Novel antibodies or antigen-binding fragments thereof derived from plasma cells in cancer tissues, and uses thereof

Abstract

The present invention relates to novel antibodies or antigen-binding fragments thereof derived from plasma cells in cancer tissues of cancer patients, and uses thereof, and does not bind to normal cells and exhibits the activity of specifically binding to antigens of cancer cells. Therefore, the antibodies or antigen-binding fragments thereof can be effectively used in the diagnosis of cancer and, when bound to drugs such as an anticancer agent, the antibodies or antigen-binding fragments thereof can also be effectively used in the prevention or treatment of cancer and the like. In addition, the antibodies or antigen-binding fragments thereof are human antibodies or antigen-binding fragments thereof derived from plasma cells in the cancer tissues of cancer patients, and thus can be safely used in the diagnosis, prevention, or treatment of human breast cancer without having to undergo humanization in which specific amino acids of the variable region of the antibody are substituted in order to avoid or minimize an immune response in humans.

Description

Novel antibodies derived from plasma cells in cancer tissue or their antigen-binding fragments, and their uses

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0154663 filed November 4, 2024, and all contents disclosed in the document of said Korean Patent Application are incorporated herein as part of this specification.

[0003] Technology field

[0004] The present invention relates to a novel antibody derived from plasma cells in cancer tissue or an antigen-binding fragment thereof, and the use thereof.

[0005]

[0006] Recently, as the effectiveness of immunotherapy for cancer patients has been verified, attempts to expand its application to anticancer treatment are continuing. However, one of the biggest challenges in anticancer immunotherapy is discovering targets or antibodies that are not expressed in normal cells but are specifically expressed only in cancer cells. This is because B cells recognize antigens through the B cell receptor (BCR), and since the diversity of the BCR can be set very high due to V(D)J recombination, somatic hypermutation, class switch recombination, etc., it is difficult to discover targets or antibodies that are expressed only in cancer cells.

[0007] Plasma cells refer to B cells that have differentiated to produce large quantities of antibodies matching the structure of the B cell receptor (BCR) in response to stimulation by antigens and T cells. Theoretically, the types of antibodies that can be produced by human plasma cells are due to the diversity of BCRs, 10 13 It is known to be more than [number].

[0008] Meanwhile, since the advancement of sequencing technology enabled single-cell RNA sequencing, it has been revealed that specific tumor-infiltrating T cells perform major functions in various cancers. However, regarding plasma cells that differentiate from B cells to produce antibodies, research on tumor-infiltrating plasma cells present within tumor microtissues is currently lacking. Plasma cell infiltration within tumor microtissues has been reported to be associated with favorable prognoses in various cancers, which may imply that plasma cells perform anti-tumor functions by producing specific antibodies.

[0009] Against this technical background, the applicant performed single-cell sequencing on the cancer tissue of a cancer patient and performed transcriptome sequencing of plasma cells. Through this, antibodies secreted from plasma cells within the cancer tissue were derived, and it was confirmed that these antibodies bind specifically only to cancer tissue. Therefore, the novel antibody of the present invention is expected to be useful for the diagnosis, prevention, or treatment of cancer.

[0010]

[0011] One objective of the present invention is to provide a novel antibody or an antigen-binding fragment thereof that is derived from plasma cells within the cancer tissue of a cancer patient and can specifically bind to cancer cell antigens.

[0012] Another object of the present invention is to provide a gene encoding the antibody or its antigen-binding fragment, an expression vector comprising the gene, and a transformant.

[0013] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody or an antigen-binding fragment thereof.

[0014] Another objective of the present invention is to provide a method for selecting antibodies derived from plasma cells within tumor tissue, such as the antibody or its antigen-binding fragment.

[0015]

[0016] To achieve the above objective, one aspect of the present invention provides an antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region comprising a CDR1-H having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, a CDR2-H having the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, and a CDR3-H having the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6; and a light chain variable region comprising a CDR1-L having the amino acid sequence of SEQ ID NO. 7 or SEQ ID NO. 8, a CDR2-L having the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10, and a CDR3-L having the amino acid sequence of SEQ ID NO. 11 or SEQ ID NO. 12.

[0017] In addition, to achieve the above objective, another aspect of the present invention provides an antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 13 or SEQ ID NO. 14; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 15 or SEQ ID NO. 16.

[0018] In addition, to achieve the above objective, another aspect of the present invention provides an antibody or an antigen-binding fragment thereof comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO. 17 or SEQ ID NO. 18; and a light chain comprising the amino acid sequence of SEQ ID NO. 19 or SEQ ID NO. 20.

[0019] In addition, to achieve the above objective, another aspect of the present invention provides a gene encoding the antibody or the antigen-binding fragment thereof, an expression vector comprising the gene, and a transformant.

[0020] In addition, to achieve the above objective, another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody or an antigen-binding fragment thereof.

[0021] In addition, to achieve the above objective, another aspect of the present invention provides a method for selecting antibodies derived from plasma cells within tumor tissue, such as the antibody or its antigen-binding fragment.

[0022]

[0023] The novel antibody or its antigen-binding fragment of the present invention is derived from plasma cells within the cancer tissue of a cancer patient and possesses activity that specifically binds to antigens on cancer cells without binding to normal cells. Therefore, the antibody or its antigen-binding fragment can be effectively used for the diagnosis of cancer, and when combined with a drug such as an anticancer agent, it can also be effectively used for the prevention or treatment of cancer. Furthermore, since the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment derived from plasma cells within the cancer tissue of a cancer patient, it can be safely used for the diagnosis, prevention, or treatment of cancer in humans without the need to undergo a humanization process that involves replacing specific amino acids in the variable region of the antibody to evade or minimize the immune response in humans.

[0024] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the following description.

[0025]

[0026] Figure 1 shows a schematic diagram of B cells and T cells in tumor tissue and plasma cells that secrete antibodies.

[0027] Figure 2 shows a schematic diagram of performing scRNA-GEX sequencing and scRNA-BCR sequencing from cancer tissues of three breast cancer patients.

[0028] Figure 3 shows a plasma cell cluster identified through scRNA-GEX sequencing.

[0029] Figure 4 shows immunoglobulin pairs in plasma cells and B cells.

[0030] Figure 5 shows the immunoglobulin frequency and binding type in plasma cells and B cells.

[0031] Figures 6a to 6c show a list of differentially expressed genes in IgG-positive plasma cells compared to IgM-positive plasma cells or Naive B cells.

[0032] Figure 7 shows the strength of ligand-receptor interactions between B cells, CD4 T cells, and cancer cells.

[0033] Figure 8 shows MHC-II pathway-related interactions between CD4 T cells and cancer cells, and between CD4 T cells and B cells.

[0034] Figure 9 shows that the MHC-II pathway is activated by HLA-DRB1 and HLA-DRA, etc., within tumor tissue.

[0035] Figure 10 shows the expression levels of CD40-CD40LG ligand and receptor genes in B cells and CD4 T cells.

[0036] Figure 11 shows the results of cluster analysis for B cells.

[0037] Figure 12 shows the results of a virtual time trajectory analysis for naive B cells.

[0038] Figure 13 shows the IGHG1 expression and HLA-DRA expression levels in B cells and plasma cells.

[0039] Figure 14 shows the results of the plasma cell clone analysis and six plasma cell clones for antibody production.

[0040] Figure 15 shows the results of a virtual time trajectory analysis for the Ab#1 clone.

[0041] Figure 16 shows the clonal frequency of B cells and plasma cells.

[0042] Figure 17 shows six antibodies derived from a plasma cell clone.

[0043] Figure 18 shows the results of immunostaining on immune and epithelial cells obtained from the patient's tonsil tissue.

[0044] Figure 19a shows the results of immunostaining of the Ab#1 antibody in cancer tissue.

[0045] Figure 19b shows the results of immunostaining of the Ab#1 antibody in normal cells surrounding the cancer tissue.

[0046] Figure 20 shows the results of immunostaining of the Ab#2 antibody in cancer tissue.

[0047] Figure 21 shows the results of immunostaining of antibodies Ab#3 to Ab#6 in cancer tissue.

[0048]

[0049] First, the terms used in the specification of the present invention will be explained.

[0050] The term "antibody" as used in the present invention refers to an immunoglobulin molecule having a structure in which one light chain is connected to each of two heavy chains by disulfide bonds by disulfide bonds, and a multimer thereof. The light chain comprises two regions, namely, one variable region (light chain variable region, VL) and one constant region (light chain constant region, CL), and the heavy chain comprises four regions, namely, one variable region (heavy chain variable region, VH) and three constant regions (heavy chain constant regions, CH; CH1, CH2, and CH3). The constant regions of the light chain and heavy chain serve to confer biological properties such as binding between light chains and / or heavy chains, secretion, complement binding, and binding with Fc receptors (FcR), while the variable regions of the light chain and heavy chain determine recognition of antigens and binding specificity. In particular, the binding specificity of antibodies is due to structural complementarity between antibody binding sites and epitopes. Since such antibody binding sites are composed mainly of residues derived from three hypervariable regions called complementarity determining regions (CDRs) included in the variable regions of heavy and light chains, such complementarity determining regions are referred to as amino acid sequences that define the binding specificity of antibodies. These three CDRs are positioned between four relatively well-conserved regions called framework regions (FRs), which are connected in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in the direction from the N-terminus to the C-terminus. At this time, the three CDRs included in the heavy chain variable region are respectively called CDR1-H, CDR2-H, and CDR3-H, and the three CDRs included in the light chain variable region are respectively called CDR1-L, CDR2-L, and CDR3-L.

[0051] The term "antigen-binding fragment" as used in the present invention refers to any polypeptide or glycoprotein comprising a part of an intact antibody, in particular, an antigen-binding site or variable region of an intact antibody. Such an antigen-binding fragment may be produced by recombinant DNA techniques or by enzymatically or chemically degrading an intact antibody. Examples of such antigen-binding fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, etc., as well as bispecific and multispecific antibodies formed therefrom.

[0052] The above Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain), and has one antigen-binding site. The above Fab' differs from the above Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The above F(ab')2 is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. The above Fv refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, while in a single-chain Fv, the heavy chain variable region and the light chain variable region are generally connected by covalent bonds via a peptide linker or directly at the C-terminus, so they can form a dimer-like structure similar to the two-chain Fv. The above antigen-binding fragment can be produced using a proteolytic enzyme (for example, if the whole antibody is restricted to papain, Fab can be obtained, and if it is restricted to pepsin, F(ab')2 fragment can be obtained), or through genetic recombination technology, but is not limited thereto.

[0053] The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may include hydrophilic amino acids such as glycine (G) and / or serine (S). The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), for example (GnS)m (where n and m are each 1 to 10), but is not limited thereto.

[0054]

[0055] The present invention will be described in detail below.

[0056]

[0057] 1. A novel antibody or its antigen-binding fragment

[0058] One aspect of the present invention provides a novel antibody or an antigen-binding fragment thereof.

[0059] In the present invention, the antibody or its antigen-binding fragment may be derived from cancer tissue, and more specifically, may be derived from plasma cells within the cancer tissue.

[0060] The antibody or its antigen-binding fragment of the present invention is derived from plasma cells within the cancer tissue of a cancer patient and may have activity that does not bind to normal cells but specifically binds to antigens on cancer cells. Therefore, the antibody or its antigen-binding fragment can be effectively used for the diagnosis of cancer, and when the antibody or its antigen-binding fragment is combined with a drug such as an anticancer agent, it can also be effectively used for the prevention or treatment of cancer.

[0061] The above-mentioned plasma cell refers to a B cell differentiated to produce large quantities of antibodies matching the structure of a B cell receptor (BCR) upon stimulation by an antigen and a T cell. The antibody or its antigen-binding fragment of the present invention is derived by a method of analyzing the sequence of antibodies secreted by plasma cells present within a tumor microtissue by sequencing the transcriptomes of the plasma cells.

[0062] Furthermore, since the antibody or its antigen-binding fragment of the present invention is a human antibody or its antigen-binding fragment derived from plasma cells within the breast cancer tissue of a breast cancer patient, it can be safely used for the diagnosis, prevention, or treatment of human breast cancer without the need to undergo a humanization process that involves replacing specific amino acids in the variable region of the antibody to evade or minimize the immune response in humans.

[0063] The antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain variable region comprising CDR1-H having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, CDR2-H having the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, and CDR3-H having the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6; and a light chain variable region comprising CDR1-L having the amino acid sequence of SEQ ID NO. 7 or SEQ ID NO. 8, CDR2-L having the amino acid sequence of SEQ ID NO. 9 (DAS) or SEQ ID NO. 10 (LGS), and CDR3-L having the amino acid sequence of SEQ ID NO. 11 or SEQ ID NO. 12.

[0064] The antibody or its antigen-binding fragment of the present invention may further comprise a heavy chain constant region and / or a light chain constant region of an antibody derived from, for example, a human or other mammal, provided that the heavy chain constant region and / or light chain constant region of the antibody derived from a human or mammal is not impaired in its characteristic of specifically binding to breast cancer cell antigens, said antibody or its antigen-binding fragment may be used without limitation in its type or amino acid sequence.

[0065] The antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 13 or SEQ ID NO. 14; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 15 or SEQ ID NO. 16.

[0066] In addition, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 17 or SEQ ID NO. 18; and a light chain comprising the amino acid sequence of SEQ ID NO. 19 or SEQ ID NO. 20.

[0067] In the present invention, the antigen-binding fragment of the antibody may be a single-chain variable fragment (scFv).

[0068] In one embodiment of the present invention, among six types of antibodies derived from plasma cells in breast cancer tissue of a breast cancer patient, two types of antibodies (Ab#1 and Ab#2) derived from plasma cells that underwent clonal expansion were confirmed to have activity that specifically binds to the cell membrane of breast cancer cells.

[0069] Among them, the Ab#1 antibody comprises a heavy chain variable region including CDR1-H having the amino acid sequence of SEQ ID NO. 1, CDR2-H having the amino acid sequence of SEQ ID NO. 3, and CDR3-H having the amino acid sequence of SEQ ID NO. 5, and a light chain variable region including CDR1-L having the amino acid sequence of SEQ ID NO. 7, CDR2-L(DAS) having the amino acid sequence of SEQ ID NO. 9, and CDR3-L having the amino acid sequence of SEQ ID NO. 11. In addition, the Ab#1 antibody comprises a heavy chain variable region including the amino acid sequence of SEQ ID NO. 13 and a light chain variable region including the amino acid sequence of SEQ ID NO. 15. In addition, the Ab#1 antibody comprises a heavy chain including the amino acid sequence of SEQ ID NO. 17 and a light chain including the amino acid sequence of SEQ ID NO. 19.

[0070] Meanwhile, the Ab#2 antibody comprises a heavy chain variable region including CDR1-H having the amino acid sequence of SEQ ID NO. 2, CDR2-H having the amino acid sequence of SEQ ID NO. 4, and CDR3-H having the amino acid sequence of SEQ ID NO. 6, and a light chain variable region including CDR1-L having the amino acid sequence of SEQ ID NO. 8, CDR2-L(LGS) having the amino acid sequence of SEQ ID NO. 10, and CDR3-L having the amino acid sequence of SEQ ID NO. 12. Additionally, the Ab#1 antibody comprises a heavy chain variable region including the amino acid sequence of SEQ ID NO. 14 and a light chain variable region including the amino acid sequence of SEQ ID NO. 16. Furthermore, the Ab#1 antibody comprises a heavy chain including the amino acid sequence of SEQ ID NO. 18 and a light chain including the amino acid sequence of SEQ ID NO. 20.

[0071] The amino acid sequences described above may include variants having different sequences by deletion, insertion, substitution, or a combination thereof of amino acid residues, within a range that does not affect the structure, function, activity, etc. of the polypeptide containing them. Additionally, the amino acid sequences may include amino acids that have undergone conventional modifications known in the art, and said amino acid modifications may be, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. The antibody or antigen-binding fragment thereof of the present invention includes not only the amino acid sequences described above, but also those having substantially the same amino acid sequence or variants thereof. The meaning of having substantially the same amino acid sequence as above may include, but is not limited to, an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the amino acid sequence described above.

[0072] In addition, the amino acid sequences described above may include conservative substitutions in which an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitution among amino acid residues having basic side chains, such as lysine, arginine, and histidine, corresponds to conservative substitution. Furthermore, amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains, such as threonine, valine, and isoleucine; Substitutions among amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, are also likewise conservative substitutions.

[0073]

[0074] 2. A novel antibody or a gene associated with its antigen-binding fragment, and a vector and a transformant containing the same

[0075] Another aspect of the present invention provides a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention.

[0076] In one embodiment of the present invention, a gene encoding a heavy chain variable region of the antibody of the present invention or its antigen-binding fragment and a gene encoding a light chain variable region of the antibody of the present invention or its antigen-binding fragment may be provided, respectively. The gene encoding the variable region of the heavy chain or light chain may undergo various modifications to the encoding region within a range that does not alter the amino acid sequence of the variable regions expressed from the encoding region, and may undergo various mutations in a portion excluding the encoding region within a range that does not affect gene expression. Ultimately, as long as the gene of the variable regions encodes a protein having equivalent activity, one or more nucleic acid bases may be substituted, deleted, inserted, or a combination thereof, and such a mutated gene is also included within the scope of the present invention.

[0077] In addition, another embodiment of the present invention provides a gene encoding each of the CDRs included in the heavy chain variable region of the antibody of the present invention or its antigen-binding fragment. Specifically, a gene encoding CDR1-H, a gene encoding CDR2-H, and a gene encoding CDR3-H are each provided individually. In particular, the gene encoding CDR1-H may encode the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, the gene encoding CDR2-H may encode the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, and the gene encoding CDR3-H may encode the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6.

[0078] In addition, another embodiment of the present invention provides a gene encoding each of the CDRs included in the light chain variable region of the antibody of the present invention or its antigen-binding fragment. Specifically, a gene encoding CDR1-L, a gene encoding CDR2-L, and a gene encoding CDR3-L are each provided individually. In particular, the gene encoding CDR1-L may encode the amino acid sequence of SEQ ID NO. 7 or SEQ ID NO. 8, the gene encoding CDR2-L may encode the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10, and the gene encoding CDR3-L may encode the amino acid sequence of SEQ ID NO. 11 or SEQ ID NO. 12.

[0079] The gene encoding the above CDRs may undergo various modifications to the coding region within a range that does not alter the amino acid sequence of the CDRs expressed from the coding region, and may also undergo various mutations in a portion excluding the coding region within a range that does not affect gene expression. Ultimately, as long as the gene encoding the above CDRs encodes a protein having equivalent activity, one or more nucleic acid bases may be substituted, deleted, inserted, or a combination thereof, and such mutated genes are also included within the scope of the present invention.

[0080] In addition, another embodiment of the present invention provides a gene encoding a heavy chain variable region and a light chain variable region of the antibody of the present invention or its antigen-binding fragment. Specifically, the gene encoding the heavy chain variable region may encode the amino acid sequence of SEQ ID NO. 13 or SEQ ID NO. 14, and the gene encoding the light chain variable region may encode the amino acid sequence of SEQ ID NO. 15 or SEQ ID NO. 16.

[0081] In addition, another embodiment of the present invention provides a gene encoding a heavy chain and a light chain of the antibody of the present invention or its antigen-binding fragment. Specifically, the gene encoding the heavy chain may encode the amino acid sequence of SEQ ID NO. 17 or SEQ ID NO. 18, and the gene encoding the light chain may encode the amino acid sequence of SEQ ID NO. 19 or SEQ ID NO. 20. For example, the base sequence encoding the amino acid sequence of SEQ ID NO. 17 may include the base sequence of SEQ ID NO. 21, the base sequence encoding the amino acid sequence of SEQ ID NO. 18 may include the base sequence of SEQ ID NO. 22, the base sequence encoding the amino acid sequence of SEQ ID NO. 19 may include the base sequence of SEQ ID NO. 23, and the base sequence encoding the amino acid sequence of SEQ ID NO. 20 may include the base sequence of SEQ ID NO. 24, but is not limited thereto.

[0082] The gene of the present invention may include a base sequence substantially identical to the base sequence encoding the amino acid sequences. The substantially identical base sequence includes, for example, cases where the same amino acid can be synthesized when transcribed and translated, and may be a base sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the listed base sequences, but is not limited thereto.

[0083] The gene encoding the above antibody or its antigen-binding fragment may include an optimized nucleotide sequence depending on the type of organism to be introduced and expressed and the expression system, such as transcription or translation, of said organism. This is due to the degeneracy of codons, and accordingly, various combinations of nucleotide sequences capable of encoding the expressed protein may exist, all of which are included within the scope of the present invention. The modification of the polynucleotide according to the above codon optimization may be determined according to the type of organism to be applied by expressing the antibody or its antigen-binding fragment of the present invention; for example, the gene of the present invention may be a gene modified to be optimized for codon selection in mammals or primates, and more specifically, may be a gene modified to be optimized for use in humans.

[0084]

[0085] In addition, another aspect of the present invention provides a recombinant expression vector comprising a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention, and a transformant in which said expression vector is introduced into a host cell.

[0086] The recombinant expression vector of the present invention comprises a gene encoding all or part of the antibody of the present invention or its antigen-binding fragment, and said gene may be operably linked to a promoter.

[0087] The above expression vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and virus vectors.

[0088] The above recombinant expression vector may appropriately combine expression regulatory sequences, such as promoters, terminators, and enhancers, or sequences for secretion, according to the purpose, depending on the type of host cell intended to express or produce all or part of the antibody of the present invention or its antigen-binding fragment.

[0089] The above expression vector may additionally include a selection marker for selecting the host cell into which the vector is introduced, and if it is a replicable expression vector, it may include a replication origin.

[0090] In addition, the recombinant expression vector may include a sequence to facilitate the purification of the expression protein; specifically, a gene encoding a separation and purification tag may be linked to a gene encoding all or part of the antibody of the present invention or its antigen-binding fragment to enable operation. In this case, the separation and purification tag may be used alone or by sequentially linking two or more of the following: GST, poly-Arg, FLAG, histidine-tag (His-tag), c-myc, etc. The gene encoding all or part of the antibody of the present invention or its antigen-binding fragment may be cloned through a restriction enzyme cleavage site. If a gene encoding a protein cleavage enzyme recognition site is used in the vector, it may be linked in frame with the gene encoding all or part of the antibody of the present invention or its antigen-binding fragment, so that when all or part of the antibody of the present invention or its antigen-binding fragment is obtained and then cleaved with a protein cleavage enzyme, all or part of the antibody of the present invention or its antigen-binding fragment in its original form can be produced.

[0091] In addition, a recombinant expression vector comprising a gene encoding all or part of the antibody or antigen-binding fragment thereof of the present invention may be introduced into the transformant of the present invention.

[0092] Transformers can be produced by transforming the recombinant expression vector according to the present invention into any one suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells, and animal cells, depending on the purpose of expression. For example, the host cell may be E. coli (E. coli BL21(DE3), DH5α, etc.) or yeast cells (genus Saccharomyces, genus Pichia, etc.). At this time, depending on the type of host cell, a person skilled in the art can easily select appropriate culture methods and culture medium conditions from known technologies in the field.

[0093] In addition, the present invention provides a method for producing an antibody that specifically binds to a cancer cell antigen or an antigen-binding fragment thereof, comprising the step of culturing the transformant.

[0094] The method for introducing a recombinant expression vector for the production of a transformant of the present invention may use known techniques, namely thermal shock, electric shock, etc.

[0095] Since the protein expressed from the above transformant is the antibody of the present invention or its antigen-binding fragment, mass production of the antibody of the present invention or its antigen-binding fragment can be facilitated by mass culturing the above transformant to express the gene.

[0096]

[0097] 3. Uses of novel antibodies or their antigen-binding fragments

[0098] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0099] In the present invention, the cancer may include all cancers that fall within the medical category of cancer, and the specific disease name is not particularly limited; however, specifically, the cancer may be one or more selected from the group consisting of gastric cancer, colorectal cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, liver cancer, cervical cancer, breast cancer, ovarian cancer, head and neck cancer, bone cancer, leukemia, carcinoid cancer, prostate cancer, lung cancer, bladder cancer, endometrial cancer, melanoma, kidney cancer, testicular cancer, glioma, thyroid cancer, skin cancer, and lymphoma, and more specifically, may be breast cancer, but is not limited thereto.

[0100] The antibody or its antigen-binding fragment of the present invention is derived from plasma cells within the cancer tissue of a cancer patient, and said antibody or its antigen-binding fragment may have activity that specifically binds to cancer cell antigens. Accordingly, said antibody or its antigen-binding fragment can be effectively used for the diagnosis of cancer, and when said antibody or its antigen-binding fragment is combined with a drug such as an anticancer agent, it can also be effectively used for the prevention or treatment of cancer.

[0101] The above prevention refers to all measures intended to suppress or delay the onset of cancer. Furthermore, the above treatment refers to all measures intended to improve or alleviate the symptoms of manifested cancer, and the desirable effects of the above treatment include prevention of the occurrence or recurrence of cancer, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the cancer, prevention of metastasis, reduction of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis.

[0102] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or additive in addition to the antibody or antigen-binding fragment thereof of the present invention.

[0103] The meaning of "pharmaceuticalally acceptable" above is that the subject of application (prescription) does not possess toxicity beyond what is acceptable without inhibiting the activity of the active ingredient. The above carrier refers to a compound that facilitates the addition of the antibody of the present invention or its antigen-binding fragment into a cell or tissue, and the above additive refers to any components necessary to prepare the pharmaceutical composition into the required formulation, such as excipients, disintegrants, binders, flavorings, preservatives, lubricants, stabilizers, viscosities, etc.

[0104] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above pharmaceutical composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao gelatin, laurin gelatin, glycerogelatin, etc. may be used as bases for suppositories, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0105] The above pharmaceutical composition may be administered parenterally or orally, such as external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, nasal injection, enteral injection, local injection, sublingual injection, or intrathoracic injection.

[0106] The above pharmaceutical composition may be provided by mixing it with a conventionally known pharmaceutical composition for the prevention or treatment of cancer or a newly developed pharmaceutical composition for the prevention or treatment of cancer. If the above pharmaceutical composition further comprises a pharmaceutical composition for the prevention or treatment of cancer, it is important that an amount is mixed such that maximum effect can be obtained with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0107] In addition, the above pharmaceutical composition may be administered alone or in combination with other cancer treatments. That is, the above pharmaceutical composition may be administered in conjunction with a known composition or other cancer treatment having a preventive or therapeutic effect against cancer, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0108]

[0109] In addition, another aspect of the present invention provides a method for preventing or treating cancer comprising administering the antibody of the present invention or its antigen-binding fragment, or the pharmaceutical composition, to a person in need.

[0110] The above-mentioned individual may be a mammal, for example, a human, and in particular, a patient. Additionally, the above-mentioned 'necessary individual' refers to an individual who has cancer or is likely to have cancer.

[0111] Additionally, the above composition may be administered in a single dose or divided into multiple doses. When fully considering these factors, it is important to administer the minimum dose sufficient to obtain maximum effect without side effects, and this dosage can be easily determined by an expert in the field. The dosage of the above pharmaceutical composition is not specifically limited but varies according to various factors including the patient's health status and weight, the severity of the disease, the type of drug, the route of administration, and the time of administration. In particular, the above pharmaceutical composition may be administered in a single daily dose or multiple doses via routes typically permitted to mammals, including humans, such as orally, rectally, intravenously, subcutaneously, intraperitoneally, intrauterinely, or intrarebrovascularly.

[0112] The antibody or its antigen-binding fragment of the present invention, or the pharmaceutical composition, may be administered in a pharmaceutically effective amount. The "pharmaceuticalally effective amount" refers to an amount that provides a sufficient preventive or therapeutic effect with a reasonable benefit / risk ratio applicable to all medical treatments. Furthermore, the "pharmaceutically effective amount" varies depending on various factors, including the severity of the disease to be treated, the age and gender of the patient, the type of disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration, the secretion rate, the duration of treatment, co-administration of drugs, and other parameters known in the art.

[0113]

[0114] In addition, another aspect of the present invention provides a health functional food composition for the prevention or improvement of cancer comprising the antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0115] The term "improvement" above may refer to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms. In this case, the health functional food may be used for the prevention or improvement of cancer, either simultaneously with or separately from a drug for treatment, either before or after the onset of the disease.

[0116] In the above-mentioned health functional food, the active ingredient may be added directly to the food or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverages, the above-mentioned health functional food may be added in an amount of about 15% by weight or less, specifically about 10% by weight or less, with respect to the raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0117] The above-mentioned health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, by further including one or more of a carrier, a diluent, an excipient, and an additive. Foods to which a compound according to one aspect may be added include various types of food, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

[0118] Specific examples of the above carrier, excipient, diluent, and additive may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0119] In addition to containing the active ingredient mentioned above, the above-mentioned health functional food may contain other ingredients as essential components without special limitations. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, such as in ordinary beverages. Examples of the above-mentioned natural carbohydrates may be monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by the choice of a person skilled in the art.

[0120] In addition to the above, the health functional food may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.

[0121]

[0122] 4. Selection method for plasma cell-derived antibodies in tumor tissue

[0123] Another aspect of the present invention provides a method for selecting plasma cell-derived antibodies within tumor tissue, such as the antibody of the present invention or an antigen-binding fragment thereof.

[0124] The above method comprises: a) obtaining a tumor tissue sample and performing single-cell transcriptome sequencing; b) analyzing the transcriptome sequencing data obtained in step a) to identify a plasma cell cluster; and c) selecting clones having full-length immunoglobulin sequences from the plasma cell clusters.

[0125] In step a) above, the tumor tissue sample may be derived from at least one cancer tissue selected from the group consisting of lung cancer, liver cancer, kidney cancer, glioma, melanoma, gastric cancer, neuroblastoma, colorectal cancer, breast cancer, uterine cancer, pancreatic cancer, chronic myeloid leukemia, prostate cancer, and head and neck cancer, but is not limited thereto.

[0126] For example, in one embodiment of the present invention, a breast cancer tissue sample from a breast cancer patient is obtained, and by going through steps a) to c), an antibody capable of specifically binding to breast cancer cells is derived; however, even when a cancer tissue sample other than breast cancer is obtained, the same method can be applied to derive an antibody that specifically binds to other cancer tissues.

[0127] In step a) above, single-cell transcriptome sequencing can be performed using the scRNA-GEX sequencing (single-cell RNA sequencing gene expression) and / or scRNA-BCR sequencing (scRNA-seq B cell receptor sequencing) methods.

[0128] The above step b) can be performed as a process of identifying plasma cell clusters by analyzing the transcriptome sequencing data obtained in the above step a) to confirm whether markers specific to each immune cell, such as plasma cells and B cells, are expressed, and by confirming whether markers specifically expressed in plasma cells are expressed.

[0129] In this case, the plasma cells may be plasma cells differentiated by stimulation of CD4 T cells and cancer cells. Stimulation of CD4 T cells can contribute to the process of B cells differentiating into plasma cells, and stimulation of cancer cells can contribute to the process of differentiation into plasma cells that secrete antibodies capable of specifically binding to cancer cell antigens. Therefore, it is desirable for the plasma cells to differentiate by receiving stimulation from both CD4 T cells and cancer cells. The existence of such intercellular stimulation or interaction can be verified using programs such as CellChat.

[0130] In addition, the plasma cells may be clonal expanded plasma cells. Clonal expansion refers to the process in which immune cells, such as B cells and T cells, are replicated in large quantities into clones with identical genetic characteristics after being activated by an antigen.

[0131] In one embodiment of the present invention, it was confirmed that among plasma cell clusters within the tumor microenvironment, only the antibodies Ab#1 and Ab#2 derived from a plasma cell population that has undergone clonal expansion possess activity that specifically binds to tumor tissue. Therefore, by selecting only the clusters that have undergone clonal expansion among the plasma cell clusters, antibodies capable of specifically binding to tumors can be efficiently selected.

[0132] The selection of the above-mentioned cloned plasma cells can be performed by selecting the plasma cells that form the largest clone, or by performing pseudotime trajectory analysis to select the most differentiated plasma cells.

[0133] Meanwhile, in step b) above, the plasma cells may refer to plasma cells within the tumor microenvironment. Plasma cells within the tumor microenvironment may include both plasma cells infiltrated into the tumor tissue and plasma cells present around the tumor tissue.

[0134]

[0135] The present invention will be explained in detail below through examples.

[0136] However, the following examples are intended to specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0137]

[0138] [Example 1]

[0139] Single-cell transcriptional profiling analysis of plasma cells in the tumor microenvironment

[0140] For immunotherapy that specifically kills only cancer cells by combining an anticancer drug with an antibody that specifically binds to cancer cells, it is essential to discover antibodies that can specifically bind only to cancer cells.

[0141] To this end, based on the possibility that plasma cells present in the tumor microenvironment can produce antibodies that specifically bind to cancer cells during an anti-tumor immune response, an experiment was conducted to analyze antibodies produced by plasma cells in tumor tissue (Fig. 1).

[0142] First, breast cancer tissue samples were obtained from two HR-positive (hormone receptor-positive) breast cancer patients and one triple-negative breast cancer patient, and scRNA-GEX sequencing (single-cell RNA sequencing gene expression) and scRNA-BCR sequencing (scRNA-seq B cell receptor sequencing) were performed (Fig. 2). HR-positive breast cancer patients were defined as tissues positive for the estrogen receptor (ER) and / or progesterone receptor (PR) by performing immunohistochemical staining on the obtained cancer tissues. This study was conducted with the approval of the Institutional Review Board of Asan Medical Center (IRB no. 2015-0438). The multiplexed scRNA sequencing libraries of each sample were prepared for 10x Genomics single-cell 5' gene expression and V(D)J sequencing according to the protocols provided by the 10x Genomics chromium single-cell immunoprofiling platform. Subsequently, sequencing data for a total of 5,735 cells were generated from a multiplexing library sequenced on an Illumina NovaSeq 6000. Of the 5,735 sequenced cells, a total of 5,131 cells that passed quality control were used for the final analysis. Genotype, gene expression count data, and demultiplexing based on the BCR repertoire through full-length BCR sequence reconstruction were performed using Cell Ranger v6.1.1.

[0143] As a result of performing scRNA-GEX sequencing on a total of 5,131 cells, various immune cells and cancer cells were identified, and among them, B cells and plasma cells could be identified through the expression of markers specific to B cells and plasma cells, and it was confirmed that plasma cells accounted for 1 to 5% of the total number of cells in the tissue sample (Fig. 3).

[0144] In addition, analysis of immunoglobulin chains using Dandelions via scRNA-BCR sequencing confirmed that plasma cell clusters mostly contained a single chain pair, and that immunoglobulins were detected in plasma cell and B cell clusters (Fig. 4). Furthermore, the IGHM-IGKC heavy and light chain pair was most frequently detected in B cells, while the IGHG1-IGKC heavy and light chain pair was most frequently detected in plasma cells (Fig. 5). Moreover, a list of genes involved in the isotype conversion from IgM to IgG in naive B cells was identified (Figs. 6a to 6c).

[0145]

[0146] [Example 2]

[0147] Identification of plasma cells involved in immune responses within tumor tissue

[0148] Next, to determine whether the plasma cells analyzed in Example 1 above are involved in the immune response within the tumor tissue, an analysis of ligand-receptor interactions between B cells, CD4 T cells, and cancer cells within the tumor tissue was performed. Cell-to-cell interactions were analyzed using scRNA sequencing data via CellChat (version 2.1.0).

[0149] As a result, strong interactions between pre-differentiated B cells, CD4 T cells, and cancer cells within the tumor tissue were confirmed (Fig. 7). Specifically, the MHC-II pathway was found to be activated by interactions between cancer cells, CD4 T cells, and B cells, while the MHC-I pathway was found to be activated by interactions between cancer cells and CD8 T cells (Fig. 8). At this time, the MHC-II pathway was confirmed to be activated through interactions between various cells within the tumor tissue where the expression of HLA-DRB1 and HLA-DRA was significantly high (Fig. 9). Meanwhile, regarding the CD40-CD40LG ligand and receptor genes, which are activated by CD4 T cells to differentiate B cells into plasma cells, CD40 is predominantly expressed in B cells and CD40LG is predominantly expressed in CD4 T cells, confirming that plasma cell differentiation can be performed through the interaction of the two cells (Fig. 10).

[0150] Through the above experiment, it was confirmed that B cells can differentiate into plasma cells through strong interactions between undifferentiated B cells and CD4 T cells, and that the differentiated plasma cells can produce antibodies that specifically bind to cancer cells through strong interactions between CD4 T cells and cancer cells. In other words, the plasma cells identified in Example 1 above may be cancer-related plasma cells induced by an immune response within the tumor tissue.

[0151]

[0152] [Example 3]

[0153] Identification of plasma cell populations exhibiting clonal expansion and synthesis of recombinant antibodies

[0154] Cluster analysis was performed on the B cell and plasma cell clusters analyzed in Example 1 above to classify the cell populations into naive B cell, B cell, and plasma cell populations (Fig. 11). Subsequently, pseudotime trajectory analysis was performed to confirm the process of naive B cells differentiating into B cells and finally into plasma cells (Fig. 12). The pseudotime trajectory analysis was performed using Monocle2 (version 2.30.0) to infer cell differentiation from B cells into plasma cells. At this time, it was confirmed that during the process of naive B cells differentiating into plasma cells, IGHG1 expression increased while HLA-DRA expression gradually decreased (Fig. 13), which indicates that the plasma cells were differentiated by an immune response within the tumor tissue.

[0155] Next, clonal analysis of plasma cells was performed using Dandelion, and the results showed that most of the cells forming the clones were plasma cells (Fig. 14). Based on this, six plasma cell clones with the full Ig sequence were selected. Among these, Ab#1 produced from the plasma cell that formed the largest clone originated from the most differentiated plasma cell in virtual time trajectory analysis, and said plasma cells were found not only in HR-positive breast cancer patients but also in triple-negative breast cancer patients (Fig. 15). Cells with the same clonal type were found more frequently in plasma cells than in B cells, and among them, the plasma cell clone expressing Ab#1 being the largest may indicate the occurrence of clonal expansion (Fig. 16).

[0156] Since antibodies produced from plasma cells exhibiting clonal expansion among multiple clones have the potential to contribute to a potent immune response within tumor tissue, recombinant antibodies were synthesized by cloning the antibody sequences of six plasma cell clones (Fig. 17). For antibody synthesis, variable regions of the heavy and light chains identified by scRNA-BCR sequencing were first synthesized, and then cloned into a pCEP4 expression vector (Invitrogen, USA) containing the leader sequence of the human immunoglobulin kappa chain and the rabbit IgG constant region. Subsequently, the expression vector was transfected into Expi293F (Gibco, USA) cells to produce proteins. 40 mL of 25 kDa linear polyethyleneimine (PEI, Polysciences, USA) was used as the transfection reagent, and the cells were cultured in Expi293F expression medium (Gibco, USA) for 5 days after expression. Rabbit IgG protein was purified from the culture supernatant using affinity chromatography with MabSelect SuRe resin (Cytiva, USA). Protein integrity and purity were evaluated via SDS-PAGE, and protein concentration was measured using the bicinchoninic acid assay (BCA assay).

[0157] Among the antibodies prepared by the method described above, the sequences of the Ab#1 and Ab#2 antibodies exhibiting clonal expansion were analyzed. As a result, it was confirmed that the Ab#1 antibody contains a heavy chain variable region including CDR1-H having the amino acid sequence of SEQ ID NO. 1, CDR2-H having the amino acid sequence of SEQ ID NO. 3, and CDR3-H having the amino acid sequence of SEQ ID NO. 5, and a light chain variable region including CDR1-L having the amino acid sequence of SEQ ID NO. 7, CDR2-L(DAS) having the amino acid sequence of SEQ ID NO. 9, and CDR3-L having the amino acid sequence of SEQ ID NO. 11. In addition, it was confirmed that the heavy chain variable region of the Ab#1 antibody contains the amino acid sequence of SEQ ID NO. 13, and the light chain variable region contains the amino acid sequence of SEQ ID NO. 15. In addition, it was confirmed that the heavy chain of the Ab#1 antibody contains the amino acid sequence of SEQ ID NO. 17, and the light chain contains the amino acid sequence of SEQ ID NO. 19.

[0158] It was confirmed that the Ab#2 antibody comprises a heavy chain variable region including CDR1-H having the amino acid sequence of SEQ ID NO. 2, CDR2-H having the amino acid sequence of SEQ ID NO. 4, and CDR3-H having the amino acid sequence of SEQ ID NO. 6, and a light chain variable region including CDR1-L having the amino acid sequence of SEQ ID NO. 8, CDR2-L(LGS) having the amino acid sequence of SEQ ID NO. 10, and CDR3-L having the amino acid sequence of SEQ ID NO. 12. In addition, it was confirmed that the heavy chain variable region of the Ab#2 antibody comprises the amino acid sequence of SEQ ID NO. 14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 16. In addition, it was confirmed that the heavy chain of the Ab#2 antibody comprises the amino acid sequence of SEQ ID NO. 18, and the light chain comprises the amino acid sequence of SEQ ID NO. 20.

[0159]

[0160] [Example 4]

[0161] Immunohistochemical staining analysis of cancer tissue using recombinant antibodies

[0162] Immunohistochemical staining was performed using the six types of recombinant antibodies synthesized in Example 3 above to verify whether the antibodies could specifically bind to cancer cells. Formalin-fixed paraffin-embedded (FFPE) tumor tissue was used, and immunohistochemical staining of the synthesized antibodies was performed using the OptiView 3, 3'-diaminobenzidine immunohistochemical detection kit on a BenchMark XT automated immunohistochemistry device (Ventana Medical Systems, Tucson, AZ, USA). As a negative control, immunohistochemical staining results for immune and epithelial cells obtained from a patient's tonsil tissue were used (Fig. 18).

[0163] First, as a result of staining tumor tissue using the Ab#1 antibody, it was confirmed that staining occurred along the cell membrane in the cancer cells of patients 1 and 3, where plasma cells were present, but no staining was observed in the cancer cells of patient 2 (Fig. 19a). In addition, no immunostaining was observed in the normal epithelial cells surrounding the tumor in all three patients (Fig. 19b).

[0164] Next, the tumor tissue was stained using the Ab#2 antibody, and staining along the cell membrane was observed in the cancer cells of patients 1 and 3, but no associated plasma cells were identified in patient 3, and no staining was observed in the cancer cells of patient 2 (Fig. 20).

[0165] Unlike the above antibodies, no immunostaining was detected in the cancer tissue for the remaining antibodies Ab#3 to Ab#6, which did not undergo clonal expansion, and only immunostaining was observed in cancer-infiltrating lymphocytes for the antibody Ab#5, which was derived from the plasma cells of patient 2 (Fig. 21).

[0166] Through the above experiment, it was confirmed that only antibodies Ab#1 and Ab#2 produced from plasma cells that underwent clonal expansion can specifically bind to cancer cells, and that these antibodies can be effectively utilized for targeted anticancer therapy and diagnosis.

[0167]

[0168] Although representative embodiments of the present invention have been described above by way of example, the scope of the present invention is not limited to such specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of this application.

Claims

1. A heavy chain variable region comprising a CDR1-H having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, a CDR2-H having the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, and a CDR3-H having the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6; and A light chain variable region comprising a CDR1-L having the amino acid sequence of SEQ ID NO. 7 or SEQ ID NO. 8, a CDR2-L having the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10, and a CDR3-L having the amino acid sequence of SEQ ID NO. 11 or SEQ ID NO. 12; comprising Antibody or its antigen-binding fragment.

2. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 13 or SEQ ID NO. 14; and A light chain variable region comprising the amino acid sequence of SEQ ID NO. 15 or SEQ ID NO. 16; comprising Antibody or its antigen-binding fragment.

3. A heavy chain comprising the amino acid sequence of SEQ ID NO. 17 or SEQ ID NO. 18; and A light chain comprising the amino acid sequence of SEQ ID NO. 19 or SEQ ID NO. 20; comprising Antibody or its antigen-binding fragment.

4. In any one of claims 1 to 3, An antibody or its antigen-binding fragment, wherein the antigen-binding fragment of the above antibody is a single-chain variable fragment (scFv).

5. In any one of claims 1 to 3, The antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment derived from cancer tissue.

6. In any one of claims 1 to 3, The antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment derived from plasma cells within cancer tissue.

7. In any one of claims 1 to 3, The antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to cancer cell antigens.

8. A gene encoding the antibody of any one of claims 1 to 3 or the antigen-binding fragment thereof.

9. A recombinant expression vector containing the gene of claim 8.

10. A transformant in which the recombinant expression vector of claim 9 is introduced into a host cell.

11. A method for producing an antibody that specifically binds to a cancer cell antigen or an antigen-binding fragment thereof, comprising the step of culturing the transformant of claim 9.

12. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, an antibody of any one of claims 1 to 3 or a binding fragment thereof.

13. In Claim 12, A pharmaceutical composition for the prevention or treatment of cancer, wherein the above cancer is breast cancer. 14.a) A step of obtaining a tumor tissue sample and performing single-cell transcriptome sequencing; b) a step of identifying plasma cell clusters by analyzing the transcriptome sequencing data obtained in step a) above; and c) a step of selecting a clone having a full-length immunoglobulin sequence from the above plasma cell cluster; comprising, Method for screening plasma cell-derived antibodies within tumor tissue.

15. In Claim 14, In step b) above, the plasma cells are differentiated by stimulation of CD4 T cells and cancer cells.

16. In Claim 14, A method in which, in step b) above, the plasma cell is a clonally expanded plasma cell.

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