CN109096395B

CN109096395B - Blocking type CD47 nano antibody and application thereof

Info

Publication number: CN109096395B
Application number: CN201710472025.5A
Authority: CN
Inventors: 安康; 安文琪; 马小伟; 范蓓; 张宝献; 潘若文
Original assignee: Hualan Genetic Engineering Co ltd; Hualan Biological Engineering Inc
Current assignee: Hualan Genetic Engineering Co ltd; Hualan Biological Engineering Inc
Priority date: 2017-06-20
Filing date: 2017-06-20
Publication date: 2022-06-24
Anticipated expiration: 2037-06-20
Also published as: CN109096395A; WO2018233575A1

Abstract

The invention provides a blocking type CD47 nano antibody and application thereof. Specifically, the invention provides a nanobody against integrin-associated protein (CD 47). Specifically, the antibody not only can specifically bind to human CD47, but also can effectively block the interaction between CD47 and a ligand SIRPa thereof. The invention also discloses the coding sequence of the antibody and the application thereof, in particular the application in treating and/or preventing, or diagnosing CD47 related diseases such as tumor.

Description

Blocking type CD47 nano antibody and application thereof

Technical Field

The invention belongs to the technical field of biomedicine or biopharmaceutical, and relates to a blocking type nano antibody aiming at an extracellular domain of an integrin-associated protein (CD 47). Also discloses its coding sequence, relative preparation method and its application, especially the application in curing and/or preventing, or diagnosing CD47 relative diseases such as tumor.

Background

CD47, also known as integrin-associated protein (IAP), is an important member of the immunoglobulin superfamily and is widely expressed in cells of different tissues. The chemical identity of CD47 is a 50kD membrane glycoprotein comprising 1 extracellular Ig-like variable domain, 5 highly hydrophobically extended transmembrane segments and 1 short selectively spliced carboxy-terminal cytoplasmic tail. The multiple regulation and control functions of the CD47 molecule in the collective body are mainly based on the interaction with signal regulatory protein a, thrombospondin and integrin, and are involved in the regulation of transmembrane migration and phagocytosis of immune cells such as neutrophils, monocytes and T cells. CD47 and inhibitory receptor signal regulatory protein a are mutually receptors and ligands, and can form a CD47-SIRPa signal complex which has the functions of mediating bidirectional signal regulation and regulating various immune response processes. In malignant tumors such as leukemia, non-Hodgkin lymphoma, bladder cancer and breast cancer, CD47 is highly expressed on the surface of tumor cells, indicating poor clinical prognosis. Tumor cells evade tumor immunity by thereby "eating me" signals. However, blocking the interaction of CD47 and SIRPa by using an anti-CD 47 antibody has the effect of targeted therapy. The three drugs currently in Phase I are Hu5F9-G4 by Forty Seven, CC-90002 by Celgene, and TTI-621 by Trillium, respectively. The CD47 antibody project of Trillium is a SIRPa-Fc fusion protein with similar CD47 affinity (nM grade) to Hu5F 9-G4. The molecular weight of the SIRPa-Fc is smaller than about 80kDa, and the SIRPa-Fc has better penetrability and tissue distribution compared with 150kDa of an antibody molecule; the affinity of SIRPa-Fc for red blood cells is far lower than that of Hu5F9-G4, which indicates that SIRPa-Fc has better safety.

The traditional monoclonal antibody has large molecular weight and is difficult to permeate into tissues, and the production period of the monoclonal antibody is long and the humanization is difficult, so that the search for the antibody with smaller molecular weight is particularly important. In addition to small molecule antibodies engineered based on traditional monoclonal antibodies, such as antigen binding fragments (Fab), single chain antibodies (scFv), a naturally occurring, currently discovered minimum antigen binding fragment is found in the body of camelidae and sharehidae in nature. This antibody was discovered in 1989 by Muydermans, free university of Brussel, et al, who, when isolating antibodies in camel serum, first discovered a heavy chain antibody lacking both the light chain CL and the constant region CH1, consisting of only the N-terminal variable region (VHH), the hinge region and two constant regions (CH1, CH2), the variable region (VHH) of which is called a nanobody. The molecular weight of the nano antibody is only about 15kDa, and the nano molecular size and the unique structure of the nano antibody endow the nano antibody with various characteristics superior to the traditional antibody, such as high stability, good water solubility, simple humanization, high targeting property, strong penetrability and the like. The nano antibody has the advantages of the traditional antibody and a small molecular drug due to the special structural property, and almost perfectly overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody. Such nanobodies, which have a molecular weight of only conventional antibody 1/10, are gradually emerging forces in the diagnosis and treatment of new generation antibodies. Therefore, the application of the nano-antibody technology to research and develop CD47 therapeutic antibody medicaments has wide prospects.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a nano antibody aiming at CD47 extracellular domain, which can block the interaction between CD47 and a ligand SIRPa thereof, and simultaneously provides a coding sequence, a preparation method and an application thereof in diagnosis and treatment.

In a first aspect of the invention, there is provided a VHH chain of an anti-CD 47 nanobody, the VHH chain comprising complementarity determining regions CDRs comprising CDR1 shown in SEQ ID No. 5, CDR2 shown in SEQ ID No. 6, and CDR3 shown in SEQ ID No. 7 (or consisting of CDR1, CDR2 and CDR 3).

In another preferred embodiment, the CD47 is human CD 47.

In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, and more preferably 1) amino acid and capable of retaining high affinity binding to CD47 and blocking the interaction between CD47 and SIRPa.

In another preferred embodiment, said VHH chain further comprises framework regions FR, said

CDRs

1, 2 and 3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

In another preferred example, the framework region FR consists of FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 2, FR3 shown in SEQ ID NO. 3 and FR4 shown in SEQ ID NO. 4 (or contains the FR1, FR2, FR3 and FR 4).

In another preferred example, the amino acid sequence of the VHH chain of the anti-CD 47 nano antibody is shown in SEQ ID NO. 8.

In addition, a heavy chain variable region of an anti-human CD47 antibody is provided, the heavy chain variable region comprising three complementarity determining regions CDR1, CDR2, and CDR3, and 3 CDRs comprising CDR1 as shown in SEQ ID No. 5, CDR2 as shown in SEQ ID No. 6, and CDR3 as shown in SEQ ID No. 7.

In a second aspect of the invention, there is provided an anti-CD 47 nanobody, which is a nanobody against the CD47 epitope and has a VHH chain having an amino acid sequence as set forth in SEQ ID No. 8.

In a third aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the VHH chain of an anti-CD 47 nanobody according to the first aspect of the invention, or an anti-CD 47 nanobody according to the second aspect of the invention.

In another preferred embodiment, the polynucleotide has the nucleotide sequence shown in SEQ ID No. 9.

In another preferred embodiment, the polynucleotide comprises DNA or RNA.

In a fourth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the third aspect of the invention.

In a fifth aspect of the invention, there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having a polynucleotide according to the third aspect of the invention integrated into its genome.

In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is selected from the group consisting of: escherichia coli, yeast cells.

In a sixth aspect of the present invention, there is provided a method for producing an anti-CD 47 nanobody, comprising the steps of:

(a) culturing the host cell of the fifth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture comprising the anti-CD 47 nanobodies; and

(b) isolating or recovering the anti-CD 47 nanobody from the culture.

In another preferred example, the anti-CD 47 nanobody has an amino acid sequence shown in SEQ ID No. 8.

In a seventh aspect of the invention, there is provided an immunoconjugate comprising:

(a) a VHH chain of an anti-CD 47 nanobody according to the first aspect of the invention, or an anti-CD 47 nanobody according to the second aspect of the invention; and

(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

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

In another preferred embodiment, the conjugated moiety is a detectable label.

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or nanoparticles of any form, and the like.

In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g. bivalent) VHH chain of an anti-CD 47 nanobody according to the first aspect of the invention, an anti-CD 47 nanobody according to the second aspect of the invention.

In another preferred embodiment, the multivalent is that the immunoconjugate comprises a plurality of repeats in its amino acid sequence of the VHH chain of the anti-CD 47 nanobody of the first aspect of the invention, and the anti-CD 47 nanobody of the second aspect of the invention.

In an eighth aspect of the invention, there is provided a use of the anti-CD 47 nanobody of the second aspect of the invention for preparing (a) a reagent for detecting CD47 molecule; (b) a medicine for treating tumor.

In another preferred embodiment, the detection comprises flow detection and cell immunofluorescence detection.

In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) the VHH chain of an anti-CD 47 nanobody of the first aspect of the invention, or an anti-CD 47 nanobody of the second aspect of the invention, or an immunoconjugate of the seventh aspect of the invention; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostatic cancer, colorectal cancer, breast cancer, large intestine cancer, prostatic cancer, cervical cancer, lymph cancer, adrenal gland tumor, or bladder tumor.

In a tenth aspect of the invention, there is provided a use of one or more of the anti-CD 47 nanobodies of the second aspect of the invention:

(i) for detecting human CD47 molecules;

(ii) for streaming detection;

(iii) for cellular immunofluorescence detection;

(iv) for the treatment of tumors;

(v) can be used for tumor diagnosis.

In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

In an eleventh aspect of the present invention, there is provided a recombinant protein having:

(i) the sequence of a heavy chain variable region VHH according to the first aspect of the invention or the sequence of a nanobody according to the second aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag and an HA tag.

In another preferred embodiment, the recombinant protein specifically binds to CD47 protein.

In a twelfth aspect of the invention, there is provided use of a VHH chain according to the first aspect of the invention, a nanobody according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention, for the preparation of a medicament, a reagent, a detection plate or a kit;

wherein the reagent, assay plate or kit is for: detecting CD47 protein in the sample;

wherein the medicament is for treating or preventing a tumor expressing CD47 protein (i.e., CD47 positive).

In another preferred embodiment, the tumor comprises: melanoma, gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, or adrenal gland tumor.

In a thirteenth aspect of the present invention, there is provided a method for detecting CD47 protein in a sample, the method comprising the steps of:

(1) contacting the sample with a nanobody according to the second aspect of the present invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD47 protein in the sample.

In a fourteenth aspect of the present invention, there is provided a method for treating a disease, the method comprising administering the nanobody of the second aspect of the present invention or the immunoconjugate of the seventh aspect of the present invention to a subject in need thereof.

In another preferred embodiment, the subject comprises a mammal, such as a human.

In a fifteenth aspect of the present invention, there is provided an anti-CD 47 nanobody VHH chain framework region FR consisting of FR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 3, and FR4 shown in SEQ ID No. 4.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is phage display CD47 nanobody immunizationAnd constructing a library and screening results. In the figure, A is the PCR amplification result of target fragments of VHH-CH1-CH2 in the library construction process, and fragments with the size of about 700bp are recovered by tapping; in the figure, B is the PCR amplification result of the VHH gene fragment, and the fragment with the size of about 400bp is purified for subsequent library construction; in the figure C is a library capacity detection diagram of the constructed library, the constructed library is coated with a plate after being diluted by a gradient, and 1/5 gradient dilution 10 is taken³10 times of⁴10 times of⁵Doubling the number of clones, determining the size of the library by counting the number of single clones, calculating the library capacity of 1.2X10⁹A CFU; in the figure, D is an insertion rate detection graph of the library, and DNA bands of gel holes from left to right are respectively: the first path is DNA molecular marker, the rest pore path is PCR product for detecting inserted fragment, the PCR product band is about 500bp, and the insertion rate of the library reaches 100%.

Fig. 2 is the result of flow cytometry to detect CD47 nanobody blocking function. The result shows that a CD47 nano antibody (the amino acid sequence is shown in SEQ ID NO.: 8) can block the interaction of CD47 and SIRPa.

Fig. 3 is an IC50 for flow cytometry detection of CD47 nanobodies versus positive control antibodies. The results show that the IC50 of the CD47 nano antibody is 1.316nM, while the IC50 of the positive control antibody (CELGENE) is 4.391nM, and the blocking effect of the candidate CD47 nano antibody is better than that of the control antibody.

FIG. 4 shows the manner of adding the detection solution in one embodiment of the present invention.

FIG. 5 shows the result of the affinity detection of CD47 nanobody. The CD47 nanobody had an affinity of 4.92E-10M as determined by ForteBio's Octet System.

FIG. 6 is the species-specific results of ELISA detection of CD47 nanobody. The CD47 nano antibody only interacts with human SIRPa, does not interact with CD47 of rats and mice, and the candidate CD47 nano antibody has better species specificity.

FIG. 7 shows the result of agglutination of erythrocytes by CD47 nanobody. The result shows that the nano antibody does not cause agglutination reaction of red blood cells.

Detailed Description

The inventor successfully obtains an anti-CD 47 nano antibody through extensive and intensive research and a large number of screens, and experimental results show that the CD47 nano antibody obtained by the invention can be effectively combined with CD47 and can also block the interaction between CD47 and a ligand SIRPa thereof.

Specifically, the invention utilizes human-derived CD47 antigen protein to immunize camels to obtain a high-quality immune nano antibody gene library. Then CD47 protein molecules are coupled on an enzyme label plate to display the correct space structure of the CD47 protein, and the antigen in the form is used for screening an immune nano antibody gene library (a camel heavy chain antibody phage display gene library) by utilizing a phage display technology, so that the CD47 specific nano antibody gene is obtained. Then the gene is transferred into escherichia coli, so that a nano antibody strain which can be efficiently expressed in the escherichia coli and has high specificity is obtained.

As used herein, the terms "nanobody of the invention", "anti-CD 47 nanobody of the invention", "CD 47 nanobody of the invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to CD47, including human CD 47. Particularly preferred are nanobodies with the amino acid sequence of the VHH chain as shown in SEQ ID No. 8.

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning and refer to the variable region of a cloned antibody heavy chain, and a single domain antibody (VHH) is constructed that consists of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Typically, single domain antibodies (VHHs) consisting of only one heavy chain variable region are constructed by obtaining an antibody naturally lacking the light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention to form conjugates. The invention also includes a cell surface marker or antigen conjugated to the anti-CD 47 protein antibody or fragment thereof.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to a polypeptide that specifically binds to CD47 protein, e.g., a protein or polypeptide having a heavy chain variable region. They may or may not contain the initial methionine.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions, called variable regions (CDRs), located in the variable region of the heavy chain, which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the invention includes molecules having an antibody heavy chain variable region with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having a binding activity to CD47 protein, which comprises the above-mentioned CDR regions. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, fragments of the nanobodies of the invention are also encompassed by the present invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide.

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the mature polypeptide encode polypeptides having the same biological functions and activities as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli,competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. drug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. a therapeutic agent (e.g., cisplatin) or any form of nanoparticle, and the like.

Pharmaceutical composition

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the invention can be directly used for binding CD47 protein molecules, thus being used for treating tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the nanobody (or its conjugate) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will also take into account such factors as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.

Labeled nanobodies

In a preferred embodiment of the invention, the nanobody carries a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, coloured labels or fluorescent labels.

The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-CD 47 nanobody is labeled with colloidal gold to obtain a gold-labeled nanobody.

The anti-CD 47 nano antibody has good specificity and high titer.

Detection method

The invention also relates to a method for detecting the CD47 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of CD47 protein in the solubilized sample.

The sample used in the detection method of the present invention is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.

Reagent kit

The present invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention also provides a detection kit for detecting the level of CD47, which comprises an antibody for recognizing CD47 protein, a lysis medium for dissolving a sample, general reagents and buffers required by detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.

Applications of

As described above, the nanobody of the present invention has wide biological and clinical application values, and its applications relate to various fields such as diagnosis and treatment of CD 47-related diseases, research in basic medicine, and biological research. One preferred application is for clinical diagnosis and targeted therapy against CD 47.

The main advantages of the invention include:

(a) the nano-antibody of the invention is directed to the CD47 protein with correct space structure.

(b) The nano antibody has strong affinity.

(c) The production of the nano antibody is simple and convenient.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1: construction and screening of phage display CD47 nano antibody library

Library construction: briefly, (1) 1mg of hCD47(ECD) -Fc antigen and Freund's adjuvant are mixed in equal volume, a Xinjiang bactrian camel is immunized once a week for 3 times, and B cells are stimulated to express antigen-specific nano antibodies; (2) after 3 times of immunization, extracting 100mL camel peripheral blood lymphocytes and extracting total RNA; (3) synthesizing cDNA and amplifying VHH by nested PCR (the first PCR result is shown in figure 1A, the fragment with the size of about 700bp is recovered by tapping, the second PCR result is shown in figure 1B, and the fragment with the size of about 400bp is purified for subsequent library construction); (4) mu.g of pMECS phage display vector (supplied by Biovector) and 10. mu.g of VHH were digested with restriction enzymes Pst I and Not I and the two fragments were ligated; (5) the ligation products are transformed into electrotransformation competent cells TG1, a CD47 nano antibody library is constructed and the library capacity is determined, wherein the size of the library capacity is 1.2 multiplied by 10⁹CFU (results are shown in fig. 1C). Meanwhile, 24 clones were randomly picked for colony PCR detection, and FIG. 1D shows colony PCR results, which indicate that the insertion rate of the constructed library is 100%.

Screening and identifying antibodies: briefly, (1) dissolve in 100mM NaHCO ₃10 μ g of hCD47(ECD) -Fc antigen (10 μ g of Fc in NaHCO) at pH 8.2₃As a control) were coupled to NUNC enzyme plates and left overnight at 4 ℃; (2) adding 100 μ L of 0.1% BSA the next day, blocking for 2h at room temperature; (3) after 2h, 100. mu.L phage (2X 10) was added¹¹CFU immune camel nanometer antibody phage display gene library) and reacting for 1h at room temperature; (4) washing 5 times with 0.05% PBS + Tween-20 to wash away non-specific phage; (5) phage specifically binding to CD47 were dissociated with 100mM triethanolamine and infected with E.coli TG1 cells grown in log phase, cultured at 37 ℃ for 1h, phage generated and purified for the next round of screening, and the same screening process was repeated for 3 rounds to enrich positive clones. (6) From the phage-enriched cell culture dish, 96 single colonies were selected and inoculated into TB medium containing 100. mu.g/mL ampicillin (2.3 g KH in 1L TB medium)₂PO₄，12.52g K₂HPO₄12g peptone, 24g yeast extract, 4mL glycerol), after growth to logarithmic phase, 1mM final concentration of IPTG was added and incubated overnight at 28 ℃. (7) Crude antibody was obtained by permeation and transferred to an antigen coated ELISA plate and left at room temperature for 1 h. (8) Unbound antibody was washed away with PBST, and a murine anti-HA antibody (COVENCE) was added and left at room temperature for 1 h. (9) Unbound antibody was washed away with PBST, goat anti-mouse alkaline phosphatase-labeled antibody was added, and the mixture was allowed to stand at room temperature for 1 h. (10) Unbound antibody was washed away with PBST, and absorbance was read on an ELISA instrument at 405nm by adding an alkaline phosphatase developing solution. (11) When the OD value of the sample well is more than 3 times larger than that of the control well (Ratio +/- > 3), the sample well is judged to be a positive clone well. (12) The positive cloning hole bacteria were shaken in 100u g/mL Amp LB liquid to extract plasmid and sequencing.

Example 2: HEK293F system expression CD47 nano antibody and blocking function identification thereof

Eukaryotic HEK293F expresses CD47Nb-Fc fusion protein: (1) cloning the CD47Nb sequence with correct sequencing result into a pFUSE-IgG4 vector (purchased from Invivogen), and extracting a pFUSE-IgG4-Nb plasmid by using an Omega plasmid macroextraction kit; (2) culturing HEK293F cells to OD 2.0X 10⁶Per mL; (3) mixing the plasmid and PEI as transfection reagent at a ratio of 1:3, standing for 20min, adding into HEK293F cell at 37 deg.C and 6% CO₂Culturing in a shaking incubator for 5-6 days; (4) collecting cell supernatant, and combining with Protein A beads at room temperature for 1 h; (5) after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M Glycine pH 3.0; (6) the eluted proteins were ultrafiltered into PBS, samples were taken after yield determination for SDS-PAGE detection, and the remaining proteins were stored in a freezer at-80 ℃.

Flow cytometry to identify blocking function of the nanobody: briefly, (1) preparation of hsrpa (ecd) -Fc-Biotin, protein Biotin protocol referred to Biotin reagent instructions; (2) taking 5X 10 samples of each⁵Stably transferring CD47 cells into 0.5% BSA-PBS buffer, adding 5 μ g of the purified CD47 nano antibody, setting a negative control (hIgG1) and a blank group (PBS), simultaneously adding 5 μ g of hSIRPa (ECD) -Fc-Biotin to all samples, and incubating for 20min at 4 ℃; (3) the cells were washed 2 times with PBS, SA-PE from eBioscience was added, incubated at 4 ℃ for 20min, and the cells were washed 2 times with PBS and then detected with a flow cytometer (BD FACS Calibur).

The results of the detection are shown in FIG. 2. The result shows that the CD47 nano antibody (the amino acid sequence is shown as SEQ ID NO. 8) can block the interaction of CD47 and SIRPa.

Example 3: IC50 for detecting CD47 nano antibody by flow cytometry

(1) Taking 5X 10 samples of each⁵CD47 cells were transfected in 0.5% BSA-PBS buffer, and CD47 nM antibody and control antibody were added in a gradient (antibody dilution gradient 25nM, 20nM, 15nM, 10nM, 5nM, 3.33nM, 2.5nM, 1.67nM, 1.25nM, 1.0nM, 0.83nM, 0.625nM), 100uL was added to each sample while setting a negative control (hIgG4), 5. mu.g hSIRPa (ECD) -Fc-Biotin was added to all samples at the same time, and incubation was carried out at 4 ℃ for 20 min; (2) washing the cells 2 times with PBS, adding SA-PE from eBioscience, incubating at 4 deg.C for 20min, washing the cells 2 times with PBS and flowingCytometry (BD FACS Calibur) assay, data processing was performed using graphpad prism 6 software.

The results are shown in FIG. 3, where the CD47 nanobody had an IC50 of 1.316nM, while the control antibody (Celgene) had an IC50 of 4.39 nM.

Example 4: affinity detection of CD47 Nanobody

(1) CD47 nanobody (Nb-Fc) was 2-fold gradient diluted from 200nM with PBST, respectively: 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125 nM. The antigenic proteins hCD47(ECD) -Fc and Fc were diluted to 20ug/mL, respectively. (2) The various test solutions were added as indicated in FIG. 4. (3) On-machine detection was performed using ForteBio's Octet System.

The detection results are shown in fig. 5: the affinity of the CD47 nano antibody is 4.92 multiplied by 10^-10M。

Example 5: species-specific detection of CD47 nanobody

(1) The CD47 nano antibody is expressed by an escherichia coli system, the expressed nano antibody contains His-tag and HA-tag, and the method is described in Zhu min et al, 2014, Nanoscale Research Letters, 9: 528 of the raw material; (2) envelope antigen proteins CD47 (human), CD47 (murine), CD47 (monkey): 0.5. mu.g per well (5. mu.g/mL, 100. mu.L), coated with IgG4 as a control, overnight at 4 ℃; (3) the next day, washing with PBST 3 times, adding 200 μ L of 1% BSA at room temperature and blocking for 2 h; (4) PBST is washed for three times, 100uL of CD47 nano antibody with the concentration of 10 mug/mL is added, and reaction is carried out for 1h at room temperature; (5) unbound antibody was washed away with PBST, and a mouse anti-HA antibody (COVENCE) was added and left at room temperature for 1 h; (6) washing away the unbound antibody by PBST, adding goat anti-mouse alkaline phosphatase labeled antibody, and standing at room temperature for 1 h; (7) unbound antibody was washed off with PBST, and absorbance was read on an ELISA instrument at 405nm by adding an alkaline phosphatase developing solution. And judging the specificity of the nano antibody according to the absorption value.

The detection results are shown in fig. 6: the CD47 nano antibody can interact with human-derived and monkey-derived CD47, but not with murine-derived CD47, and has better species specificity.

Example 6: agglutination reaction of CD47 nano antibody to red blood cell

Because the surface of the red blood cell has high expression of CD47, the red blood cell can be more easily and preferentially combined with CD47 antibody drugs, and the drugs are concentrated on the surface to play a role of a 'water storage tank'. Therefore, anemia is likely to occur in this case. Only after the medicine enters the body, the medicine firstly needs to break through the 'absorption pool' effect of the platelets on the CD47 antibody, and can effectively reach the action position to play a role. The CD47 nanobody, positive control antibody (CELGENE), negative control (IgG4) were serially diluted (4000nM, 1000nM, 250nM, 62.5nM, 15.625nM, 3.906nM, 0.976nM, 0nM), respectively. The antibodies diluted in the gradient are respectively added into the erythrocyte suspension of the monkey, and the result is observed after the reaction is carried out for 30min at 37 ℃.

The results show (fig. 7) that neither the CD47 nanobody nor the control antibody caused agglutination of erythrocytes. The results of the control antibody (Celgene) were consistent with those reported by Penka S.Petrova et al, 2016, Clin.cancer.Res.23 (4).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Nuode Biotech Co., Ltd

<120> blocking type CD47 nano antibody and application thereof

<130> P2017-1222

<160> 9

<170> PatentIn version 3.5

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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ccagggaagg agcgcgaggg ggtcgcagct atttatcctg atgctggtat cacgttctat 180

accgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa cacgctgttt 240

ctgcaaatga acagcctgaa acctgaagac actgccacgt actactgtgc ggcagcgcct 300

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Claims

1. A VHH chain of an anti-CD 47 nanobody, wherein said VHH chain comprises complementarity determining regions CDRs comprising the amino acid sequences set forth in SEQ ID NOs: 5, CDR1 shown in SEQ ID NO: 6, and the CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.

2. The VHH chain according to claim 1, further comprising a framework region FR consisting of the amino acid sequence of SEQ ID NO: 1, FR1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: 4 FR4 shown.

3. The VHH chain according to claim 1, wherein the amino acid sequence of the VHH chain of the anti-CD 47 nanobody is represented by SEQ ID NO 8.

4. An anti-CD 47 nanobody, which is a nanobody against the CD47 epitope and has a VHH chain having the amino acid sequence shown in SEQ ID No. 8.

5. A polynucleotide encoding a protein selected from the group consisting of: the VHH chain of the anti-CD 47 nanobody of claim 1, or the anti-CD 47 nanobody of claim 4.

6. The polynucleotide of claim 5, having the nucleotide sequence set forth in SEQ ID NO. 9.

7. An expression vector comprising the polynucleotide of claim 5.

8. A host cell comprising the expression vector of claim 7, or having the polynucleotide of claim 5 integrated into its genome.

9. A method of producing an anti-CD 47 nanobody, comprising the steps of: (a) culturing the host cell of claim 8 under conditions suitable for nanobody production, thereby obtaining a culture comprising the anti-CD 47 nanobody; and (b) isolating or recovering said anti-CD 47 nanobody from said culture.

10. An immunoconjugate, comprising:

(a) the VHH chain of the anti-CD 47 nanobody of claim 1, or the anti-CD 47 nanobody of claim 4; and (b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

11. The immunoconjugate of claim 10, wherein the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radiolabel, an MRI or CT contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine, an antibody Fc fragment, an antibody scFv fragment, a viral particle, a liposome, a prodrug-activating enzyme, a chemotherapeutic agent, or any form of nanoparticle.

12. The immunoconjugate of claim 10, wherein the conjugate is selected from the group consisting of: gold nanoparticles/nanorods or nanomagnets.

13. Use of the anti-CD 47 nanobody of claim 4 for the preparation of a reagent for the detection of CD47 molecules.

14. A recombinant protein, said recombinant protein having:

(i) the sequence of the heavy chain variable region VHH of claim 1 or the sequence of the nanobody of claim 4; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

15. Use of a VHH chain according to claim 1, a nanobody according to claim 4, or an immunoconjugate according to claim 10 for the preparation of a reagent, a detection plate or a kit;

wherein the reagent, assay plate or kit is for: detecting the CD47 protein in the sample.

16. A method for the in vitro non-diagnostic detection of CD47 protein in a sample, said method comprising the steps of:

(1) contacting a sample with the nanobody of claim 4;