Patent Application
20170058021
The present invention relates to the field of biochemistry and pharmaceutical technologies, particularly to nanobodies that bind to human pulmonary surfactant protein A (SP-A) with specificity.
In the beginning of 20th century, the Nobel Prize winner German scientist Paul Ehrlich proposed the idea of “magic bullet” for future drug development, i.e., an ideal drug that would selectively destroy diseased cells without affecting healthy cells. In the past several decades, scientists have been exploring to develop such ideal drugs.
In the 1970s, targeted drug delivery system was developed and widely used for the treatment of cancer. Meanwhile, with the advancement in research, new targeted drug delivery carriers has emerged, the routes of administration has been broadened, and targeted drug delivery system has been expanded to treat many diseases other than cancer.
Developing targeted drugs for respiratory diseases is one of the hotspots, and it is primarily focused on the following areas:
Starting from the earlier 1950s, inhaled corticosteroids have been used for the treatment of asthma and COPD. Since then, with the improvement in inhaled drugs and devices, inhaled corticosteroids have become the main therapeutic agents for the treatment of asthma and COPD. However, inhaled drugs are mainly suitable for topical treatment of airways diseases, and are not effective against parenchyma and interstitial lung diseases due to low bioavailability.
Passive Lung-Targeting Drugs Through Drug Carriers.
1. Currently, a variety of drug carriers such as liposomes, microparticles, microspheres have been used in the research of lung-targeted drug delivery. However, these passive targeting drugs have poor tissue selectivity, and cannot avoid significant residue in the liver, spleen and other organs. Therefore, they don't achieve optimal targeting effect.
The ligand-receptor or antigen-antibody binding is a special recognition mechanism of the human body, and it has been reported that the mechanism could achieve active drug targeting to enhance drug efficacy and reduce the side effects. For example, a composite drug made of paclitaxel liposome and a monoclonal antibody against the epidermal growth factor has anti-tumor effect 25 times greater then that of the drug without the monoclonal antibody. Thus, to achieve ideal active lung targeting effect, it is critical to find a receptor in the lung tissue with high specificity and prepare a targeting ligand with high affinity. Studies have shown that pulmonary alveolar type II epithelial cells which account for 16% of the total cells in lung parenchyma have proliferation and secretion functions. Type II cells can synthesize and secrete pulmonary surfactant. The main components of the pulmonary surfactant are lipids (90%) and proteins (10%), and the proteins are specific pulmonary surfactant proteins (SP). SP has been named as SP-A, SP-B, SP-C, SP-D, SP-A based on the discovery order, and SP-A was first discovered and has strong expression in pulmonary alveolar type II epithelial cells with abundant signals, and is rarely expressed in other tissues. Thus, SP-A is highly lung-specific, and is an ideal receptor in the lung tissue with specificity.
In addition to high affinity, an ideal targeting ligand should be small molecular weight, high tissue penetration, and weak immunogenicity. Antigen-antibody binding is the strongest recognition mechanism, and therefore antibody is the preferred ligand. However, although of high affinity, full antibodies are not ideal ligands due to their large molecular weight (with a relative molecular weight of 150,000), weak tissue penetration and strong immunogenicity. With the development of antibody and gene engineering technologies, antibody fragments (Fab, ScFv) now have the advantages of small molecular weight and weak immunogenicity, but they has lower stability and affinity than full antibodies.
In 1993, scientists from Belgian first reported the existence of Heavy Chain antibody (HCAbs) without the light chain in the blood of camelids. The variable domain (VHH) of the heavy chains of HCAbs has a complete and independent antigen-binding capacity, and if cloned, a single domain antibodies in the nanometer scale which are known as Nanobodies® (Nbs) can be obtained. Nanobody has many advantages as a ligand: 1) small molecular weight, strong tissue penetration, and high affinity. It has a molecular weight of only 15,000 which is the least molecular weight among the known antibody molecules; its ability to penetrate tissues is significantly superior to full antibody, and its affinity with specific antigen is of nmol scale. 2) Stable structure. It can maintain stability even if stored at 37° C. for a week, under high temperature (90° C.), or under strong denaturing conditions such as being exposed to chaotropic agent, protease and strong PH value. 3) Weak immunogenicity. As its gene has high homology with human VH III family, it has weak immunogenicity and good biocompatibility. Because of these advantages, nanobody has been studied extensively as a new antibody drug, but its use as targeted ligand for SP-A has not been reported.
The present invention provides a solution for the above-mentioned deficiencies of the prior art. The prior application CN104109207A discloses nanobodies that bind to rat's pulmonary surfactant protein A (SP-A), and the applicant continues to work on the nanobodies that bind to human pulmonary surfactant protein A (SP-A).
The present invention provides nanobodies that bind to human pulmonary surfactant protein A (SP-A) as well as the preparing methods and use of the same.
The present invention also provides nucleic acid encoding nanobodies that bind to pulmonary surfactant protein A.
The technical solutions are as follows:
In accordance with the first aspect of the present invention, a lung-targeting nanobody is provided. The nanobody comprises an amino acid sequence having the formula of Q(x)2LVESGG(x)2V (x)2G(x) SL(x) LS(x)24E (x)n2KG(x)4S(x)n3T(x)2Y(x) C(x)n4S(x)n5V(x)n6R; wherein x is any amino acid; n2˜n6 are positive integers; 1≦n2≦21; 1≦n3≦19; 1≦n4≦50; 1≦n5≦22; 1≦n6≦8. Preferably, 17≦n2≦21; n3 is 18 or 19; 16≦n4≦50; 17≦n5≦22; n6 is 7 or 8.
In accordance with another embodiment of the present invention, the nanobody comprise an amino acid sequence having the formula of Q(X1)LVESGG(X2)V(X3)G (X4)SL(X5) LS(X6) E (X7) KG(X8) S(X9) (T(X10) Y(X11) C(X12) S(X13) V(X14)R, wherein
X1 is selected from a group consisting of LQ and VK;
X2 is selected from a group consisting of GS, GL and DL;
X3 is selected from a group consisting of QS and QP;
X4 is G;
X5 is selected from a group consisting of I, S, R and T;
X6 is selected from a group consisting of
X7 is selected from a group consisting of
X9 is selected from a group consisting of
X11 is any amino acid or NULL;
X12 is selected from a group consisting of
X13 is selected from a group consisting of
X14 is selected from a group consisting of SGSAGTAC, PYPDPLEP.
Preferably, X11 is Y, or V.
In accordance with another embodiment of the present invention, the nanobody comprises an amino acid sequence comprising any of SEQ ID NOs 16 to 30.
In accordance with the second aspect of the present invention, the present invention provides nucleic acids encoding the lung-targeting nanobody. Said nucleic acids encode the nanobody described in claim 1.
In accordance with an embodiment of the present invention, the nucleic acid comprise a polynucleotide sequence comprising any of SEQ ID NOs 1 to 15.
In accordance with the third aspect of the present invention, the present invention provides a method of preparing the antibody, comprising the steps of:
In the method, preferably, the nanobody library in step 1 is pre-built anti pulmonary surfactant protein A nanobody libraries, by affinity selection.
Technical route of the method is shown in
In accordance with the fourth aspect of the present invention, the present invention provides the use of nanobody as targeted ligand for SP-A.
In accordance with a preferred embodiment of the present invention, the specific target of the nanobodies is pulmonary surfactant protein A (SP-A).
SP-A was the first discovered pulmonary surfactant protein, has strong expression in pulmonary alveolar type II epithelial cells with abundant signals, and is rarely expressed in other tissues. SP-A is highly lung-specific, and is an ideal lung-specific receptor. In accordance with embodiments of the present invention, alpacas was immunized with SP-A, an antibody library was built, affinity selection was employed to screen and identify genes with lung-targeting specificity, and SP-A nanobodies with high affinity were obtained by prokaryotic expression. In vivo and in vitro experiments were conducted to verify the nanobody has high specificity for targeting lung tissue.
Specifically, constructed anti pulmonary surfactant protein A (SP-A) nanobody library is incubated in fresh frozen sections of human lung, after several rounds of affinity selection, human lung tissue SP-A nanobody libraries is built, and 15 nanobodies strains which could bind human lung SP-A efficiently are screened out. Sequencing analysis showed they were all VHH sequences (nanobody sequences).
Nb4 had the highest affinity, and were selected as the preferred embodiments for prokaryotic expression to obtain nanobodies with a molecular weight of about 190,000 and a size of nanometer scale. In in vitro Werstern Blot and ELISA experiments, Nb4 showed good affinity with hSP-A, immunohistochemistry and in vivo imaging results showed its lung-targeting specificity as it could bind to natural SP-A in the lung tissue.
In accordance to another embodiment of the present invention, synthetic method was used to obtain the polypeptide of the human lung tissue nanobody.
To further optimize the human lung tissue nanobody of the present invention, the active region of the polypeptide sequences of the selected clones were tested. Wherein the polypeptide of Nb4 (without the MQAQKAG part) is obtained by synthetic method. Testing results showed that the functional polypeptides of Nb4 still has good lung-targeting distribution specificity after the removal of MQAQKAG.
The present invention provides human pulmonary surfactant protein A nanobody (hSPA-Nb) against the human pulmonary surfactant protein A (SP-A). And through a variety of methods are verified human lung tissue SPA-Nb prepared by the invention has a good lung-targeting distribution specificity. The operation flow of the present invention is shown in
In accordance with embodiments of the present invention, the human lung tissue SPA-Nb coding sequence refers to the nucleotide sequence of the SPA-Nb polypeptide, such as the sequences from SEQ ID NO.16 to SEQ ID NO.30 and its degenerate sequence. The degenerate sequence refers to sequences from SEQ ID NO.16 to SEQ ID No. 30 wherein one or more codons were substituted.
Corresponding amino acid codon see
The SPA-Nb coding sequences also include variants of SEQ ID NO.16 to SEQ ID No. 30 that encoding proteins with the same functions as SPA-Nb. Such variants include (but are not limited to): the deletion, insertion or substitution of a plurality (usually 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) of nucleotides, and the adding at the 5 ‘and/or 3’ end of a plurality of (typically less than 60, preferably less than 30, more preferably less than 10, the top for 5 or less) nucleotides.
Once the SPA-Nb coding sequence is obtained, large quantities of the recombinant sequences can be obtained. This is usually done by cloning the sequence into a vector, and transferred to the cells, then using conventional methods to isolate the sequences from the proliferated host cell.
In addition, the sequences can also be obtained by synthetic methods, as the length of the inhibitory factor of the nanobodies of the present invention is short. Typically, a number of small fragments can be synthesized first, and a long fragment can be formed by linking the small fragments.
In accordance with the present invention, various forms of vectors known in the art, such as those that are commercially available, can be used. For example, using a commercially available vector, the nucleotide sequence encoding the polypeptide of the invention can be operably linked to expression control sequence to form a protein expression vector.
As used herein, the term “operably linked” means the situation where part of the DNA sequence can affect the activity of other part of the DNA sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
In accordance with embodiments of the present invention, the term “host cell” includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cells, such as CHO cells, COS cells and the like.
The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, total protein extracted from fresh human lung tissue serves as an antigen which verify antibody targeting and specificity. These fragments or functional regions can be prepared using recombinant or synthesized by synthetic peptide synthesizer. Antibodies that bind unmodified human lung SPA gene product could be produced by immunizing animals with gene products of prokaryotic cells (such as E. coli); antibodies binding to post-translationally modified forms thereof can be acquired by immunizing animals with gene products produced by eukaryotic cells (e.g., yeast or insect cells).
The technical solution of the present invention has the following technical effects compared with the prior art:
The present invention provides nanobodies that bind to human pulmonary surfactant protein A (hSP-A) with specificity. The present invention take fresh frozen sections of lung as antigen, gene sequences with high affinity with hSP-A were obtained by constructing an SP-A antibody library and affinity selection, and nanobodies with high affinity and small molecule weight were obtained by induced expression of the gene sequences through a prokaryotic expression vector. Immunohistochemistry and in vivo imaging in nude mice showed the nanobodies have high specificity for targeting lung tissue. By providing nanobodies with lung-targeting specificity, the present invention provides tools for further research on lung-targeting ligands for targeted drug delivery for human lung diseases.
The present invention is further illustrated using the following embodiments, but any of the embodiments or its combinations thereof should not be construed as a limitation to the scope of the present invention.
1.1 the Preparation of Human Pulmonary Surfactant Protein A (hSP-A)
Grind 5 mg fresh human lung tissue with the mixture of protein lysate and PMSF in a tissue grinder for 3 min (60 HZ, 90S), centrifuged supernatant, measuring protein content (BCA).
1.2 hSP-A Identification:
Purified hSP-A was isolated by SDS-PAGE and transferred onto nitrocellulose membrane. It was sealed in 20% goat serum and incubated for 2 hours, then immune serum containing mouse polyclonal antibody against hSP-A (at room temperature for 2 hours, and washed 3 times with PBS) and serum containing anti-mouse IgG-HRP (at room temperature for 1 hours, washed 3 times with PBS) were added sequentially. Scanning of fluorescence scanner and photographs of the camera displays the target bands are around 35 Kd, 70 Kd, 120 Kd, multiple bands (
Elisa test was performed to measure the immunological activity of the purified protein. An Elisa plate with 96 wells were coated with purified hSP-A and an irrelevant protein, and incubated overnight at 4° C. The next day, it was sealed in 3% skim milk and incubated at 37° C. for an hour, then immune serum containing hSP-A monoclonal antibody (at room temperature for 2 hours, and washed 3 times with PBS) and serum containing goat anti-mouse IgG-HRP (at room temperature for 1 hours, washed 3 times with PBS) were added sequentially. TMB was added last to develop the image, and sulfuric acid was added to stop the reaction. The OD value of each well was measured using the chromogenic microplate, which showed that, compared with the control group, both purified hSP-A and SP-A monoclonal antibody had obvious binding activity (
Affinity selection technique was employed to screen the VHH antibody library with acetone fixed fresh frozen sections of human lung.
Preliminary screening by indirect Phage Elisa showed that 15 sequences had affinity value greater three times the affinity value for the negative control group, and these 15 sequences were positive clones (
3.1 Construction of hSPA-Nb Prokaryotic Expression Vector
The 15 clones selected by Phage ELISA were sent for sequencing (
Recombinant plasmid with correct sequence was transformed into E. coli BL21 (DE3), the expression conditions were optimized, and protein expression was induced at 25° C., 0.8 mmol/L IPTG. The expressed product was purified with nickel affinity chromatography and molecular sieve. SDS-PAGE electrophoresis showed that the expressed nanobody had a molecular weight of 19 kDa (
The 15 clones obtained by the present invention are effective lung-targeting ligands as their nucleotide sequences and amino acid sequences specifically bind to SP-A, which are listed below:
To further verify the affinity between hSPA-Nb and human pulmonary surfactant protein A, and whether hSPA-Nb has lung-specificity, Western blot and ELISA were used to preliminarily measure the antigen specificity of hSPA-Nb, and immunohistochemistry and in vivo imaging were used to verify its lung-specificity in vivo.
Purified human lung tissue SPA-Nb4, irrelevant nanobody (H1N1 nanobodies) and commercial anti-human SP-A monoclonal antibody were selected as the primary antibody to test the affinity between SPA-Nb4 and hSPA using Western blot and ELISA (using the same method described in section 1.2). The results showed that Nb4 had significant binding specificity with hSPA (
When A549 (lung), L-02 (liver), 293T (kidney) cells were grown and cover the cell plates to 95%-100%, PBS washed 3 times, incubated in fixative 30 min, PBS washed 3 times, 0.2% Triton X-100 permeabilization 5 min, blocked for 1 h by 20% goat serum, diluted primary antibody (human lung tissues Nb4-Fitc) for the experimental group, anti-human SP-A monoclonal antibody as a positive control group, and H1N1-Fitc nanobodies as a negative control group) was dropped on. The secondary antibody was anti-mouse-IgG-APC. The results showed that Nb4 and SPA monoclonal antibody (SPA-monopoly-ant) had significant binding effect with human lung tissue (shown as green/red), wherein the human lung tissue Nb4 binding ability is similar with SPA-monopoly-ant. All three antibodies had no obvious binding effect with human heart, liver, spleen, kidney, muscle tissues, nor had the negative control group (
The fresh human lung, liver, spleen, kidney and other tissue sections were fixed, diluted primary antibody (human lung tissues Nb4 for the experimental group, SP-A monoclonal antibody as a positive control group, and H1N1 nanobodies as a negative control group) was dropped on. The secondary antibody was His-IgG-HRP or anti-mouse-IgG-HRP. The results showed that human lung tissues Nb4 and SPA monoclonal antibody (SPA-monopoly-ant) had significant binding effect with human lung tissue (shown as brown), wherein Nb4 binding ability is similar with SPA-monopoly-ant. All three antibodies had no obvious binding effect with human heart, liver, spleen, kidney, muscle tissues, nor had the negative control group (
Sequence homology analysis showed that there is a high degree of homology between the amino acid sequence of human and mouse rSPA. Since it is easier to obtain in vivo imaging using nude mice, nude mice were chosen for testing specificity in vivo. Five-week-old nude mice were chosen, and after continuous inhalation anesthesia, 200 ul FITC-labeled nanobody was injected intravenously at the tail, and the dose was 1 mg/kg of the animal body weight. The nude mices were imaged at 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours after the injection, respectively. At the same time, 200 ul H1N1-Fitc nanobody was injected intravenously at the tail as the negative control group (
The above experiment was repeated using the functional region of the polypeptides of synthetic human lung tissues Nb4 and Nb17 (without the MQAQKAG portion). It was found that the synthetic polypeptides also binded to hSPA with specificity, and clustered around the lung in vivo testing.
Sequence homology comparative analysis was conducted on the selected 15 sequences, and it was found that human lung tissues Nb23, Nb25, Nb27, Nb29 and Nb39 had the same polypeptide sequence, human lung tissues Nb28 and Nb4 had high sequence similarity; while the rest of the sequences were quite different.
To further verify that the 15 nanobody sequences exhibits lung-targeting affinity with SP-A, 8 clones (excluding those with the same sequence as Nb4) were expressed and purified in accordance with the method described in Examples 5 and 6. Soluble expressions of these nanobdies were obtained, where Nb1 has the least protein expression concentration of 3 mg/L, while the rest of nanbodies have an average protein expression concentration of 8 mg/L.
In Western blot and ELISA, affinity was clearly shown in all 6 proteins, and the OD450 value in ELISA for 5 nanobodies, namely human lung tissues Nb11, Nb15, Nb17, Nb6 and Nb43 were 2 times greater than that of the negative control group. Immunohistochemical staining showed that these clones had strong affinity. All clones showed significant differences with the negative control group.
In vivo specificity testing in mice showed that five nanbodies, namely Nb11, NB15, NB17, NB6 and Nb43 had specificity similar to that of Nb17; while there were variations in the clustering effect, all the images exhibited obvious clustering in the lung.
Above mentioned specific embodiments of the present invention are presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Thus, equality of changes and modifications without departing from the spirit and scope of the invention shall fall within the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510086499.7 | Feb 2015 | CN | national |
This application is a U.S. National Phase of PCT/CN2015/076119, filed Apr. 8, 2015, which claims priority to Chinese Patent Application No. 201510086499.7, filed Feb. 16, 2015.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/076119 | 4/8/2015 | WO | 00 |
