Mutant protein of single-chain variable fragment with improved stability

Abstract

The present invention relates to a mutant protein of a single-chain variable fragment having improved stability and, more specifically, to a mutant protein of a single-chain variable fragment having excellent binding affinity to immune checkpoint molecules and superior in vivo stability, in which a heavy-chain variable region including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3 and a light-chain variable region including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6 are linked by a stability-enhancing linker of SEQ ID NO: 7.

Description

Mutant protein of a single-strand variable fragment with improved stability

[0001] The present invention relates to a novel anti-PD-L1 single-chain variable fragment (scFv) variant protein and its uses.

[0002]

[0003] The PD-1 (Programmed Death-1) receptor and its ligand, PD-L1 (Programmed Death-Ligand 1), are immune checkpoint proteins implicated in the suppression of immune system responses associated with chronic infections, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 modulates immune responses by binding to the inhibitory receptor PD-1, which is expressed on the surface of T-cells, B-cells, and monocytes. The formation of the PD-L1 / PD-1 complex negatively modulates T-cell receptor signaling, leading to the subsequent downregulation of T-cell activation and the suppression of anti-tumor immune activity.

[0004] Immune checkpoint inhibitors targeting PD-1 and PD-L1 have achieved innovative success in the field of cancer treatment, leading to the development of various drugs utilizing them. Major drugs currently approved by the FDA and on the market include Keytruda, Opdivo, Tecentriq, Bavencio, Imfinzi, Libtayo, and Tuoyi. However, most of the approved treatments are immunoglobulin G (IgG)-based therapies, which are monoclonal antibodies. They have a very large molecular weight of approximately 150 kDa, which limits their penetration into solid tumors and makes it difficult to deliver the drug deep into tumor tissue.

[0005] To overcome these limitations, single-chain variable fragments (scFvs) can be used. scFvs are the heavy chain variable region (V) of an antibody. H ) and light chain variable region (V LIt is an antibody fragment of approximately 25–30 kDa in size connected by a linker. Compared to immunoglobulin G (IgG) antibodies, it has the advantage of excellent tissue penetration due to its smaller size and does not induce Fc receptor-mediated immune responses because it lacks an Fc region. In addition, it is advantageous in terms of reducing manufacturing costs as it can be produced using a microorganism-based expression system.

[0006] However, scFv has disadvantages such as a very short half-life in the body and reduced stability due to aggregation, which necessitates repeated administration or limits therapeutic efficacy. Consequently, despite their utility, clinical application is currently limited to specific cases such as hematological cancers and macular degeneration, necessitating the development of new scFv-based therapies to address these issues.

[0007]

[0008] [Prior Art Literature]

[0009] [Patent Literature]

[0010] (Patent Document 0001) Korean Registered Patent No. 10-2719839 (Oct. 21, 2024)

[0011]

[0012] The present invention aims to provide a novel single-chain variable fragment variant protein having excellent binding affinity to immune checkpoint molecules and stability in vivo.

[0013] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the above single-chain variable fragment variant protein.

[0014]

[0015] 1. A heavy chain variable region (V) comprising CDRH1 of SEQ ID NO. 1, CDRH2 of SEQ ID NO. 2, and CDRH3 of SEQ ID NO. 3. H A light chain variable region (V) comprising ) and CDRL1 of SEQ ID NO. 4, CDRL2 of SEQ ID NO. 5, and CDRL3 of SEQ ID NO. 6 L) A single-stranded variable fragment variant protein linked by the stability-enhancing linker of SEQ ID NO. 7.

[0016] 2. In the above 1, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 8, a single chain variable fragment variant protein.

[0017] 3. In the above 2, the light chain variable region comprises the amino acid sequence of SEQ ID NO. 9, a single-strand variable fragment variant protein.

[0018] 4. A single-stranded variable fragment variant protein of 1 above, wherein a bioactive substance is fused to the C-terminus.

[0019] 5. In the above 4, the physiologically active substance is a single-chain variable fragment variant protein, which is any one of the chemo-anticancer agents selected from the group consisting of paclitaxel, docetaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, irinotecan, and topotecan.

[0020] 6. The single-stranded variable fragment variant protein of 1, having a length of 150 to 350 aa.

[0021] 7. A pharmaceutical composition for the treatment or prevention of cancer comprising any one of the single-strand variable fragment variant proteins of 1 to 6 above.

[0022] 8. A pharmaceutical composition for the treatment or prevention of cancer, wherein the above 7 further comprises bevacizumab.

[0023] 9. A pharmaceutical composition for the prevention or treatment of cancer according to 7 above, wherein the cancer is any one selected from the group consisting of non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, pro-anal cancer, endometrial cancer, vaginal cancer, vulvar cancer, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, prostate cancer, kidney cancer, and ureteral cancer.

[0024]

[0025] The single-chain variable fragment variant protein of the present invention has excellent binding affinity to immune checkpoint molecules and stability in vivo.

[0026] The single-chain variable fragment variant protein of the present invention allows serum albumin to bind between the heavy chain variable region and the light chain variable region, so the N-terminus or C-terminus can be utilized for the delivery of physiologically active substances such as anticancer agents.

[0027] The single-chain variable fragment variant protein of the present invention exhibits excellent anticancer effects when administered alone or in combination.

[0028]

[0029] Figure 1 shows the SDS-PAGE analysis results for the expression and purification of PDL1-ABD-HL and PDL1-ABD-LH variants. (a) is the analysis result of PDL1-ABD-HL and (b) is the analysis result of PDL1-ABD-LH (Lad: molecular weight standard, BI: cell lysate sample before induction, AI: cell lysate sample after induction, CL: total cell lysate sample, FT: column pass-through, W1: wash buffer, W2: elution buffer, E: sample after purification, P: insoluble precipitate).

[0030] Figure 2 shows the results of anti-PD-L1 ELISA analysis of PDL1-ABD-HL and PDL1-ABD-LH variants according to the concentration of protein coated on an immune plate. (a) to (d) are, in order, cases where the coating protein concentrations are 0.5 µl / mL, 1.0 µg / mL, 1.5 µg / mL, and 2.0 µg / mL (x-axis is sample concentration, y-axis is absorbance according to sample concentration).

[0031] Figure 3 shows the results of analyzing the stability of PDL1-ABD stored for 7 days. (a) shows the absorption spectrum of PDL1-ABD in PBS solution, and (b) shows the absorption spectrum of PDL1-ABD in a solution containing albumin (HSA, Human Serum Albumin). (c) shows the results of comparing the aggregation index calculated based on this.

[0032]

[0033] The present invention provides a variant protein of a single-chain variable fragment with improved stability.

[0034] The present invention provides a variant protein of a single-strand variable fragment having excellent binding affinity to immune checkpoint molecules and in vivo stability, wherein a heavy chain variable region comprising CDRH1 of SEQ ID NO. 1, CDRH2 of SEQ ID NO. 2, and CDRH3 of SEQ ID NO. 3, and a light chain variable region comprising CDRL1 of SEQ ID NO. 4, CDRL2 of SEQ ID NO. 5, and CDRL3 of SEQ ID NO. 6 are connected by a stability-enhancing linker of SEQ ID NO. 7.

[0035] "Antibody" refers to immunoglobulin. Antibodies generally have a structure consisting of two heavy chains and two light chains stabilized by a pair of disulfide bonds.

[0036] "Heavy chain" is a variable region domain V containing a variable amino acid sequence sufficient to confer specificity to the antigen. H and is interpreted to mean including both the full-length heavy chain and its fragments, which contain the hinge and the three invariant region domains CH1, CH2, and CH3. Additionally, "light chain" refers to the variable region domain V containing a variable amino acid sequence sufficient to confer specificity to the antigen. L It is interpreted to mean including both the full-length light chain and its fragments, including the invariant domain CL. The variable domain fragment (Fv) is a heavy chain variable domain (V H ) and light chain variable region (V LIt is a region that directly participates in antigen binding and gives diversity to the antibody. The complementarity-determining region (CDR) is the region that comes into direct contact with the antigen and has diverse variations, while the framework region (FR) is a region with relatively few variations other than the CDR. There are three CDRs in the variable regions of the light chain and heavy chain, respectively, and they are called heavy chain CDR1 to 3 and light chain CDR1 to 3 in order from the N-terminus.

[0037] In this specification, "CDR" is interpreted as a complementarity determining region present in the heavy chain variable region and the light chain variable region of the antibody. The CDRs present in the heavy chain variable region are denoted as CDRH1, CDRH2, and CDRH3 in order from the N-terminus, and the CDRs present in the light chain variable region are denoted as CDRL1, CDRL2, and CDRL3 in order from the N-terminus.

[0038] A single-chain variable fragment (scFv) is an antigen-binding fragment of an antibody having a heavy-chain variable region or its antigen-binding region and a light-chain variable region or its antigen-binding region, with each region connected by a linker.

[0039] In this specification, a single-chain variable fragment is a heavy-chain variable region (V H )-Linker-Light Chain Variable Region(V L It refers to a form of a single protein chain that has a composition joined by ), is generally small in size (about 25 kDa), and contains two or fewer disulfide bonds.

[0040] The single-chain variable fragment is not limited to having a specific amino acid length as long as it is capable of fusing a stability-enhancing linker according to the purpose of the present invention.

[0041] The single-stranded variable fragment is, for example, having an amino acid length of 100 aa or more, 110 aa or more, 120 aa or more, 130 aa or more, 140 aa or more, 150 aa or more, 160 aa or more, 170 aa or more, 180 aa or more, 190 aa or more, 200 aa or more, 210 aa or more, 220 aa or more, 230 aa or more, 240 aa or more, 250 aa or more, 260 aa or more, 270 aa or more, 280 aa or more, 290 aa or more, 300 aa or more, 310 aa or more, 320 aa or more, 330 aa or more, 340 aa or more, 350 aa or more, 360 aa or more, 370 aa or more, 380 aa or more, 390 It may be aa or more, 400 aa or more, 410 aa or more, 420 aa or more, 430 aa or more, 440 aa or more, 450 aa or more, 460 aa or more, 470 aa or more, 480 aa or more, 490 aa or more, or 500 aa or more.

[0042] The single-stranded variable fragment is, for example, having an amino acid length of 500 aa or less, 490 aa or less, 480 aa or less, 470 aa or less, 460 aa or less, 450 aa or less, 440 aa or less, 430 aa or less, 420 aa or less, 410 aa or less, 400 aa or less, 390 aa or less, 380 aa or less, 370 aa or less, 360 aa or less, 350 aa or less, 340 aa or less, 330 aa or less, 320 aa or less, 310 aa or less, 300 aa or less, 290 aa or less, 280 aa or less, 270 aa or less, 260 aa or less, 250 aa or less, 240 aa or less, 230 aa or less, 220 aa or less, 210 It may be aa or less, 200 aa or less, 190 aa or less, 180 aa or less, 170 aa or less, 160 aa or less, 150 aa or less, 140 aa or less, 130 aa or less, 120 aa or less, 110 aa or less, or 100 aa or less.

[0043] The single-chain variable fragment may have a length of, for example, 150 to 350 aa, 160 to 340 aa, 170 to 330 aa, 180 to 320 aa, 190 to 310 aa, or 200 to 300 aa.

[0044] Heavy chain variable region (V H ) and light chain variable region (V L ) is not limited to having a specific amino acid length.

[0045] Heavy chain variable region (V H ) and light chain variable region (V L Each ) refers to, for example, the length of the amino acid being 50 aa or more, 55 aa or more, 60 aa or more, 65 aa or more, 70 aa or more, 75 aa or more, 80 aa or more, 85 aa or more, 90 aa or more, 95 aa or more, 100 aa or more, 105 aa or more, 110 aa or more, 115 aa or more, 120 aa or more, 125 aa or more, 130 aa or more, 135 aa or more, 140 aa or more, 145 aa or more, 150 aa or more, 155 aa or more, 160 aa or more, 165 aa or more, 170 aa or more, 175 aa or more, 180 aa or more, 185 aa or more, 190 aa or more, 195 aa or more, 200 aa It may be 205 aa or more, 210 aa or more, 215 aa or more, 220 aa or more, 225 aa or more, 230 aa or more, 235 aa or more, 240 aa or more, 245 aa or more, or 250 aa or more.

[0046] Heavy chain variable region (V H ) and light chain variable region (V LEach ) is, for example, of the length of the amino acid being 250 aa or less, 245 aa or less, 240 aa or less, 235 aa or less, 230 aa or less, 225 aa or less, 220 aa or less, 215 aa or less, 210 aa or less, 205 aa or less, 200 aa or less, 195 aa or less, 190 aa or less, 185 aa or less, 180 aa or less, 175 aa or less, 170 aa or less, 165 aa or less, 160 aa or less, 155 aa or less, 150 aa or less, 145 aa or less, 140 aa or less, 135 aa or less, 130 aa or less, 125 aa or less, 120 aa or less, 115 aa or less, 110 aa or less, 105 It may be aa or less, 100 aa or less, 95 aa or less, 90 aa or less, 85 aa or less, 80 aa or less, 75 aa or less, 70 aa or less, 65 aa or less, 60 aa or less, 55 aa or less, or 50 aa or less.

[0047] The heavy chain variable region and the light chain variable region may each have a length of, for example, 70 to 150 aa, 80 to 140 aa, 90 to 130 aa, or 100 to 120 aa.

[0048] Amino acids referred to by abbreviations in this specification are described as follows according to IUPAC-IUB nomenclature: Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V.

[0049] In one embodiment, the single-chain variable fragment may be a single-chain variable fragment targeting PD-L1.

[0050] In one embodiment, the single-chain variable fragment may be derived from atezolizumab that binds to PD-L1.

[0051] In one embodiment, the atezolizumab-derived single-strand variable fragment comprises a heavy-strand variable region (V) including SEQ ID NOs. 1, 2, and 3. H A light chain variable region (V) comprising ) and sequence numbers 4, 5, and 6 L It may include ). Polypeptides having the amino acid sequences of SEQ ID NOs 1 to 6 may each have the CDR function of an anti-PD-L1 antibody as described in Table 1 below.

[0052] Classification Amino Acid Sequence Sequence Number Heavy Chain CDRCDRH1AASGFTFS1CDRH2AWISPYGGSTYYADSVKGRF2CDRH3CARRHWPGGFDYW3 Light Chain CDRCDRL1RASQDVSTAVAW4CDRL2SASFLY5CDRL3QQYLYHPAT6

[0053] The heavy and light variable regions of a single-strand variable fragment can be connected by a peptide linker. The linker is not limited to a specific sequence as long as it can maintain the flexibility and structural stability of the single-strand variable fragment. For example, (GS) n , (GGS) n , (GGGGGS) n Or it may be composed of a combination thereof. The above n is any natural number and may be from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The order and repetition of these combinations are not limited and may be a combination of two or more types.

[0054] The above linker may further include an albumin binding domain (ABD).

[0055] In this specification, the term "stability-enhancing linker" refers to a linker containing an albumin binding site within the linker.

[0056] In this specification, the term "albumin binding domain (ABD)" refers to a portion that interacts with an albumin protein, and may be, for example, an antibody, an antibody analog, a protein domain, a protein motif, a peptide, a compound, an aptamer, an oligonucleotide, a sugar, etc.

[0057] The albumin binding site may have an amino acid length of 10 to 150 aa, 30 to 100 aa, or 30 to 50 aa.

[0058] It is preferable that the albumin binding site bound within the linker be fused at a position spaced at least 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa from the heavy chain variable region and the light chain variable region, respectively. For example, it may be fused between the 7th to 14th amino acid of a linker having a length of 16 to 20 aa and an adjacent amino acid.

[0059] If the albumin binding site is separated from the heavy chain variable region and the light chain variable region by at least 3 aa and is not fused, interference with the adjacent complementarity determining region (CDR) may occur, which may reduce the binding affinity to the antibody.

[0060] The albumin binding site bound within the linker is located in the opposite direction to the position of the complementarity determining region (CDR) of the heavy and light variable regions. Here, "opposite direction" means, for example, that if the complementarity determining region (CDR) is located below the single-strand variable fragment variant protein, the albumin binding site is located above it. Furthermore, "opposite direction" means, for example, that if the complementarity determining region (CDR) is located to the left of the single-strand variable fragment variant protein, the albumin binding site is located to the right.

[0061] Albumin may be human serum albumin (HSA).

[0062] The variant protein of the present invention can delay the metabolism and degradation of the variant protein and increase permeability to cancer cells by binding to serum albumin through the albumin binding site bound within the linker when administered in vivo.

[0063] When the albumin binding site is fused into the linker of the single-stranded variable fragment, a bioactive substance can be additionally bound to the N-terminus or C-terminus of the single-stranded variable fragment. Additionally, when the albumin binding moiety is fused at a location other than the C-terminus of the single-stranded variable fragment, a bioactive substance can be bound to the C-terminus of the single-stranded variable fragment. In this case, the single-stranded variable fragment variant protein of the present invention functions as a carrier to deliver the bioactive substance to a target.

[0064] A bioactive substance refers to any substance capable of exhibiting an anticancer effect when administered into the body. A bioactive substance may be, for example, a chemical anticancer agent. A bioactive substance may be any one selected from the group consisting of paclitaxel, docetaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, irinotecan, and topotecan.

[0065] The albumin binding site may be fused to the center of the linker amino acid sequence of the single-stranded variable fragment.

[0066] The albumin binding site may be, for example, an albumin protein domain, an affibody, or a peptide. It is small in size compared to an antibody (IgG) and fused to scFv to form a single polypeptide chain. It may not contain disulfide bonds in its structure.

[0067] The albumin binding site may be a part of a specific protein or an artificially designed domain.

[0068] For example, this may be a small triple-stranded protein domain found in various surface proteins expressed by Gram-positive bacteria. Specifically, it may be derived from streptococcal protein G or protein PAB of Finegoldia magna, or modified therefrom to further increase albumin binding affinity.

[0069] For example, this may be a VNAR (V domain of cartilage oligomeric matrix protein, chondrocyte-derived) derived from the cartilage oligomeric matrix protein of a sea shark, or one modified from it to further increase albumin binding affinity.

[0070] In one embodiment, the stability-enhancing linker may have the amino acid sequence of GGGGSGGSTLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGSTSGGGGS (Sequence No. 7).

[0071] The N-terminus of the above single-strand variable fragment may further include a start restriction sequence.

[0072] In one embodiment, the start limit sequence may be MG.

[0073] The C-terminus of the above single-strand variable fragment may further include a His-Tag sequence for the isolation and purification of the single-strand variable fragment variant.

[0074] In one embodiment, the histag sequence may be HHHHHH.

[0075] In one embodiment, the single-strand variable fragment protein comprises a heavy-strand variable region (V) including SEQ ID NOs. 1, 2, and 3. H A light chain variable region (V) comprising ) and sequence numbers 4, 5, and 6 L ) may be a single-strand variable fragment connected by a stability-enhancing linker of sequence number 7.

[0076] In one embodiment, the heavy chain variable region and the light chain variable region may include the sequence number of [Table 2].

[0077] Classification Amino Acid Sequence Sequence Number Heavy Chain Variable Region EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAA8 Light Chain Variable Region DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK9

[0078] The single-strand variable fragment of the present invention may be produced in a microorganism that expresses a sequence encoding the corresponding protein.

[0079] Microorganisms known in the field may be used without limitation. For example, it may be Escherichia coli, but is not limited thereto.

[0080] When producing a variant protein using a microbial system, expression is facilitated if the protein does not contain post-translational modifications (PTMs) and has few or no disulfide bonds. Antibody forms such as IgG contain numerous PTMs and disulfide bonds, and additional binding and purification processes are required when using compounds. The variant of the present invention is in the form of a single fused polypeptide and can facilitate expression in a microbial system due to its characteristics of having no PTMs and few disulfide bonds.

[0081] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the single-chain variable fragment variant protein.

[0082] Cancer includes, for example, brain cancer, head and neck cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, uterine cancer, vascular tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma, laryngeal cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenocystic carcinoma, adenoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytoma, vulvar adenocarcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, Cystodenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymocyte, epithelial cell carcinoma, orbital cancer, focal nodular hyperplasia, gallbladder cancer, pyloric carcinoma, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasm, intrasquamous cell neoplasm, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, arthral cancer, pelvic cancer, giant cell carcinoma, colorectal cancer, lymphoma, malignant mesothelial tumor, medulloblastoma, medulloepithelial tumor, meningeal cancer, mesothelial carcinoma, metastatic carcinoma, oral cancer, mucoepidermal carcinoma, multiple myeloma, Muscle cancer, nasal duct cancer, nervous system cancer, non-epithelial skin cancer, non-Hodgkin lymphoma, oculomotor cell carcinoma, oligodendroglioma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, leiomyoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, striatal muscle carcinoma, mesothelial sublayer carcinoma, T-cell leukemia, tongue cancer, ureteral cancer, urethral cancer, cervical cancer, uterine body cancer, vaginal cancer, VIPoma, vulvar cancer,It may have been selected from a group consisting of well-differentiated carcinomas and Wilms tumors, but is not limited thereto.

[0083] In this invention, "prevention of cancer" refers to any act that suppresses or delays the occurrence, spread, and recurrence of cancer.

[0084] In this invention, "treatment of cancer" refers to any act of causing the death of cancer cells or improving or beneficially altering the symptoms of cancer.

[0085] The pharmaceutical composition of the present invention may comprise an active ingredient alone, or may further comprise one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0086] The active ingredient of the pharmaceutical composition of the present invention may include the above single-chain variable fragment variant proteins.

[0087] In the present invention, the term "pharmaceuticalally acceptable carrier" refers to a carrier or diluent that does not significantly irritate a living organism and does not impair the biological activity and properties of the administered component. The pharmaceutically acceptable carrier in the present invention may be used as saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added to form an injectable solution suitable for injection into tissues or organs. Additionally, it may be formulated into a dry preparation (particularly a lyophilized preparation) that can become an injectable solution by adding an isotonic sterile solution, or, in some cases, sterile water or physiological saline solution. Furthermore, a target organ-specific antibody or other ligand may be conjugated to the carrier to act specifically on a target organ.

[0088] Additionally, the composition of the present invention may further include a filler, an excipient, a disintegrant, a binder, or a lubricant. Additionally, the composition of the present invention may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.

[0089] In one example of a formulation, the pharmaceutical composition may be in an injectable form and may be administered intravenously, but is not limited thereto.

[0090] In the present invention, the composition can be administered in a pharmaceutically effective amount.

[0091] The term "effective dose" as used in the present invention refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects, and such level may be determined by factors including the patient's health condition, type and severity of cancer, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0092] In the present invention, the pharmaceutical composition may be accompanied by instructions associated with the packaging in a form directed by a government agency governing the manufacture, use, and sale of drugs, as needed, said instructions indicating approval by a private agency regarding the form of the composition or administration to humans or animals, and may, for example, be a label approved by the U.S. Food and Drug Administration for the prescription of drugs.

[0093] The present invention provides a method for treating cancer comprising the step of administering the pharmaceutical composition of the present invention to an individual.

[0094] In the present invention, "individual" is a human or a non-human animal (e.g., a mammal).

[0095] In one embodiment, the individual may be a normal person.

[0096] In one embodiment, the individual may be an individual requiring administration of the variant protein of the present invention.

[0097] In one embodiment, the individual may be an individual that has developed cancer or is likely to develop cancer.

[0098] In the present invention, "administration" means introducing a specific substance to an individual by an appropriate method.

[0099] The method of the present invention may include the step of administering the pharmaceutical composition of the present invention to an individual. Additionally, the administered pharmaceutical composition of the present invention may be an effective amount.

[0100] The active ingredient included in the pharmaceutical composition of the present invention is, for example, 1 μg / kg / day to 300 μg / kg / day when administered parenterally, and preferably 1 μg / kg / day to 100 μg / kg / day.

[0101] The administration route of the pharmaceutical composition of the present invention may be administered via various oral or parenteral routes as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, or orally, intra-arterially, intramedullaryly, intrathecally, transdermally, intranasally, locally, intrapulmonaryly, or rectally, but is not limited thereto.

[0102] The pharmaceutical composition of the present invention may be provided as a solid dosage form for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0103] The pharmaceutical composition of the present invention may be provided as a liquid formulation for oral administration, such as a suspension, liquid formulation, emulsion, or syrup.

[0104] The pharmaceutical composition of the present invention may be provided as a formulation for parenteral administration, such as a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, or a suppository.

[0105] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other conventional therapeutic agents; when administered in combination, it may be administered sequentially or simultaneously, and may be administered as a single or multiple doses, which can be easily determined by a person skilled in the art.

[0106] In one embodiment, the pharmaceutical composition of the present invention may be administered in combination with bevacizumab.

[0107] The present invention will be explained in more detail below with reference to examples.

[0108]

[0109] Examples

[0110] Design of single-strand variable fragment variant proteins

[0111] Based on the stability-enhancing linker, the heavy chain variable region (V) of scFv H ) and light chain variable region (V L Examples 1 and 2 were designed by varying the order of ) (Table 3). The amino acid sequence of the scFv protein is V linked by a stability-enhancing linker. H and V L In the order of, N-terminal V H and C-terminal V L It is a region or N-terminal V L and C-terminal V H It consists of a region. The sequence 'MG' was inserted at the N-terminus as the restriction site for the NcoI enzyme, and the hexahistidine tag (Hig-tag) sequence HHHHHH was inserted at the C-terminus as a protein purification tag. A SpeI restriction site encoding the amino acid residue 'TS' was used for the insertion of the albumin binding domain.

[0112] Classification Name Sequence Number Example 1 PDL1-ABD-HL10 Example 2 PDL1-ABD-LH11

[0113] Experimental Example

[0114] 1. Protein Expression and Purification

[0115] 1-1. Plasmid

[0116] Among the variant proteins constructed by the above design, Examples 1 and 2 were produced as proteins through cytoplasmic expression in a bacterial host. Specifically, the gene encoding the above amino acid sequence was optimized using ExpOptimizer (NovoPro Bioscience, Shanghai, China), synthesized and subcloned by Macrogen (Seoul, South Korea), and inserted into the NcoI / KpnI restriction site in the pBAD vector to generate the pBAD_PDL1scFv plasmid.

[0117] The above plasmid was transformed into E. coli TOP10 host cells. The transformed cells were cultured in standard 2xYT medium containing 100 µg / ml ampicillin at 37°C for 16 hours while shaking at 200 rpm. Cells were inoculated into the same fresh medium and cultured until the optical density at 600 nm (OD600) reached 0.5–0.6. Protein expression was induced by L-(+)-arabinose at a final concentration of 0.2% (w / v). After lowering the temperature to 23°C, the culture medium was incubated for 24 hours; the grown cells were harvested by centrifugation at 8,000 rpm at 4°C, and the cell pellets were stored at -80°C. Pre-induction (BI) and post-induction (AI) samples for each variant were collected, centrifuged at 13,000 rpm for 1 minute, and then resuspended in PBS (pH 7.4) containing 2M urea.

[0118] The above examples tagged with His were purified by metal-affinity chromatography using Ni-NTA. Cell pellets were lysed in lysis buffer (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) with 1 mg / mL lysozyme and 5 mg / mL DNase and incubated on ice for at least 5 minutes. The solution was sonicated for a total of 15 minutes using 1-second pulse, 2-second rest, and 28% amp (500 W, 20 kHz), and this process was repeated after a 5-minute rest. Subsequently, the mixture was subjected to 10,000 at 4°C for 20 minutes. The sample was centrifuged at g, and the supernatant was incubated with Ni-NTA agarose resin at 4°C for 30 minutes. The incubated resin was loaded onto a polypropylene column with a filter, washed with a wash buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0), and eluted with an elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Using a PD-10 desalting column according to the manufacturer's protocol, the buffer was exchanged with PBS (pH 7.4). Consequently, purified proteins of 11.58 mg / L and 3.24 mg / L were obtained from PDL1-ABD-HL and PDL1-ABD-LH, respectively, stored at 4°C, and analyzed by SDS-PAGE.

[0119]

[0120] 1-2. SDS-PAGE Analysis

[0121] The prepared proteins, including resuspended pre-induction samples (BI), post-induction samples (AI), total cell samples (CL), column pass-through (FT), wash buffer (W1), elution buffer (W2), purified protein (E), and insoluble precipitate (P), were analyzed using SDS-PAGE (Sodiuim Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). All samples were treated with 2 containing 100 mM dithiothreitol (DTT) for reduction. The samples were mixed with a sample loading dye (0.2% bromophenol blue, 4% SDS, 20% glycerol, 100 mM Tris-HCl, pH 6.8). DTT was excluded to prepare unreduced samples. The samples were boiled at 100°C for 10 minutes and then placed on a 12% SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue (0.25% Coomassie blue, 50% ethanol, 10% acetic acid) and estinate (50% ethanol, 10% acetic acid). The gel was imaged and visualized using a ChemiDoc XRS+ system (Bio-Rad, Hercules, California, USA).

[0122] Figure 1 shows the results of SDS-PAGE analysis of cell lysates collected during expression. Figure 1a shows the analysis results of PDL1-ABD-HL and Figure 1b shows the analysis results of PDL1-ABD-LH. In the AI ​​sample, a band with a molecular weight of 31.9 kDa was detected, which corresponds to the molecular weights of PDL1-ABD-HL and PDL1-ABD-LH.

[0123]

[0124] 2. Analysis of binding affinity for PD-L1

[0125] The anti-PD-L1 targeting efficiency of the purified PDL1-ABD variants was analyzed via anti-PD-L1 ELISA (Enzyme-Linked Immunosorbent Assay) according to chain sequence. Specifically, human PD-L1 Fc chimeras (100 µL per well at concentrations of 0.5 µg / mL, 1.0 µg / mL, 1.5 µg / mL, and 2.0 µg / mL) were coated onto immunoplatters by incubating overnight at 4°C in coating buffer (PBS, pH 7.4). The plates were washed three times by shaking with PBST (0.05% Tween 20) at 200 µL / well. Subsequently, the plates were blocked by adding blocking buffer (5% skim milk) to PBST at 200 µL / well and incubating at room temperature, followed by four washes. The purified proteins of each PDL1-ABD-HL and PDL1-ABD-HL were incubated in PBS (pH 7.4) at room temperature. Cultured proteins were prepared in blocking buffer to create a 50 nM stock, which was serially diluted three-fold. The prepared samples (100 µL / well) were loaded onto plates and incubated at room temperature, after which unbound proteins were washed four times. Finally, the samples were incubated with 100 µL / well of HRP-conjugated anti-rabbit IgG antibody diluted 1:3000 in blocking buffer. The wells were washed four times. Detection was performed by adding 100 µL / well of TMB, after which the reaction was quenched with 2 M HCl. Bound proteins were quantified by measuring absorbance at 450 nm.

[0126] The experimental results are shown in Figure 2 and Table 2. Similar concentration-dependent curves were observed for all cases where the concentration of the coating protein coated on the immune plate was 0.5 µg / mL, 1.0 µg / mL, 1.5 µg / mL, and 2.0 µg / mL (Figures 5a to 5d), and it was confirmed that the binding affinity remained stable even at the lowest concentration of the coating protein, 0.5 µg / mL (Figure 5a). This indicates that the insertion of the stability-enhancing linker did not cause significant distortion in the structure of PDL1scFv, and consequently, the anti-PD-L1 binding affinity of the PDL1-ABD variant was maintained.

[0127] In addition, it was confirmed that the binding affinity of heavy chains PDL1-ABD-HL or PDL1-ABD-LH did not show a significant difference depending on the order of the chains (Table 4).

[0128] Coating protein concentration (ug / mL) 0.5 1.0 1.5 2.0 EC50 PDL1-ABD-HL 28.6 41.4 54.0 43.3 PDL1-ABD-LH 54.3 47.5 43.8 42.8

[0129]

[0130] 3. Stability Analysis

[0131] To evaluate stability in vivo, the stability of PDL1-ABD in a solution containing HSA was analyzed. Specifically, the purified protein of Example 1 prepared in the experimental example was incubated at 37°C for 7 days in PBS (pH 7.4) or PBS containing 10 μM of HSA (pH 7.4) (n=5). The absorption spectrum of each sample was analyzed, and based on this, the aggregation index was calculated using the following formula.

[0132]

[0133] Figure 3 shows the results of the stability analysis. (a) and (b) show the absorption spectra of Example 1 according to wavelength in PBS and PBS containing 10 µM HSA, respectively. (c) is a graph showing the results using the above formula, confirming that aggregation was significantly reduced in the presence of HSA. This confirms that aggregation formation was significantly reduced under PBS conditions containing HSA compared to the control group (PBS), demonstrating that stability can be maintained even in an environment where HSA is present at high concentrations in vivo.

Claims

1. A heavy chain variable region (V) comprising CDRH1 of SEQ ID NO. 1, CDRH2 of SEQ ID NO. 2, and CDRH3 of SEQ ID NO. 3 H A light chain variable region (V) comprising ) and CDRL1 of SEQ ID NO. 4, CDRL2 of SEQ ID NO. 5, and CDRL3 of SEQ ID NO. 6 L ) A single-stranded variable fragment variant protein linked by the stability-enhancing linker of SEQ ID NO.

7.

2. The single-strand variable fragment variant protein of Claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.

8.

3. The single-strand variable fragment variant protein of Claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO.

9.

4. A single-strand variable fragment variant protein according to Claim 1, wherein a physiologically active substance is fused to the C-terminus.

5. In claim 4, the physiologically active substance is a single-chain variable fragment variant protein, which is any one of the chemo-anticancer agents selected from the group consisting of paclitaxel, docetaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, irinotecan, and topotecan.

6. The single-strand variable fragment variant protein of claim 1 having a length of 150 to 350 aa.

7. A pharmaceutical composition for the treatment or prevention of cancer comprising a single-chain variable fragment variant protein of any one of claims 1 to 6.

8. A pharmaceutical composition for the treatment or prevention of cancer according to claim 7, further comprising bevacizumab.

9. A pharmaceutical composition for the prevention or treatment of cancer according to claim 7, wherein the cancer is any one selected from the group consisting of non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, pro-anal cancer, endometrial cancer, vaginal cancer, vulvar cancer, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, prostate cancer, kidney cancer, and ureteral cancer.

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