KR-20260065538-A - MUTANT SINGLE-CHAIN VARIABLE FRAGMENT WITH IMPROVED STABILITY

Abstract

The present invention relates to a variant protein of a single-strand variable fragment with improved stability, and more specifically, to a variant protein of a single-strand variable fragment having excellent binding affinity to immune checkpoint molecules and in vivo stability by connecting a heavy-strand 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-strand variable region comprising CDRL1 of SEQ ID NO. 4, CDRL2 of SEQ ID NO. 5, and CDRL3 of SEQ ID NO. 6 with a stability-enhancing linker of SEQ ID NO. 7.

Inventors

• 권인찬
• 권나현
• 김영채

Assignees

• 광주과학기술원

Dates

Publication Date

20260508

Application Date

20251028

Priority Date

20241029

Claims (9)

1. A single-strand variable fragment variant protein comprising a heavy chain variable region (V H ) 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 (V L ) including CDRL1 of SEQ ID NO. 4, CDRL2 of SEQ ID NO. 5, and CDRL3 of SEQ ID NO. 6, linked by a stability-enhancing linker of SEQ ID NO. 7.
2. A single-strand variable fragment variant protein according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 8.
3. A single-strand variable fragment variant protein according to 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 bioactive 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 chemical 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.

Description

Mutant single-chain variable fragment with improved stability The present invention relates to a novel anti-PD-L1 single-chain variable fragment (scFv) variant protein and its uses. 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. 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. To overcome these limitations, single-chain variable fragments (scFv) can be used. scFv is an antibody fragment approximately 25–30 kDa in size formed by linking the heavy chain variable region (V H ) and the light chain variable region (V L ) of an antibody with 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. Additionally, it is advantageous in terms of reducing manufacturing costs as it can be produced using a microorganism-based expression system. 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. 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). 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 the 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). 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. The present invention provides a variant protein of a single-chain variable fragment with improved stability. 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. "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. "Heavy chain" is interpreted to mean the entire length heavy chain and its fragments, including the variable region domain VH , which contains a sufficient variable amino acid sequence to confer specificity to the antigen, and the three constant region domains CH1, CH2, and CH3, as well as the hinge. Additionally, "light chain" is interpreted to mean the entire length light chain and its fragments, including the variable region domain VL and the constant region domain CL, which contain a sufficient variable amino acid sequence to confer specificity to the antigen