Anti-PD-l1 single-chain variable fragment and single-chain variable fragment-drug conjugate comprising same

Abstract

The present invention relates to an anti-PD-L1 single-chain variable fragment and a single-chain variable fragment-drug conjugate comprising same and, more specifically, to an anti-PD-L1 single-chain variable fragment having PD-L1 binding ability and a single-chain variable fragment-drug conjugate comprising same, the anti-PD-L1 single-chain variable fragment comprising a heavy chain variable region (VH), a light chain variable region (VL), and a linker connecting same to each other, wherein, on the basis of the Kabat number system, at least one of the amino acid at position 42 of the VH and the amino acid at position 41 of the VL is substituted with an unnatural amino acid, thereby allowing a drug to be conjugated at an optimized position with a high conjugation yield through a click chemistry reaction and allowing preparation of a single-chain variable fragment-drug conjugate in which the drug conjugation number (DAR) is uniformly controlled, and to a single-chain variable fragment-drug conjugate comprising the same.

Description

Anti-PD-L1 single-chain variable fragment and single-chain variable fragment-drug conjugate containing the same

[0001] The present invention relates to an anti-PD-L1 single-chain variable fragment having an optimized drug conjugation site and an antibody-drug conjugate comprising the same.

[0002] Antibody-drug conjugates (ADCs) are being actively researched as next-generation anticancer therapies capable of selectively delivering drugs to tumor cells by combining the high target specificity of antibodies with cytotoxic drugs. Most ADCs developed to date are based on full-length antibodies, particularly immunoglobulin G (IgG).

[0003] IgG is a protein with a large molecular weight of approximately 150 kDa, and it has been pointed out that it has limitations in sufficiently penetrating tumor tissues, particularly solid tumors. In the tumor microenvironment, the diffusion of large proteins can be restricted due to high cell density and abnormal vascular structures, and these characteristics can reduce the therapeutic efficiency of IgG-based ADCs.

[0004] Accordingly, attempts have been made to utilize antibody fragments with relatively small sizes. Single-chain variable fragments (scFvs) are structures in which the variable heavy chain (VH) and variable light chain (VL) regions of an antibody are connected by short peptide linkers; due to their small molecular weight of approximately 25–30 kDa, they can exhibit excellent penetration into tumor tissues. However, problems have been reported regarding scFvs, such as lower structural stability compared to full-length antibodies, a shorter in vivo half-life, and a susceptibility to protein aggregation. These characteristics are recognized as significant technical challenges in the development of scFv-based drug delivery systems.

[0005] Meanwhile, in traditional ADC manufacturing methods, the method of conjugating drugs using lysine or cysteine ​​residues present in antibody proteins has been widely used. However, this method tends to result in drugs binding randomly at various locations within the antibody molecule, leading to the generation of a mixture of various species with different drug-associated numbers (DARs). This heterogeneity not only impairs the quality control and reproducibility of ADCs but also causes problems that make it difficult to predict efficacy and toxicity.

[0006] Therefore, there is a need for a technology to design single-chain variable fragments that can efficiently conjugate drugs while maintaining antigen binding ability and protein stability, and in particular, to identify a suitable site for drug conjugation within scFv.

[0007] The present invention aims to provide an anti-PL-L1 single-chain variable fragment having a drug conjugation site to which a drug can be efficiently conjugated while maintaining antigen binding ability and structural stability, and an antibody-drug conjugate comprising the same.

[0008] 1. A single-chain variable fragment having PD-L1 binding ability and comprising a heavy chain variable region (VH), a light chain variable region (VL), and a linker connecting VH and VL, wherein, based on the Kavat numbering system, at least one of the amino acid at the 42nd position of VH (VH42) and the amino acid at the 41st position of VL (VL41) is substituted with a non-natural amino acid.

[0009] 2. In the above 1, the non-natural amino acid is a single-chain variable fragment selected from the group consisting of frTet, pAzF, pEPA, and pEhF.

[0010] 3. In the above 1, the non-natural amino acid is a single-chain variable fragment that is combined via a click chemical reaction with a drug having a functional group selected from the group consisting of transcyclooctene, norbornene, azide, dibenzocyclooctine, bicyclononine, and cyclooctine.

[0011] 4. In 1 above, the linker comprises a single-chain variable fragment having the structure of the following structural formula 1:

[0012] [Structural Formula 1]

[0013] S1-ABD-S2

[0014] (In the formula, S1 and S2 are each independently spacers composed of 1 to 30 amino acids, and ABD is an albumin binding site composed of 5 to 100 amino acids).

[0015] 5. A single-strand variable fragment according to 1 above, wherein the heavy chain variable region has the amino acid sequence represented by SEQ ID NO. 1 and the light chain variable region has the amino acid sequence represented by SEQ ID NO. 2.

[0016] 6. A single-chain variable fragment-drug conjugate in which any one of the single-chain variable fragments 1 to 5 above is conjugated to a drug.

[0017] 7. In the above 6, a single-chain variable fragment-drug conjugate comprising the structure of the following structural formula 2:

[0018] [Structural Formula 2]

[0019] Single-chain variable fragment-[connector]-[cleavage linker]-drug

[0020] (In the formula, the linker is a chemical structure formed by the click chemical reaction between a non-natural amino acid of a single-chain variable fragment and a drug, and the cleavage linker is a peptide cleaved by a lysosomal enzyme).

[0021] 8. In the above 6, the drug is a single-chain variable fragment-drug conjugate selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), DM1, DM4, calciemacin, pyrrolobenzodiazepine (PBD), SN-38, DXd, doxorubicin, paclitaxel, camptothecin, irinotecan, topotecan, exatecan, etoposide, vincristine, vinblastine, imatinib, sorafenib, sunitinib, and lenvatinib.

[0022] 9. In 7 above, the cleavage linker portion is composed of glutamic acid-valine-alanine (Glu-Val-Ala), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamic acid-glycine-glycine-phenylalanine-glycine (Glu-Gly-Gly-Phe-Gly), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly), alanine-alanine-asparagine (Ala-Ala-Asn), asparagine-asparagine (Asn-Asn), and glutamine-asparagine (Gln-Asn). A single-chain variable fragment-drug conjugate comprising any one amino acid linker selected from the group.

[0023] 10. A pharmaceutical composition for the treatment or prevention of cancer comprising the single-chain variable fragment-drug conjugate of 6 above.

[0024] 11. A pharmaceutical composition for the treatment or prevention of cancer, wherein the cancer is any one selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, kidney cancer, bladder cancer, head and neck cancer, triple-negative breast cancer, gastric cancer, gastroesophageal junction cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, ovarian cancer, pancreatic cancer, and esophageal cancer.

[0025] The single-chain variable fragment of the present invention can efficiently conjugate drugs using a click chemical reaction.

[0026] The single-chain variable fragment of the present invention can bind a drug at an optimized location with a high binding yield.

[0027] The single-chain variable fragment of the present invention can produce a single-chain variable fragment-drug conjugate with a uniformly controlled number of drug conjugates (DAR), thereby improving the reproducibility of the manufacturing process and quality uniformity.

[0028] The single-chain variable fragment of the present invention is designed so that a drug is conjugated to a specific location where a non-natural amino acid is introduced, thereby reducing heterogeneity that may occur in the existing random conjugation method using lysine or cysteine ​​residues.

[0029] The single-chain variable fragment of the present invention can stably produce a single-chain variable fragment-drug conjugate having a uniform structure, thereby ensuring the efficacy and safety of the drug.

[0030] The single-chain variable fragment of the present invention can maintain excellent antigen binding ability to PD-L1 even when bound to a drug and albumin, thereby improving selective binding to target cells and drug delivery efficiency.

[0031] The single-chain variable fragment of the present invention can bind to albumin in the body by including an albumin binding domain, thereby effectively extending the residence time and half-life of the single-chain variable fragment in the blood.

[0032] The single-chain variable fragment of the present invention has a relatively small molecular weight, so it exhibits excellent tissue penetration into tumor tissue, particularly solid tumors.

[0033] The single-chain variable fragment according to the present invention and the single-chain variable fragment-drug conjugate containing the same can be usefully utilized in the treatment of various diseases, including anticancer treatment.

[0034] Figure 1 is a diagram showing the overall structure of PDL1Albu of the embodiment and the positions of VH42 and VL41 within the structure.

[0035] Figure 2 is a diagram showing the VH and VL structures of atezolizumab and the positions of VH42 and VL41 within those structures.

[0036] Figure 3 shows (a) the absorption spectrum of TCO-DXd and (b) the absorption spectrum of aPDL1 Albu-DXd after synthesis and purification.

[0037] Figure 4 shows the dye inhibition band intensity in the G44 variant and G226 variant of single-chain variable fragment-drug conjugate A.

[0038] Figure 5 shows the dye inhibition band intensity in the G44 variant and G226 variant of single-chain variable fragment-drug conjugate B.

[0039] The present invention provides an anti-PD-L1 single-chain variable fragment and a single-chain variable fragment-drug conjugate comprising the same.

[0040] The present invention provides an anti-PD-L1 single-chain variable fragment and a single-chain variable fragment-drug conjugate comprising the same, which has PD-L1 binding ability and includes a heavy chain variable region (VH), a light chain variable region (VL), and a linker connecting them, wherein at least one of the amino acid at the 42nd position of the VH region and the amino acid at the 41st position of the VL region is substituted with a non-natural amino acid, thereby enabling the binding of a drug to an optimized position with a high conjugation yield through a click chemical reaction and enabling the production of a single-chain variable fragment-drug conjugate with a uniformly controlled number of drug conjugations (DAR).

[0041] Antibodies refer to immunoglobulins. Antibodies generally have a structure consisting of two heavy chains and two light chains stabilized by a pair of disulfide bonds.

[0042] The heavy chain means that it includes the full-length heavy chain and its fragments, including a variable region domain VH containing a sufficient variable amino acid sequence to confer specificity to the antigen and three constant region domains CH1, CH2 and CH3, and hinges.

[0043] The light chain means that it includes both the full-length light chain and fragments thereof, including a variable region domain VL and a constant region domain CL that contain a sufficient variable amino acid sequence to confer specificity to the antigen.

[0044] The variable fragment (Fv) is a region that provides diversity to the antibody by directly participating in antigen binding, consisting of a heavy chain variable region (VH) and a light chain variable region (VL).

[0045] The complementarity determining region (CDR) is the complementarity determining region present in the heavy chain variable region and the light chain variable region of the antibody. The CDR is the region that comes into direct contact with the antigen and exhibits diverse variations. The framework (FR) is a region other than the CDR that has relatively few variations. The CDRs present in the heavy chain variable region are denoted as HCDR1, HCDR2, and HCDR3 in order from the N-terminus, and the CDRs present in the light chain variable region are denoted as LCDR1, LCDR2, and LCDR3 in order from the N-terminus.

[0046] The single-chain variable fragment (scFv) of the present invention refers to 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, wherein each region is connected by a linker.

[0047] The single-strand variable fragment of the present invention targets PD-L1. PD-L1 is an immune checkpoint protein expressed on the surface of tumor cells and immune cells, and is known to inhibit T cell activity through interaction with PD-1. Therefore, an antibody or antibody fragment targeting PD-L1 can induce an anti-tumor immune response by blocking the immune checkpoint.

[0048] In the single-strand variable fragment, at least one of the amino acid at the 42nd position of the VH region (VH42) and the amino acid at the 41st position of the VL region (VL41) is substituted with a non-natural amino acid based on the Kavat numbering system.

[0049] In this invention, the amino acid residue positions of the heavy chain variable region (VH) and light chain variable region (VL) are defined based on the Kavat numbering system. The Kavat numbering system is a system established by comparing and analyzing various antibody sequences, and it is a method of defining the amino acid positions of antibody variable regions by number according to unified standards.

[0050] According to the Kavat numbering system, amino acid residues at structurally corresponding positions can be represented by the same number even if they are different antibody sequences. Therefore, when a specific residue is defined in this specification based on the Kavat numbering system, it refers to a structurally corresponding position in various antibodies, rather than being limited to a specific antibody sequence.

[0051] The amino acid residue numbers of the heavy chain variable region of atezolizumab (Sequence No. 1) and their corresponding kavat numbers are as shown in Table 1 below.

[0052]

[0053] The amino acid residue numbers of the light chain variable region of atezolizumab (Sequence No. 2) and their corresponding kavat numbers are as shown in Table 2 below.

[0054]

[0055] In the single-chain variable fragment of the present invention, at least one of the amino acid at the 42nd position of the VH region (VH42) and the amino acid at the 41st position of the VL region (VL41) is substituted with a non-natural amino acid based on the Kavat numbering system.

[0056] VH42 and VL41 are residues located in the framework regions of VH and VL, respectively. Since they are situated at a distance greater than a certain level from the CDR region responsible for antigen binding, their influence on antigen binding ability is limited. Furthermore, VH42 and VL41 are exposed on the surface of the antibody variable region, enabling chemical binding with external molecules without significantly impairing the stability of the antibody structure.

[0057] In one embodiment, the amino acid at the VH42 position may be substituted with a non-natural amino acid.

[0058] In one embodiment, the amino acid at the VL41 position may be substituted with a non-natural amino acid.

[0059] In one embodiment, the amino acids at the VH42 and VL41 positions can both be substituted with non-natural amino acids.

[0060] Non-natural amino acids may be amino acids containing a tetrazine group, amino acids containing an azide group, or amino acids containing an alkyne group.

[0061] In one embodiment, the non-natural amino acid is any one selected from the group consisting of frTet, pAzF, pEPA, and pEhF.

[0062] frTet has tetrazine introduced as shown in Chemical Formula 1 below, so it can be site-specifically conjugated with a drug to which transcyclooctene (TCO) has been introduced through the reverse electron demand Diels-Elder (IEDDA) reaction as shown in Reaction Scheme 1.

[0063] [Chemical Formula 1]

[0064]

[0065] [Reaction Equation 1]

[0066]

[0067] pAzF contains an azide functional group as shown in Chemical Formula 2 below, so it can be site-specifically conjugated with a drug to which dibenzocyclooctane (DBCO) has been introduced through a strain-induced azide-alkyne cyclization addition reaction (SPAAC).

[0068] [Chemical Formula 2]

[0069]

[0070] [Reaction Equation 2]

[0071]

[0072] pEPA and pEhF each contain an azide functional group, so they can be site-specifically conjugated with drugs to which dibenzocyclooctane (DBCO), bicyclononine (BCN), or difluorocyclooctane (DIFO) have been introduced via SPAAC.

[0073] In one embodiment, a non-natural amino acid can be combined through a click chemical reaction with a drug having a functional group selected from the group consisting of transcyclooctene, norbornene, azide, dibenzocyclooctine, bicyclononine, and cyclooctine.

[0074] The single-stranded variable fragment has a structure in which VH and VL are connected by a linker. Since the single-stranded variable fragment consists of a single polypeptide chain, it maintains the antigen-binding site of the antibody while having a small total molecular weight, resulting in excellent tissue penetration.

[0075] In one embodiment, the single-strand variable fragment may have a structure in which VH and VL are 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.

[0076] In one embodiment, the linker may be composed of (GS)n, (GGS)n, (GGGGGS)n, or a combination thereof. Here, n is any natural number, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Additionally, the linker is not limited to the order of combination and the number of repetitions of the above sequences, and may be in the form of a combination of two or more types of sequences.

[0077] The linker may include an albumin binding domain (ABD). An albumin binding domain refers to a part that interacts with an albumin protein, and may be, for example, an antibody, antibody analog, protein domain, protein motif, peptide, compound, aptamer, oligonucleotide, sugar, etc.

[0078] In one embodiment, the albumin binding site may be a peptide capable of binding to serum albumin. The albumin binding peptide may be fused to a single-strand variable fragment or other protein to increase its half-life in the body.

[0079] In one embodiment, the albumin binding site may consist of 5 to 100, 10 to 80, or 20 to 60 amino acids.

[0080] In one embodiment, the albumin binding site may include the peptide sequence of Table 3 below.

[0081]

[0082] In addition, the albumin binding site may include variants capable of binding to serum albumin while having sequence identity of 80%, 85%, 90%, 95%, or 98% or more with the above sequence.

[0083] In one embodiment, the albumin binding site may be a peptide that binds to human serum albumin (HSA).

[0084] In one embodiment, the linker may include the structure of the following structural formula 1:

[0085] [Structural Formula 1]

[0086] S1-ABD-S2

[0087] (In the formula, S1 and S2 are each independently spacers composed of 1 to 30 amino acids, and ABD is an albumin binding site composed of 5 to 100 amino acids).

[0088] The linker of structural formula 1 can confer albumin binding function while maintaining the antigen binding ability of the single-chain variable fragment, and accordingly, can contribute to an increase in half-life in the body, improved drug delivery efficiency, and increased anticancer efficacy.

[0089] The linker of structural formula 1 can be located between VH and VL of a single-chain variable fragment, and in one embodiment, the linker can be arranged in the form of VH-linker-VL.

[0090] Spacers S1 and S2 provide spatial clearance between each domain of the single-strand variable fragment, which can minimize structural interference of the protein and help the single-strand variable fragment form an appropriate three-dimensional structure.

[0091] The spacer is not limited to a specific sequence and may be a flexible peptide including, for example, glycine (Gly), serine (Ser), alanine (Ala), or a combination thereof.

[0092] In one embodiment, the spacer is based on a repeating sequence including glycine (Gly) and serine (Ser), but may be a modified sequence additionally including amino acids such as alanine (Ala), threonine (Thr), asparagine (Asn), or glutamine (Gln) as needed.

[0093] In one embodiment, the spacer may include a repeating sequence of the form (Gly-Ser)n, where n may be an integer from 1 to 10.

[0094] In one embodiment, S1 and S2 may each independently have any one amino acid sequence selected from the group consisting of SGGGGS, GGGGS, GGGGSGGGGS, GGSGGS, GSGSGS, GGGSG, GGGGSGGS, GGSGGSG, GSGGGGS, and GGSGSGGS.

[0095] In one embodiment, S1 and S2 may each independently have any one amino acid sequence selected from the group consisting of GGGGSGGST, GSTSGGGGS, AGGGGSGGS, GGAGGS, GGSGAS, GGSGTS, GGSGNS, GGSGQS, GGSAGS, GGSQGS, GGTGGS, GGNGGS, and GGQGGS.

[0096] Spacers S1 and S2 may each independently be flexible peptides composed of 1 to 30, 3 to 20, or 5 to 10 amino acids.

[0097] Single-strand variable fragments are not limited to having specific amino acid sequences and lengths as long as they have the ability to bind to PD-L1.

[0098] In one embodiment, the single-chain variable fragment may consist of 200 to 500, 200 to 400, or 200 to 300 amino acids.

[0099] A single-chain variable fragment can include an array of either VH-linker-VL or VL-linker-VH.

[0100] In one embodiment, the single-strand variable fragment may be derived from atezolizumab, known as an antibody that binds to PD-L1. For example, the single-strand variable fragment may be configured in the form of a scFv containing the VH and VL sequences of atezolizumab.

[0101] In one embodiment, the single-strand variable fragment may include a tablet tag.

[0102] The single-strand variable fragment may be produced within a microorganism that expresses the sequence encoding the corresponding protein.

[0103] Microorganisms known in the field may be used without limitation. For example, it may be Escherichia coli, but is not limited thereto.

[0104] The present invention provides a single-chain variable fragment-drug conjugate in which the single-chain variable fragment described above is conjugated with a drug.

[0105] In one embodiment, the single-chain variable fragment-drug conjugate may include the structure of the following structural formula 2:

[0106] [Structural Formula 2]

[0107] Single-chain variable fragment-[connector]-[cleavage linker]-drug

[0108] (In the formula, the linker is a chemical structure formed by the click chemical reaction between a non-natural amino acid of a single-chain variable fragment and a drug, and the cleavage linker is a peptide cleaved by a lysosomal enzyme).

[0109] The drug may be any one selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), DM1, DM4, calchiemaicin, pyrrolobenzodiazepine (PBD), SN-38, DXd, doxorubicin, paclitaxel, camptothecin, irinotecan, topotecan, exatecan, etoposide, vincristine, vinblastine, imatinib, sorafenib, sunitinib, and lenvatinib.

[0110] The linkage is a chemical structure formed by a click chemical reaction between a non-natural amino acid of a single-chain variable fragment and a drug, for example, it may be the chemical structure of reaction schemes 1 and 2.

[0111] The cleavage linker portion is any one amino acid linker selected from the group consisting of glutamic acid-valine-alanine (Glu-Val-Ala), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamic acid-glycine-glycine-phenylalanine-glycine (Glu-Gly-Gly-Phe-Gly), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly), alanine-alanine-asparagine (Ala-Ala-Asn), asparagine-asparagine (Asn-Asn), and glutamine-asparagine (Gln-Asn). It is possible.

[0112] The present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising the single-chain variable fragment-drug conjugate described above.

[0113] The present invention provides a method for treating cancer comprising the single-chain variable fragment-drug conjugate described above.

[0114] The cancer may be any one selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, kidney cancer, bladder cancer, head and neck cancer, triple-negative breast cancer, gastric cancer, gastroesophageal junction cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, ovarian cancer, pancreatic cancer, and esophageal cancer.

[0115]

[0116] Examples

[0117] 1. Computational Analysis for Drug Conjugation Site Search

[0118] Computational analysis was performed to explore suitable sites for drug conjugation on antibody fragment proteins.

[0119] The single-chain variable fragment (PDL1Albu) of the example has a structure in which the atezolizumab-derived heavy-chain variable region (VH) and light-chain variable region (VL) are connected by a linker (S1-ABD-S2) containing an albumin binding site (ABD), as shown in Table 4 below, and a His-Tag is attached to the C-terminus of VL.

[0120]

[0121] The structural model of the PDL1Albu protein was predicted using AlphaFold2. Based on the predicted structural model, the solvent accessibility of each amino acid residue was analyzed. Solvent accessibility analysis was performed using the PyMOL program (Version 2.4.1, Schrodinger, New York, NY, USA).

[0122] In addition, to evaluate the effect of specific residue mutations on protein stability, mutation stability analysis was performed using the PyRosetta program (PyRosetta 4, The PyRosetta Team at Johns Hopkins University, Baltimore, MD, USA). The mutation stability analysis was substituted using tyrosine (Y) and tryptophan (W), natural amino acids similar in structure to pAzF and frTet. The effect of mutations in each residue on protein structural stability was predicted, and candidate sites where structural stability was maintained were selected. Finally, candidate drug conjugation sites were selected from residues satisfying the following criteria: a) residues that maintain protein stability upon the introduction of mutations (Y, W), b) residues with high solvent accessibility, c) residues not located in the functional or structurally important regions of the antibody, and d) residues not located near the CDR, which is important for antigen binding. Based on the above criteria, candidate sites for drug conjugation within the antibody fragment were derived as shown in Table 5 below.

[0123]

[0124]

[0125] 2. Production of expression vector

[0126] To express PDL1Albu, the corresponding gene was inserted into the pBAD expression vector.

[0127] To introduce reactive functional groups for drug conjugation, mutagenesis was performed by substituting codons at selected residue positions with amber codons. This allowed the non-natural amino acids para-azidophenylalanine (pAzF) or 4-(1,2,4,5-tetrazine-3-yl)phenylalanine (frTet) to be introduced at specific positions.

[0128] In this way, the pBAD-PDL1Albu-XAmb expression vector was constructed. Here, X represents a specific residue position where a non-natural amino acid is introduced, and Amb indicates that the codon at that position has been substituted with an amber codon.

[0129] The above expression vector was used for the expression of PDL1Albu-XpAzF and PDL1Albu-XfrTet protein variants.

[0130]

[0131] 3. Protein Expression and Purification

[0132] To express wild-type PDL1Albu, the pBAD-PDL1Albu plasmid was transformed into E. coli TOP10 cells.

[0133] Transformed cells were cultured in 2xYT medium containing 100 μg / mL ampicillin at 37°C and 200 rpm. Subsequently, when the OD600 of the culture medium reached approximately 0.5, 0.2% (w / v) L-(+)-arabinose was added to induce protein expression. After induction of expression, culture was carried out at 23°C for approximately 24 hours.

[0134] An amber codon repression system was used for the expression of variants into which non-natural amino acids were introduced.

[0135] To prepare the PDL1Albu-frTet variant, the corresponding pBAD expression vector was co-transformed into E. coli C321.ΔA.exp cells with the pDule-C11RS plasmid. When the OD600 reached approximately 0.5, protein expression was induced by adding 1 mM frTet and 0.4% (w / v) L-(+)-arabinose.

[0136] To prepare the PDL1Albu-pAzF variant, E. coli C321.ΔA.exp cells were co-transformed with the pEVOL-pAzF plasmid. When the OD600 reached approximately 0.5, protein expression was induced by adding 1 mM pAzF and 0.4% (w / v) L-(+)-arabinose.

[0137] The expressed cells were harvested by centrifuging at 8,000 rpm under 4℃ conditions.

[0138] The harvested cell pellet was suspended in lysis buffer (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) with the addition of 1 mg / mL lysozyme. Subsequently, the cells were lysed using sonication. Sonication was performed under conditions of repeating 1-second pulse and 2-second rest cycles.

[0139] After cell lysis, the lysate was centrifuged at 10,000 g at 4°C for 20 minutes to recover the supernatant. The recovered supernatant was reacted with Ni-NTA agarose resin to bind His-tag proteins.

[0140] The protein-bound resin was mounted on a column and washed with a washing buffer (20 mM imidazole). Subsequently, the protein was eluted using an elution buffer containing 250 mM imidazole. The purified protein was stored at 4°C, and protein expression and purification were confirmed via SDS-PAGE analysis.

[0141]

[0142] 4. Preparation of Single-Chain Variable Fragment-Drug Conjugates

[0143] Single-chain variable fragment-drug conjugates were prepared using PDL1Albu-frTet and PDL1Albu-pAzF. TCO-DXd and DBCO-MMAE were used as the drugs, respectively, and GGFG and Val-Cit-PAB were used as the cleavage linkers, respectively.

[0144] A single-chain variable fragment-drug conjugate A was prepared by conjugating PDL1Albu-frTet with TCO-GGFG-DXd of the following chemical formula 3 using an IEDDA reaction.

[0145] [Chemical Formula 3]

[0146]

[0147] A single-chain variable fragment-drug conjugate B was prepared by conjugating PDL1Albu-pAzF with DBCO-PEG4-Val-Cit-PAB-MMAE of the following chemical formula 4 using the SPAAC reaction.

[0148] [Chemical Formula 4]

[0149]

[0150] The single-chain variable fragment and the drug were mixed at a final molar ratio of 1:2 or 1:4, and the IEDDA reaction was carried out for about 30 minutes, while the SPAAC reaction was carried out overnight at 4°C under rotational conditions.

[0151] The conjugates formed after the reaction were confirmed by SDS-PAGE analysis. The bands of unconjugated and conjugated proteins were quantitatively analyzed using the Volume Tools function of Image Lab software (Bio-Rad).

[0152] In addition, to evaluate the conjugation efficiency more accurately, an experiment was performed to label unreacted proteins by reacting the generated conjugate with an excess amount of TCO-Dye or DBCO-Dye.

[0153]

[0154] 5. Analysis of Conjugation Efficiency of Single-Chain Variable Fragment-Drug Conjugates A and B

[0155] After performing the drug conjugation reaction, the conjugation efficiencies of single-chain variable fragment-drug conjugate A and single-chain variable fragment-drug conjugate B were compared through SDS-PAGE analysis and dye inhibition analysis.

[0156] Under conditions where the drug response time was set to a sufficiently long duration, the dye inhibition band intensity was low in most variants; this is interpreted as being due to the drug being conjugated to most proteins during the sufficient response time, leaving almost no residual reaction groups.

[0157] On the other hand, under conditions where the drug response time was set short, differences in dye inhibition band intensity were observed among variants, which is considered to reflect differences in drug conjugation efficiency at each location.

[0158] Analysis results showed that the dye inhibition band intensity was lowest in the G44 and G226 variants of both single-chain variable fragment-drug conjugate A and single-chain variable fragment-drug conjugate B (Figs. 4 and 5), and the intensity of the shifted band was also highest in the SDS-PAGE analysis. These results indicate that the drug conjugation efficiency is highest at that location.

[0159] The positions with the highest conjugation efficiency correspond to the VH42 and VL41 positions, respectively, based on the Kavat numbering system. Furthermore, the same trend was observed in both single-strand variable fragment-drug conjugate A and single-strand variable fragment-drug conjugate B, implying that high drug conjugation efficiencies are commonly observed at the VH42 and VL41 positions in different click chemistry modes, namely the IEDDA reaction and the SPAAC reaction. These results suggest that VH42 and VL41 are common positions within the antibody fragment structure that are favorable for drug conjugation.

Claims

1. Having PD-L1 binding ability, It includes a heavy chain variable region (VH), a light chain variable region (VL), and a linker connecting the VH and the VL, and A single-strand variable fragment in which, based on the Kavat numbering system, at least one of the amino acid at the 42nd position of VH (VH42) and the amino acid at the 41st position of VL (VL41) is substituted with a non-natural amino acid.

2. The non-natural amino acid of claim 1 is a single-chain variable fragment selected from the group consisting of frTet, pAzF, pEPA, and pEhF.

3. The non-natural amino acid of claim 1, wherein the non-natural amino acid is a single-chain variable fragment that is combined through a click chemical reaction with a drug having a functional group selected from the group consisting of transcyclooctene, norbornene, azide, dibenzocyclooctine, bicyclononine, and cyclooctine.

4. The linker of claim 1 comprises a single-chain variable fragment having the structure of the following structural formula 1: [Structural Formula 1] S1-ABD-S2 (In the formula, S1 and S2 are each independently spacers composed of 1 to 30 amino acids, and ABD is an albumin binding site composed of 5 to 100 amino acids).

5. A single-strand variable fragment according to Claim 1, wherein the heavy chain variable region has an amino acid sequence represented by SEQ ID NO. 1 and the light chain variable region has an amino acid sequence represented by SEQ ID NO.

2.

6. A single-chain variable fragment-drug conjugate in which the single-chain variable fragment of any one of claims 1 to 5 is conjugated to a drug.

7. The single-chain variable fragment-drug conjugate of Claim 6 comprising the structure of the following structural formula 2: [Structural Formula 2] Single-chain variable fragment-[connector]-[cleavage linker]-drug (In the formula, the linker is a chemical structure formed by a click chemical reaction between the non-natural amino acid of the single-chain variable fragment and the drug, and the cleavage linker is a peptide cleaved by a lysosomal enzyme).

8. The drug of claim 6, wherein the drug is a single-chain variable fragment-drug conjugate selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), DM1, DM4, calciemacin, pyrrolobenzodiazepine (PBD), SN-38, DXd, doxorubicin, paclitaxel, camptothecin, irinotecan, topotecan, exatecan, etoposide, vincristine, vinblastine, imatinib, sorafenib, sunitinib, and lenvatinib.

9. In claim 7, the cutting linker portion comprises glutamic acid-valine-alanine (Glu-Val-Ala), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamic acid-glycine-glycine-phenylalanine-glycine (Glu-Gly-Gly-Phe-Gly), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly), alanine-alanine-asparagine (Ala-Ala-Asn), asparagine-asparagine (Asn-Asn) and A single-chain variable fragment-drug conjugate comprising any one amino acid linker selected from the group consisting of glutamine-asparagine (Gln-Asn).

10. A pharmaceutical composition for the treatment or prevention of cancer comprising the single-chain variable fragment-drug conjugate of Claim 6.

11. A pharmaceutical composition for the treatment or prevention of cancer according to claim 10, wherein the cancer is any one selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, kidney cancer, bladder cancer, head and neck cancer, triple-negative breast cancer, gastric cancer, gastroesophageal junction cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, ovarian cancer, pancreatic cancer, and esophageal cancer.

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