 (ECD)-Fc-Biotin albumen, 4 DEG C of incubation 20min are added；

(2) PBS washs 2 cells, and the SA-PE of eBioscience is added, and 4 DEG C of incubations 20min, PBS wash 2 cells Upper machine testing afterwards, testing result is as shown in figure 3, Nb43 shows good blocking activity.

Embodiment 8: humanization blocking-up type PD-L1 nano antibody Fc fusion protein is anti-in dendritic cells-T cell mixing lymph The activation of CD4+T cell is measured in answering

(1) white from healthy donor periphery pyknohemia using human lymphocyte separating liquid (Tianjin Chao Yang) density gradient centrifugation Separating peripheral blood mononuclear cells PBMC in cell；

(2) the 1640 culture medium culture 1-2h of RPMI for using serum-free, removes not adherent cell, by cell culture in containing In the RPMI of 10%FBS, 10ng/mL GM CSF and 20ng/mL IL 4；

(3) it cultivates 5-6 days, the TNF-α of 10ng/mL is added and is incubated for the dendritic cells for obtaining maturation for 24 hours；

(4) dendritic cells obtained are resuspended in RPMI complete medium, 2 × l0⁵/mL.Then in 96 hole U-shaped base plates (Costar:3799) 50 μ L are added in every hole in, cultivate in incubator；

(5) using magnetic bead separation kit (Mi 1tenyi Bio tee:130-096-533) to specifications method from CD4 is separated in another donor PBMC⁺T cell；

(6)l×l0⁴A dendritic cells and l × 10⁵A CD4⁺T cell mixing, is resuspended and is added in RPMI complete medium Enter 96 well culture plates, every hole is added 50 μ L cells and mixes liquid: every hole is added 100 μ L and is diluted in the humanization in RPMI complete medium PD-L1 nano antibody (SEQ ID NO.:14) Fc fusion protein (MY1909), culture remove supernatant after 5-7 days, utilize IFN-γ It is horizontal that ELISA detection kit (ebioscience) detects IFN-γ in supernatant；

As a result as shown in Figure 4, it is seen that PD-L1 nano antibody Fc fusion protein can enhance CD4 in mixed lymphocyte reaction (MLP)⁺ The interferon of T cell is secreted, i.e. PD-L1 blocking-up type nano antibody Fc fusion protein enhances the activation of T cell.

Embodiment 9: inhibitory activity of the humanization blocking-up type PD-L1 nano antibody Fc fusion protein to tumour growth

Using immune deficiency NOD/SCID (non-obese patients with type Ⅰ DM/severe combined immunodeficiency) mice study, it is living in vivo Property.Peripheral blood by K-1735 A375 and people to NOD/SCID mouse subcutaneous transplanting expression human PD-L 1 is single Nucleus PBMC tests to complete.Detailed step is as follows:

(1) A375 and PBMC are mixed before the injection, subcutaneous injection；

(2) humanization blocking-up type PD-L1 nano antibody (SEQ ID NO.:14) Fc fusion protein (MY1909) connects in tumour First time intraperitoneal injection for 24 hours after kind, weekly administration is primary later, dosage 2mg/kg；

(3) PBS is as negative control, and the TECENTRIQ of Roche Holding Ag is as positive control (benchmark, BMK).Often 11 mouse of a experimental group.Twice a week observe tumour formation, and use vernier caliper measurement tumour major diameter and minor axis, calculate swell Knurl product, draws tumor growth curve figure (Fig. 5).Fusion protein (antibody) MY1909 can under 2mg/kg dosage as the result is shown To significantly inhibit tumour growth.

Inhibitory activity of the embodiment 10:MY1909 to tumour growth

Using the activity in vivo of C57BL/6 mouse subcutaneous transplanting tumor model research MY1909.By to C57BL/6 mouse The colon carcinoma cell line MC38 (MC38-hPD-L1) of expression human PD-L 1 is inoculated to construct test model, evaluates MY1909 pairs The inhibitory activity of its tumour.Detailed step is as follows:

(1) MC38-hPD-L1 tumour cell (1 × 10 is inoculated in tested C57BL/6 right side of mice⁶/ only, cell training It supports in the DMEM culture medium containing 10% heat-inactivated fetal bovine serum (ExCell Biology) and 50 μ g/mL hygromycin B, 1×10⁶Cell is resuspended using 100 μ L PBS).When the mouse lotus knurl gross tumor volume that is averaged reaches about 64mm³When, by 24 mice with tumor 3 experimental groups are randomized into, every group 6, the grouping same day is defined as the 0th day.

(2) 3 groups of mouse gave Isotype control (Isotype control) (01 group) of hIgG1 respectively in the 0th day and are used as feminine gender Control, (02 group) of Analogue to Atezolizumab (Roche) be used as positive control, MY1909 (03 group), test sample is all It is diluted to 0.5mg/mL, dosage 5mg/kg using PBS, is injected intraperitoneally, administration frequency 2 times a week, is administered 3 weeks altogether.

(3) after grouping, the gross tumor volume of animal is measured in experiment twice a week.Gross tumor volume is public by calliper to measure Formula is TV=0.5a × b2, and wherein a is the major diameter of tumour, and b is the minor axis of tumour.It draws tumor growth curve figure (Fig. 6), investigates Inhibiting effect (table 4) of each group test sample to tumour growth.MY1909 can significantly press down under 5mg/kg dosage as the result is shown Tumour growth processed.

Antitumous effect of the table 4.MY1909 in subcutaneous MC38-hPD-L1 tumor model

Remarks: a. average value ± SEM；

B. Relative tumor proliferation rate T/C (%) is antitumor activity evaluation index: formula: T/C%=Ti/Ci*100%；

Ti and Ci is respectively the mean tumour volume of processing group and control group Day i days

C. Tumor growth inhibition (Tumor growth inhibition, TGI): formula: %TGI=(1- (Ti-T0)/ (Ci-C0)) * 100%；

Ti and Ci is respectively the mean tumour volume of processing group and control group Day i days；

T0 and C0 is respectively 0 day mean tumour volume of processing group and control group Day

D. compared with the 1st group.Data have carried out SQRT conversion before using independent sample T check analysis.2nd group and 3rd group is compared, P > 0.05.

(4) the 20th day after GP TH, compared with Isotype hIgG1 control treatment group, MY1909 single therapy group Inhibition rate of tumor growth (TGI%) be 58.19%, have the function of statistically significant anti-MC38-hPD-L1 tumour growth (P<0.05).In MY1909 single therapy group, 5 completed tumor regressions, (the 34th day) is without tumour growth when ending experimental endpoints Sign.Show that MY1909 can significantly inhibit tumour growth under 5mg/kg dosage.

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.

Sequence table

<110>Hualan Bio-Engineering Co Ltd.

Hua Lan genetic engineering Co., Ltd

<120>a kind of anti-PD-L1 humanization nano antibody and its application

<130> P2018-0872

<150> 2017104713177

<151> 2017-06-20

<160> 15

<170> PatentIn version 3.5

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<212> PRT

<213>artificial sequence (artificial sequence)

<400> 14

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Ala Ser Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Ile Ala Thr Val

35 40 45

Ser Gly Ala Ala Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe

50 55 60

Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Ile Asn

65 70 75 80

Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Asp

85 90 95

Asp Tyr Tyr Ala Phe Leu Ser Arg Gly Ala Arg Asp Phe Arg Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 366

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 15

caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

tcctgcgccg cctccaggtt caccgcctcc atgggctggt tcaggcaggc ccccggcaag 120

ggcctggagg gcatcgccac cgtgtccggc gccgcctcca ccaactacgc cgactccgtg 180

aagggcaggt tcaccatctc caaggacaac tccaagaaca ccctgtacct gcagatcaac 240

tccctgaggg acgaggacac cgccgtgtac tactgcgccg ccgacgacga ctactacgcc 300

ttcctgtcca ggggcgccag ggacttcagg tactggggcc agggcaccct ggtgaccgtg 360

tcctcc 366

Claims (10)

1. a kind of VHH chain of anti-PD-L1 nano antibody, which is characterized in that the VHH chain includes complementary determining region CDR, described Complementary determining region CDR includes CDR1 shown in SEQ ID NO.:5, CDR2 and SEQ ID NO.:7 shown in SEQ ID NO.:6 Shown in CDR3.

2. a kind of anti-PD-L1 nano antibody, which is characterized in that it is the nano antibody for PD-L1 epitope, and is had such as The VHH chain of amino acid sequence shown in SEQ ID NO.:8 or SEQ ID NO.:14.

3. a kind of polynucleotides, which is characterized in that the polynucleotide encoding protein selected from the group below: described in claim 1 Anti- PD-L1 nano antibody VHH chain or anti-PD-L1 nano antibody as claimed in claim 2.

4. a kind of expression vector, which is characterized in that the expression vector contains polynucleotides as claimed in claim 3.

5. a kind of host cell, which is characterized in that the host cell contains expression vector as claimed in claim 4 or its base Because being integrated with polynucleotides as claimed in claim 3 in group.

6. a kind of method for generating anti-PD-L1 nano antibody, which is characterized in that comprising steps of

(a) under conditions of being suitble to generate nano antibody, host cell described in claim 5 is cultivated, to obtain containing described The culture of anti-PD-L1 nano antibody；And

(b) the anti-PD-L1 nano antibody is separated or recycled from the culture.

7. a kind of polypeptide, which is characterized in that the polypeptide contains:

(a) the VHH chain of anti-PD-L1 nano antibody as described in claim 1 or as claimed in claim 2 PD-L1 nanometers anti- Antibody；

(b) optional, with the polypeptide in conjunction with or the modification marker selected from the group below that is coupled: chemical markers or biomarker Object.

8. a kind of immune conjugate, which is characterized in that the immune conjugate contains:

(a) the VHH chain of anti-PD-L1 nano antibody as described in claim 1 or as claimed in claim 2 PD-L1 nanometers anti- Antibody；

(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or enzyme.

9. the purposes of anti-PD-L1 nano antibody as claimed in claim 2, which is characterized in that be used to prepare (a) for detecting PD- The reagent of L1 molecule；(b) for blocking preparation of the PD-L1 in conjunction with PD-1；(c) for treating the drug of tumour.

10. a kind of pharmaceutical composition, which is characterized in that contain:

(i) the VHH chain of anti-PD-L1 nano antibody as described in claim 1 or as claimed in claim 2 PD-L1 nanometers anti- Antibody or immune conjugate as claimed in claim 8；And

(ii) pharmaceutically acceptable carrier.