Ultrastable antibody fragments with novel disulfide bridges

Abstract

The present invention relates to a recombinant library of particles displaying multiple single-chain variable fragments (scFv) against various antigens, such as HER2. The displayed single-chain variable fragments are characterized by improved stability due to non-natural interdomain disulfide bridges at novel positions. The present invention relates to methods for constructing such libraries, their use to obtain stabilized scFv with desired antigen-binding properties, and to such scFv. In some embodiments, the interdomain disulfide bridge is artificially introduced. In further embodiments, the artificially introduced disulfide bridge is formed between a cysteine ​​residue in CDR-H3 at position −4, counting from the conserved tryptophan H103 in FR-H4, and a cysteine ​​residue in CDR-L1 at position L34, according to the Kabat numbering scheme.

Description

[Technical Field]

[0001] The present invention relates to recombinant natural antibody fragments, more particularly to single-chain variable fragments with improved stability due to non-native interdomain disulfide bridges at novel positions. The invention also relates to recombinant expression libraries of such antibody fragments, methods for constructing such libraries, and their use for selecting stable antibody fragments with desired antigen-binding properties. [Background technology]

[0002] Single-chain fragment variable regions (scFvs) offer many advantages over full-length antibodies. They can be expressed cost-effectively and with high yields in microorganisms. Their small size also allows for the access of cryptic (hidden) epitopes. Furthermore, due to their small size, scFvs also have improved pharmacokinetic properties. scFv fragments penetrate tissues (such as tumors) more rapidly and evenly and have faster clearance, potentially making them beneficial for radiotherapy and in vivo diagnostic applications. Their small size allows for screening and selection by in vitro display methods, such as phage display, which avoids animal immunization. Furthermore, scFvs enable the production of various types of highly engineered antibody formats by genetically conjugating them to other entities, such as antibody fragments or intact antibodies.

[0003] Unfortunately, conventional scFv fragments are prone to variable and dynamic oligomerization and / or domain swapping, where two or more scFvs pair intermolecularly. Oligomerization has many effects on the functionality and stability of scFvs. For example, it increases the tendency of scFvs to aggregate, which can lead to various problems in handling and storage. Furthermore, oligomeric scFv molecules alter the affinity and activity for antigens due to avidity effects, complicating the medical use of scFvs and their in vitro use and diagnostic assay development. Oligomers can also crosslink targets in vivo, potentially resulting in altered pharmacodynamic effects.

[0004] High stability is one of the most important requirements for antibodies and antibody fragments in biomedical applications. Unfortunately, many scFvs are less stable than the corresponding full-length antibodies, and many scFvs have a V H -V L They are prone to denaturation and aggregation caused by exposure of less hydrophobic residues at the interface. In general, scFvs are also less thermostable than the corresponding intact antibodies or Fab fragments.

[0005] Stabilization of scFvs can be achieved, at least to some extent, by engineering stabilizing disulfide bridges into the scFv molecule. Indeed, to stabilize scFvs, V H and V L Previous studies have introduced disulfide bridges at various positions in scFv. However, this approach results in altered stability, and there is still a need for a means to improve the stability of scFv. Summary of the Invention

[0006] The present invention provides a heavy chain variable domain (V) comprising complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3). H ) and a light chain variable domain (V) containing complementarity determining regions CDR-L1, CDR-L2, and CDR-L3. L) and V H and V L are linked by a peptide linker in either orientation, CDR-L1 and CDR-H3 are linked to each other via an interdomain disulfide bridge, and V H or V L have been engineered to lack the naturally conserved intradomain disulfide bridges. In this context, the naturally conserved intradomain disulfide bridges are specifically designated V, VB, VC, VD, VE, VF, VH, VF ... L and / or V H Preferably, the native intradomain disulfide bridge is absent from the variable domain, which is located after the peptide linker.

[0007] In some embodiments, the interdomain disulfide bridges are artificially introduced.

[0008] In some further embodiments, the artificially introduced disulfide bridge is formed between a cysteine ​​residue in CDR-H3 at position -4, counting from the conserved tryptophan H103 in FR-H4, and a cysteine ​​residue in CDR-L1 at position L34, according to the Kabat numbering scheme.

[0009] In some embodiments, the peptide linker is at least 12 amino acids in length.

[0010] In some embodiments, V H and / or V L The CDR sequences of V are derived from natural diversity, excluding residue −4 from the conserved tryptophan H103 in FR-H4 and the cysteine ​​at residue L34 in CDR-L1 according to the Kabat numbering scheme. H and / or V LThe CDR sequences of V are designed completely or partially in silico, but in some further embodiments, H and / or VL In both cases, a cysteine ​​residue in CDR-H3 at position -4 and a cysteine ​​residue in CDR-L1 at position L34, counting from the conserved tryptophan H103 in FR-H4, according to the Kabat numbering scheme, must be present.

[0011] 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 of the ds-scFv 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 in this order, and 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 SEQ ID NO:8, in that order. In some further embodiments, the framework is a functionally equivalent conservative sequence variant of the referenced sequences. According to some aspects of the invention, i.e., ds-scFvs lacking other native intra-domain disulfide bridges, the framework has been engineered to not contain both the native cysteines at positions corresponding to residue 22 of SEQ ID NO:1 and residue 30 of SEQ ID NO:3, or the native cysteines at positions corresponding to residue 23 of SEQ ID NO:5 and residue 32 of SEQ ID NO:7.

[0012] In some embodiments, the V of the ds-scFv H The domain comprises the amino acid sequence set forth in SEQ ID NO: 48, LThe domain comprises the amino acid sequence shown in SEQ ID NO: 49, or a functionally equivalent conservative sequence variant of the described sequence. According to some aspects of the present invention, i.e., a ds-scFv lacking other native intra-domain disulfide bridges, said SEQ ID NO: 48 has been engineered to not contain both native cysteines at positions corresponding to residues 22 and 98, thereby forming a V H Alternatively, SEQ ID NO: 49 may be engineered to not contain both of the native cysteines at positions corresponding to residues 23 and 88, thereby preventing the formation of native intradomain disulfide bridges within V L Prevents the formation of native intradomain disulfide bridges within the

[0013] In some further embodiments described above, the ds-scFv is an anti-HER2 ds-scFv. Preferably, the anti-HER2 ds-scFv comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 12, CDR-H2 having the amino acid sequence of SEQ ID NO: 13, CDR-H3 having the amino acid sequence of SEQ ID NO: 14, CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and CDR-L3 having the amino acid sequence of SEQ ID NO: 17. In some more specific embodiments, the anti-HER2 ds-scFv comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0014] The present invention also provides molecular entities comprising one or more ds-scFv units according to various embodiments of the invention, and the use of ds-scFvs according to any embodiment of the invention to construct such molecular entities. In some embodiments, the molecular entities are bispecific antibodies, diabodies, multispecific antibodies, CAR-T cells, bispecific T-cell engagers (BiTEs), or constructs comprising a ds-scFv of the invention fused to a moiety such as the fragment crystallizable (Fc) portion of an antibody or an alternative protein scaffold-based affinity reagent such as a designed ankyrin protein (DARPin), nanobody, or affibody. The molecular entity may also be, for example, a pharmaceutically active agent, drug, radioisotope, enzyme, or chelator.

[0015] In a still further aspect, the present invention provides a nucleic acid molecule encoding a ds-scFv according to any embodiment of the invention.

[0016] In yet a further aspect, the present invention provides particles that display on their surface a ds-scFv according to any embodiment of the invention, and libraries of such particles, wherein the particles display a plurality of different ds-scFvs of the invention. Preferably, the particles are phage particles, yeast cells, bacterial cells, mammalian cells, or ribosomes.

[0017] Furthermore, the present invention provides a method for generating a library of particles displaying a plurality of different ds-scFvs according to various embodiments of the invention, comprising generating a library of particles displaying different heavy chain variable domains (V) that all have a cysteine ​​residue at position -4, counting from the conserved tryptophan H103, according to the Kabat numbering scheme. H and manipulating a plurality of first nucleic acids encoding different light chain variable domains (V) polypeptides, all having a cysteine ​​residue at position L34 according to the Kabat numbering scheme. Land engineering only one or more second nucleic acids encoding the polypeptide. Each of the multiple first nucleic acids is then cloned into an expression vector in any order, along with only one second nucleic acid or one of the multiple second nucleic acids, including a nucleic acid encoding an intervening peptide linker, including, but not limited to, a peptide linker such as one comprising or consisting of SEQ ID NOs: 9-11. When multiple second nucleic acids are used, the combination of the first and second nucleic acids is random. The cloning results in a plurality of different vectors, which are then expressed on particles, thereby generating a first library of particles, each particle comprising a V encoded by a first nucleic acid and the second nucleic acid. H and V L Preferably, the first library of particles is a phage display library.

[0018] In this method, either the first nucleic acid or the second nucleic acid is engineered to lack native intradomain disulfide bridges in the encoded polypeptide. In this context, native intradomain disulfide bridges are specifically those located at positions V, V, VB, VC, VD, VE, VF, VH, VF ... L and / or V H Preferably, the native intradomain disulfide bridge is absent from the variable domain located after the peptide linker in the encoded polypeptide.

[0019] In some embodiments, either the first nucleic acid or the 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 involved in interdomain disulfide bridge formation and the remaining intradomain disulfide bridges remain unaltered.

[0020] In some embodiments, the method may include introducing additional diversity into a subset of said first library of particles having desired target binding properties, thereby generating a second library of particles, preferably a library of cellular particles, more preferably a library of yeast or mammalian cells, even more preferably a library of mammalian cells. H or V L The second library may be introduced by mutagenesis or domain shuffling of either the variable domains located after the peptide linker. Preferably, shuffling of the variable domains located after the peptide linker is used. In particular, the mutagenesis or domain shuffling preferably results in the reintroduction of cysteines that form natural intradomain disulfide bridges in the polypeptides displayed by the second library.

[0021] In some embodiments, all or a portion of the plurality of first nucleic acids and / or one second nucleic acid, or all or a portion of the plurality of second nucleic acids, are optionally artificially designed and / or synthetically derived. In some other embodiments, optionally all or a portion of the first nucleic acid and / or one second nucleic acid, or all or a portion of the plurality of second nucleic acids, are derived from natural diversity, except for engineered nucleotides corresponding to residue −4 from tryptophan H103 in FR-H4 and the cysteine ​​in CDR-H3 at residue L34 of CDR-L1 in the encoded polypeptide.

[0022] Further aspects, embodiments and details are described in the following figures, detailed description and examples. [Brief explanation of the drawings]

[0023] The accompanying drawings are provided to provide a further understanding of the invention, constitute a part of this specification, illustrate embodiments of the invention, and together with the description, serve to explain the principles of the invention.

[0024] [Figure 1]FIG. 1 is a schematic diagram of the scFv constructs used in the examples. [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 bridges migrate faster in the non-reduced form (N). Lady Blue protein gel stain was used to stain the gel. 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 run in the non-reduced form. [Figure 3] SDS-PAGE of purified scFv variants LH_SS-, HL_SS-, LH_SSC, HL_SSC, HL_SSC, HL_S-C, LH_S-C, LH_SS+, and HL_SS- expressed in ExpiCHO is shown. Variants with interdomain disulfide bridges grow faster in the non-reduced form (N). Lady Blue protein gel stain was used to stain the gel. 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 run in the non-reduced form. [Figure 4] Immunoreactivity of scFv constructs against HER2 is shown. Each bar represents the mean ± standard deviation of triplicate values. Specific signals were calculated by subtracting the signal obtained from streptavidin wells from the signal obtained from wells containing biotinylated HER2. Concentrations of scFv: 10 nM (black), 100 nM (patterned), and 300 nM (white). [Figure 5]Figure 1 shows binding of scFv constructs produced in E. coli to HER2 as detected by biolayer interferometry (BLI) (OctetRED384). The y-axis represents binding (nm) and the x-axis represents time (s). [Figure 6] Figure 1 shows binding of scFv constructs produced in the Expi-CHO™ system to HER2 as detected by biolayer interferometry (BLI) (OctetRED384). The y-axis represents binding (nm) and the x-axis represents time (s). [Figure 7] Figures 7A and 7B show derivative plots showing the midpoints of the melting transitions (Tm) of scFvs expressed in the LH (7A) and HL (7B) orientations: LH_SS- and HL_SS- (filled circles), LH_S-C and HL_S-C (open triangles), LH_SSC and HL_SSC (filled squares), and LH_SS+ and HL_SS+ (open diamonds). The negative control (buffer only) is marked as a straight line in the figure. [Figure 8] Figure 1 shows the effect of signal sequence and scFv construct on phage display levels, as assessed by binding to protein L. The specific signal, reported 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. [Figure 9] Figure 1 shows the influence of signal sequence and scFv construct on binding to HER2. All phage stocks were used in the study with 5x109 cfu / ml phage. Each bar represents the mean ± standard deviation of triplicate values. Black: pEB32x, white: pEB3V3 with a modified pelB signal sequence. [Figure 10]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 seq rev, respectively. PCR A and B products were digested with LguI to form cohesive ends, ligated, and the scFv library was generated using a modified FASTR reaction. [Figure 11] Binding of loop library phage stocks to protein L in a phage immunoassay is shown, indicating phage display levels of scFv. Each bar represents the mean ± standard deviation of triplicate values. [Figure 12] Figure 1 shows the results of a phage immunoreactivity assay of an scFv library panned against biotinylated HER2 as a specific signal for HER2. Each bar represents the mean ± standard deviation of triplicate values. Black: panning round 1, patterned: panning round 2, white: panning round 3. [Figure 13] Figure 1 shows that individual clones isolated from the library after panning round 3 generate specific signals 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 streptavidin wells from the signal obtained from wells containing biotinylated HER2. [Figure 14] Figure 1 shows single clone sequence alignment of the CDR-H3 loop region of HER2-specific clones isolated after panning round 3. The randomized region is in bold. A non-native cysteine ​​(at position H100B in CDR-H3 of template ds-ScFv HL_S-C) is indicated by an *. DETAILED DESCRIPTION OF THE INVENTION

[0025] (definition) Before describing the present invention, it is to be understood that this disclosure is not limited to any particular compositions, reagents, devices, protocols, or methodology described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] 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.

[0027] It should be further noted that certain features of the present disclosure, which are for clarity described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are for brevity described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. Furthermore, any feature, detail, or embodiment disclosed in the context of ds-scFvs provided herein also applies, where applicable and even if not repeated, to the libraries of such ds-scFvs provided herein, and vice versa.

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and "the" mean one or more. Thus, unless otherwise specified, singular nouns also have the meaning of the corresponding plural nouns, and vice versa. Thus, the terms "a," "one or more," and "at least one" can be used interchangeably.

[0029] The term "and / or" in phrases such as "X and / or Y" should be understood to mean either "X and Y" or "X or Y" and should be interpreted as providing explicit support for both meanings or either meaning.

[0030] The terms "comprising," "including," and "having" may be used interchangeably.

[0031] As used herein, the term "antibody" refers to the immunoglobulin G structure, which contains two identical heavy chains (approximately 50-60 kDa) and two identical light chains (approximately 23 kDa) organized into globular structural motifs called Ig-fold domains. The heavy chains contain three constant Ig-fold domains (C H1 , C H2 , and C H3 ) and one variable Ig-fold domain (V H ), and the light chain has one constant (C L ) and one variable (V L ) Ig-fold domains. Each domain consists of two antiparallel β-sheets formed from 7-9 antiparallel β-strands connected by loops. Ig-fold domains are stabilized by highly conserved intradomain disulfide bridges formed between cysteine ​​residues in the two antiparallel β-sheets. Typically, C H1 and C H2 Two or four covalent disulfide bridges in the hinge region between the two heavy chains connect the heavy and light chains, while the heavy and light chains are C H1 and C L As used herein, "V" refers to a group of amino acids that are connected via a covalent disulfide bridge linking the amino acids. H " and "V L " and "V H Domain" and "V L The term "domain" is interchangeable with the term "domain."

[0032] As used herein, the term "complementarity-determining region" (CDR) refers to the highly variable regions in the variable domains of an antibody. Each variable domain has three CDRs, L In the domain, there are CDR-L1, CDR-L2, and CDR-L3, and V HIn the domain, there are CDR-H1, CDR-H2, and CDR-H3. All CDRs are collectively involved in antigen recognition and binding. However, CDR-H3 is usually considered the most important CDR involved in antigen binding because it is the most variable in loop length and sequence. As used herein, the terms "CDR" and "CDR loop" are interchangeable.

[0033] As used herein, the term "framework" (FR) refers to the non-CDR portions of a variable domain (β-sheets and non-hypervariable loops). It provides structural support for the antigen-binding site but also influences the CDR loop conformation. In the primary structure of an antibody (i.e., its linear amino acid sequence), the term "framework" refers to the amino acid sequences intervening between the CDRs. Thus, each variable domain has four framework regions, the V L The domains are FR-L1, FR-L2, FR-L3 and FR-L4. H The domains are FR-H1, FR-H2, FR-H3 and FR-H4.

[0034] As used herein, the term "humanized framework" refers to a framework of human origin that has typically been engineered to contain several amino acid changes compared to the original framework of human origin. Since the amino acid modifications are non-natural to the original framework of human origin, they can be represented as amino acids of non-human origin. Typically, humanized frameworks are used in humanized antibodies, i.e., antibodies in which CDR regions of non-human origin are incorporated into a framework of human origin. The purpose of humanizing a framework, i.e., introducing amino acids of non-human origin, is to ensure that the functional properties of the humanized antibody correspond to those of the parent non-human antibody from which the CDR sequence is derived. Those skilled in the art are familiar with methods for humanizing a given framework for a given parent non-human antibody.

[0035] Human Immunoglobulin V L or V HDomains exist in different subtypes, as is generally known in the art. L For V, the framework essentially contributes to the framework of subtype kappa I, as is well known to those skilled in the art. H For example, the framework essentially corresponds to a subtype III framework, as is also well known to those skilled in the art. In some further embodiments, the framework is a "consensus framework," i.e., a framework that is consistent with each existing V L and V H Human immunoglobulin V within subtypes L or V H The framework may represent the most frequently occurring amino acid residues in the framework sequence.

[0036] As used herein, the term "conservative sequence variant" refers to an amino acid sequence containing modifications that do not significantly alter the structural or functional properties of the antibody in question. Conservative amino acid sequence variants include variants resulting from amino acid substitutions with similar amino acid additions. As is well known in the art, the similarity may be determined based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. Conservative amino acid sequence variants also include variants containing small amino acid deletions and / or insertions. Preferably, conservative sequence variants encompassed by the present invention can be referred to as "functionally equivalent conservative sequence variants." Those skilled in the art can readily determine whether a given sequence variant is functionally equivalent.

[0037] All numbering of amino acid positions comprising variable domains and identification of CDRs used herein are according to the Kabat numbering scheme unless otherwise specified. The Kabat numbering scheme is well known to those skilled in the art. Thus, for example, the expression "H100B-L34" refers to a polypeptide having a cysteine ​​in CDR-H3 at position H100B and a cysteine ​​in CDR-L1 at position L34 according to the Kabat numbering scheme. Similarly, the expression "H44-L100" refers to a polypeptide having a cysteine ​​in FR-H2 at position H44 and a cysteine ​​in FR-L4 at position L100 according to the Kabat numbering scheme. It should be noted that the position assignments according to the Kabat numbering scheme refer to specific amino acid positions defined by sequence conservation, and do not refer to the actual amino acid positions in the linear amino acid sequence of a given antibody.

[0038] More generally, the H100B is a V H It corresponds to amino acid position -4, counting from highly conserved tryptophan H103 in the polypeptide, and this tryptophan is the first amino acid of FR-H4. Such an indirect definition for the CDR-H3 cysteine ​​moiety involved in the non-native interdomain disulfide bridge is necessary because, as described later in this specification, the length of the CDR-H3 loop can vary, thereby altering the Kabat numbering of the cysteine ​​residues. In particular, an expression such as "position -4, counting from tryptophan H103 in FR-H4" is not affected by sequence variations before said position -4.

[0039] It should be noted that in the context of the present invention, the last two amino acids of FR-H3 according to the Kabat numbering scheme are considered to be the first two amino acid residues of CDR-H3. Thus, FR-H3 is shorter by two amino acid residues than conventionally considered by the Kabat numbering scheme, but the CDR-H3 loop is longer by two amino acid residues. In other words, using Kabat numbering, FR-H3 is defined herein by amino acid residues H66-H92, and CDR-H3 is defined by amino acid residues H93-H102. The corresponding conventional definitions are H66-H94 and H95-H102, respectively. It should be noted that the Kabat numbering scheme allows for length variation within CDR-H3 without affecting the definition of CDR-H3 by Kabat numbering. In other words, regardless of any variation in loop length, CDR-H3 is comprised of amino acids H93-H102 according to the Kabat numbering scheme.

[0040] As used herein, the term "fragment variable" (Fv) refers to a V L Domain and V H Fv fragments refer to antibody fragments consisting of V domains. L and V H Because the domains are not covalently linked to each other via peptide linkers or disulfide bonds, they are inherently unstable.

[0041] As used herein, the term "single-chain fragment variable" (scFv) refers to a V fragment consisting of Vs connected by a flexible peptide linker that stabilizes the structure. L and V H This refers to a recombinant Fv fragment composed of a single domain. One of the most significant drawbacks of conventional scFvs, which prevents them from reaching their full potential, is that their stability can still be problematic, despite the presence of stabilizing peptide linkers. Furthermore, many conventional scFvs are prone to aggregation and variable and dynamic oligomerization. Furthermore, they are less thermostable than the corresponding complete antibodies.

[0042] As used herein, the term "disulfide-stabilized Fv fragment" (dsFv) refers to a V fragment consisting of V fragments connected to each other via disulfide bridges. L and V H It refers to an antibody fragment composed of domains.

[0043] As used herein, the term "disulfide-stabilized single-chain fragment variable" (ds-scFv) refers to a group of Fv fragments connected in either orientation by a flexible peptide linker (V L -Linker-V H or V H -Linker-V L ), further stabilized by an artificial interdomain disulfide bridge, V L and V H The term "Fv fragment" refers to a recombinant Fv fragment composed of two or more domains. In the present invention, the stabilizing interdomain disulfide bridge is located at a novel position between the CDR-H3 loop and the CDR-L1 loop. Due to its location, the interdomain disulfide bridge of the present invention may also be referred to as an H3 / L1 interloop disulfide bridge.

[0044] As used herein, the term "disulfide bridge" refers to a covalent bond between two sulfur atoms (-SS-) formed by the coupling of two thiol (-SH) groups. Cysteine, one of the 20 proteinogenic amino acids, has an SH group in its side chain and can readily dimerize to cysteine ​​in aqueous solution by forming a disulfide bond.

[0045] Two types of disulfide bridges are relevant in the present invention, depending on their location. The first type, i.e., the "intradomain disulfide bridge", is H and V L This refers to the highly conserved native disulfide bridge formed between cysteine ​​residues in the two antiparallel β-sheets within V. H and V L In both of these, the intradomain disulfide bridges are located in the first and third framework regions, i.e., V H So, let's compare the FR-H1 and FR-H3. LNow, connect FR-L1 and FR-L3. V H In V, the bridge-forming cysteines are at positions H22 and H92 according to the Kabat numbering scheme, but L In the , cysteines are at positions L23 and L88.

[0046] The second type of disulfide bridge, i.e., the "interdomain disulfide bridge", is V H and V L " refers to a non-natural disulfide bridge between the CDR-H3 and CDR-L1 loops, more specifically between the CDR-H3 and CDR-L1 loops. The disulfide bridge is "artificial" in the sense that the cysteine ​​residues that form the bridge have been artificially introduced (i.e., engineered) into the scFv in question by recombinant techniques, thereby resulting in the corresponding ds-scFv. It should be understood that there is nothing artificial or non-natural about the cysteine ​​residues themselves or the disulfide bridges formed between cysteine ​​residues. As used herein, the term "artificial" is interchangeable with the term "non-natural." In the context of the ds-scFvs of the present invention comprising a stabilizing disulfide bridge between the CDR-L1 and CDR-H3 loops, the term "inter-domain disulfide bridge" is interchangeable with the term "inter-loop disulfide bridge."

[0047] According to the above description, the ds-scFv of the present invention has three disulfide bridges (V H One inside, V L One in, and V H and V L However, some embodiments of the present invention relate to so-called intermediate ds-scFvs that contain only one inter-domain disulfide bridge and one intra-domain disulfide bridge, as described in detail herein below.

[0048] As used herein, the term "transition temperature" (Tm) refers to the temperature at which 50% of a macromolecule, e.g., an antibody, is denatured and is considered to be a standard parameter for describing the thermal stability of proteins.

[0049] As used herein, the term "trastuzumab" (also known as hu4D5) refers to a recombinant humanized version of the murine anti-HER2 antibody 4D5. Trastuzumab is approved for the treatment of HER2 receptor-positive breast cancer and gastric cancer. An scFv fragment of trastuzumab (hu4D5-8) also exists.

[0050] As used herein, the term "human epidermal growth factor receptor 2" (HER2, also known as neu) refers to a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. It is an oncogene found 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 grades of breast cancer. It is an important biomarker and target for treatment of approximately 30% of breast cancer patients. Overexpression of HER2 is also known to occur in invasive forms of ovarian cancer, gastric cancer, lung adenocarcinoma, and uterine cancer.

[0051] As used herein, the term "recombinant expression library" refers to a collection of antibodies or antibody fragments, such as ds-scFvs of the invention containing one interdomain and two intradomain disulfide bridges or intermediate ds-scFvs containing one interdomain and one intradomain disulfide bridge, displayed on a heterologous host particle (such as a phage particle, a ribosome, or a cell surface, e.g., yeast, bacterial, or mammalian cells) 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, diversity may depend on the display system in question. For example, it may be difficult to obtain a diversity of more than 1E5 in a mammalian cell expression library.

[0052] As used herein, the "E" in an expression such as "1E5" is an exponent and indicates that the number is to be multiplied by 10 to the power of the "subsequent number." In other words, for example, the expression "1E5" means "1 x 10 5 " and is equal to 100,000.

[0053] (Detailed explanation) The present invention relates to recombinant native antibody fragments, more particularly single-chain variable fragments with improved stability due to an interdomain disulfide bridge at a novel position, namely between CDR-H3 and CDR-L1. Preferably, the interdomain disulfide bridge is formed via cysteines at position -4 and L34, counting from tryptophan H103, according to the Kabat numbering scheme.

[0054] In addition to the novel stabilizing interdomain disulfide bridge, the ds-scFv of the present invention contains two native intradomain disulfide bridges, one in V H Inside, one V L However, in some aspects of the invention, the ds-scFv of the invention are engineered to contain only one of the native intra-domain disulfide bridges.

[0055] V lacks native intradomain disulfide bridges H or V L Engineering either one of the cysteine ​​residues into a single domain can be achieved by mutating one or, preferably, both of the cysteine ​​residues normally involved in disulfide bridge 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 herein below, which relate to ds-scFvs of the invention engineered to contain only one native intra-domain disulfide bridge, including the specifically mentioned SEQ ID NOs.

[0056] The CDR loops of the present ds-scFvs can vary in both amino acid composition and length. CDR diversity can be derived from natural or non-natural sources, or both. Natural sources include B cells from immunized or non-immunized human or animal subjects, while non-natural diversity can be designed in silico and genetic material can be synthesized. Natural and in silico designed CDR diversity can also be combined, with or without CDR randomization. Furthermore, in some embodiments, the CDR sequences correspond to the CDR sequences of existing antibodies or antigen-binding fragments thereof, such as scFvs or Fabs. In particular, the source of one or more heavy chain CDRs can be different from the source of one or more light chain CDRs.

[0057] In some embodiments, the ds-scFv of the invention comprise a CDR-H3 region whose length is at least 12 amino acids, and in some particular embodiments, 13 amino acids. Such CDR-H3 regions are particularly suitable for incorporation of the stabilizing interdomain disulfide bridge of the invention between CDR-L1 and CDR-H3.

[0058] For CDR randomization, one or more CDR loops are typically randomized at positions most likely to contribute to antigen recognition and binding. Therefore, the preferred order of randomization is typically 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 the preferred order is not intended to be limiting, and CDR loops can be selected independently of each other for randomization. There is no limit to the number of amino acid positions randomized. It should be understood that randomization is not limited to amino acid substitutions at given amino acid positions, but may also include amino acid insertions and / or deletions at those positions, thereby potentially altering the CDR loop length.

[0059] 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 diversity. Preferably, the amino acid modification is located upstream from the cysteine ​​at residue -4, counting from tryptophan H103 conserved in FR-H4 according to the Kabat numbering scheme. It is important that the CDR randomization does not include the cysteine, because otherwise, the formation of interdomain disulfide bridges will be impossible. For CDR-H1, if present, the number of randomized amino acid positions is typically at least one, preferably two or more. For CDR-H2, if present, the number of randomized amino acid positions is typically at least two, preferably four or more, more preferably six or more.

[0060] For CDR-L1, the number of randomized amino acid positions, if present, is preferably at least one. It is important that CDR randomization does not involve the cysteine ​​residue at position L34 of CDR-L1 according to the Kabat numbering scheme, because otherwise the formation of a stabilizing interdomain disulfide bridge would be impossible. For CDR-L3, the number of randomized amino acid positions, if present, is typically at least two. For CDR-L2, the number of randomized amino acid positions, if present, is typically one or more.

[0061] The CDR regions of the present ds-scFv can be embedded in any suitable 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-grafting studies. The amino acid sequences of the trastuzumab framework are set forth in SEQ ID NOS: 1-8, such that SEQ ID NOS: 1 represents FR-H1, 2 represents FR-H2, 3 represents FR-H3, 4 represents FR-H4, 5 represents FR-L1, 6 represents FR-L2, 7 represents FR-L3, and 8 represents FR-L4. Thus, in some embodiments, the ds-scFv of the present invention comprises a VFv comprising the framework regions of SEQ ID NOS: 1, 2, 3, and 4, in that order. H domain and the framework regions of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 in this order. VL Includes domain. V H In the domains, the cysteine ​​residues that form native intradomain disulfide bridges are located in FR-H1 and FR-H3, more specifically at position 22 in SEQ ID NO: 1 and at position 30 in SEQ ID NO: 3, which correspond to positions H22 and H92, respectively, according to the Kabat numbering scheme in ds-scFv. L In the domain, the cysteine ​​residues forming the native intra-domain disulfide bridges are located at FR-L1 and FR-L3, more specifically at position 23 of SEQ ID NO: 5 and at position 32 of SEQ ID NO: 7, which correspond to positions L23 and L88 according to the Kabat numbering scheme in ds-scFv, respectively. According to some embodiments of the present invention, i.e., ds-scFvs lacking other native intra-domain disulfide bridges, the framework is H or V L Either of the above has been engineered to not contain one or both of the native intra-domain disulfide bridge-forming cysteine ​​residues.

[0062] However, the framework need not be 100% identical to the sequences disclosed above and may be varied so long as the functional or structural properties of the ds-scFv remain essentially unchanged. Thus, in some embodiments, one or more of the 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 to the above sequences. Furthermore, the framework may be based on or essentially correspond to any framework encoded by V and J gene segments. Consensus human framework regions may also be used, for example, as described in U.S. Pat. No. 6,300,064.

[0063] In particular, when the framework of trastuzumab, or a functionally equivalent conservative sequence variant of said framework, is used, tryptophan H103 according to the Kabat numbering scheme used to define 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.

[0064] In some embodiments using the trastuzumab framework, the ds-scFv of the invention comprises a V of SEQ ID NO: 48, mediated by a peptide linker. H and V set forth in SEQ ID NO: 49 Lin any order. In such embodiments, the CDR loops can have various amino acid compositions and lengths depending on their target antigens, with CDR-H1 being 5 amino acids long, CDR-H2 being 16 to 19 amino acids long, preferably 17 amino acids long, CDR-H3 being 12 to 21 amino acids long, preferably 13 amino acids long, CDR-L1 being 11 amino acids long, CDR-L2 being 7 amino acids long, and CDR-L3 being 9 to 11 amino acids long, preferably 9 amino acids long. The fourth-to-last amino acid in CDR-H3 (i.e., the amino acid residue at position -4, counting from the first amino acid of FR-L4) must be cysteine. The last amino acid in CDR-L1 (i.e., the amino acid residue at position -1, counting from the first amino acid of FR-L2) must also be cysteine. Functionally equivalent conservative sequence variants of the above ds-scFvs are also encompassed by the present invention.

[0065] In these embodiments where the CDR-H3 is composed of 13 amino acids, the amino acid at position -4, counting from tryptophan H103, corresponds to the amino acid at position H100B according to the Kabat numbering scheme. In some embodiments, the cysteine ​​residues forming the artificial disulfide bridge are at positions H100B and L34 according to the Kabat numbering scheme.

[0066] It will be understood that the ds-scFv of the present invention can be non-human (e.g., mouse, rabbit, goat), chimeric, humanized (non-human CDRs incorporated into humanized framework regions), or fully human (both the framework and CDRs are derived from a human antibody). However, particularly for therapeutic purposes, humanized and fully human ds-scFvs are preferred. For diagnostic and some other non-therapeutic purposes, the ds-scFv need not be humanized or fully human, but may be non-human or chimeric ds-scFv as well. In particular, V H The source and type of V L It may be different from that of

[0067] V in this ds-scFv Hand V L The domains are connected by a flexible linker peptide, typically 15-20 amino acids in length. However, in some embodiments, the linker may be 12-15 amino acids long or even shorter, while in some other embodiments, the linker may be longer than 20 amino acids, e.g., 25 or even 30 amino acids. The linker minimizes oligomerization of ds-scFvs while maintaining a distance between the C-terminus of one variable domain and the N-terminus of the other domain that favors proper folding and formation. It is generally believed that short linkers (typically 12-15 amino acids or less) prevent physical association of two V domains within the same polypeptide, resulting in the formation of multimers, while long linkers (typically longer than 20 amino acids) may favor proteolysis or weak domain association in scFvs.

[0068] In some embodiments, multimers of the present ds-scFvs may be desirable. For example, it is envisioned that the present ds-scFvs may be used to generate diabodies, i.e., bispecific antibodies composed of 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 none of the scFv chains can form functional scFvs by themselves, thereby inducing the formation of diabodies composed of different scFv partners. Linkers of only 1-4 amino acids result in the formation of primarily trimeric and tetrameric structures.

[0069] Typically, in ds-scFvs, the linker peptide is primarily composed of glycine and serine residues. The 15-amino acid (glycine-serine) peptide linker (also designated (Gly-Ser) or (GGGGS) linker) shown in SEQ ID NO: 9 is the most widely used. However, longer linkers, such as the 20-amino acid (Gly-Ser) or (GGGGS) shown in SEQ ID NO: 10, may be used, e.g., to minimize oligomerization. In some embodiments, other residues, such as the charged residues glutamic acid (Glu) and / or lysine (Lys), may also be incorporated into the linker, e.g., to enhance solubility. In some embodiments, amino acid residues such as alanine (Ala) and / or threonine (Thr) may be incorporated into the linker, as in the case of the linker GGGGSGAGGSGGGGTGGGGS (SEQ ID NO: 11) used in this example.

[0070] V in this ds-scFv H Domains and V L The order of the domains, also called orientation, is V L -Linker-V H (LH, V L -V H Also written as V H -Linker-V L (HL, V H -V L However, individual scFvs may perform better in one configuration than the other, e.g., with respect to their binding properties. Furthermore, in some embodiments, expression yields may vary depending on the configuration, with the HL orientation usually being preferred.

[0071] In some embodiments, the ds-scFv of the present invention may comprise one or more additional peptide tags for various purposes, such as to facilitate purification, isolation, immobilization, and / or detection. A variety of peptide tags suitable for such and other purposes are readily available in the art. Non-limiting examples of such peptide tags are described herein below.

[0072] The stabilized ds-scFv molecules of the present invention have improved stability compared to corresponding conventional scFv molecules. Protein stability is typically measured by reversible protein unfolding, either by heat or by chaotropes such as guanidine hydrochloride or urea, using methods known in the art. Thus, in some embodiments, a measure of protein stability is thermostability, i.e., resistance to irreversible unfolding due to heat stress. In some embodiments, a measure of protein stability is pH dependence, i.e., resistance to protein unfolding due to pH fluctuations. Additionally, a measure of protein stability may also be resistance to proteases. In particular, for antibodies, stability may be measured as stability in serum.

[0073] Thermal stability can be measured using a number of non-limiting biophysical or biochemical techniques known in the art. Perhaps the most common method for measuring protein thermal shifts is differential scanning fluorescence (DSF) or thermofluorometry, which utilizes small fluorescent molecules whose fluorescence is enhanced when bound to exposed hydrophobic surfaces, such as those created by protein unfolding. 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 for the determination of thermal unfolding transitions. The temperature at which a protein unfolds is an indicator of overall protein stability.

[0074] In other embodiments, thermostability can be measured biochemically. An exemplary biochemical method for assessing thermostability is a heat challenge assay, in which a composition whose thermostability is to be determined is exposed to a range of elevated temperatures for a set period of time. 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 an expression library displaying them) are subjected to a range of elevated temperatures, e.g., for about 1 to about 1.5 hours. The activity of the test composition is then assayed by a relevant biochemical assay. Preferably, the assay is a binding assay performed to determine any changes in the target molecule binding properties of the heat-challenged composition. The binding assay may be, for example, a functional or quantitative ELISA assay. The temperature at which antigen-binding properties are lost is indicative of overall thermostability.

[0075] In some embodiments, thermal stability can be assessed by measuring the melting point (Tm) of a test composition using any of the techniques described above. The melting point is the temperature at the midpoint of the thermal transition curve at which 50% of the composition's molecules are denatured, as determined, for example, by unfolding or loss of antigen binding. Tm is considered the standard parameter for describing the thermal stability of a protein.

[0076] In some embodiments, the ds-scFv of the invention also have a thermal stability that is about 1°C, about 1.25°C, about 1.5°C, about 1.75°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C or more greater than a control molecule, such as a corresponding conventional scFv molecule.

[0077] Surprisingly, this interdomain disulfide bridge between the CDR-H3 and CDR-L1 loops significantly improved the thermal stability of this ds-scFv over H44-L100, a disulfide bridge previously suggested to serve as a universal position for scFv stabilization.

[0078] The Ds-scFv of the present invention can be used for a variety of research, diagnostic, and therapeutic purposes, largely due to their antigen-binding specificity. Furthermore, they can be utilized as building blocks for various molecular entities, including, but not limited to, engineered therapeutic proteins such as bispecific antibodies and CAR T cells.

[0079] Bispecific T cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies containing two scFvs directed against different antigens. One scFv targets the cluster of differentiation protein complex (CD3) on T cells, while the other scFv targets disease-specific antigens, forming a link between T cells and diseased cells, such as tumor cells. In close proximity to diseased cells, T cells destroy them by programming them to undergo apoptosis. It is envisioned that the ds-scFvs of the present invention are suitable as building blocks for constructing BiTes.

[0080] It is also envisioned that the ds-scFv of the present invention can be utilized to form various other types of bivalent, bispecific, or multispecific antibody constructs. For example, it can be expressed as a fusion to either the N- or C-terminus of either the light or heavy chain of a full-length antibody construct. Alternatively, one or both Fab arms of a bispecific antibody can be replaced with a ds-scFv of the present invention while maintaining the Fc portion (i.e., a crystallizable fragment) for immunomodulatory properties. This results in an antibody construct that is significantly smaller than an intact IgG antibody, yet is expected to have essentially unchanged immunomodulatory properties and a long half-life due to the presence of the Fc portion. Thus, the ds-scFv of the present invention is suitable as a building block for constructs comprising a fusion of a ds-scFv with the Fc portion of an antibody. Further bispecific constructs can be generated by fusing the ds-scFv of the present invention to alternative protein scaffold-based affinity reagents, such as Design Ankyrin Proteins (DARPins), nanobodies, and affibodies. Such binder molecules are readily available in the art.

[0081] It is further envisioned that the ds-scFv of the present invention can also be used in CAR T cell therapy. CAR T cell therapy is a type of cancer immunotherapy that uses a patient's T cells to find and kill tumor cells. CAR T cells are engineered T cells that express an artificial T cell receptor (CAR) on their cell surface. These artificial T cell receptors are chimeric and contain both an antigen-binding domain and a T cell activation domain. In addition to the antigen-binding domain (the ds-scFv of the present invention) and the T cell activation domain, the CAR also consists of a hinge, transmembrane, and costimulatory domain, as is well known to those skilled in the art. When a CAR has a ds-scFv that binds to a cancer-associated antigen, binding of the ds-scFv portion of the receptor can activate T cells and kill cancer cells.

[0082] In some further embodiments, the ds-scFv of the present invention may be included in a molecular entity such as a pharmaceutically active agent, a drug, a radioisotope, an enzyme (e.g., alkaline phosphatase), or a chelator, to name a few non-limiting examples. Depending on the type of molecular entity, a given ds-scFv may be conjugated or recombinantly fused to said molecular entity using means and methods readily available in the art.

[0083] The present invention also encompasses nucleic acid molecules encoding the ds-scFv of the present invention and various embodiments thereof.

[0084] Referring now to specific embodiments, the present invention provides anti-HER2 ds-scFv variants derived from trastuzumab scFv variants by introducing a stabilizing interdomain disulfide bridge of the invention at a novel position, i.e., between CDR-H3 and CDR-L1, more specifically between position -4, counting from H103, and position L34 according to the Kabat numbering scheme. In some embodiments, position -4, counting from H103, is position H100B.

[0085] Surprisingly, the introduction of the interdomain disulfide bridge of the present invention improved the thermal stability of phage-displayed ds-scFv and soluble ds-scFv proteins by 10°C compared to the thermal stability of the original scFv lacking the interdomain disulfide bridge. Importantly, the interdomain disulfide bridge at position H44-L100 according to the Kabat numbering scheme had significantly less impact on thermal stability than the interloop disulfide bridge of the present invention. The Tm values ​​of the comparative ds-scFv with the H44-L100 modification were only 3-4°C higher than those of the original scFv, but 7°C lower than the Tm values ​​of the mutants with the novel interloop disulfide bridge at position H100B-L34 examined in this study.

[0086] Furthermore, the anti-HER2 scFv with the added interdomain disulfide bridge retained the antigen-binding properties of the parent scFv in both orientations.

[0087] In some embodiments, the anti-HER2 ds-scFv comprises essentially the same CDR sequences as trastuzumab, except for the cysteine ​​residues that form the interdomain disulfide bridges of the present invention. Thus, in some embodiments, the anti-HER2 ds-scFv comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 12, CDR-H2 having the amino acid sequence of SEQ ID NO: 13, CDR-H3 having the amino acid sequence of SEQ ID NO: 14, CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or conservative sequence variants of the above 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 set forth in SEQ ID NO: 18 and SEQ ID NO: 19, respectively. The cysteine ​​residues that form the artificial interdomain disulfide bridges are located at position 10 of SEQ ID NO: 14 and position 11 of SEQ ID NO: 15.

[0088] Thus, in some embodiments, the anti-HER2 ds-scFv of the invention comprises a V comprising the CDR sequences: SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14. H and a VL domain comprising CDR sequences: SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, H Domain and V L The domains may be in either orientation. In some further embodiments, such V H Domain and V L The domains are separated by peptide linkers, for example peptide linkers selected from the peptide linkers of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functionally equivalent sequence variants thereof disclosed above.

[0089] In some further embodiments, the anti-HER2 ds-scFv of the invention comprises a V H domain and a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32.L The cysteine ​​residues that form the artificial interdomain disulfide bridges are at position 106 of SEQ ID NO:31 and at position 34 of SEQ ID NO:32, while the cysteines that form the natural intradomain disulfide bridges are at positions 22 and 96 of SEQ ID NO:31 and at positions 23 and 88 of SEQ ID NO:32. In some further embodiments, such V H and V L The domains are separated by peptide linkers, for example peptide linkers selected from the peptide linkers of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functionally equivalent sequence variants thereof disclosed above.

[0090] In some still 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 still other 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 bridges are at positions 106 and 174 of SEQ ID NO: 20 and positions 34 and 235 of SEQ ID NO: 21. The cysteine ​​residues that form the intradomain disulfide bridges are at positions 22, 96, 163, 228 of SEQ ID NO: 20 and positions 23, 88, 151, 225 of SEQ ID NO: 21. The DNA sequence encoding SEQ ID NO: 20 is set forth in SEQ ID NO: 45, and the DNA sequence encoding SEQ ID NO: 21 is set forth in SEQ ID NO: 46.

[0091] In some embodiments, the CDR-H3 of an anti-HER2 ds-scFv may contain one or more amino acid alterations (substitutions, deletions, and / or insertions) between H93 and H100A according to the Kabat numbering scheme (corresponding to amino acid residues 1-9 of SEQ ID NO: 14, amino acid residues 97-105 of SEQ ID NO: 31, amino acid residues 97-105 of SEQ ID NO: 20, or amino acid residues 226-234 of SEQ ID NO: 21), while the anti-HER2 ds-scFv still exhibits specific binding to HER2, preferably with a Kd of <10 nM. In other words, the amino acid alterations in CDR-H3 are located upstream from the cysteine ​​at residue -4 from the tryptophan H103 conserved in FR-H4. These amino acid alterations are preferably located at positions corresponding to amino acid residues 3-9 of SEQ ID NO: 14, amino acid residues 99-105 of SEQ ID NO: 31, amino acid residues 99-105 of SEQ ID NO: 20, or amino acid residues 228-234 of SEQ ID NO: 21.

[0092] Alternatively or additionally, the anti-HER2 ds-scFv of the present invention may also comprise one or more amino acid modifications (substitutions, deletions and / or insertions) in the CDR-H1 loop and / or the CDR-H2 loop compared to the amino acid sequences disclosed herein.

[0093] Independent of the presence or absence of amino acid changes in CDR-H1, CDR-H2 and / or CDR-H3, the anti-HER2 ds-scFv of the present invention may, in some embodiments, comprise one or more amino acid modifications (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 comprise one or more amino acid modifications (substitutions, deletions and / or insertions) compared to the amino acid sequences of the CDR-L2 loop disclosed herein.

[0094] As will be readily understood by those skilled in the art, the requirement for the above amino acid modifications is that the anti-HER2 ds-scFv still exhibits specific binding to HER2, preferably with a Kd of <10 nM. The presence or absence of such binding specificity can be easily determined by means and methods available in the art. Typically, the order of priority for diversifying 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. To facilitate such diversification, one of the two intradomain disulfide bridges can be eliminated by substituting one or both of the cysteine ​​residues normally involved in bridge formation (including those specifically mentioned for a given SEQ ID NO:) in accordance with what is described elsewhere herein. The intradomain disulfide bridge may be reintroduced later, again in accordance with what is described elsewhere in the disassembly procedure.

[0095] In some embodiments, the anti-HER2 ds-scFv of the present invention are provided for use in the treatment of cancer, particularly breast cancer or gastric cancer, and in some further embodiments may be used for specific antibody, BiTE, or CAR T cell therapy.

[0096] In particular, the stabilizing interdomain disulfide bridge-forming cysteine ​​residues of the invention can be engineered into any existing scFv by techniques well known in the art (including, for example, site-directed mutagenesis). In vitro display techniques may also be applied to generate ds-scFv of the invention.

[0097] Thus, an embodiment of the present invention relates to a recombinant library of particles displaying a plurality of the present ds-scFvs against various antigens, such as HER2. Further embodiments relate to methods for constructing and using such libraries to obtain novel ds-scFvs with desired antigen-binding properties. Thus, ds-scFvs against desired antigens can be easily generated from the present libraries by antigen screening, without the need for immunization of host animals and hybridoma production, thereby substantially reducing the time and effort typically required for antibody production by conventional methods.

[0098] Thus, the present invention provides a method for constructing a recombinant expression library, more specifically, a library of particles that display the ds-scFv of the present invention thereon, thereby linking the genotype and phenotype of the vesicles. 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 some other embodiments use both phage display libraries and mammalian cell display libraries.

[0099] To construct the display library of the present invention, codons for stabilizing interdomain disulfide bridge-forming cysteine ​​residues are inserted into V domains having the desired diversity using methods well known in the art. H and V LThe CDRs are introduced into appropriate positions in the nucleic acid encoding the CDRs. Preferably, the nucleic acid is a DNA molecule. Diversity can be derived from natural or non-natural sources. For example, diversity can be derived from immune libraries constructed from variable domain genes isolated from B cells derived from immunized animals or humans. Alternatively, diversity can be derived from variable domain genes isolated from non-immunized donors, which can be derived from naive libraries. In some further embodiments, diversity can be derived from fully synthetic libraries, usually derived from non-immune sources, and subjected to in silico computerized design and gene synthesis. In synthetic libraries, humanized frameworks or frameworks derived from human antibodies are combined with randomized CDRs at positions most likely to contribute to antigen recognition and binding. In semi-synthetic libraries, natural and in silico designed CDR diversity is combined, with or without CDR randomization. CDR randomization can be achieved, for example, by PCR-based assembly of synthetic oligos, as is well known in the art.

[0100] Due to its important role in antigen recognition and binding, the present display library of ds-scFv is, in some preferred embodiments, a CDR-H3 library. Such libraries can be constructed by diversifying the CDR-H3 loop length and / or amino acid composition 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.

[0101] Additionally, the other CDR loops may vary in length. In some embodiments, particularly those using the framework of trastuzumab or a functionally equivalent conservative sequence variant thereof, CDR-H1 is typically 5 amino acids in length, CDR-H2 is typically 16-19, preferably 17, amino acids in length, and CDR-H3 is typically 12-21 amino acids in length. In some embodiments, CDR-H3 is 13 amino acids in length. Meanwhile, CDR-L1 is typically 11 amino acids in length, CDR-L2 is typically 7 amino acids in length, and CDR-L3 is typically 9-11 amino acids in length. In some embodiments, CDR-L3 is 9 amino acids in length. In some embodiments, one or more of the CDR loops may comprise one or more randomized amino acids, in accordance with what is described elsewhere herein.

[0102] Because the length of CDR-H3 can vary, the amino acid position of the cysteine ​​residue partially involved in the artificial disulfide bridge must be defined indirectly via its 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 contains the V H and the V variant shown in SEQ ID NO: 49 L More generally, the recombinant expression library is based on variants of V, VII, VIII, VIIIA, VIIIB, VIIIC, VIIID, VIIIE, VIIIE, VIIIF, VIIIG, VIIIG, VIIIH, VIIIH, VIIII, VIIIH, VIIII, VIIIH, VIIIIH, VIII ...H, VIIIH, VIIIH, VIIIH, VIIIH, H and V comprising the framework regions shown in SEQ ID NOs: 5 to 8 LIn accordance with the above, the sequences 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 cysteines, 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 cysteines, thereby preventing the formation of one of the two naturally occurring intra-domain disulfide bridges. For the sequences SEQ ID NO: 48 and SEQ ID NO: 49, this means that one or both of the amino acid residues at positions 22 and 98 of SEQ ID NO: 48 can be engineered not to be cysteines, or one or both of the amino acid residues at positions 23 and 88 of SEQ ID NO: 49 can be engineered not to be cysteines, thereby preventing the formation of one of the two naturally occurring intra-domain disulfide bridges.

[0103] As in the context of ds-scFv above, any suitable framework can be used to generate the recombinant expression library of the present invention. Preferably, the framework is a humanized framework or a framework derived from a human antibody. In certain embodiments, the framework is that of trastuzumab or a conservative or other variant thereof, as described above. In some other embodiments, the framework is a human consensus framework.

[0104] Depending on the source of the CDRs and / or frameworks, the ds-scFvs displayed by the recombinant expression libraries of the present invention may be depicted as recombinant non-human, chimeric, humanized, fully human, or artificial (in silico designed), as will be understood by those skilled in the art. H The source and design of V L The source and design may differ from that of the original equipment.

[0105] V derived from the desired diversity and containing stabilizing interdomain disulfide bridge-forming cysteine ​​residues in CDR-H3 and CDR-L1 H and V Lis located at position -4, counting from H103 of FR-H4, and at position L34 of CDR-L1, according to the Kabat numbering scheme, and preferably V H or V L Once nucleic acid molecules have been engineered that lack one or both of the native cysteines involved in intradomain disulfide bridge formation within either of the ds-scFvs, they are cloned into an expression vector to generate a library of ds-scFvs. The choice of expression vector will depend on the type of display library to be generated, as will be readily understood by those skilled in the art. A wide variety of suitable expression vectors are commercially available.

[0106] In phage display, nucleic acids encoding ds-scFvs are fused to phage coat protein genes in phage or phagemid vectors, allowing the phage to display the scFvs on their surface. The most widely used phage display format utilizes the filamentous bacteriophage M13, where proteins of interest are fused to the phage coat protein pIII, enabling high-frequency monovalent display. In this approach, phagemid vectors carrying randomly cloned nucleic acids encoding many different ds-scFvs are electroporated into E. coli, followed by infection with helper phage to generate a phage library, with each phage displaying a different ds-scFv on its surface. The art is replete with suitable vectors, helper phage, and other means for constructing phage display libraries.

[0107] For mammalian cell display, nucleic acids encoding ds-scFvs with membrane-anchored segments can be integrated into the genome of mammalian cells, allowing the cells to display ds-scFvs on their surface. Various mammalian cell lines, including but not limited to CHO (Chinese Hamster Ovary) and HEK293 (Human Embryonic Kidney) cells, can be used for this purpose. Various strategies exist for integrating ds-scFv expression constructs into the genome of cells, including the use of homologous recombination and landing pad target site-specific recombinases. The efficiency of homologous recombination can be improved, for example, by using Zing Finger or TALE effector nucleases or DNA double-strand breaks induced by CRISPR / Cas systems. Cells displaying ds-scFvs with specific binding specificities can be enriched and isolated by antigen-coated magnetic bead-based separation or FACS (fluorescence-activated cell sorting)-based screening using fluorescently labeled antigens, or any other technique suitable for this purpose.

[0108] Once a recombinant expression library, such as a phage display library, is generated, it can be used to select ds-scFvs with desired antigen-binding properties by a process called panning, which typically involves several rounds of selection against one or more target antigens, preferably immobilized on a solid surface (e.g., beads or a microtiter plate). After incubation with the target antigen, unreacted phage are removed, for example, by extensive washing. Bound phage are then eluted and concentrated by amplification in suitable host cells (typically bacterial cells) before the next round of selection. These steps are typically repeated two to four times to obtain the phage most specific for the desired target antigen. In some embodiments, the stringency of selection may be increased with each selection round to enrich for ds-scFvs with high affinity and specificity.

[0109] In some embodiments, the panning process may also include 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 stock is used for positive selection against the target antigen.

[0110] After the panning rounds, the enrichment of antigen-specific phage binders is usually tested, typically by using a suitable immunoassay. Individual clones are then screened from the enriched pool of antigen-specific phage binders. Positive clones may then be further characterized, for example, by determining their antigen-binding properties and / or by sequencing.

[0111] Generally, phage display libraries allow for greater diversity than mammalian cell display libraries. In other words, the repertoire of different variants in phage display libraries can be significantly larger than that in mammalian cell display libraries. Therefore, the former library may be preferable to the latter library.

[0112] On the other hand, the presence of three disulfide bridges in the ds-scFv to be displayed, i.e., one interdomain disulfide bridge of the present invention and two naturally occurring intradomain disulfide bridges, may reduce the display rate (i.e., yield) on the phage surface. Because mammalian cells can well express molecules with three disulfide bridges, it is thought that the display efficiency will not be significantly reduced in mammalian cell display libraries.

[0113] Surprisingly, it has now been recognized that the benefits of phage display and mammalian cell display libraries can be combined. To this end, the present invention utilizes an interdomain disulfide bridge and only one naturally occurring intradomain disulfide bridge (instead of the two intradomain disulfide bridges specific to scFvs, according to the Kabat numbering scheme, V L between the cysteines at positions L23 and L88, and between the cysteines at positions V H In V, the missing intradomain disulfide bridge (located between the cysteines at positions H22 and H92) is first created. This allows the phage to express molecules with two disulfide bridges at a high rate, making it possible to create a display library with very high diversity without compromising display efficiency. The missing intradomain disulfide bridge is located between the cysteines at positions H22 and H92. H or V L In the former option, V H may, in some embodiments, have the amino acid sequence set forth in SEQ ID NO: 34, and in the latter option, V L may, in some embodiments, have the amino acid sequence set forth in SEQ ID NO: 33. In some preferred embodiments, the intradomain disulfide bridge is from the second V domain, i.e., the V domain when the ds-scFv is in the LH orientation. H domain, and when the ds-scFv is in the HL orientation, V L In some embodiments, the latter option (HL orientation) is preferred.

[0114] Once the most promising ds-scFvs are obtained from a large phage display library, the missing intradomain disulfide-bridge-forming cysteines are replaced by V L Shuffling (intradomain disulfide bridges V L (if missing from V) or H Shuffling (intradomain disulfide bridges V HThe ds-scFv library containing all three disulfide bridges, i.e., the non-native interdomain disulfide bridge of the present invention and the two native intradomain disulfide bridges, can then be subjected to selection with the target antigen using mammalian cell display technology. This approach increases the success rate of obtaining a suitable ds-scFv specific for the target antigen because it can take advantage of the greater diversity of the phage display library, despite the lower display efficiency.

[0115] Therefore, in one embodiment of the present invention, first V H High diversity is introduced into the CDRs of V L is not diversified or is diversified to a limited extent, e.g., contains variants >1E1>1E2>1E3>1E4>1E5. The first library so generated contains, e.g., V >1E8 or >1E9 or >1E10. H In some embodiments, the orientation of the ds-scFv in such libraries may be V H -V L and V L lacks native intra-domain disulfide bridges. In some further embodiments, such libraries are then expressed on filamentous phage and selected against the target antigen.

[0116] In some embodiments, V L Shuffling may then be used to introduce further diversity into the library. In other words, the antigen-specific Vs enriched from the phage display library H The domains are diversified V containing natural intradomain disulfide bridges. L The resulting second library may then be subjected to antigen-specific selection using mammalian cell display technology.

[0117] It should be noted that in the approach described above using the preferred embodiments, other cell-based display technologies, such as yeast display, may be used in place of mammalian cell display, although mammalian cell display is generally preferred. L -V H Orientation V H -V L In such cases, the native intradomain disulfide bridges are V H The first library is V L Although there is high diversity in the CDRs of V H is not diversified or is diversified to a limited extent. H Shuffling is used to introduce additional diversity into the library while simultaneously resolving natural intradomain disulfide bridges. H and the second library so generated can then be subjected to target antigen-specific selection using cell-based display technology.

[0118] In some embodiments, further diversity may be introduced into the first library described above, preferably by mutagenesis techniques readily available in the art, such as random mutagenesis, e.g., by error-prone PCR. L or V H It may be used instead of, or in addition to, shuffling.

[0119] Furthermore, diversity may be introduced into the first and / or second library, preferably the first library, by CDR randomization using means and methods readily available in the art, also in accordance with what has been discussed elsewhere herein (which will not be repeated here).

[0120] As explained above, it may be advantageous to combine phage display and mammalian cell display approaches, but it will be appreciated that target-specific ds-scFvs of the invention containing all three disulfide bridges can be successfully obtained from either phage display libraries (albeit with lower yields) or mammalian cell libraries (albeit with lower diversity).

[0121] Each display particle carries the genetic information for the recombinant polypeptide to be displayed on its surface. This feature makes it possible to identify nucleic acids encoding ds-scFvs exhibiting the desired specificity by selecting particles carrying ds-scFvs from a potentially very complex recombinant library. The nucleic acid from the best clones can then be isolated, inserted into an appropriate expression vector, and transfected or transformed into a suitable expression host to produce the ds-scFv according to standard recombinant techniques.

[0122] Many types of suitable expression vectors are available, including, but not limited to, plasmids and modified viruses, which are primarily contained in host cells as autonomous DNA molecules or integrated into genomic DNA. The vector system must be compatible with the host cell used, as 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 and include, but are not limited to, promoters, leaders, polyadenylation, and signal sequences.

[0123] Expression vectors can be transfected or transformed into host cells by standard techniques commonly used for introducing exogenous nucleic acids into prokaryotic or eukaryotic host cells, including, but not limited to, electroporation, nucleofection, sonoporation, magnetofection, heat shock, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0124] Ds-scFv can be expressed in a variety of expression systems, including, but not limited to, prokaryotic host cells such as bacteria (e.g., Escherichia coli, Bacillus), yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae), and fungi (e.g., filamentous fungi), as well as 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 containing a nucleic acid, preferably DNA, encoding the ds-scFv of the invention are cultured under conditions suitable for the production of the ds-scFv, and the resulting ds-scFv is then recovered. Ds-scFv of the invention may also be produced by in vitro protein expression according to protocols known in the art.

[0125] Bacterial cell expression systems, such as E. coli, are the most rapid and inexpensive form and therefore, in some cases, are preferred for producing ds-scFvs. In such embodiments, ds-scFvs are preferably targeted to the periplasmic space, which contains chaperones and disulfide isomer enzymes that enable correct protein folding and disulfide bridge formation, respectively. Secretion can be directed to the periplasmic space with the aid of a signal peptide (SP), such as pelB, linked to the N-terminus of the ds-scFv using standard recombinant techniques. Non-limiting examples of suitable signal peptides include those set forth in SEQ ID NOs: 22, 23, 25, and 28. ds-scFvs can also be expressed at high yields in the cytoplasm of E. coli or other bacterial cells. However, due to the reducing environment of the cytoplasm, disulfide bonds cannot form in most prokaryotes. Therefore, ds-scFvs 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 the Origami™ B host strain. Soluble expression of disulfide-bonded proteins in the cytoplasm of bacterial cells such as E. coli is possible using a system known as CyDisCo. This system is based on co-expressing the protein of interest with sulfhydryl oxidase and disulfide bond isomerase.

[0126] In some embodiments, it may be desirable to express the ds-scFv of the present invention as a fusion to one or more peptide or small protein tags that facilitate purification, isolation, immobilization, and / or detection. Non-limiting examples of affinity tags suitable for purification or immobilization 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 such as GFP, and enzyme tags that generate colored products upon contact with a chromogenic substrate. Non-limiting examples of suitable enzyme tags include alkaline phosphatase (AP) and (horseradish) peroxidase (HRP). Other tags, such as biotin, avidin, and streptavidin, can also be used for detection purposes. They can be detected using biotin / avidin / streptavidin-binding proteins conjugated to enzymes, fluorophores, or other reporter molecules. Vectors and other means and methods for producing the present ds-scFv as fusion proteins are readily available in the art. [Example]

[0127] Example 1: Anti-HER2-ds-scFv constructs The anti-HER2 scFv variants used in the examples were designed and then the genes were ordered as cloned genes from Twist Bioscience (USA).

[0128] In the construct, an artificial disulfide bridge 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 bridge, V L and V H Each of the V domains contained a native intradomain disulfide bridge. LIn V, the intradomain disulfide bridge was between cysteines at positions L23 and L88 according to the Kabat numbering scheme (corresponding to positions 23 and 88, respectively, of SEQ ID NO: 32). H In ScFv, the intradomain disulfide bridge was between cysteines at positions H22 and H92 according to the Kabat numbering scheme (corresponding to positions 22 and 96, respectively, of SEQ ID NO: 31). L -V H ) orientation and HL(V H -V L Constructs with all three disulfide bridges (LH_SSC and HL_SSC, respectively) and constructs in which the native intradomain disulfide bridge was removed from the second domain in the scFv were tested. In LH_S-C, the intradomain disulfide bridge was H (Domain Order V L -V H ) and in HL_S-C, V L From (Domain Order V H -V L ) were removed. Wild-type scFv (LH_SS- and HL_SS-) with only the native intradomain disulfide bridges were used as controls. In addition, V at positions H44-L100 was removed. L and V H The aforementioned constructs containing a disulfide bridge between V and V were expressed in both LH and HL orientations and evaluated as references. L and V H A schematic representation of the amino acid sequences is shown in Figure 1 and Table 1, respectively. All scFv constructs were V L and V H A 20aa glycine-serine peptide linker GGGGSGAGGSGGGGTGGGGS (SEQ ID NO: 11) was included between the two.

[0129] The construct used for expression of 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 (MKSLLPTAAAGLLLAAQPAMA; SEQ ID NO: 22), Lac promoter, and chloramphenicol resistance gene. A His6 tag was introduced from the vector to the C-terminus of the scFv.

[0130] The constructs used in Example 6 to study phage display were cloned into the Sfil site in the phagemid vector pEB32x (Huovinen et al., 2013) and pEB3V3, which is identical to the vector pEB32x but contains a modified pelB signal peptide MKYLLPTVVVGLLLLAAQPAMA (SEQ ID NO: 23) encoded by atg aag tac ctt cta ccg acg gta gtc 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) and is encoded by atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc gcg gcc cag ccg gcc atg gcg (SEQ ID NO: 26). From these vectors, scFvs are expressed fused to the C-terminal domain of the phage coat protein pIII. The vectors contained the Lac promoter, the pelB signal peptide, and a chloramphenicol resistance gene for antibiotic selection.

[0131] The scFv construct used in Example 3 for expression of scFv in mammalian cells was designed to contain the Kozak sequence gccgccacc and signal peptide MVLQTQVFISLLWISGAYG (SEQ ID NO: 28 human Ig kappa chain V-IV region B17) at the N-terminus and a His6 tag at the C-terminus, as described by Vazquez-Lombardi et al., 1997. The gene was ordered from Twist Bioscience cloned between the EcoRI and XbaI sites in the expression vector pTwistCMV Betglobin WPRE Neo (Twist Bioscience).

[0132] [Table 1(1)] [Table 1(2)] [Table 1(3)] * Numbering according to the Kabat numbering scheme (Prof Andrew CR Martin's Group at UCL 2022). CDR-H3 and CDR-L1 loops are shown in bold. Substitutions of amino acid residues (Val, Ala, Gly, or Gln) by Cys are marked in italics and underlined. Substitutions of cysteine ​​residues by Val and Ala are double underlined.

[0133] Example 2: Expression of anti-HER2 ds-scFv in E. coli The ScFv fragments LH_SS-, LH_SSC, HL_SSC, HL_S-C, and LH_S-C were expressed in the periplasmic space of E. coli in the XL1-Blue strain using vector pAK400. ScFvs were produced in shake flask cultures in 300 ml of SB medium containing 0.5% glucose, 10 μg / ml tetracycline, and 25 μg / ml chloramphenicol. Cultures were grown at 37°C and 300 rpm and induced with 200 μM IPTG to an OD of 0.5-1.0. Then, scFvs were produced overnight at 26°C and 250 rpm. Cells were harvested by centrifugation (15 min, 7000 x g, 4°C). To release periplasmic proteins from the cells, the pellet was resuspended in 30 ml of 20 mM phosphate buffer pH 7.4, 300 mM NaCl, 0.4 mg / ml lysozyme, 10 mM MgCl2, and 25 U / ml nuclease. After 30 minutes of incubation at room temperature, the sample was freeze-thawed three times. The cell lysate was clarified by centrifugation (20 minutes at 2000 g), and the supernatant was filtered through a 0.22 μm filter. ScFv was purified from the cell lysate by Ni-NTA affinity chromatography using 0.5 ml of Ni-NTA HisPur resin (Thermo Scientific), followed by preparative size-exclusion chromatography (SEC) using a Superdex® 75 10 / 300 GL column (Cytiva, USA) with PBS pH 7.4 as the elution buffer. The concentration of scFv in the SEC fractions was quantified using OctetRED384 (ForteBio, USA), which utilizes biolayer interferometry (BLI) technology. A streptavidin biosensor was coated with biotinylated protein L, which binds to scFv. Purity was analyzed by SDS-PAGE.

[0134] After Ni-NTA purification, the product contained many impurities, so size-exclusion chromatography (SEC) purification was performed. After SEC purification, the yield of scFv with interloop disulfide bridges was less than one-tenth of that of the wild-type (LH_SS-). The yields of scFv after Ni-NTA and SEC purification are shown in Table 2.

[0135] [Table 2] a Expression yields were determined by Octet using biolayer interferometry (BLI) technology. Streptavidin biosensors were coated with biotinylated protein L, which binds to scFv. Values ​​represent values ​​from one independent measurement.

[0136] The formation of interdomain disulfide bridges 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 was analyzed by SDS-PAGE using both reducing and nonreducing sample buffers. The results are shown in Figure 2.

[0137] The purity levels of the scFv versions LH_SSC and LH_S-C remained low even after SEC purification. Therefore, the possibility of interloop disulfide formation could not be assessed. The SEC fraction of LH_SS- also contained some minor impurities. HL_SSC and HL_S-C could be purified by Ni-NTA and SEC. Nonreducing SDS-PAGE analysis suggested the formation of interloop disulfide bridges, as the proteins migrated faster (~23 kDa) than their reduced analogs (~28 kDa) (Figure 2).

[0138] Example 3: Expression of anti-HER2 ds-scFv in mammalian cells The 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™ cells (Thermo Scientific) from Vector pTwistCMV Betglobin WPRE Neo (Twist Bioscience).

[0139] ExpiCHO cells were transiently transfected with 3 μg of plasmid using ExpiFectamine™ CHO (Thermo Scientific) transfection reagent to produce scFv according to the manufacturer's maximum titer protocol.

[0140] ExpiCHO-S™ cells were cultured in 6-well plates (Nunclon Delta Surface, Thermo Scientific) covered with breathable sealing tape (Nunc™ Sealing Tapes, Thermo Scientific) with Ex-piCHO™ Expression Medium (Thermo Scientific) in a culture volume of 2 ml on an orbital shaker at 125 rpm in a +37°C incubator at ≥80% relative humidity and 8% CO2.

[0141] The day after transfection, 12 μl of Expi-Fectamine™ CHO Enhancer and 320 μl of ExpiCHO™ Feed were added to the cells per well. The plates were transferred to a +32°C incubator with a humidified atmosphere of 5% CO2 in air and orbital shaking at 125 rpm. Five days after transfection, the second volume of ExpiCHO™ Feed was added to the wells (320 μl / well). The plates were immediately transferred to a +32°C incubator with a humidified atmosphere of 5% CO2 in air and orbital shaking. Culture supernatants were harvested 13 days after transfection by centrifugation at 5,000 × g for 30 minutes, and then the supernatants were filtered using a 0.22 μm filter. ScFv was purified from the cell lysate by Ni-NTA affinity chromatography using a HisPur™ Ni-NTA spin column with a 0.2 ml resin bed (Thermo Scientific). The elution fractions containing the majority of the protein were combined, and the buffer was changed to PBS pH 7.4 using a 10K MWCO Slide-A-Lyzer™ G2 dialysis cassette (Thermo Scientific). The yield of scFv after protein purification from ExpiCHO cells was measured by A280 and is shown in Table 3.

[0142] All scFv variants could be expressed in ExpiCHO-S™ cells (Table 3). These included the trastuzumab scFv fragments expressed in both orientations (LH_SS- and HL_SS-), trastuzumab variants with an interloop disulfide bridge at position H100B-L34 (LH_SSC and HL_SSC), trastuzumab variants with an interloop disulfide bridge at position H100B-L34 but no intradomain disulfide bridge (LH_S-C and HL_S-C), and trastuzumab variants with a disulfide bridge at position H44-L100 (LH_SS+ and HL_SS+). Based on the literature, H44-L100 has been suggested to serve as a universal position for scFv stabilization (Weatherhill et al. 2012).

[0143] The yield of scFv expressed in the HL orientation was 2- to 20-fold higher than that of scFv expressed in the LH orientation (Table 3).

[0144] [Table 3] a Expression yield was determined by absorption method: A 280nm = 1 corresponds to an scFv concentration of 0.58 mg / ml. The molar extinction coefficient of LH_SS- was determined by Vector NTI and used to analyze all scFv constructs. Values ​​represent values ​​from one independent measurement.

[0145] The formation of interdomain disulfide bridges was assessed by SDS-PAGE analysis by running samples of the purified scFv versions in reducing and non-reducing sample buffer. The gels were stained with Ready Blue Protein Gel Stain. The results are shown in Figure 3. Proteins with interdomain disulfide bridges migrate faster in non-reduced form in SDS-PAGE gels due to denser packaging than similar variants lacking interdomain disulfide bridges.

[0146] Mammalian-expressed Ni-NTA-purified scFvs were free of any other proteins as impurities based on reduced SDS-PAGE analysis (Figure 3, wells marked "R"). However, in the non-reduced sample, LH_SSC, HL_SSC, HL_S-C, LH_SS+, and HL_SS+ bands with higher molecular weights (~40 and ~60 kDa) were observed, indicating the possibility of disulfide bridge formation between two or more scFvs forming dimers and oligomers, respectively.

[0147] Positions H100B-L34 allowed complete inter-loop disulfide bridge formation, as evidenced by nonreducing 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 sample did not contain a form lacking disulfide bridges. However, positions H44-L100 only partially allowed inter-domain disulfide bridge formation, since a form lacking inter-domain disulfide bridges could be detected when samples were run under nonreducing conditions (~28 kDa) (Figure 3).

[0148] Example 4: Antigen-binding properties of anti-HER2 ds-scFv 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 streptavidin-coated microtiter wells for 30 minutes, and the plate was washed four times. Purified scFv was then added and incubated for 1 hour. After four washes, bound scFv was detected with an anti-His tag antibody, PentaHis (QIAGEN), labeled with Eu-N1 chelate (PerkinElmer, Finland). The plate was washed four times, DELFIA enhancement solution was added, and the plate was incubated for 10 minutes. Time-resolved fluorescence was then read using a Victor multilabel counter (PerkinElmer, Finland). The results are shown in Figure 4.

[0149] As can be seen from Figure 4, the binding of the scFv mutants with interloop disulfide bridges (LH_SSC, HL_SSC, HL_S-C, and LH_S-C) to HER2 was generally observed to be similar to that of the wild-type (LH_S-C) at the concentrations used in the assay. A slight 1.5-fold increase in binding of HL_SSC and HL_S-C at 10 nM was observed compared to the binding of the wild-type (LH_SS-). LH_SSC produced a signal similar to that of the wild-type, while LH_S-C showed a 1.5-fold decrease in signal compared to that of the wild-type.

[0150] Binding kinetics and preliminary Kd data of purified scFv were obtained using the Octet RED384 (ForteBio) system. Streptavidin-coated biosensors (ForteBio) were loaded with 200 ng / ml biotinylated HER2 (His, Avitag, Acro Biosystems, USA) for 600 seconds. ScFv association was followed for 600 seconds, and dissociation was followed for 7200 seconds. Measurements were performed in a 100 μl volume in a 384-well filter-bottom plate (ForteBio) at 30°C with a shaker speed of 1000 rpm and a sensor offset of 4 or 6 mm. All measurements were performed in PBS pH 7.4, 0.1% BSA, and 0.05% Tween-20. K D , k a and k d was calculated using Octet Data analysis software 8.2. The results are shown in Figures 5 and 6 and Tables 4 and 5.

[0151] Introduction of the interloop disulfide bridge at H100B-L34 maintained the high binding affinity of wild-type HER2 (Figures 5 and 6, Tables 4 and 5). Trastuzumab scFv (LH_SS+ and HL_SS+) with the interdomain disulfide bridge at H44-L100 also showed high binding affinity (K at subnanomolar levels). d values), which is consistent with previous results obtained for various scFvs stabilized by an interdomain disulfide bridge at H44-L100 (Weatherhill et al. 2012; Benschop et al. 2019).

[0152] [Table 4] a K D , k a , and k d Values ​​are determined by monovalent analysis at two scFv concentrations of 10 and 100 nM and represent values ​​from one independent experiment. b k dThe window for value calculation was 0 to 1600 s.

[0153] [Table 5] K D , k a , and k d The value is a Three scFv concentrations of 10, 60 and 200 nM by monovalent analysis; b Four scFv concentrations of 0.2, 2, 10, and 200 nM in bivalent analysis; c Five scFv concentrations of 0.2, 0.6, 10, 60, and 200 nM in monovalent analysis; d Five scFv concentrations of 0.2, 2, 10, 60, and 200 nM in monovalent analysis; e Determined by monovalent analysis at three scFv concentrations of 10, 60 and 200 nM. Values ​​in the table represent values ​​from a single independent experiment.

[0154] Example 5: Thermostability of anti-HER2 ds-scFv Thermal stability was measured by thermofluorescence assay on a CFX96 Real-Time System equipped with a C1000 Thermal Cycler and Bio-Rad CFX CFX Manager 3.1 software. The total reaction volume was 25 μl. For the assay, 22.5 μl of 5 μM scFv (or 0.35-2 μM for low-yield proteins) in PBS was mixed with 2.5 μl of 50x SYPRO Orange dye (Sigma-Aldrich) diluted in PBS prior to assay from a 5000x stock. Samples were heated in a PCR system from +25 to +95°C in +0.5°C increments, and fluorescence was measured in fluorescence resonance energy transfer (FRET) mode. The midpoint temperature of thermal denaturation (T m ) were identified by melting peak analysis, plotting the first derivative of fluorescence emission (-d(RFU) / dT) as a function of temperature. The results are shown in Figure 7, and the Tm's are listed in Table 6.

[0155] Based on the results in Table 6, the scFv variants (LH_SSC and HL_SSC) with inter-loop disulfide bridges at positions H100B-L34 were significantly more stable than the wild-type (LH_SS- and HL_SS-) without inter-domain disulfide bridges. m The values ​​+78.5°C and +77.5°C were +11°C and +9.7°C higher than the corresponding parent scFv, respectively. L Removal of the intradomain disulfide bridge (HL_S-C) from the wild-type (T m = 67.8°C), the thermal stability was slightly reduced (T m =65.7℃).

[0156] The interdomain disulfide bridges at positions H44-L100 (LH SS+ and HL SS+) had a significantly less effect on thermal stability than the interloop disulfide bridge at H100B-L34. m The values ​​were only 3–4°C higher than those of the wild type but 7°C lower than those of the mutant with an interloop disulfide bridge at the novel H100B-L34 position.

[0157] [Table 6] The midpoint temperature for thermal denaturation of scFv (T m ) is determined by thermofluorescence. Values ​​represent the mean ± standard deviation of three replicate measurements.

[0158] Example 6: Phage display of anti-HER ds-scFv Phage display of ds-scFv variants was studied in the phagemid vectors pEB32x and pEB3V3. Phage production was performed in E. coli XL1-Blue cells. Cells containing the phagemid were inoculated into 20 ml of SB containing 0.5% glucose, 10 μg / ml tetracycline, and 25 μg / ml chloramphenicol and incubated at 37°C and 300 rpm. When the OD600 reached 0.4, VCS M13 helper phage was added to 20x the number of infections, and the culture was incubated for 30 min without shaking. Cells were harvested by centrifugation (10 min, 3200 g, 4°C) and resuspended in 20 ml of SB containing 10 μg / ml tetracycline and 25 μg / ml chloramphenicol but no glucose. After shaking at 30°C for 1 hour, 30 μg / ml kanamycin and 100 μM IPTG were added, and phage were produced overnight at 26°C and 300 rpm. Cells were removed by centrifugation, and phage were precipitated from the supernatant by adding 1 / 5 volume of 20% PEG8000, 2.5 M NaCl. Phage were pelleted by centrifugation (20 min, 10,000 g, 4°C). The pellet was resuspended in TBS and centrifuged to remove residual precipitate, after which PEG / NaCl phage precipitation was repeated.

[0159] Phage display of scFvs was analyzed by immunoassay to measure phage binding to biotinylated Protein L (Figure 8) (ProSpec, biotinylated with Ez-link NHS-PEG4 biotin, Thermo Scientific) and biotinylated HER2 (Figure 9). Protein L is known to bind conformationally specific to the κ1V gene family framework used in ds-scFvs. Phage binding to biotinylated HER2 was measured. All reagents were diluted in Kaivogen red assay buffer. Biotinylated Protein L (50 ng / well) and HER2 (10 ng / well) were added to streptavidin-coated microtiter plate wells, incubated with gentle shaking at room temperature for 30 minutes, and washed four times. 1e9 phage were added to wells in triplicate, and phage binding to streptavidin wells was measured as a negative control. Phages were allowed to bind for 1 hour at room temperature with shaking. The plates were then washed, and Eu-N1-labeled anti-phage antibody (125 ng / ml) was added and incubated for 1 hour to detect bound phages. The plates were washed four times again, and DELFIA enhancement solution was added and incubated for 10 minutes, after which time-resolved fluorescence was read using a Victor 1420 Multilabel Counter (PerkinElmer, Finland). For the assay, phage titers were measured by immunoassay based on phage binding to uncoated maxisorb plates. Bound phages were detected with Eu-labeled anti-phage antibody as described above. These results are shown in Figures 8 and 9.

[0160] The highest phage display, measured from phage binding to protein L, was observed with the wild-type scFv (LH_SS-), which contains only the natural intradomain disulfide bridges (Figure 8). Adding an artificial interdomain disulfide bridge between CDRs -L1 and -H3 in the LH_SSC phage, which has both natural intradomain disulfide bridges, did not result in the display of the scFv on the phage surface. However, V L Intradomain disulfide bridges in VH -V L When the domain order was removed from the ds-scFv (HL_S-C mutant), phage display was established. Phages displaying the folded scFv also recognized HER2. The signals from binding to protein L and HER2 were 27% and 53%, respectively, of the signals from the corresponding wild-type phage (Figures 8 and 9).

[0161] Example 7: Construction and validation of CDR-H3 libraries The possibility of using disulfide-stabilized scFvs as frameworks in antibody libraries was investigated. The ds-scFv variant HL_S-C (Figures 8 and 9), which was shown to be functionally displayed on phage, was used as a framework in a library in which the WGGDGFY (SEQ ID NO: 47) sequence in the CDR-H3 loop was diversified with 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.

[0162] The library was constructed by oligonucleotide-guided PCR mutagenesis. The scFv was amplified with two PCR fragments, adding an LguI site for ligating the fragments in FASTR-type cloning. An NNS codon was added to one of the fragments with a randomized oligo (Figure 10). The two fragments were then PCR-purified, digested with LguI, and ligated into the complete scFv with T4 DNA ligase. The product was then further amplified, PCR-purified, digested with SfiI, and cloned into the vector pEB32x. The primers used for library construction are listed in Table 7, and the library construction strategy is illustrated in Figure 10. Transformation of the ligation library into E. coli XL1-Blue cells yielded a library size ranging from 5.7 x 10 to 4.4 x 10 transformants.

[0163] [Table 7] aThe hybridizing region is underlined, and the LguI recognition site is in bold.

[0164] To address the potential of utilizing a novel phage antibody library with interloop disulfide bonds as a source of discovery for novel binders to HER2, the loop library was enriched against HER2 through three successive rounds of selection against the target antigen by panning. Biotinylated HER2 antibodies were immobilized on streptavidin-coated M280 Dynabeads. After four washes, phage were allowed to bind for 1 hour. The beads were washed four times, and bound phage were eluted with trypsin. The eluted phage were infected into E. coli XL1-Blue cells, and the cells were grown with VCS M13 helper phage until an OD600 of 0.4 was reached. A new phage stock was then generated. Phage were produced overnight at 26°C and 250 rpm, then purified from the culture medium and subjected to two rounds of PEG / NaCl precipitation.

[0165] The unselected phage libraries were analyzed for binding to protein L by immunoassay. The results are shown in Figure 11. Phage stocks produced after each panning round were tested for binding to HER2 by phage immunoassay. The results are shown in Figure 12. Forty-eight clones from each library were picked after three rounds of panning, and single phage clones were generated overnight in 96-well cell culture plates. Cells were removed by centrifugation, and the culture supernatant containing the phage was tested for binding to HER2 in a phage immunoassay. HER2-binding phage was detected with a europium-labeled anti-VCS M13 antibody. The results are shown in Figure 13. Randomly selected pairs of clones from each library that showed binding to HER2 were sequenced. Some of the sequences are shown in Figure 14.

[0166] Each of the unselected CDR-H3 randomized loop libraries showed binding to protein L (Figure 11), indicating that scFvs with alternative loop lengths were also correctly folded and displayed on phage.

[0167] When the libraries were panned against HER2, the specific signal in the phage immunoassay gradually increased through the panning rounds for each loop library (Figure 12), indicating an enrichment of (specific) binders to HER2.

[0168] After three rounds of panning, the numbers of HER2-positive clones (S / B>5) in the 13, 14, 15, 16, 17, and 19 aa loop libraries were 22 / 48, 30 / 48, 28 / 48, 26 / 48, and 42 / 19, respectively (Figure 13). Sequencing of several randomly selected HER2-positive individual clones from the 13, 14, and 15 loop libraries (Figure 14) showed that new sequences were obtained.

[0169] It is obvious to those skilled in the art that with the advancement of technology, the basic idea of ​​the invention can be implemented in various ways, therefore the invention and its embodiments are not limited to the above-described examples, but rather may vary within the scope of the claims.

[0170] (References) Weatherill, EE, Cain, KL, Heywood, SP, Compson, JE, Heads, JT, Adams, R. & Humphreys, DP (2012) Towards a universal disulfide stabilized single chain Fv format: importance of interchain disulfide bond location and VL-VH orientation. Protein Eng Des Sel 25: 321-329 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 of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease. MAbs 11:1175-1190 Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Pluckthun A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods. 201(1):35-55. Huovinen T, Syrjanpaa M, Sanmark H, Brockmann EC, Azhayev A, Wang Q, Vehniainen M, Lamminmaki U. (2013) Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles. Protein Eng Des Sel. 26(10):683-93. Vazquez-Lombardi R, Nevoltris D, Luthra A, Schofield P, Zimmermann C, Christ D. (2018) Transient expression of human antibodies in mammalian cells. Nat. Protoc. 13(1):99-117.

Claims

1. Disulfide-stabilized single chain fragment variable (ds-scFv), A heavy chain variable domain (V) comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 Ｈ )and, A light chain variable domain (V) comprising complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 Ｌ )and, Including, The V Ｈ and the V Ｌ are linked by a peptide linker in either orientation, The CDR-L1 and the CDR-H3 are linked to each other via an interdomain disulfide bridge, and the V Ｈ or the V Ｌ either ds-scFv, which has been engineered to lack native intradomain disulfide bridges.

2. The ds-scFv of claim 1, wherein the interdomain disulfide bridge is artificial.

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

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

5. The native intradomain disulfide bridges absent from the variable domain are numbered V according to the Kabat numbering scheme. Ｌ The bridge formed between cysteine ​​residues L23 and L88 in Ｈ The ds-scFv according to any one of claims 1 to 4, wherein the bridge formed between cysteine ​​residues H22 and H92 in

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 V Ｈ and / or the V Ｌ The ds-scFv according to any one of claims 1 to 5, wherein the CDR sequences are derived from natural diversity, with the exception of residue -4 from the conserved tryptophan H103 in FR-H4 and the cysteine ​​at residue L34 in CDR-L1.

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

9. One or more of the CDR sequences comprise one or more randomized amino acids, except that residue −4 from conserved tryptophan H103 in FR-H4 and residue L34 in the CDR-L1 are cysteine ​​according to the Kabat numbering scheme, and the ds-scFv comprises V Ｌ Inside or V Ｈ 9. The ds-scFv of any one of claims 1 to 8, which contains a native intra-cysteine ​​domain disulfide bridge in either one of the domains, but not both.

10. 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, 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, and is 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 cysteines, 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 cysteines; or Ｈ The V domain comprises the amino acid sequence set forth in SEQ ID NO: 48 or a functionally equivalent conservative sequence variant thereof, Ｌ 10. The ds-scFv of any one of claims 1 to 9, wherein the domain comprises the amino acid sequence set forth in SEQ ID NO: 49 or a functionally equivalent sequence variant thereof, and is engineered such 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.

11. The V Ｈ or the V Ｌ 11. The ds-scFv of any one of claims 1 to 10, further engineered to reintroduce the native intradomain disulfide bridges that are missing in any of the domains.

12. 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, 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, or Ｈ The V domain comprises the amino acid sequence set forth in SEQ ID NO: 48 or a functionally equivalent conservative sequence variant thereof, Ｌ The ds-scFv of claim 11, wherein the domain comprises the amino acid sequence set forth 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, and preferably, the CDR-H1 has the amino acid sequence of SEQ ID NO: 12, the CDR-H2 has the amino acid sequence of SEQ ID NO: 13, the CDR-H3 has the amino acid sequence of SEQ ID NO: 14, the CDR-L1 has the amino acid sequence of SEQ ID NO: 15, the CDR-L2 has the amino acid sequence of SEQ ID NO: 16, and the CDR-L3 has the amino acid sequence of 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. 16. The molecular entity of 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 the ds-scFv of any one of claims 1 to 10 fused to a moiety selected from the group consisting of ... crystallizable fragment (Fc) portion of an antibody and an alternative protein scaffold-based affinity reagent, a pharmaceutically active agent, a drug, a radioisotope, an enzyme or a chelator.

17. 17. Use of the ds-scFv of any one of claims 1 to 16 for constructing a molecular entity selected from the group consisting of bispecific antibodies, diabodies, multispecific antibodies, CAR-T cells, bispecific T-cell engagers (BiTEs), constructs comprising the ds-scFv of any one of claims 1 to 10 fused to a moiety selected from the group consisting of bispecific antibodies, diabodies, multispecific antibodies, CAR-T cells, bispecific T-cell engagers (BiTEs), a fragment crystallizable (Fc) portion of an antibody and an alternative protein scaffold-based affinity reagent.

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 library of particles, said library displaying a plurality of different ds-scFvs according to any one of claims 1 to 16.

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

22. 13. A method for producing a library of particles, said library displaying a plurality of different ds-scFvs according to any one of claims 1 to 10 or 13, said method comprising: i) Different heavy chain variable domains (V) that all have a cysteine ​​residue at position −4, counting from the conserved tryptophan H103, according to the Kabat numbering scheme. Ｈ a.) manipulating a plurality of first nucleic acids encoding polypeptides; ii) different light chain variable domains (V) all having a cysteine ​​residue at position L34 according to the Kabat numbering scheme; Ｌ a.) manipulating one or more second nucleic acids encoding the polypeptide; iii) cloning each one of the plurality of first nucleic acids and one of the second nucleic acids into an expression vector, the first nucleic acid and the second nucleic acid being in any order and interposed by a nucleic acid encoding a peptide linker, thereby resulting in a plurality of different vectors; iv) expressing the plurality of different vectors of step iii) on particles, thereby generating a first library of particles, each particle containing a V encoded by the plurality of nucleic acids of steps i) and ii). Ｈ and V Ｌ presenting different ds-scFv polypeptides comprising the segments; Including, A method wherein a plurality of either said first nucleic acid or said second nucleic acid is engineered to lack native intra-domain disulfide bridges in said encoded polypeptide.

23. 23. The method of claim 22, wherein the native intra-domain disulfide bridge lacks the variable domain located after the peptide linker in the encoded polypeptide.

24. 24. The method of claim 22 or 23, wherein the first library of particles is a phage display library.

25. The method further comprises selecting a subset of the first library of particles having desired target binding properties, preferably V Ｈ or V Ｌ 25. The method of any one of claims 22 to 24, comprising introducing further diversity by mutagenesis or domain shuffling of either of said variable domains, preferably by shuffling of said variable domains located after said peptide linker, thereby generating a second library of particles.

26. 26. The method of claim 25, wherein the mutagenesis or domain shuffling results in the reintroduction of disulfide bridges within the native domain engineered to be absent in either the first nucleic acid or the second nucleic acid.

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

28. 24. The method of 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 synthetically obtained.

29. 29. The method of 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 derived from natural diversity excluding the nucleotide corresponding to the engineered cysteine ​​at residue L34 of CDR-L1 and residue -4 from the conserved tryptophan H103 in FR-H4 in the encoded polypeptide.

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