CN110894239B

CN110894239B - Humanized bispecific nanobody targeting EGFR dimer interface

Info

Publication number: CN110894239B
Application number: CN201911024764.3A
Authority: CN
Inventors: 李黄金; 赵林; 温碧燕
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2021-07-13
Anticipated expiration: 2039-10-25
Also published as: CN110894239A

Abstract

The invention discloses a humanized bispecific nanobody targeting an EGFR dimer interface, and the amino acid sequence of the humanized bispecific nanobody is shown in SEQ ID NO. 1. The antibody can be combined with PBMCs in an in vitro test to effectively enhance the inhibition effect on tumor cells over-expressed by EGFR. The bispecific nanobody can target an EGFR dimerization interface, an NK cell Fc gamma RIIIa receptor and human serum albumin, and has tumor inhibition activity. The method has the potential of developing a therapeutic nano antibody medicament which has the homologous and heterodimerization functions of resisting EGFR, the conventional antibody ADCC (antibody dependent cellular cytotoxicity) and long half life, and provides a new method for solving the problem of drug resistance of EGFR targeted medicaments.

Description

Humanized bispecific nanobody targeting EGFR dimer interface

Technical Field

The invention relates to the technical field of genetic engineering, and particularly relates to a humanized bispecific nanobody targeting an EGFR dimer interface.

Background

The epidermal growth factor receptor (EGFR or Her 1/ErbB 1) is an important member of the human epidermal receptor family, plays an important role in the growth and differentiation of normal epidermal cells, and is an effective target for treating many epithelial cancers. EGFR, a Receptor Tyrosine Kinase (RTK), comprises a glycosylated extracellular domain, a single hydrophobic transmembrane segment, an intracellular portion (containing a membrane proximal segment, a protein kinase domain, and a carboxyl terminus). Binding of ligands such as Epidermal Growth Factor (EGF) to cell surface EGFRs results in exposure of the dimer interface and heterodimerization with either homologous or heterologous to another EGFR family member. Dimerization results in RTKs activating and phosphorylating specific tyrosine residues, thereby initiating mitotic signaling cascades and other cellular activities. EGFR overexpression and mutation in lung, breast, gastric, colorectal, head and neck, pancreatic and glioblastoma makes EG FR an ideal target for anti-tumor therapy. Monoclonal antibodies cetuximab and panitumumab have been used as EGF R inhibitors for anticancer therapy, but these drugs have a low clinical efficacy (about 15%).

"dimerization" is a key and common step that must be followed for activation of EGFR family receptors, and it is possible to effectively prevent homo-or heterodimerization and activation of receptors through steric hindrance by antibodies by selecting, as a targeting site of antibodies, dimer interface regions highly conserved in evolution, particularly β -loops of dimer interface regions directly involved in dimer formation. Single nanobodies may have difficulty reaching the conventional antibody level in vivo in anti-tumor activity of monospecific antibodies directed against the signaling pathway due to lack of ADCC effect of conventional antibodies. ADCC is mainly dominated by NK cells, and the construction of the bispecific antibody by using the nanobody resisting NK cell surface Fc receptor FcyRIIIa (also called CD16) can effectively endow the nanobody with ADCC. The stability of the nanobody in serum is also a restrictive problem of its druggability. The albumin is used as a carrier protein, so that the serum stability of the small molecular drug can be effectively improved, and the pharmacokinetic behavior of the drug in vivo can be improved. It is a desirable strategy to mediate drug conjugation to albumin in vivo with nanobodies targeting human serum albumin, thereby improving drug half-life and pharmacokinetic behavior.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a humanized bispecific nanobody targeting an EGFR dimer interface.

The first purpose of the invention is to provide a humanized bispecific nanobody targeting an EGFR dimer interface.

The second purpose of the invention is to provide a gene for coding the humanized bispecific nanobody targeting the EGFR dimer interface.

The third object of the present invention is to provide a recombinant vector.

The fourth purpose of the invention is to provide a recombinant strain.

The fifth purpose of the invention is to provide the application of one or more of the humanized bispecific nanobody, the gene, the recombinant vector or the recombinant strain in the preparation of drugs for treating EGFR positive tumors and/or inhibiting the proliferation of EGFR positive tumor cells.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention designs a bispecific nanobody targeting an EGFR dimerization interface and an NK cell Fc gamma RIIIa receptor (CD16) by using a nanobody sequence targeting human serum albumin as a coupling medium. The bispecific nanobody (TSsdAb) adopts sdAbEGFR- (G)₄S)₃-sdAbHSA-(G₄S)₃the-sdAbFcyRIIIa-His 6 mode, wherein sdAbEGFR is the anti-EGFR screened in this laboratory^237-Single domain antibodies, sdabhas and sdabfcyriiia are reported in the literature as anti-human albumin and anti-fccyriiia, respectively, (G)₄S)₃For flexible linkers, His6 is an expression tag.

The bispecific nano antibody gene is obtained by an artificial synthesis method, engineering bacteria are constructed by a pichia pastoris X33 secretory expression system, and target protein is purified in a small scale by cation exchange chromatography and nickel column affinity chromatography. The specific binding capacity of the bispecific antibody and the cell surface EGFR is analyzed by flow cytometry by taking A431 as a target cell, and the antibody is proved to have a dose-dependent binding effect. And the killing effect of the bispecific nanobody on EGFR positive tumor cells alone and/or in combination with NK cells is found, and meanwhile, the bispecific nanobody generates a certain Antibody Dependent Cellular Cytotoxicity (ADCC) effect on cells.

Therefore, the invention claims a humanized bispecific nanobody targeting an EGFR dimer interface, and the amino acid sequence of the humanized bispecific nanobody is shown in SEQ ID NO 1.

The gene of the humanized bispecific nanobody with the coding targeting EGFR dimer interface has the nucleotide sequence shown in SEQ ID No. 2.

A recombinant vector is a plasmid connected with the gene.

Preferably, the plasmid is pGAPZ α a.

A recombinant strain is a host bacterium carrying the recombinant vector.

Preferably, the host bacterium is Pichia pastoris X33.

The invention further claims application of one or more of the humanized bispecific nanobody, the gene, the recombinant vector or the recombinant strain in preparation of drugs for treating EGFR positive tumors and/or inhibiting proliferation of EGFR positive tumor cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a humanized bispecific nanobody targeting on an EGFR dimerization interface. The antibody can be combined with PBMCs in an in vitro test to effectively enhance the inhibition effect on tumor cells expressing the EGFR. It is a bispecific nano antibody which can target EGFR dimerization interface, NK cell Fc gamma RIIIa receptor and human serum albumin, and has antitumor activity. The method has the potential of developing a therapeutic nano antibody medicament which has the homologous and heterodimerization functions of resisting EGFR, the conventional antibody ADCC (antibody dependent cellular cytotoxicity) and long half life, and provides a new method for solving the problem of drug resistance of EGFR targeted medicaments.

Drawings

FIG. 1 shows the sequence of NK-77 gene.

FIG. 2 shows the results of double digestion; m: DNA Marker DL 2000; pGAPZ α A plasmid; 2. a target gene.

FIG. 3 is a colony PCR assay; m: DNA Marker DL 2000; 1-20: and (4) single colonies.

FIG. 4 is a process for screening high expression strains; m: protein Marker; empty: an empty host; 1-16: the transformant culture supernatant.

FIG. 5 shows the purification of a protein of interest; m: portein Molecular Weight Marker (Low); 1: collecting the bacterial supernatant; 2: concentrating by 10 times with ammonium sulfate; 3: CM column pre-sample; 4: CM column breakthrough; 5: eluting the sample by a CM column; 6: ni column penetration sample; 7: eluting the sample by using a Ni column.

Fig. 6 is a flow cytometry analysis (His) 6-tagged bispecific nanobody binding capacity on a431 cell surface; a shows the effect of bispecific nanobody concentration on binding capacity. After stimulating AB431 cells with Epidermal Growth Factor (EGF) at 7. mu.g/ml, A431 cells were treated with nanobodies at different concentrations. Different color plots represent different antibody concentration sets; b histograms represent 3, 30 and 300. mu.g/ml bispecific antibody treated cells, respectively. The x-axis is the antibody concentration and the y-axis is the fluorescence intensity.

FIG. 7 shows the MTT assay for the antiproliferative effect of antibodies on different cell lines; a shows that different antibodies and PBMCs have anti-proliferation effects on different cells, EGFR dimer Nb77, EGFR-HSA-CD16, EGFR-HSA-CD16 and PBMCs have inhibition effects on A431 and H292 cells, and the effect on 3T3 cells is less obvious; b shows the antiproliferative effect of EGFR dimer Nb77, EGFR-HSA-CD16, EGFR-HSA-CD16 on A431 cells in cooperation with PBMCs, and all show the dose-dependent effect, the ordinate shows the antiproliferative rate, and the abscissa shows the antibody drug concentration (. mu.M).

Figure 8 is a humanized bispecific nanobody serum stability study targeting EGFR dimer interface.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The expression vector pGAPZ alpha A host, the cloning host E.coli DH5 alpha and the expression host Pichia X-33 glycerol seed, A431 cell, H292 cell and 3T3 cell are all preserved in the laboratory.

Single domain antibody phage library was purchased from Endomen (Beijing) Biotech Ltd, endonucleases such as Xho I, Xba I, Bln I, DL2000^TMDNA Marke, Taq DNA polymerase, T4 DNA ligase, Ampicillin (Ampicillin, Amp), was purchased from TaKaRa, a Dalibao organism.

The bispecific nanobody was purified using Ni-NTA, CM-Sepharose, Sephadex-G25(GE Healthcare, hamshire, UK).

FITC-labeled mouse anti-HIS-labeled antibody was purchased from Beijing Baiolai Boke technologies, Inc.

Human epidermal carcinoma A431 cells and human lung carcinoma NCI-H292 cells were purchased from Jennio Biotech, Inc., China.

DMEM, RPMI-1640, Fetal Bovine Serum (FBS) and supplements were purchased from HyClone, USA.

Example 1 Gene design of humanized bispecific Nanobody targeting EGFR dimer interface

According to the nano antibody HSA pm6A6 which is published by Genbank and targets human serum albumin, the nano antibody which is used for resisting NK cell surface Fc receptor Fc gamma RIIIa (CD16) and the existing nano antibody EGFR dimer Nb77 which targets EGFR dimer interface in a laboratory, the humanized bispecific nano antibody which targets EGFR dimer interface is constructed.

Wherein, the amino acid sequence of HSA pm6A6 is:

AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS (the amino acid sequence is shown in SEQ ID NO. 3);

the amino acid sequence of the nano antibody for resisting NK cell surface Fc receptor Fc gamma RIIIa (CD16) is as follows:

EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVS (the amino acid sequence is shown in SEQ ID NO. 4);

the amino acid sequence of the existing nano antibody EGFR dimer Nb77 targeting the EGFR dimer interface in the laboratory is as follows:

QVQLLESGGGLVQPGGSLRLSCAASGFKVIDETMGWVRQAPGKGLEWVSTIMDPNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGQEKGAEKLKYWGQGTLVTVS (the amino acid sequence is shown in SEQ ID NO. 5).

The above 3 kinds of nanobody sequences are connected by a connecting peptide sequence of 'GGGGSGGGGSGGGGS' in the sequence of 'EGFR dimer Nb77-HSA pm6A6-Fc gamma RIIIa (CD 16)', namely:

(the amino acid sequence is shown as SEQ ID NO. 1).

The gene coding sequence is optimized according to pichia pastoris preferred codons, a small segment of signal peptide gene sequence (AAAAGAGAGGCTGAAGCT) is added at the 5 'end, and a His label and a stop codon TAG (the nucleotide sequence of which is shown in SEQ ID NO. 2) are added at the 3' end; further, a protective base (GAGD) and an Xho I cleavage site (CTCGAG) were added to the 5 'end, an Xba I cleavage site (TCTAGA) and a protective base (CTAC) were added to the 3' end, and the total length was 1217bp, and the gene sequence was named NK-77 (shown in FIG. 1). All gene sequences are synthesized by Wuhan Kingkurui bioengineering GmbH and cloned to pUC57 vector to obtain recombinant vector pUC 57-NK-77.

Example 2 construction of recombinant plasmids containing humanized bispecific Nanobodies targeting EGFR dimer interface

First, experiment method

Primers were designed using Primer Premier 6.0 software, and synthesized by Wuhan Kingrui bioengineering, Inc. and designed as shown in Table 1.

Table 1 primer design sequences:

f: an upstream primer; r: downstream primer

Using the recombinant vector pUC57-NK-77 obtained in example 1 as a template, the target gene was amplified by PCR, and the pGAPZ α -A plasmid was extracted and subjected to restriction of enzymes and ligation of the target gene and the vector.

Second, experimental results

The target gene and the expression vector pGAPZ alpha A are subjected to double enzyme digestion by Xho I and Xba I at the same time, and after plasmid recovery, the agarose electrophoresis identification result is shown in figure 2. The vector size is 3.1Kb, and the target gene 1217 bp. After the recombinant plasmid NK-77-pGAPZ alpha A was transformed into E.coli DH5 alpha, it was spread on LB medium containing 100. mu.g/ml AMP and 20 single colonies were selected therefrom for colony PCR identification, the results are shown in FIG. 3, in which the gene sequence of strain No.4 is shown in FIG. 1 and is identical to the designed gene sequence. The resulting positive transformants were designated: NK-77-pGAPZ alpha A-DH5 alpha.

Example 3 transformation and expression of recombinant plasmids containing humanized bispecific Nanobodies targeting the EGFR dimer interface

First, experiment method

The positive transformant NK-77-pGAPZ alpha A-DH5 alpha obtained in example 2 was subjected to amplification culture, a large amount of recombinant plasmid NK-77-pGAPZ alpha A was extracted, and linearization of recombinant plasmid NK-77-pGAPZ alpha A was carried out in the reaction system: ddH₂30 mu.l of O, 20 mu.l of 10 XK buffer, 10 mu.l of BlnI and 140 mu.l of recombinant plasmid system, performing instant centrifugation for 10 seconds, and performing enzyme digestion in water bath at 37 ℃ for 2 hours to linearize the plasmid NK-77-pGAPZ alpha A.

Taking the prepared Pichia X-33 competent cells, adding linearized NK-77-pGAPZ alpha A plasmid for electrotransformation, screening high-resistance transformants by using a Zeocin YPD solid medium plate containing 1000 mu g/mL, identifying the high-resistance recombinants by colony PCR, and identifying whether the corresponding recombinants express target proteins by SDS-PAGE electrophoresis.

After the expression of the transformant is confirmed, performing shake flask fermentation to culture the transformant, centrifuging to process bacterial liquid, collecting fermentation supernatant, adding 0.385g of ammonium sulfate solid particles into each milliliter of supernatant, standing at 4 ℃ for 6 hours, centrifuging to remove the supernatant, taking precipitates, then re-suspending and dissolving the precipitates by using CM-Sepharose cation column equilibrium buffer solution, performing suction filtration on a sample by using a 0.45 mu m filter membrane, purifying a nano antibody by using CM-Sepharose cation exchange chromatography, and further purifying by using a Ni-NTA affinity chromatography column and Sephadex-g 25.

Second, experimental results

The recombinant DNA No.4 in example 2 was extracted, linearized and electrically transformed into wild-type Pichia pastoris X-33, and screened by a high-resistance plate to obtain 16 high-resistance transformants. The electrophoresis results of the supernatants obtained after 48 hours of culture in YPD medium are shown in FIG. 4.

The results showed that

transformants numbers

2, 8, 11, 14 and 16 all produced expression products of approximately 44kDa in size compared to the empty host culture supernatant. The glycerol strain of transformant No. 16 was selected for shake flask fermentation, and the antibody was concentrated and purified by CM-Sepharose cation exchange chromatography, and then purified by Ni-NTA affinity column, Sephadex-g25 to a purity of over 95% (as shown in FIG. 5). The finally obtained humanized bispecific nanobody targeting the EGFR dimer interface is named as EGFR-HSA-CD 16.

Example 4 specific binding Capacity of humanized bispecific Nanobody targeting EGFR dimer interface to cells

First, experiment method

To investigate the ability of humanized bispecific nanobodies targeting the EGFR dimer interface to specifically bind EGFR on cells, EGFR-overexpressed a431 cells were used as target cells. After treatment of a431 cells with EGF and the purified antibody EGFR-HSA-CD16 at various concentrations, the ability of the antibody to bind to the cells was analyzed by flow cytometry.

Culturing A431(EGFR overexpression) in cell culture medium containing 10% (v/v) FBS, and collecting 1 × 10⁶The cells were blocked in a 1.5ml EP tube with 2% BSA in PBS buffer at 4 ℃ for 1h, centrifuged at 1000rpm/min for 5min, washed three times with PBS and the supernatant discarded. The humanized bispecific nanobody EGFR-HSA-CD16 targeting the EGFR dimer interface prepared in example 4 was added to cells and incubated at 4 ℃ for 2 h. Experimental groups (3, 30 and 300. mu.g/ml) were set to which antibodies at different concentrations were added, while blank control groups to which the above antibodies were not added were set. After washing with PBS three times, the supernatant was discarded, and 100. mu.l of a goat anti-mouse secondary antibody diluted 500 times with FITC-labeled anti-His tag was added, followed by incubation for 30min in the dark. After three PBS washes, the supernatant was discarded, and the pellet was resuspended in 300 μ l of 4 ℃ pre-cooled PBS, transferred to a flow tube and assayed for antibody binding activity using a Coulter ELITE flow cytometer (Beckman, USA).

Second, experimental results

Flow cytometry analysis showed that the binding capacity of the humanized bispecific nanobody EGFR-HSA-CD16 targeting the EGFR dimer interface increased with increasing antibody concentration (3, 30 and 300 μ g/ml) and the signal peak in the high dose antibody group was significantly enlarged (as shown in figure 6). These results show that the binding of the humanized bispecific nanobody EGFR-HSA-CD16 targeting the EGFR dimer interface to the cell surface is specific and concentration gradient dependent.

Example 5 humanized bispecific Nanobody targeting EGFR dimer interface and anti-tumor proliferation Effect

First, experiment method

The effect of the bispecific nanobody EGFR-HSA-CD16 on the proliferation of different cell lines in vitro by the action of PBMCs (peripheral blood mononuclear cells) was examined using the MTT assay, wherein cells with different EGFR phenotypes were used.

NIH 3T3 cells (EGFR is weakly expressed), A431 cells and H292 cells (EGFR is highly expressed) are respectively cultured in a cell culture medium containing 10% (v/v) FBS until culture bottles are full, the adherent cells are digested by pancreatin containing 0.25% EDTA, and the cell suspension is planted into a 96-well cell culture plate according to 5000 cell numbers per well. After 24 hours of culture, the maintenance medium containing no FBS was replaced, and culture was continued for 24 hours. The experimental group and the control group were set up, and the medium was replaced with a medium containing different concentrations of the antibody (EGFR dimer interface targeting nanobody EGFR dimer Nb77, EGFR dimer interface targeting bispecific nanobody EGFR-HSA-CD16 prepared in example 4) while adding 5 × 10 per well⁴The pretreated PBMCs were cultured for 48h, and then the antitumor cell proliferation activity of each group was measured according to the MTT kit instructions. All experiments were performed in triplicate. Percent inhibition was calculated using the formula: (OD)_{Control group}-OD_{Experimental group})/OD_{Control group}×100％。

Second, experimental results

The antibody EGFR-HSA-CD16 has a significant inhibitory effect on EGFR-overexpressing a431 cells, while it has a weaker inhibitory effect on EGFR-weakly-expressing mouse fibroblast 3T3 cells (as human EGFR is homologous to murine EGFR dimeric interface region, nanobodies targeting the human EGFR dimeric interface region can also recognize the murine EGFR dimeric interface region) (as shown in fig. 7A). Furthermore, the ability of EGFR-HSA-CD16 to inhibit proliferation was similar to the nanobody EGFR dimer Nb77 previously constructed in this laboratory targeting the EGFR dimer interface region, both of which had dose-dependent effects (as shown in fig. 7B). Wherein, under the coaction of the EGFR-HSA-CD16 and the PBMCs, the inhibition effect of the bispecific nanobody EGFR-HSA-CD16 on tumor cells is obviously enhanced. The results showed that the nanobody had ADCC effect (antibody-dependent cellular cytotoxicity).

Example 6 humanized bispecific Nanobody serum stability Studies targeting the EGFR dimer interface

First, experiment method

The humanized bispecific nano antibody EGFR-HSA-CD16 targeting the EGFR dimer interface and the humanized monospecific nano antibody EGFR dimer Nb77 targeting the same are respectively mixed with human serum, incubated in water bath at 37 ℃ for 1d, 3d and 7d respectively, and then sampled for SDS-PAGE electrophoresis. Then, membrane transfer was performed, PBS was washed three times, and then 100 μ l of goat anti-mouse secondary antibody diluted 500 times with HRP-labeled anti-His tag was added, and incubation was performed for 30min in the dark. Adding substrate DAB, developing for 10-15min, adding 1M H₂SO₄The reaction was terminated. The stability of the antibody was photographed and analyzed.

Second, experimental results

The humanized bispecific nanobody EGFR-HSA-CD16 targeting the EGFR dimer interface did not degrade during the interaction with serum, and remained stable at 1d, 3d, and 7d, while the same targeted humanized monospecific nanobody EGFR dimer Nb77 degraded at 3d and degraded by more than half at 7d (as shown in fig. 8), indicating that the humanized bispecific nanobody EGFR-HSA-CD16 targeting the EGFR dimer interface is much more stable in serum than the same targeted humanized monospecific nanobody EGFR dimer Nb 77. The reason is that the nano antibody EGFR-HSA-CD16 has a fragment which is targeted to bind with human serum albumin, and the binding can reduce the degradation speed of EGFR-HSA-CD16, so that the half-life period of the bispecific nano antibody in serum is greatly prolonged.

Sequence listing

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Claims

1. A humanized bispecific nanobody targeting an EGFR dimer interface is characterized in that the amino acid sequence of the humanized bispecific nanobody is shown as SEQ ID NO: 1.

2. A gene for coding a humanized bispecific nanobody targeting an EGFR dimer interface is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.

3. A recombinant vector which is a plasmid to which the gene of claim 2 is ligated.

4. A recombinant strain which is a host bacterium carrying the recombinant vector of claim 3.

5. Use of one or more of the humanized bispecific nanobody of claim 1, the gene of claim 2, the recombinant vector of claim 3, the recombinant strain of claim 4 for the preparation of a medicament for the treatment of human EGFR-positive tumors and/or for inhibiting the proliferation of EGFR-positive tumor cells, wherein said tumor is human epidermal carcinoma.