CN115433281B - Humanized nanometer antibody for resisting PD-L1 and application thereof - Google Patents
Humanized nanometer antibody for resisting PD-L1 and application thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Abstract
The application discloses a humanized nanometer antibody for resisting PD-L1 and application thereof. The nano antibody provided by the application comprises 3 complementarity determining regions, wherein the amino acid sequence of CDR1 is SEQ ID No.4, the amino acid sequence of CDR2 is SEQ ID No.6, and the amino acid sequence of CDR3 is SEQ ID No.9. On the basis of the camel-derived nano-antibody for resisting PD-L1, humanized transformation and experiments prove that the affinity of the anti-PD-L1 humanized nano-antibody M2 (SEQ ID No. 11) and the anti-PD-L1 humanized nano-antibody M5 (SEQ ID No. 12) are respectively improved by 2.54,6.47 times, so that the humanized nano-antibody specifically combined with PD-L1 is obtained, the limitation of the camel-derived nano-antibody in clinical application is overcome, and the humanized nano-antibody is expected to be used for treating tumors expressed by PD-L1 or diagnosing and detecting PD-L1 proteins.
Description
Technical Field
The application relates to the field of antibodies of biotechnology, in particular to an anti-PD-L1 humanized nano antibody and application thereof.
Background
Immune checkpoints are a class of immunosuppressive molecules expressed on immune cells, regulate the degree of immune activation, and play an important role in preventing autoimmune diseases. The essence of immune checkpoints is protein molecules which are produced by immune cells and used for regulating autoimmune functions, and the immune systems can be ensured to be in a normal range when activated. Immune checkpoint immunotherapy is a therapeutic approach to kill tumor cells by modulating T cell activity through co-suppression or co-stimulatory signals.
As a typical representation of immune checkpoint inhibitors, the apoptosis receptor-1 (PD-1) inhibits T lymphocyte function by binding to its ligand PD-L1/PD-L2, regulates the immune system and promotes self tolerance by down regulating the immune system's response to human cells, inhibiting T cell inflammatory activity. Under normal physiological state, PD-1 is a double-edged sword which can prevent autoimmune diseases and prevent the immune system from killing cancer cells.
As a ligand for PD-1, apoptosis-ligand 1 (PD-L1) is a transmembrane protein having a molecular weight of 40kDa, and is involved in the inhibition of the immune system in abnormal situations (tissue transplantation, autoimmune diseases, hepatitis, etc.). The immune system responds to foreign antigens in the lymph nodes or spleen under normal physiological conditions, promoting antigen-specific cytotoxic T cells (CD 8) + T cells) proliferate while PD-1 and PD-L1 bind to transmit inhibitory signals, reducing lymph node CD8 + T cell proliferation. The expression of PD-L1 on tumors is associated with decreased survival rates of esophageal cancer, pancreatic cancer and other types of cancer, indicating that this pathway can be used as a novel tumor immunotherapeutic target and confirmed by many studies.
The nano antibody is obtained by cloning a heavy chain variable region (VHH) of a camel natural antibody, and is a novel small molecular antibody fragment. Nanobodies have unique advantages: the molecular weight is small, the tissue penetrability is good, the specificity is strong, the affinity is high, and the water solubility is good. In addition, the nano antibody is highly expressed in a prokaryotic or eukaryotic system, and has wide application prospect in immunodiagnosis and treatment.
Disclosure of Invention
The application aims to develop a humanized nano antibody capable of binding to PD-L1 and blocking the binding of PD-L1/PD-1, and the humanized nano antibody plays a role in diagnosis or treatment of tumors expressing PD-L1.
In a first aspect, the application claims a nanobody.
The nanobody claimed by the application is a nanobody against PD-L1. Firstly, immunizing a Sinkiang Bactrian camel, stimulating B cells to express an antigen-specific nanobody, and obtaining a camel-derived nanobody (SEQ ID No. 1) by adopting a phage display technology. Then, the camelid nanobody (SEQ ID No. 1) is compared with a human antibody germline database (Vbase), and the germline with the highest homology is screened for Complementarity Determining Region (CDR) transplantation. CDR grafting also includes amino acid mutations that are different from germline and 5 angstroms from the CDRs, designing different humanized nanobodies.
The application claims the anti PD-L1 nanobody, comprising 3 complementarity determining regions;
the 3 complementarity determining regions are CDR1, CDR2 and CDR3;
the amino acid sequence of the CDR1 is SEQ ID No.4;
the amino acid sequence of the CDR2 is SEQ ID No.6;
the amino acid sequence of the CDR3 is SEQ ID No.9.
Further, the nanobody comprises 4 framework regions in addition to the complementarity determining regions; the 4 framework regions are FR1, FR2, FR3 and FR4, respectively;
the amino acid sequence of the FR1 is SEQ ID No.2 or SEQ ID No.3;
the amino acid sequence of the FR2 is SEQ ID No.5;
the amino acid sequence of the FR3 is SEQ ID No.7 or SEQ ID No.8;
the amino acid sequence of FR4 is SEQ ID No.10.
Still further, the nanobody may be A1) or A2) or A3) or A4) as follows:
a1 A nano antibody with an amino acid sequence shown as SEQ ID No. 11;
a2 A nano antibody with an amino acid sequence shown as SEQ ID No. 12;
a3 A nano antibody obtained by connecting protein labels to the N end and/or the C end of an amino acid sequence shown in SEQ ID No. 11;
a4 A protein label is connected to the N end and/or the C end of the amino acid sequence shown in SEQ ID No.12, and the nano antibody is obtained.
The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a His tag, flag tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In a specific embodiment of the application, his is fused at the carbon end of the nanobody 6 And (5) a label.
In a second aspect, the application claims a biological material associated with the nanobody of the first aspect above.
The biological material claimed in the present application may be any of B1) to B12):
b1 Nucleic acid molecules encoding nanobodies as described in the first aspect above;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1);
b4 A recombinant vector comprising the expression cassette of B2);
b5 A recombinant microorganism comprising the nucleic acid molecule of B1);
b6 A recombinant microorganism comprising the expression cassette of B2);
b7 A recombinant microorganism containing the recombinant vector of B3);
b8 A recombinant microorganism comprising the recombinant vector of B4);
b9 A transgenic animal cell line comprising the nucleic acid molecule of B1);
b10 A transgenic animal cell line comprising the expression cassette of B2);
b11 A transgenic animal cell line comprising the recombinant vector of B3);
b12 A transgenic animal cell line comprising the recombinant vector of B4).
In the above biological material, the nucleic acid molecule may be DNA such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, and the like.
In the above biological material, B2) the expression cassette refers to a DNA capable of expressing the nanobody in a host cell, and the DNA may include not only a promoter for initiating transcription of the nanobody-encoding gene, but also a terminator for terminating transcription of the nanobody-encoding gene. Further, the expression cassette may further comprise regulatory elements such as enhancers.
In a specific embodiment of the application, the above expression cassette comprises a T7 promoter, a nucleic acid sequence as shown in SEQ ID No.13 or SEQ ID No.14 and a T7 terminator.
Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.
In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector.
In the above biological material, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into a pET26B vector. In one embodiment of the present application, B3) the recombinant vector is a recombinant vector obtained by introducing the nanobody encoding gene (SEQ ID No.13 or SEQ ID No. 14) into pET26B vector.
In the above biological material, the microorganism may be bacteria (e.g., E.coli), yeast, algae, or fungi.
Further, the coding sequence of CDR1 of the nanobody may be nucleotides 142 to 156 of SEQ ID No.13 or nucleotides 142 to 156 of SEQ ID No. 14. The coding sequence of CDR2 of the nanobody can be 208-228 nucleotides of SEQ ID No.13 or 208-228 nucleotides of SEQ ID No. 14. The coding sequence of CDR3 of the nanobody can be 343-399 nucleotides of SEQ ID No.13 or 343-399 nucleotides of SEQ ID No. 14.
Still further, the nucleic acid molecule of B1) may be any of the following:
c1 A DNA molecule with a nucleotide sequence shown as 67-432 of SEQ ID No.13 or 67-432 of SEQ ID No. 14;
c2 A DNA molecule with a nucleotide sequence shown as 7-432 of SEQ ID No.13 or 7-432 of SEQ ID No. 14;
c3 A DNA molecule with a nucleotide sequence shown as SEQ ID No.13 or SEQ ID No. 14;
c4 A DNA molecule that hybridizes under stringent conditions to a DNA molecule defined in any one of C1) -C3) and encodes the nanobody;
c5 A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in any one of C1) -C4) and encoding the nanobody.
Wherein the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In a third aspect, the application claims a polypeptide molecule comprising a nanobody structure as described in the first aspect above.
The polypeptide molecule claimed in the present application may specifically be any of the following:
d1 A single chain antibody comprising the nanobody of the first aspect;
d2 Fab containing nanobodies as described in the first aspect above;
d3 A whole antibody comprising a nanobody as described in the first aspect above;
d4 A fusion antibody (e.g., diabody) comprising a nanobody as described in the first aspect above;
d5 An Antibody Drug Conjugate (ADC) comprising a nanobody as described in the first aspect above.
In a fourth aspect, the application claims a kit comprising a nanobody as described in the first aspect above or a biological material as described in the second aspect above or a polypeptide molecule as described in the third aspect above.
In a fifth aspect, the application claims a method of preparing a nanobody as described in the first aspect above.
The method for preparing the nanobody according to the first aspect of the application may comprise the following steps: introducing a nucleic acid molecule encoding the nanobody of the first aspect into a host cell to obtain a recombinant host cell expressing the nanobody, and culturing the recombinant host cell to obtain the nanobody.
Further, the introduction of the nucleic acid molecule into the host cell may be accomplished by chemical or electrical transformation.
Further, after culturing the recombinant host cell, the method further comprises the steps of: firstly, obtaining periplasmic space protein by a osmotic shock method, and then purifying the periplasmic space protein by affinity chromatography and ultrafiltration liquid exchange in sequence to obtain the nano antibody.
Further, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described in the second aspect above.
Further, the host cell is a microbial cell or an animal cell. Such as bacteria (e.g., E.coli), yeast, algae, or fungi. Such as HEK293 cells, CHO cells or insect cells.
In a specific embodiment of the application, the recipient cell is in particular E.coli.
In a sixth aspect, the application claims any of the following applications:
e1 Use of a nanobody as described in the first aspect hereinbefore for the preparation of a product for detecting PD-L1;
e2 Use of a biomaterial according to the second aspect of the foregoing in the manufacture of a product for detecting PD-L1;
e3 Use of a polypeptide molecule according to the third aspect of the foregoing in the preparation of a product for detecting PD-L1;
e4 Use of a kit according to the fourth aspect of the foregoing for the preparation of a product for the detection of PD-L1;
e5 Use of the method of the fifth aspect of the foregoing in the preparation of a product for detecting PD-L1;
e6 Use of a nanobody as described in the first aspect hereinbefore for the preparation of a product which binds to PD-L1;
e7 Use of the biomaterial of the second aspect of the foregoing in the preparation of a product that binds PD-L1;
e8 Use of a polypeptide molecule according to the third aspect of the foregoing for the preparation of a product which binds to PD-L1;
e9 Use of a kit as described in the fourth aspect of the foregoing for the preparation of a product for binding to PD-L1;
e10 Use of the method of the fifth aspect of the foregoing for the preparation of a product that binds PD-L1;
e11 Use of a nanobody as described in the first aspect hereinbefore for the preparation of a product for the diagnosis or treatment of a PD-L1 expressing tumour;
e12 Use of a biomaterial as described in the second aspect of the foregoing in the manufacture of a product for the diagnosis or treatment of a PD-L1 expressing tumour;
e13 Use of a polypeptide molecule as described in the third aspect hereinbefore for the manufacture of a product for the diagnosis or treatment of a PD-L1 expressing tumour;
e14 Use of a kit as described in the fourth aspect of the foregoing for the preparation of a product for the diagnosis or treatment of a PD-L1 expressing tumor;
e15 Use of the method of the fifth aspect of the foregoing for the preparation of a product for the diagnosis or treatment of a PD-L1 expressing tumor.
Further, the tumors may include, but are not limited to: melanoma, gastric cancer, lymphoma, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, carcinoma of large intestine, or adrenal gland tumor.
On the basis of a camel-derived nano-antibody (SEQ ID No. 1) for resisting PD-L1, humanized transformation and experiments prove that the affinity of the anti-PD-L1 humanized nano-antibody M2 (SEQ ID No. 11) and the anti-PD-L1 humanized nano-antibody M5 (SEQ ID No. 12) are respectively improved by 2.54,6.47 times, so that the humanized nano-antibody specifically combined with PD-L1 is obtained, the limitation of the camel-derived nano-antibody in clinical application is overcome, and the humanized nano-antibody is expected to be used for treating tumors expressed by PD-L1 or diagnostic detection of PD-L1 proteins.
Drawings
FIG. 1 is a diagram showing the construction of an electrophoresis identification of an expression plasmid of an anti-PD-L1 humanized nanobody. The four expression plasmids were identified by NdeI and XhoI double digestion, wherein, lane 1 is pET26b-NB43, lane 2 is pET26b-M2, lane 3 is pET26b-M5, and lane 4 is pET26b-M7.
FIG. 2 is an electrophoresis diagram of SDS-PAGE proteins purified with anti-PD-L1 humanized nanobodies. Four nanobody purified proteins are identified by SDS-PAGE electrophoresis, wherein lane 1 is NB43 camelid nanobody, lane 2 is M2 humanized nanobody, lane 3 is M5 humanized nanobody, and lane 4 is M7 humanized nanobody.
FIG. 3 is a graph showing affinity detection of anti-PD-L1 humanized nanobodies. The abscissa is the reaction time(s), and the ordinate is the reaction value (RU), where a is NB43 camelid antibody, B is M2 humanized nanobody, C is M5 humanized nanobody, and D is M7 humanized nanobody. Different lines represent the binding and dissociation curves generated by different concentration gradients, with high concentration curves having large (upper) and low (lower) concentration response values.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The main reagents and their manufacturer information in the following examples are as follows:
pET26b expression vector: novagen Corp;
NdeI: NEB company;
XhoI: NEB company;
recombinant Human PD-L1/B7-H1 His-tag Protein, CF: r & D company;
Ni-NTA 6FF His tag protein purification kit: bioengineering (Shanghai) stock Co.Ltd;
amicon Ultra-0.5 centrifugal filter device; millipore Co;
PBS ph7.4 (1×): thermo company;
Sure PAGE TM Bis-Tris,10×8,4-12%,12wells: nanjing Jinsri Biotechnology Co.
The key instruments and their manufacturer information in the following examples are as follows:
full temperature shaking flask cabinet (shaking table): the Taicang laboratory equipment factory;
ultra-clean bench: suzhou Antai air technologies Co., ltd;
centrifuge: eppendorf corporation;
gel imaging system: protein Simple company;
eStain TM l1 protein staining instrument: nanjing Jinsri biotechnology Co., ltd;
NanoDrop ultramicro biological detector: thermo Fisher company;
biacore T200 intermolecular interaction analysis System: GE company.
Example 1 humanized modification of camel-derived nanobody
Firstly, a PD-L1 antigen is used for immunizing a Bactrian camel in Xinjiang, B cells are stimulated to express antigen-specific nanobodies, a phage display technology is adopted to obtain a plurality of nanobodies, and camel-derived nanobody NB43 (SEQ ID No. 1) with highest affinity is selected. Then humanized on the basis of camelid nanobody NB43 (SEQ ID No. 1), nanobody structure analysis according to IMGT numbering and definition scheme is shown in Table 1.
TABLE 1, camel-derived nanobody CDR sequence statistics
| Region(s) | Amino acid sequence | Corresponding sequence listing |
| CDR1 | RFTAS | SEQ ID No.4 |
| CDR2 | VSGAAST | SEQ ID No.6 |
| CDR3 | AADDDYYAFLSRGARDFRY | SEQ ID No.9 |
And (3) comparing camel-derived nanobody NB43 (SEQ ID No. 1) with a human antibody seed factor database (Vbase), screening out a germline with highest homology of IGHV3-66 x 01, and using the germline for CDR transplantation of the antibody. Three different humanized nanobody sequences M2, M5 and M7 were designed by CDR grafting and amino acid mutation different from germline (table 2).
TABLE 2 engineering of anti-PD-L1 humanized nanobodies
| Sequence number | M2 | M5 | M7 |
| FR1 | SEQ ID No.2 | SEQ ID No.2 | SEQ ID No.3 |
| CDR1 | SEQ ID No.4 | SEQ ID No.4 | SEQ ID No.4 |
| FR2 | SEQ ID No.5 | SEQ ID No.5 | SEQ ID No.5 |
| CDR2 | SEQ ID No.6 | SEQ ID No.6 | SEQ ID No.6 |
| FR3 | SEQ ID No.7 | SEQ ID No.8 | SEQ ID No.8 |
| CDR3 | SEQ ID No.9 | SEQ ID No.9 | SEQ ID No.9 |
| FR4 | SEQ ID No.10 | SEQ ID No.10 | SEQ ID No.10 |
EXAMPLE 2 expression and purification of anti-PD-L1 nanobodies
1. Construction of anti-PD-L1 nanobody expression plasmid
The humanized sequences of the anti-PD-L1 nanobody designed according to Table 2, cloning plasmids of four sequences of the Kirschner organism total gene were named pUC57-NB43, pUC57-M2, pUC57-M5 and pUC57-M7, respectively.
pUC57-NB43 Structure description: a recombinant plasmid obtained by inserting the encoding gene (SEQ ID No. 15) of camelid nanobody NB43 between NdeI and SmaI cleavage sites of pUC57 vector.
pUC57-M2 Structure description: the gene encoding the humanized nanobody M2 (SEQ ID No. 13) was inserted into the pUC57 vector between NdeI and SmaI cleavage sites to obtain a recombinant plasmid.
pUC57-M5 Structure description: the gene encoding the humanized nanobody M5 (SEQ ID No. 14) was inserted into the pUC57 vector between NdeI and SmaI cleavage sites to obtain a recombinant plasmid.
pUC57-M7 Structure description: the gene encoding the humanized nanobody M7 (SEQ ID No. 16) was inserted into the pUC57 vector between NdeI and SmaI cleavage sites to obtain a recombinant plasmid.
The four synthetic cloning plasmids are digested by NdeI and XhoI, are connected and cloned to pET26b vector (Novagen) framework fragments which are digested by the same, and are correctly obtained into corresponding recombinant expression plasmids through sequencing verification.
The results of the digestion identification of the four recombinant expression plasmids are shown in FIG. 1. As can be seen from the figure, the plasmid fragment after NdeI and XhoI double digestion is 5225bp, the target fragment is about 438bp, and the identification result meets the expectations.
Structural description of recombinant expression plasmid pET26b-NB 43: the recombinant vector was obtained by inserting a DNA fragment of the target gene (SEQ ID No. 15) between NdeI and XhoI, which were the cleavage sites of the pET26b vector. The 1 st to 6 th sites of SEQ ID No.15 are NdeI cleavage sites, the 7 th to 66 th sites are coding genes of signal peptide, the 67 th to 432 th sites are coding genes of NB43 camel-source nanobody, and the 433 th to 438 th sites are XhoI cleavage sites.
Structural description of recombinant expression plasmid pET26 b-M2: and (3) inserting a target gene DNA fragment (SEQ ID No. 13) between NdeI and XhoI of the enzyme cutting site of the pET26b vector. The 1 st to 6 th sites of SEQ ID No.13 are NdeI cleavage sites, the 7 th to 66 th sites are coding genes of signal peptide, the 67 th to 432 th sites are coding genes of M2 humanized nano antibody (shown as SEQ ID No. 11), and the 433 th to 438 th sites are XhoI cleavage sites.
Structural description of recombinant expression plasmid pET26 b-M5: the recombinant vector was obtained by inserting a DNA fragment of the target gene (SEQ ID No. 14) between NdeI and XhoI, which were the cleavage sites of the pET26b vector. The 1 st to 6 th sites of SEQ ID No.14 are NdeI cleavage sites, the 7 th to 66 th sites are coding genes of signal peptide, the 67 th to 432 th sites are coding genes of M5 humanized nano antibody (shown as SEQ ID No. 12), and the 433 th to 438 th sites are XhoI cleavage sites.
Structural description of recombinant expression plasmid pET26 b-M7: the recombinant vector was obtained by inserting a DNA fragment of the target gene (SEQ ID No. 16) between NdeI and XhoI, which were the cleavage sites of the pET26b vector. The 1 st to 6 th sites of SEQ ID No.16 are NdeI cleavage sites, the 7 th to 66 th sites are coding genes of signal peptide, the 67 th to 432 th sites are coding genes of M7 humanized nano antibody, and the 433 th to 438 th sites are XhoI cleavage sites.
2. anti-PD-L1 nanobody expression
The four recombinant expression plasmids of pET26b-NB43, pET26b-M2, pET26b-M5 and pET26b-M7 constructed in the first step are transformed into BL21 (DE 3), and the recombinant host cells are named as pET26b-NB43/BL21 (DE 3), pET26b-M2/BL21 (DE 3), pET26b-M5/BL21 (DE 3) and pET26b-M7/BL21 (DE 3), respectively. Four recombinant host cells were selected for monoclonal, 5ml LB kana resistance cultureThe medium was incubated at 37℃and 180rpm overnight. The following day according to 1:100 proportion expansion culture to 100ml, shaking bacteria to OD 600nm The value was about 0.6-1.0, and 1mM IPTG was added thereto for induction of expression at 28℃and 180 rpm.
3. anti-PD-L1 nanobody purification
And step two, extracting protein from the induced bacterial liquid, wherein the operation steps are as follows:
osmotic shock method: centrifuging 12000g of induced bacterial liquid for 1min, and collecting bacterial cells; adding solution I (formula: 200g/L sucrose, 1mM EDTA,30mM Tris,HCl to adjust pH to 8.0) to suspend thallus, lightly blowing with gun head, mixing, and lightly shaking with ice bath for 10min; the supernatant was removed by centrifugation at 8000g for 10min at 4 ℃; add 4ml of solution II (5 mM MgCl) 2 ) Suspending, lightly blowing and suspending by using a gun head, ice-bathing and lightly shaking for 10min; centrifuging at 12000g for 15min, and collecting supernatant to obtain periplasmic space protein.
Affinity chromatography: ni-NTA 6FF His tag protein purification kit is adopted for carrying out Ni column affinity chromatography. Periplasmic space protein supernatant and binding/washing buffer in a volume ratio of 1:1, uniformly mixing, standing, fully incubating, and purifying by a column; balancing the column by using a binding/washing buffer solution with the volume of five times that of the column, adding a periplasmic space protein supernatant binding/washing buffer solution into the column, and flowing out through the pre-packed column by gravity; washing the column with binding/washing buffer and collecting the flow-through until absorbance 280nm of the flow-through approaches baseline; the elution buffer elutes the recombinant protein on the column.
Ultrafiltration liquid exchange: the purified elution buffer was added to an Amicon Ultra-0.5 centrifugal filtration device (Millipore Co.) and centrifuged at 10000g in batches for 3min until about 150. Mu.l of solution remained; 300. Mu.l PBS (pH 7.4) was gently added, and 10000g was centrifuged to a remaining 150. Mu.l, and repeated three times; samples were collected from PBS (pH 7.4) elution ultrafiltration tubes and subjected to SDS-PAGE for detection (FIG. 2). Experiments show that the target proteins are obtained by four nano antibodies of NB43, M2, M5 and M7, the molecular weights are respectively 14.25KD,14.32KD and 14.29KD, the purity is more than 90% according to the expectations, and the nano antibodies can be used for molecular level activity evaluation experiments.
Example 3 anti-PD-L1 nanobody affinity assay
The binding affinity of the humanized designed anti-PD-L1 humanized nanobody to the recombinant human PD-L1 antigen (R & D company) was detected by surface plasmon resonance (Surface Plasmon Resonance, SPR). Coupling the conjugate recombinant human PD-L1 antigen to Series S Sensor Chip CM chip (GE company), loading Biacore T200 intermolecular interaction analysis system (GE company), and flowing the analyte anti-PD-L1 humanized nano antibody on the chip surface (the temperature is 25 ℃, the flow speed is 30 mu L/min, the binding time is 90 seconds, and the dissociation time is 150 seconds), wherein the conjugate has binding activity with the analyte, the refractive index of the gold film surface is changed, and finally the SPR angle is changed. By detecting SPR angle changes, biacore T200 Evaluation Software program software outputs raw data and performs a 1:1 binding pattern for kinetic fitting analysis to detect affinity constants of humanized antibodies to PD-L1 antigen (FIG. 3). The experimental results showed that: nanobody NB43, M2, M5 and M7 affinities were 6.772nM,2.667nM,1.047nM and 19.84nM, respectively. Wherein, compared with camelid nanobody NB43, the affinity of anti-PD-L1 humanized nanobody M2 and M5 is respectively improved by 2.54,6.47 times.
In conclusion, on the basis of the camel-derived nanobody for resisting PD-L1, humanized transformation and experiments prove that the affinity of the anti-PD-L1 humanized nanobody M2 and the anti-PD-L1 humanized nanobody M5 is respectively improved by 2.54,6.47 times, and the humanized nanobody specifically combined with PD-L1 is obtained. Humanized engineering of camel-derived nanobodies against PD-L1 is expected.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> Hualan Gene engineering Co., ltd
<120> a humanized nanobody against PD-L1 and use thereof
<130> GNCLN221430
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Claims (12)
1. An anti-PD-L1 nanobody comprising 3 complementarity determining regions and 4 framework regions;
the 3 complementarity determining regions are CDR1, CDR2 and CDR3;
the amino acid sequence of the CDR1 is SEQ ID No.4;
the amino acid sequence of the CDR2 is SEQ ID No.6;
the amino acid sequence of the CDR3 is SEQ ID No.9;
the 4 framework regions are FR1, FR2, FR3 and FR4;
the amino acid sequence of the FR1 is SEQ ID No.2;
the amino acid sequence of the FR2 is SEQ ID No.5;
the amino acid sequence of the FR3 is SEQ ID No.8;
the amino acid sequence of FR4 is SEQ ID No.10.
2. The nanobody of claim 1, wherein: the nanobody is A1) or A2) as follows:
a1 A nano antibody with an amino acid sequence shown as SEQ ID No. 12;
a2 A protein label is connected to the N end and/or the C end of the amino acid sequence shown in SEQ ID No.12, and the nano antibody is obtained.
3. A biological material associated with the nanobody of claim 1 or 2, characterized in that: the biomaterial is any one of B1) to B12):
b1 A nucleic acid molecule encoding the nanobody of claim 1 or 2;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1);
b4 A recombinant vector comprising the expression cassette of B2);
b5 A recombinant microorganism comprising the nucleic acid molecule of B1);
b6 A recombinant microorganism comprising the expression cassette of B2);
b7 A recombinant microorganism containing the recombinant vector of B3);
b8 A recombinant microorganism comprising the recombinant vector of B4);
b9 A transgenic animal cell line comprising the nucleic acid molecule of B1);
b10 A transgenic animal cell line comprising the expression cassette of B2);
b11 A transgenic animal cell line comprising the recombinant vector of B3);
b12 A transgenic animal cell line comprising the recombinant vector of B4).
4. A biomaterial according to claim 3, wherein: b1 The nucleic acid molecule is any one of the following:
c1 A DNA molecule with a nucleotide sequence shown in 67-432 of SEQ ID No. 14;
c2 A DNA molecule with a nucleotide sequence shown in positions 7-432 of SEQ ID No. 14;
c3 A DNA molecule with a nucleotide sequence shown as SEQ ID No. 14.
5. A polypeptide molecule comprising the nanobody of claim 1 or 2.
6. An antibody drug conjugate comprising the nanobody of claim 1 or 2.
7. A kit comprising a nanobody according to claim 1 or 2 or a biological material according to claim 3 or 4 or a polypeptide molecule according to claim 5 or an antibody drug conjugate according to claim 6.
8. The method for preparing the nanobody according to claim 1 or 2, comprising the steps of: introducing a nucleic acid molecule encoding the nanobody of claim 1 or 2 into a host cell to obtain a recombinant host cell expressing the nanobody, and culturing the recombinant host cell to obtain the nanobody.
9. The method of manufacturing according to claim 8, wherein: the recombinant host cell may further comprise the following steps after culturing the recombinant host cell: firstly, obtaining periplasmic space protein by a osmotic shock method, and then purifying the periplasmic space protein by affinity chromatography and ultrafiltration liquid exchange in sequence to obtain the nano antibody.
10. The method of manufacturing according to claim 8, wherein: the nucleic acid molecule encoding the nanobody is the nucleic acid molecule of claim 4.
11. The method of manufacturing according to claim 8, wherein: the host cell is a microbial cell.
12. Any of the following applications:
e1 Use of the nanobody of claim 1 or 2 in the preparation of a product for detecting PD-L1;
e2 Use of the biomaterial of claim 3 or 4 for the preparation of a product for the detection of PD-L1;
e3 Use of the polypeptide molecule of claim 5 or the antibody drug conjugate of claim 6 in the preparation of a product for detecting PD-L1;
e4 Use of the kit of claim 7 for the preparation of a product for detecting PD-L1;
e5 Use of the nanobody of claim 1 or 2 for the preparation of a product that binds to PD-L1;
e6 Use of the biomaterial of claim 3 or 4 for the preparation of a product that binds to PD-L1;
e7 Use of the polypeptide molecule of claim 5 or the antibody drug conjugate of claim 6 for the preparation of a product that binds to PD-L1;
e8 Use of the kit of claim 7 for the preparation of a product that binds to PD-L1;
e9 Use of the nanobody of claim 1 or 2 for the preparation of a product for diagnosing or treating PD-L1 expressing tumors;
e10 Use of the biomaterial according to claim 3 or 4 for the preparation of a product for the diagnosis or treatment of a PD-L1 expressing tumor;
e11 Use of the polypeptide molecule of claim 5 or the antibody drug conjugate of claim 6 in the manufacture of a product for diagnosing or treating a PD-L1 expressing tumor;
e12 Use of the kit of claim 7 for the preparation of a product for diagnosing or treating a PD-L1 expressing tumor.
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| ES2899036T3 (en) * | 2016-08-04 | 2022-03-09 | Innovent Biologics Suzhou Co Ltd | Anti-PD-L1 nanobody and its use |
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| CN108713027A (en) * | 2015-12-17 | 2018-10-26 | 詹森生物科技公司 | Specifically bind the antibody and application thereof of HLA-DR |
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