Chemical synthesis method for single-chain antibody fragments and products obtained thereby

Abstract

A new method for the complete chemical synthesis of single-chain antibody fragment (scFv) proteins is disclosed, resulting in products that are structurally close and functionally equivalent to their biological counterparts, but with the advantage of being free of impurities and having more reproducible properties. The method comprises the following general steps: (a) synthesizing subsequences individually, (b) sequentially assembling the subsequences obtained in step (a) according to the order in which they appear in the target amino acid sequence, and (c) performing oxidative folding of the assembled complete peptide molecule obtained in step (b), and dialyzing the resulting product in a buffer solution.

Description

[Technical Field]

[0001] The present invention relates to the field of preparation of antibodies for use in human therapy and diagnosis, and in summary, the present invention demonstrates the use of chemical protein synthesis as a general platform for the production of highly functional antibody fragments. [Background technology]

[0002] Biologics are drugs made from complex molecules engineered using living microorganisms, plant, or animal cells. Many are produced using recombinant DNA technology. They are sometimes called biopharmaceuticals or biological drugs. Biopharmaceuticals represent a growing segment of the pharmaceutical industry, accounting for 30% of all new drug sales for therapeutic and diagnostic applications in the United States in recent years. The increasing demand for novel biopharmaceuticals requires new production processes that offer improved capacity, space-time yield, and shorter development times, including the use of suitable expression systems. Biologics include interleukins, vaccines, and, most notably, antibodies, which bind to the surface of cells in the body and are widely used in cancer treatment.

[0003] Antibodies are important detection elements in research, diagnostics, and therapy. To date, antibodies are the fastest growing class of therapeutic protein drugs. In addition to the production of classical IgG antibody formats, a rapidly increasing number of recombinant antibodies are now available. Antibody fragments are also gaining clinical importance, and several methods are being developed to meet these demands.

[0004] Small recombinant Ab (rAb) fragments are increasingly being utilized as alternatives to whole monoclonal antibodies (mAbs) in several medical diagnostic and therapeutic applications. Various rAb formats have been designed for specific applications, including engineering modifications to antigen binding, valency, and molecular weight (MW). One of the most widespread types of rAbs is the single-chain variable fragment (scFv), which has been successfully engineered into many different Ab formats and is easily expressed in several expression systems. scFvs are complex peptide products that contain many amino acids, typically ranging from 230 to 280, and are organized into three-dimensional structures. They contain the complete antigen-binding site of an antibody, including the variable heavy (VH) and variable light (VL) domains. The VH domain is connected to the VL domain by an introduced flexible polypeptide linker.

[0005] rAb fragments have been expressed in a wide variety of hosts, including prokaryotes such as E. coli and B. subtilis, and eukaryotes such as S. cerevisiae, Pichia pastoris, insect cells, plant cells, and mammalian cells. For therapeutic purposes, large quantities of antibodies are required, sometimes exceeding one gram per patient per year. Therefore, there is a need to develop production systems for the efficient and cost-effective production of these molecules.

[0006] All of the above systems are capable of producing antibodies and antibody fragments using live microorganisms, and the resulting products are "biological drugs," which are subject to the requirements for the production of products derived from rDNA technology and the general recommendations for the quality control of biological products (e.g., GMP Directive 91 / 356 / EEC and Directive 90 / 219 / EEC on the contained use of genetically modified microorganisms).

[0007] Despite their recognized and unique utility, biologics suffer from many limitations associated with their natural origins (most of which are complex mixtures that are not easily identified or characterized); they are heat-sensitive and prone to microbial contamination, requiring sterility from the initial manufacturing process. Furthermore, compared with drugs obtained by chemical synthesis, biologics are difficult, and sometimes impossible, to fully characterize using available laboratory testing methods, and some of the components of a finished biologic remain unknown. The quality of these formulations is highly dependent on the manufacturing process; therefore, manufacturers must ensure the consistency, quality, and purity of their formulations by ensuring that the manufacturing process remains substantially the same over time. In contrast, pharmaceutical manufacturers can significantly alter their manufacturing process and then analyze the finished product to establish that it is identical to the product prior to the manufacturing change.

[0008] Furthermore, the living systems used to produce biologics can be sensitive to even the most minor changes in the manufacturing process. Small process variations can have a significantly larger impact on the properties of the finished biologic and, most importantly, how it functions in the body. To ensure that the manufacturing process remains identical over time, biologic manufacturers must strictly control the origin and properties of the starting materials and consistently implement hundreds of process controls that ensure predictable manufacturing results. Finally, biologic process controls are established individually for each unique manufacturing process / product and cannot be applied to manufacturing processes / products made by other manufacturers. These process controls may be confidential to the original manufacturer. Therefore, it is difficult or impossible for a second manufacturer to produce the "same" biologic without in-depth knowledge and experience of the original company's process.

[0009] An additional challenge faced by biologics arises from their natural variability. Various factors can compromise the consistency, safety, and efficacy of these products, particularly the following: a) All biological systems inherently undergo genetic change through mutation and selection, and foreign genes inserted into new host cells may exhibit increased genetic instability. The goal of molecular genetic studies is to establish that the correct sequences are generated and integrated into the host cell, and that both the structure and copy number of the inserted sequence are maintained within the cell from cultivation through to the end of production. Such studies can provide valuable information that should be considered in conjunction with tests performed at the protein level to ensure product quality and consistency. b) Products expressed in foreign hosts may differ structurally, biologically, or immunologically from their native counterparts. Such changes may occur at the post-translational level or during production or purification and may result in undesirable clinical effects. Therefore, their presence must be shown to be justified and consistently controlled. c) The choice of manufacturing procedures will affect the nature, extent, and amount of potential impurities in the finished product, which the purification process must be shown to be able to remove. Examples of these are endotoxins in products expressed in bacterial cells, and adventitious agents and DNA in products expressed in mammalian cells. d) Unintended variability in the production culture can result in favorable changes in the expression of other genes in the host / vector system or in changes that result in variations in the product. Such variations can result in variations in yield, changes to the product itself (e.g., the nature and degree of glycosylation) and / or quantitative and qualitative differences in the impurities present. In summary, in the field of biopharmaceuticals, procedures that ensure consistency of production conditions as well as the final product are essential. e) Extensive "scale-up" operations, such as at the fermentation and / or purification levels, occur during the progression from laboratory development to full-scale commercial production, which can have significant impacts on product quality, including effects on product conformation, yield, and / or quantitative and qualitative differences in impurities.

[0010] For these reasons, the production of biological products is typically a complex operation, requiring extensive in-process control and quality control testing during each production step.

[0011] As an alternative to biological synthesis, a completely chemical synthesis approach has been proposed for the production of numerous low-molecular-weight immunogenic peptides. For example, Non-Patent Document 1 describes the synthesis of a typical immune system-related peptide having a size of 5 kDa (corresponding to approximately 40 amino acids). Non-Patent Document 2 describes a method using solid-phase synthesis and chemoselective ligation to obtain an effector peptide of approximately 7 kDa that binds to formyl peptide receptors and stimulates immune responses. Non-Patent Document 3 describes a method for producing sequences of more than 50 amino acids using solid-phase synthesis and native chemical ligation. The document points out the need to control the secondary and tertiary structure and mentions the synthesis of a 7.3 kDa, 66-amino acid-long (66-meric) peptide. None of these documents address the synthesis of scFvs or similar complex peptides. Patent document 1 describes an antibody that binds to the c-erbB2 receptor epitope, and speculatively mentions chemical synthesis as a possible preparation method, with reference to standard manuals on peptide synthesis, but no concrete evidence is provided as to the practicality of this method for generating complex antibody structures, nor are any reproducible synthesis examples provided.

[0012] Thus, there is an unmet need for a fully chemically synthesized method capable of generating functionally effective scFvs. These structures contain a significant number of amino acids, ranging from 200 to 300. There is a particular need to achieve through chemical synthesis a valid three-dimensional structure that reflects the same function as the biological reference product, i.e., a structure that can recapitulate its immunostimulatory function. This result cannot be easily achieved by simple oxidation, especially for peptides with molecular weights in the range mentioned herein. Furthermore, the development of production systems that make these molecules available in an efficient and cost-effective manner is also necessary. These new production systems pose special challenges, particularly with regard to correct protein folding. Proteins function properly only if they are correctly folded. When proteins are produced, their conformation can change depending on concentration, interaction partners, preparation buffers, and more. Factors that affect the protein's ability to reach its final conformation can prevent the correct hydrogen bonds from forming, alter the patterns required to form these structures, and potentially adversely affect all other higher-order protein structural levels. External factors such as temperature, pH, and formulation affect hydrogen bonding. Given that structural changes can affect functionality, the ability to accurately detect small changes in secondary and tertiary structure is crucial in protein manufacturing and formulation. This is particularly true for globular proteins such as antibodies, also called immunoglobulins due to their globular appearance. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 99 / 55367 [Non-patent literature]

[0014] [Non-Patent Document 1] Scientific Reports, 7(1), 2017, pp. 1-11 [Non-patent document 2] Chemibiochem, 19(5), 2018, pp. 459-469 [Non-patent document 3] Frontiers in Bioengineering and Biotechnology 8(1), 2020, p.162 Summary of the Invention

[0015] The production of scFv fragments by fully chemical synthesis is described. This production system has the potential to become an alternative approach for the rapid and highly parallel expression of a diverse range of antibody fragments. Compared to traditional approaches to obtaining scFvs in biological systems, this synthetic system saves time, converts biological products into chemically synthesized pharmaceuticals, and frees production from the quality control required for biological products. Specifically, the method aims to synthesize scFvs whose amino acid sequence (target sequence) contains n cysteine ​​amino acids (cysteine ​​residues) separated by m amino acid subsequences. The process includes the following steps: (a) individually synthesizing all of the subsequences, including leading cysteine ​​residues, if present; (b) sequentially assembling the subsequences obtained in step (a) in the order of their appearance in the target amino acid sequence; and (c) performing oxidative folding of the assembled structure obtained in step (b) and dialyzing the resulting product in a buffer solution. [Brief explanation of the drawings]

[0016] [Figure 1] Primary structure and synthesis scheme of scFv Ab1(D2B). [Figure 2] Synthetic Route for Segments 1 and 2. [Figure 3] Synthetic pathway for segments 3, 4, and 5. [Figure 4] Assembly of Ab1(D2B) molecules by native chemical ligation and subsequent folding. [Figure 5](A) HPLC elution profile of the D2B folding reaction. HPLC conditions: Column: Imtakt Intrada WP-RP 3 μm (4.6 × 250 mm); Gradient elution: 5–20% B (1 min) / 20–35% B (1–20 min); Temperature: 80°C; Flow rate: 1.0 mL / min. a) Unfolded Ab1 (D2B) compound 26, b) 1 day after the folding reaction, c) Purified folded Ab1 (D2B) compound 27. (B) ESI MS spectrum of folded D2B compound 27. ESI MS:m / z Calculated value C1207H1823N337O381S7(27375.26)[M+10H]10+ 2738.53, [M+11H]11+ 2489.66, [M+12H]12+ 2282.27; Measurements 2738.43, 2489.34, 2282.19. [Figure 6] Binding of scFv Ab3 was assessed by ELISA at a concentration of 4.0 μg / ml using human recombinant TNFα (red bar) as the positive antigen and BSA (orange bar) as the unrelated protein. Synthetic scFv Ab3 was resuspended and dialyzed in various ways: A) resuspended in water, B) resuspended in water and dialyzed against water, C) resuspended in selected buffer, D) resuspended in water and dialyzed against selected buffer, E) resuspended in water, dialyzed against water, and dialyzed against selected buffer. scFv Ab3 RREF was used as a reference. [Figure 7] SDS-PAGE samples using acrylamide gel (4-12%): A) ScFv Ab1; B) ScFv Ab2; C) Ab3. Samples: 1) synthetic ScFv; 2) ScFv RREF; M) marker. [Figure 8] SEC chromatograms performed on A) synthetic ScFv Ab1, B) ScFv Ab1 RREF, C) synthetic ScFv Ab2, D) ScFv Ab2 RREF, E) synthetic ScFv Ab3, F) ScFv Ab3 RREF. [Figure 9]FACS analysis was performed on positive and negative cells. A) Reactivity of scFv Ab1: 1) positive cell line (PC3-PIP) and 2) negative cell line (HCC1937). B) Reactivity of scFv Ab2: 1) positive cell line (MDAMB361) and 2) negative cell line (MDAMB468). [Figure 10] Binding of scFvs assessed by ELISA at various concentrations: A) scFv Ab1 against PC3-PIP (positive cells) and PC3 WT (negative cells); B) scFv Ab2 against MDAMB361 (positive cells) and MDAMB468 (negative cells); C) scFv Ab3 against human recombinant TNFα (positive antigen) and BSA (irrelevant protein). [Figure 11] Sensorgrams of kinetic analysis: (A) synthetic scFv Ab1 or (B) scFv Ab1 RREF sample injection of 50 to 3.2 nM; (C) synthetic scFv Ab2 or (D) scFv Ab2 RREF sample injection of 12.5 to 0.8 nM; and (E) purified synthetic scFv Ab3 or (F) scFv Ab3 RREF sample injection of 100 to 12.5 nM. DETAILED DESCRIPTION OF THE INVENTION

[0017] The methods of the present invention rely solely on chemical synthesis, i.e., they do not require any biological processes (e.g., processes using microorganisms, plant / animal cells, or parts thereof), and do not employ recombinant technology. The method aims to obtain scFv antibody fragments having a desired amino acid sequence (target sequence) that is the known sequence of a biological reference product, i.e., a naturally occurring or biologically produced scFv. The method is applicable to the production of scFvs containing up to 350 amino acids. Particularly advantageous and unexpected is the effectiveness of the method for obtaining target sequences at the upper end of these ranges, e.g., 100-350, or more specifically, 150-200, 150-250, 150-300, 200-300, 250-300, or 230-280 amino acids, which correspond to more spatially complex structures. However, the method is also applicable to the synthesis of target sequences containing fewer amino acids, such as 100-150 or 100-200.

[0018] Compared with a biological reference product (RREF), the scFv produced by the present method share binding specificity and exhibit substantially the same binding kinetics. This functional equivalence indicates that the present method is suitable for reproducing the primary structure (amino acid sequence) and secondary and tertiary structures (three-dimensional folding) of the biological reference product. This allows the scFv obtained by the present invention to be used for substantially the same applications / functions as the reference biological scFv. Correct folding of the scFv in its secondary and tertiary structures is generally caused by the formation of disulfide bonds between cysteine ​​residues present in the primary structure, and is achieved by contacting the primary structure peptide with an appropriate folding buffer system, followed by further treatment of the resulting product under defined conditions.

[0019] The target sequence for scFv in this method contains one or more cysteine ​​amino acids (cysteine ​​residues) variably distributed along the sequence; these are preferably internal, but may also be present as terminal amino acids at one or both ends of the peptide. Thus, the target sequence is defined herein as a linear amino acid sequence containing n cysteine ​​residues separated by a subsequence of m amino acids; m and n vary within ranges typical for scFvs: in particular, n can be 1-10, preferably 2-6; m can be 0-100, preferably 5-80; m = 0 corresponds to a peptide with two adjacent cysteine ​​amino acids, which is also included in the present invention. The values ​​of m and n are selected under the condition that the resulting target sequence does not exceed 350 amino acids, e.g., within one or more of the subranges defined above.

[0020] The method comprises the following general steps: a. individually synthesizing all of said subsequences, including the preceding cysteine ​​residue, if present; b. sequentially assembling the subsequences obtained in step a in the order in which they appear in the target amino acid sequence; c. performing oxidative folding on the assembled structure obtained in step b, and dialyzing the resulting product in a buffer; Includes:

[0021] Step a consists of independent synthetic substeps, each producing a subsequence of the target sequence, each of which can be carried out independently of the other by manual or automated peptide synthesis.

[0022] Manual synthesis of a subsequence can advantageously begin with currently available or easily prepared complementary fragments of the same sequence; these fragments are subjected to a condensation reaction to form an amide (peptide) bond between the free carboxy and amino groups of each amino acid to be linked; condensation selectivity can be achieved by prior protection of functional groups of the fragments that will not be involved in the linkage; protecting groups / reactions are not limited and can be appropriately selected from those currently available to those skilled in the art. For example, free amino groups can be protected by Fmoc (9-fluorenylmethyloxycarbonyl) derivatization; free carboxy groups can be protected by forming an appropriate ester group with trityl alcohol or benzyl alcohol, for example. Furthermore, if glutamic acid or glutamine is present as the N-terminal amino acid of a fragment, it can be converted to pyroglutamic acid or pyroglutamine, respectively, by a conventional ring-closing reaction; the original amino acid can then be restored by a conventional ring-opening reaction. Furthermore, the 20 naturally occurring amino acids, excluding alanine, phenylalanine, glycine, isoleucine, leucine, methionine, proline and valine, have functional groups in their side chains that need to be protected to prevent co-reaction; suitable protecting groups can be selected from those available in the prior art; for example, t-butyl protection is suitable for Asp, Glu, Ser, Thr and Tyr; t-butyloxycarbonyl (Boc) is suitable for Lys and Trp; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) is suitable for Arg; trityl is suitable for Asn, Cys, Gln and His; and 3-methyl-3-pentyl (Epe) is suitable for Asp.

[0023] Preferably, the condensation reaction is carried out on a resin (e.g., HMPB-Chem Matrix) to which a first fragment, in which groups not involved in the condensation reaction are protected, is bound; the resin / fragment coupling reaction conveniently utilizes the free carboxyl group to be protected, thereby allowing the coupling reaction to serve the dual purpose of coupling and protection; the second fragment, in which groups not involved in the condensation are protected, is then eluted onto the resin, where it undergoes a condensation reaction with the first fragment; thus, a resin-bound extended fragment is obtained; further segments can be added to the (appropriately deprotected) amino terminus of the resin-bound peptide by the same series of steps as above; the final added segment contains a cysteine ​​residue as the N-terminal amino acid, which marks the start of the subsequence of interest; the cysteine ​​residue is also present in a protected form (e.g., in cyclic thiazolidine form). The resin-bound intact subsequence is then cleaved and recovered: this is achieved by eluting the resin with a suitable solvent (e.g., 1.0% TFA in DCM) at the appropriate pH, which hydrolyzes the peptide / resin ester bond and releases and recovers the subsequence; the carboxyl group previously bound to the resin is now in its free form and undergoes thioesterification (e.g., conversion to a phenylthioester); and finally, any remaining protecting groups are removed.

[0024] Automated synthesis can follow similar steps as above, including the relevant protecting group addition / deprotection reactions, but is carried out on a suitable peptide synthesizer (e.g., Protein Technologies Inc., AZ, USA).

[0025] In step b, the subsequences obtained in step a are assembled, i.e., linearly ligated, in the order in which they appear in the target sequence. The subsequences to be ligated from step a, except for the first subsequence, contain their first cysteine ​​amino acid residue in its amino-protected form; the carboxyl-terminal group of the subsequence obtained from step a is in thioester form. Step b is carried out by native chemical ligation, followed by restoration of the cysteine ​​residue from its protected form to its original form, usually by reaction with an amine such as methoxamine. The native chemical ligation reaction proceeds in the presence of an aryl thiol catalyst, chemoselectively and regioselectively generating a thioester-linked intermediate. This intermediate rapidly and spontaneously rearranges by intramolecular S,N-acyl transfer to form a native amide bond at the ligation site (Dowson, PE, Muir, TW, Clark-Lewis, I., Kent, SBH Science, 1994, 266, 776).

[0026] Thus, the native chemical ligation in step b comprises the following substeps: (a) dissolving the subsequences to be ligated in a ligation buffer, (b) stirring overnight, (c) terminating the reaction, (d) adjusting the final pH to a range of 3 to 5, and (e) purifying the resulting mixture. The ligation buffer used in step (a) is preferably a phosphate buffer at pH 7, to which a mixture of mercaptophenylacetic acid, guanidine hydrochloride, tris(2-carboxyethyl)phosphine hydrochloride, and ascorbic acid or a salt thereof is added; in step (b), stirring is preferably carried out at room temperature; in step (c), terminating the reaction is preferably carried out by adding a mercaptoalkanesulfonate, optionally followed by the addition of hydroxylamine; in step (d), pH adjustment is preferably carried out by adding HCl; and in step (e), purification is preferably carried out by HPLC.

[0027] In step c, oxidative folding is performed by adding the peptide to be folded to a folding buffer, stirring at room temperature for 1 day, and then purifying the product. The folding buffer is preferably a Tris buffer supplemented with trehalose and guanidine hydrochloride, with a final pH in the range of 7-9. The resulting scFv is then dialyzed against a buffer, i.e., an aqueous buffer system; this step has been found to be important for ensuring the required level of binding specificity of the recovered scFv; in fact, simply resuspending the oxidative folding reaction mixture in an aqueous system (water or an aqueous buffer) or dialysis against water alone has not yielded a product with sufficient binding specificity.

[0028] Preferably, the buffer used maintains the pH of the dialysate in the range of 4.6 to 8.0, preferably 7.4±0.5, as obtained with, for example, a phosphate-based buffer; alternative buffers, for example, acetate, citrate, glycine, or histidine, can be used. In addition to the buffer, the dialysate can contain substances commonly used in antibody suspensions, such as: (i) salts (e.g., sodium chloride or potassium chloride), preferably at a concentration of 3 to 200 mM; (ii) chelating agents (e.g., EDTA), preferably at a concentration of 1 to 15 mM; (iii) amino acids (e.g., glycine, glutamic acid, methionine, proline), preferably at a concentration of 1 to 300 mM; (iv) sugars (e.g., maltose, mannitol, sorbitol, sucrose, trehalose), preferably at a concentration of 12 to 300 mM; or (iv) surfactants (e.g., polysorbates), preferably at a concentration of 0.1 to 1.6 wt%. After (or preferably before) dialysis in said buffer, the oxidatively folded product can be subjected to further dialysis in water, ie pure unbuffered water.

[0029] The techniques applied in dialysis (equipment, operating conditions, etc.) are per se well known to those skilled in the art (see Practical Skills in Biomolecular Sciences, 3rd ed. Essex: Pearson Education Limited. p. 379. ISBN 978-0-13-239115-3) and will be relied upon for the purposes of the present invention; details of the dialysis process are given in the experimental section.

[0030] A preferred, non-limiting application of this method is directed to the sequence of D2B (abbreviated herein as Ab1 (SEQ ID NO: 1)), the sequence of trastuzumab (abbreviated herein as Ab2 (SEQ ID NO: 2)), or the sequence of adalimumab (abbreviated herein as Ab3 (SEQ ID NO: 3)). Compared to the corresponding biologically derived scFv (see RREF), the product obtained according to the present invention has the advantage of being free of biological contaminants (viruses, endotoxins, etc.), i.e., it is obtained with a higher biological purity and does not require the complex step of biological purification; moreover, it offers good batch-to-batch reproducibility due to the ease of standardization of the chemical synthesis step compared to the biological step.

[0031] The scFv obtained by this method can be used directly for therapy for the same immunological purposes as the reference biological product, or can be used as starting material for the preparation of complete antibodies (containing conventionally linked variable and constant regions) by conventional methods.

[0032] The present invention is further illustrated by the following non-limiting examples, in which the methods presented are for illustrative purposes only and can be freely modified to produce these or different products within the scope of the present invention. [Example]

[0033] [experiment] We report a procedure for the synthesis of scFvs and evidence that their activity is comparable to that of corresponding scFvs of biological origin obtained in prokaryotic systems (reference biological scFvs abbreviated as RREF). The following scFv candidates were used as models: Ab1: SEQ ID NO: 1-D2B: A mouse MAb belonging to IgG1, with the variable domains VH belonging to the mouse mVH3 subgroup and the light chain (VK) belonging to the mouse VK12,13 subgroup. Ab2: SEQ ID NO:2 - Herceptin (transtuzumab): a recombinant humanized antibody belonging to the IgG1 human isotype. The variable domains are VH belonging to the human hVH3 subgroup and the light chain (VK) belonging to the human VK1 subgroup. Ab3: SEQ ID NO: 3 - Humira (adalimumab): The first approved antibody selected from a phage display library to be approved by the FDA is a recombinant human antibody belonging to the IgG1 human isotype. The variable domains are VH in the human hVH3 subgroup and the light chain (VK) in the human VK1 subgroup.

[0034] 1. Synthesis of scFv While this example details the synthesis of the scFv of SEQ ID NO:1 (D2B) for illustrative purposes, the same techniques and reagents were used to prepare other scFv sequences, particularly the scFvs of SEQ ID NO:2 (transtuzumab) and SEQ ID NO:3 (adalimumab), and equivalence to the corresponding biologically derived reference products was obtained and successfully confirmed in the experimental section. In particular, those skilled in the art will recognize that these techniques and reagents are not specific to the exemplified scFv, but are applicable to different scFvs, within the peptide size (number of amino acids) limitations referenced herein.

[0035] Abbreviation Boc, t-butoxycarbonyl; tCu, t-butyl; DCM, dichloromethane; DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-(3H)-one; DIC, N,N'-diisopropylcarbodiimide; DIPEA, diisopropylethylamine; DMF, N,N'-dimethylformamide; DMS, dimethyl sulfide; EDT, 1,2-ethanedithiol; Epe, 3-methyl-3-pentyl; ESI MS, electrospray ionization mass spectrometry; Et2O, diethyl ether; Fmoc, 9-fluorenylmethyloxycarbonyl; GnHCl, guanidine hydrochloride; HCTU, 1-[bis(dimethylamino)methylene]-5-chlorobenzotriazolium 3-oxide hexafluorophosphate; HMPB, 4-(4-hydroxymethyl-3-methoxyphenyl)butyryl; HPLC, high-performance liquid chromatography; MESNa, sodium 2-mercaptoethanesulfonate; MPAA, 4-mercaptophenylacetic acid; MSNT, 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole; NMP, N-methyl-2-pyrrolidone; Oxymapure pure), ethyl 2-cyano-2-(hydroxyimino)acetate; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; RP-HPLC, reversed-phase HPLC; SPPS, solid-phase peptide synthesis; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TFA, trifluoroacetic acid; Thz, thiazolidine-4-carboxylic acid; TIS, triisopropylsilane; Trt, trityl.

[0036] Analysis and purification Preparative HPLC was performed using a Shimadzu Prominence LC-20AD HPLC system (Shimadzu Corporation, Kyoto, Japan) using Osaka Soda Capcell Pak C. 18 UG120 5 μm (20 × 250 mm) column, Waters Xselect CSH C 18The analysis was performed using a 5 μm (20 × 250 mm) column and an Agilent ZORBAX 300SB-CN 7 μm (21.2 × 250 mm) column with a binary mixture of eluents A (0.1% TFA in HO) and B [CHCN / HO / TFA (v / v, 90 / 10 / 0.09)] at a flow rate of 10 mL / min. Separation was performed using a linear gradient as described and detection was at 220 nm. Analytical HPLC was performed on a Shimadzu Prominence LC-20AD HPLC system equipped with an Imtakt Intrada WP-RP 3 μm (4.6 × 250 mm) column using a binary mixture of eluents A and B. The analysis was performed using a linear gradient at a flow rate of 1.0 mL / min and detection was at 220 nm. ESI MS experiments were performed on a Synapt HDMS mass spectrometer (Waters, MA, USA).

[0037] Rationale for the chemical synthesis of scFv D2B scFv D2B consists of 254 amino acid residues and is bounded by two disulfide bonds (C 22 -C 96 and C 161 -C 226 The entire molecule was divided into five segments separated by cysteine ​​residues and assembled by native chemical ligation as shown in Figure 1. The resulting reduced molecule then underwent oxidative folding under denaturing conditions to form the complete scFv D2B molecule.

[0038] Loading of C-terminal Fmoc amino acid onto resin Fmoc amino acids were loaded onto HMPB-ChemMatrix resin and used for peptide elongation as follows: The C-terminal amino acid was introduced onto HMPB-ChemMatrix resin (0.45 mmol / g) using the standard loading procedure with Fmoc-AA / 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) / N-methylimidazole (5 / 5 / 5 equivalents) in CHCl for 3 h. Each peptide segment with a glycine at the C-terminus, i.e., 2, 6, 7, 11, 12, 17, and 18, was prepared in the form of a fully protected peptide with a free α-carboxy group and used for segment condensation on the solid support.

[0039] Automated Peptide Synthesis Fmoc solid-phase peptide synthesis (SPPS) was performed using a Prelude automated peptide synthesizer (Protein Technologies, AZ, USA). Peptide chains were elongated using a standard Fmoc protocol in N,N-dimethylformamide (DMF) by coupling with Fmoc amino acids (except Cys derivatives) / 1-[bis(dimethylamino)-methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) / DIPEA (5.3 / 5.0 / 10 equiv.) or Fmoc-Cys(Trt) / HCTU / 2,3,6-trimethylpyridine (5.3 / 5.0 / 20 equiv.) (15 min). To improve and suppress insufficient solvation of the peptide-resin complex during peptide elongation, the following sequence was incorporated with the respective pseudoproline dipeptide unit: Ile 52 -Ser 53 , Asp 73 -Thr 74 , Asp 90 -Thr 91 , Val 117 -Thr 118 , Gly 135 -Ser 136 , Val 157 -Thr 158 , Ser 197 -Ser 198 , Tyr209 -Ser 210 and Ala 222 -Thr 223 。N α Deprotection of the N-Fmoc group was carried out using 20% piperidine / DMF (5 min × 2). During peptide synthesis, all washes after coupling and deprotection were performed with DMF. The following side-chain protecting groups were used: t-butyl (tBu) for Asp, Glu, Ser, Thr, and Tyr; t-butoxycarbonyl (Boc) for Lys and Trp; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg; trityl (Trt) for Asn, Cys, Gln, and His; and 3-methyl-3-pentyl (Epe) for Asp 55、194、219 which is known to effectively reduce the aspartimide side reaction resulting from repeated base treatment with 20% piperidine / DMF.

[0040] Manual synthesis The protected segments 2, 6, 7, 11, 12, 16, 17, and 18 used in convergent solid-phase peptide synthesis (CSPPS) by segment condensation on a solid support were manually synthesized on a 4 mmol scale using Fmoc-Gly loaded 2-chlorotrityl resin. Peptide elongation was carried out using a coupling protocol with Fmoc-AA, Boc-Thz-OH, or L-pyroglutamic acid (<E) / oxyma pure / N,N'-diisopropylcarbodiimide (5 / 5 / 5 equivalents) in NMP at room temperature for 2 h. Deprotection was carried out using 20% piperidine / DMF (5 min × 2). Each protected segment was detached from the resin by treatment with 1% TFA in CH2Cl2 (20 mL, 5 min × 5), and the entire filtrate was collected in pyridine (5 mL). The solvent was removed under reduced pressure on a rotary evaporator. The residue was precipitated from 2% aqueous NaHCO3, the precipitate was washed with water, and then freeze-dried from water to obtain the crude product in the form of a fully protected peptide with a free α-carboxy group. Each crude product was used in the next reaction without further purification.

[0041] Figure 2 Compound 3 Protected peptide 1 on the resin, prepared on a peptide synthesizer at a 0.20 mmol scale, was treated with 20% piperidine in DMF (5 min × 2) to remove the N-terminal Fmoc group. After washing with DMF and CHCl, it was coupled with protected peptide segment 2 (0.36 g, 0.24 mmol) in NMP (8.0 mL) in the presence of 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT, 0.14 g, 0.48 mmol) and DIPEA (0.12 mL, 0.72 mmol) at room temperature overnight. Protected segment 3 was cleaved from the resin by treatment with 1% TFA in CHCl (10 mL, 5 min × 14). The entire filtrate was recovered in pyridine (5.0 mL), and the solvent was removed under reduced pressure on a rotary evaporator. The residue was precipitated from water, the precipitate was washed with water, and then lyophilized from water to give crude protected peptide 3 (0.41 g).

[0042] compound 4 To a solution of crude protected peptide 3 (0.41 g, 0.13 mmol) and thiophenol (0.41 mL, 3.8 mmol) in DMF (10 mL), PyBOP (0.33 g, 0.64 mmol) and DIPEA (0.11 mL, 0.64 mmol) were added sequentially at −15° C., and the mixture was stirred overnight. The reaction was terminated by the addition of TFA (1.0 mL) at −15° C., and the mixture was then allowed to warm to room temperature. The solvent was removed under reduced pressure on a rotary evaporator at 40° C. The resulting residue was treated with a TFA / HO / triisopropylsilane (TIS) / dimethyl sulfide (DMS) (v / v, 95:5:3:3) cleavage cocktail (8.0 mL) at room temperature. After 4 hours, diethyl ether (EtO) was added to the reaction mixture to obtain a precipitate, which was washed with EtO and collected by centrifugation (×10,000 rpm, 10 min) twice before drying. Purification was performed using preparative HPLC [column, Osaka Soda Capcell Pak C 18 The mixture was then purified using a 30-minute gradient of 35-45% B in a UG120 (20 x 250 mm); flow rate, 10 mL / min; temperature, 60 °C) to separate peptide thioester 4 (segment 1). <E 1 -Y 95) (55 mg). ESI MS: m / z calculated C 394 H 575 N 103 O 118 S3(2271.66):[M+2H] 2+ 1136.83, [M+3H] 3+ 758.22; measurements 1136.61, 758.07.

[0043] compound 8 Resin-bound protected peptide 5 (0.20 mmol), obtained after removal of the N-terminal Fmoc group, was coupled with protected segment 6 (0.38 g, 0.24 mmol) in the presence of DEPBT (0.14 g, 0.48 mmol) and DIPEA (0.12 mL, 0.72 mmol) in NMP (8.0 mL) overnight at room temperature. The resulting peptide resin was treated with 20% piperidine in DMF to remove the N-terminal Fmoc group, followed by double coupling with protected segment 7 (0.57 g, 0.24 mmol) in the presence of DEPBT (0.14 g, 0.48 mmol) and DIPEA (0.12 mL, 0.72 mmol) in NMP. The resulting D2B(35-95)-protected peptide resin was transferred to the automated synthesizer, and peptide elongation resumed. After completion of chain assembly of the D2B(22-95) peptide, the protected segment 8 was cleaved from the resin by treatment with 1% TFA in CHCl (10 mL, 5 min × 20). The entire filtrate was taken up in pyridine (10 mL), and the solvent was removed under reduced pressure on a rotary evaporator. The residue was precipitated from 2% aqueous NaHCO, and the precipitate was washed with water and then lyophilized from water to give crude protected peptide 8 (1.8 g).

[0044] compound 9 To a solution of crude protected peptide 8 (0.89 g, 61 μmol) and thiophenol (0.18 mL, 1.8 mmol) in DMF (10 mL), PyBOP (0.16 g, 0.31 mmol) and DIPEA (52 μL, 0.31 mmol) were added sequentially at −15°C, and the mixture was stirred overnight. The reaction was terminated by the addition of TFA (0.50 mL) at −15°C, and the mixture was then allowed to warm to room temperature. The solvent was removed under reduced pressure on a rotary evaporator at 40°C. The residue was treated with a TFA / HO / TIS / DMS (v / v, 95:5:3:3) cleavage cocktail (16 mL) at room temperature. After 4 h, diethyl ether (EtO) was added to the reaction mixture to obtain a precipitate, which was washed with EtO and collected by centrifugation (×10,000 rpm, 10 min) twice before drying. Purification was performed using preparative HPLC [column: Waters Xselect CSH C 18 5 μm (20 × 250 mm); flow rate, 10 mL / min; temperature, 60 °C) with a gradient of 5–25% B (0–1 min) / 25–50% B (1–20 min) to obtain peptide thioester 9 (segment 2 C 22 -Y 95 ) (41 mg). ESI MS: m / z calculated C 394 H 575 N 103 O 118 S3(8738.72):[M+4H] 4+ 2185.48, [M+5H] 5+ 1748.74, [M+6H] 6+ 1457.45; measurements 2185.48, 1748.55, 1457.32.

[0045] Figure 3 compound 13 Resin-bound protected peptide 10 (0.20 mmol), obtained after removal of the N-terminal Fmoc group, was coupled with protected segment 11 (0.46 g, 0.24 mmol) in the presence of DEPBT (0.14 g, 0.48 mmol) and DIPEA (0.12 mL, 0.72 mmol) in NMP (8.0 mL) overnight at room temperature. The resulting peptide resin was treated with 20% piperidine in DMF to remove the N-terminal Fmoc group, followed by double coupling with protected segment 12 (0.39 g, 0.24 mmol) in the presence of DEPBT (0.14 g, 0.48 mmol) and DIPEA (0.12 mL, 0.72 mmol) in NMP. Protected peptide segment 3 [D2B(96-160)] was cleaved from the resin by treatment with 1% TFA in CHCl (10 mL, 5 min × 20). The entire filtrate was taken up in pyridine (5.0 mL) and the solvent was removed under reduced pressure on a rotary evaporator. The residue was precipitated from 2% aqueous NaHCO3, the precipitate was washed with water, and then lyophilized from water to give crude protected peptide 13 (0.30 g).

[0046] compound 14 To a solution of crude protected peptide 13 (0.47 g, 55 μmol) and thiophenol (0.18 mL, 1.7 mmol) in DMF (15 mL), PyBOP (0.14 g, 0.28 mmol) and DIPEA (48 μL, 0.28 mmol) were added sequentially at −15°C, and the reaction mixture was stirred overnight. The reaction was terminated by the addition of TFA (0.10 mL) at −15°C, and the mixture was then allowed to warm to room temperature. The solvent was removed under reduced pressure on a rotary evaporator at 40°C. The residue was treated with a TFA / HO / TIS / DMS (v / v, 95:5:3:3) cleavage cocktail (16 mL) at room temperature. After 4 h, EtO was added to the reaction mixture to obtain a precipitate, which was washed with EtO and collected by centrifugation (×10,000 rpm, 10 min) twice before drying. Purification was performed by preparative HPLC [column, Agilent ZORBAX 300SB-CN 7 μm (21.2 × 250 mm); flow rate, 10 mL / min; temperature, 80 °C] using a gradient of 5–30% B (0–1 min) / 30–47.4% B (1–20 min) to obtain peptide thioester 14 (segment 3 C96 -T 160 ) (46 mg). ESI MS: m / z calculated C 272 H 409 N 71 O 96 S3(6305.85):[M+3H] 3+ 2102.95, [M+4H] 4+ 1577.46, [M+5H] 5+ 1262.17; measurements 2102.89, 1577.17, 1262.14.

[0047] compound 19 Protected segment 4 (C 161 -Y 225 The synthesis of resin-bound protected peptide 15 (0.40 mmol) was carried out by sequential assembly of protected segment 16 (0.79 g, 0.48 mmol) and protected segment 17 (1.3 g, 0.48 mmol) using the DEPBT (0.29 g, 0.96 mmol) / DIPEA (0.25 mL, 1.4 mmol) method in NMP (8.0 mL) at room temperature overnight. Resin-bound protected D2B (170-225), obtained after removal of the N-terminal Fmoc group, was double-coupled with segment 18 (0.67 g, 0.48 mmol) using the same procedure described above to give resin-bound segment 4 (C 161 -Y 225 ) was obtained. The protected peptide segment 19 was cleaved from the resin by treatment with 1% TFA in CHCl (20 mL, 5 min × 15). The entire filtrate was taken up in pyridine (10 mL), and the solvent was removed under reduced pressure on a rotary evaporator. The residue was precipitated from 2% aqueous NaHCO, and the precipitate was washed with water and then lyophilized from water to give crude protected peptide 19 (2.7 g).

[0048] compound 20 To a solution of crude protected peptide 19 (2.7 g, 0.24 mmol) and thiophenol (0.76 mL, 7.1 mmol) in DMF (15 mL), PyBOP (0.62 g, 1.2 mmol) and DIPEA (0.21 mL, 1.2 mmol) were added sequentially at −15°C, and the mixture was stirred overnight. The reaction was terminated by the addition of TFA (1.0 mL) at −15°C, and the mixture was then allowed to warm to room temperature. The solvent was removed under reduced pressure on a rotary evaporator at 40°C. The residue was treated with a TFA / HO / TIS / DMS (v / v, 95:5:3:3) cleavage cocktail (20 mL) at room temperature. After 4 h, EtO was added to the reaction mixture to obtain a precipitate, which was washed with EtO and collected by centrifugation (×10,000 rpm, 10 min) twice before drying. Purification was performed by preparative HPLC [column: Waters Xselect CSH C18 5 μm (20 × 250 mm); flow rate: 10 mL / min; temperature: 60 °C] using a gradient of 5–25% B (0–1 min) / 25–39.5% B (1–11 min) to obtain peptide thioester 20 segment 4 (C 161 -Y 225 ) (99 mg). ESI MS: m / z calculated C 318 H 479 N 87 O 98 S3(7185.02):[M+5H] 5+ 1438.00, [M+6H] 6+ 1198.50, [M+7H] 7+ 1027.43; measurements 1438.02, 1198.53, 1027.48.

[0049] compound 22 Resin-bound protected peptide 21 (0.40 mmol) was treated with a TFA / H2O / TIS / DMS:1,2-ethanedithiol (EDT) (v / v, 95:5:3:3:2) cleavage cocktail (20 mL) at room temperature for 3 h. Et2O was added to the reaction mixture, resulting in a precipitate, which was washed twice with Et2O and dried. Purification was performed by preparative HPLC [column: Waters Xselect CSH C18 5 μm (20 × 250 mm); flow rate: 10 mL / min; 60 °C] using a gradient of 5–15% B (0–1 min) / 15–22% B (1–20 min) to obtain compound 22 segment 5 (C 226 -H 254 ), (86 mg) was obtained. ESI MS: m / z calculated C 148 H 221 N 51 O 38 S(3354.78)[M+3H] 3+ 1119.26, [M+4H] 4+ 839.70, [M+5H] 5+ 671.96; measurements 1119.26, 839.70, 671.75.

[0050] Figure 4 compound 23 Segment 5 (compound 22, 58 mg, 17 μmol) and peptide thioester compound 20 (99 mg, 14 μmol) were dissolved in freshly prepared ligation buffer [0.2 M NaHPO, pH 7.8, 13 mL containing 50 mM 4-mercaptophenylacetic acid (MPAA) and 8 M guanidine hydrochloride (GnHCl)]. Then, 0.5 M tris(2-carboxyethyl)phosphine hydrochloride (TCEP) / HO (pH 7, 0.70 mL) and 0.5 M L-ascorbic acid sodium salt / HO (0.7 mL) were added to the peptide solution, sequentially. The reaction mixture was stirred overnight at room temperature, then terminated by the addition of sodium 2-mercaptoethanesulfonate (MESNa, 0.46 g, 2.8 mmol), 0.5 M L-ascorbic acid sodium salt / HO (0.70 mL), and 0.5 M TCEP / HO (pH 7, 0.70 mL). O-Methylhydroxylamine hydrochloride (0.13 g, 1.5 mmol) was added to the ligation mixture, and the mixture was then adjusted to pH 3.8 with 2 M HCl. After stirring overnight at room temperature, the mixture was purified by preparative HPLC (Waters Xselect CSH C18 5 μm column, 20 × 250 mm; flow rate, 10 mL / min; temperature, 60 °C) using a gradient of 5–20% B (0–1 min) / 20–34.7% B (1–15 min) to give compound 23 (53 mg). ESI MS:m / z Calculated value C 459 H 694 N 138 O 136 S3(10417.16)[M+10H] 10+ 1042.76, [M+11H] 11+ 948.06, [M+12H] 12+ 869.13;Measurements 1042.68, 948.09, 869.14.

[0051] compound 24 Segment (4-5) compound 23 (53 mg, 5.1 μmol) and segment 3-thioester compound 14 (46 mg, 7.3 μmol) were dissolved in freshly prepared ligation buffer [0.2 M NaHPO, pH 7.8, 4.5 mL containing 50 mM MPAA and 8 M GnHCl]. Then, 0.5 M TCEP / HO (pH 7, 0.25 mL) and 0.5 M L-ascorbic acid sodium salt / HO (0.25 mL) were added sequentially to the peptide solution. After stirring the reaction mixture at room temperature for 1 day, the reaction was terminated by the addition of MESNa (0.16 g, 1.0 mmol), 0.5 M L-ascorbic acid sodium salt / HO (0.25 mL), and 0.5 M TCEP / HO (pH 7, 0.25 mL). O-Methylhydroxylamine hydrochloride (0.13 g, 1.5 mmol) was added to the ligation mixture, and the mixture was then adjusted to pH 3.8 with 2 M HCl. After stirring the reaction mixture for 5 h, the mixture was purified by preparative HPLC [column: Imtakt Intrada WP-RP 3 μm (4.6 × 250 mm); flow rate: 1.0 mL / min; temperature: 80 °C] using a gradient of 5-20% B (0-1 min) / 20-29.5% B (1-9 min) to give segment (3 + 4 + 5), compound 24 (35 mg). ESI MS: m / z calculated C 724 H 1097 N 209 O 232 S5(16601.27)[M+7H] 7+ 2372.61, [M+8H] 8+ 2076.16, [M+9H] 9+ 1845.59; measurements 2372.35, 2076.18, 1845.46.

[0052] compound 25 To a solution of segment (3-4-5) compound 24 (35 mg, 2.1 μmol) and segment 2-thioester compound 9 (41 mg, 4.7 μmol) in freshly prepared ligation buffer (0.2 M NaHPO, pH 7.9, 1.8 mL containing 50 mM MPAA and 8 M GnHCl), 0.5 M TCEP / HO (pH 7, 0.10 mL) and 0.5 M sodium L-ascorbate / HO (0.10 mL) were added sequentially. After stirring the reaction mixture overnight at room temperature, the reaction was terminated by the addition of MESNa (66 mg, 3.9 mmol), 0.5 M sodium L-ascorbate / HO (0.1 mL), and 0.5 M TCEP / HO (pH 7, 0.1 mL). O-Methylhydroxylamine hydrochloride (52 mg, 0.6 mmol) was added to the ligation mixture, which was then adjusted to pH 3.8 with 2 M HCl. The mixture was stirred overnight at room temperature and purified by HPLC (column: Imtakt Intrada WP-RP 3 μm (4.6 × 250 mm); flow rate: 1.0 mL / min; 80 °C) using a gradient of 5-20% B (0-1 min) / 20-27.1% B (1-10 min) to give segment (2-3-4-5), compound 25 (7.0 mg). ESI MS: m / z calculated C 1111 H 1666 N 312 O 350 S7(25217.80)[M+27H] 27+ 934.99, [M+28H] 28+ 901.64, [M+29H] 29+ 870.58; measurements 934.93, 901.46, 870.61.

[0053] compound 26 To a solution of segment (2-3-4-5) compound 25 (7.0 mg, 0.30 μmol) and segment 1-thioester 4 (2.0 mg, 0.60 μmol) in freshly prepared ligation buffer (0.2 M NaHPO, pH 7.9, 0.3 mL containing 50 mM MPAA and 8 M GnHCl), 0.5 M TCEP / HO (pH 7, 15 μL) and 0.5 M L-ascorbic acid sodium salt / HO (15 μL) were added sequentially. After stirring the reaction mixture at room temperature for 1 day, the reaction was terminated by the addition of MESNa (2.0 mg, 12 μmol), 0.5 M L-ascorbic acid sodium salt / HO (15 μL), and 0.5 M TCEP / HO (pH 7, 15 μL). The reaction mixture was purified by HPLC [column: Imtakt Intrada WP-RP 3 μm (4.6 × 250 mm); flow rate: 1.0 mL / min; temperature: 80 °C] using a gradient of 5–20% B (0–1 min) / 20–27.9% B (1–10 min) to give segment (1-2-3-4-5), compound 26 (4.0 mg). Figure 5A(a) ESI MS: m / z calculated C 1207 H 1827 N 337 O 381 S7(27379.29)[M+15H] 15+ 1826.29, [M+16H] 16+ 1712.21, [M+17H] 17+ 1611.55; measurements 1826.26, 1712.47, 1611.46.

[0054] compound 27 The (1-2-3-4-5) segment, compound 26 (4.0 mg, 0.15 μmol) was dissolved in folding buffer (4.0 mL of 0.1 M Tris-HCl, pH 8.6, containing 0.2 M trehalose and 8 M GnHCl). The solution was stirred at room temperature for 1 day. The folding reaction mixture was then purified by RP-HPLC [column: Imtakt Intrada WP-RP 3 μm (4.6 × 250 mm); flow rate: 1.0 mL / min; temperature: 80 °C] using a gradient of 5-20% B (0-1 min) / 20-26.3% B (1-9 min) to obtain the folded scFv D2B compound 27 (1.0 mg) (Figure 5A(c)). ESI MS: m / z calculated C 1207 H 1823 N 337 O 381 S7(27375.26)[M+10H] 10+ 2738.53, [M+11H] 11+ 2489.66, [M+12H] 12+ 2282.27; measurements 2738.43, 2489.34, 2282.19. Figure 5B

[0055] Protein concentration measurement The folded protein ScFv full-length compound 27 (1.0 mg) was dissolved in water (1.0 mL), and the protein concentration of the solution (0.047 mM, 1.3 mg / mL) was determined by measuring its absorbance at 280 nm using a NanoDrop 2000c spectrophotometer, with a calculated extinction coefficient of 51590 M. -1 cm -1 and molecular weight 27375.26. The solution was divided into five aliquots (200 μL each), lyophilized, and four aliquots were used for assay, analysis, and dialysis as described below.

[0056] dialysis Two-step dialysis was performed (Practical Skills in Biomolecular Sciences, 3rd ed. Essex: Pearson Education Limited. p. 379. ISBN 978-0-13-239115-3). We used SpectraPro2 dialysis membranes (MCWO 12-14 KDa) manufactured by Spectrumlabs. The sample was resuspended in water and extensively dialyzed against 100 volumes of water. A second dialysis step was performed in a buffer solution, the optimal buffer being 10 mM sodium hydrogen phosphate, 150 mM sodium chloride, and 3 mM EDTA, adjusted to pH 7.4. Dialysis in a buffer solution was found to be essential for the correct folding of the scFv. Indeed, as shown by the data in Figure 6 (ScFv SEQ ID NO:3), binding of scFv when resuspended in water or buffer or dialyzed against water alone exhibited nonspecific binding and was visible on BSA (an unrelated protein). Comparable results were obtained in this study using scFv Ab1 and Ab2.

[0057] The synthetically produced scFvs have the expected molecular weight of the biological references, as shown by SDS-PAGE (Figure 7: A) scFv Ab1; B) scFv Ab2, and C) scFv Ab3). The small weight difference between the two is due to the fact that the scFv RFEE contains two tags (Myc and His), while only the His tag is present in the synthetic product.

[0058] 2. Size Exclusion Chromatography Test Size exclusion chromatography (SEC) is a well-established technique for separating proteins and polymers by molecular weight, making it ideal for measuring aggregates in the quality control of biopharmaceuticals. SEC profiles (Figure 8 A. scFv Ab1; B. scFv Ab2; C. scFv Ab3) demonstrated the absence of aggregates in the synthetically produced scFvs, as well as the absence of dimers, which are present in trace amounts in the scFvs RREF. Notably, only the synthetically produced scFvs achieved nearly 100% homogeneity.

[0059] Measurements were performed on a Series 200 HPLC system (PerkinElmer) consisting of a solvent pump, autosampler, and UV detector. Data were analyzed using TC Nav software. A Superdex 75 5 / 15 GL (15 × 5 mm) column (Cytiva) was used. The salt buffer consisted of 10 mM sodium diphosphate and 150 mM sodium chloride adjusted to pH 7.4. The injection volume was 10 μl, the flow rate was 0.3 ml / min, and the detection wavelength was 280 nm.

[0060] 3. Functional analysis of scFv molecules Next, the antigen binding of the synthesized scFv fragments was analyzed by FACS\ELISA and surface plasmon resonance using a Biocore T200.

[0061] FACS (Fluorescence Activated Cell Sorting) Test Chemically synthesized scFv or prokaryotically produced scFv RREF (ScFv Ab1 SEQ ID NO:1; ScFv Ab2 SEQ ID NO:2) (10 μg / mL) was incubated with tumor cells (5×10 5 ) and the mixture was incubated on ice for 30 minutes. Binding was detected with an anti-His Tag antibody (produced in mouse) for 30 minutes on ice. Total antibody binding was revealed with the antibody anti-mouse IgG-Alexa488 for 30 minutes on ice. Five thousand cells for each sample were analyzed on a FACS Canto using DIVA software. The results are presented in Figure 9 (A scFv Ab1 and B scFv Ab2). All tested antibodies showed specificity for antigen-positive cells (Figures 9A1 and 9B1) and for antigen-negative cells (Figures 9A2 and 9B2).

[0062] ELISA (enzyme-linked immunosorbent assay) test For the test, 5 x 10 4Tumor cells were plated in 96-well plates and fixed with glutaraldehyde (0.1% in PBS, 5 min) and glycine (0.1 M in PBS, 10 min).

[0063] Plates were saturated with 1% BSA in PBS for 2 hours.

[0064] All scFv antibodies were tested at two-fold dilutions ranging from 2.5 to 0.0025 μg / ml. Plates were incubated for 1 hour at room temperature. Binding was detected with antibody anti-His Tag (produced in mouse) for 1 hour at room temperature, followed by HPR-conjugated antibody anti-mouse IgG. Color was developed with 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) and measured at 450 nm after blocking with 50 μL of 1 M sulfuric acid.

[0065] The results are summarized in Figure 10A for scFv Ab1, Figure 10B for scFv Ab2, and Figure 10C for scFv Ab3, showing the binding of scFvs produced in different systems at different concentrations on positive cells (PC3-PIP for scFv Ab1 and MDAMB361 for scFv Ab2, respectively) or positive antigen (TNFα for scFv Ab3), as well as on negative cells (PC3 for scFv Ab1 and MDAMB468 for scFv Ab2, respectively) or unrelated protein (BSA for scFv Ab3). These graphs demonstrate that the curves for scFvs obtained by chemical synthesis exhibit, within experimental deviation limits, virtually identical binding behavior to the product scFv RREF; the same conclusions can be drawn for the series of curves associated with negative cells or unrelated proteins.

[0066] Surface plasmon resonance test using Biacore T200 Biacore is a surface plasmon resonance-based device aimed at characterizing biomolecular interactions (in this case, antibodies and antigens) (Jason-Moller L et al Curr Protoc Protein Sci. 2006 Sep; Chapter 19: Unit 19.13).

[0067] The synthesized scFv and scFv RREF were tested using standard kinetic methods to measure the affinity of scFvs generated in different systems.

[0068] In the standard kinetics, the soluble antigens purified on the sensor chip CM5 (hPSMA for scFv Ab1; hHER2-ECD for scFv Ab2; hTNFα for scFv Ab3, respectively) were immobilized on the CM5 sensor chip for the experiment (Figure 11).

[0069] The results are reported in Tables I, II, and III together with the chi-square value (Chi 2 ) and p-value indicating the statistical fitness of the experiment (Chi 2 <RU maximum 10%, and p-value < 16).

[0070]

Table 1

[0071]

Table 2

[0072]

Table 3

[0073] The sensorgram curves of all the tested scFvs, i.e., those biologically synthesized versus chemically synthesized according to the present invention, consistently overlap, indicating behavioral equivalence.

[0074] In conclusion, the experimental results presented in this experimental section show that the complete chemical synthesis of scFv is achievable and reliable, resulting in the same primary structure and substantially the same secondary structure as the reference biological product, and maintaining substantially the same biological activity. Thus, the object of the present invention is achieved.

[0075] Sequence Listing SEQ ID NO: 1 (scFv D2B) 254aa TIFF2026505450000004.tif32147 SEQ ID NO: 2 (scFv Ab2; scFv transtuzumab) 252aa TIFF2026505450000005.tif29147 SEQ ID NO: 3 (scFv Ab3; scFv adalimumab) 252aa TIFF2026505450000006.tif30147

Claims

1. 1. A method for synthesizing a single chain variable antibody fragment (scFv) in which the amino acid sequence (target sequence) contains n cysteine ​​residues separated by subsequences of m amino acids, the process comprising the steps of: a. individually synthesizing all of said subsequences, including their preceding cysteine ​​residues, if present; b. Sequentially assembling the subsequences obtained in step a according to the order in which they appear in the target amino acid sequence; c. carrying out oxidative folding of the assembled structure obtained in step b, and dialyzing the resulting scFv in a buffer; A method comprising:

2. 2. The method of claim 1, wherein n is 1 to 10, preferably 2 to 6, and m is 0 to 100, preferably 5 to 80.

3. 3. The method of claim 1 or 2, wherein the target sequence comprises up to 350 amino acids, more preferably 100 to 300 amino acids, even more preferably 200 to 300 amino acids.

4. The method according to any one of claims 1 to 3, wherein step a) can be carried out by manual or automated peptide synthesis.

5. 5. The method of claim 4, wherein the manual peptide synthesis comprises condensation on a resin and the automated peptide synthesis comprises peptide elongation carried out in a peptide synthesizer.

6. 6. The method according to any one of claims 1 to 5, wherein in step a) (a) the preceding cysteine ​​residue is present as its N-protected precursor, and (b) the final carboxy group of the resulting subsequence is further converted into a thioester form.

7. 7. The method of claim 6, wherein the thioester is a phenyl thioester and the N-protected cysteine ​​precursor is an N-protected cyclic cysteine.

8. 8. The method of any one of claims 1 to 7, wherein in step b, the assembly is carried out by native chemical ligation, followed by restoration of the original cysteine ​​residue, if present as its N-protected precursor.

9. 9. The method of any one of claims 1 to 8, wherein the native chemical ligation comprises: (a) dissolving the sequences to be ligated in a ligation buffer; (b) stirring overnight; (c) stopping the reaction; (d) adjusting the pH of the reaction mixture to a range of 3 to 5; and (e) purifying the resulting mixture.

10. 10. The method of claim 9, wherein in (a), the ligation buffer is a phosphate buffer at pH 7 to which a mixture of mercaptophenylacetic acid, guanidine hydrochloride, tris(2-carboxyethyl)phosphine hydrochloride, and ascorbic acid or a salt thereof is added; in (c), quenching is carried out by the addition of a mercaptoalkanesulfonate, optionally followed by the addition of hydroxylamine; and step (d) is carried out by the addition of HCl.

11. 11. The method according to any one of claims 1 to 10, wherein the oxidative folding is carried out by adding the peptide to be folded to a folding buffer, followed by stirring at room temperature for 1 day.

12. 12. The method of claim 11, wherein the folding buffer is a Tris buffer supplemented with trehalose and guanidine hydrochloride and having a final pH in the range of 7 to 9.

13. The method according to any one of claims 1 to 12, wherein in step c, the dialysis is carried out in a buffer solution having a pH comprised between 4.6 and 8.0, preferably a pH of 7.4±0.

5.

14. 14. The method of claim 13, wherein the buffer is a phosphate buffer.

15. The method of any one of claims 1 to 14, wherein step c comprises a further dialysis in water.

16. The method according to any one of claims 1 to 15, wherein the final product obtained from step c is purified by HPLC.

17. The method of any one of claims 1 to 16, wherein the target sequence is SEQ ID NO: 1 (scFV D2B), SEQ ID NO: 2 (scFV transtuzumab) or SEQ ID NO: 3 (scFV adalimumab).

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