KR102896511B1 - Antibody specifically binding to SIRPα variant - Google Patents

Abstract

The present invention relates to an antibody that specifically binds to a SIRPα variant, and more particularly, to an antibody or antigen-binding fragment thereof that specifically binds to a SIRPα variant or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2, and a method for detecting a SIRPα variant in a sample using the same.
The antibody or antigen-binding fragment thereof according to the present invention exhibits an effect capable of accurately confirming and quantifying the presence of a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 during the process of expressing, purifying, and studying the same.

Description

Antibody specifically binding to SIRPα variant

The present invention relates to an antibody that specifically binds to a SIRPα variant, and more particularly, to an antibody or antigen-binding fragment thereof that specifically binds to a SIRPα variant or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2, and a method for detecting a SIRPα variant in a sample using the same.

SIRPα (Signal-regulatory protein alpha) is a single-transmembrane molecule of the Ig superfamily present in myeloid cells such as macrophages, dendritic cells, neutrophils, and glial cells. The extracellular domain consists of one IgV domain and two IgC domains, and 10 types of mutants have been reported in humans for the IgV domain, which is the binding site for CD47. On the other hand, the intracellular domain contains immunoreceptor tyrosine-based inhibition motifs (ITIM), and upon binding to CD47, binding to the tyrosine dephosphorylation enzymes SHP-1 and SHP-2 is induced, thereby transmitting an inhibitory signal.

The physiological phenomenon of SIRPα-CD47 interaction has been described as one in which CD47 on erythrocytes binds to SIRPα on macrophages, transmitting a "don't eat me" signal, thereby avoiding unnecessary phagocytosis of erythrocytes. Furthermore, it has been suggested that in a tumor microenvironment, CD47, which is highly expressed on tumor cells, binds to SIRPα on macrophages or dendritic cells, thereby inhibiting their phagocytosis. It is anticipated that this inhibition of phagocytosis will subsequently lead to inhibition of tumor antigen presentation to T cells and, consequently, to suppression of the tumor immune response.

To date, inhibition of the SIRPα-CD47 interaction with antibodies against CD47, a SIRPα ligand, has been reported to enhance tumor cell phagocytosis. This phenomenon has been observed even when anti-SIRPα antibodies are used, under conditions where anticancer antibodies with effector activity attract tumor cells to immune cells are used in combination. Furthermore, in a syngeneic mouse tumor model using anti-CD47 antibodies, in addition to the antitumor effect, it has been suggested that they induce tumor immunity.

Meanwhile, strategies focusing on disrupting the interaction between CD47 and SIRPα, such as administration of agents that block CD47 or SIRPα, have been examined as potential anticancer therapies since high CD47 expression on tumor cells was found to be a negative prognostic factor for survival in acute myeloid leukemia (AML) and several solid cancers.

As part of this therapy, various SIRPα mutants with significantly improved affinity for CD47 compared to wild-type SIRPα have been developed, and their effectiveness as competitive antagonists of CD47-SIRPα binding has been confirmed.

Therefore, there is an increasing demand for SIRPα variant-specific antibodies as a means to accurately confirm and quantify the presence of SIRPα variants during the process of expressing, purifying, and studying SIRPα variants.

Accordingly, the inventor of the present invention conducted repeated research to develop an antibody that specifically binds to a SIRPα variant or fragment thereof including the amino acid sequence of SEQ ID NO: 2, and discovered that antibodies having a specific CDR (complementarity determining region) sequence exhibit very high binding specificity and binding affinity to the SIRPα variant, thereby having very high utility, thereby completing the present invention.

Accordingly, an object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

Another object of the present invention is to provide a polynucleotide encoding the antibody or an antigen-binding fragment thereof, a vector comprising the polynucleotide, and a cell transformed with the vector.

Another object of the present invention is to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to a SIRPα variant or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2, which comprises the step of culturing the cell under conditions in which a polynucleotide is expressed to produce a polypeptide comprising light and heavy chain variable regions, and the step of recovering the polypeptide from the cell or the culture medium in which the cell or the polypeptide is cultured.

Another object of the present invention is to provide a method for detecting a SIRPα variant comprising an amino acid sequence of SEQ ID NO: 2 in a sample, comprising the following steps:

(a) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and

(b) a step of detecting binding of the sample to the antibody or antigen-binding fragment thereof.

In order to achieve the above-described object of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof, a vector comprising the polynucleotide, and a cell transformed with the vector.

In order to achieve another object of the present invention, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to a SIRPα variant or fragment thereof comprising an amino acid sequence of SEQ ID NO: 2, comprising the steps of culturing the cell under conditions in which a polynucleotide is expressed to produce a polypeptide comprising light and heavy chain variable regions, and recovering the polypeptide from the cell or a culture medium in which the cell or the polypeptide is cultured.

In order to achieve another object of the present invention, the present invention provides a method for detecting a SIRPα variant comprising an amino acid sequence of SEQ ID NO: 2 in a sample, comprising the following steps:

(a) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and

(b) a step of detecting binding of the sample to the antibody or antigen-binding fragment thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

In the present invention, the SIRPa (also referred to as SIRP-alpha, SIRPα, CD172a or SHPS-1) is a protein expressed in monocytes, most subpopulations of tissue macrophages, granulocytes, DC subsets of lymphoid tissues, some myeloid progenitor cells, and also expressed in neurons at various levels, and comprises an amino acid sequence of SEQ ID NO: 1. The amino acid sequence of wild-type SIRPα comprising the amino acid sequence of SEQ ID NO: 1 is known as NCBI accession No. AAH33092, NP_001317657, NP_001035112, NP_001035111, NP_542970, XP_024307604, XP_005260727, XP_047295872, XP_047295871, XP_011527475, etc., but is not limited thereto.

The antibody provided by the present invention is an antibody that specifically binds to a SIRPα variant, more specifically, a SIRPα variant including the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, and may be characterized in that it does not bind to a wild-type SIRPα including the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, but exhibits high binding specificity to a SIRPα variant including the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

In the present invention, the “SIRPα variant” may mean a protein in which the amino acid sequence of the wild-type SIRPα according to the reference number, excluding the amino acid sequence of the sequence number 1, is connected to the C-terminus of the sequence number 2.

In the present invention, the “fragment” may mean a protein in which a part of the remaining amino acid sequence, excluding the amino acid sequence of SEQ ID NO. 1, in the amino acid sequence of SEQ ID NO. 2 and the amino acid sequence of the wild-type SIRPα according to the reference number is connected to the C-terminus of SEQ ID NO. 2.

In the present invention, “antibody” is also called immunoglobulin (Ig), and is a general term for proteins that selectively act on antigens and participate in the body’s immunity. Whole antibodies found in nature are generally composed of two pairs of light chains (LC) and heavy chains (HC), which are polypeptides composed of multiple domains, or are composed of two pairs of HC/LC structures as their basic units. There are five types of heavy chains that constitute mammalian antibodies, indicated by the Greek letters α, δ, ε, γ, and μ, and depending on the type of heavy chain, they constitute different types of antibodies, such as IgA, IgD, IgE, IgG, and IgM. There are two types of light chains that constitute mammalian antibodies, indicated by λ and κ.

The heavy and light chains of antibodies are structurally divided into variable and constant regions according to the variability of the amino acid sequence. The constant region of the heavy chain is composed of three or four heavy chain constant regions, such as CH1, CH2, and CH3 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the type of antibody, and the light chain is composed of one constant region, CL. The variable regions of the heavy and light chains are each composed of one domain, the heavy chain variable region (VH) or the light chain variable region (VL). The light and heavy chains are connected by a single covalent disulfide bond with their variable and constant regions aligned side by side, and the heavy chains of the two molecules bound to the light chain are connected by two covalent disulfide bonds to form the entire antibody. Whole antibodies specifically bind to antigens through the variable regions of the heavy and light chains, and since whole antibodies are composed of two pairs of heavy and light chains (HC/LC), one molecule of whole antibodies has bivalent monospecificity, binding to the same two antigens through the two variable regions. The antibody variable region that binds to the antigen is called the antigen-binding site of the antibody, and the part of the antigen surface recognized by the antibody is called the epitope.

The variable region of an antibody, which contains the antigen-binding site, is subdivided into a framework region (FR) with low sequence variability and a complementary determining region (CDR), which is a hypervariable region with high sequence variability. VH and VL each have three CDRs and four FRs, arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. The CDR with the highest sequence variability within the variable region of an antibody is the site that directly binds to the antigen and is most important for the antigenic specificity of the antibody.

In one aspect of the present invention, the antibody or antigen-binding fragment thereof according to the present invention may preferably be characterized by comprising (i) a heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 4, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 5; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 7, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 8.

In another aspect of the present invention, the antibody or antigen-binding fragment thereof according to the present invention may be characterized by preferably comprising (i) a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 10.

The antibody or antibody fragment according to the present invention is not limited in type as long as it has the above-described CDR, VH and VL, or light chain and heavy chain configuration, and the antibody may be an antibody in the form of IgG, IgA, IgM, IgE, or IgD. Preferably, it may be an antibody in the form of IgG.

In addition, in the present invention, the antigen-binding fragment of an antibody means a fragment of an antibody that maintains specific binding affinity to a SIRPα variant or a fragment thereof including the amino acid sequence of SEQ ID NO: 2, and specifically, may be in the form of Fab, F(ab)2, Fab', F(ab')2, Fv, diabody, scFv, etc. Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, and is composed of one variable region and one constant region of each of the heavy and light chains. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and has a form in which two Fabs are linked by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in the form of a heavy chain hinge added to a Fab obtained by reducing and separating the disulfide bond of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment composed only of the variable regions of each of the heavy and light chains. A scFv (single chain variable fragment) is a recombinant antibody fragment in which the VH and VL are linked by a flexible peptide linker. A diabody is a fragment in which the VH and VL of an scFv are linked by a very short linker, so that they cannot bind to each other, but instead bind to the VL and VH of another scFv of the same type, forming a dimer.

In addition, the antibody according to the present invention may be a monoclonal antibody derived from a single B cell, or a polyclonal antibody derived from a plurality of B cells, and is preferably a monoclonal antibody which is a group of antibodies in which the amino acid sequences of the heavy and light chains of the antibodies are substantially identical. The antibody or antibody fragment according to the present invention may be derived from any animal, including mammals including humans, birds, etc., and may be a chimeric antibody or antibody fragment having antibody sequences of different species together. The antibody or antibody fragment of the present invention may be conjugated to an enzyme, a fluorescent substance, a radioactive substance, a protein, etc., but is not limited thereto.

The present invention also provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof.

In the present invention, 'polynucleotide' may be described as an oligonucleotide or a nucleic acid, and includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The polynucleotide may be single-stranded or double-stranded. The polynucleotide means a base sequence encoding an antibody composed of a CDR configuration specific for a polypeptide comprising the amino acid of the above-mentioned SEQ ID NO: 2, or a heavy chain and a light chain having a VH and VL configuration.

A polynucleotide encoding the antibody of the present invention or an antigen-binding fragment thereof can be obtained by methods well known in the art. For example, based on a DNA sequence encoding part or all of the heavy and light chains of the antibody or the corresponding amino acid sequence, the polynucleotide can be synthesized using an oligonucleotide synthesis technique well known in the art, such as the polymerase chain reaction (PCR) method.

The present invention also provides a vector comprising the polynucleotide.

The 'vector' of the present invention is used for the purpose of cloning or expressing the polynucleotide of the present invention for the recombinant production of the antibody of the present invention or an antigen-binding fragment thereof, and generally includes at least one of a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably, it may be a vector including the polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.

A plasmid, a type of vector, is a linear or circular double-stranded DNA molecule into which foreign polynucleotide fragments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA fragments can be incorporated into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors, including those of bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell by introduction into the host cell and are thereby replicated together with the host genome.

In the present invention, the above-mentioned "vector" may be understood to have the same meaning as an "expression vector," which is a type of vector capable of expressing the polynucleotide. A polynucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence. The regulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence may, for example, exert its influence directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). The regulatory sequences include promoters, enhancers, and other expression control elements. The vector of the present invention may preferably be pOptiVEC™-TOPO and pcDNA™3.3-TOPO.

The present invention also provides a cell transformed with the above vector.

The type of cell of the present invention is not particularly limited as long as it can be used to express a polynucleotide encoding an antibody or a fragment thereof included in the expression vector of the present invention. The cell (host cell) transformed with the expression vector according to the present invention may be a prokaryote (e.g., Escherichia coli), a eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, insect cell, or a hybridoma derived therefrom). Preferably, it may be a cell derived from a mammal including a human.

Suitable prokaryotes for this purpose include gram-negative or gram-positive organisms, for example Enterobacteriaceae, for example Escherichia, for example E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans and Shigella, and Bacilli, for example B. subtilis and B. Bacillus licheniformis, Pseudomonas, for example, P. aeruginosa, and Streptomyces. The cell of the present invention is not particularly limited as long as it is capable of expressing the vector of the present invention, but is preferably E. coli.

The eukaryotic cell of the present invention is most commonly Saccharomyces cerevisiae. However, many other genera, species and strains may be used, including but not limited to Schizosaccharomyces pombe, Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. Thermotolerans (K. thermotolerans and K. marxianus); yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, e.g. Schwanniomyces occidentalis; and filamentous fungi, e.g. Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, e.g. A. nidulans and A. niger. It is available.

The above “transformation” refers to a change in the genotype of a host cell due to the introduction of a foreign polynucleotide, and refers to the introduction of the foreign polynucleotide into the host cell, regardless of the method used for the transformation. The foreign polynucleotide introduced into the host cell may be maintained by integration into the genome of the host cell or may be maintained without integration, and the present invention encompasses both.

The recombinant expression vector can be introduced into cells for producing antibodies or fragments thereof and transformed by methods known in the art, including but not limited to transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and any known method for introducing nucleic acids into cells.

In addition, the cell of the present invention is a cultured cell that can be transformed or transfected with the polynucleotide of the present invention or a vector containing the same, and which can subsequently be expressed in the host cell. A recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed. The cell of the present invention may also be a cell that contains the polynucleotide of the present invention, but does not express it at a desired level unless a regulatory sequence is introduced into the cell so as to be operably linked to the polynucleotide.

The cells of the present invention can be cultured in various media. Commercially available media, such as Ham's F1O (Sigma-Aldrich Co., St. Louis, MO), minimal essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.), are suitable for culturing cells. The media may, if necessary, contain hormones and/or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or an equivalent energy source.

The present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to a SIRPα variant or fragment thereof comprising an amino acid sequence of SEQ ID NO: 2, comprising the steps of culturing the cell under conditions in which a polynucleotide is expressed to produce a polypeptide comprising light and heavy chain variable regions, and recovering the polypeptide from the cell or a culture medium in which the cell or the polypeptide is cultured.

The cells of the production method of the present invention are as described above, and include a polynucleotide encoding the antibody of the present invention. The polypeptide of the production method may be the antibody of the present invention or a fragment thereof itself, or may be one to which an amino acid sequence other than the antibody of the present invention or a fragment thereof is additionally bound.

In this case, the antibody or fragment thereof of the present invention can be removed using methods well known to those skilled in the art. The culture medium composition and culture conditions may vary depending on the type of cell, and these can be appropriately selected and controlled by those skilled in the art.

The antibody molecule may be accumulated within the cytoplasm of a cell, secreted from the cell, or targeted to the periplasm or the extracellular medium (supernatant) by an appropriate signal sequence, and preferably targeted to the periplasm or the extracellular medium. Furthermore, it is preferable to refold the produced antibody molecule using a method well known to those skilled in the art and to make it have a functional conformation. The recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cell, and this can be appropriately selected and controlled by those skilled in the art.

The polypeptide may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide is produced intracellularly, the cell may be disrupted as a first step to release the protein. Particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, may be included in any preceding step to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants. Antibodies produced from cells may be purified, for example, using hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. The antibodies of the present invention are preferably purified by affinity chromatography.

The present invention also provides a method for detecting a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 in a sample, comprising the following steps:

(a) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and

(b) a step of detecting binding of the sample to the antibody or antigen-binding fragment thereof.

Binding of SIRPα variants and antibodies can be detected, quantified, and/or determined using a variety of assays available to those skilled in the art. While any suitable means for performing the assays is within the scope of the present invention, specific examples include fluorescence-activated cell sorting (FACS), ELISA, Western blotting, and immunohistochemistry (IHC). Preferred methods include IHC and FACS.

In the present invention, the sample may be a biological sample. A "biological sample" may be any sample that can be collected from an individual.

Preferred biological samples include samples such as blood samples, plasma samples, or lymph samples if the cancer is a liquid tumor.

Desirable biological samples include those obtained from a biopsy or surgical resection if the cancer is a solid tumor.

Preferably, the biological sample is a biological fluid, such as serum, whole blood cells, tissue samples, or biopsies of human origin. The sample may include, for example, biopsied tissue.

The antibody or antigen-binding fragment thereof according to the present invention exhibits an effect capable of accurately confirming and quantifying the presence of a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 during the process of expressing, purifying, and studying the same.

Figure 1 shows the results of an ELISA assay to evaluate whether two types of antibodies prepared in an example of the present invention can detect SIRPα variants.
Figure 2 shows the results of Western blotting to evaluate whether two types of antibodies prepared in an example of the present invention can detect SIRPα variants.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

1. Production of SIRPα variant (vSIRPα)-specific monoclonal antibodies

A monoclonal antibody specific for a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2 was produced according to the following steps:

- Hybridoma production through splenocyte fusion after inoculating Balb/c mice with the protein of sequence number 2

- Ensuring monoclonality through subcloning

Two types of antibodies were selected according to the above method.

2. Indirect ELISA assay using anti-vSIRPα antibody

Using two types of antibodies produced according to the above method, it was confirmed by indirect ELISA assay whether vSIRPα according to sequence number 2 could be detected.

The experimental conditions were as follows:

- Coating: 1.0 ug/ml, 100 ul vSIRPα antigen (SEQ ID NO: 2) in PBS

- Primary antibodies: Two types of antibodies developed

- Secondary antibody: Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific (min

The results are shown in Fig. 1. As shown in Fig. 1, the two types of antibodies produced were confirmed to detect vSIRPα containing the amino acid sequence of sequence number 2.

3. Antibody sequence analysis

The sequences of the two antibodies produced above were confirmed to be identical, sharing a parental clone, confirming that they are a single antibody. The sequence information for these antibodies is shown in Table 1 below.

5. Western blotting using anti-vSIRPα antibody

It was confirmed by Western blotting whether the two types of antibodies produced in the present invention could specifically detect vSIRPα containing the amino acid sequence of SEQ ID NO: 2. Antibodies against wild-type SIRPα were used as a control group.

The results for this are shown in Fig. 2.

As shown in Fig. 2, it was confirmed that both types of antibodies detect vSIRPα containing the amino acid sequence of sequence number 2 well, but do not detect wild-type SIRPα (wtSIRPα).

The antibody or antigen-binding fragment thereof according to the present invention has a very high potential for industrial application because it exhibits the effect of accurately confirming and quantifying the presence of a SIRPα variant during the process of expressing, purifying, and studying a SIRPα variant comprising the amino acid sequence of SEQ ID NO: 2.

Attach an electronic file of the sequence list

Claims (11)

An antibody or antigen-binding fragment thereof that specifically binds to a SIRPα (Signal-regulatory protein alpha) variant or fragment thereof comprising an amino acid sequence represented by SEQ ID NO: 2, wherein the antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 4, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 5; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 6, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 7, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 8.
delete In claim 1, the antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 10.
An antibody or antigen-binding fragment thereof, characterized in that it comprises:
An antibody or antigen-binding fragment thereof, characterized in that the antibody in claim 1 is a monoclonal antibody.
In claim 1, the antibody or antigen-binding fragment thereof is characterized in that the antibody is selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.
An antibody or antigen-binding fragment thereof, characterized in that in claim 1, the antibody fragment is selected from the group consisting of diabody, Fab, Fab', F(ab)2, F(ab')2, Fv, and scFv.
A polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 and 3 to 6.
A vector comprising the polynucleotide of claim 7.
Cells transformed with the vector of Article 8, excluding cells existing in the human body and human embryos.
A method for producing an antibody or antigen-binding fragment thereof that specifically binds to a SIRPα variant or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2, comprising the step of culturing the cell of claim 9 under conditions in which a polynucleotide is expressed to produce a polypeptide comprising light and heavy chain variable regions, and the step of recovering the polypeptide from the cell or the culture medium in which the cell or the polypeptide is cultured.
A method for detecting a SIRPα variant comprising an amino acid sequence of SEQ ID NO: 2 in a sample, comprising the following steps:
(a) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 and 3 to 6; and
(b) a step of detecting binding of the sample to the antibody or antigen-binding fragment thereof.