WO2018233575A1 - Cd47-blocking nanobody and use thereof - Google Patents

Abstract

Disclosed is a CD47-blocking nanobody. Not only can the antibody specifically bind to human CD47, same can also effectively block the interaction between CD47 and a ligand thereof, SIRPa. Also disclosed is the use of the antibody in the treatment and/or prevention, or diagnosis of CD47-related diseases, such as tumours.

Description

Blocking CD47 Nanobody and use thereof Technical field

The invention belongs to the field of biomedical or biopharmaceutical technology and relates to a blocking type Nanobody directed against the extracellular segment of the integrin-related protein (CD47) molecule. Also disclosed are coding sequences thereof, related methods of preparation, and uses thereof, particularly in the treatment and/or prevention, or diagnosis of CD47-associated diseases, such as tumors.

Background technique

CD47, also known as integrin-associated protein (IAP), is an important member of the immunoglobulin superfamily and is widely expressed in different tissue cells. The chemical property of CD47 is a 50 kD membrane glycoprotein comprising an extracellular Ig variable domain, five highly hydrophobic extended transmembrane segments, and a short selectively spliced carboxyl terminal cytoplasmic tail. The multiple regulation of CD47 molecules in the collective is based on interactions with signal-regulated protein a, thrombospondin, and integrin, and is involved in transmembrane migration and phagocytosis of immune cells such as neutrophils, monocytes, and T cells. Adjustment of function. CD47 and the inhibitory receptor signal-modulating protein a are receptors and ligands, which form a CD47-SIRPa signal complex, which has the function of mediating two-way signal regulation and regulating various immune processes. In malignant tumors such as leukemia, non-Hodgkin's lymphoma, bladder cancer and breast cancer, CD47 is highly expressed on the surface of tumor cells, suggesting a poor clinical prognosis. Tumor cells evade tumor immunity by taking the "Don't Eat Me" signal. However, blocking the interaction of CD47 and SIRPa by using an anti-CD47 antibody has the effect of targeted treatment. The three drugs currently in Phase I are Forty Seven's Hu5F9-G4, Celgene's CC-90002 and Trillium's TTI-621. Trillium's CD47 antibody project is a SIRPa-Fc fusion protein with similar CD47 affinity (nM level) to Hu5F9-G4. SIRPa-Fc has a molecular weight of about 80 kDa, which has better penetrability and tissue distribution than 150 kDa of antibody molecules; SIRPa-Fc has much lower affinity for red blood cells than Hu5F9-G4, indicating that it may be better. Security.

Traditional monoclonal antibodies have large molecular weights and are difficult to penetrate into tissues, and the production cycle of monoclonal antibodies is long and humanization is difficult. Therefore, it is particularly important to find antibodies with smaller molecular weights. In addition to antigen-binding fragments (Fab), single-chain antibodies (scFv) and other small molecule antibodies engineered based on traditional monoclonal antibodies, there is a naturally occurring minimal antigen-binding fragment found in the natural family Camelidae and Shark. This antibody was discovered in 1989 by Muyldermans et al. at the Free University of Brussels. They first discovered a heavy chain antibody in the isolation and detection of antibodies in camel serum. The antibody lacks two light chain CLs and a constant region CH1, only by N. The terminal variable region (VHH), the hinge region and two constant regions (CH1, CH2), the variable region (VHH) is called a Nanobody. The nano-antibody has a molecular weight of only about 15kDa, and its nanometer molecular size and unique structure give it superior characteristics to traditional antibodies, such as high stability, good water solubility, simple humanization, high targeting, and penetration. Strong and so on. Due to its special structural properties, Nanobodies combine the advantages of traditional antibodies and small molecule drugs, and almost completely overcome the shortcomings of traditional antibody development cycle, low stability and harsh storage conditions. Nanobodies with a molecular weight of only 1/10 of conventional antibodies are emerging as a new force in the diagnosis and treatment of next-generation antibodies. Therefore, the application of nano-antibody technology to develop CD47 therapeutic antibody drugs has broad prospects.

Summary of the invention

OBJECTS OF THE INVENTION: The present invention provides a Nanobody directed against the extracellular domain of CD47, which is capable of blocking the interaction of CD47 with its ligand SIRPa, and provides the coding sequence, preparation method, and Use in diagnostic treatment.

In a first aspect of the invention, there is provided a VHH chain of an anti-CD47 Nanobody, the VHH chain comprising a complementarity determining region CDR comprising the CDR1 set forth in SEQ ID NO.: 5, SEQ ID CDR2 shown by NO.: 6 and CDR3 shown by SEQ ID NO.: 7 (or consisting of CDR1, CDR2 and CDR3).

In another preferred embodiment, the CD47 is human CD47.

In another preferred embodiment, the amino acid sequence of any one of the above amino acid sequences further comprises at least one of adding, deleting, modifying and/or substituting, such as 1-3, preferably 1-2, more preferably 1) amino acid and retains a high affinity binding to CD47, blocking the derivative sequence of CD47 and SIRPa interaction.

In another preferred embodiment, the VHH chain further comprises a framework region FR, the CDR1, CDR2 and CDR3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

In another preferred embodiment, the framework region FR is represented by FR1 shown in SEQ ID NO.: 1, FR2 shown in SEQ ID NO.: 2, FR3 shown in SEQ ID NO.: 3, and SEQ ID. NO.: FR4 composition shown in 4 (or containing the above-mentioned FR1, FR2, FR3 and FR4).

In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-CD47 Nanobody is as shown in SEQ ID NO.: 8.

Furthermore, 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 the three CDRs comprising SEQ ID NO. CDR1 shown in 5, CDR2 shown in SEQ ID NO.: 6, and CDR3 shown in SEQ ID NO.: 7.

In a second aspect of the invention, there is provided an anti-CD47 Nanobody which is a Nanobody directed against a CD47 epitope and which has a VHH chain of the amino acid sequence set forth in SEQ ID NO.: 8.

Furthermore, an anti-CD47 antibody which is an antibody against a CD47 epitope and has a VH chain of the amino acid sequence shown in SEQ ID NO.: 8 is also provided.

In a third aspect, the present invention provides a polynucleotide encoding a protein selected from the group consisting of the VHH chain of the anti-CD47 Nanobody of the first aspect of the invention, or the second aspect of the invention Said anti-CD47 Nanobody.

In another preferred embodiment, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9.

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

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

In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or a combination thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.

According to a fifth aspect of the invention, a host cell comprising the expression vector of the fourth aspect of the invention, or a polynucleotide thereof according to the third aspect of the invention, is integrated into the host cell.

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 E. coli, yeast cells.

According to a sixth aspect of the invention, a method for producing an anti-CD47 Nanobody, comprising the steps of:

(a) cultivating the host cell of the fifth aspect of the invention under conditions suitable for the production of a Nanobody, thereby obtaining a culture comprising the anti-CD47 Nanobody;

(b) isolating or recovering said anti-CD47 Nanobody from said culture.

In another preferred embodiment, the anti-CD47 Nanobody has the amino acid sequence set forth in SEQ ID NO.: 8.

According to a seventh aspect of the present invention, there is provided an immunoconjugate comprising:

(a) a VHH chain of an anti-CD47 Nanobody according to the first aspect of the invention, or an anti-CD47 Nanobody according to the second aspect of the invention;

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

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

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

In another preferred embodiment, the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- A drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.

In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of an anti-CD47 Nanobody of the first aspect of the invention, an anti-antibody according to the second aspect of the invention CD47 Nanobody.

In another preferred embodiment, the polyvalent means a VHH chain comprising a plurality of repeating anti-CD47 Nanobodies according to the first aspect of the invention in the amino acid sequence of the immunoconjugate, the present invention The anti-CD47 Nanobody described in the two aspects.

In an eighth aspect of the invention, there is provided the use of the anti-CD47 Nanobody of the second aspect of the invention for the preparation of (a) an agent for detecting a CD47 molecule; (b) a medicament for treating a tumor.

In another preferred embodiment, the detection comprises flow detection, cellular immunofluorescence detection.

According to a ninth aspect of the invention, a pharmaceutical composition comprising:

(i) the VHH chain of the anti-CD47 Nanobody of the first aspect of the invention, or the anti-CD47 Nanobody of the second aspect of the invention, or the immunoconjugate of the seventh aspect of the invention;

(ii) a pharmaceutically acceptable carrier.

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

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating a tumor, the tumor being selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, Colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.

In a tenth aspect of the invention, there is provided the use of one or more of the anti-CD47 Nanobodies of the second aspect of the invention:

(i) for detecting human CD47 molecules;

(ii) for flow detection;

(iii) for cellular immunofluorescence detection;

(iv) for the treatment of tumors;

(v) for tumor diagnosis.

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

According to an eleventh aspect of the present invention, a recombinant protein comprising:

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

(ii) an optional tag sequence that facilitates 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 a CD47 protein.

A twelfth aspect of the invention provides the use of the VHH chain according to the first aspect of the invention, the Nanobody of the second aspect of the invention, or the immunoconjugate of the seventh aspect of the invention, Used to prepare medicaments, reagents, test plates or kits;

Wherein the reagent, the detection plate or the kit is used for: detecting the CD47 protein in the sample;

Wherein the agent is for treating or preventing a tumor expressing a CD47 protein (ie, CD47-positive).

In another preferred embodiment, the tumor comprises: melanoma, gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer , adrenal tumors, or a combination thereof.

According to a thirteenth aspect of the invention, a method for detecting a CD47 protein in a sample, the method comprising the steps of:

(1) contacting the sample with the Nanobody of the second aspect of the invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein formation of a complex means that a CD47 protein is present in the sample.

In a fourteenth aspect of the invention, a method of treating a disease comprising administering a Nanobody of the second aspect of the invention or the immunoconjugate of the seventh aspect of the invention to a subject in need thereof.

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

According to a fifteenth aspect of the invention, the framework region FR of the anti-CD47 Nanobody VHH chain is provided, and the framework region FR of the VHH chain is represented by FR1, SEQ ID NO.: 2 of SEQ ID NO.: FR2 shown, FR3 shown in SEQ ID NO.: 3, FR4 composition shown in SEQ ID NO.: 4 (or containing FR1, FR2, FR3 and FR4 as described).

In a sixteenth aspect of the invention, a CAR-T cell comprising a chimeric antigen receptor CAR, the antigen binding domain of the CAR having a VHH chain according to the first aspect of the invention is provided Or the Nanobody of the second aspect of the invention.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows the construction and screening results of a phage display CD47 Nanobody immunological library. In the figure, A is the PCR amplification result of the target fragment of VHH-CH1-CH2 during the construction of the library, and the fragment is about 700 bp in size; in the figure, B is the PCR amplification result of the VHH gene fragment, and the purification size is about 400 bp. fragments for subsequent library construction; FIG capacity C is detected FIG construct libraries, library were plated after serial dilutions, showing taken 1/5 10 ^3-fold dilutions, ^{104-fold, 105-fold} The number of clones was determined by calculating the number of clones. The library capacity was calculated to be 1.2× ^{10 9} CFU. In the figure, D is the insertion rate detection map of the library. The DNA bands from left to right are: The channel is a DNA molecular marker, and the remaining channels are PCR products for detecting the inserted fragment. The PCR product band is about 500 bp, and the insertion rate of the library reaches 100%.

Figure 2 is the result of flow cytometry to detect the blocking function of CD47 Nanobody. The results indicate that a CD47 Nanobody (amino acid sequence as shown in SEQ ID NO.: 8) is capable of blocking the interaction of CD47 with SIRPa.

Figure 3 is a flow cytometry assay for IC50 of CD47 Nanobody and positive control antibody. The results showed that the IC50 of the CD47 Nanobody was 1.316 nM, while the IC50 of the positive control antibody (CELGENE) was 4.391 nM. The blocking effect of the candidate CD47 Nanobody was better than that of the control antibody.

Figure 4 shows the manner in which the test solution is added in one embodiment of the present invention.

Figure 5 is the result of affinity detection of CD47 Nanobody. The affinity of the CD47 Nanobody was determined to be 4.92E-10M using a ForteBio's Octet System.

Figure 6 is a species-specific result of ELISA for detection of CD47 Nanobody. The CD47 Nanobody interacts only with human SIRPα and does not interact with CD47 in rats and mice. The candidate CD47 Nanobody has better species specificity.

Figure 7 is the result of agglutination of erythrocytes by CD47 Nanobody. The results show that the Nanobody does not cause agglutination of red blood cells.

Detailed ways

The inventors have successfully obtained an anti-CD47 Nanobody through extensive screening through extensive and in-depth research. The experimental results show that the CD47 Nanobody obtained by the present invention can effectively bind to CD47 and block CD47 and its ligand. The interaction of SIRPa.

Specifically, the present invention utilizes a human CD47 antigen protein to immunize a camel to obtain a high quality immune nanobody gene library. Then, the CD47 protein molecule was coupled to the ELISA plate to display the correct spatial structure of the CD47 protein. The antigen of this form was screened by the phage display technology to screen the immuno Nanobody gene library (the camelid heavy chain antibody phage display gene library), thereby obtaining CD47-specific Nanobody gene. This gene was further transferred to Escherichia coli to obtain a highly specific Nanobody strain which was highly expressed in Escherichia coli.

As used herein, the terms "nanobody of the invention", "anti-CD47 Nanobody of the invention", "CD47 Nanobody of the invention" are used interchangeably and refer to a nanoparticle that specifically recognizes and binds to CD47 (including human CD47). antibody. Particularly preferred is 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 isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region 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 to the variable region of the heavy chain . Particular amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the terms "single domain antibody (VHH)", "nanobody" have the same meaning and refer to the variable region of a cloned antibody heavy chain, constructing a single domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR). The variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure. The CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules that are combined with the antibodies or fragments thereof of the invention to form Conjugate. The invention also encompasses cell surface markers or antigens that bind to the anti-CD47 protein antibody or fragment thereof.

As used herein, the term "heavy chain variable region" and the "V _H" are used interchangeably.

As used herein, the terms "variable region" are used interchangeably with "complementarity 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 CDR3.

In another preferred embodiment, the complementarity determining region CDR comprises CDR1 set forth in SEQ ID NO.: 5, CDR2 set forth in SEQ ID NO.: 6, and CDR3 set forth in SEQ ID NO.: 7 (or Consists of CDR1, CDR2 and CDR3).

GYTYSSYCMG (SEQ ID NO.: 5)

IYPDAGIT (SEQ ID NO.: 6)

AAAPPSVPCRLVVARYNY (SEQ ID NO.: 7)

In another preferred embodiment, the VHH chain further comprises a framework region FR, the CDR1, CDR2 and CDR3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

In another preferred embodiment, the framework region FR is represented by FR1 shown in SEQ ID NO.: 1, FR2 shown in SEQ ID NO.: 2, FR3 shown in SEQ ID NO.: 3, and SEQ ID. NO.: FR4 composition shown in 4 (or containing the above-mentioned FR1, FR2, FR3 and FR4).

QVQLQESGGGSVQAGGSLRLSCAAS (SEQ ID NO.: 1)

WFRQAPGKEREGVAA (SEQ ID NO.: 2)

FYTDSVKGRFTISRDNAKNTLFLQMNSLKPEDTATYYC (SEQ ID NO.: 3)

WGQGTQVTVSS (SEQ ID NO.: 4)

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 invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to a polypeptide which specifically binds to a CD47 protein, such as a protein or polypeptide having a heavy chain variable region. . They may or may not contain an initial methionine.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention encompasses any protein or protein conjugate having a heavy chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), so long as the variable region is linked to the heavy chain of an antibody of the invention The variable regions are identical or at least 90% homologous, preferably at least 95% homologous.

In general, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, referred to as variable regions (CDRs), which are divided into four framework regions (FR), four FR amino acid sequences. Relatively conservative, not directly involved in the binding reaction. These CDRs form a cyclic structure in which the β-sheets formed by the FRs 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. The amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.

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 antigen. Thus, the invention includes those molecules having an antibody heavy chain variable region with a CDR, as long as the CDRs thereof have 90% or more (preferably 95% or more, optimally 98% or more) homology to the CDRs identified herein. Sex.

The present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by 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 substantially retains the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It 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 mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed by the 6His tag). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

The antibody of the present invention refers to a polypeptide comprising the above CDR regions having CD47 protein binding activity. The term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein. The term also encompasses active fragments and active derivatives of the antibodies of the invention.

Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions. The encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.

The invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof. In addition to the nearly full length polypeptide, the invention also includes fragments of the Nanobodies of the invention. Typically, the fragment will have at least about 50 contiguous amino acids, 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 of the antibody of the invention.

In the present invention, "conservative variant of the antibody of the present invention" means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.

The present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

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

The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.

In another preferred embodiment, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method. One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

Once the relevant sequences are obtained, the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods. The biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.

At present, it has been possible to obtain a DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.

Transformation of host cells 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 absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, 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 cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.

The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, 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 (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or capable of producing detectable products. Enzyme.

Therapeutic agents that can be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides; 2. biotoxic; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; Particles; 6. liposomes; 7. nanomagnetic particles; 8. drug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (eg , cisplatin) or any form of nanoparticles, etc.

Pharmaceutical composition

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

The pharmaceutical composition of the present invention can be directly used for binding to a CD47 protein molecule, and thus can be used for treating tumors. In addition, other therapeutic agents can be used simultaneously.

The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-described Nanobody of the present invention (or a conjugate thereof) and pharmaceutically An acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched to 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 and solutions are preferably prepared under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, 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 per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

Labeled Nanobody

In a preferred embodiment of the invention, the Nanobody is provided with a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.

The colloidal gold label can be carried out by methods known to those skilled in the art. In a preferred embodiment of the invention, the anti-CD47 Nanobody is labeled with colloidal gold to provide a colloidal gold-labeled Nanobody.

The anti-CD47 Nanobody of the present invention has good specificity and high potency.

Detection method

The invention also relates to methods of detecting CD47 protein. The method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of CD47 protein in the dissolved sample.

In the detection method of the present invention, the sample to be used is not particularly limited, and a representative example is a cell-containing sample present in the cell preservation solution.

Kit

The present invention also provides a kit comprising the antibody (or a fragment thereof) or a test plate of the present invention. In a preferred embodiment of the present 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 the sample, detecting a common reagent and a buffer required, such as various buffers, detection Mark, detect substrates, etc. The test kit can be an in vitro diagnostic device.

application

As described above, the Nanobody of the present invention has a wide range of biological application value and clinical application value, and its application relates to various fields such as diagnosis and treatment of diseases related to CD47, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy against CD47.

The main advantages of the invention include:

(a) The Nanobody of the present invention is highly specific for human CD47 protein having the correct spatial structure.

(b) The nanobody of the present invention has a strong affinity.

(c) The production of the Nanobody of the present invention is simple.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

Example 1: Construction and screening of phage display CD47 Nanobody library

Preparation of antigen: (1) The nucleotide sequence of hCD47 was synthesized on pCDNA3.1(-) vector, and then its extracellular domain was subcloned into pFUSE vector; (2) extracted with Omega plasmid extract kit. pFUSE-hCD47 (ECD) plasmid; (3) HEK293F cells were cultured to an OD of 2.0×10 ⁶ cells/mL; (4) The plasmid was mixed with the transfection reagent PEI 1:3 and allowed to stand for 20 min, then added to HEK293F cells were cultured for 5 days at 37 ° C in a 6% CO ₂ shaker incubator; (5) Cell supernatants were collected and combined with Protein A column for 1 h at room temperature; (6) phosphate buffered with pH 7.0 After washing the column with liquid, the protein was eluted with 0.1 M pH 3.0 Glycine; (7) The eluted protein was ultrafiltered into PBS to obtain hCD47(ECD)-Fc antigen.

Library construction: Briefly, (1) 1 mg hCD47 (ECD)-Fc antigen was mixed with Freund's adjuvant in an equal volume to immunize a Xinjiang Bactrian camel once a week for 3 times to stimulate B cell expression antigen specific (2) After the end of 3 immunizations, extract 100 mL of camel peripheral blood lymphocytes and extract total RNA; (3) synthesize cDNA and amplify VHH by nested PCR (first PCR results are shown in Figure 1A, tapping A fragment of about 700 bp in size was recovered, and the second PCR result is shown in Figure 1B. A fragment of about 400 bp in size was purified for subsequent library construction); (4) 20 μg of pMECS phage was digested with restriction endonucleases Pst I and Not I. The display vector (supplied by Biovector) and 10 μg of VHH were ligated into two fragments; (5) the ligation product was transformed into electroporation competent cell TG1, and a CD47 nanobody library was constructed and the storage capacity was determined. The storage capacity was 1.2×10 ⁹ CFU (results such as Figure 1C)). At the same time, 24 clones were randomly picked for colony PCR detection. Figure 1D shows the results of colony PCR. The results showed that the insertion rate of the constructed library was 100%.

Antibody Screening Identification: Briefly, (1) 10 μg of hCD47(ECD)-Fc antigen (10 μg Fc in NaHCO ₃ as a control) dissolved in 100 mM NaHCO ₃ , pH 8.2 was coupled to a NUNC plate, placed at 4 ° C. (2) Add 100 μL of 0.1% BSA on the next day and block for 2 h at room temperature; (3) 2 h, add 100 μL of phage (2×10 ¹¹ CFU immunized camelid nano-antibody phage display gene library) for 1 h at room temperature; (4 Wash 5 times with 0.05% PBS + Tween-20 to wash away non-specific phage; (5) Dissociate phage specifically binding to CD47 with 100 mM triethanolamine, and infect E. coli grown in log phase TG1 cells were cultured for 1 h at 37 ° C, and phage were generated and purified for the next round of screening. The same screening process was repeated for 3 rounds to allow positive clones to be enriched. (6) From the cell culture dishes containing phage after enrichment, 96 single colonies were selected and inoculated into TB medium containing 100 μg/mL ampicillin (2.3 L KH ₂ PO ₄ , 12.52 g in 1 L TB medium) K ₂ HPO ₄ , 12 g peptone, 24 g yeast extract, 4 mL glycerol), after growth to log phase, was added to a final concentration of 1 mM IPTG and incubated overnight at 28 °C. (7) A crude antibody was obtained by an infiltration method, and the antibody was transferred to an antigen-coated ELISA plate and allowed to stand at room temperature for 1 hour. (8) Unbound antibody was washed away with PBST, and mouse anti-HA antibody (COVENCE) was added and allowed to stand at room temperature for 1 h. (9) Unbound antibody was washed away with PBST, goat anti-mouse alkaline phosphatase-labeled antibody was added, and left at room temperature for 1 h. (10) The unbound antibody was washed away with PBST, and an alkaline phosphatase coloring solution was added thereto, and the absorption value was read at a wavelength of 405 nm on an ELISA apparatus. (11) When the sample well OD value is more than 3 times the control well OD value (Ratio +/- > 3), it is judged as a positive clone hole. (12) The bacteria of the positive clone well were shaken in an LB liquid containing 100 μg/mL Amp to extract a plasmid and perform sequencing.

Example 2: Expression of CD47 Nanobody by HEK293F System and Identification of Its Blocking Function

The eukaryotic cell HEK293F expresses the CD47Nb-Fc fusion protein: (1) The CD47Nb sequence with the correct sequencing result was cloned into the pFUSE-IgG4 vector (purchased from Invivogen), and the pFUSE-IgG4-Nb plasmid was extracted with the Omega plasmid extract kit; The HEK293F cells were cultured to an OD of 2.0×10 ⁶ cells/mL; (3) the plasmid and the transfection reagent PEI were uniformly mixed 1:3, and then allowed to stand for 20 min, and then added to HEK293F cells at 37 ° C, 6% CO ₂ Incubate for 5-6 days in a shaker incubator; (4) Collect the cell supernatant and bind it with Protein A beads for 1 h at room temperature; (5) Wash the beads with phosphate buffer pH 7.0, then use 0.1 M pH 3.0 Glycine The protein was eluted; (6) The eluted protein was ultrafiltered into PBS, and the yield was measured and sampled for SDS-PAGE, and the remaining proteins were stored in a -80 ° C refrigerator.

Flow cytometry to identify the blocking function of Nanobodies: Briefly, (1) preparation of hSIRPa (ECD)-Fc-Biotin, protein biotin method reference biotin reagent instructions; (2) 5 × 10 ⁵ per sample CD47 stably transfected cells were added to 0.5% BSA-PBS buffer, 5 μg of the above purified CD47 Nanobody was added, and a negative control (hIgG1) and a blank group (PBS) were set, and 5 μg of hSIRPα(ECD)-Fc- was simultaneously added to all samples. Biotin was incubated at 4 °C for 20 min; (3) cells were washed twice with PBS, added with eBioscience SA-PE, incubated at 4 °C for 20 min, washed twice with PBS, and detected by flow cytometry (BD FACS Calibur).

The test results are shown in Figure 2. The results indicate that the CD47 Nanobody (amino acid sequence as shown in SEQ ID NO.: 8) is capable of blocking the interaction of CD47 with SIRPa.

Example 3: Flow cytometry to detect IC50 of CD47 Nanobody

(1) Take 5×10 ⁵ CD47 stable cells in 0.5% BSA-PBS buffer for each sample, add gradient dilution CD47 Nanobody and control antibody (antibody dilution gradient is 25nM, 20nM, 15nM, 10nM, 5nM, 3.33nM, 2.5nM, 1.67nM, 1.25nM, 1.0nM, 0.83nM, 0.625nM), add 100uL to each sample, and set a negative control (hIgG4), and add 5μg hSIRPa(ECD)-Fc-Biotin to all samples simultaneously. Incubate at 4 °C for 20 min; (2) Wash the cells twice with PBS, add eBioscience SA-PE, incubate at 4 °C for 20 min, wash the cells twice with PBS, and measure with flow cytometry (BD FACS Calibur) using graphpad prism 6 software. Perform data processing.

The results are shown in Figure 3. The IC50 of the CD47 Nanobody was 1.316 nM, while the IC50 of the control antibody (Celgene) was 4.39 nM.

Example 4: Affinity Detection of CD47 Nanobody

(1) CD47 Nanobody (Nb-Fc) was subjected to 2-fold gradient dilution from 200 nM with PBST: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM. The antigenic proteins hCD47 (ECD)-Fc and Fc were each diluted to 20 ug/mL. (2) Add various test solutions as indicated in Figure 4. (3) On-board inspection with ForteBio’s Octet System.

The results of the assay are shown in Figure 5: the affinity of the CD47 Nanobody was 4.92 x 10 ^-10 M.

Example 5: Species-specific detection of CD47 Nanobody

(1) The CD47 Nanobody is expressed by an E. coli system, and the expressed Nanobody contains His-tag and HA-tag. For the method, see Zhu min et al., 2014, Nanoscale Research Letters, 9: 528; (2) coated antigenic protein. CD47 (human), CD47 (mouse), CD47 (monkey): 0.5 μg per well (5 μg/mL, 100 μL), coated with IgG4 as control, overnight at 4 °C; (3) washed 3 times with PBST the next day, added 200 μL of 1% BSA was blocked at room temperature for 2 h; (4) PBST was washed three times, 100 uL of 10 μg/mL CD47 Nanobody was added, and reacted for 1 h at room temperature; (5) Unbound antibody was washed away with PBST, and mouse anti-HA antibody was added. (COVENCE), placed at room temperature for 1 h; (6) wash away unbound antibody with PBST, add goat anti-mouse alkaline phosphatase-labeled antibody, and leave it at room temperature for 1 h; (7) wash away unbound with PBST The antibody was added to an alkaline phosphatase color developing solution, and the absorption value was read at 405 nm on an ELISA apparatus. The specificity of the Nanobody was judged based on the absorption value.

The results are shown in Figure 6. The CD47 Nanobody is capable of interacting with human and monkey-derived CD47, but not with murine CD47, which has better species specificity.

Example 6: Agglutination of erythrocytes by CD47 Nanobody

Since the surface of the red blood cells has high expression of CD47, it is easier to preferentially bind to the drug of the CD47 antibody, and the drug is concentrated on the surface to function as a "reservoir". Therefore, this situation is prone to anemia. Only when the drug enters the body, it is necessary to break through the "absorption pool" effect of the platelet on the CD47 antibody in order to effectively reach the action site. CD47 Nanobody, positive control antibody (CELGENE), negative control (IgG4) were serially diluted (4000 nM, 1000 nM, 250 nM, 62.5 nM, 15.625 nM, 3.906 nM, 0.976 nM, 0 nM). The gradient-diluted antibodies were separately added to the monkey's red blood cell suspension, and the reaction was observed at 37 ° C for 30 min.

The results showed that (Fig. 7), neither the CD47 Nanobody nor the control antibody caused an agglutination reaction of red blood cells. The experimental results of the control antibody (Celgene) are consistent with those reported by Penka S. Petrova et al., 2016, Clin. Cancer. Res. 23 (4).

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

Claims (10)

1. A VHH chain of an anti-CD47 Nanobody, wherein the VHH chain comprises a complementarity determining region CDR comprising CDR1 set forth in SEQ ID NO: 5, and SEQ ID NO: CDR2, and CDR3 set forth in SEQ ID NO:7.
2. The VHH chain according to claim 1, wherein said VHH chain further comprises a framework region FR, said framework region FR comprising FR1 shown in SEQ ID NO: 1, and FR2 shown in SEQ ID NO: FR3 shown in SEQ ID NO: 3, and FR4 shown in SEQ ID NO: 4.
3. An anti-CD47 Nanobody characterized by being a Nanobody directed against a CD47 epitope and having a VHH chain of the amino acid sequence set forth in SEQ ID NO.: 8.
4. A polynucleotide, which comprises a protein selected from the group consisting of the VHH chain of the anti-CD47 Nanobody of claim 1, or the anti-CD47 Nanobody of claim 3.
5. The polynucleotide according to claim 4, which has the nucleotide sequence shown as SEQ ID NO.: 9.
6. An expression vector comprising the polynucleotide of claim 4.
7. A host cell comprising the expression vector of claim 6, or a polynucleotide thereof according to claim 4 integrated in the genome.
8. A method for producing an anti-CD47 Nanobody, comprising the steps of: (a) cultivating the host cell of claim 7 under conditions suitable for producing a Nanobody, thereby obtaining a culture comprising the anti-CD47 Nanobody And (b) isolating or recovering said anti-CD47 Nanobody from said culture.
9. An immunoconjugate characterized in that the immunoconjugate comprises:

   (a) the VHH chain of the anti-CD47 Nanobody of claim 1, or the anti-CD47 Nanobody of claim 3; and (b) a coupling moiety selected from the group consisting of: a detectable label, a drug , toxins, cytokines, radionuclides, enzymes, or a combination thereof.
10. Use of the anti-CD47 Nanobody of claim 3, for the preparation of (a) an agent for detecting a CD47 molecule; and (b) a medicament for treating a tumor.

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