Hyperstable antibody fragments with novel disulfide bonds

Abstract

The present invention relates to a library of recombinant particles that displays a plurality of single chain variable fragments (scFv) against various antigens (e.g., HER2). The single chain variable fragments shown are characterized by improved stability resulting from disulfide bonds between non-natural domains at new locations. The invention also relates to methods of constructing such libraries, to the use of the libraries to obtain stabilized scFvs with desired antigen binding properties and to such scFvs.

Description

Technical Field

[0001] The present invention relates to recombinant antibody fragments, more specifically, to single-chain variable fragments with improved stability due to non-native inter-domain disulfide bonds at novel locations. The present invention also relates to recombinant expression libraries of such antibody fragments and methods for constructing such libraries and their use for selecting stable antibody fragments with desired antigen binding properties. Background of the Invention

[0003] Single-chain variable fragments (scFv) have several advantages over full-length antibodies. They can be expressed in microorganisms in high yields and in a cost-effective manner. They are small and therefore can reach cryptic (hidden) epitopes. In addition, due to their small size, they exhibit improved pharmacokinetic properties - scFv fragments can penetrate tissues (such as tumors) faster and more evenly, and are cleared faster, which may be beneficial for radiotherapy and in vivo diagnostic applications. The small size of scFv allows it to be screened and selected by in vitro display methods such as phage display, thereby avoiding animal immunization. In addition, scFv can be used to produce various types of improved antibody formats by genetically conjugating it to other entities (such as antibody fragments or complete antibodies).

[0004] Unfortunately, conventional scFv fragments are prone to variable dynamic oligomerization and / or domain exchange phenomena, in which two or more scFvs produce intermolecular pairing. Oligomerization has many effects on the functionality and stability of scFv. For example, it increases the aggregation tendency of scFv, which brings various handling and storage problems. It also complicates the medical use of scFv as well as its in vitro applications and diagnostic test development, because the affinity and activity of oligomeric scFv molecules for antigens are changed due to avidity effects. Oligomers can also cross-link with targets in vivo, resulting in altered pharmacodynamic effects.

[0005] High stability is one of the most important requirements for antibodies and antibody fragments in biopharmaceutical applications. Unfortunately, many scFvs are less stable than their full-length counterparts: many scFvs are prone to V H -V L Exposure of hydrophobic residues at the interface leads to denaturation and aggregation. In general, the thermal stability of scFv is also lower than that of the corresponding intact antibody or Fab fragment.

[0006] Stabilization of scFv can be achieved (at least to some extent) by engineering disulfide bonds for stabilization into the scFv molecule. H and V L The scFv is stabilized by introducing disulfide bonds at various positions in the scFv. However, the stability obtained by this method is variable, and thus there is still a need for methods of providing scFvs with improved stability. Summary of the invention

[0007] The present invention provides a disulfide bond stabilized single chain variable fragment (ds-scFv), which comprises a heavy chain variable domain (V H ), and a light chain variable domain (V L ), where V H and V L connected by a peptide linker in any orientation, and wherein CDR-L1 and CDR-H3 are linked to each other by an interdomain disulfide bond, and wherein V H or V L has been engineered to lack naturally conserved intradomain disulfide bonds. In this context, native intradomain disulfide bonds specifically refer to those in V according to the Kabat numbering scheme. L The disulfide bond formed between cysteine ​​residues L23 and L88 in V H Preferably, the variable domain following the peptide linker lacks disulfide bonds within the native domain.

[0008] In some embodiments, interdomain disulfide bonds are artificially introduced.

[0009] In some further embodiments, the artificially introduced disulfide bond is formed between a cysteine ​​residue in CDR-H3 at position -4 from the conserved tryptophan H103 in FR-H4 and a cysteine ​​residue at position L34 in CDR-L1 according to the Kabat numbering scheme.

[0010] In some embodiments, the peptide linker is at least 12 amino acids in length.

[0011] In some embodiments, V H and / or V L The CDR sequences of are obtained from natural diversity, except for a cysteine ​​at residue -4 from the conserved tryptophan H103 of FR-H4 and a cysteine ​​at residue L34 of CDR-L1 according to the Kabat numbering scheme. In some other embodiments, V H and / or V L The CDR sequences of V H and / or V LOne or more CDR sequences of the sequences contain one or more randomized amino acids. In both cases, a cysteine ​​residue in CDR-H3 at position -4 from the conserved tryptophan H103 in FR-H4 and a cysteine ​​residue at position L34 in CDR-L1 must be present according to the Kabat numbering scheme.

[0012] In some embodiments, the ds-scFv comprises a humanized framework or a framework derived from a human antibody. In some more specific embodiments, the V H The structural domain comprises a framework containing FR-H1 of SEQ ID NO: 1, FR-H2 of SEQ ID NO: 2, FR-H3 of SEQ ID NO: 3, and FR-H4 of SEQ ID NO: 4 in order, and V L The domain comprises a framework containing, in order, FR-L1 of SEQ ID NO:5, FR-L2 of SEQ ID NO:6, FR-L3 of SEQ ID NO:7, and FR-L4 of SEQ ID NO:8. In some further embodiments, the framework is a functionally equivalent conservative sequence variant of the sequences. According to certain aspects of the invention, i.e., a ds-scFv lacking another disulfide bond within a native domain, the framework has been engineered so that it does not contain two native cysteines at positions corresponding to residue 22 of SEQ ID NO:1 and residue 30 of SEQ ID NO:3, or does not contain two native cysteines at positions corresponding to residue 23 of SEQ ID NO:5 and residue 32 of SEQ ID NO:7.

[0013] In some embodiments, the V of the ds-scFv is H The domain comprises the amino acid sequence shown in SEQ ID NO:48, and V L The domain comprises the amino acid sequence set forth in SEQ ID NO:49, or a functionally equivalent conservative sequence variant of said sequence. According to some aspects of the invention, i.e., a ds-scFv lacking another disulfide bond within a native domain, said SEQ ID NO:48 has been engineered to not contain two native cysteines at positions corresponding to residues 22 and 98, thereby preventing H The native intradomain disulfide bonds are formed within the V domain, or the SEQ ID NO: 49 has been engineered to not contain the two native cysteines at positions corresponding to residues 23 and 88, thereby preventing L Formation of native intradomain disulfide bonds within.

[0014] In some further embodiments of the above, the ds-scFv is an anti-HER2 ds-scFv. Preferably, the anti-HER2 ds-scFv comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 12, a CDR-H2 having an amino acid sequence of SEQ ID NO: 13, a CDR-H3 having an amino acid sequence of SEQ ID NO: 14, a CDR-L1 having an amino acid sequence of SEQ ID NO: 15, a CDR-L2 having an amino acid sequence of SEQ ID NO: 16, and a CDR-L3 having an amino acid sequence of SEQ ID NO: 17. In some more specific embodiments, the anti-HER2 ds-scFv comprises an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0015] The present invention also provides a molecular entity comprising one or more ds-scFv units of various embodiments of the present invention, and the use of ds-scFv of any embodiment of the present invention for constructing such molecular entities. In some embodiments, the molecular entity is a bispecific antibody, a double antibody (diabody), a multispecific antibody, a CAR-T cell, a bispecific T cell engager (BiTE), or a construct comprising a ds-scFv of the present invention fused with a fragment such as an antibody (Fc) part or other affinity agents based on protein scaffolds (e.g., Design Ankyrin Protein (DARPin), nanobody or affibody). Molecular entities can also be, for example, pharmaceutically active agents, drugs, radioisotopes, enzymes or chelating agents.

[0016] In yet another aspect, the present invention provides a nucleic acid molecule encoding a ds-scFv of any embodiment of the present invention.

[0017] In a further aspect, the invention provides particles displaying on their surface a ds-scFv of any embodiment of the invention, and libraries of such particles, the libraries displaying a plurality of different ds-scFvs of the invention. Preferably, the particle is a phage particle, a yeast cell, a bacterial cell, a mammalian cell or a ribosome.

[0018] In addition, the present invention provides a method for preparing a library of particles, wherein the library displays a plurality of different ds-scFvs according to various embodiments of the present invention. The method comprises engineering a plurality of ds-scFvs encoding different heavy chain variable domains (V H ) polypeptides, all heavy chain variable domain polypeptides having a cysteine ​​residue at position -4 from the conserved tryptophan H103 according to the Kabat numbering scheme, and engineered to encode different light chain variable domains (V L) polypeptide, all light chain variable domain polypeptides have a cysteine ​​residue at position L34 according to the Kabat numbering scheme. Each of the plurality of first nucleic acids is then cloned with only one second nucleic acid or one of the plurality of second nucleic acids in any order into an expression vector to include a nucleic acid encoding an inserted peptide linker, including but not limited to a peptide linker, such as a peptide linker comprising or consisting of SEQ ID NO: 9-11. If multiple second nucleic acids are used, the combination of the first and second nucleic acids is random. Cloning generates multiple different vectors, which are then expressed on particles, thereby generating a first particle library, each particle displaying a different ds-scFv polypeptide, the polypeptide comprising the V encoded by the first and second nucleic acids. H and V L Preferably, the first particle library is a phage display library.

[0019] In the method, the first or second nucleic acid is engineered to lack a native intradomain disulfide bond in the encoded polypeptide. In this context, a native intradomain disulfide bond specifically refers to a disulfide bond in V according to the Kabat numbering scheme. L The disulfide bond formed between cysteine ​​residues L23 and L88 in V H Preferably, the variable domain located after the peptide linker in the encoded polypeptide lacks a disulfide bond within the native domain.

[0020] In some embodiments, the first or second nucleic acid, or both, can be engineered such that the encoded polypeptide has one or more CDR loops with one or more randomized amino acids, provided that the cysteines participating in interdomain disulfide bond formation and the remaining intradomain disulfide bonds remain unchanged.

[0021] In some embodiments, the method may include introducing further diversity into a subset of the first particle library having desired target binding properties, thereby creating a second particle library, preferably a cell particle library, more preferably a yeast or mammalian cell library, and even more preferably a mammalian cell library. For example, V H or V L The further diversity is introduced by mutagenesis or domain shuffling. Preferably, shuffling of the variable domains after the peptide linker is employed. It is noteworthy that mutagenesis or domain shuffling preferably results in the reintroduction of cysteines that form native intra-domain disulfide bonds in the polypeptides displayed by the second library.

[0022] In some embodiments, all or part of the plurality of first nucleic acids, and / or all or part of the one second nucleic acid or the plurality of second nucleic acids (as the case may be), may be artificially designed and / or synthesized. In some other embodiments, all or part of the plurality of first nucleic acids, and / or all or part of the one second nucleic acid or the plurality of second nucleic acids (as the case may be), may be derived from natural diversity, except for the engineered nucleotides corresponding to the cysteine ​​in CDR-H3 at residue -4 from tryptophan H103 in FR-H4 and the cysteine ​​at residue L34 of CDR-L1 in the encoded polypeptide.

[0023] Further aspects, embodiments and details are set forth in the following figures, detailed description and examples.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are included to provide a further understanding of the present invention and constitute a part of this specification, illustrate embodiments of the present invention and together with the description help explain the principles of the present invention. In the drawings:

[0026] Figure 1 is a schematic diagram of the scFv constructs used in the Examples.

[0027] Figure 2 SDS-PAGE of purified scFv variants LH_SS-, LH_SSC, HL_SSC, HL_S-C and LH_S-C expressed in E. coli is shown. Variants with interdomain disulfide bonds migrate faster in non-reduced form (N). The gel was stained with Ready Blue Protein Gel Stain. Imaging was performed using ChemiDoc, ImageLab 5.2.1 software (Bio-Rad). Precision Plus Protein Dual Color Standards (Bio-Rad) were used as markers (lane 1). R = sample reduced with 5% β-mercaptoethanol. N = sample swimming in non-reduced form.

[0028] Figure 3SDS-PAGE of purified scFv variants LH_SS-, HL_SS-, LH_SSC, HL_SSC, HL_S-C, LH_S-C, LH_SS+ and HL_SS- expressed in ExpiCHO is shown. Variants with interdomain disulfide bonds migrate faster in non-reduced form (N). The gel was stained using Ready Blue Protein Gel Stain. Imaging was performed using ChemiDoc, ImageLab 5.2.1 software (Bio-Rad). Precision Plus Protein Dual Color Standards (BioRad) were used as markers (lane 1). R = sample reduced with 5% β-mercaptoethanol. N = sample run in non-reduced form.

[0029] Figure 4 The immunoreactivity of scFv constructs to HER2 is shown. Each bar represents the mean ± standard deviation of 3 values. The specific signal was calculated by subtracting the signal obtained from the streptavidin wells from the signal obtained from the wells containing biotinylated HER2. Concentrations of scFv: 10 nM (black), 100 nM (checkered) and 300 nM (white).

[0030] Figure 5 The binding of scFv constructs produced in E. coli to HER2 is shown as detected by biolayer interferometry (BLI) (OctetRED384). The y-axis represents binding (nm) and the x-axis represents time (s).

[0031] Figure 6 Detection of Expi-CHO by biolayer interferometry (BLI) (OctetRED384) is shown TM Binding of scFv constructs generated by the system to HER2. The y-axis represents binding (nm) and the x-axis represents time (s).

[0032] Fig. 7A and 7B Derivative plots of the melting transition midpoints (Tm) of scFvs expressed in the LH (7A) and HL (7B) orientations are shown. LH_SS- and HL_SS- (black circles), LH_S-C and HL_S-C (white triangles), LH_SSC and HL_SSC (black squares), LH_SS+ and HL_SS+ (white diamonds). Negative controls (buffer only) are marked as straight lines in the graphs.

[0033] Figure 8Shown are the effects of the signal sequence and scFv constructs on the phage display levels assessed by binding to protein L. The specific signal expressed as time-resolved fluorescence counts represents the mean ± standard deviation of triplicate values. Black: phagemid vector pEB32x, white: phagemid vector pEB3V3 with a modified pelB signal sequence.

[0034] Fig. 9 The effect of signal sequence and scFv construct on binding to HER2 is shown. All phage stocks used in the test were 5x10 9 cfu / ml phage. Each bar represents the mean ± standard deviation of three values. Black: pEB32x, white: pEB3V3 with modified pelB signal sequence.

[0035] Fig.10 Figure 1 is a schematic diagram of scFv library construction. CDR-H3 was randomized with NNS codons. Two PCR products A and B were generated using forward primer WO375 and reverse primer HL_S-C Rev and forward primer HL_S-C 13-19aa loop and reverse primer HS076 new seqrev, respectively. PCR products A and B were digested with LguI to form sticky ends and ligated by a modified FASTR reaction to create a scFv library.

[0036] Fig.11 Binding of loop library phage stocks to protein L in phage immunoassays is shown, illustrating the level of phage display of scFvs. Each bar represents the mean ± standard deviation of three values.

[0037] Fig.12 The results of phage immunoreactivity assays of scFv libraries panned against biotinylated HER2 (as a specific signal for HER2) are shown. Each bar represents the mean ± standard deviation of three values. Black: panning round 1; checkered: panning round 2; white: panning round 3.

[0038] Fig.13 Shows that a single clone isolated from the library after round 3 of panning produced a specific signal for HER2. Each square represents a single clone / well in a 96-well plate. The values ​​shown were calculated by subtracting the signal obtained from the streptavidin wells from the signal obtained from the wells containing biotinylated HER2.

[0039] Fig.14Shown is a monoclonal sequence alignment of the CDR-H3 loop region of HER2-specific clones isolated after round 3 panning. The randomized region is shown in bold. The non-natural cysteine ​​(located at position H100B in CDR-H3 of template ds-scFv HL_S-C) is indicated by *.

[0040] definition

[0041] Before describing the present invention, it should be understood that the present disclosure is not strictly limited to any specific compositions, reagents, devices, protocols or methods described herein, as these may vary. It should also be understood that the terminology used herein is only used to describe specific embodiments and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0042] It should also be noted that unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] It should also be noted that certain features of the disclosure described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosure described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable subcombination. In addition, if applicable and even if not repeated, any feature, detail or embodiment disclosed in the context of a ds-scFv provided herein also applies to the libraries of such ds-scFv provided herein, and vice versa.

[0044] As used herein and in the appended claims, the singular forms "a," "an," and "the" mean one or more. Therefore, unless otherwise indicated, singular nouns also have the meaning of the corresponding plural nouns, and vice versa. Therefore, the terms "a," "an," "one or more," and "at least one" can be used interchangeably.

[0045] The term "and / or" in phrases such as "X and / or Y" should be understood as "X and Y" or "X or Y" and should be considered to provide clear support for both meanings or either meaning.

[0046] The terms "comprising," "including," and "having" can be used interchangeably.

[0047] The term "antibody" as used herein refers to the structure of immunoglobulin G comprising two identical heavy chains (~50-60 kDa) and two identical light chains (~23 kDa), which are composed of a globular structural motif called an Ig fold domain. There are three constant Ig fold domains (C H1 , C H2 and C H3) and a variable Ig fold domain (V H ), while the light chain consists of a constant (C L ) and a variable (V L ) Ig fold domains. Each domain consists of two antiparallel β-sheets, which are composed of seven to nine antiparallel β-strands connected by loops. The Ig fold domain is stabilized by highly conserved intradomain disulfide bonds formed between cysteine ​​residues in the two antiparallel β-sheets. Typically, the C H1 and C H2 Two or four covalent disulfide bonds in the hinge region connect the two heavy chains, while the heavy and light chains are connected by C H1 and C L The term "V" as used herein H ” and “V L ” can be used together with the term “V H Domain" and "V L domain" are used interchangeably.

[0048] The term "complementarity determining region" (CDR) as used herein refers to the highly variable region in the variable domain of an antibody. There are three CDRs in each variable domain: V L CDR-L1, CDR-L2 and CRD-L3 in the domain, as well as V H CDR-H1, CDR-H2 and CDR-H3 in the structural domain. All CDRs participate in antigen recognition and binding together. However, CDR-H3 is generally considered to be the most important CDR involved in antigen binding because its loop length and sequence variation are the largest. The terms "CDR" and "CDR loop" used herein can be used interchangeably.

[0049] The term "framework" (FR) as used herein refers to the non-CDR portion of the variable domain (β-sheet and non-hypervariable loops). It provides structural support for the antigen binding site, but also affects the conformation of the CDR loops. In the primary structure of an antibody (i.e., its linear amino acid sequence), the term "framework" refers to the amino acid sequence between the CDRs. Thus, there are four framework regions in each variable domain: V L FR-L1, FR-L2, FR-L3 and FR-L4 in the domain, and V H FR-H1, FR-H2, FR-H3 and FR-H4 in the structural domain.

[0050] The term "humanized framework" as used herein refers to a framework of human origin, which is usually engineered to include some amino acid changes compared to the original human-derived framework. Since amino acid changes are non-natural for the original human-derived framework, they can be expressed as amino acids of non-human origin. Typically, a humanized framework is used in a humanized antibody, i.e., an antibody in which the CDR region of non-human origin is integrated into a human-derived framework. The framework is humanized, i.e., amino acids of non-human origin are introduced, with the goal of ensuring that the functional properties of the humanized antibody correspond to the functional properties of the parent non-human antibody from which the CDR sequence is derived. Those skilled in the art know how to humanize a given framework for a given parent non-human antibody.

[0051] Human immunoglobulin V L or V H Domains exist in different subtypes, which is generally known in the art. In one embodiment, for V L , the framework substantially corresponds to the framework of the kappa I subtype known to those skilled in the art. H , the framework substantially corresponds to the framework of subtype III known to those skilled in the art. In some further embodiments, the framework may be a "shared framework", i.e., a framework representing each existing V L and V H Human immunoglobulin V L or V H The framework of the most frequently occurring amino acid residues in the framework sequence.

[0052] The term "conservative sequence variant" as used herein refers to an amino acid sequence comprising modifications that do not significantly change the structural or functional properties of the antibody in question. Conservative amino acid sequence variants include variants produced by amino acid substitutions of similar amino acids. As is well known in the art, the similarity can be determined based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues involved. Conservative amino acid sequence variants also include variants comprising small amino acid deletions and / or insertions. Preferably, the conservative sequence variants encompassed by the present invention can be expressed as "functionally equivalent conservative sequence variants". Those skilled in the art can easily determine whether a given sequence variant is functionally equivalent.

[0053] Unless otherwise indicated, all numbering of amino acid positions in the variable domains used herein and identification of CDRs are performed according to the Kabat numbering scheme. The Kabat numbering scheme is well known to those skilled in the art. Thus, for example, the expression "H100B-L34" refers to a polypeptide with cysteine ​​at the H100B position in CDR-H3 and cysteine ​​at the L34 position in CDR-L1 according to the Kabat numbering scheme. Similarly, the expression "H44-L100" refers to a polypeptide with cysteine ​​at the H44 position in FR-H2 and cysteine ​​at the L100 position in FR-L4 according to the Kabat numbering scheme. It should be noted that the positional assignment according to the Kabat numbering scheme refers to certain amino acid positions defined by sequence conservation, rather than the actual amino acid positions in the linear amino acid sequence of a given antibody.

[0054] In more general terms, H100B refers to V H The amino acid position -4 in a polypeptide from the highly conserved tryptophan H103, wherein tryptophan is the first amino acid of FR-H4. Such an indirect definition of the CDR-H3 cysteine ​​that participates in a non-native interdomain disulfide bond is required because, as described later in this specification, the length of the CDR-H3 loop may vary, thereby changing the Kabat numbering of this cysteine ​​residue. It is noteworthy that the expression "position -4 from tryptophan H103 in FR-H4" etc. is not affected by changes in the sequence before said position -4.

[0055] It is noteworthy that in the context of the present invention, the last two amino acids of FR-H3 according to the Kabat numbering scheme are regarded as the first two amino acid residues of CDR-H3. Therefore, FR-H3 is two amino acid residues shorter than the Kabat numbering scheme is generally considered, while the CDR-H3 loop is two amino acid residues longer than the Kabat numbering scheme is generally considered. In other words, using Kabat numbering, FR-H3 is defined by amino acid residues H66-H92 here, while CDR-H3 is defined by amino acid residues H93-H102. The corresponding conventional definitions are H66-H94 and H95-H102, respectively. It is noteworthy that the Kabat numbering scheme allows for length variations within CDR-H3 without affecting the definition of CDR-H3 by Kabat numbering. In other words, regardless of any changes in loop length, according to the Kabat numbering scheme, CDR-H3 comprises amino acids H93-H102.

[0056] The term "variable fragment" (Fv) as used herein refers to a fragment comprising V L and V H The Fv fragment itself is unstable because the V L and V HThe domains are not covalently bound to each other via peptide linkers or disulfide bonds.

[0057] The term "single-chain variable fragment" (scFv) as used herein refers to a fragment comprising V and V fragments connected by a flexible peptide linker to stabilize the structure. L and V H One of the biggest drawbacks of conventional scFvs is that, despite having a stabilized peptide linker, their stability can still be an issue, which prevents them from realizing their full potential. In addition, many conventional scFvs are prone to aggregation and are prone to variable dynamic oligomerization. In addition, their thermal stability is lower than that of the corresponding intact antibodies.

[0058] As used herein, the term "disulfide-stabilized Fv fragment" (dsFv) refers to a fragment comprising V fragments linked to each other by disulfide bonds. L and V H Domain-specific antibody fragments.

[0059] As used herein, the term "disulfide-stabilized single-chain variable fragment" (ds-scFv) refers to a single-chain variable fragment comprising a V L and V H The recombinant Fv fragment of the V L and V H The domains are connected in any direction (V L -Connector-V H or V H -Connector-V L ) and further stabilized by an artificial interdomain disulfide bond. In the present invention, the stabilized interdomain disulfide bond is located at a new position between the CDR-H3 and CDR-L1 loops. Due to its location, the interdomain disulfide bond of the present invention can also be expressed as an H3 / L1-interloop disulfide bond.

[0060] The term "disulfide bond" as used herein refers to a covalent bond formed between two sulfur atoms (-S-S-) coupled by two sulfhydryl (-SH) groups. Cysteine, as one of the 20 proteinogenic amino acids, has an SH group on its side chain and can easily dimerize to cystine by forming a disulfide bond in aqueous solution.

[0061] In terms of their location, two types of disulfide bonds are relevant to the present invention. The first type, i.e., "intradomain disulfide bonds", refers to those between V H and V L A highly conserved native disulfide bond is formed between cysteine ​​residues in two antiparallel β-sheets. H and V L In the structure, the first and third framework regions are connected by a disulfide bond, i.e., V H FR-H1 and FR-H3 and V LFR-L1 and FR-L3 in V H In V, the cysteines that form disulfide bonds are located at positions H22 and H92, while in L In the present invention, cysteines are located at positions L23 and L88 (according to the Kabat numbering scheme).

[0062] The second type of disulfide bond, the "interdomain disulfide bond", is the H and V L The term "inter-domain disulfide bond" is used interchangeably with the term "inter-ring disulfide bond" in the context of the ds-scFv of the present invention comprising a stabilized disulfide bond between the CDR-L1 and CDR-H3 loops. The disulfide bond is "artificial" because the cysteine ​​residues that form the disulfide bond are artificially introduced (i.e., engineered) into the scFv in question by recombinant technology to produce the corresponding ds-scFv. It should be understood that there is nothing artificial or non-natural in the cysteine ​​residues themselves or in the disulfide bonds formed between cysteine ​​residues. As used herein, the term "artificial" is interchangeable with the term "non-natural". In the context of the ds-scFv of the present invention comprising a stabilized disulfide bond between the CDR-L1 and CDR-H3 loops, the term "inter-domain disulfide bond" is interchangeable with the term "inter-ring disulfide bond".

[0063] According to the above, the ds-scFv of the present invention contains three disulfide bonds, one at V H Inside, one in V L Inside, one in V H and V L However, some embodiments of the present invention relate to so-called intermediate ds-scFvs, which comprise only one interdomain disulfide bond and one intradomain disulfide bond, as explained in more detail later in this specification.

[0064] The term "transition temperature" (Tm) as used herein refers to the temperature at which 50% of a macromolecule (eg, an antibody) is denatured, and is considered a standard parameter for describing the thermal stability of proteins.

[0065] The term "trastuzumab" (also known as hu4D5) as used herein refers to a recombinant humanized version of the murine anti-HER2 antibody 4D5. Trastuzumab has been approved for the treatment of HER2 receptor-positive breast cancer and gastric cancer. A scFv fragment of trastuzumab (hu4D5-8) also exists.

[0066] The term "human epidermal growth factor receptor 2" (HER2, also known as neu) as used herein refers to a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. It is an oncogene present on the surface of all breast cells. Amplification or overexpression of HER2 has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. It has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients. Overexpression of HER2 is also known to occur in aggressive forms of ovarian cancer, gastric cancer, lung adenocarcinoma, and uterine cancer.

[0067] As used herein, the term "recombinant expression library" refers to a collection of antibodies or antibody fragments, such as a ds-scFv of the invention comprising one interdomain disulfide bond and two intradomain disulfide bonds or an intermediate ds-scFv comprising one interdomain disulfide bond and one intradomain disulfide bond, displayed on a heterologous host particle (e.g., on a phage particle, on a ribosome, or on a cell surface, such as a yeast cell, a bacterial cell, or a mammalian cell), or expressed in vitro. The number of different antibodies or antibody fragments in such a library is typically >1E4, more preferably >1E5, even more preferably >1E6, even more preferably >1E7, even more preferably >1E8, even more preferably >1E9, and most preferably >1E10. As is known in the art, the diversity may depend on the display system in question. For example, it may be difficult to obtain a diversity higher than 1E5 in a mammalian cell expression library.

[0068] As used herein, the "E" in a statement such as "1E5" refers to an exponent, indicating that the number should be multiplied by the power of 10, the power exponent being the number that follows. In other words, for example, the statement "1E5" is equivalent to the statement "1x10 5 ”, which is equal to 100,000. DETAILED DESCRIPTION OF THE INVENTION

[0070] The present invention relates to recombinant antibody fragments, more specifically to single chain variable fragments having improved stability due to an interdomain disulfide bond located at a novel position, i.e., between CDR-H3 and CDR-L1. Preferably, the interdomain disulfide bond is formed by cysteines at position -4 and at position L34, counting from tryptophan H103 according to the Kabat numbering scheme.

[0071] In addition to the novel stabilized interdomain disulfide bond, the ds-scFv of the present invention may also contain two native intradomain disulfide bonds, one at V H Inside, one in V L However, in some aspects of the invention, the ds-scFv of the invention has been engineered to contain only one of the disulfide bonds within the native domain.

[0072] V Hor V L The engineered protein lacks disulfide bonds within its native domain, which can be achieved by mutating one or preferably both of the cysteine ​​residues that normally participate in disulfide bond formation using techniques readily available in the art. More specifically, one or both of the cysteine ​​residues are substituted with another (i.e., non-cysteine) amino acid. In some embodiments, the non-cysteine ​​amino acid is selected from Phe, Met, Tyr, and Gly, more preferably from Leu and Ile, and even more preferably from Ala and Val. This applies to all cases mentioned below involving ds-scFvs that have been engineered to contain only one disulfide bond within the native domain, including the specifically mentioned SEQ ID NO.

[0073] The CDR loops of the ds-scFv of the present invention can vary in both amino acid composition and length. The diversity of CDRs can be derived from natural or non-natural sources, or both. Natural sources include B cells of immune or non-immune human or animal subjects, while non-natural diversity can be designed by computer and synthesized genetic material. Natural and computer-designed CDR diversity can also be combined with or not combined with CDR randomization. In addition, in some embodiments, the CDR sequence corresponds to the sequence of an existing antibody or its antigen-binding fragment, such as scFv or Fab. It is worth noting that the source of one or more heavy chain CDRs may be different from the source of one or more light chain CDRs.

[0074] In some embodiments, the ds-scFv of the invention comprises a CDR-H3 region of at least 12 amino acids in length, and in some specific embodiments, 13 amino acids in length. Such a CDR-H3 region is particularly suitable for inserting a stabilizing interdomain disulfide bond of the invention between CDR-L1 and CDR-H3.

[0075] For CDR randomization, one or more CDR loops are usually randomized at the position that is most likely to contribute to antigen recognition and binding. Therefore, the priority of the randomization is usually CDR-H3, one or both of CDR-H1 and CDR-H2, one or both of CDR-L1 and CDR-L3 and CDR-L2. It should be noted that it is not intended to limit the priority, and the CDR loop can be independently selected for randomization. The number of randomized amino acid positions is not limited. It should be understood that randomization is not limited to the amino acid substitution of a given amino acid position, and can also involve the amino acid insertion and / or deletion of the position, thereby causing potential changes in the length of the CDR loop.

[0076] For CDR-H3, the number of randomized amino acid positions is preferably at least three, more preferably at least four, and even more preferably at least six, depending on the desired degree of diversification. Preferably, the amino acid change is located at a position upstream of the cysteine ​​at residue-4 from the conserved tryptophan H103 of FR-H4 according to the Kabat numbering scheme. It is important that the CDR randomization does not involve the cysteine, because otherwise the interdomain disulfide bond cannot be formed. For CDR-H1, the number of randomized amino acid positions is generally at least one, preferably two or more (if any). For CDR-H2, the number of randomized amino acid positions is generally at least two, preferably four or more (if any), and more preferably six or more (if any).

[0077] For CDR-L1, the number of randomized amino acid positions is preferably at least one (if any). Importantly, CDR randomization does not involve the cysteine ​​residue at position L34 in CDR-L1 according to the Kabat numbering scheme, otherwise a stabilized interdomain disulfide bond cannot be formed. For CDR-L3, the number of randomized amino acid positions is usually at least two (if any). For CDR-L2, the number of randomized amino acid positions is usually one or more (if any).

[0078] The CDR regions of the ds-scFv of the present invention can be embedded in any appropriate framework. At least for therapeutic applications, the framework is preferably a humanized framework or a framework derived from a human antibody. In some embodiments, the framework is the framework of trastuzumab (also known as hu4D5), which has been successfully used in various CDR transplantation studies. The amino acid sequence of the trastuzumab framework is shown in SEQ ID NO.1-8, wherein SEQ ID NO: 1 represents FR-H1, SEQ ID NO: 2 represents FR-H2, SEQ ID NO: 3 represents FR-H3, SEQ ID NO: 4 represents FR-H4, SEQ ID NO: 5 represents FR-L1, SEQ ID NO: 6 represents FR-L2, SEQ ID NO: 7 represents FR-L3, and SEQ ID NO: 8 represents FR-L4. Therefore, in some embodiments, the ds-scFv of the present invention comprises V H Domain and V L domain, where V H The structural domain comprises the framework regions of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 in order, V L The structural domain comprises the framework regions of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 in order. HIn the V domain, the cysteine ​​residues that form disulfide bonds within the native domain are located at FR-H1 and FR-H3, more specifically at position 22 in SEQ ID NO: 1 and position 30 in SEQ ID NO: 3, corresponding to positions H22 and H92 in ds-scFv according to the Kabat numbering scheme, respectively. L In the structural domain, the cysteine ​​residues that form the disulfide bonds within the native domain are located in FR-L1 and FR-L3, more specifically at position 23 in SEQ ID NO:5 and position 32 in SEQ ID NO:7, corresponding to positions L23 and L88 in the ds-scFv according to the Kabat numbering scheme, respectively. According to certain aspects of the invention, i.e., ds-scFvs lacking another disulfide bond within the native domain, the framework has been engineered to not contain V H or V L One or both of the cysteine ​​residues that form disulfide bonds within the above-mentioned native domains.

[0079] However, the framework need not be 100% identical to the sequences disclosed above, but may vary, as long as the functional or structural properties of the ds-scFv remain substantially unchanged. Thus, in some embodiments, one or more framework regions may be functionally equivalent conservative sequence variants of the above sequences, or may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the above sequences. In addition, the framework may be based on or substantially correspond to any framework encoded by the V and J gene segments. A consensus human framework region may also be used, for example as described in U.S. Pat. No. 6,300,064.

[0080] In particular, when the framework of trastuzumab, or a functionally equivalent conservative sequence variant of the framework, is employed, tryptophan H103 according to the Kabat numbering scheme for defining the position of the artificially introduced cysteine ​​residue in the CDR-H3 loop corresponds to the first amino acid of FR-H4, i.e. the tryptophan at amino acid position 1 of SEQ ID NO: 4.

[0081] In some embodiments using the trastuzumab framework, the ds-scFv of the present invention may comprise the V sequences shown in SEQ ID NO: 48 in any order. H and V shown in SEQ ID NO:49 L, a peptide linker is inserted in the middle. In such embodiments, the CDR loops may have different amino acid compositions and lengths, depending on their target antigens, with a length of 5 amino acids for CDR-H1, 16-19 amino acids for CDR-H2, preferably 17 amino acids for CDR-H3, 12-21 amino acids for CDR-L1, preferably 13 amino acids for CDR-L2, 7 amino acids for CDR-L3, and 9-11 amino acids for CDR-L3, preferably 9 amino acids. The fourth to last amino acid in CDR-H3 (i.e., the amino acid residue at position -4 from the first amino acid of FR-L4) must be cysteine. The last amino acid of CDR-L1 (i.e., the amino acid residue at position -1 from the first amino acid of FR-L2) must also be cysteine. The present invention also encompasses functionally equivalent conservative sequence variants of the above-mentioned ds-scFv.

[0082] In those embodiments where CDR-H3 comprises 13 amino acids, amino acid position -4, counted from tryptophan H103, corresponds to amino acid position H100B according to the Kabat numbering scheme. In some embodiments, the cysteine ​​residues that form the artificial disulfide bond are located at positions H100B and L34 according to the Kabat numbering scheme.

[0083] It should be understood that the ds-scFv of the present invention can be non-human (e.g., mouse, rabbit, goat), chimeric, humanized (non-human CDRs are integrated into humanized framework regions), or fully human (both framework and CDRs are derived from human antibodies). However, humanized and fully human ds-scFv are preferred, especially for therapeutic purposes. For diagnostic and some other non-therapeutic purposes, ds-scFv does not have to be humanized or fully human, but can also be non-human or chimeric ds-scFv. It is worth noting that V H The source and type can be related to V L The sources and types are different.

[0084] V in the ds-scFv of the present invention H and V LThe domains are connected by a flexible linker peptide, which is generally 15-20 amino acids long. However, in some embodiments, the length of the linker can be 12-15 amino acids or even shorter, and in some other embodiments, the length of the linker can be greater than 20 amino acids, such as 25 amino acids or even 30 amino acids. The linker keeps the C-terminus of a variable domain and the N-terminus of another domain at a certain distance to facilitate correct folding and formation of an antigen binding site, while also minimizing the oligomerization of ds-scFv. It is generally believed that short linkers (generally 12-15 amino acids or less) will prevent the physical combination of two V domains in the same polypeptide and cause the formation of polymers, while long linkers (generally longer than 20 amino acids) can facilitate proteolysis or weak domain binding in scFv.

[0085] In some embodiments, multimers of the ds-scFv of the present invention may be desired. For example, it is envisioned that the ds-scFv of the present invention can be used to generate diabodies, i.e., bispecific antibodies comprising two scFvs with different antigen binding specificities, one or both of which are ds-scFvs of the present invention. In such embodiments, the linker peptide should be short, typically 5-10 amino acids, so that both scFv chains cannot form functional scFvs by themselves, thereby inducing the formation of diabodies comprising different scFv partners. Only 1-4 amino acid linkers mainly lead to the formation of trimer and tetramer constructs.

[0086] Usually in ds-scFv, the joint peptide mainly includes glycine and serine residues.The most widely used is the peptide joint of 15 amino acids (glycine 4-serine) 3 shown in SEQ IDNO:9, also expressed as (Gly4-Ser) 3 or (GGGGS) 3 joint.However, longer joints can be used, for example, 20 amino acids (Gly4Ser) 4 or (GGGGS) 4 shown in SEQ ID NO:10, to minimize oligomerization.In some embodiments, other residues (for example, charged residue glutamic acid (Glu) and / or lysine (Lys)) can also be incorporated into the joint, for example, to enhance solubility.In some embodiments, amino acid residues such as alanine (Ala) and / or threonine (Thr) can be incorporated into the joint, such as the joint GGGGSGAGGSGGGGTGGGGS (SEQ ID NO:11) used in the present embodiment.

[0087] V in the ds-scFv of the present invention H and V L The order (also called orientation) of the domains can be V L -Connector-V H (LH, also denoted as V L -VH ) or V H -Connector-V L (HL, also denoted as V H -V L ). However, a single scFv may perform better in one configuration than in another, for example in terms of binding properties. In addition, in some embodiments, expression yields may vary depending on the configuration, so the HL orientation is generally preferred.

[0088] In some embodiments, the ds-scFv of the present invention may include one or more additional peptide tags for various purposes, such as to facilitate purification, separation, immobilization and / or detection. Various peptide tags suitable for such and other purposes are readily available in the art. Non-limiting examples of such peptide tags will be listed later in this specification.

[0089] Compared with the corresponding conventional scFv molecules, the stabilized ds-scFv molecules of the present invention have improved stability. Protein stability is usually measured by reversibly unfolding the protein with heat or a chaotropic agent (e.g., guanidine hydrochloride or urea) using methods known in the art. Therefore, in some embodiments, the measurement of protein stability is thermal stability, i.e., resistance to irreversible unfolding caused by thermal challenges. In some embodiments, the measurement of protein stability is pH-dependent, i.e., resistance to protein unfolding caused by pH changes. In addition, the measurement of protein stability can also be tolerance to proteases. In particular, for antibodies, stability can also be measured as stability in serum.

[0090] Thermal stability can be measured using a variety of non-restrictive biophysical or biochemical techniques known in the art. The most commonly used method for measuring protein thermal changes may be differential scanning fluorimetry (DSF) or thermofluorescence, which utilizes small fluorescent molecules, and when these small fluorescent molecules are combined with exposed hydrophobic surfaces (such as hydrophobic surfaces produced by protein unfolding), their fluorescence can be enhanced. Alternatively, thermal stability can be determined by other analytical techniques, such as differential scanning calorimetry (DSC) and temperature-dependent circular dichroism spectroscopy (CD). All of these biophysical techniques allow determination of thermal unfolding transitions. The temperature of protein unfolding indicates overall protein stability.

[0091] In other embodiments, thermal stability can be measured biochemically. An exemplary biochemical method for evaluating thermal stability is a thermal challenge assay, in which the composition to be determined to have thermal stability is exposed to a set time in a series of elevated temperatures. For example, in some embodiments, one or more test compositions (e.g., ds-scFv and / or reference scFv molecules of the present invention, or particles of expression libraries showing them) are placed in a series of elevated temperatures, for example, for about 1 to about 1.5 hours. The activity of the test composition is then determined by a related biochemical assay. Preferably, the assay is a combination assay for determining any changes in the target molecule binding properties of the thermal challenge composition. The combination assay can be, for example, a functional or quantitative ELISA assay. The temperature at which the antigen binding properties are lost indicates overall thermal stability.

[0092] In some embodiments, thermal stability can be assessed by measuring the melting temperature (Tm) of the test composition using any of the above techniques. The melting temperature is the temperature at the midpoint of the thermal transition curve where 50% of the composition molecules are denatured, such as determined by unfolding or loss of antigen binding. Tm is considered a standard parameter for describing the thermal stability of proteins.

[0093] In some embodiments, the ds-scFv of the invention has a thermal stability that is about 1, about 1.25, about 1.5, about 1.75, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 degrees Celsius or more higher than a control molecule (e.g., a corresponding conventional scFv molecule).

[0094] Surprisingly, the interdomain disulfide bond of the present invention between CDR-H3 and CDR-L1 loops renders the thermal stability of the present ds-scFv significantly better than H44-L100, a disulfide bond previously proposed as a general location for scFv stabilization.

[0095] The ds-scFv of the present invention can be used for various research, diagnostic and therapeutic purposes, mainly depending on their antigen binding specificity. In addition, they can be used as building blocks for various molecular entities, including but not limited to engineered therapeutic proteins, such as bispecific antibodies and CAR T-cells.

[0096] Bispecific T cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies that contain two scFvs directed against different antigens. Another scFv targets the protein complex cluster of differentiation (CD3) on T cells, while another scFv targets a disease-specific antigen, thereby forming a connection between T cells and diseased cells (e.g., tumor cells). In the vicinity of diseased cells, T cells destroy them by programming them to undergo apoptosis. It is understood that the ds-scFvs of the present invention are suitable as building blocks for constructing BiTes.

[0097] It is also understood that the ds-scFv of the present invention can be used to form various other types of bivalent or bispecific or multispecific antibody constructs. For example, it can be expressed as the N-terminus or C-terminus of the light chain or heavy chain fused to the full-length antibody construct. Alternatively, one or two Fab arms of a bispecific antibody can be replaced with the ds-scFv of the present invention, while retaining the Fc portion (i.e., a crystallizable fragment) to provide immunomodulatory properties. This results in an antibody construct that is much smaller than a complete IgG antibody, but due to the presence of the Fc portion, it is expected to still have substantially unchanged immunomodulatory properties and a longer half-life. Therefore, the ds-scFv of the present invention is suitable as a building block for a fusion comprising a ds-scFv and an antibody Fc portion. By fusing the ds-scFv of the present invention with other affinity reagents based on protein scaffolds (e.g., Design Ankyrin Protein (DARPin), nanobody or affibody, etc.), further bispecific constructs can be created. Such binding molecules are readily available in the art.

[0098] It is further understood that the ds-scFv of the present invention can also be used in CAR T-cell therapy. CAR T-cell therapy is a cancer immunotherapy that uses the patient's T cells to find and kill tumor cells. CAR T-cells are engineered T cells that express artificial T cell receptors CAR on the cell surface. These artificial T cell receptors are chimeric-they contain antigen binding domains and T cell activation domains. In addition to the antigen binding domain (ds-scFv of the present invention) and the T cell activation domain, CAR also contains hinges, transmembrane and co-stimulatory domains, which are well known to those skilled in the art. When CAR has a ds-scFv that binds to a cancer-associated antigen, the binding of the ds-scFv portion of the receptor can activate T cells to kill cancer cells.

[0099] In some further embodiments, the ds-scFv of the present invention can be contained in a molecular entity, for example, a pharmaceutically active agent, a drug, a radioisotope, an enzyme (e.g., alkaline phosphatase), or a chelating agent, to name a few non-limiting examples. Depending on the type of molecular entity, a given ds-scFv can be conjugated or recombinantly fused to the molecular entity using existing means and methods in the art.

[0100] The present invention also encompasses nucleic acid molecules encoding the ds-scFv of the present invention and various embodiments thereof.

[0101] Turning now to a specific embodiment, the present invention provides anti-HER2 ds-scFv variants. They are scFv variants derived from trastuzumab by introducing the stabilizing interdomain disulfide bond of the present invention into a new position, namely between CDR-H3 and CDR-L1, more specifically, between the -4 position and the L34 position from H103 according to the Kabat numbering scheme. In some embodiments, the -4 position from H103 is position H100B.

[0102] Surprisingly, the introduction of the interdomain disulfide bonds of the present invention increased the thermal stability of ds-scFv displayed on phage and soluble ds-scFv proteins by 10°C compared to the original scFv without the interdomain disulfide bonds. Importantly, the interdomain disulfide bonds located at positions H44-L100 according to the Kabat numbering scheme had a significantly lower impact on thermal stability than the inter-loop disulfide bonds of the present invention. The Tm value of the comparable ds-scFv with H44-L100 modification was only 3-4°C higher than the original scFv, but 7°C lower than the Tm value of the variant with the inter-loop disulfide bond at the novel H100B-L34 position examined in this study.

[0103] Furthermore, the anti-HER2 scFv with added interdomain disulfide bonds retained the antigen-binding properties of its parent scFv in both orientations.

[0104] In some embodiments, the anti-HER2 ds-scFv comprises a CDR sequence substantially identical to trastuzumab, except for the cysteine ​​residues that form the interdomain disulfide bonds of the present invention. Thus, in some embodiments, the anti-HER2 ds-scFv comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 12, a CDR-H2 having an amino acid sequence of SEQ ID NO: 13, a CDR-H3 having an amino acid sequence of SEQ ID NO: 14, a CDR-L1 having an amino acid sequence of SEQ ID NO: 15, a CDR-L2 having an amino acid sequence of SEQ ID NO: 16, and a CDR-L3 having an amino acid sequence of SEQ ID NO: 17, or conservative sequence variants of said CDR sequences, provided that the HER2 binding properties of the anti-HER2 ds-scFv are not lost. For comparison, the CDR-H3 and CDR-L1 of trastuzumab are shown in SEQ ID NO: 18 and SEQ ID NO: 19, respectively. The cysteine ​​residues forming the artificial interdomain disulfide bond are at position 10 in SEQ ID NO:14 and at position 11 in SEQ ID NO:15.

[0105] Thus, in some embodiments, the anti-HER2 ds-scFv of the invention comprises V H Domain and V L Domain, V H The structural domain comprises CDR sequences SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, V L The structural domain comprises CDR sequences SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, V H Domain and V L In some further embodiments, such V H Domain and V L The domain is inserted into a peptide linker, for example, a peptide linker selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and functionally equivalent sequence variants thereof.

[0106] In some further embodiments, the anti-HER2 ds-scFv of the invention comprises V H Domain and V L Domain, V H The domain comprises or consists of the amino acid sequence shown in SEQ ID NO:31, V LThe domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 32. The cysteine ​​residues that form the disulfide bonds between the artificial domains are located at position 106 in SEQ ID NO: 31 and position 34 in SEQ ID NO: 32; while the cysteines that form the disulfide bonds within the natural domains are located at positions 22 and 96 in SEQ ID NO: 31 and positions 23 and 88 in SEQ ID NO: 32. In some further embodiments, such V H and V L The domain is inserted into a peptide linker, for example, a peptide linker selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and functionally equivalent sequence variants thereof.

[0107] In some further embodiments, the anti-HER2 ds-scFv is in the HL orientation and has the amino acid sequence of SEQ ID NO: 20. In some further embodiments, the anti-HER2 ds-scFv is in the LH orientation and has the amino acid sequence of SEQ ID NO: 21. In these embodiments, the cysteine ​​residues that form the artificial interdomain disulfide bonds are located at positions 106 and 174 in SEQ ID NO: 20, and positions 34 and 235 in SEQ ID NO: 21. The cysteine ​​residues that form intradomain disulfide bonds are located at positions 22, 96, 163, 228 in SEQ ID NO: 20, and positions 23, 88, 151, 225 in SEQ ID NO: 21. The DNA sequence encoding SEQ ID NO: 20 is shown in SEQ ID NO: 45, and the DNA sequence encoding SEQ ID NO: 21 is shown in SEQ ID NO: 46.

[0108] In some embodiments, the CDR-H3 of the anti-HER2 ds-scFv may comprise one or more amino acid changes (substitutions, deletions and / or insertions) between H93-H100A according to the Kabat numbering scheme (corresponding to amino acid residues 1-9 in SEQ ID NO: 14, amino acid residues 97-105 in SEQ ID NO: 31, amino acid residues 97-105 in SEQ ID NO: 20, or amino acid residues 226-234 in SEQ ID NO: 21), provided that the anti-HER2 ds-scFv still exhibits specific binding to HER2, preferably with a Kd of <10 nM. In other words, the amino acid changes in CDR-H3 are upstream of the cysteine ​​at residue -4, counting from the conserved tryptophan H103 of FR-H4. These amino acid changes are preferably at positions corresponding to amino acid residues 3-9 in SEQ ID NO:14, amino acid residues 99-105 in SEQ ID NO:31, amino acid residues 99-105 in SEQ ID NO:20, or amino acid residues 228-234 in SEQ ID NO:21.

[0109] Alternatively or additionally, the anti-HER2 ds-scFv of the invention may also contain one or more amino acid changes (substitutions, deletions and / or insertions) in the CDR-H1 loop and / or in the CDR-H2 loop compared to the amino acid sequences disclosed herein.

[0110] Regardless of the presence or absence of amino acid changes in CDR-H1, CDR-H2, and / or CDR-H3, in some embodiments, the anti-HER2 ds-scFv of the invention may contain one or more amino acid changes (substitutions, deletions, and / or insertions) in CDR-L1 and / or CDR-L3 compared to the amino acid sequences disclosed herein. In some embodiments, the CDR-L2 loop may also contain one or more amino acid changes (substitutions, deletions, and / or insertions) compared to the amino acid sequences of the CDR-L2 loop disclosed herein.

[0111] It is readily understood by those skilled in the art that the requirement for the above amino acid changes is that the anti-HER2 ds-scFv still shows specific binding to HER2, preferably with Kd <10 nM. The presence or absence of the binding specificity can be easily determined by means and methods available in the art. Generally, the priority order for diversifying the CDR loops is CDR-H3, one or both of CDR-H1 and CDR-H2, one or both of CDR-L1 and CDR-L3, and CDR-L2. In order to promote the diversification, one of the disulfide bonds within the two domains can be eliminated by replacing one or both of the cysteine ​​residues that are usually involved in disulfide bond formation, including those specifically mentioned for a given SEQ ID NO, as described elsewhere in this specification. The intradomain disulfide bonds can be subsequently reintroduced, as described elsewhere in this specification.

[0112] In some embodiments, the anti-HER2 ds-scFv of the invention is used to treat cancer, particularly breast cancer or gastric cancer. In some further embodiments, they can be used for bispecific antibodies, BiTE or CAR T-cell therapy.

[0113] It is noteworthy that the cysteine ​​residues of the present invention that form stabilizing interdomain disulfide bonds can be engineered into any existing scFv by techniques well known in the art, including, for example, site-directed mutagenesis. In vitro display technology can also be used to generate the ds-scFv of the present invention.

[0114] Thus, one aspect of the invention relates to a recombinant particle library displaying a plurality of ds-scFvs of the invention against various antigens (e.g., HER2). Further aspects relate to methods for constructing such libraries and their uses to obtain novel ds-scFvs with desired antigen binding properties. Thus, ds-scFvs against desired antigens can be easily generated from the library of the invention by antigen screening without the need for host animal immunization and hybridoma production, thereby greatly shortening the time and effort typically required to produce antibodies by conventional means.

[0115] Thus, the present invention provides a method for constructing a recombinant expression library, more specifically a library of particles on which the ds-scFv of the present invention is displayed, thereby linking the genotype and phenotype of the particles. The particles forming the library can be phage particles, ribosomes, yeast cells, bacterial cells or mammalian cells. In some preferred embodiments, the library is a phage display library, while other embodiments use both phage display and mammalian cell display libraries.

[0116] To construct the display library of the present invention, codons for cysteine ​​residues that form stabilizing interdomain disulfide bonds are introduced into V sequences encoding genes with the desired diversity using methods known in the art.H and V L The diversity may be derived from natural or non-natural sources. For example, the diversity may be derived from an immune library constructed from variable domain genes isolated from B cells derived from immunized animals or humans. Alternatively, the diversity may be derived from a naive library ( In some further embodiments, diversity can be derived from fully synthetic libraries that are usually derived from non-immune sources and computer-designed and gene-synthesized. In synthetic libraries, humanized frameworks or frameworks derived from human antibodies are combined with CDRs that are randomized at positions that are most likely to contribute to antigen recognition and binding. In semi-synthetic libraries, natural and computer-designed CDR diversity is combined with or without CDR randomization. CDR randomization can be achieved, for example, by PCR-based synthetic oligonucleotide assembly, which is well known in the art.

[0117] Due to its important role in antigen recognition and binding, the ds-scFv display library of the present invention is a CDR-H3 library in some preferred embodiments. Such libraries can be constructed by diversifying the length and / or amino acid composition of the CDR-H3 loop of existing antibodies or antigen-binding fragments thereof. In some embodiments, the length of the CDR-H3 loop is preferably 12 to 21 amino acids.

[0118] The lengths of other CDR loops may also be different. In some embodiments, especially in embodiments employing the trastuzumab framework or its functionally equivalent conservative sequence variants, CDR-H1 is usually 5 amino acids long, CDR-H2 is usually 16-19 amino acids long, preferably 17 amino acids long, and CDR-H3 is usually 12-21 amino acids long. In some embodiments, CDR-H3 is 13 amino acids long. In turn, CDR-L1 is usually 11 amino acids long, CDR-L2 is usually 7 amino acids long, and CDR-L3 is usually 9-11 amino acids long. In some embodiments, CDR-L3 is 9 amino acids long. In some embodiments, one or more CDR loops may include one or more randomized amino acids, as described elsewhere in this specification.

[0119] Since CDR-H3 can vary in length, the amino acid position of the cysteine ​​residue participating in the artificial disulfide bond must be defined indirectly by the position relative to the highly conserved tryptophan residue at position 1 of FR-H4. This definition applies regardless of the framework used. In some embodiments, the recombinant expression library is based on the V sequence shown in SEQ ID NO: 48. H Variants and V shown in SEQ ID NO:49 LMore generally, in some embodiments, the recombinant expression library is based on V sequences comprising the framework regions shown in SEQ ID NOs: 1-4. H and V comprising the framework regions shown in SEQ ID NOs: 5-8 L Variants. According to the above, the sequence can be engineered so that one or both of amino acid residue 22 of SEQ ID NO:1 and amino acid residue 30 of SEQ ID NO:3 are not cysteine, or one or both of amino acid residue 23 of SEQ ID NO:5 and amino acid residue 32 of SEQ ID NO:7 are not cysteine, thereby preventing the formation of one of the disulfide bonds within the two native domains. For the sequences SEQ ID NO:48 and SEQ ID NO:49, this means that they can be engineered so that one or both of the amino acid residues at positions 22 and 98 of SEQ ID NO:48 are not cysteine, or one or both of the amino acid residues at positions 23 and 88 of SEQ ID NO:49 are not cysteine, thereby preventing the formation of one of the disulfide bonds within the two native domains.

[0120] As with the case of ds-scFv above, any suitable framework can be used to create a recombinant expression library of the present invention. Preferably, the framework is a humanized framework or a framework derived from a human antibody. In some specific embodiments, the framework is a framework of trastuzumab or a conservative variant thereof or other variants, as described above. In some other embodiments, the framework is a human consensus framework.

[0121] Depending on the source of the CDR and / or framework, the ds-scFv displayed by the recombinant expression library of the present invention can be represented as recombinant non-human, chimeric, humanized, fully human or artificial (computer-designed), as understood by those skilled in the art. H The source and design can be related to V L different.

[0122] Once the encoding V H and V L The nucleic acid molecules are engineered and then cloned into expression vectors to create a ds-scFv library. H and V L A nucleic acid molecule derived from the desired diversity and comprising cysteine ​​residues at CDR-H3 and CDR-L1 that form stabilizing interdomain disulfide bonds, preferably at position -4 from H103 in FR-H4 and at position L34 in CDR-L1 according to the Kabat numbering scheme, and preferably lacking residues that participate in V H or V LOne or two native cysteines in the domains of the PCR product are disulfide bonded. The choice of expression vector depends on the type of display library to be created, as will be readily appreciated by those skilled in the art. A variety of suitable expression vectors are commercially available.

[0123] For phage display, the nucleic acid encoding the ds-scFv is fused to the phage coat protein gene in the phage or phagemid vector, so that the phage displays the scFv on its surface. The most widely used phage display format utilizes the filamentous phage M13 and fuses the target protein to the phage coat protein pill, thereby achieving high-frequency monovalent display. In this method, a phagemid vector with randomly cloned nucleic acids encoding a large number of different ds-scFvs is electroporated into E. coli and then infected with a helper phage to produce a phage library, each of which displays a different ds-scFv on its surface. There are multiple suitable vectors, helper phages, and other methods for constructing phage display libraries in the art.

[0124] For mammalian cell display, nucleic acids encoding ds-scFvs with membrane anchor fragments can be integrated into the genome of mammalian cells, so that cells display ds-scFvs on their surfaces. Various mammalian cell lines can be used for this purpose, including but not limited to CHO (Chinese hamster ovary) and HEK293 (human embryonic kidney) cells. There are various strategies for integrating ds-scFv expression constructs into the genome of cells; these include, for example, site-specific recombinases using homologous recombination and targeted landing pads. For example, zinc fingers or TALE effector nucleases or CRISPR / Cas systems that induce DNA double-strand breaks can be used to increase the efficiency of homologous recombination. Cells displaying ds-scFvs with certain binding specificities can be enriched and isolated by separation based on magnetic beads coated with antigens or screening based on FACS (fluorescence activated cell sorting) using fluorescently labeled antigens, or by any other suitable technology for this purpose.

[0125] Once a recombinant expression library (e.g., a phage display library) is generated, it can be used to select ds-scFvs with desired antigen binding properties by a method called panning, which typically includes several rounds of selection for one or more target antigens, preferably immobilized on a solid surface (e.g., beads or microtiter plates). After incubation with the target antigen, unreactive phages are removed, for example, by extensive washing. The bound phages are sequentially eluted and enriched by amplification in appropriate host cells (usually bacterial cells) before the next round of selection. These steps are typically repeated two to four times to obtain the most specific phages for the desired target antigen. In some embodiments, the selection stringency can be increased for each round of selection to enrich for ds-scFvs with high affinity and specificity.

[0126] In some embodiments, the panning process may also involve one or more rounds of negative selection. To this end, a negative selection protein (i.e., a non-target antigen) is contacted with the phage display library. Phages that bind to the non-target antigen are removed, and the remaining phage reserves are used for positive selection against the target antigen.

[0127] After multiple rounds of panning, it is usually tested whether antigen-specific phage binders have been enriched, usually by using an appropriate immunoassay. Individual clones are then screened from the enriched pool of antigen-specific phage binders. Positive clones can then be further characterized, for example by determining their antigen binding properties and / or by sequencing.

[0128] Typically, phage display libraries allow greater diversity than mammalian cell display libraries. In other words, the collection of different variants in a phage display library can be significantly larger than the collection of different variants in a mammalian cell display library. Therefore, the former library is sometimes preferred over the latter.

[0129] On the other hand, the presence of three disulfide bonds in the ds-scFv to be displayed, i.e., one interdomain disulfide bond of the present invention and two naturally occurring intradomain disulfide bonds, can reduce the display efficiency (i.e., yield) on the phage surface. It is understood that in mammalian cell display libraries, the display efficiency is not significantly reduced because mammalian cells are able to express molecules with three disulfide bonds very well.

[0130] It has now surprisingly been found that the advantages of phage display and mammalian cell display libraries can be combined. To this end, first a scFv library having the interdomain disulfide bonds of the invention and only one naturally occurring intradomain disulfide bond (instead of the two intradomain disulfide bonds native to scFv, according to the Kabat numbering scheme, at V L Between the cysteines at positions L23 and L88, and V H This enables the creation of display libraries with very high diversity without compromising display efficiency, as phage can express molecules with two disulfide bonds at a high rate. The missing intradomain disulfide bonds can be engineered to be present in V H or V L For the former option, V H In some embodiments, it may have the amino acid sequence shown in SEQ ID NO: 34; and for the latter option, V LIn some embodiments, it may have the amino acid sequence shown in SEQ ID NO: 33. In some preferred embodiments, in the second V domain, that is, in the V domain when the ds-scFv is in the LH orientation. H The lack of intradomain disulfide bonds in the domain and the V L Intradomain disulfide bonds are absent in the domain. In some embodiments, the latter option (HL orientation) is preferred.

[0131] Once the most promising ds-scFv is obtained from a large phage display library, it can be synthesized by using V L Reorganization (if V L lack of intradomain disulfide bonds) or V H Reorganization (if V H The missing cysteine ​​that forms the intradomain disulfide bond can be reintroduced by using a method that is not suitable for the present invention. The ds-scFv library thus obtained, which contains all three disulfide bonds (i.e., the non-native interdomain disulfide bond of the present invention and the two native intradomain disulfide bonds), can then be selected using the target antigen using mammalian cell display technology. This method increases the success rate of obtaining appropriate ds-scFvs specific for the target antigen because, despite the lower display efficiency of the phage display library, its greater diversity can be utilized.

[0132] Therefore, in one embodiment of the present invention, high diversity is first introduced into V H CDR, while V L The first library thus created may contain, for example, >1E8 or >1E9 or >1E10 V H Variants. In some embodiments, the orientation of the ds-scFv in such libraries is V H -V L , V L Lack of native intradomain disulfide bonds. In some further embodiments, such libraries are subsequently expressed on filamentous phage and selected against the target antigen.

[0133] In some embodiments, V L Shuffling is used to introduce additional diversity into the library. In other words, antigen-specific V enriched from the phage display library H The domains can be combined with diverse V containing disulfide bonds within the native domains L The resulting second library can then be selected for antigen specificity using mammalian cell display technology.

[0134] It is worth noting that in the methods described in the above preferred embodiments, other cell-based display technologies, such as yeast display, can be used instead of mammalian cell display, although mammalian cell display is generally preferred. L -V H Direction other than V H -V L direction. In this case, V H The native intradomain disulfide bonds are missing in the first library, and L There is high diversity in the CDRs of V H Not diversified or diversified to a limited extent. Then you can use V H Shuffling was used to introduce additional diversity into the library, while also reintroducing native intradomain disulfide bonds into the V H , the second library thus created is then subjected to target antigen-specific selection using cell-based display technology.

[0135] In some embodiments, further diversity can be introduced by mutagenesis techniques readily available in the art (e.g., random mutagenesis, e.g., by error-prone PCR), preferably into the first library described above. In some embodiments, mutagenesis can be used instead of V L or V H Reorganize or remove V L or V H Shuffling also employs mutagenesis.

[0136] In addition, diversity can be introduced into the first and / or second library, preferably into the first library, by CDR randomization using means and methods readily available in the art, which is consistent with what is described elsewhere in this specification and will not be repeated here.

[0137] It should be understood that, although combining phage display and mammalian cell display methods can be beneficial as described above, target-specific ds-scFvs of the present invention containing all three disulfide bonds can also be successfully obtained from phage display libraries (although with lower yields) or mammalian cell libraries (although with lower diversity).

[0138] Each display particle carries the genetic information for the recombinant polypeptide displayed on its surface. This feature allows the identification of nucleic acids encoding ds-scFvs exhibiting the desired specificity by selecting particles carrying the nucleic acid from a potentially very complex recombinant library. The best cloned nucleic acid can then be isolated, inserted into a suitable expression vector, and transfected or transformed into a compatible expression host to produce ds-scFvs according to standard recombinant techniques.

[0139] Many types of suitable expression vectors are available, including but not limited to plasmids and modified viruses, which are retained in the host cell as autonomous DNA molecules or integrated into the genomic DNA. The vector system must be compatible with the host cell used, which is well known in the art. Preferably, the DNA encoding the ds-scFv of the present invention is operably linked to one or more heterologous expression control sequences that allow expression of the ds-scFv. Suitable control sequences are readily available in the art, including but not limited to promoters, leader sequences, polyadenylation sequences, and signal sequences.

[0140] The expression vector can be transfected or transformed into the host cell by standard techniques commonly used to introduce exogenous nucleic acid into prokaryotic or eukaryotic host cells, including but not limited to electroporation, nucleofection, sonoporation, magnetofection, heat shock, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0141] ds-scFv can be expressed in various expression systems, including but not limited to prokaryotic host cells, such as bacteria (e.g., E. coli, Bacillus), yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae) and fungi (e.g., filamentous fungi), and eukaryotic hosts, such as plant cells, insect cells (e.g., Sf9) and mammalian cells (e.g., CHO cells). Host cells transfected with an expression vector comprising a nucleic acid (preferably DNA) encoding a ds-scFv of the present invention will be cultured under conditions suitable for producing ds-scFv, and the obtained ds-scFv will then be recovered. The ds-scFv of the present invention can also be produced by in vitro protein expression according to protocols known in the art.

[0142] Bacterial cell expression systems (e.g., E. coli) are the fastest and cheapest form and are therefore preferred for production of ds-scFv in some cases. In such embodiments, ds-scFv is preferably targeted to its oxidized periplasmic space, which contains molecular chaperones and disulfide isomerases that allow for proper protein folding and disulfide bond formation, respectively. Secretion can be directed to the periplasmic space with the help of a signal peptide (SP) such as pelB, for example, by linking the signal peptide to the N-terminus of the ds-scFv using standard recombinant techniques. Non-limiting examples of suitable signal peptides include those shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:28. ds-scFv can also be expressed in high yields in the cytoplasm of E. coli or other bacterial cells. However, due to the reducing environment of the cytoplasm, disulfide bonds cannot be formed in most prokaryotes. Therefore, ds-scFv must be recovered from inclusion bodies, where they accumulate and refold, which can be time-consuming and inefficient. However, there are some exceptions, such as Origami. TM B host strains, etc. In addition, soluble expression of disulfide-bonded proteins can be achieved in the cytoplasm of bacterial cells (e.g., E. coli) using a system called CyDisCo, which is based on co-expression of the target protein with a sulfhydryl oxidase and a disulfide isomerase.

[0143] In some embodiments, it may be desirable to express the ds-scFv of the present invention as a fusion with one or more peptide or small protein tags to facilitate purification, separation, fixation and / or detection. Non-limiting examples of suitable affinity tags for purification or fixation purposes include polyhistidine tags (His-tags), hemagglutinin tags (HA-tags), glutathione-S-transferase tags (GST-tags), and biotin tags. Suitable detection tags include, but are not limited to, Myc-tags, FLAG-tags, fluorescent proteins (e.g., GFP), and enzyme tags that produce colored products when in contact with chromogenic substrates. Non-limiting examples of suitable enzyme tags include alkaline phosphatase (AP) and (horseradish) catalase (HRP). Other tags (e.g., biotin, avidin, and streptavidin) may also be used for detection. They may be detected with biotin / avidin / streptavidin binding proteins conjugated to enzymes, fluorophores, or other reporter molecules. Vectors, other means, and methods for producing ds-scFv of the present invention as fusion proteins are readily available in the art. Example

[0144] Example 1. Anti-HER2-ds-scFv construct

[0145] The anti-HER2 scFv variants used in the examples were designed and then the genes were ordered from Twist Bioscience (USA) in the form of cloned genes.

[0146] In the construct, an artificial disulfide bond was introduced between the CDR-H3 loop (position H100B according to Kabat numbering, corresponding to position 106 in SEQ ID NO: 31) and the CDR-L1 loop (position L34 according to Kabat numbering, corresponding to position 34 in SEQ ID NO: 32) of the scFv fragment of trastuzumab (also known as hu4D5). In addition to the added inter-loop disulfide bond, V L and V H Each has a natural intradomain disulfide bond. L In V, the intradomain disulfide bond is located between the cysteines at positions L23 and L88 according to the Kabat numbering scheme (corresponding to positions 23 and 88 in SEQ ID NO: 32, respectively). H In the embodiment, the intradomain disulfide bond is located between the cysteines at positions H22 and H92 according to the Kabat numbering scheme (corresponding to positions 22 and 96 in SEQ ID NO: 31, respectively). L -V H ) and HL(V H -V L ) orientation. Constructs with all three disulfide bonds (LH_SSC and HL_SSC, respectively) and constructs in which the native intradomain disulfide bonds had been removed from the second domain in the scFv were tested. In LH_S-C, the intradomain disulfide bonds were removed from V H Remove (domain sequence V L -V H ), while in HL_S-C, the intradomain disulfide bonds are from V L Remove (domain sequence V H -V L ). A wild-type scFv with only native intradomain disulfide bonds (LH_SS- and HL_SS-) was used as a control. L and V H A construct containing a disulfide bond between positions H44-L100 was expressed in both LH and HL orientations and evaluated as a reference. L and V H The schematic diagrams of the amino acid sequences are as follows Figure 1 and as shown in Table 1. All scFv constructs were L and V HThere is a 20aa glycine-serine peptide linker GGGGSGAGGSGGGGTGGGGS (SEQ ID NO: 11) in between.

[0147] The construct used for expressing ds-scFv in E. coli in Example 2 was designed to contain SfiI cloning sites at both ends. The ScFv gene was cloned into the SfiI site of the periplasmic expression vector pAK400 (Krebber et al., 1997), which contains the pelB signal peptide (MKSLLPTAAAGLLLLAAQPAMA; SEQ ID NO: 22), the Lac promoter and the chloramphenicol resistance gene. A His6-tag was introduced into the C-terminus of the scFv of the vector.

[0148] The constructs used for phage display studies in Example 6 were cloned into the SfiI site of the phagemid vectors pEB32x (Huovinet. al., 2013) and pEB3V3. pEB3V3 is identical to the vector pEB32x, except that it contains a modified pelB signal peptide MKYLLPTVVVGLLLLAAQPAMA (SEQ ID NO: 23), encoded by atg aag tac ctt cta ccg acg gtagtc gtt gga ttg tta tta ctc gcg gcc cag ccg gcc atg gcg (SEQ ID NO: 24). pEB32x contains the pelB signal peptide MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 25), encoded by atg aaa tac ctattg cct acg gca gcc gct gga ttg tta tta ctc gcg gcc cag ccg gcc atg gcg (SEQ ID NO: 26). scFv is expressed from these vectors as a fusion to the C-terminal domain of the phage coat protein pill. The vectors have the Lac promoter, pelB signal peptide, and chloramphenicol resistance gene for antibiotic selection.

[0149] The scFv construct used for expressing scFv in mammalian cells in Example 3 was designed to contain the Kozak sequence gccgccacc and the signal peptide MVLQTQVFISLLLWISGAYG (SEQ ID NO: 28, human Ig kappa chain V-IV region B17) at the N-terminus (as described by Vazquez-Lombardi et al., 1997) and a His6-tag at the C-terminus. The gene was ordered from TwistBioscience and cloned between the EcoRI and XbaI sites in the expression vector pTwistCMV Betglobin WPRE Neo (Twist Bioscience).

[0150] Table 1. Description of the scFv constructs evaluated in the study and their V H and V L In scFv, V H and V L Connected by a peptide linker GGGGSGAGGSGGGGTGGGGS (SEQ ID NO: 11).

[0151]

[0152]

[0153]

[0154] *Numbering is according to the Kabat numbering scheme (Professor Andrew CR Martin's group at UCL, 2022). CDR-H3 and CDR-L1 loops are in bold. Amino acid residues (Val, Ala, Gly or Gln) replaced by Cys are indicated by Italics and Underline Wire Replacement of cysteine ​​residues with Val and Ala Double underscore Mark.

[0155] Example 2. Expression of anti-HER2 ds-scFv in Escherichia coli

[0156] ScFv fragments LH_SS-, LH_SSC, HL_SSC, HL_S-C and LH_S-C are expressed in the periplasmic space of the XL1-Blue strain of Escherichia coli using pAK400 plasmid. ScFv is cultured in 300ml SB medium (containing 0.5% glucose, 10 μg / ml tetracycline and 25 μg / ml chloramphenicol) in a shake flask. The culture is grown at 37°C, 300rpm, and induced with 200 μM IPTG when OD600 is 0.5-1.0, and scFv is produced overnight at 26°C, 250rpm subsequently. Cells are harvested by centrifugation (15 minutes, 7000g, 4°C). In order to release periplasmic proteins from cells, the precipitate is resuspended in 30ml 20mM phosphate buffer pH 7.4, 300mM NaCl, 0.4mg / ml lysozyme, 10mM MgCl2, 25U / ml nuclease. After incubation at room temperature for 30 min, samples were freeze-thawed three times. Cell lysates were clarified by centrifugation (20 min at 2000 g) and used The supernatant was filtered through a filter. ScFv was purified from the cell lysate by Ni-NTA affinity chromatography using 0.5 ml Ni-NTA HisPur resin (Thermo Scientific) and then used Preparative size exclusion chromatography (SEC) was performed on a 75 10 / 300GL column (Cytiva, USA) using PBS pH 7.4 as elution buffer. The scFv concentration in the SEC fraction was quantified by OctetRED384 (ForteBio, USA) using the biolayer interferometry (BLI) technique. In the measurement, the streptavidin biosensor was coated with biotinylated protein L, which binds to the scFv. Purity was analyzed by SDS-PAGE.

[0157] After Ni-NTA purification, the product contained many impurities, so size exclusion chromatography (SEC) purification was required. After SEC purification, the yield of scFv with inter-ring disulfide bonds was more than 10 times lower than that of wild type (LH_SS-). The yield of scFv after Ni-NTA and SEC purification is shown in Table 2.

[0158] Table 2. scFv yields after protein purification in E. coli.

[0159]

[0160] a Expression yields were determined by Octet using the biolayer interferometry (BLI) technique. Streptavidin biosensors were coated with biotinylated protein L that bound the scFv. The values ​​represent values ​​from one independent measurement.

[0161] The formation of interdomain disulfide bonds in scFvs (LH_SS-, LH_SSC, HL_SSC, HL_S-C and LH_S-C) expressed in E. coli and purified by Ni-NTA and SEC were analyzed by SDS-PAGE using reducing and non-reducing sample buffers. These results are shown in Figure 2 middle.

[0162] Even after SEC purification, the purity level of the scFv versions LH_SSC and LH_S-C was still low. Therefore, possible inter-ring disulfide bond formation could not be assessed. The SEC fraction of LH_SS- also contained some minor impurities. HL_SSC and HL_S-C were able to be purified by Ni-NTA and SEC. Non-reducing SDS-PAGE analysis indicated the formation of inter-ring disulfide bonds, as the protein migrated faster (~23 kDa) than its reduced analog (~28 kDa) ( Figure 2 ).

[0163] Example 3. Expression of anti-HER2 ds-scFv in mammalian cells

[0164] ScFv fragments LH_SS-(hu4D5 scFv), HL_SS-(hu4D5 scFv), LH_SSC, HL_SSC, HL_S-C, LH_S-C, LH_SS+ and HL_SS+ were expressed in ExpiCHO TM The protein was expressed in cells (Thermo Scientific) from the vector pTwistCMVBetglobin WPRE Neo (Twist Bioscience).

[0165] Using ExpiFectamine TM ExpiCHO cells were transiently transfected with 3 μg of plasmid using the CHO (Thermo Scientific) transfection reagent and scFv was produced according to the manufacturer's maximum titer protocol.

[0166] ExpiCHO-S TM Cells were cultured in 2 ml volume in six-well plates (Nunclon Delta Surface, Thermo Scientific) with ExpiCHO TM Expression medium (Thermo Scientific) and sealed with breathable sealing tape (Nunc TM The cells were covered with 1% PEGylated Sealing Tapes (Thermo Scientific) and cultured in an incubator at +37°C on an orbital shaker at 125 rpm, relative humidity ≥ 80%, 8% CO2.

[0167] On the second day after transfection, add 12 μl of ExpiFectamine to each well of cells. TM CHO Enhancer and 320 μl ExpiCHO TM Feed. Transfer the plate to a +32°C incubator with 5% CO2 in humidified air and orbital shaking at 125 rpm. On day 5 post-transfection, add a second volume of ExpiCHO TM Feed (320 μl / well). Immediately transfer the plate to a +32°C incubator with humidified air containing 5% CO2 and orbital shaking. On day 13 after transfection, harvest the culture supernatant by centrifugation at 5000 x g for 30 minutes and use The supernatant was filtered using a HisPur with a 0.2 ml resin bed. TM ScFv was purified from cell lysate by Ni-NTA affinity chromatography using Ni-NTA spin columns (Thermo Scientific). The eluted fractions containing the majority of the protein were pooled and purified using a 10K MWCO Slide-A-Lyzer TM The buffer was exchanged to PBS pH 7.4 using a G2 dialysis cassette (Themro Scientific). The scFv yield was measured by A280 after protein purification from ExpiCHO cells, as shown in Table 3.

[0168] All scFv variants were expressed in ExpiCHO-S TM Cells are expressed (Table 3). These variants include trastuzumab scFv fragments (LH_SS- and HL_SS-) expressed in two directions, trastuzumab variants (LH_SSC and HL_SSC) with inter-ring disulfide bonds at H100B-L34 positions, trastuzumab variants (LH_S-C and HL_S-C) with inter-ring disulfide bonds at H100B-L34 positions and trastuzumab variants (LH_S-C and HL_S-C) without intra-domain disulfide bonds, and trastuzumab variants (LH_SS+ and HL_SS+) with disulfide bonds at H44-L100 positions. According to the literature, H44-L100 is considered to be a universal position for scFv stabilization (Weatherhill et al., 2012).

[0169] The yields of scFvs expressed in the HL orientation were 2-20 times higher than those expressed in the LH orientation (Table 3).

[0170] Table 3. scFv yields after protein purification from ExpiCHO cells.

[0171]

[0172] a The expression yield was determined by absorbance method: A280nm =1, corresponding to a scFv concentration of 0.58 mg / ml. The molar extinction coefficient of LH_SS- was determined by Vector NTI and used for the analysis of all scFv constructs. The values ​​are from one independent measurement.

[0173] Interdomain disulfide bond formation was assessed by SDS-PAGE analysis by electrophoresis of samples of purified scFv versions in reducing and non-reducing sample buffer. The gel was stained with Ready Blue Protein Gel Stain. These results are shown in Figure 3 Proteins with interdomain disulfide bonds migrate faster in non-reducing SDS-PAGE gels because they are packed more tightly than similar variants lacking interdomain disulfide bonds.

[0174] According to reducing SDS-PAGE analysis, the mammalian expressed Ni-NTA purified scFv does not contain any other protein impurities ( Figure 3 , wells are labeled with "R"). However, LH_SSC, HL_SSC, HL_S-C, LH_SS+, and HL_SS+ bands with higher molecular weights (~40 and ~60 kDa) were observed in the non-reduced samples, indicating the possible formation of disulfide bonds between two or more scFvs to form dimers and oligomers, respectively.

[0175] The H100B-L34 position enables full inter-loop disulfide bond formation, as confirmed by non-reducing SDS-PAGE analysis of LH_SSC, HL_SSC, HL_S-C, and LH_S-C: the scFv migrated faster in the gel (~23 kDa) and the samples did not contain any forms without disulfide bonds. However, the H44-L100 position is only partially capable of inter-domain disulfide bond formation, as forms without inter-domain disulfide bonds could be detected when the samples were electrophoresed under non-reducing conditions (~28 kDa). Figure 3 ).

[0176] Example 4. Antigen Binding Properties of Anti-HER2 ds-scFv

[0177] The antigen binding properties of purified anti-HER2 ds-scFv were analyzed by time-resolved immunofluorescence assay. In the assay, biotinylated HER2 was bound to a streptavidin-coated microtiter plate for 30 minutes, and the plate was washed four times. The purified scFv was then added and incubated for 1 hour. After four washes, the bound scFv was detected by the anti-His tag antibody 5-his (QIAGEN) labeled with Eu-N1 chelate (PerkinElmer, Finland). The plate was washed four times, DELFIA enhancement solution was added and incubated for 10 minutes, and then the time-resolved fluorescence was read with a Victor multilabel counter (PerkinElmer, Finland). These results are shown in Figure 4 middle.

[0178] from Figure 4 As can be seen in the Figures, at the concentrations used in the assay, the binding of the scFv variants with inter-loop disulfide bonds (LH_SSC, HL_SSC, HL_S-C, and LH_S-C) to HER2 was generally similar to that of the wild type (LH_S-C). A modest 1.5-fold increase in binding of HL_SSC and HL_S-C at 10 nM concentration was observed compared to the wild type (LH_SS-). The signal generated by LH_SSC was similar to that of the wild type, while the signal of LH_S-C was reduced by 1.5-fold compared to the wild type.

[0179] Using the Octet RED384 (ForteBio) system, the binding kinetics and preliminary Kd data of the purified scFv were obtained. 200ng / ml biotinylated HER2 (His, Avitag, Acro Biosystems, USA) was loaded onto a streptavidin-coated biosensor (ForteBio) for 600 seconds. The ScFv then bound for 600 seconds and dissociated for 7200 seconds. The measurements were performed at 30°C in a 100μl volume in a 384-well slanted bottom plate (ForteBio), with an oscillator speed of 1000rpm and a sensor offset of 4 or 6mm. All measurements were performed in PBS pH7.4, 0.1% BSA, 0.05% Tween-20. K was calculated using Octet data analysis software 8.2 D , k a and k d These results are as follows Figure 5 and Figure 6 As shown in Tables 4 and 5.

[0180] The introduction of an inter-loop disulfide bond in H100B-L34 maintained the high binding affinity of the wild-type to HER2 ( Figure 5 and Figure 6, Table 4 and Table 5). The trastuzumab scFv with interdomain disulfide bonds in H44-L100 (LH_SS+ and HL_SS+) also showed that high binding affinity (K d values ​​were in the subnanomolar range), which is consistent with previous results obtained using various scFvs stabilized by the interdomain disulfide bonds in H44-L100 (Weatherhill et al., 2012; Benschop et al., 2019).

[0181] Table 4. ExpiCHO-S assayed by Octet TM K of cell-expressed scFv D , k a and k d value

[0182]

[0183] a K D , k a and k d Values ​​were determined by monovalent analysis of two scFv concentrations (10 and 100 nM). The values ​​represent values ​​from one independent experiment. b k d The value is calculated in a window of 0-1600 seconds.

[0184] Table 5. K values ​​of scFv expressed in E. coli as determined by Octet D , k a and k d value

[0185]

[0186] K D , k a and k d The value is determined by: a Three scFv concentrations of 10, 60, and 200 nM were analyzed by monovalence; b Four scFv concentrations of 0.2, 2, 10, and 200 nM were analyzed by bivalency; c Five scFv concentrations of 0.2, 0.6, 10, 60, and 200 nM were analyzed by monovalence; d Five scFv concentrations of 0.2, 2, 10, 60 and 200 nM were analyzed by monovalence; and e Three scFv concentrations of 10, 60 and 200 nM were analyzed by monovalence. The values ​​in the table represent values ​​from a single independent experiment.

[0187] Example 5. Thermal stability of anti-HER2 ds-scFv

[0188] In a CFX96 real-time system equipped with a C1000 thermal cycler and Bio-Rad CFX Manager 3.1 software, thermal stability was measured using the Thermofluor assay. The total reaction volume was 25 μl. For the assay, 22.5 μl 5 μM scFv in PBS (or 0.35-2 μM for low-yield proteins) was mixed with 2.5 μl 50x SYPRO Orange dye (Sigma-Aldrich) diluted in PBS before the assay from a 5000x stock solution. The sample was heated from +25 to +95 °C (+0.5 °C increments) using a PCR system, and fluorescence in fluorescence resonance energy transfer (FRET) mode was measured. The midpoint temperature (T) of thermal denaturation was determined by melting peak analysis. m ), which plots the first derivative of fluorescence emission as a function of temperature (-d(RFU) / dT). These results are shown in Figure 7 and the Tm values ​​are shown in Table 6.

[0189] According to the results in Table 6, the scFv variants with an inter-loop disulfide bond at the H100B-L34 position (LH_SSC and HL_SSC) are significantly more stable than the wild type without an inter-domain disulfide bond (LH_SS- and HL_SS-). m The values ​​were +78.5°C and +77.5°C, respectively, which were +11°C and +9.7°C higher than the corresponding original scFv. L Removal of intradomain disulfide bonds in (HL_S-C) leads to thermal stability (T m =65.7°C) was slightly lower than that of the wild type (Tm=67.8°C).

[0190] The effect of the interdomain disulfide bonds (LH_SS+ and HL_SS+) at the H44-L100 position on thermal stability is not as significant as that of the inter-ring disulfide bonds at H100B-L34. m The values ​​were only 3-4°C higher than the wild type, but 7°C lower than the variant with the inter-loop disulfide bond in the new position H100B-L34.

[0191] Table 6. Thermal stability of single chain variable fragment variable constructs

[0192]

[0193]

[0194] The midpoint temperature of thermal denaturation (T m ) was determined by thermofluorescence. Values ​​represent the mean ± standard deviation of three repeated measurements.

[0195] Example 6. Phage display of anti-HER ds-scFv

[0196] Phage display of ds-scFv variants was studied in phagemid vectors pEB32x and pEB3V3. Phage was produced in E. coli XL1-Blue cells. Cells containing phagemid were inoculated into 20ml SB containing 0.5% glucose, 10 μg / ml tetracycline and 25 μg / ml chloramphenicol, and incubated at +37°C, 300rpm. When OD600 reached 0.4, VCS M13 helper phage was added to 20x multiplicity of infection, and the culture was incubated for 30 minutes without shaking. Cells were collected by centrifugation (10 minutes, 3200g, 4°C) and resuspended in 20ml SB containing 10 μg / ml tetracycline and 25 μg / ml chloramphenicol but not containing glucose. After shaking for 1 hour at 30°C, 30 μg / ml kanamycin and 100 μM IPTG were added, and phage was produced overnight at 26°C, 300rpm. Cells were removed by centrifugation and phages were precipitated from the supernatant by adding 1 / 5 volume of 20% PEG8000, 2.5M NaCl. Phages were precipitated by centrifugation (20 min, 10000 g, 4C). The precipitate was resuspended in TBS and any remaining precipitate was removed by centrifugation, and the phage precipitation with PEG / NaCl was repeated.

[0197] Phage display of scFv was analyzed by immunoassay, in which the binding of phage to biotinylated protein L ( Figure 8 ) (ProSpec, biotinylated with Ez-link NHS-PEG4 biotin, Thermo) and biotinylated HER2 ( Fig. 9) binding. Protein L is known to bind to the Kappa 1V gene family framework used in ds-scFv in a conformation-specific manner. The binding of phage to biotinylated HER2 was also measured. All reagents were diluted in Kaivogen red assay buffer. Biotinylated protein L (50ng / well) and HER2 (10ng / well) were added to streptavidin-coated microtiter plate wells, incubated for 30 minutes with slow shaking at room temperature, and then washed four times. 1e9 phage was added to the wells in triplicate, and the binding of phage to streptavidin wells was measured as a negative control. Phage was shaken at room temperature for 1 hour. The plate was then washed and Eu-N1-labeled anti-phage antibody (125ng / ml) was added and incubated for 1 hour to detect the bound phage. The plate was washed again 4 times, DELFIA enhancement solution was added and incubated for 10 minutes, and then time-resolved fluorescence was read by Victor 1420 multi-label counter (PerkinElmer, Finland). For the assay, phage titers were measured by immunoassay based on phage binding to uncoated maxisorb plates. Bound phage were detected using Eu-labeled anti-phage antibodies as described above. These results are shown in Figure 8 and Fig. 9 middle.

[0198] The highest phage display observed, measured by phage binding to protein L, was for the wild-type scFv (LH_SS-) containing only the native intradomain disulfide bonds. Figure 8 ). In LH_SSC phage, which has two natural intradomain disulfide bonds, an artificial interdomain disulfide bond was added between CDR-L1 and -H3, resulting in no scFv display on the phage surface. H -V L Domain sequence of ds-scFv with V removed L When the intradomain disulfide bonds in HL_S-C variant were removed, phage display was established. The phage displaying the folded scFv also recognized HER2. The signals from binding to protein L and HER2 were 27% and 53% of the corresponding wild-type phage, respectively ( Figure 8 and Fig. 9 ).

[0199] Example 7. Construction and validation of CDR-H3 library

[0200] The possibility of using disulfide-stabilized scFv as a framework for antibody libraries was investigated. The ds-scFv variant HL_S-C ( Figure 8 and Fig. 9) was used as the framework in the library, where the WGGDGFY (SEQ ID NO: 47) sequence of the CDR-H3 loop was diversified using NNS codons. In addition, various CDR-H3 loop lengths (13, 14, 15, 17 and 19 amino acids) were introduced into the library with similar randomization. Libraries with different loop lengths were constructed and studied separately.

[0201] The library was constructed by oligonucleotide directed PCR mutagenesis. ScFv was amplified in two PCR fragments to which LguI sites were added for ligation into the fragments in FASTR-type clones. NNS codons were added to one of the fragments by randomized oligonucleotides ( Fig.10 The two fragments were then PCR purified, digested with LguI and ligated into the complete scFv using T4 DNA ligase. The product was then further amplified, PCR purified, digested with SfiI and cloned into the vector pEB32x. The primers used in the library construction are shown in Table 7. The library construction strategy is as follows Fig.10 When the ligated library was transformed into E. coli XL1-Blue cells, 5.7x10 6 -4.4x10 7 The library size of each transformant.

[0202] Table 7. Primers used in PCR reactions

[0203]

[0204]

[0205] a The hybridization region is underlined. The LguI recognition site is in bold.

[0206] To explore the possibility of using a new phage antibody library with inter-loop disulfide bonds as a source for the discovery of novel binders against HER2, a loop library against HER2 was enriched by three consecutive rounds of panning against the target antigen. Biotinylated HER was immobilized on streptavidin-coated M280 dynabeads. Phages were allowed to bind for 1 h after four washes. The beads were washed four times and bound phages were eluted with trypsin. New phage stocks were generated by infecting E. coli XL1-Blue cells with the eluted phages and, after the cells were grown to an OD600 of 0.4, with VCS M13 helper phages. After overnight production at 26°C, phages were purified in the culture medium at 250 rpm by two subsequent PEG / NaCl precipitations.

[0207] The unselected phage library was analyzed by immunoassay for binding to protein L. These results are shown in Fig.11 The phage pool generated after each round of panning was tested for binding to HER2 by phage immunoassay. These results are shown in Fig.12 48 clones were picked from each library after three rounds of panning and single phage clones were generated overnight in 96-well cell culture plates. Cells were removed by centrifugation and culture supernatants containing phage were tested for binding to HER2 in a phage immunoassay. Phage binding to HER2 was detected by Europium-labeled anti-VCS M13 antibody. These results are shown in Fig.13 Several randomly selected clones from each library that showed binding to HER2 were sequenced. Some sequences are shown in Fig.14 middle.

[0208] Each unselected CDR-H3 randomized loop library showed binding to protein L ( Fig.11 ), indicating that scFvs with alternative loop lengths were also correctly folded and displayed on phage.

[0209] When the library was panned against HER2, the specific signal in the phage immunoassay gradually increased with each round of panning of the ring library ( Fig.12 ), indicating that (specific) binders to HER2 were enriched.

[0210] After three rounds of panning, the numbers of HER2-positive clones (S / B>5) in the 13, 14, 15, 16, 17, and 19aa loop libraries were 22 / 48, 30 / 48, 28 / 48, 26 / 48, 42 / 19 ( Fig.13 ). Some HER2-positive independent clones were randomly selected from the 13, 14, and 15 ring libraries for sequencing ( Fig.14 ), and found that a new sequence was obtained.

[0211] For those skilled in the art, as technology advances, the basic idea of ​​the invention can be implemented in various ways. Therefore, the present invention and its embodiments are not limited to the above-described embodiments, but can be varied within the scope of the claims.

[0212] References

[0213] Weatherill,E.E.,Cain,K.L.,Heywood,S.P.,Compson,J.E.,Heads,J.T.,Adams,R.&Humphreys,D.P.(2012)Towards a universal disulfide stabilisedsingle chainFv format:importance of interchain disulphide bond location andV L -V H orientation.Protein Eng Des Sel 25:321–329

[0214] Benschop,R.J.,Chow,C.K.,Tian,Y.,Nelson,J.,Barmettler,B.,Atwell,S.,Clawson,D.,Chai,Q.,Jones,B.,Fitchett,J.,Torgerson,S.,Ji,Y.,Bina,H.,Hu,N.,Ghanem,M.,Manetta,J.,Wroblewski,V.J.,Lu,J.&Allan,B.W.(2019)Development oftibulizumab,a tetravalent bispecific antibody targeting BAFFand IL-17A forthe treatment of autoimmune disease.MAbs 11:1175–1190

[0215] Krebber A,Bornhauser S,Burmester J,Honegger A,Willuda J,BosshardHR,Plückthun A.(1997)Reliable cloning of functional antibody variabledo-mains fromhybridomas and spleen cell repertoires em-ploying a reengineeredphage displaysystem.J.Immunol.Methods.201(1):35-55.

[0216] Huovinen T, M,Sanmark H,Brockmann EC,Azhayev A,WangQ, M, U.(2013)Two ScFv antibody libraries derivedfromidentical V L -V H framework with different binding site designs displaydistinctbinding profiles.Protein Eng Des Sel.26(10):683-93.

[0217] Vazquez-Lombardi R,Nevoltris D,Luthra A,Schofield P,Zimmermann C,Christ D.(2018)Transient expression of human antibodies in mammaliancells.Nat.Protoc.13(1):99-117.

Claims

1. A disulfide-stabilized single-chain variable fragment (ds-scFv) comprising: - A heavy chain variable domain (V H ),as well as - A light chain variable domain (V L ), Where V H and V L connected by a peptide linker in any orientation, and wherein CDR-L1 and CDR-H3 are linked to each other via an interdomain disulfide bond, and wherein V H or V L It has been engineered to lack native intradomain disulfide bonds.

2. The ds-scFv of claim 1, wherein the interdomain disulfide bonds are artificial.

3. The ds-scFv according to claim 1 or 2, wherein the interdomain disulfide bond is formed between a cysteine ​​residue at position -4 in CDR-H3, counted from the conserved tryptophan H103 in the framework region (FR-H4) following CDR-H3, and a cysteine ​​residue at position L34 in CDR-L1, all numbering being according to the Kabat numbering scheme.

4. The ds-scFv according to any one of claims 1 to 3, wherein the native intradomain disulfide bond lacking in the variable domain is located after the peptide linker.

5. The ds-scFv according to any one of claims 1 to 4, wherein the native intra-domain disulfide bonds lacking in the variable domain are numbered according to the Kabat numbering scheme in V L The disulfide bond formed between cysteine ​​residues L23 and L88 in V H A disulfide bond is formed between the cysteine ​​residues H22 and H92.

6. The ds-scFv according to any one of claims 1 to 4, wherein the peptide linker is at least 12 amino acids in length.

7. The ds-scFv according to any one of claims 1 to 5, wherein V H and / or V L The CDR sequences of were obtained from natural diversity, except for a cysteine ​​at residue -4 from the conserved tryptophan H103 of FR-H4 and a cysteine ​​at residue L34 of CDR-L1.

8. The ds-scFv according to any one of claims 1 to 6, wherein V H and / or V L The CDR sequences are designed completely or partially in silico.

9. The ds-scFv according to any one of claims 1 to 8, wherein one or more of the CDR sequences contain one or more randomized amino acids, provided that residue -4 from the conserved tryptophan H103 of FR-H4 and residue L34 of CDR-L1 according to the Kabat numbering scheme are cysteine, and the ds-scFv has a CDR sequence of 1 or more randomized amino acids. L Internal or V H Contains but not both contain native cysteine ​​intradomain disulfide bonds.

10. The ds-scFv according to any one of claims 1 to 9, wherein the V H The domain comprises a framework comprising FR-H1 of SEQ ID NO: 1, FR-H2 of SEQ ID NO: 2, FR-H3 of SEQ ID NO: 3, and FR-H4 of SEQ ID NO: 4, and the V L The domain comprises a framework comprising FR-L1 of SEQ ID NO:5, FR-L2 of SEQ ID NO:6, FR-L3 of SEQ ID NO:7, and FR-L4 of SEQ ID NO:8, wherein the framework is engineered such that one or both of amino acid residue 22 of SEQ ID NO:1 and amino acid residue 30 of SEQ ID NO:3 are not cysteine, or one or both of amino acid residue 23 of SEQ ID NO:5 and amino acid residue 32 of SEQ ID NO:7 are not cysteine; or wherein V H The domain comprises the amino acid sequence shown in SEQ ID NO: 48 or a functionally equivalent conservative sequence variant thereof, and V L The domain comprises the amino acid sequence shown in SEQ ID NO:49 or a functionally equivalent sequence variant thereof, which is engineered so that one or both of the amino acid residues at positions 22 and 98 of SEQ ID NO:48 are not cysteine, or so that one or both of the amino acid residues at positions 23 and 88 of SEQ ID NO:49 are not cysteine.

11. The ds-scFv according to any one of claims 1 to 10, further engineered so that V H or V L The native intradomain disulfide bonds missing in the PCR product were reintroduced.

12. The ds-scFv according to claim 11, wherein the V H The structural domain comprises a framework comprising FR-H1 of SEQ ID NO: 1, FR-H2 of SEQ ID NO: 2, FR-H3 of SEQ ID NO: 3, and FR-H4 of SEQ ID NO: 4, and the V L The structural domain comprises a framework comprising FR-L1 of SEQ ID NO: 5, FR-L2 of SEQ ID NO: 6, FR-L3 of SEQ ID NO: 7, and FR-L4 of SEQ ID NO: 8; or wherein said V H The domain comprises the amino acid sequence shown in SEQ ID NO: 48 or a functionally equivalent conservative sequence variant thereof, and the V L The domain comprises the amino acid sequence shown in SEQ ID NO: 49 or a functionally equivalent sequence variant thereof.

13. The ds-scFv according to any one of claims 1 to 12, wherein the ds-scFv is an anti-HER2 ds-scFv, preferably wherein CDR-H1 has the amino acid sequence SEQ ID NO: 12, CDR-H2 has the amino acid sequence SEQ ID NO: 13, CDR-H3 has the amino acid sequence SEQ ID NO: 14, CDR-L1 has the amino acid sequence SEQ ID NO: 15, CDR-L2 has the amino acid sequence SEQ ID NO: 16 and CDR-L3 has the amino acid sequence SEQ ID NO:

17.

14. The ds-scFv of claim 13, wherein the anti-HER2 ds-scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.

15. A molecular entity comprising one or more ds-scFv units according to any one of claims 1 to 14.

16. A molecular entity according to claim 15, wherein the molecular entity is a bispecific antibody, a diabody, a multispecific antibody, a CAR-T cell, a bispecific T cell engager (BiTE), a construct comprising a ds-scFv according to any one of claims 1-10 fused to a portion selected from an antibody fragment crystallizable (Fc) portion and other protein scaffold-based affinity reagents, a pharmaceutically active agent, a drug, a radioisotope, an enzyme or a chelator.

17. Use of the ds-scFv of any one of claims 1 to 16 for constructing a molecular entity selected from the following: a bispecific antibody, a diabody, a multispecific antibody, a CAR-T cell, a bispecific T cell engager (BiTE), and a construct, wherein the construct comprises the ds-scFv of any one of claims 1 to 10 fused to a portion selected from the Fc portion of an antibody and other protein scaffold-based affinity reagents.

18. A nucleic acid molecule encoding the ds-scFv of any one of claims 1 to 16.

19. A particle displaying the ds-scFv of any one of claims 1 to 16 on its surface.

20. A particle library displaying a plurality of different ds-scFvs according to any one of claims 1 to 16.

21. The particle of claim 19 or the library of particles of claim 20, wherein the particle is a bacteriophage particle, a yeast cell, a bacterial cell, a mammalian cell or a ribosome.

22. A method for preparing a particle library displaying a plurality of different ds-scFvs according to any one of claims 1 to 10 or 13, the method comprising: i) Encoding different heavy chain variable domains (V H ) polypeptides, all of which have a cysteine ​​residue at position -4 from the conserved tryptophan H103 according to the Kabat numbering scheme; ii) Encoding different light chain variable domains (V L ) polypeptides, each of which has a cysteine ​​residue at position L34 according to the Kabat numbering scheme; iii) cloning each of the plurality of first nucleic acids and one second nucleic acid into an expression vector, wherein the first and second nucleic acids may be arranged in any order and a nucleic acid encoding a peptide linker is inserted, thereby producing a plurality of different vectors; and iv) expressing the plurality of different vectors of step iii) on particles, thereby generating a first library of particles, each particle displaying a different ds-scFv polypeptide comprising a V encoded by the plurality of nucleic acids of steps i) and ii) H and V L Segment, where The plurality of first or second nucleic acids are engineered to lack native intradomain disulfide bonds in the encoded polypeptide.

23. The method of claim 22, wherein the variable domain of the encoded polypeptide following the peptide linker lacks a native intradomain disulfide bond.

24. The method according to claim 22 or 23, wherein: The first particle library is a phage display library.

25. The method according to any one of claims 22 to 24, wherein the method comprises introducing further diversity into a subset of the first particle library having the desired target binding properties, preferably by mutagenesis or V H or V L The second particle library is created by domain shuffling of the variable domains, preferably by shuffling of the variable domains after a peptide linker.

26. The method of claim 25, wherein the mutagenesis or domain shuffling results in the reintroduction of a disulfide bond within a native domain that was engineered to be deleted in the first or second nucleic acid.

27. The method according to claim 25 or 26, wherein the particles of the second particle library are cell particles, preferably yeast or mammalian cells, more preferably mammalian cells.

28. The method according to any one of claims 18 to 23, wherein all or part of each of the plurality of nucleic acids in step i) and / or step ii) is artificially designed and / or synthesized.

29. The method according to any one of claims 22 to 28, wherein all or part of each of the plurality of nucleic acids in step i) and / or step ii) is obtained from natural diversity, except for the nucleotides corresponding to the engineered cysteines at residue -4 from the conserved tryptophan H103 of FR-H4 and residue L34 of CDR-L1 in the encoded polypeptide.

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