Novel antibody framework

Abstract

The present invention relates to a novel antibody framework with advantageous properties. Specifically disclosed is an isolated antibody or a functional fragment thereof comprising a VH domain and a VL domain, wherein the VL domain comprises: (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) framework region IV, wherein the framework region IV is a human Vλ germline sequence for framework region IV, wherein the CDR domains CDR1, CDR2 and CDR3 are specific to a target antigen and are selected from (i) CDR domains CDR1, CDR2 and CDR3 from a parent rodent antibody; (ii) CDR domains CDR1, CDR2 and CDR3 from a parent human or humanized antibody comprising a Vκ domain.

Description

Technical Field

[0001] The present invention relates to novel antibody frameworks with advantageous properties. Background Art

[0002] The present invention relates to a novel chimeric human antibody light chain framework comprising framework regions I to III from Vκ and framework region IV from Vλ, having advantageous properties such as high stability and reduced aggregation tendency.

[0003] Over the past four decades, since the development of the first monoclonal antibodies (“mAbs”; & Milstein, Nature. 256 (1975) 495-7), antibodies have become an increasingly important class of biomolecules for research, diagnosis and treatment purposes. Initially, antibodies were obtained exclusively by immunizing animals with the antigen of interest. Although antibodies of non-human origin can be used in research and diagnosis, in therapeutic methods, human antibodies may recognize non-human antibodies as foreign matter and produce an immune response to non-human antibody drug products, thereby making the non-human antibody drug products less effective or ineffective. Therefore, recombinant methods have been established to make non-human antibodies have lower immunogenicity.

[0004] Initial efforts to convert non-human mAbs into less immunogenic therapeutics required engineering chimeric antibodies consisting of variable domains from animals (e.g., rodents) and human constant regions (Boulianne et al., Nature 312, (1984) 643-646). Additional approaches have focused on humanizing rodent mAbs by introducing CDRs into human variable domain scaffolds (Jones et al., Nature 321 (1986) 522-525; Riechmann et al., Nature 332 (1988) 323-7) or by resurfacing variable domains (Roguska et al., Proc. Natl. Acad. Sci. USA 91 (1994) 969-973).

[0005] For humanization by CDR loop grafting, the human acceptor framework is selected based on homology to the donor framework (e.g., Roguska et al., Protein Engineering 9 (1996) 895-904; WO / 2008 / 144757 (for rabbits)) or based on preferred stability characteristics (Ewert et al., Methods 34 (2004) 184-199). The latter concept has been used to humanize rabbit antibodies into a common variable domain framework (US 8,193,235).

[0006] In the case of any selected approach, the resulting mAb or functional fragment ideally retains the desired pharmacodynamic properties of the donor mAb while displaying drug-like biophysical properties and minimal immunogenicity. With respect to the biophysical properties of mAbs or their functional fragments, aggregation propensity has become a major concern for the developability of therapeutic molecules, primarily for the following three reasons:

[0007] First, protein aggregates generally show a higher potential to trigger an immune response in the host, which can lead to the formation of anti-drug antibodies and ultimately drug neutralizing antibodies (Joubert et al., J. Biol. Chem. 287 (2012) 25266-25279).

[0008] Second, aggregates affect production yield because the workload of their removal increases (Cromwell et al., AAPS Journal 8 (2006), Article 66).

[0009] Third, off-target effects can be observed. The problem of oligomer formation is more obvious for applications where monovalent binding is preferred (including bispecific (or multispecific) antibody forms in which each target and construct only have monovalent), because the formation of oligomers in these cases will produce protein aggregates with multivalent binding properties, which may cause off-target effects. An example of this non-specific activity is the use of a construct with a single CD3ε-binding domain in the form of a bispecific antibody. This form can, for example, bind one of its two binding domains to a cancer antigen, and bind its second CD3ε-binding domain to recruit cytotoxic T cells. Due to the need for cross-linking of monovalent CD3ε-binding moieties to induce signal transduction by CD3ε, T cells will only be stimulated when multiple bispecific constructs are connected by binding to the target cell surface, and therefore adopt the characteristics of cross-linked molecules, thereby resulting in a specific T cell response exclusively for cancer cells. On the contrary, the oligomers of this construct will exhibit the characteristics of cross-linked bispecific antibodies, and therefore activate cytotoxic T cells (even when not bound to cancer cells), thereby causing systemic activation of T cells. This nonspecific and systemic activation of T cells leads to elevated cytokine levels, which can cause adverse effects.

[0010] Additionally, a reliable and universally applicable acceptor framework would be beneficial in achieving a robust method for humanizing non-human antibodies, as cloning, expression, and purification methods could be standardized.

[0011] In order to meet the above-mentioned criteria for humanization of non-human mAbs, published methods propose to use the human consensus variable domain framework sequence as an acceptor scaffold for transplanting non-human complementary determining regions. Based on the following assumption: for each amino acid position in the protein, the residues that contribute to protein stability have been enriched in the germline sequence pool during evolution, it is generally accepted that the closer the resulting humanized variable domain is to the human germline consensus sequence in the corresponding variable domain family, the higher the expected stability. As described by Steipe (Steipe et al., J. Mol. Biol. 1994 240 (1994) 188-92) and by ( This concept, reviewed by Carter et al., J. Mol. Biol. 305 (2001) 989-1010, has been widely accepted and has found widespread application. Non-limiting examples are: (a) using consensus sequence variable domains for humanization of non-human antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289); (b) using consensus sequence variable domains to construct CDR libraries for in vivo screening of stable target binding antibodies (Knappik et al., J. Mol. Biol. 296 (2000) 57-86); and (c) knowledge-based approaches: improving the stability of antibody variable domains by exchanging non-consensus residues for consensus residues (Steipe, loc. cit.).

[0012] Furthermore, the stability of different variable domain families is described with VH3, which is the most stable variable heavy chain domain. Importantly, in the case of variable light chain domains, the Vκ family is preferred over the Vλ family (Ewert, loc. cit.). Specifically, the human consensus sequences of VH3 and Vκ1 have been described as having favorable biophysical properties (Ewert, loc. cit.) and are particularly suitable for humanization of antibodies from non-human sources (used in Carter, loc. cit.).

[0013] In this regard, there are several publications in which the human Vκ1-VH3 consensus framework hu-4D5 has been used for the humanization of rodent and rabbit antibodies (Rader, J. Biol. Chem. 275 (2000) 13668-13676; WO / 2005 / 016950; WO2008 / 004834). Alternatively, naturally occurring sequences belonging to the same family as hu-4D5 have been used to generate stable humanized single-chain (scFv) fragments from rabbit sources (US 8,293,235; Borras et al., J. Biol. Chem. 285 (2010) 9054-9066).

[0014] Importantly, it has been noted that natural selection always evolves stable variable domains in the context of full-length antibodies, where the variable domains are adjacent to and contact the constant domains 1. Therefore, it is entirely possible that certain non-consensus residues confer better stability to isolated variable domains, for example in the context of single-chain Fv (scFv) fragments. In support of this hypothesis, non-consensus mutations that contribute to stability have been described in U.S. Patent Application US 2009 / 0074780.

[0015] Furthermore, antibody stability is critical to the preparation, purification, shelf life and subsequent cost of antibody therapeutic products. Even minor improvements in one or more of these parameters may be highly relevant to the question of whether the research and development of antibody drugs is becoming commercially viable.

[0016] Therefore, despite the fact that many attempts have been made to solve the problem of obtaining humanized antibody drugs from non-human antibodies, there is still a large unmet need to develop novel human antibody frameworks with favorable properties such as high stability and reduced aggregation tendency, wherein the human antibody framework contains as few mutations as possible, and ideally no mutations, when compared to the naturally occurring sequence, so as to minimize the risk of generating immunogenic sequences. Such stable human frameworks can also be used to stabilize fully human antibodies or fragments thereof, for example, by loop grafting or simply by exchanging components that contribute to stability between the parent antibody and the stable framework.

[0017] The prior art has not currently achieved or proposed a solution to this problem which has been provided by the present invention, i.e. a novel chimeric human antibody light chain framework comprising framework regions I to III from Vκ and framework region IV from Vλ, which has advantageous properties such as high stability and reduced aggregation tendency. Summary of the invention

[0018] The present invention relates to a novel chimeric human antibody light chain framework comprising framework regions I to III from Vκ and framework region IV from Vλ, which has advantageous properties such as high stability, reduced aggregation tendency and minimal immunogenic potential.

[0019] Therefore, in the first aspect, the present invention provides an isolated antibody or a functional fragment thereof comprising a VH domain and a VL domain, wherein the VL domain comprises: (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) framework region IV, wherein framework region IV is a human Vλ germline sequence for framework region IV, wherein the CDR domains CDR1, CDR2 and CDR3 are specific for a target antigen and are selected from (i) CDR domains CDR1, CDR2 and CDR3 from a parent rodent antibody; (ii) CDR domains CDR1, CDR2 and CDR3 from a parent human or humanized antibody comprising a Vκ domain.

[0020] In some embodiments, the CDR domains CDR1, CDR2, and CDR3 are from a parent human or humanized antibody approved for therapeutic use or otherwise commercialized.

[0021] In some embodiments, the CDR domains CDR1, CDR2, and CDR3 are from a parental mouse or rat antibody.

[0022] In some embodiments, the Vλ germline sequence is selected from: SEQ ID NOs. 16 to 22.

[0023] In some embodiments, the Vκ framework regions I to III are (i) framework regions present in the sequence of SEQ ID NO: 23, or (ii) framework regions comprising no more than five mutations compared to each framework region present in the sequence of SEQ ID NO: 23, and wherein the VH domain includes framework regions I to IV, which are (i) framework regions present in the sequence of SEQ ID NO: 30, or (ii) framework regions comprising no more than five mutations compared to each framework region present in the sequence of SEQ ID NO: 30.

[0024] In some embodiments, the amino acid residue at position AHo101 of framework region III of the VL domain is glutamic acid.

[0025] In some embodiments, the Vκ framework regions I to III are framework regions present in a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 8.

[0026] In some embodiments, the Vκ framework regions I to III are present in the sequence SEQ ID NO:8.

[0027] In some embodiments, the framework regions I to IV are a combination of framework regions present in a sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0028] In some embodiments, the isolated antibody or its functional fragment is selected from: IgG antibody, Fab fragment, scFv fragment, single-chain diabody, tandem scDb, linear dimeric scDb, cyclic dimeric scDb, bispecific T cell engager, tandem three scFv, three antibodies, three-chain antibodies, bispecific Fab2, two mini antibodies, four antibodies, scFv-Fc-scFv fusion, two diabodies, DVD-Ig, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusion, and knob-in-hole structure and double antibody, wherein the knob-in-hole structure is a bispecific IgG prepared by KiH technology, and the double antibody is a bispecific IgG prepared by Duobody technology.

[0029] In some embodiments, the IgG-scFv fusion is selected from: a scFv linked to the C-terminus of an IgG light chain, a scFv linked to the N-terminus of an IgG light chain, a scFv linked to the N-terminus of an IgG heavy chain, a scFv linked to the C-terminus of an IgG heavy chain, a scFv linked to the N-terminus of both an IgG heavy chain and a light chain, and a dsscFv linked to the C-terminus of an IgG heavy chain.

[0030] In a second aspect, the present invention provides a pharmaceutical composition comprising the isolated antibody or a functional fragment thereof according to the first aspect.

[0031] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0032] In a third aspect, the present invention provides a nucleic acid sequence or a set of nucleic acid sequences, wherein the nucleic acid sequence or the set of nucleic acid sequences (i) encodes the antibody VL domain of the isolated antibody or its functional fragment according to the first aspect, or the isolated antibody or its functional fragment according to the first aspect.

[0033] In a fourth aspect, the present invention provides a vector or a vector set, wherein the vector or the vector set comprises the nucleic acid sequence or the nucleic acid sequence set of the third aspect.

[0034] In a fifth aspect, the present invention provides a host cell, wherein the host cell comprises the nucleic acid sequence or a set of nucleic acid sequences of the third aspect, or the vector or a set of vectors of the fourth aspect.

[0035] In some embodiments, the host cell is an expression host cell.

[0036] In a sixth aspect, the present invention provides a method for producing the antibody VL domain of the isolated antibody or its functional fragment of the first aspect, or the isolated antibody or its functional fragment of the first aspect, the method comprising the following steps: expressing the nucleic acid sequence or a set of nucleic acid sequences of the third aspect, or the vector or a set of vectors of the fourth aspect, or the host cell of the fifth aspect.

[0037] In a seventh aspect, the present invention provides a method for producing a humanized antibody, the method comprising the following steps:

[0038] a. separating at least one antibody of interest from an animal that has been immunized with an antigen of interest; and

[0039] b. Cloning the VL CDR region of the at least one antibody of interest into the nucleic acid sequence encoding the antibody VL domain of the isolated antibody or functional fragment thereof of the aforementioned first aspect.

[0040] In some embodiments, the method further comprises one or more of the following steps:

[0041] aa. Isolating affinity-matured memory B cells that interact with the antigen of interest by using fluorescence-activated cell sorting clonal isolation;

[0042] ab. Cultivating single B cells in a co-culture system that does not require immortalization of single B cell clones;

[0043] ac. screening B cell culture supernatants in a cell-based ELISA to identify at least one antibody that binds to the antigen of interest; and / or

[0044] ad. Cloning the VH CDR region of at least one antibody into a nucleic acid sequence encoding a human antibody VH domain.

[0045] In an eighth aspect, the present invention provides a method for producing a nucleic acid sequence encoding the antibody VL domain of the isolated antibody or its functional fragment of the aforementioned first aspect, or one or more nucleic acid sequences encoding the isolated antibody or its functional fragment of the aforementioned first aspect, the method comprising combining in one or more steps the nucleic acid sequences encoding: (i) human Vκ1 framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3 from a parent antibody specific for the target antigen; and (iii) framework region IV, which is a human Vλ germline sequence for framework region IV.

[0046] In some embodiments, the method uses one of the following methods:

[0047] i. in a nucleic acid construct comprising a nucleic acid sequence encoding a human or humanized Vκ1 domain, replacing Vκ1 framework region IV by framework region IV, wherein framework region IV is a human Vλ germline sequence for framework region IV;

[0048] ii. inserting in one or more steps one or more nucleic acid sequences encoding the following into a nucleic acid construct comprising a nucleic acid sequence encoding framework region IV: (i) human Vκ1 framework regions I to III; and (ii) CDR domains CDR1, CDR2, and CDR3 from a parent antibody specific for an antigen of interest, wherein the framework region IV is a human Vλ germline sequence for framework region IV;

[0049] iii. mutating the nucleic acid sequence encoding framework region IV in the nucleic acid sequence encoding a human or humanized Vκ1 domain to generate a framework region IV, wherein the framework region IV is a human Vλ germline sequence for framework region IV;

[0050] iv. in one or more steps in a nucleic acid construct comprising a nucleic acid sequence encoding a light chain domain comprising human Vκ1 framework regions I to III, CDR domains CDR1, CDR2 and CDR3, and framework region IV, replacing one or more nucleic acid sequences encoding the CDR domains by one or more nucleic acid sequences encoding corresponding one or more CDR domains from a parent antibody specific for an antigen of interest, wherein the framework region IV is a human Vλ germline sequence for framework region IV.

[0051] In some embodiments, the Vλ germline sequence is selected from: SEQ ID NOs. 16 to 22.

[0052] In some embodiments, the nucleic acid construct is a recombinant vector.

[0053] In a ninth aspect, the present invention provides a nucleic acid sequence or a set of nucleic acid sequences, which can be obtained by the method of the eighth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The variable domain topology outlining the framework and CDR regions is shown.

[0055] Figure 2 Sequence alignment of the variable domain sequences of hu-4D5 (Carter, 1992) and the alternative frameworks tested is shown. Differences from hu-4D5 are indicated in black. For hu-4D5, only the framework regions Vκ Frameworks I to IV (SEQ ID NOs: 34 to 37) and VH Frameworks I to IV (SEQ ID NOs: 38 to 41) are shown.

[0056] Figure 3 Shown is a comparison of the normalized time-resolved loss of monomer content of scFv1 to scFv10.

[0057] Figure 4 Shown are models of the interaction between framework III residue AHo101 and the lambda connecting region (framework IV); VLκ (upper panel; PDB ID: 1FVC) and VLλ (lower panel; PDB ID 2A9M).

[0058] Figure 5 Shown are superimposed normalized SE-HPLC chromatograms of two scFv constructs of a humanized mouse monoclonal antibody with a native / original variable domain (grey) and a VL domain containing a lambda framework IV (black). The normalized scFv monomer peak is marked with an asterisk (*), while oligomer and aggregate peaks are highlighted with brackets. DETAILED DESCRIPTION

[0059] Compared to existing methods for generating human frameworks for humanizing / stabilizing non-human antibodies or stabilizing human antibodies, the present invention is characterized by the fact that the present invention relates to replacing the κ connecting segment in the κ variable light chain domain with a λ connecting segment (framework region IV), thereby generating a κ-λ chimeric variable light chain domain with improved protein stability and reduced aggregation tendency. The present invention also relates to mutations of the κ consensus residues at position AHo101 (framework region III) and replacement of the κ consensus residues with λ consensus residues to support packaging of the λ connecting segment in the κ-λ chimeric variable light chain domain, thereby further improving protein stability and further reducing aggregation tendency.

[0060] Thus, in a first aspect, the present invention relates to an antibody VL domain comprising: (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) framework region IV selected from:

[0061] a. A human Vλ germline sequence for framework region IV, in particular a Vλ germline sequence selected from the following list: SEQ ID NO.16 to 22;

[0062] b. A Vλ-based sequence which is (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, in particular SEQ ID NO.17; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, in particular a Vλ consensus sequence selected from the following list: SEQ ID NOs.16 and 17; and

[0063] c. a Vλ-based sequence having one or two mutations, particularly one mutation, compared to the closest human Vλ germline sequence for framework region IV;

[0064] Provided that if in case b. or c., framework region IV has the sequence FGQGTKLTVLG (SEQ ID No. 15),

[0065] (w) the human Vκ framework regions I to III are different from the framework regions shown in the following clone list: FW1.4gen (SEQ ID NO:4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt and 43-FW1.4opt;

[0066] (x) the human Vκ framework regions I to III are different from the sequences obtainable by rearrangement of the framework region sequences as shown in the following clone list: FW1.4gen (SEQ ID NO:4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt and 43-FW1.4opt;

[0067] (y) the human Vκ framework regions I to III are different from the sequence obtainable by mutating the sequence FW1.4gen (SEQ ID NO:4) at one or more positions 15, 22, 48, 57, 74, 87, 88, 90, 92, 95, 97 and 99 (AHo numbering); or

[0068] (z) the human Vκ framework regions I to III comprise no more than five mutations compared to the corresponding regions in the human Vκ sequence of SEQ ID No: 8, in particular less than five, less than four, less than three, in particular only one or no mutations compared to the human Vκ sequence of SEQ ID No: 8.

[0069] In the context of the present invention, clones 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt and 43-FW1.4opt refer to the clones listed in Borras et al. (loc. cit.). These clones are variants of the VL domain FW1.4 gen (SEQ ID NO: 4) that differ from those of FW1.4 gen (SEQ ID NO: 4) at certain positions in the VL framework region, as shown in Table 5.

[0070] In a specific embodiment, the framework region IV is not FGQGTKLTVLG (SEQ ID No. 15).

[0071] In the context of the present invention, the term "antibody" is used as a synonym for "immunoglobulin" (Ig), which is defined as a protein belonging to the IgG, IgM, IgB, IgA, or IgD class (or any subclass thereof), and includes all conventionally known antibodies and functional fragments thereof. A "functional fragment" of an antibody / immunoglobulin is defined as a fragment of an antibody / immunoglobulin that retains an antigen binding region (e.g., a variable region of IgG). The "antigen binding region" of an antibody is typically present in one or more hypervariable regions of the antibody, i.e., CDR-1, CDR-2, and / or CDR-3 regions; however, variable "framework" regions may also play an important role in antigen binding, such as by providing a scaffold for the CDRs.

[0072] In the context of the present invention, the numbering system proposed by Honegger & Plückthun (Honegger & Plückthun, J. Mol. Biol. 309 (2001) 657-670) is used unless otherwise specifically mentioned. In addition, the following residues are defined as CDR regions: CDR-L1: L24-L42; CDR-L2: L58-L72; CDR-L3: L107-L138; CDR-H1: H27-H42; CDR-H2: H57-H76; CDR-H3: H109-H138. Preferably, the "antigen-binding region" includes at least amino acid residues 4 to 149 of the variable light (VL) chain and 5 to 144 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 149 of VL and 4 to 146 of VH, and particularly preferably the entire VL and VH chains (amino acids 1 to 149 of VL and 1 to 149 of VH; according to Figure 2 serial number). Figure 2 The framework and CDR regions are indicated in the text. The preferred class of immunoglobulins used in the present invention is IgG. The "functional fragments" of the present invention include F(ab')2 fragments, Fab fragments, and scFv domains. F(ab')2 or Fab can be engineered to minimize or completely eliminate the intermolecular disulfide interactions that exist between the CH1 and CL domains. The antibodies of the present invention or their functional fragments can be part of a bifunctional or multifunctional construct as further described in paragraphs

[0055] to

[0058] .

[0073] In the context of the present invention, the term "Vκ" and "Vλ" refer to a family of antibody light chain sequences grouped according to sequence identity and homology. The method for determining sequence homology is, for example, by using a homology search matrix such as BLOSUM (Henikoff, S. & Henikoff, JG, Proc. Natl. Acad. Sci. USA 89 (1992) 10915-10919), and the method for grouping sequences according to homology is well known to those of ordinary skill in the art. For both Vκ and Vλ, different subfamilies can be identified (see, for example, Knappik, the above citation, which groups Vκ into Vκ1 to Vκ4 and Vλ into Vλ1 to Vλ3).

[0074] In the context of the present invention, the term "sequence obtainable by rearrangement of the framework region sequences as found in the list of clones: ..." refers to a sequence that can be generated by using (i) the amino acid residues present at a given position in all the sequences included in said list (see Borras, loc. cit.), or (ii) the amino acid residues at the positions optimized in Borras (Borras, loc. cit.), any of which is present at one of the different positions in said sequence (positions 15, 22, 40, 49, 58, 69, 70, 72, 74, 77, 79 and 81 in Borras (Borras, loc. cit.); corresponding to AHo positions 15, 22, 48, 57, 74, 87, 88, 90, 92, 95, 97 and 99).

[0075] In one embodiment of the invention, the amino acid residue at position AHo101 (position 101 according to the numbering system of Honegger and Plückthun) of framework region III is the amino acid residue present at said position in the human Vλ consensus sequence, in particular wherein the amino acid residue is different from phenylalanine, more in particular wherein the amino acid residue is glutamic acid.

[0076] In one embodiment of the present invention, the Vκ framework regions I to III belong to a Vκ domain subfamily selected from Vκ1, Vκ2, Vκ3 and Vκ4, in particular to the Vκ1 family.

[0077] In the context of the present invention, the Vκ domain subfamily is represented by the consensus sequences shown in SEQ ID NOs: 23 to 26. When using the method listed in paragraph

[0039] , a given antibody variable light chain domain is considered to belong to the Vκ domain subfamily if it shows the highest degree of homology with the Vκ domain subfamily.

[0078] In a specific embodiment, the Vκ framework regions I to III comprise no more than five mutations compared to (a) the closest human germline sequence, or (b) one of the consensus sequences SEQ ID NOs:23 to 26, in particular SEQ ID NO:23; in particular less than five, less than four, less than three, in particular only one or no mutations compared to (a) the closest human germline sequence, or (b) one of the consensus sequences SEQ ID NOs:23 to 26, in particular SEQ ID NO:23.

[0079] In one embodiment of the present invention, the Vκ framework regions I to III are framework regions present in a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 8, in particular SEQ ID NO: 8.

[0080] In one embodiment of the present invention, the CDR domains CDR1, CDR2 and CDR3 are independently selected from: (i) CDR domains CDR1, CDR2 and CDR3, which are derived from a parent non-human antibody specific for an antigen of interest, in particular from a parent rabbit antibody or from a parent rodent antibody, in particular a parent mouse or rat antibody; (ii) CDR domains CDR1, CDR2 and CDR3, which are derived from a parent human or humanized antibody comprising a Vκ domain, in particular from an antibody approved for treatment or otherwise commercialized; (iii) CDR domains CDR1, CDR2 and CDR3, which are derived from a CDR domain according to (i) or (ii), in particular a CDR domain obtained by optimizing one or more CDR domains according to (i) or (ii); and (iv) a CDR domain to be replaced by one or more CDR domains according to (i), (ii) and / or (iii).

[0081] In one embodiment of the invention, framework regions I to IV are a combination of framework regions as present in a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0082] In a second aspect, the present invention relates to an isolated antibody or a functional fragment thereof, which comprises the antibody VL domain according to the present invention.

[0083] In specific embodiments, the antibody VL domain has binding specificity for a target of interest.

[0084] As used herein, a binding molecule is "specific for", "specifically recognizes", or "specifically binds to" a target such as human CD3, i.e., such binding molecule is able to distinguish such target biomolecule from one or more reference molecules, since binding specificity is not an absolute, but a relative property. In its most general form (and when no defined reference is mentioned), "specific binding" refers to the ability of a binding molecule to distinguish a target biomolecule of interest from unrelated biomolecules as determined according to specificity determination methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA tests, and peptide scanning. For example, a standard ELISA assay can be performed. Scoring can be performed by standard color development (e.g., secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). The reaction in certain wells is scored, for example, by optical density at 450 nm. A typical background (= negative reaction) can be about 0.1 OD; a typical positive reaction can be about 1 OD. This means that the ratio between positive scores and negative scores can be 10 times or more. Typically, determination of binding specificity is not performed using a single reference biomolecule, but rather using a panel of about three to five unrelated biomolecules such as milk powder, BSA, transferrin, and the like.

[0085] In the context of the present invention, the term "about" or "approximately" means between 90% and 110% of a given value or range.

[0086] However, "specific binding" can also refer to the ability of a binding molecule to distinguish between a target biomolecule and one or more closely related biomolecules used as reference points. In addition, "specific binding" can relate to the ability of a binding molecule to distinguish between different parts of its target antigen, such as different domains, regions or epitopes of a target biomolecule, or one or more important amino acid residues or one or more stretches of amino acid residues of a target biomolecule.

[0087] In the context of the present invention, the term "epitope" refers to the portion of a given target biomolecule that is required for specific binding between the target biomolecule and a binding molecule. An epitope can be continuous, i.e., formed by adjacent structural elements present in the target biomolecule, or discontinuous, i.e., formed by structural elements at different positions in the primary sequence of the target biomolecule, such as in the amino acid sequence of a protein that is a target, but closely adjacent in the three-dimensional structure adopted by the target biomolecule, such as in a body fluid.

[0088] In one embodiment of the present invention, the isolated antibody or functional fragment thereof is selected from the group consisting of: IgG antibody, Fab fragment and scFv fragment.

[0089] In another specific embodiment of the present invention, the isolated antibody or functional fragment thereof is a bispecific construct in the form of an antibody, wherein the bispecific construct is selected from the group consisting of single-chain diabodies, tandem scDb, linear dimeric scDb, cyclic dimeric scDb, bispecific T cell engagers, tandem tri-scFv, triabodies, triabodies, bispecific Fab2, di-minibodies, tetrabodies, scFv-Fc-scFv fusions, di-diabodies, DVD-Ig, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions, and knob-in-hole structures and double antibodies, wherein the knob-in-hole structure is a bispecific IgG prepared by KiH technology, and the double antibody is a bispecific IgG prepared by Duobody technology. Preferably, the IgG-scFv fusion is selected from: a scFv coupled to the C-terminus of an IgG light chain, a scFv coupled to the N-terminus of an IgG light chain, a scFv coupled to the N-terminus of an IgG heavy chain, a scFv coupled to the C-terminus of an IgG heavy chain, a scFv coupled to the N-terminus of both an IgG heavy chain and a light chain, and a dsscFv coupled to the C-terminus of an IgG heavy chain. Particularly suitable for use herein are single-chain diabodies (scDbs), in particular bispecific monomeric scDbs.

[0090] The bispecific scDb, specifically the bispecific monomeric scDb, specifically comprises two variable heavy chain domains (VH) or fragments thereof, and two variable light chain domains (VL) or fragments thereof, which are connected by linkers L1, L2 and L3 in the following order: VHA-L1-VLB-L2-VHB-L3-VLA, VHA-L1-VHB-L2-VLB-L3-VLA, VLA-L1-VLB-L2-VHB-L3-VHA, VLA-L1- VHB-L2-VLB-L3-VHA, VHB-L1-VLA-L2-VHA-L3-VLB, VHB-L1-VHA-L2-VLA-L3-VLB, VLB-L1-VLA-L2-VHA-L3-VHB or VLB-L1-VHA-L2-VLA-L3-VHB, wherein the VLA and VHA domains together form an antigen binding site for a first antigen and VLB and VHB together form an antigen binding site for a second antigen.

[0091] The linker L1 is specifically a peptide with 2-10 amino acids, more specifically 3-7 amino acids, and most specifically 5 amino acids, and the linker L3 is specifically a peptide with 1-10 amino acids, more specifically 2-7 amino acids, and most specifically 5 amino acids. The intermediate linker L2 is specifically a peptide with 10-40 amino acids, more specifically 15-30 amino acids, and most specifically 20-25 amino acids.

[0092] The bispecific constructs of the present invention can be produced using any conventional antibody production method known in the art (see, e.g., Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14 for bispecific construct production; Hornig, N. & Leger, O., Pathobiology 74 (2007) 3-14 for bispecific diabodies and tandem scFvs). A., Methods Mol. Biol. 907 (2012) 713-727 and WO 99 / 57150). Specific examples of suitable methods for preparing the bispecific constructs of the present invention also include Genmab (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750) technologies. Methods for producing bispecific antibodies comprising a functional antibody Fc portion are also known in the art (see, for example, Zhu et al., Cancer Lett. 86 (1994) 127-134); and Suresh et al., Methods Enzymol. 121 (1986) 210-228).

[0093] These methods generally involve the production of monoclonal antibodies: for example, by using hybridoma technology to fuse myeloma cells with spleen cells of mice that have been immunized with the desired antigen (see, for example, Yokoyama et al., Curr. Protoc. Immunol. Chapter 2, Unit 2.5, 2006), or by using recombinant antibody engineering (antibody library cloning or phage display / yeast display) (see, for example, Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8); using known molecular cloning techniques to combine the antigen-binding domains of two different monoclonal antibodies or fragments or portions thereof to give a bispecific construct.

[0094] In one embodiment of the present invention, the isolated antibody or its functional fragment comprises a VH domain, which belongs to a VH domain subfamily selected from the group consisting of VH1A, VH1B, VH2, VH3, VH4, VH5 and VH6, in particular to VH domain subfamily VH3 or VH4, in particular to VH domain subfamily VH3.

[0095] In the context of the present invention, a VH domain subfamily is represented by the consensus sequences shown in SEQ ID NOs: 27 to 33. When using the method set out in paragraph

[0039] , a given antibody variable heavy chain domain is considered to belong to a VH domain subfamily if it shows the highest degree of homology with said VH domain subfamily.

[0096] In a specific embodiment, the VH domain comprises no more than five mutations compared to (a) the closest human germline sequence, or (b) one of the consensus sequences SEQ ID NO:28 to 34, in particular SEQ ID NO:30; in particular less than five, less than four, less than three, in particular only one or no mutations compared to (a) the closest human germline sequence, or (b) one of the consensus sequences SEQ ID NO:27 to 33, in particular SEQ ID NO:30.

[0097] In a third aspect, the present invention relates to a pharmaceutical composition comprising the isolated antibody or a functional fragment thereof of the present invention, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0098] In a fourth aspect, the present invention relates to a nucleic acid sequence or a collection of nucleic acid sequences, which encodes the antibody VL domain of any one of the present invention, or the isolated antibody or its functional fragment thereof of the present invention; and / or relates to one or more nucleic acid sequences obtainable by the method according to the ninth aspect of the present invention.

[0099] In a fifth aspect, the present invention relates to a vector or a collection of vectors, said vector comprising the nucleic acid sequence or a collection of nucleic acid sequences of the present invention.

[0100] In a sixth aspect, the present invention relates to a host cell (especially an expression host cell), which comprises the nucleic acid sequence or a collection of nucleic acid sequences of the present invention, or the vector or a collection of vectors of the present invention.

[0101] In the seventh aspect, the present invention relates to a method for producing any one of the antibody VL domains of the present invention, the isolated antibody of the present invention or a functional fragment thereof, which comprises the following steps: expressing the nucleic acid sequence or a set of nucleic acid sequences of the present invention, the vector or a set of vectors of the present invention, or the host cell of the present invention (especially an expression host cell).

[0102] In an eighth aspect, the present invention relates to a method for producing humanized rabbit antibodies or rodent antibodies, especially mouse or rat antibodies, comprising the following steps:

[0103] a) immunizing rabbits or rodents (especially mice or rats) with the antigen of interest;

[0104] b) isolating at least one antibody of interest; and

[0105] c) cloning the VL CDR region of the at least one antibody of interest into the nucleic acid sequence encoding the antibody VL domain of the present invention.

[0106] In a specific embodiment, the framework region IV in the antibody VL domain is not FGQGTKLTVLG (SEQ ID No. 15) when generating a humanized rabbit antibody.

[0107] In a specific embodiment, the method of the present invention further comprises one or more of the following steps:

[0108] aa. Cloning and isolating affinity-matured memory B cells that interact with an antigen of interest, particularly by using fluorescence-activated cell sorting;

[0109] ab. Cultivation of single B cells in a co-culture system that does not require immortalization of single B cell clones;

[0110] ac. screening B cell culture supernatants in a cell-based ELISA to identify at least one antibody that binds to the antigen of interest; and / or

[0111] ad. Cloning the VH CDR region of at least one antibody into a nucleic acid sequence encoding a human antibody VH domain.

[0112] Methods for humanization of rabbit or rodent antibodies are well known to those of ordinary skill in the art (see, e.g., Borras, loc. cit.; Rader et al., The FASEB Journal, express article 10.1096 / fj.02-0281fje, published online on Oct. 18, 2002; Yu et al., (2010) A Humanized Anti-VEGF Rabbit Monoclonal Antibody Inhibits Angiogenesis and Blocks Tumor Growth in Xenograft Models. PLoS ONE 5(2):e9072. doi:10.1371 / journal.pone.0009072). Thus, immunization of rabbits or rodents can be performed by using an antigen of interest, such as a protein, or in the case of a peptide or protein antigen, by DNA immunization of rabbits with a nucleic acid, such as a plasmid, encoding the peptide or protein of interest.

[0113] In the ninth aspect, the present invention relates to a method for producing a nucleic acid sequence encoding the antibody VL domain of the present invention, or one or more nucleic acid sequences encoding an isolated antibody or a functional fragment thereof according to the present invention, comprising combining nucleic acid sequences encoding (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) framework region IV, the framework region IV being selected from:

[0114] a. A human Vλ germline sequence for framework region IV, in particular a Vλ germline sequence selected from the following list: SEQ ID NO.16 to 22;

[0115] b. A Vλ-based sequence which is (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, in particular SEQ ID NO.17; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, in particular a Vλ consensus sequence selected from the following list: SEQ ID NOs.16 and 17; and

[0116] c. a Vλ-based sequence having one or two mutations, particularly one mutation, compared to the closest human Vλ germline sequence for framework region IV;

[0117] In particular, use one of the following methods:

[0118] i. replacing the Vκ framework region IV in a nucleic acid construct (particularly a recombinant vector) comprising a nucleic acid sequence encoding a human or humanized Vκ domain by a framework region IV selected from:

[0119] a. A human Vλ germline sequence for framework region IV, in particular a Vλ germline sequence selected from the following list: SEQ ID NO.16 to 22;

[0120] b. A Vλ-based sequence which is (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, in particular SEQ ID NO.17; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, in particular a Vλ consensus sequence selected from the following list: SEQ ID NOs.16 and 17; and

[0121] c. a Vλ-based sequence having one or two mutations, particularly one mutation, compared to the closest human Vλ germline sequence for framework region IV;

[0122] ii. inserting in one or more steps one or more nucleic acid sequences encoding the following into a nucleic acid construct, in particular a recombinant vector, comprising a nucleic acid sequence encoding framework region IV: (i) human Vκ framework regions I to III; and (ii) CDR domains CDR1, CDR2 and CDR3, wherein the framework region IV is selected from:

[0123] a. A human Vλ germline sequence for framework region IV, in particular a Vλ germline sequence selected from the following list: SEQ ID NO.16 to 22;

[0124] b. A Vλ-based sequence which is (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, in particular SEQ ID NO.17; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, in particular a Vλ consensus sequence selected from the following list: SEQ ID NOs.16 and 17; and

[0125] c. a Vλ-based sequence having one or two mutations, particularly one mutation, compared to the closest human Vλ germline sequence for framework region IV;

[0126] iii. mutating the nucleic acid sequence encoding framework region IV in the nucleic acid sequence encoding a human or humanized Vκ domain to generate a framework region IV selected from:

[0127] a. A human Vλ germline sequence for framework region IV, in particular a Vλ germline sequence selected from the following list: SEQ ID NO.16 to 22;

[0128] b. A Vλ-based sequence which is (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, in particular SEQ ID NO.17; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, in particular a Vλ consensus sequence selected from the following list: SEQ ID NOs.16 and 17; and

[0129] c. a Vλ-based sequence having one or two mutations, in particular one mutation, compared to the closest human Vλ germline sequence for framework region IV; or

[0130] iv. in one or more steps, in a nucleic acid construct, in particular a recombinant vector, comprising a nucleic acid sequence encoding a light chain domain comprising human Vκ framework regions I to III, CDR domains CDR1, CDR2 and CDR3 and framework region IV, replacing one or more of the nucleic acid sequences encoding the CDR domains with one or more nucleic acid sequences encoding the corresponding one or more CDR domains from an antibody of interest, wherein the framework region IV is selected from:

[0131] a. A human Vλ germline sequence for framework region IV, in particular a Vλ germline sequence selected from the following list: SEQ ID NO.16 to 22;

[0132] b. A Vλ-based sequence which is (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, in particular SEQ ID NO.17; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, in particular a Vλ consensus sequence selected from the following list: SEQ ID NOs.16 and 17; and

[0133] c. a Vλ-based sequence having one or two mutations, particularly one mutation, compared to the closest human Vλ germline sequence for framework region IV;

[0134] In a specific embodiment of the method of the present invention, wherein in case (b) or (c) framework region IV has the sequence FGQGTKLTVLG (SEQ ID No. 15),

[0135] (w) the human Vκ framework regions I to III are different from the framework regions present in the following clone list: FW1.4gen (SEQ ID NO:4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt and 43-FW1.4opt;

[0136] (x) the human Vκ framework regions I to III are different from the sequences obtainable by rearrangement from the framework region sequences listed in the following clone list: FW1.4gen (SEQ ID NO:4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt and 43-FW1.4opt;

[0137] (y) the human Vκ framework regions I to III are different from the sequence obtainable by mutating the sequence FW1.4gen (SEQ ID NO:4) at one or more positions 15, 22, 48, 57, 74, 87, 88, 90, 92, 95, 97 and 99 (AHo numbering); or

[0138] (z) the human Vκ framework regions I to III comprise no more than five mutations compared to the corresponding regions in the human Vκ sequence of SEQ ID No: 8, in particular less than five, less than four, less than three, in particular only one or no mutations compared to the human Vκ sequence of SEQ ID No: 8.

[0139] In a specific embodiment, the framework region IV is not FGQGTKLTVLG (SEQ ID No. 15) when one or more of the CDR domains are of rabbit origin.

[0140] Example

[0141] The following examples illustrate the invention without limiting its scope.

[0142] Example 1: Construction of scFv constructs with exemplary rabbit CDRs

[0143] Our goal was to identify variable domains that showed improved stability with respect to unfolding and aggregation propensity. In addition, these domains should be as close as possible to the human germline antibody repertoire to minimize the risk of eliciting an immune response in humans. Surprisingly, we found that the VH3 consensus framework is composed of framework regions I to III from the consensus Vκ1 and framework region IV from a different Vλ domain (see Figure 1 The combination of chimeric VL domains composed of ) produces scFv constructs with excellent biophysical properties.

[0144] The starting point of our invention was the human VH3 and Vκ1 consensus framework Hu-4D5 as published in 1992 (Carter, loc. cit.). We grafted the CDRs of an exemplary rabbit anti-TNFα antibody (WO / 2009 / 155723) into the hu-4D5 variable domains as described (Rader 2000, loc. cit.; WO 2005 / 016950; WO 2008 / 004834). The humanized variable domains were joined by a flexible peptide linker as described by Borras, loc. cit., resulting in a single-chain Fv (scFv) fragment (scFv1).

[0145] In addition to hu-4D5, another published framework solution was tested, namely framework FW1.4gen (scFv2), for which extensive biophysical data have been published (Borras, loc. cit.). Framework FW1.4gen includes several amino acid substitutions when compared to hu-4D5, thus deviating from the human consensus. In VH, such differences may result from affinity maturation, on the one hand because it is derived from a human cDNA library, containing the sequence of mature antibodies that have undergone somatic hypermutation, rather than germline sequences, and on the other hand because the authors have introduced several mutations for the purpose of accommodating rabbit CDRs. In VL, few or no mutations were intentionally introduced, suggesting that most of the differences result from the use of somatic hypermutation in the germline sequences, or may be an artifact of the library cloning procedure.

[0146] Experimental determination of thermal unfolding of two constructs (scFv1 and scFv2, respectively) based on hu-4D5 or FW1.4gen showed the superior performance of scFv1, i.e. the framework has a higher homology with the human consensus sequence and is therefore consistent with the reference. Surprisingly, this superior stability did not translate into a higher stability with respect to monodispersity during storage under stress conditions (see Table 4). On the contrary, scFv2 showed better stability here.

[0147] To rationalize the observed differences in stability, sequence variations within the framework were more closely examined. A five-amino acid fragment was identified within the framework IV region that was transformed into the consensus sequence of Vκ1 in FW1.4gen (scFv3) (see Figure 3 ) can lead to impaired stability of the monomeric state in stress stability studies.

[0148] This finding was highly unexpected as it contradicts the common understanding that a consensus sequence (in this case, that of Vκ1) would present the most favorable solution with respect to the stability of the corresponding variable domain. Based on this finding, we intended to further examine the determinants of domain stability in the model system. Since the five amino acids within the framework region IV of FW1.4 make the linker segment (framework region IV) similar to a λ-type framework region IV sequence rather than a κ-type, we hypothesized that a λ-type linker segment might be more favorable for the stability of the Vκ1-VH3 variable domain than a κ-type linker segment.

[0149] Indeed, we found that the complete replacement of Vκ1 framework region IV with the corresponding Vλ framework region (scFv5, scFv9) resulted in a more favorable stability profile than scFv1 or scFv2 in terms of both the midpoint of thermal unfolding (Table 3) and the stability of the monomeric state during stress stability studies (Table 4). In addition, the introduction of different truncated Vλ framework region IV germline motifs into the background of the Vκ1 consensus framework (scFv7, scFv10) showed some stabilizing effects. Importantly, the complete replacement of Vκ1 framework region IV with Vλ region IV may be less immunogenic, since all individual framework regions remain consistent with the germline, or consistent with the germline consensus, in the case of this approach.

[0150] In addition, the position of AHo101 in framework III was determined by comparing the structural models of the Vκ and Vλ variable domains that contribute to the packing of framework region IV ( Figure 4 ) were used to identify. The introduction of Vλ consensus residues at this position (scFv4, scFv6, scFv8) resulted in further improvements in the domain stability during thermal unfolding (Table 3) and stress stability (Table 4).

[0151] In summary, it has been found that in the context of scFv variable domain constructs, superior stability characteristics are achieved by combining consensus framework region IV from different Vλ germline genes with framework regions I to III of Vκ sequences. The stability of these artificial and chimeric variable domains is further improved by the F101E modification (according to the AHo numbering scheme) in framework region III of the variable light chain domain, which is located spatially close to framework region IV.

[0152] Importantly, based on the public information ( Protein Engineering, Design & Selection 25 (2012) 485–505), the preferred properties of the variable domain are also expected to be translated into other antibody formats.

[0153] Exemplary rabbit binders were identified from the reference (WO 2009 / 155723) and their CDRs were grafted into the corresponding variable domains of the human consensus VH3 / Vκ1 framework (Rader 2000, loc. cit.; WO / 2005 / 016950; WO 2008 / 004834; US 8,293,235) using the CDR definitions as disclosed in Borras et al. (Borras, loc. cit.). For loop grafting of the rabbit CDRs, the CDRs were grafted according to Honegger (Honegger & Plückthun, loc. cit.; see Figure 2 ) were numbered in the humanized variable domains. The scFv construct was generated by connecting VL and VH with a flexible Gly4-Ser linker to generate a NH2-VL-linker-VH-COOH configuration.

[0154] method

[0155] Construct design and fabrication

[0156] The resulting amino acid sequence was synthesized from scratch and cloned into an expression vector modified based on the pET26b (+) backbone (Novagen) for E. coli expression. The expression construct was transformed into E. coli strain BL12 (DE3) (Novagen), and in 2YT culture medium (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual), the cell culture was used as a starting culture. The expression culture was inoculated, and the expression culture was incubated in a baffled flask at 37 ° C and 200 rpm. Once OD600 nm reaches 1, protein expression was induced by adding IPTG to a final concentration of 0.5 mM. After overnight expression, cells were harvested by centrifugation at 4000 g. For the preparation of inclusion bodies, the cell pellet was resuspended in IB resuspension buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% Triton (Triton) X-100). The cell slurry was supplemented with 1 mM DTT, 0.1 mg / mL lysozyme, 10 mM leupeptin, 100 μM PMSF and 1 μM pepstatin. The cells were dissolved by 3 ultrasonic homogenization cycles while cooling the cells on ice. 0.01 mg / mL DNA enzyme was then added and the homogenate was incubated for 20 minutes at room temperature. The inclusion bodies were settled by centrifugation at 10,000 g and 4 ° C. The IB was resuspended in IB resuspension buffer and homogenized by ultrasonic treatment before another centrifugation. A total of at least 3 washing steps were performed with IB resuspension buffer and then 2 washings were performed with IB washing buffer to produce the final IB.

[0157] For protein refolding, the separated IBs were resuspended in solubilization buffer (100mM Tris / HCl pH 8.0, 6M Gdn-HCl, 2mM EDTA) at a ratio of 5mL / g wet IBs. Solubilization was incubated at room temperature for 30 minutes until DTT was added to a final concentration of 20mM, and the incubation was continued for another 30 minutes. After solubilization was complete, the solution was clarified by centrifugation at 8500g and 4°C for 10 minutes. Refolding was performed by rapid dilution in refolding buffer (usually: 100mM Tris-HCl pH 8.0, 4.0M urea, 5mM cysteine, 1mM cystine) at a final protein concentration of 0.5g / L of dissolved protein. The refolding reaction was routinely incubated for a minimum of 14 hours. The resulting protein solution was concentrated and exchanged by diafiltration with a native buffer (50mM citrate-phosphate pH 6.4, 200mM NaCl). The refolded protein is purified by size exclusion chromatography on a suitable resin material (e.g., Superdex 75, GE Healthcare). The separated monomer fractions are analyzed by size exclusion HPLC, SDS-PAGE is used to analyze the purity, and UV / Vis spectroscopy is used to analyze the protein content. The protein concentration is adjusted to the desired level, and stability analysis is performed.

[0158] Comparison of structural models

[0159] The three-dimensional structures of the variable domains VLκ and VLλ were compared using examples of structural models obtained in the PDB (PDB ID 1FVC and PDB ID 2A9M, respectively). Analysis of the packing of the VL framework region IV revealed differences in the orientation of the side chains from position 147 upwards. In addition, the orientation of the amino acid side chains at position 101 is different in the Vκ and Vλ structures. Figure 4 As shown, the different packing of the central amino acid (position 101) is obvious. In the case of Vκ, phenylalanine 101 is indicated in the core of the domain in the λ variable domain, however, the glutamic acid at position 101 is exposed to the solvent, and V147 (arrow) is instead positioned on the hydrophobic core. Based on this situation, variable domains containing amino acid exchange F101E (SEQ ID No: 8, 11 and 13) were generated to accommodate the Vλ-specific packing of the Vλ framework IV.

[0160] Example 2: Determination of biophysical data of scFv constructs

[0161] Thermal unfolding

[0162] The transition midpoint of the thermal unfolding of the tested constructs was determined by differential scanning fluorimetry (DSF), essentially as described by Niesen (Niesen et al., Nat Protoc. 2 (2007) 2212-21). The DSF assay was performed on a qPCR instrument (e.g., MX3005p, Agilent Technologies). The samples were diluted in a 25 μL total volume of buffer (citrate-phosphate pH 6.4, 0.25 M NaCl) containing a final concentration of 5xSYPRO orange. In a buffer exploratory experiment, the pH dependence of the unfolding temperature was determined for all constructs and comparable pH characteristics were observed. Samples were measured in triplicate, and the program was set so that the temperature ramped from 25°C to 96°C. Fluorescence signals were obtained, and raw data were analyzed with GraphPad Prism (GraphPad Software Inc.).

[0163] Stress stability research

[0164] Through two weeks and under 37 ℃ of storage, analyze the oligomerization of protein by size exclusion-high performance liquid chromatography (SE-HPLC), and analyze the degradation of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Before research, sample is concentrated into 1,10 and 40g / L, and determine t0 time point.By separating sample on Shodex KW-402.5-4F (Showa Denko) and evaluating gained chromatogram, quantify monomer content.In order to calculate the relative percentage of protein monomer, the area of ​​monomer peak is divided by the total area of ​​the peak that cannot be attributed to sample matrix.Carry out SDS-PAGE analysis with Any kD Mini-ProteanTGX gel (Bio-Rad Laboratories) to assess protein degradation, and utilize Coomassie brilliant blue to dye.At different time points, monitor protein concentration by carrying out UV-Vis spectroscopy with Infinity reader M200 Pro (Tecan Group Ltd.) equipped with Nanoquant flat plate.

[0165] Example 3: Construction of scFv constructs from commercially available humanized mouse monoclonal antibodies

[0166] In order to confirm that the proposed concept has wide applicability to the stabilization of antibody variable domains, two single-chain Fv constructs were produced based on commercially available humanized mouse monoclonal antibodies containing Vκ light chains. For the first construct, the original variable domain sequence was used for the amino acid sequence of IgG as disclosed, and for the second construct, a modified sequence was used, in which the framework region IV of the variable light domain was replaced with the corresponding sequence from the lambda germline gene. In the scFv construct, the variable domain is connected by a flexible (Gly4Ser)4 linker (SEQ ID NO:1) with 20 amino acids, thereby producing the configuration of NH2-VL-linker-VH-COOH as used in the model constructs scFv1 to scFv10 (see Table 2). Expression and refolding of scFv molecules were performed as described above, and the refolded protein was purified by affinity chromatography on protein L resin. Analysis of the purified protein by SE-HPLC revealed significant differences in the producibility of the constructs, which were manifested as significantly increased monomer content of the constructs containing the lambda framework IV in VL (see Table 2). Figure 5 ). In addition, thermal unfolding analysis of constructs with the original sequence and molecules containing the lambda framework IV in VL yielded unfolding midpoints of 69.9° C. and 71.2° C., respectively. This observation is consistent with the results described in paragraph

[0082] .

[0167] Example 4: Construction of scFv constructs with shorter linker sequences

[0168] To analyze the effect of the linker sequence, a shorter (Gly4Ser)3 linker (SEQ ID NO: 34) with 15 amino acids was used to make an alternative construct in the same manner as described above in Example 1. In addition to the linker, the two constructs scFv11 and scFv12 correspond to the constructs scFv1 and scFv5, respectively (see Table 2). Stress stability studies were performed as described above in Example 2. As shown in Table 4, the construct scFv12 containing the lambda framework IV in the VL of the shorter linker showed increased stability relative to the construct with the common kappa light chain scFv11. The overall stability of the molecule is lower than that of the corresponding construct with a shorter linker. It is well known that scFv stability can be increased by replacing the 15-mer linker with a 20-mer or 25-mer linker (see and Plückthun, J. Mol. Biol. 305 (2001) 989-1010). Therefore, these findings are consistent with the results using a 20-mer linker and confirm that the stability of the variable domain is improved by introducing the lambda framework IV into the VL.

[0169] Table 1: Protein sequence list

[0170]

[0171]

[0172]

[0173]

[0174] (In SEQ ID NOs: 23 to 33, the CDR regions are in bold and italics)

[0175] Table 2: Combinations of variable domains in different scFv constructs

[0176]

[0177]

[0178] Table 3: Transition midpoints of thermal unfolding for all constructs determined by differential scanning fluorimetry

[0179]

[0180] Table 4: Monomer loss during storage

[0181]

[0182]

[0183] Note: Italicized entries are from scFv constructs with Vκ-type framework IV regions

[0184] Table 5: List of clones and sequence changes listed in Borras et al. (loc. cit.)

[0185] All clones listed below have the sequence of FW1.4gen (SEQ ID NO:4), but differ at the positions indicated in bold below (position numbering according to Borras et al., loc. cit.)

[0186]

[0187]

[0188] *****

[0189] The present invention is not limited in scope by the specific embodiments described herein. In fact, those skilled in the art will understand various modifications of the present invention in addition to those described herein based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0190] All patents, applications, publications, test methods, references, and other materials cited herein are incorporated by reference to the extent permitted by applicable patent law. Sequence Listing <110> Numa Medical Technologies Ltd. <120> Novel antibody framework <130> 15PA02028PCN-DI <150> EP13003264.2 <151> 2013-06-26 <160> 42 <170> PatentIn Version 3.5 <210> 1 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Artificial joint <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 2 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Artificial VL sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 3 <211> 122 <212> PRT <213> artificial sequence <220> <223> Artificial VH series <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Gly 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ile Ser Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Pro Asp Asp Ser Asn Ser Met Gly Thr Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 4 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 5 <211> 122 <212> PRT <213> artificial sequence <220> <223> Artificial VH series <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gly 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Val Ile Ile Ser Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Pro Asp Asp Ser Asn Ser Met Gly Thr Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 6 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 7 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 8 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Glu Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 9 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 10 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gln Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 11 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Glu Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 12 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 13 <211> 111 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Glu Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 14 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Gly 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ile Ser Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Pro Asp Asp Ser Asn Ser Met Gly Thr Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial VL framework sequence <400> 15 Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 1 5 10 <210> 16 <211> 11 <212> PRT <213> Homo sapiens <400> 16 Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 1 5 10 <210> 17 <211> 11 <212> PRT <213> Homo sapiens <400> 17 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1 5 10 <210> 18 <211> 11 <212> PRT <213> Homo sapiens <400> 18 Phe Gly Gly Gly Thr Gln Leu Ile Ile Leu Gly 1 5 10 <210> 19 <211> 11 <212> PRT <213> Homo sapiens <400> 19 Phe Gly Glu Gly Thr Glu Leu Thr Val Leu Gly 1 5 10 <210> 20 <211> 11 <212> PRT <213> Homo sapiens <400> 20 Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly 1 5 10 <210> 21 <211> 11 <212> PRT <213> Human (Homo sapiens) <400> 21 Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly 1 5 10 <210> 22 <211> 11 <212> PRT <213> Human (Homo sapiens) <400> 22 Phe Gly Gly Gly Thr Gln Leu Thr Ala Leu Gly 1 5 10 <210> 23 <211> 108 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 24 <211> 113 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 24 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Angry <210> 25 <211> 109 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 26 <211> 114 <212> PRT <213> artificial sequence <220> <223> Artificial VL sequence <400> 26 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Light Arg <210> 27 <211> 119 <212> PRT <213> artificial sequence <220> <223> Artificial VH series <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gly Tyr Cys Ser Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 28 <211> 117 <212> PRT <213> artificial sequence <220> <223> Artificial VH series <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 29 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 29 Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile His Asn Ile Gly Glu Ala Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Tyr Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Gly Ser Gly Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 31 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Gly Gly Gly Gly Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 32 <211> 119 <212> PRT <213> artificial sequence <220> <223> Artificial VH series <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Gly Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 33 <211> 119 <212> PRT <213> artificial sequence <220> <223> Artificial VH series <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Asp Pro Gly Gly Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 34 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Artificial VL sequence <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 35 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Artificial VL sequence <400> 35 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 36 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Artificial VL sequence <400> 36 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210> 37 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial VL sequence <400> 37 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 38 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 39 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 39 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 40 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 40 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 20 25 30 <210> 41 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial VH sequence <400> 41 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 42 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Artificial joint <400> 42 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 20

Claims

1. An isolated antibody or a functional fragment thereof comprising a VH domain and a VL domain, characterized in that: The VL domain comprises: (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) framework region IV, wherein framework region IV is a human Vλ germline sequence for framework region IV, and the Vλ germline sequence is selected from: SEQ ID NO.16 to 22, wherein the CDR domains CDR1, CDR2 and CDR3 are specific to the target antigen and are selected from the CDR domains CDR1, CDR2 and CDR3 from the parent mouse or rat antibody.

2. The isolated antibody or functional fragment thereof according to claim 1, characterized in that The Vκ framework regions I to III are (i) framework regions present in the sequence of SEQ ID NO: 23, or (ii) framework regions comprising no more than five mutations compared to each framework region present in the sequence of SEQ ID NO: 23, and wherein the VH domain includes framework regions I to IV, which are (i) framework regions present in the sequence of SEQ ID NO: 30, or (ii) framework regions comprising no more than five mutations compared to each framework region present in the sequence of SEQ ID NO:

30.

3. The isolated antibody or functional fragment thereof according to claim 1, characterized in that The amino acid residue at position AHo101 of the framework region III of the VL domain is glutamic acid.

4. The isolated antibody or functional fragment thereof according to claim 1, characterized in that The Vκ framework regions I to III are framework regions present in sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO:

8.

5. The isolated antibody or functional fragment thereof according to claim 4, characterized in that The Vκ framework regions I to III are present in the sequence SEQ ID NO:

8.

6. The isolated antibody or functional fragment thereof according to claim 1, characterized in that The framework regions I to IV are a combination of framework regions present in the sequences selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:

13.

7. The isolated antibody or functional fragment thereof according to claim 1, characterized in that The isolated antibody or its functional fragment is selected from: IgG antibody, Fab fragment, scFv fragment, single-chain diabody, tandem scDb, linear dimeric scDb, cyclic dimeric scDb, bispecific T cell engager, tandem three scFv, three antibodies, three-chain antibodies, bispecific Fab2, two mini antibodies, four antibodies, scFv-Fc-scFv fusion, two diabodies, DVD-Ig, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusion, and knob-in-hole structure and double antibody, wherein the knob-in-hole structure is a bispecific IgG prepared by KiH technology, and the double antibody is a bispecific IgG prepared by Duobody technology.

8. The isolated antibody or functional fragment thereof according to claim 7, characterized in that The IgG-scFv fusion is selected from: a scFv linked to the C-terminus of an IgG light chain, a scFv linked to the N-terminus of an IgG light chain, a scFv linked to the N-terminus of an IgG heavy chain, a scFv linked to the C-terminus of an IgG heavy chain, a scFv linked to the N-terminus of both an IgG heavy chain and a light chain, and a dsscFv linked to the C-terminus of an IgG heavy chain.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the isolated antibody or a functional fragment thereof according to any one of claims 1 to 8.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

11. A nucleic acid sequence or a collection of nucleic acid sequences, characterized in that: The nucleic acid sequence or set of nucleic acid sequences (i) encodes the antibody VL domain of the isolated antibody or its functional fragment according to any one of claims 1 to 8, or the isolated antibody or its functional fragment according to any one of claims 1 to 8.

12. A carrier or a carrier collection, characterized in that: The vector or a collection of vectors comprises the nucleic acid sequence or a collection of nucleic acid sequences according to claim 11.

13. A host cell, characterized in that The host cell comprises the nucleic acid sequence or a collection of nucleic acid sequences according to claim 11, or the vector or a collection of vectors according to claim 12.

14. The host cell according to claim 13, characterized in that The host cell is an expression host cell.

15. A method for producing an antibody VL domain of an isolated antibody or a functional fragment thereof according to any one of claims 1 to 8, or an isolated antibody or a functional fragment thereof according to any one of claims 1 to 8, the method comprising the following steps: Expressing the nucleic acid sequence or the collection of nucleic acid sequences according to claim 11, or the vector or the collection of vectors according to claim 12, or the host cell according to claim 13 or 14.

16. A method for producing a humanized antibody, characterized in that: The method comprises the following steps: a. separating at least one antibody of interest from a mouse or rat that has been immunized with an antigen of interest; and b. cloning the VL CDR region of the at least one antibody of interest into the nucleic acid sequence encoding the antibody VL domain of the isolated antibody or functional fragment thereof according to any one of claims 1 to 8.

17. The method according to claim 16, characterized in that The method further comprises one or more of the following steps: aa. Isolating affinity-matured memory B cells that interact with the antigen of interest by using fluorescence-activated cell sorting clonal isolation; ab. Cultivating single B cells in a co-culture system that does not require immortalization of single B cell clones; ac. screening B cell culture supernatants in a cell-based ELISA to identify at least one antibody that binds to the antigen of interest; and / or ad. The VH CDR region of at least one antibody is cloned into a nucleic acid sequence encoding a human antibody VH domain.

18. A method for producing a nucleic acid sequence encoding an antibody VL domain of an isolated antibody or a functional fragment thereof according to any one of claims 1 to 8, or one or more nucleic acid sequences encoding an isolated antibody or a functional fragment thereof according to any one of claims 1 to 8, the method comprising combining in one or more steps nucleic acid sequences encoding: (i) human Vκ1 framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3 from a parent mouse or rat antibody specific for an antigen of interest; and (iii) framework region IV, which is a human Vλ germline sequence for framework region IV, and the Vλ germline sequence is selected from: SEQ ID NO. 16 to 22.

19. The method according to claim 18, characterized in that The method uses one of the following methods: i. In a nucleic acid construct comprising a nucleic acid sequence encoding a human or humanized Vκ1 domain, replacing Vκ1 framework region IV by framework region IV, wherein framework region IV is a human Vλ germline sequence for framework region IV, wherein the Vλ germline sequence is selected from: SEQ ID NO. 16 to 22; ii. inserting one or more nucleic acid sequences encoding the following into a nucleic acid construct comprising a nucleic acid sequence encoding framework region IV in one or more steps: (i) human Vκ1 framework regions I to III; and (ii) CDR domains CDR1, CDR2 and CDR3 from a parent antibody specific for an antigen of interest, wherein the framework region IV is a human Vλ germline sequence for framework region IV, the Vλ germline sequence being selected from the group consisting of: SEQ ID NOs. 16 to 22; iii. mutating the nucleic acid sequence encoding framework region IV in the nucleic acid sequence encoding a human or humanized Vκ1 domain to generate a framework region IV, wherein the framework region IV is a human Vλ germline sequence for framework region IV; iv. in one or more steps, in a nucleic acid construct comprising a nucleic acid sequence encoding a light chain domain comprising human Vκ1 framework regions I to III, CDR domains CDR1, CDR2 and CDR3, and framework region IV, one or more nucleic acid sequences encoding the CDR domains are replaced by one or more nucleic acid sequences encoding corresponding one or more CDR domains from a parent mouse or rat antibody specific for an antigen of interest, wherein the framework region IV is a human Vλ germline sequence for framework region IV, and the Vλ germline sequence is selected from: SEQ ID NO.16 to 22.

20. The method according to claim 18, characterized in that The nucleic acid construct is a recombinant vector.

21. A nucleic acid sequence or a collection of nucleic acid sequences, characterized in that: The nucleic acid sequence or the collection of nucleic acid sequences can be obtained by the method according to claim 18.

Images