Novel stable antibody variable domain framework combinations

Abstract

To provide novel antibody variable domain combinations with advantageous properties.SOLUTION: The present invention provides novel combinations of human antibody heavy chain domains with chimeric human antibody light chain frameworks, comprising framework regions I to III derived from VK and framework region IV derived from Vλ, with advantageous properties, such as high stability, reduced aggregation propensity, and improved binding affinity.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This invention relates to a novel combination of antibody variable regions having advantageous properties. [Background technology]

[0002] The present invention relates to a novel combination of a human antibody heavy chain domain and a chimeric human antibody light chain framework, comprising VK-derived framework regions I-III and Vλ-derived framework region IV, and possessing advantageous properties such as high stability, reduced aggregation tendency, and improved binding affinity.

[0003] In the past 40 years since the development of the first monoclonal antibodies ("mAbs," Koehler & Milstein, Nature, 256(1975) 495-7), antibodies have become an increasingly important class of biomolecules for research, diagnostic, and therapeutic purposes. Initially, antibodies were obtained exclusively by immunizing animals with the corresponding antigen of interest. While non-human antibodies can be used in research and diagnostics, in therapeutic approaches, the human body may recognize non-human antibodies as foreign substances, triggering an immune response to non-human antibody drugs and reducing or rendering them ineffective. Therefore, recombinant methods have been proposed to reduce the immunogenicity of non-human antibodies.

[0004] The first attempts to convert non-human mAbs into less immunogenic therapeutics required the manipulation of chimeric antibodies consisting of an animal (e.g., rodent) variable domain and a human constant region (Boulianne et al., Nature 312, (1984) 643-646). Further approaches aimed at humanizing rodent mAbs by introducing a complementarity-determining region (CDR) within a human variable domain scaffold (Jones et al., Nature 321 (1986) 522-525; Riechmann et al., Nature 332 (1988) 323-7), or by resurfacing the variable domain (Roguska et al., Proc.NATl.Acad.Sci. USA 91 (1994) 969-973).

[0005] For humanization of CDRs by loop grafting, the human acceptor framework is selected based on homology with the donor framework (e.g., Roguska et al., Protein Engineering 9(1996) 895-904; International Publication No. 2008 / 144757 (for rabbits)) or based on a suitable stability profile (Ewert et al., Methods 34(2004) 184-199). The latter concept has been used for humanization of rabbit antibodies to a universal variable domain framework (U.S. Patent No. 8,193,235).

[0006] The mAbs or functional fragments obtained by any selected approach ideally exhibit drug-like biophysical properties and minimal immunogenicity while retaining the desired pharmacodynamic properties of the donor mAb. Regarding the biophysical properties of the mAb, or its functional fragment, the tendency to aggregate has been a major concern for the development of therapeutic molecules, primarily for three reasons:

[0007] Firstly, protein aggregates generally show a high potential to induce an immune response in the host that leads to the formation of anti-drug antibodies and ultimately drug-neutralizing antibodies (Joubert et al., J. Biol. Chem. 287(2012) 25266-25279).

[0008] Secondly, aggregates affect manufacturing yield due to the increased effort required to remove them (Cromwell et al., AAPS Journal 8 (2006), Article 66).

[0009] Thirdly, off-target effects may be observed. Concerns regarding oligomerization are even more pronounced in applications where monovalent binding is preferred, such as bispecific (or multispecific) antibody formats having only one valence per target and construct, because oligomerization in these cases results in protein aggregates with multivalent binding properties, which can lead to off-target effects. An example of such nonspecific activity is the use of constructs having a single CD3ε-binding domain in a bispecific antibody format. Such a format may, for example, bind one of its two binding domains to a cancer antigen and its second CD3ε-binding domain to recruit cytotoxic T cells. Since crosslinking of the monovalent CD3ε-binding moiety is required to induce CD3ε-mediated signaling, T cells will only be stimulated if they are involved with multiple bispecific constructs bound to the surface of target cells—and thus adopt the properties of the crosslinking molecule—resulting in a T cell response specific only to cancer cells. Conversely, oligomers of such constructs exhibit the properties of crosslinked bispecific antibodies and therefore activate cytotoxic T cells even without binding to cancer cells, thereby resulting in systemic T cell activation. Such nonspecific and systemic activation of T cells can lead to elevated cytokine levels and potentially cause adverse effects.

[0010] Antibody stability is crucial to production, purification, and shelf life, and consequently, to the commercial cost of antibody therapeutics. Even slight improvements in one or more of these parameters are highly relevant to whether the research and development of antibody drugs is commercially viable.

[0011] Furthermore, since cloning, expression, and purification methods can be standardized, a reliable and universally applicable acceptor framework is beneficial in enabling robust methods for humanizing non-human antibodies.

[0012] To meet the above criteria for humanization of non-human mAbs, published methodologies propose the use of human consensus variable domain framework sequences as acceptor scaffolds for the engraftment of non-human complementarity-determining regions. It is generally understood that, based on the assumption that residues contributing to protein stability are abundant in the evolutionary germline sequence pool for each amino acid position in a protein, the closer the resulting humanized variable domain is to the human germline sequence of its respective variable domain family, the more stable it is expected to be. This concept, described by Steipe (Steipe et al., J.Mol.Biol. 240(1994) 188-92) and reviewed by Woern (Woern et al., J.Mol.Biol. 305(2001) 989-1010), is widely accepted and has found broad applications. Non-limiting examples include (a) the use of consensus sequence variable domains for humanization of non-human antibodies (Carter et al., Proc.NATl.Acad.Sci. USA 89(1992) 4285-4289); (b) the use of consensus sequence variable domains for constructing CDR libraries for in vitro screening of stable target-binding antibodies (Knappik et al., J.Mol.Biol. 296(2000) 57-86); and (c) a knowledge-based approach to improving the stability of antibody variable domains by replacing non-consensus residues with consensus residues (Steipe et al., J Mol Biol, 240(1994), 188-92).

[0013] Furthermore, the stability of different variable domain families is demonstrated by VH3, the most stable variable heavy domain. Importantly, for variable light chain domains, the VK family is preferred over the Vλ family (Ewert et al., J Mol Biol, 325 (2003) 531-53). Specifically, the human consensus sequences of VH3 and VK1 possess favorable biophysical properties (Ewert et al., J Mol Biol, 325 (2003) 531-53) and are particularly suitable for the humanization of non-human antibodies (used in Carter et al., Proc.NATl.Acad.Sci. USA 89 (1992) 4285-4289).

[0014] In line with this, several publications describe the use of the human Vκ1-VH3 consensus framework hu-4D5 in the humanization of rodent and rabbit antibodies (Rader, J. Biol. Chem. 275(2000) 13668-13676; International Publication No. 2005 / 016950; International Publication No. 2008 / 004834). Alternatively, combinations of VH and VL sequences belonging to the same family as hu-4D5 were originally derived from recombinant clonal immunorepertory repertoire and have been used to generate stable humanized single-chain (scFv) fragments derived from rabbits (US Patent No. 8,293,235; Borras et al., J. Biol. Chem. 285(2010) 9054-9066).

[0015] To further optimize such a framework, a chimeric human antibody light chain domain, comprising framework regions I-III from the VK light chain and framework region IV from the Vλ light chain, was identified to possess advantageous properties, such as high stability and reduced aggregation tendency (International Publication No. 2014 / 206561).

[0016] However, for certain rabbit antibodies, the use of the VK1 / VH3 combination was associated with a significant decrease in antigen-binding affinity of the humanized variable domain, which requires engraftment at the donor framework site. While the overall homology of the VK1 / VH3 combination to the rabbit variable domain appears to be the greatest, we cannot rule out the possibility that other combinations might better support engraftment of rabbit CDRs.

[0017] Therefore, despite the fact that many attempts have already been made to address the problem of obtaining humanized antibody drug substances from non-human antibodies, a great unmet need remains to develop novel human antibody domains, or combinations of antibody domains, that possess favorable properties, such as high stability, reduced aggregation tendency, and improved binding affinity. Here, the human antibody framework contains as few mutations as possible, and ideally no mutations, compared to naturally occurring sequences, in order to reduce the risk of creating immunogenic sequences as much as possible. Such a stable human framework can also be used to stabilize a full human antibody or a fragment thereof, for example, by loop transplantation, or simply by exchanging stability-contributing components between the parent antibody and the stable framework.

[0018] The solution to this problem provided by the present invention, namely a novel combination of a VH domain and a chimeric human antibody light chain domain, comprising framework regions I-III from VK and framework region IV from Vλ, having advantageous properties such as high stability, reduced aggregation tendency, and improved binding affinity, has not been achieved or suggested in the prior art. [Overview of the project]

[0019] The present invention relates to a novel combination of a specific VH domain and a specific chimeric human antibody light chain domain, comprising framework regions I-III from VK and framework region IV from Vλ, having advantageous properties such as high stability, reduced aggregation tendency, minimal immunogenicity, and improved binding affinity.

[0020] Therefore, in a first aspect, the present invention relates to an antibody or a functional fragment thereof, comprising: (a) Variable light chain, Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determination region, where: (i) The LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity, preferably at least 95% sequence identity, with respect to the corresponding framework region obtained from the VK1 sequence described in SEQ ID NO: 41; or the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51; and (ii) A variable light chain in which LFW4 is a Vλ-based sequence that is at least 90% identical to sequence number 63, preferably LFW4 is the sequence described in sequence number 63, sequence number 79, or sequence number 80, and more preferably LFW4 is the sequence described in sequence number 63; Furthermore, (b) Variable heavy chain, Here, the variable heavy chain includes the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determination region. Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 82% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21, or at least 75% sequence identity with respect to the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1, resulting in a variable heavy chain.

[0021] In an alternative embodiment, the present invention relates to an antibody or a functional fragment thereof, comprising: (a) Variable light chain, Here, the variable light chain contains the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region, and each LCDR represents a light chain complementarity-determining region, where: (iii) the LFW1, LFW2, and LFW3 regions together show at least 80% sequence identity, preferably at least 95% sequence identity, to the corresponding framework region obtained from the Vκ1 sequence set forth in SEQ ID NO: 41; or to the corresponding framework region obtained from the Vκ3 sequence set forth in SEQ ID NO: 51; and (iv) the LFW4 is a Vλ-based sequence that is at least 90% identical to SEQ ID NO: 63, preferably the LFW4 is the sequence set forth in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, more preferably the LFW4 is the sequence set forth in SEQ ID NO: 63, variable light chain; And, (b) a variable heavy chain, Here, the variable heavy chain contains the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region, and each HCDR represents a heavy chain complementarity-determining region, where the HFW1, HFW2, HFW3, and HFW4 regions together show at least 85% sequence identity, preferably 95% sequence identity, to the corresponding framework region obtained from the VH4 sequence set forth in SEQ ID NO: 21; or to the corresponding framework region obtained from the VH1A sequence set forth in SEQ ID NO: 1, variable heavy chain.

[0022] In a second aspect, the present invention relates to a pharmaceutical composition comprising an antibody of the present invention, or a functional fragment thereof, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0023] In a third aspect, the present invention relates to a nucleic acid sequence or collection of nucleic acid sequences encoding an antibody of the present invention, or a functional fragment thereof.

[0024] In a fourth aspect, the present invention relates to a vector or collection of vectors comprising a nucleic acid sequence or a collection of nucleic acid sequences of the present invention.

[0025] In a fifth aspect, the present invention relates to a host cell, specifically an expression host cell, comprising a nucleic acid sequence or collection of nucleic acid sequences of the present invention, or a vector or collection of vectors of the present invention.

[0026] In a sixth aspect, the present invention relates to a method for producing an antibody or a functional fragment thereof, comprising the steps of expressing a nucleic acid sequence or collection of nucleic acid sequences of the present invention, or a vector or collection of vectors of the present invention, or a host cell of the present invention, specifically an expression host cell.

[0027] In a seventh aspect, the present invention relates to a method for humanizing a non-human antibody, specifically a rabbit or rodent antibody, comprising the following steps. (a) In one or more steps, nucleic acid sequences encoding the variable heavy chain (VH) CDR and variable light chain (VL) CDR of the non-human antibody are cloned into one or more nucleic acid sequences encoding the antibody or a functional fragment thereof described in the present invention, wherein at least the VH CDR3 and VL CDR3 of the non-human antibody are cloned.

[0028] In the eighth aspect, the present invention relates to a method for optimizing a target parent antibody, comprising the following steps: (a) In one or more steps, the nucleic acid sequences encoding the VH CDR and VL CDR of the parent antibody are cloned into one or more nucleic acid sequences encoding the antibody or a functional fragment thereof according to the present invention, wherein at least the VH CDR3 and VL CDR3 of the parent antibody are cloned.

[0029] In the ninth aspect, the present invention relates to a method for generating a diverse collection of antibodies or functional fragments thereof, comprising the following steps: (a) Cloning one or more diverse collections of nucleic acid sequences encoding one or more diverse collections of VH CDRs and / or VL CDRs into one or more nucleic acid sequences encoding the antibody or a functional fragment thereof as described in the present invention, in one or more steps. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows that λ-capped (upper half) or uncapped (i.e., including Vk Framework IV; lower half, gray), VK1, VK2, and VK3 consensus variable light chains combine with VH1A, VH1B, VH2, VH3, VH4, VH5, and VH6 consensus variable heavy chain domains to lead to the 42 constructs investigated in this study. [Figure 2] Figure 2 shows the expression yield of the capped (white bars) framework variant compared to the uncapped (gray bars) control. [Figure 3] Figure 3 shows the mean thermal denaturation temperatures (Tm) determined by differential scanning fluorescence (DSF) analysis of capped framework variants (white bars) and uncapped framework objects (gray bars), measured in five phosphate-citrate buffer solutions containing 0.15 M NaCl in the pH range of 3.5–7.5. [Figure 4A]Figure 4 shows a comparison of normalized monomer content (relative to initial monomer content) measured by SE-HPLC after storage at 37°C and 10 mg / mL for 2 weeks for capped variants (white bars) and uncapped variants (gray bars) (Figure 4A). The reference sample of the capped variant VK1 / VH3 showed a 39% less decrease in monomer content compared to the uncapped variant. Underlined values ​​indicate framework combinations that show less monomer loss than the reference VK1 / VH3 germline combination compared to the uncapped control during incubation. The monomer content (%) of the capped framework variants (white bars) and uncapped framework variants (gray bars) is shown in Figure 4B. Several frameworks show less monomer content loss than the reference, as indicated by the baseline (VK1 / VH3). [Figure 4B] Figure 4 shows a comparison of normalized monomer content (relative to initial monomer content) measured by SE-HPLC after storage at 37°C and 10 mg / mL for 2 weeks for capped variants (white bars) and uncapped variants (gray bars) (Figure 4A). The reference sample of the capped variant VK1 / VH3 showed a 39% less decrease in monomer content compared to the uncapped variant. Underlined values ​​indicate framework combinations that show less monomer loss than the reference VK1 / VH3 germline combination compared to the uncapped control during incubation. The monomer content (%) of the capped framework variants (white bars) and uncapped framework variants (gray bars) is shown in Figure 4B. Several frameworks show less monomer content loss than the reference, as indicated by the baseline (VK1 / VH3). [Figure 5]Figure 5 shows the absolute percentage monomer content of TNF-specific regulatory molecules in capped framework variants (white bars) and uncapped framework controls (gray bars) after storage at 37°C for 2 weeks at 10 mg / mL. [Modes for carrying out the invention]

[0031] Detailed description of the invention This disclosure relates to antibodies and fragments thereof, comprising novel combinations of specific VH domains and specific chimeric human antibody light chain domains, which have advantageous properties, such as high stability, reduced aggregation tendency, minimal immunogenicity, and improved binding affinity, comprising framework regions I-III from VK and framework region IV from Vλ. Surprisingly, combinations of human variable VL / VH consensus frameworks have been found, comprising a humanized consensus (i) framework regions I-III from VK1 or VK3, and (ii) framework region IV from Vλ, and (iii) a combinatorial pair of a VH1A, VH1B, or VH4 heavy chain domain, which exhibit at least equivalent or superior biophysical properties compared to VK1 / VH3 (capped or uncapped) while fully retaining specificity and antigen-binding affinity. The inventors have further demonstrated that introducing two mutations at specific framework positions (T24K and T84S) of VH4 not only improves biophysical properties but also improves affinity. Furthermore, the inventors have demonstrated for the first time that, in addition to selecting the optimal combination of VH and VL frameworks, incorporating a specific λ cap (sk17, SEQ ID NO: 63) into the VL consensus domain also leads to further improvements in the biophysical properties of each antibody or its functional fragment. Surprisingly, antibodies containing sk17 were found to have improved not only storage and thermal stability but also affinity.

[0032] Therefore, in a first aspect, the present invention relates to an antibody or a functional fragment thereof, comprising: (a) Variable light chain, Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determination region, where: (i) The LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity, preferably at least 95% sequence identity, with respect to the corresponding framework region obtained from the VK1 sequence described in SEQ ID NO: 41; or the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51; and (ii) A variable light chain in which LFW4 is a Vλ-based sequence that is at least 90% identical to sequence number 63, preferably LFW4 is the sequence described in sequence number 63, sequence number 79, or sequence number 80, and more preferably LFW4 is the sequence described in sequence number 63; Furthermore, (b) Variable heavy chain, Here, the variable heavy chain includes the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determination region. Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 82% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21, or at least 75% sequence identity with respect to the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1, resulting in a variable heavy chain.

[0033] According to this disclosure, the framework regions taken from the VK1 sequence by sequence number 41 are LFW1', LFW2', LFW3', and LFW4', respectively, as indicated by sequence numbers 42, 43, 44, and 45. The framework regions taken from the VK3 sequence by sequence number 51 are LFW1', LFW2', LFW3', and LFW4', respectively, as indicated by sequence numbers 52, 53, 54, and 55. The framework regions taken from the VH4 sequence by sequence number 21 are LFW1', LFW2', LFW3', and LFW4', respectively, as indicated by sequence numbers 22, 23, 24, and 25. The framework regions taken from the VH4 sequence by sequence number 26 are LFW1', LFW2', LFW3', and LFW4', respectively, as indicated by sequence numbers 27, 28, 29, and 30. The framework regions taken from the VH1A sequence by sequence number 1 are LFW1', LFW2', LFW3', and LFW4', respectively, indicated by sequence numbers 2, 3, 4, and 5. The framework regions taken from the VH1A sequence by sequence number 6 are LFW1', LFW2', LFW3', and LFW4', respectively, indicated by sequence numbers 7, 8, 9, and 10.

[0034] In certain embodiments of such a configuration, the HFW4 region has (i) a sequence contained in the human heavy chain germline J segment, or (ii) a sequence contained in a rearranged human VH sequence. In certain embodiments, the HFW4 sequence is WGQGTLVTVSS, or a sequence exhibiting at least 70%, at least 80%, or at least 90% sequence identity to WGQGTLVTVSS.

[0035] In an alternative embodiment, the present invention relates to an antibody or a functional fragment thereof, comprising: (a) Variable light chain, Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determination region, where: (v) The LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity, preferably at least 95% sequence identity, with respect to the corresponding framework region obtained from the VK1 sequence described in SEQ ID NO: 41; or the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51; and (vi) A variable light chain in which LFW4 is a Vλ-based sequence that is at least 90% identical to sequence number 63, preferably LFW4 is the sequence described in sequence number 63, sequence number 79, or sequence number 80, and more preferably LFW4 is the sequence described in sequence number 63; Furthermore, (b) Variable heavy chain, Here, the variable heavy chain includes the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determination region. Herein, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity, preferably 95% sequence identity, with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21, or the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1, thus forming a variable heavy chain.

[0036] In one embodiment, the present invention relates to an antibody or a functional fragment thereof, comprising: (a) Variable light chain, Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determination region, where: (i) The LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity, preferably at least 95% sequence identity, for example at least 97% sequence identity, and optionally at least 86%, preferably at least 90%, more preferably at least 96% sequence homology with respect to the corresponding framework region obtained from the VK1 sequence described in SEQ ID NO: 41; or the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51; and (ii) A variable light chain in which LFW4 is a Vλ-based sequence that is at least 90% identical to sequence number 63, preferably LFW4 is the sequence described in sequence number 63, sequence number 79, or sequence number 80, and more preferably LFW4 is the sequence described in sequence number 63; Furthermore, (b) Variable heavy chain, Here, the variable heavy chain includes the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determination region. Here, the above HFW1, HFW2, HFW3, and HFW4 regions are combined. (i) With respect to the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1, the sequence exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95%, and optionally at least 85%, for example at least 90%, at least 95%, at least 96%, preferably at least 90%, more preferably at least 96% homology; or (ii) With respect to the corresponding framework region obtained from the VH1B sequence described in Sequence ID No. 6, the sequence exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95%, and optionally at least 85%, for example at least 90%, at least 93%, at least 96%, preferably at least 93%, more preferably at least 96% homology; or (iii) A variable heavy chain exhibiting at least 82%, at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95% sequence identity, and optionally at least 90%, preferably at least 95% homology, with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21.

[0037] In certain such embodiments, the HFW4 region has (i) a sequence contained in the human heavy chain germline J segment, or (ii) a sequence contained in a rearranged human VH sequence. In certain embodiments, the HFW4 sequence is WGQGTLVTVSS, or a sequence exhibiting at least 70%, at least 80%, or at least 90% sequence identity to WGQGTLVTVSS.

[0038] In an alternative embodiment, the HFW1, HFW2, HFW3, and HFW4 regions are combined to represent the following: (iv) With respect to the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1, the sequence exhibits at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 95%, more preferably at least 97%, and optionally at least 90%, for example at least 90%, at least 95%, at least 96%, preferably at least 90%, more preferably at least 96% homology; or (v) exhibiting at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the corresponding framework region obtained from the VH1B sequence described in Sequence ID No. 6, and optionally at least 90%, more preferably at least 93%, more preferably at least 96% homology; or (vi) The sequence identity is at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95%, with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21, and optionally at least 90%, more preferably at least 95% homology.

[0039] In the context of this invention, the term “antibody” is used as a synonym for “immunoglobulin” (Ig), defined as a protein belonging to class IgG, IgM, IgE, IgA, IgY, or IgD (or its subclasses), and includes all conventionally known antibodies. Naturally occurring “antibodies” are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FWs). Each VH and VL consists of three CDRs and four FWs arranged from the amino terminus to the carboxyl terminus in the following order: FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0040] The term “antibody fragment” refers to at least a portion of an intact antibody or its recombinant variant, while the term “functional fragment” or “functional antibody fragment” refers to an antibody fragment containing at least one antigen-binding domain, e.g., the antigen-determining variable region of an intact antibody, sufficient to confer recognition and specific binding to a target, e.g., an antigen, of the functional antibody fragment. Examples of functional antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies, e.g., sdAb(either VL or VH), camelid VHH domains, and antibody fragments, such as two or more Fab fragments linked by disulfide crosslinks at a hinge region, or two or more bivalent fragments containing, e.g., two isolated CDRs or other epitope-binding fragments of linked antibodies, or multispecific molecules formed from such fragments. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be transplanted into scaffolds based on polypeptides, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide minibodies). The "antigen-binding region" or "antigen-binding domain" of an antibody is usually found in one or more hypervariable regions of the antibody, namely the CDR1, CDR2, and / or CDR3 regions. However, variable "framework" regions also play an important role in antigen binding, for example, by providing a scaffold for the CDR. The constant region of an antibody mediates the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, or chimeric antibodies.Antibodies may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0041] The "complementarity-determining region" ("CDR") is defined in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). This refers to an amino acid sequence with boundaries determined using one of many well-known schemes, including those described by the "IMGT" numbering scheme. For example, in the classical form, Kabat assigns the CDR amino acid residues of the heavy chain variable domain (VH) 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues of the light chain variable domain (VL) The base is numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89- It is assigned the number 97 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of human VL.In IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), while the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering by "Kabat"). In IMGT, the CDR of an antibody can be determined using the program IMGT / DomainGap Align.

[0042] In the context of this invention, unless otherwise specified, the numbering system proposed by Honegger & Plueckthun is used (Honegger & Plueckthun, J. Mol. Biol. 309 (2001) 657-670). Furthermore, the following residues are defined as CDRs (according to AHo's numbering scheme): LCDR1 (also called CDR-L1): L24-L42; LCDR2 (also called CDR-L2): L58-L72; LCDR3 (also called CDR-L3): L107-L138; HCDR1 (also called CDR-H1): H27-H42; HCDR2 (also called CDR-H2): H57-H76; HCDR3 (also called CDR-H3): H108-H138. To clarify, the numbering system by Honegger & Plueckthun incorporates sequence gaps to account for the length diversity found in naturally occurring antibodies, particularly in the different VH and VL subfamilies, and especially in CDRs. Therefore, not all positions 1-149 in a given antibody variable domain are typically occupied by amino acid residues. To clarify, the framework regions according to the numbering system by Honegger & Plueckthun are as follows: For variable light chains, LFW1 (or VL framework region I): L1-L23; LFW2 (or VL framework region II): L43-L57; LFW3 (or VL framework region III): L73-L106; and LFW4 (or VL framework region IV): L139-L149; and for variable heavy chains, HFW1 (or VH framework region I): L1-L26; HFW2 (or VH framework region II): L43-L56; HFW3 (or VH framework region III): L77-L107; and HFW4 (or VH framework region IV): L139-L149.

[0043] Preferably, the “antigen-binding region” comprises at least amino acid residues 4-149 of the variable light chain (VL) and amino acid residues 5-144 of the variable heavy chain (VH) (numbered respectively by Honegger & Plueckthun), more preferably amino acid residues 3-149 of the VL and 4-146 of the VH, and particularly preferably the complete VL and VH chain (amino acid positions 1-149 of the VL and 1-149 of the VH). The framework region and CDR are shown in Table 7. A preferred class of immunoglobulin for use in the present invention is IgG. The “functional fragments” of the present invention include the domains of F(ab')2 fragment, Fab fragment, Fv, and scFv. F(ab')2 or Fab is designed to minimize or completely eliminate intermolecular disulfide interactions that occur between the CH1 domain and the CL domain. The antibodies or functional fragments of the present invention may be part of a bifunctional or multifunctional construct, as further described in paragraphs

[0083] to

[0087] .

[0044] In the context of this invention, the terms "VH" (variable heavy chain), "VK", and "Vλ" refer to families of antibody heavy and light chain sequences that are grouped according to sequence identity and homology. For example, methods for determining sequence homology using homology lookup matrices, such as BLOSUM (Henikoff, S. & Henikoff, JG, Proc. NATl. Acad. Sci. USA 89 (1992) 10915-10919), and grouping sequences by homology are well known to those skilled in the art. Different subfamilies of VH, VK, and Vλ can be identified, for example, as shown in Knappik et al., J. Mol. Biol. 296 (2000) 57-86, which group VH1A, VH1B, and VH2-VH6, VK1-VK4, and Vλ1-Vλ3. In vivo, antibody VK, Vλ, and VH chains are the result of random rearrangement of germline K chain V and J segments, germline λ chain V and J segments, and heavy chain V, D, and J segments, respectively. The subfamily to which a given antibody variable chain belongs is determined by the corresponding V segment and, in particular, by framework regions FW1-FW3. Thus, any VH sequence, characterized in this application by only a specific set of framework regions HFW1-HFW3, can be combined with any HFW4 sequence, for example, an HFW4 sequence taken from one of the heavy chain germline J segments, or an HFW4 sequence taken from a rearranged VH sequence. In certain embodiments, the HFW4 sequence is WGQGTLVTVSS.

[0045] Preferably, the antibody or functional fragment of the present invention is an isolated antibody or a functional fragment thereof. As used herein, the term “isolated antibody” means a polypeptide or its protein that, by its origin or manipulation, (i) exists in a host cell as part of the expression product of an expression vector, or (ii) is bound to a protein or other chemical component other than those bound in nature, or (iii) does not occur in nature. “Isolated” further means a protein that is: (i) chemically synthesized; or (ii) expressed in a host cell and produced from the relevant protein by gel chromatography, etc. The term “isolated antibody” also means an antibody that substantially does not contain other antibodies with different antigen specificity (for example, an isolated antibody that specifically binds to IL23R substantially does not contain antibodies that specifically bind to antigens other than IL23R). However, an isolated antibody that specifically binds to IL23R may cross-react with other antigens, such as IL23R molecules of other species. Furthermore, an isolated antibody may substantially not contain other cellular material and / or chemicals.

[0046] "Affinity" refers to the combined strength of non-covalent interactions between a single binding site or molecule, such as an antibody or a functional fragment thereof, and its binding partner, such as an antigen. Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair, such as the interaction between a single antibody-binding domain and its antigen. Generally, affinity is expressed using the dissociation constant (K). D Affinity can be expressed as ). Affinity can be measured by common methods known in the art, including those described herein.

[0047] The following terms are used to describe the sequence relationship between two or more polynucleotide or amino acid sequences: “sequence identity” or “percentage of sequence identity,” and “sequence homology” or “percentage of sequence homology.” As used herein, “sequence identity” is determined by calculating the maximum number of amino acid residues that are identical between two polypeptide sequences, where gaps and / or insertions may be considered to allow for the greatest degree of sequence overlap. For example, two completely identical 100-mer polypeptides have 100% sequence identity. If they differ by one mutation, i.e., one polypeptide contains a deletion of one amino acid, their sequence identity is 99% (99 out of 100 positions are identical). In other words, the "sequence identity ratio" is calculated by comparing two sequences that are optimally aligned on the comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the sequence identity ratio. "Sequence homology" is the degree of similarity when comparing two sequences.If necessary or desirable, optimal alignment of sequences for comparison can be achieved using, for example, the Smith-Waterman local homology algorithm (Adv. Appl. Math. 2:482 (1981)), the Needleman-Bunsch homology alignment algorithm (J. MoI. Biol. 48:443-53 (1970)), the Pearson-Lippmann homology search method (Proc. NATl. Acad. Sci. USA 85:2444-48 (1988)), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or visual inspection (generally, Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York). This can be derived by (see 1999). Unless otherwise specified herein, the degree of sequence homology referred to herein is determined by the use of the Dayhoff PAM matrix (MO Dayhoff, R. Schwartz, BC Orcutt: A model of Evolutionary Change in Proteins, pages 345-352; in: Atlas of protein sequence and structure, National Biomedical Research Foundation, 1979).

[0048] In one embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 85%, at least 90%, at least 95%, at least 97%, preferably 95%, and more preferably 97% sequence identity with respect to the corresponding framework region obtained from the VH1A sequence described in SEQ ID NO: 1. Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, and more preferably at least 95% sequence identity with respect to the corresponding framework region obtained from the VH1A sequence described in SEQ ID NO: 1.

[0049] In further embodiments, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 90%, for example, at least 90%, at least 93%, at least 95%, or at least 96% sequence homology, preferably at least 93%, and more preferably at least 96%, with respect to the corresponding framework region obtained from the VH1A sequence described in SEQ ID NO: 1. Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 85% sequence homology, for example, at least 90%, at least 95%, or at least 96%, preferably at least 90%, and more preferably at least 96%, with respect to the corresponding framework region obtained from the VH1A sequence described in SEQ ID NO: 1.

[0050] In one embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90%, and more preferably at least 95% sequence identity with respect to the corresponding framework region obtained from the VH1B sequence described in SEQ ID NO: 6. Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 93%, at least 95%, and at least 97% sequence homology, preferably at least 93%, and more preferably at least 95% sequence homology with respect to the corresponding framework region obtained from the VH1B sequence described in SEQ ID NO: 6.

[0051] In further embodiments, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 90%, preferably at least 93%, and more preferably at least 96% sequence homology to the corresponding framework region obtained from the VH1B sequence described in SEQ ID NO: 6. Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 85%, preferably at least 90%, and more preferably at least 96% sequence identity to the corresponding framework region obtained from the VH1B sequence described in SEQ ID NO: 6.

[0052] In another embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 85%, at least 90%, at least 95%, or at least 97% sequence identity, preferably at least 90%, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21. Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 82%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity, preferably at least 90%, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21. In one embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 85%, at least 90%, at least 95%, and at least 97% sequence identity, preferably at least 90%, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, wherein the variable heavy chain comprises two mutants T24K and T84S (see SEQ ID NO: 26). Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 82%, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity, preferably at least 90%, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, wherein the variable heavy chain comprises two mutants T24K and T84S (see SEQ ID NO: 26). In another embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 85%, at least 90%, at least 95%, and at least 97% sequence identity, preferably at least 90%, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, wherein the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun).Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 82%, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity, preferably at least 90%, with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21, wherein the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun).

[0053] In further embodiments, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 85%, at least 90%, at least 95%, at least 97%, and preferably at least 90% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 26, and preferably where the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun). Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 90% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 26, wherein the variable heavy chain preferably contains lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun).

[0054] In one embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 90%, for example at least 95%, at least 97%, and preferably at least 90% sequence homology to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21. Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 90%, for example at least 95%, at least 97%, and preferably at least 90% sequence homology to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21. In further embodiments, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 90%, for example at least 95%, at least 97%, preferably at least 90%, sequence homology to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, and where the variable heavy chain comprises two mutants T24K and T84S (see SEQ ID NO: 26). Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 90%, for example at least 95%, at least 97%, preferably at least 90%, sequence homology to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, and where the variable heavy chain comprises two mutants T24K and T84S (see SEQ ID NO: 26). In another embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain comprising HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 90%, for example at least 95%, at least 97%, preferably at least 90%, sequence homology to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, wherein the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun).Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 90%, for example, at least 95%, at least 97%, preferably at least 90%, sequence homology to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21, where the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun).

[0055] In further embodiments, the antibody or functional fragment of the present invention comprises a variable heavy chain including HFW1, HFW2, HFW3, and HFW4 regions, where together these regions exhibit at least 90% sequence homology, for example at least 93%, preferably at least 96%, to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 26, preferably where the variable heavy chain includes lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun). Alternatively, the HFW1, HFW2, and HFW3 regions together exhibit at least 90% sequence homology, for example at least 93%, preferably at least 96%, to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 26, preferably where the variable heavy chain includes lysine at AHo position 24 and serine at AHo position 84 (numbering by Honegger & Plueckthun).

[0056] In one embodiment, the antibody or functional fragment of the present invention comprises a variable light chain including LFW1, LFW2, and LFW3 regions, where together these regions exhibit at least 80% sequence identity, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, with respect to the corresponding framework region obtained from the VK sequence described in SEQ ID NO: 41.

[0057] In further embodiments, the antibody or functional fragment of the present invention comprises a variable light chain including LFW1, LFW2, and LFW3 regions, where together these regions exhibit at least 86%, for example, at least 90%, at least 95%, and preferably at least 96% sequence identity with respect to the corresponding framework region obtained from the VK sequence described in SEQ ID NO: 41.

[0058] In another embodiment, the antibody or functional fragment of the present invention comprises a variable light chain including LFW1, LFW2, and LFW3 regions, where together these regions exhibit at least 80%, for example, at least 85%, at least 90%, at least 93%, at least 95%, and at least 97% sequence identity, preferably at least 93%, with respect to the corresponding framework region obtained from the VK sequence described in SEQ ID NO: 41.

[0059] In further embodiments, the antibody or functional fragment of the present invention comprises a variable light chain including LFW1, LFW2, and LFW3 regions, where together these regions exhibit at least 86%, preferably at least 90%, at least 95%, and at least 96% sequence homology, preferably at least 90%, more preferably at least 96%, to the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51.

[0060] In one embodiment, the antibody or functional fragment of the present invention comprises a variable light chain containing LFW4, where the LFW4 region is a Vλ-based sequence that is at least 90% identical to SEQ ID NO: 63. Preferably, the LFW4 region is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80. In a preferred embodiment, the LFW4 region is the sequence described in SEQ ID NO: 63.

[0061] Preferably, when both the LFW4 regions of the antibody heavy chain and light chain contain Cys mutations, the formation of interchain disulfide bonds further improves the biophysical properties of the corresponding antibody. In certain embodiments, the antibody heavy chain contains the Cys mutation G51C (numbered by Honegger & Plueckthun). In certain embodiments, the LFW4 region is the sequence described in SEQ ID NO: 79 or SEQ ID NO: 80. In one embodiment, the antibody or functional fragment of the present invention includes: (a) Variable light chains comprising LFW4 as described in Sequence ID No. 79 or Sequence ID No. 80; and, (b) A variable heavy chain comprising HFW1, HFW2, HFW3, and HFW4 regions, wherein the HFW1, HFW2, HFW3, and HFW4 regions together exhibit (i) at least 85% sequence identity, more preferably at least 90% sequence identity, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 26; or (ii) at least 85% sequence identity, preferably at least 95% sequence identity, more preferably at least 97% sequence identity, with respect to the corresponding framework region obtained from the VH1A sequence described in SEQ ID NO: 1; or (iii) at least 85% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, with respect to the corresponding framework region obtained from the VH1B sequence described in SEQ ID NO: 6; wherein the variable heavy chain contains cysteine ​​at position AHo 51 (numbered by Honegger & Plueckthun).

[0062] In the embodiment of the embodiment shown in (b) above, the HFW1, HFW2, and HFW3 regions together exhibit at least 82%, for example, at least 85%, at least 90%, and at least 95% sequence homology with respect to the corresponding framework regions of the VH sequences of sequence numbers 21, 26, 1, and 6.

[0063] In a preferred embodiment, the antibody or functional fragment of the present invention comprises a variable light chain including HFW1, HFW2, HFW3, and HFW4 regions, where together the HFW1, HFW2, and HFW3 regions exhibit at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 97%, preferably at least 93%, and more preferably at least 95%, to the corresponding framework region obtained from the VK1 / sk17 sequence described in SEQ ID NO: 57.

[0064] In a preferred embodiment, the antibody or functional fragment of the present invention comprises a variable light chain including HFW1, HFW2, HFW3, and HFW4 regions, where together the HFW1, HFW2, and HFW3 regions exhibit at least 80% sequence identity, for example, at least 81%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity, preferably at least 93%, and more preferably at least 95%, to the corresponding framework region obtained from the VK3 / sk17 sequence described in SEQ ID NO: 61.

[0065] In preferred embodiments, the antibody or functional fragment of the present invention is the VH1A sequence described in SEQ ID NO: 1, the VH1B sequence described in SEQ ID NO: 6, the VH4 sequence described in SEQ ID NO: 21, or the VH4 sequence described in SEQ ID NO: 26. mut The variable heavy chain includes HFW1, HFW2, HFW3, and HFW4 regions obtained from the sequence. Preferably, the variable heavy chain of the antibody of the present invention includes the following: (a) HFW1 as described in Sequence ID No. 2, HFW2 as described in Sequence ID No. 3, HFW3 as described in Sequence ID No. 4, and HFW4 as described in Sequence ID No. 5; or (b) HFW1 as described in Sequence ID No. 7, HFW2 as described in Sequence ID No. 8, HFW3 as described in Sequence ID No. 9, and HFW4 as described in Sequence ID No. 10; or (c) HFW1 as described in Sequence ID No. 22, HFW2 as described in Sequence ID No. 23, HFW3 as described in Sequence ID No. 24, and HFW4 as described in Sequence ID No. 25; or (d) HFW1 as described in Sequence ID No. 27, HFW2 as described in Sequence ID No. 28, HFW3 as described in Sequence ID No. 29, and HFW4 as described in Sequence ID No. 30.

[0066] In a preferred embodiment, the antibody or functional fragment of the present invention is V as described in SEQ ID NO: 41. Κ The antibody comprises a variable light chain containing LFW1, LFW2, and LFW3 regions, obtained from sequence 1 or from the VK3 sequence described in Sequence ID No. 51. Preferably, the variable light chain of the antibody of the present invention comprises: (a) LFW1 as described in Sequence ID No. 42, LFW2 as described in Sequence ID No. 43, and LFW3 as described in Sequence ID No. 44; or (b) LFW1 as described in Sequence ID No. 52, LFW2 as described in Sequence ID No. 53, and LFW3 as described in Sequence ID No. 54.

[0067] In a particular embodiment, the antibody or its functional fragment has the following framework sequence: (a) The HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85%, for example at least 90%, at least 95%, and at least 97% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, and optionally at least 90%, for example at least 93%, at least 95%, preferably at least 95% sequence homology, and the LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK sequence described in SEQ ID NO: 41. The sequence identity is, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86%, for example at least 90%, at least 95%, preferably at least 96%, and thereafter LFW4 is a Vλ-based sequence that is at least 90% identical to SEQ ID NO: 63, preferably LFW4 is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, and more preferably LFW4 is the sequence described in SEQ ID NO: 63; especially, (i) where the variable heavy chain includes HFW1 described in SEQ ID NO: 22, HFW2 described in SEQ ID NO: 23, HFW3 described in SEQ ID NO: 24, and HFW4 described in SEQ ID NO: 25, and the variable light chain includes LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63; or (ii) where the variable heavy chain includes HFW1 described in SEQ ID NO: 27, HFW2 described in SEQ ID NO: 28, HFW3 described in SEQ ID NO: 29, and HFW4 described in SEQ ID NO: 30, and the variable light chain includes LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63; or (b) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85%, for example at least 90%, at least 95%, at least 97% sequence identity, preferably at least 90% sequence identity, and optionally at least 90%, for example at least 93%, at least 95%, preferably at least 95% sequence homology with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, and here, the LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51. Identity, for example, sequence identity of at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93% sequence identity, and optionally at least 86%, preferably at least 90% sequence homology, at least 95%, at least 96%, preferably at least 80%, more preferably at least 96%, and where LFW is a Vλ-based sequence and is at least 90% identical to SEQ ID NO: 63, preferably LFW4 is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, more preferably LFW4 is the sequence described in SEQ ID NO: 63; especially: (i) where the variable heavy chain includes HFW1 described in SEQ ID NO: 22, HFW2 described in SEQ ID NO: 23, HFW3 described in SEQ ID NO: 24, and HFW4 described in SEQ ID NO: 25, and the variable light chain includes LFW1 described in SEQ ID NO: 52, LFW2 described in SEQ ID NO: 53, LFW3 described in SEQ ID NO: 54, and LFW4 described in SEQ ID NO: 63; or (ii) where the variable heavy chain includes HFW1 described in SEQ ID NO: 27, HFW2 described in SEQ ID NO: 28, HFW3 described in SEQ ID NO: 29, and HFW4 described in SEQ ID NO: 30, and the variable light chain includes LFW1 described in SEQ ID NO: 52, LFW2 described in SEQ ID NO: 53, LFW3 described in SEQ ID NO: 54, and LFW4 described in SEQ ID NO: 63; or (c) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit sequence homology of at least 85%, for example at least 90%, at least 95%, sequence homology of at least 97%, and optionally at least 90%, for example at least 95%, preferably at least 95%, with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, and here the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84, and here the LFW1, LFW2, and LFW3 regions together exhibit sequence homology of the corresponding framework region obtained from the VK1 sequence described in SEQ ID NO: 41 The LFW4 exhibits at least 80% sequence identity with respect to the framework region, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86%, for example, at least 90%, at least 95%, preferably at least 96%, and where LFW4 is a Vλ-based sequence and is at least 90% identical to SEQ ID NO: 63, preferably LFW4 is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, and more preferably LFW4 is the sequence described in SEQ ID NO: 63; especially: Here, the variable heavy chain includes HFW1 described in SEQ ID NO: 27, HFW2 described in SEQ ID NO: 28, HFW3 described in SEQ ID NO: 29, and HFW4 described in SEQ ID NO: 30; the variable light chain includes LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63; or (d) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85%, for example, at least 90%, at least 95%, at least 97%, preferably at least 84%, sequence identity, and optionally at least 90%, for example, at least 93%, at least 95%, preferably at least 93%, sequence homology with respect to the corresponding framework region obtained from the VH4 sequence described in SEQ ID NO: 21, and here the variable heavy chain contains lysine at AHo position 24 and serine at AHo position 84, and here the LFW1, LFW2, and LFW3 regions together exhibit the corresponding framework region obtained from the VK3 sequence described in SEQ ID NO: 51 The sequence exhibits at least 80% sequence identity with respect to the 'k' region, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86%, preferably at least 90%, at least 95%, at least 96%, preferably at least 90%, and more preferably at least 96%, where LFW4 is a Vλ-based sequence and is at least 90% identical to SEQ ID NO: 63, preferably LFW4 is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, and more preferably LFW4 is the sequence described in SEQ ID NO: 63; especially: Here, the variable heavy chain includes HFW1 described in SEQ ID NO: 27, HFW2 described in SEQ ID NO: 28, HFW3 described in SEQ ID NO: 29, and HFW4 described in SEQ ID NO: 30; the variable light chain includes LFW1 described in SEQ ID NO: 52, LFW2 described in SEQ ID NO: 53, LFW3 described in SEQ ID NO: 54, and LFW4 described in SEQ ID NO: 63; or (e) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity, for example at least 90%, at least 95%, at least 97%, preferably at least 95%, more preferably at least 97%, and optionally at least 90% sequence homology, for example at least 93%, at least 95%, at least 96%, preferably at least 95%, more preferably at least 96%, and here the LFW1, LFW2, and LFW3 regions together The sequence exhibits at least 80% sequence identity, for example at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86%, for example at least 90%, at least 95%, preferably at least 96%, with respect to the corresponding framework region obtained from the VK1 sequence described in SEQ ID NO: 41, wherein LFW4 is a Vλ-based sequence and is at least 90% identical to SEQ ID NO: 63, preferably LFW4 is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, and more preferably LFW4 is the sequence described in SEQ ID NO: 63; especially: The above variable heavy chain includes HFW1 described in SEQ ID NO: 2, HFW2 described in SEQ ID NO: 3, HFW3 described in SEQ ID NO: 4, and HFW4 described in SEQ ID NO: 5, and the above variable light chain includes LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63; or (f) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity, for example at least 90%, at least 93%, at least 95%, and at least 97% sequence identity, preferably at least 95%, more preferably at least 97%, and optionally at least 90% sequence homology, for example at least 93%, at least 95%, and at least 96%, preferably at least 95%, more preferably at least 96%, and here the LFW1, LFW2, and LFW3 regions together exhibit the sequence described in SEQ ID NO: 51 The LFW4 exhibits at least 80% sequence identity with respect to the corresponding framework region obtained from the VK3 sequence, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86%, preferably at least 90%, at least 95%, at least 96%, preferably at least 90%, and more preferably at least 96%, wherein the LFW4 is a Vλ-based sequence and is at least 90% identical to SEQ ID NO: 63, preferably the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, and more preferably the sequence described in SEQ ID NO: 63; especially: The above variable heavy chain includes HFW1 described in SEQ ID NO: 2, HFW2 described in SEQ ID NO: 3, HFW3 described in SEQ ID NO: 4, and HFW4 described in SEQ ID NO: 5, and the above variable light chain includes LFW1 described in SEQ ID NO: 52, LFW2 described in SEQ ID NO: 53, LFW3 described in SEQ ID NO: 54, and LFW4 described in SEQ ID NO: 63; or (g) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity, for example at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95%, and optionally at least 90%, preferably at least 93%, more preferably at least 96% sequence homology with respect to the corresponding framework region obtained from the VH1B sequence described in SEQ ID NO: 6, and here the LFW1, LFW2, and LFW3 regions together exhibit at least 85% sequence identity, for example at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 90%, more preferably at least 95%, and here the LFW1, LFW2, and LFW3 regions together exhibit at least 96% sequence homology with respect to the VK1 sequence described in SEQ ID NO: 41 The sequence exhibits at least 80% sequence identity with respect to the corresponding framework region, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86%, for example, at least 90%, at least 95%, preferably at least 96%, where LFW4 is a Vλ-based sequence and is at least 90% identical to SEQ ID NO: 63, preferably LFW4 is the sequence described in SEQ ID NO: 63, SEQ ID NO: 79, or SEQ ID NO: 80, and more preferably LFW4 is the sequence described in SEQ ID NO: 63; especially: The above variable heavy chain includes HFW1 described in SEQ ID NO: 7, HFW2 described in SEQ ID NO: 8, HFW3 described in SEQ ID NO: 9, and HFW4 described in SEQ ID NO: 10, and the above variable light chain includes LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63; or (h) Here, the HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity, for example, at least 90%, at least 93%, at least 95%, at least 97% sequence identity, preferably at least 90%, more preferably at least 95%, and optionally at least 90%, preferably at least 93%, more preferably at least 96% sequence homology, and here the LFW1, LFW2, and LFW3 regions together exhibit at least 85% sequence identity, for example, at least 90%, at least 93%, at least 95%, at least 97%, at least 90%, more preferably at least 95%, at least 90%, more preferably at least 96%, and here the LFW1, LFW2, and LFW3 regions together exhibit at least 85% sequence identity, for example, at least 90%, at least 93%, at least 95%, more preferably at least 97%, at least 90%, more preferably at least 95%, at least 90%, more preferably at least 93%, at least 96%, and here the LFW1, LFW2, and LFW3 regions together exhibit at least 85% sequence identity, at least 90%, at least 93%, at least 95%, more preferably at least 97%, at least 90%, more preferably at least 93%, at least 90%, more preferably at least 96%, at least 90%, more preferably at least 93%, at least 90%, more preferably0%, more preferably at least 90%, more preferably at least 90%, more preferably at least 90%, more preferably at least 9 The sequence exhibits at least 80% sequence identity with respect to the work region, for example, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, preferably at least 93%, and optionally at least 86% sequence homology, preferably at least 90%, more preferably at least 95%, and even more preferably at least 96%, wherein LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63, preferably LFW4 is the sequence described in sequence number 63, sequence number 79, or sequence number 80, and more preferably LFW4 is the sequence described in sequence number 63; especially: The above variable heavy chain includes HFW1 described in SEQ ID NO: 7, HFW2 described in SEQ ID NO: 8, HFW3 described in SEQ ID NO: 9, and HFW4 described in SEQ ID NO: 10, and the above variable light chain includes LFW1 described in SEQ ID NO: 52, LFW2 described in SEQ ID NO: 53, LFW3 described in SEQ ID NO: 54, and LFW4 described in SEQ ID NO: 63.

[0068] In alternative embodiments of the embodiments shown in (a) to (h) above, the HFW1, HFW2, and HFW3 regions together exhibit at least 82%, for example, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity with respect to the corresponding framework region of the respective VH sequence, preferably at least 90% sequence identity, and optionally at least 90%, for example, at least 93%, and preferably at least 95% sequence homology.

[0069] Preferably, the antibody of the present invention, or a functional fragment thereof, comprises the following: (a) Variable light chain, Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determination region. (b) Variable heavy chain, Here, the variable heavy chain includes the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determination region. And here: (i) The above LFW1, LFW2, LFW3, HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 93% sequence identity, e.g., at least 95%, at least 97%, and optionally at least 95% sequence homology, with respect to the corresponding framework regions obtained from the VK1 / sk17 sequence described in SEQ ID NO: 57 and the VH1A sequence described in SEQ ID NO: 1, and here ●The above LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK1 / sk17 sequence described in Sequence ID No. 57; and ● The above LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63; and ● The above HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity with respect to the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1; or (ii) The LFW1, LFW2, LFW3, HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 90% sequence identity, e.g., at least 95%, at least 97%, and optionally at least 92% sequence homology, with respect to the corresponding framework regions obtained from the VK1 / sk17 sequence described in SEQ ID NO: 57 and the VH1B sequence described in SEQ ID NO: 6, and here ●The above LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK1 / sk17 sequence described in Sequence ID No. 57; and ● The above LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63; and ● The above HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity with respect to the corresponding framework region obtained from the VH1B sequence described in Sequence ID No. 6; or (iii) The LFW1, LFW2, LFW3, HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 75% sequence identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity, and optionally at least 80%, e.g., at least 85%, at least 90%, at least 95%, and at least 97% sequence homology, and here ●The above LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK1 / sk17 sequence described in Sequence ID No. 57; and ● The above LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63; and ● The above HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21; or (iv) The LFW1, LFW2, LFW3, HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity, e.g., at least 90%, at least 95%, at least 97%, and optionally at least 80% sequence homology, e.g., at least 85%, at least 90%, at least 95%, at least 97%, with respect to the corresponding framework regions obtained from the VK3 / sk17 sequence described in SEQ ID NO: 61 and the VH1A sequence described in SEQ ID NO: 1, and here ●The above LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK3 / sk17 sequence described in Sequence ID No. 61; and ● The above LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63; and ● The above HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity with respect to the corresponding framework region obtained from the VH1A sequence described in Sequence ID No. 1; or (v) The LFW1, LFW2, LFW3, HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 90% sequence identity, e.g., at least 95%, at least 97%, and optionally at least 90% sequence homology, e.g., at least 95%, at least 97%, with respect to the corresponding framework regions obtained from the VK3 / sk17 sequence described in SEQ ID NO: 61 and the VH1B sequence described in SEQ ID NO: 6, and here ●The above LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK3 / sk17 sequence described in Sequence ID No. 61; and ● The above LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63; and ● The above HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity with respect to the corresponding framework region obtained from the VH1B sequence described in Sequence ID No. 6; or (vi) The LFW1, LFW2, LFW3, HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity, and optionally at least 75% sequence homology, e.g., at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% sequence homology, and here ●The above LFW1, LFW2, and LFW3 regions together exhibit at least 80% sequence identity with respect to the corresponding framework region obtained from the VK3 / sk17 sequence described in Sequence ID No. 61; and ● The above LFW4 is a Vλ-based sequence and is at least 90% identical to sequence number 63; and ● The above HFW1, HFW2, HFW3, and HFW4 regions together exhibit at least 85% sequence identity with respect to the corresponding framework region obtained from the VH4 sequence described in Sequence ID No. 21.

[0070] In alternative embodiments of the embodiments shown in (i) to (vi) above, the HFW1, HFW2, and HFW3 regions together exhibit at least 82%, for example, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity with respect to the corresponding framework region of the respective VH sequence, preferably at least 90% sequence identity, and optionally at least 90%, for example, at least 93%, and preferably at least 95% sequence homology.

[0071] In a preferred embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain containing HFW1 described in SEQ ID NO: 22, HFW2 described in SEQ ID NO: 23, HFW3 described in SEQ ID NO: 24, and HFW4 described in SEQ ID NO: 25; and a variable light chain containing LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63. In a more preferred embodiment, the antibody or functional fragment of the present invention comprises a variable heavy chain containing HFW1 described in SEQ ID NO: 27, HFW2 described in SEQ ID NO: 28, HFW3 described in SEQ ID NO: 29, and HFW4 described in SEQ ID NO: 30; and a variable light chain containing LFW1 described in SEQ ID NO: 42, LFW2 described in SEQ ID NO: 43, LFW3 described in SEQ ID NO: 44, and LFW4 described in SEQ ID NO: 63.

[0072] In certain embodiments, the antibody or functional fragment of the present invention, specifically VK1 λ-capped / VH1A, VK1 λ-capped / VH1B, VK1 λ-capped / VH4, and VK1 λ-capped / VH4 mut , VK3 λ-capped / VH1A, VK3 λ-capped / VH1B, VK3 λ-capped / VH4, and VK3 λ-capped / VH4 mut The antibody or functional fragment selected from the list has the following characteristics: When expressed in scFv (single-chain variable fragment antibody) antibody format, the average midpoint of the thermal denaturation temperature (Tm) is determined by differential scanning fluorescence (DSF) analysis (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221) in five phosphate-citrate buffers containing 0.15 M NaCl in the pH range of 3.5 to 7.5. The midpoint of the thermal denaturation transition of the scFv construct is determined by differential scanning fluorescence analysis using the fluorescent dye SYPRO® orange. Samples under the relevant excipient conditions are prepared by adding stock excipients prepared in the relevant buffers to a final protein concentration of 50 μg ml. -1 The samples are prepared as follows. In the buffer scouting experiment, the samples are diluted in final scFv buffer at various pH values ​​(pH 3.4, 4.4, 5.4, 6.4, and 7.2) containing a final concentration of 5×SYPRO® Orange to a total volume of 100 μl. Along with the unknown sample, the scFv DSF reference is measured as an internal control. 25 microliters of the prepared sample are added in triplicate to white-walled AB gene PCR plates. The assay is performed on a qPCR machine used as a thermal cycler, and fluorescence emission is detected using a custom dye calibration routine in the software. The PCR plates containing the test samples are heated from 25°C to 96°C in 1°C increments, with a 30-second pause after each temperature increase. The total assay time is approximately 2 hours. Tm is calculated by the GraphPad Prism software using mathematical second derivatives to calculate the inflection point of the curve. The reported Tm is the average of the three measurements.

[0073] In preferred embodiments, the antibody or its functional fragment has a mean midpoint of a thermal denaturation temperature (Tm) at least 65°C, preferably at least 69°C. The protein is analyzed for oligomerization by SE-HPLC during storage at 37°C for 14 days in 50 mM citrate-phosphate pH 6.4 and 150 mM NaCl. Before the study, 10 g of the sample was prepared. -1 The sample is concentrated, and the d0 time point is determined. Monomer content is quantified by separating the sample with a Shodex KW-402.5-4F column and evaluating the resulting chromatogram. In calculating the relative proportion of protein monomers, the area of ​​the monomer peak is divided by the total area of ​​peaks not attributable to the sample matrix. In a preferred embodiment, the antibody or its functional fragment is concentrated to a concentration of 10 g -1 When stored at 37°C for 2 weeks in 50 mM citrate-phosphate pH 6.4 and 150 mM NaCl, it exhibits a loss of monomer content of 15%, 12%, 10%, 7%, 5%, or less than 2%, preferably less than 5%, more preferably less than 2%.

[0074] In certain embodiments, the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 together are placeholder regions that are specific to the target of interest or can be replaced by corresponding complementarity-determining regions from a donor antibody that is specific to the target of interest.

[0075] In one embodiment of the present invention, the above-mentioned CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are: (i) CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 derived from parental non-human antibodies, specifically from parental rabbit antibodies or parental rodent antibodies, specifically from parental mouse or rat antibodies, having specificity for the antigen of interest; (ii) CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 derived from parental human or humanized antibodies, specifically from antibodies approved for therapeutic use or commercially available; (iii) CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 obtained from CDR domains obtained by optimization of one or more CDR domains according to (i) or (ii); and (iv) Selected independently from the CDR domains to be replaced by one or more CDR domains according to (i), (ii), and / or (iii). In the case of (iv), any sequence can be used as the CDR sequence that is suitable for the intended use of each variable domain. For example, if the antibody variable domain containing the above CDR sequence must be expressed before the intended substitution of the above CDR sequence, a CDR derived from a well-expressed antibody variable domain may be selected. If no expression has to be performed, any sequence compatible with the desired substitution procedure can be used. Due to its function as a placeholder CDR, in such situations the above CDR sequence does not need to support actual antigen binding.

[0076] In certain embodiments, the antibody or functional fragment thereof has binding specificity to the target of interest. As such, the antibody or functional fragment of the present invention comprises complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which together are specific to the target of interest.

[0077] As used herein, the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are specific to a given target of interest if together they possess the ability to specifically bind to a given target of interest, such as an antigen. As used herein, the terms “specifically bind” or “binding specificity” refer to the ability of an individual antibody containing the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 to react with one antigenic determinant and not with a different antigenic determinant. Antibody binding affinity is the strength of the reaction between a single antigenic determinant and the antibody. In particular, the term “the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are specific to a given target of interest” refers to complementarity-determining regions produced and / or selected for a given target of interest, and they together possess the ability to recognize the given target of interest. As used herein, the term “recognize” refers to finding and interacting with a corresponding target of interest, particularly its three-dimensional structural epitope (e.g., binding, blocking, inhibiting, antagonizing, stimulating (agonizing)).

[0078] As used herein, a binding molecule is "specific to / for," "specifically recognizes," or "specifically binds" to a target, such as human IL23R, etc., if such a binding molecule can distinguish one or more reference molecules from such a target biomolecule. Binding specificity is relative, not absolute. In its most common form (and where no defined reference is mentioned), "specific binding" refers to the ability of a binding molecule to distinguish a biomolecule unrelated to the biomolecule of interest, as determined, for example, according to specificity assay methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) assays, and peptide scans. For example, a standard ELISA assay can be performed. Scoring can be performed by standard colorimetric assays (e.g., secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). The reaction in a particular well is scored by optical density, for example, at 450 nm. A typical background (=negative reaction) may be around 0.1 OD; a typical positive reaction may be around 1 OD. This means that the ratio of positive to negative scores can be more than 10 times. In further examples, an SPR assay can be performed in which a difference of at least 10 times, preferably at least 100 times, between the background and the signal indicates specific binding. Generally, the determination of binding specificity is performed using a set of about 3 to 5 unrelated biomolecules, such as powdered milk, BSA, and transferrin, rather than a single reference biomolecule.

[0079] However, “specific binding” may also refer to the ability of the binding molecule to distinguish between a target biomolecule and one or more related biomolecules used as a reference point. Furthermore, “specific binding” may also refer to the ability of the binding molecule to distinguish between different parts of its target antigen, such as different domains, regions, or epitopes of the target biomolecule, or one or more important amino acid residues or stretches of amino acid residues of the target biomolecule.

[0080] In the context of this invention, the term "epitope" refers to a portion of a given target biomolecule necessary for specific binding between the target biomolecule and the binding molecule. Epitopes can be continuous, i.e., formed by adjacent structural elements present on the target biomolecule, or discontinuous, i.e., formed by structural elements located at different positions in the primary sequence of the target biomolecule, for example, in the amino acid sequence of the target protein, but close to the three-dimensional structure adopted by the target biomolecule in a bodily fluid.

[0081] In one embodiment of the present invention, the isolated antibody or its functional fragment is selected from IgG antibodies, Fab, and scFv fragments. Preferably, the antibody or its functional fragment of the present invention is an scFv antibody fragment. A "single-chain Fv" or "scFv" or "sFv" antibody fragment comprises the VH and VL domains of the antibody, where these domains are present on a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the sFv to form a desired structure for target binding. A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of the antibody, where these domains are present on a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH domain and the VL domain, which allows the scFv to form a desired structure for antigen binding (see, for example, Plueckthun, The pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315).

[0082] In a particular embodiment, the functional fragment is an scFv format including the linker described in sequence number 64 or sequence number 65, preferably sequence number 64.

[0083] In another specific embodiment of the present invention, an isolated antibody or a functional fragment thereof Single-chain diabody (scDb), tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), bispecific T cell engager (BiTE; tandem di-scFv), tandem tri-scFv, tribody (Fab-(scFv)2) or bibody (Fab-(scFv)1), triplicate antibody, scDb-scFv, bispecificate Fab2, dimini antibody (di-miniantibody), tetrabody, scFv-Fc-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusion, e.g., bsAb (scFv bound to the C-terminus of the light chain), Bs1Ab (scFv bound to the N-terminus of the light chain) v), Bs2Ab (scFv bound to the N-terminus of the heavy chain), Bs3Ab (scFv bound to the C-terminus of the heavy chain), Ts1Ab (scFv bound to the N-terminus of both the heavy and light chains), Ts2Ab (dsscFv bound to the C-terminus of the heavy chain), and Knob-into-Hole (KiHs) (bispecific IgG prepared by KiH technology), MATCH (International Publication No. 2016 / 0202457; Egan A multispecific construct, such as a bispecific construct or a multivalent construct, such as a bivalent construct, is an antibody format selected from any suitable multispecific, such as bispecific, format known in the art, including, as an example, T., et al., mAbs 9 (2017) 68-84, and duobody (bispecific IgG prepared by duobody technology) (MAbs. 2017 Feb / Mar;9(2):182-212. doi: 10.1080 / 19420862.2016.1268307). Single-stranded diabodies (scDb), in particular bispecific monomer scDb, are especially preferred for use herein.

[0084] The term "diabody" refers to an antibody fragment having two antigen-binding sites, where these fragments contain VH bound to VL within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domains pair with complementary domains on another chain to produce two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are fully described, for example, by European Patent No. 404097, International Publication No. 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described by Hudson et al., Nat. Med. 9:129-134 (2003).

[0085] Bispecific scDbs, especially bispecific monomeric scDbs, are 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- The present invention particularly comprises two variable heavy chain domains (VH) or fragments thereof and a variable light chain domain (VL) or fragment thereof, linked by linkers L1, L2, and L3 in the order L2-VHA-L3-VHB or VLB-L1-VHA-L2-VLA-L3-VHB, where the VLA and VHA domains together form an antigen-binding site for a first antigen, and the VLB and VHB together form an antigen-binding site for a second antigen.

[0086] Linker L1 is specifically a peptide of 2-10 amino acids, more specifically a peptide of 3-7 amino acids, and most specifically a peptide of 5 amino acids; L3 linker is specifically a peptide of 1-10 amino acids, more specifically a peptide of 2-7 amino acids, and most specifically a peptide of 5 amino acids; the intermediate linker L2 is specifically a peptide of 10-40 amino acids, more specifically a peptide of 15-30 amino acids, and most specifically a peptide of 20-25 amino acids.

[0087] In one embodiment of the present invention, the isolated antibody or its functional fragment is a multispecific and / or polyvalent antibody in the MATCH format described in International Publication No. 2016 / 0202457; Egan T., et al., mAbs 9 (2017) 68-84.

[0088] The bispecific, bivalent, multispecific and / or polyvalent constructs of the present invention can be produced using any convenient antibody production method known in the art (for example, for the production of bispecific constructs, see Fischer, N. & Leger, O., Pathobiology 74(2007)3-14; for bispecific diabodies and tandem scFv, see Hornig, N. & Faerber-Schwarz, A., Methods Mol. Biol. 907(2012)713-727, and International Publication No. 99 / 57150). Specific examples of preferred methods for preparing the bispecific constructs of the present invention include, in particular, the techniques of 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). Methods for producing bispecific antibodies containing a functional antibody Fc moiety 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).

[0089] These methods typically involve the generation of monoclonal antibodies, for example, fusing mouse-derived spleen cells immunized with a desired antigen to myeloma cells using hybridoma technology (see, e.g., Yokoyama et al., Curr. Protoc. Immunol. Chapter 2, Unit 2.5, 2006), or recombinant antibody technology (repertory cloning or phage display / yeast display) (see, e.g., Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8), and combinations of antigen-binding domains or fragments or parts thereof of two different monoclonal antibodies to generate bispecific constructs using known molecular cloning techniques.

[0090] In a second aspect, the present invention relates to a pharmaceutical composition comprising the antibody or a functional fragment thereof, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0091] The term "pharmaceutically acceptable" means a compound, material, composition, and / or dosage form that is suitable for use in contact with human or animal tissue, within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio.

[0092] The pharmaceutical compositions according to this disclosure may further routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, auxiliary immunostimulants, such as adjuvants and cytokines, and optionally other therapeutic agents. The compositions may also contain antioxidants and / or preservatives. Examples of antioxidants include thiol derivatives (e.g., thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, sulfites (e.g., sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate), and nordihydroguaiaretinic acid. Suitable preservatives may include, for example, phenol, chlorobutanol, benzyl alcohol, methylparaben, propylparaben, benzalkonium chloride, and cetylpyridinium chloride.

[0093] In certain embodiments provided herein, the antibody or its functional fragment may be isolated, prepared, expressed, or created by recombinant means such as an antibody expressed using a recombinant expression vector transfected into a host cell, a recombinant, an antibody isolated from a combinatorial antibody library, or an antibody prepared, expressed, created, or isolated by other means including, for example, synthesis, genetic manipulation, or splicing of a DNA sequence encoding a human immunoglobulin sequence, such as a human immunoglobulin gene sequence.

[0094] Therefore, in a third aspect, the present invention relates to a nucleic acid sequence or collection of nucleic acid sequences encoding the antibody or a functional fragment thereof.

[0095] In a fourth aspect, the present invention relates to a vector or collection of vectors comprising a nucleic acid sequence or collection of nucleic acid sequences of the present invention. The term “vector” or “expression vector” means a polynucleotide, most commonly a DNA plasmid, comprising a nucleotide sequence encoding the antibody or a fragment thereof of the present invention for recombinant expression in a host cell, preferably a mammalian cell. The vector may or may not be replicable in the cell. Once a polynucleotide encoding the variable heavy chain and / or variable light chain, or a fragment thereof, of the antibody described herein is obtained, a vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide comprising an antibody encoding a nucleotide sequence is described herein. Using methods well known to those skilled in the art, an expression vector comprising the antibody coding sequence and appropriate transcription and translational regulatory signals can be constructed. These methods include, for example, in vitro recombinant DNA technology, synthesis technology, and in vivo genetic recombination.

[0096] An expression vector can be introduced into a host cell by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce the antibody or fragment thereof described herein. Accordingly, the present invention relates to a host cell comprising a nucleic acid sequence or collection of nucleic acid sequences of the present invention, or a vector or collection of vectors of the present invention, in particular an expression host cell. In certain embodiments, the host cell comprises a vector comprising a polynucleotide encoding both the variable heavy chain and the variable light chain of the antibody or fragment thereof. In certain embodiments, the host cell comprises two different vectors: a first vector comprising a polynucleotide encoding the variable heavy chain or fragment of the antibody, and a second vector or fragment thereof comprising a polynucleotide encoding the variable light chain of the antibody. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding the variable heavy chain or fragment of the antibody, and the second host cell comprises a second vector comprising a polynucleotide encoding the variable light chain or functional fragment of the antibody.

[0097] In a sixth aspect, the present invention relates to a process for expressing a nucleic acid sequence or collection of nucleic acid sequences of the present invention, or a vector or collection of vectors of the present invention, or a method for producing an antibody or functional fragment thereof of the present invention, including a host cell of the present invention, particularly an expressing host cell.

[0098] In a seventh aspect, the present invention relates to a method for humanizing a non-human antibody, specifically a rabbit or rodent antibody, and includes the following steps: (a) In one or more steps, the nucleic acid sequences encoding the variable heavy chain (VH) CDR and variable light chain (VL) CDR of the non-human antibody are cloned into one or more nucleic acid sequences encoding the antibody or a functional fragment thereof, wherein at least the VH CDR3 and VL CDR3 of the non-human antibody are cloned.

[0099] Methods for humanizing rabbit or rodent antibodies are well known to those skilled in the art (see, for example, Borras, loc. cit.; Rader et al, The FASEB Journal, express article 10.1096 / fj.02-0281fje, published online October 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). Immunization of rabbits or rodents can be carried out by rabbit DNA immunization using the antigen of interest itself, e.g., a protein, or, in the case of a peptide or protein antigen, a nucleic acid encoding the peptide or protein of interest, e.g., a plasmid.

[0100] In certain embodiments, the method further includes cloning of the VH CDR2 and / or VL CDR1 regions. In certain embodiments, the method includes cloning of both the VH CDR2 and VL CDR1 regions.

[0101] In certain embodiments, the method further includes cloning of the VH CDR1 and / or VL CDR2 regions. In certain embodiments, the method includes cloning of both the VH CDR1 and VL CDR2 regions.

[0102] In a particular embodiment, the method further includes one or more of the following steps: (aa) Immunization of non-human animals, particularly rabbits or rodents, with the antigen of the choice; and (ab) Isolation of at least one target antibody.

[0103] In a particular embodiment, the method further includes one or more of the following steps: (ac) Clonal isolation of affinity mature memory B cells that interact with the target antigen, particularly by using fluorescence-activated cell sorting; (ad) Culture of single B cells in a co-culture system that does not require immortalization of single B cell clones; (ae) Screening of B cell culture supernatants in cell-based ELISA to identify at least one antibody that binds to the target antigen; and / or (af) Cloning of the VH CDR of at least one antibody to a nucleic acid sequence encoding the human antibody VH domain.

[0104] In one embodiment, the framework region of the non-human antibody exhibits the highest homology to the corresponding framework regions of combinations of the human VH family selected from VH1A (SEQ ID NO: 1), VH1B (SEQ ID NO: 6), and VH4 (SEQ ID NO: 21), and the human VL family selected from VK1 (SEQ ID NO: 41) and VK3 (SEQ ID NO: 51). In another embodiment, the framework region of the non-human antibody has the highest homology to both (i) a framework region of a combination of the human VH family selected from VH2 (SEQ ID NO: 11), VH3 (SEQ ID NO: 16), VH5 (SEQ ID NO: 31), and VH6 (SEQ ID NO: 36), and the human VL family selected from VK1 (SEQ ID NO: 41), VK2 (SEQ ID NO: 46), VK3 (SEQ ID NO: 51), and VK4 (SEQ ID NO: 81); or (ii) a framework region of a combination of the human VH family selected from VH1A (SEQ ID NO: 1), VH1B (SEQ ID NO: 6), VH2 (SEQ ID NO: 11), VH3 (SEQ ID NO: 16), VH4 (SEQ ID NO: 21), VH5 (SEQ ID NO: 31), and VH6 (SEQ ID NO: 36), and the human VL family selected from VK2 (SEQ ID NO: 46), and VK4 (SEQ ID NO: 81).

[0105] In certain embodiments, the framework region of the non-human antibody exhibits the highest homology to the corresponding framework regions of combinations of the human VH family selected from VH2 (SEQ ID NO: 11), VH3 (SEQ ID NO: 16), VH5 (SEQ ID NO: 31), and VH6 (SEQ ID NO: 36), as well as the human VL family selected from VK2 (SEQ ID NO: 46) and VK4 (SEQ ID NO: 81).

[0106] In the eighth embodiment, the present invention relates to a method for optimizing a target parent antibody, comprising the following steps: (a) In one or more steps, the nucleic acid sequences encoding the VH CDR and VL CDR of the parent antibody are cloned into one or more nucleic acid sequences encoding the antibody or a functional fragment thereof according to the present invention, wherein at least the VH CDR3 and VL CDR3 of the parent antibody are cloned.

[0107] In certain embodiments, the method further includes cloning of the VH CDR2 and / or VL CDR1 regions. In certain embodiments, the method includes cloning of both the VH CDR2 and VL CDR1 regions.

[0108] In certain embodiments, the method further includes cloning of the VH CDR1 and / or VL CDR2 regions. In certain embodiments, the method includes cloning of both the VH CDR1 and VL CDR2 regions.

[0109] In one embodiment, the framework region of the parent antibody has the highest homology overall to the corresponding framework regions of combinations of the human VH family selected from VH1A (SEQ ID NO: 1), VH1B (SEQ ID NO: 6), and VH4 (SEQ ID NO: 21), and the human VL family selected from VK1 (SEQ ID NO: 41) and VK3 (SEQ ID NO: 51). In another embodiment, the framework region of the parent antibody has the highest homology overall to the corresponding framework region of a combination of (i) the human VH family selected from VH2 (SEQ ID NO: 11), VH3 (SEQ ID NO: 16), VH5 (SEQ ID NO: 31), and VH6 (SEQ ID NO: 36), and the human VL family selected from VK1 (SEQ ID NO: 41), VK2 (SEQ ID NO: 46), VK3 (SEQ ID NO: 51), and VK4 (SEQ ID NO: 81); or (ii) the human VH family selected from VH1A (SEQ ID NO: 1), VH1B (SEQ ID NO: 6), VH2 (SEQ ID NO: 11), VH3 (SEQ ID NO: 16), VH4 (SEQ ID NO: 21), VH5 (SEQ ID NO: 31), and VH6 (SEQ ID NO: 36), and the human VL family selected from VK2 (SEQ ID NO: 46), and VK4 (SEQ ID NO: 81).

[0110] In certain embodiments, the framework region of the parent antibody exhibits the highest homology overall to the corresponding framework regions of combinations of the human VH family selected from VH2 (SEQ ID NO: 11), VH3 (SEQ ID NO: 16), VH5 (SEQ ID NO: 31), and VH6 (SEQ ID NO: 36), as well as the human VL family selected from VK2 (SEQ ID NO: 46) and VK4 (SEQ ID NO: 81).

[0111] In the ninth aspect, the present invention relates to a method for generating a diverse collection of antibodies or functional fragments thereof, comprising the following steps: (a) Cloning one or more diverse collections of nucleic acid sequences encoding one or more diverse collections of VH CDRs and / or VL CDRs into one or more nucleic acid sequences encoding an antibody or a functional fragment thereof according to the present invention in one or more steps.

[0112] In a particular embodiment, a diverse collection of nucleic acid sequences encoding a diverse collection of VH CDR3s is cloned.

[0113] In certain embodiments, a diverse collection of nucleic acid sequences encoding a diverse collection of VL CDR3s is cloned.

[0114] In certain embodiments, a diverse collection of nucleic acid sequences encoding a diverse collection of VH CDR2 and / or a diverse collection of nucleic acid sequences encoding a diverse collection of VL CDR1 are cloned. In certain embodiments, both a diverse collection of VH CDR2 and a diverse collection of VL CDR1 regions are cloned.

[0115] In certain embodiments, a diverse collection of nucleic acid sequences encoding a diverse collection of VH CDR1 and / or a diverse collection of nucleic acid sequences encoding a diverse collection of VL CDR2 are cloned. In certain embodiments, both a diverse collection of VH CDR1 and a diverse collection of VL CDR2 are cloned. [Examples]

[0116] The following examples illustrate the present invention without limiting its scope. Example 1:

[0117] This study aimed to identify combinations of human variable VL / VH consensus frameworks consisting of combinatorial pairs of humanized consensus VL and VH domains that exhibit similar or superior biophysical properties compared to VK1 / VH3 while fully retaining specificity and antigen-binding affinity. Consensus germline sequences were used based on the assumption that residues contributing to protein stability were enriched during evolution in the germline sequence pool (Steipe et al., J Mol Biol, 240 (1994) 188-92). This reduces the risk of immunogenic reactions because aggregation (Joubert et al., J Biol Chem, 287 (2012) 25266-79) and non-human framework residues within the acceptor framework may dehumanize the framework region and increase the appearance of human T cell epitopes. In addition to the VK / VH (VK1, VK2, VK3, VH1A, VH1B, VH2, VH3, VH4, VH5, VH6) permutations, Numab stabilization techniques (so-called λ-caps) were applied to all VK domains to evaluate the potential stabilization contribution to non-VK1 / VH3 framework combinations and their potential impact on production yield and antigen-binding affinity. The λ-cap technique involves substituting framework region IV of the VK family consensus variable light region with a Vλ family germline sequence (λ-cap).

[0118] Regarding stability, the superiority of λ-capped K1 / VH3 consensus scFv over λ-capped VK1 / VH3 consensus scFv has been demonstrated previously (International Publication No. 2014 / 206561).

[0119] To identify the most appropriate human acceptor antibody variable region scfold for humanization and stabilization of rabbit antibody variable domains, we initiated testing of combinations of VK family light chain consensus sequences (VK1, VK2, and VK3), all human VH consensus sequences (VH1A, 1B, 2, 3, 4, 5, and 6), and different human germline λ-type light chain framework IV sequences (λ-capped, specifically SEQ ID NO: 62). For this purpose, rabbit CDR sets that specifically bind to the human interleukin-23 receptor (IL23R) (SEQ ID NOs: 66 to 71) were transplanted into all consensus framework combinations shown in Figure 1 (upper half) to allow direct comparison of humanized scFv fragments based solely on the characteristics of different consensus acceptor frameworks. Furthermore, regulatory molecules (uncapped) lacking the kappa-to-lambda substitution in framework region IV were generated for each VL / VH combination (Figure 1, lower half) and compared with their respective capped variants. Next, the results were confirmed with alternative rabbit CDR sets. Surprisingly, certain non-VK1 / VH3 combinations yielded higher expression levels, denaturation temperatures, and / or greater monomer stability from the λ cap than VK1 / VH3 scaffolds. Several VK / VH3 combinations and λ caps were clearly superior to λ-capped VK1 / VH3 combinations in terms of thermal denaturation and monomer stability, which was unexpected from a prior art perspective. Furthermore, we identified suitable lambda germline sequences for use as lambda caps to optimize stability and maintain antigen-binding affinity.

[0120] result: In the first step, the newly synthesized VL and VH gene sequences were cloned into recent expression vectors by domain shuffling and successfully expressed in E. coli. All constructs were expressed in scFv format with VL-VH domain orientation and a 20-amino acid glycine-serine (G4S) 4-linker (SEQ ID NO: 64) between domains. The ScFv molecules were purified using an appropriate purification strategy (protein L affinity chromatography followed by size exclusion chromatography), and since the monomer content of all constructs exceeded 95%, the quality of all scFv used for subsequent analysis was initially equivalent. Overall, when comparing biophysical properties (aggregation and thermal stability) and functional properties (SPR), the fully consensus constructs exhibited considerably more consistently inferior properties than the λ-capped controls. We found that scFv molecules containing VK(1-3), in which framework region IV is completely replaced with a Vλ germline sequence (λ-cap) and VH(1-6) consensus domain, generally exhibit a more favorable stability profile compared to the corresponding fully consensus (uncapped) molecules. Specifically, λ-capped scFv variants were largely superior across different germline families in terms of productivity, thermal denaturation midpoint, and monomeric stability during storage at various temperatures.

[0121] Several examples will be discussed in more detail below.

[0122] Example 2: Determination of biophysical data of scFv constructs Expression level: Single-stranded variable fragments were generated using common conditions, as described in Egan et al., MAbs 9 (2017) 68-84. Expression was performed in E. coli, and the antibody fragments accumulated in inclusion bodies, which were then solubilized and refolded in common refolding buffer, as described in Egan et al., MAbs 9 (2017) 68-84. The yield per liter of expression culture for all molecules is shown in Figure 2. Most surprisingly, these results show consistently improved productivity of λ-capped scaffolds (white bars) compared to their respective uncapped controls (gray bars), with two exceptions (VK2 / VH5 and VK3 / VH4).

[0123] The production of the two molecules (VK2 / VH1B and VK2 / VH2) was only possible through λ-capping of their light chains; the uncapped, complete consensus variant was not producible at all. Furthermore, VH3, including the framework combination, exhibited the highest yield within the group of molecules sharing the same subfamily of VK chains (VK1, VK2, VK3). Apart from the VK1 / VH3 combination, the VK1 / VH4, VK2 / VH4, and VK3 / VH1B combinations benefited most from replacing framework region IV with the Vλ germline, as their yield increased most significantly upon λ-capping within their respective groups sharing the same light chain framework.

[0124] thermal stability The midpoint of thermal denaturation (Tm) was determined for all molecules by differential scanning fluorescence (DSF) analysis described above in five phosphate-citrate buffer solutions containing 0.15 M NaCl in the pH range of 3.5–7.5 (Egan et al., MAbs 9 (2017) 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221). When expressed in scFv (single-chain variable fragment antibody) antibody format, as determined by differential scanning fluorescence (DSF) analysis (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221) as described above, the average midpoint of the thermal denaturation temperature (Tm) is at least 60°C, at least 65°C, at least 70°C, at least 80°C, or at least 90°C. Figure 3 shows the average pH data over the measured pH range, including the standard deviation. Lambda-capped scFv variants (white bars) consistently show higher average melting temperatures than uncapped (gray bars) variants, with the exception of the VK3 / VH5 combination, which has a significantly higher average Tm than the uncapped variants. Surprisingly, several low-expression framework combinations exhibit very high thermal stability, especially with lambda caps (e.g., VK3 / VH4), suggesting that the midpoint of thermal denaturation does not correlate with the expression level of the molecule. All combinations of VK1 and VK3 light chains, as well as the variable heavy chains VH1A, VH1B, and VH4, yield variable regions with higher denaturation temperatures than VK1 / VH3. VK1 / VH4 exhibits the highest melting temperature, while VK2 / VH5 and VK3 / VH1B show the highest relative increase in Tm upon capping.

[0125] Storage stability All producible molecules underwent a 2-week storage stability test at a concentration of 10 mg / mL at 37°C. The monomer content of the lambda-capped variants after storage was compared to that of the uncapped variants. Across all combinations, light chain capping significantly increased the monomer content between 32% (VK1 / VH1B) and 57% (VK3 / VH1A) after 2 weeks of storage at 37°C, compared to the uncapped full consensus scFv. The production of the three framework combinations was only possible by stabilizing the light chain with lambda capping. This was because sufficient quantities of the uncapped control could not be produced for this study (VK2 / VH1B, VK2 / VH6) or could not be quantitatively precipitated before the start of the study (VK3 / VH6). The VK2 / VH2 combination could not be produced in sufficient quantities for this study.

[0126] Surprisingly, when benchmarked against a lambda-capped Vκ1 / VH3 reference molecule, many VL-VH germline combinations show a significant increase in monomer content upon capping compared to the reference (circled values). Furthermore, some combinations exhibit less loss of monomer content when stored at 37°C for two weeks, and can therefore be considered more stable overall than the reference combination. This is the case for the lambda-capped combinations Vκ1 / VH1A, Vκ1 / VH4, Vκ1 / VH5, Vκ2 / VH1A, Vκ2 / VH1B, Vκ2 / VH3, Vκ2 / VH5, Vκ3 / VH1A, Vκ3 / VH1B, Vκ3 / VH4, and Vκ3 / VH5.

[0127] Affinity measurement To functionally characterize the humanized rabbit variable domains, the affinity of each scFv to IL23R was measured by surface plasmon resonance (SPR). Surprisingly, most molecules retained high antigen-binding affinity, which was not exclusively expected for CDRs, and the donor framework residues were not transplanted into the human acceptor scaffold.

[0128] Furthermore, since both the capped and uncapped variants show at least similar affinity or even a slight overall advantage over the capped variant, it can be concluded that light-chain capping does not adversely affect target affinity. When comparing the capped and uncapped variants and within each molecule sharing the same light-chain domain, the kinetics of antibody-target interaction are similar. Only the off-rate values (k d ) of the VK3 constructs show slightly higher variability, and VK3 / VH1A and VK3 / VH5 show slightly faster off-rates than their VK1 or VK2 controls. VK1 / VH3 shows a slightly slower off-rate than all other molecules. This is also reflected in the overall best dissociation constants. However, in some combinations, the fast on-rate compensates for the off-rate drawback (e.g., VK1 / VH4), and an essentially the same overall affinity as the scFv containing VH3 is obtained. Table 1 summarizes the affinity for human IL23R for all combinations of capped and uncapped frameworks investigated in this study.

[0129]

Table 1

[0130] Table 2 summarizes all data obtained with human IL23R-specific scFv fragments. Each framework variant was compared with the uncapped (full consensus) control in four different categories (yield, average Tm, loss of monomer content, and K D ), and the increments were calculated. Values in bold indicate that the performance of each framework combination is superior compared to the VK1 / VH3 reference.

[0131] Example 3: Confirmation test using TNF-specific molecules Based on the outstanding overall performance of Vκ1 / VH4 and Vκ3 / VH4 IL23R-specific molecules, these framework combinations were selected as particularly useful for direct humanization by simple engraftment of rabbit anti-human TNF CDRs (SEQ ID NOs. 72 to 77). The objective of this study was to confirm the results obtained with IL23R-specific CDRs (SEQ ID NOs. 66 to 71). As a reference for a control confirmation study, Vκ1 / VH3 and Vκ3 / VH3 framework combinations were used for engraftment of the same anti-TNF CDR set.

[0132] [Table 2]

[0133] The results for the selected (human IL23R CDR transplant) framework combinations, namely VK1 / VH3, VK1 / VH4, VK3 / VH3, and VK3 / VH4, were confirmed by transplantation of human TNF-specific CDRs into these frameworks. Furthermore, mutant variants of the VK1 / VH4 framework-based molecule, containing amino acids that branch off from the consensus sequence at specific framework positions (2 / 81 of VH4), were included in this study.

[0134] Confirming the trend observed above for IL23R-specific CDRs, where affinity is not affected by CDR transplantation into VH4 framework combinations, the capped Vκ1 / VH4 consensus molecule showed even higher affinity for TNF-specific CDRs than the capped VΚ1 / VH3 consensus molecule. Furthermore, the affinity of VΚ1 / VH4 to human TNF could be further improved by introducing two mutations at specific framework locations (T24K and T84S; SEQ ID NO: 78).

[0135] Therefore, in certain embodiments, the present invention relates to the VH4 described in Sequence ID No. 26. mut This relates to antibodies containing sequences or their functional fragments.

[0136] Importantly (see Table 3), both VK1 / VH4 molecules (even the unmutated consensus FW) exhibit higher affinity for human TNF than the VK1 / VH3 variant. Therefore, this framework appears particularly suitable for the direct humanization of rabbit antibodies by transplantation of CDR sets and is superior to the current state of technology (VK1 / VH3).

[0137] Since molecules containing the VH3 domain exhibit better productivity than their corresponding VH4-containing molecules, a comparison of production yields for TNF-specific molecules confirms previously obtained data for IL23R-specific molecules (Table 4). However, the general process for scFv production (particularly the refolding process) was originally developed for VK1 / VH3 framework-based molecules, which may bias the productivity results and favor VH3 containing VL / VH framework combinations. Production yields for VH4 containing framework combinations appear to be further optimizeable using adapted production processes.

[0138] Storage stability tests using TNF-specific molecules confirm the previously observed stabilizing effect of lambda capping, as the monomer content after 2 weeks of storage under stress conditions is again clearly superior for all capped molecules compared to the monomer content of the control molecule (VK1 / VH3 consensus) used in this study. Overall, the stability of molecules containing VH4 is comparable to that of molecules containing VH3, and the VK3 / VH4 framework combination is the most stable (see Figure 5).

[0139] [Table 3]

[0140] [Table 4]

[0141] Example 4: Sk12 vs. Sk17 λ-cap As described above, in addition to selecting the optimal combination of VL and VH frameworks, the incorporation of a λ cap into the VL consensus domain also results in a significant improvement in the biophysical properties of each scFv molecule. Various germline sequences in the FW region 4 of the lambda-type light chain also appear likely to yield different properties when used as a λ cap. When comparing different lambda-type FW 4 regions, we identified sequence sk17 (SEQ ID NO: 63) as particularly stable (Table 5) in light of a VK1 / VH3 framework combination containing a different set of TNF-specific CDRs than those used in the above study and in international publication 2014 / 206561. The two molecules differ only in the sequence of their λ caps and were named sk12 (SEQ ID NO: 62) and sk17 (SEQ ID NO: 63). In terms of storage stability and thermal stability, molecules containing sk17 λ caps are superior to molecules containing sk12 λ caps.

[0142] Table 6 provides further evidence that sk17 is superior to sk12. The same IL17-specific rabbit CDR loop was transplanted into four different Vκ1-possessing framework combinations. Constructs 1-3 contained the same Vκ1 with an sk17λ cap and a different variable heavy chain (correspondingly VH1A, VH4, or VH1B), while construct 4 contained a Vκ1 light chain with an sk12λ cap and a VH3 heavy chain. All molecules possessing sk17 were superior in terms of target binding affinity to molecules possessing the current state of the art Vκ1(sk12) / VH3 framework combination (construct 4). Furthermore, molecules possessing Vκ1 / VH4 (construct 2) exhibited superior thermal stability, as the midpoint of thermal denaturation measured by DSF measurements was significantly elevated. Therefore, sk17 appears to be particularly useful for stabilization by lambda capping.

[0143] [Table 5]

[0144] [Table 6]

[0145] The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0146] To the extent permitted under the respective patent laws, all patents, applications, publications, test methods, documents, and other materials cited herein are incorporated herein by reference.

[0147] [Table 7-1] [Table 7-2] [Table 7-3]

[0148] [Table 8]

Claims

1. Antibodies or antigen-binding fragments containing the following: (a) Variable light chain, Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determination region. The LFW1, LFW2, and LFW3 regions together exhibit sequence identity with respect to the corresponding framework region having sequence numbers 42-44 of the Vκ1 sequence described in sequence number 41; or with respect to the corresponding framework region having sequence numbers 52-54 of the Vκ3 sequence described in sequence number 51; Furthermore, (b) Variable heavy chain, Here, the variable heavy chain includes the region HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determination region, where The HFW1, HFW2, HFW3, and HFW4 regions together exhibit sequence identity with respect to the corresponding framework regions having sequence numbers 2 to 5 of the VH1A sequence described in sequence number 1; The antibody or its antigen-binding fragment is characterized in that the Vκ-based LFW1, LFW2, and LFW3 regions are combined with a Vλ-based LFW4 sequence which is identical to sequence number 63.

2. The antibody or antigen-binding fragment according to claim 1, wherein the CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are independently selected from (i) CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 derived from a parental non-human antibody having specificity for the antigen of interest; and (ii) CDR domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 derived from a parental human antibody containing a Vκ domain.

3. A pharmaceutical composition comprising the antibody or antigen-binding fragment described in claim 1, and a pharmaceutically acceptable carrier and / or excipient.

4. A nucleic acid or collection of nucleic acids encoding the antibody or its antigen-binding fragment as described in claim 1.

5. A vector or collection of vectors comprising the nucleic acid or collection of nucleic acids described in claim 4.

6. A method for producing an antibody or antigen-binding fragment according to claim 1, comprising the step of expressing a nucleic acid or collection of nucleic acids according to claim 4, or a vector or collection of vectors according to claim 5.

7. A method for humanizing non-human antibodies, A method comprising one or more steps of cloning nucleic acids encoding variable heavy chain CDRs CDR1, CDR2, and CDR3, and variable light chain CDRs CDR1, CDR2, and CDR3 of the non-human antibody into one or more nucleic acids encoding the antibody or its antigen-binding fragment according to claim 1.

8. The method according to claim 7, further comprising one or more of the following steps: (aa) Immunization of non-human animals with the target antigen; (ab) Isolation of at least one target antibody; (ac) Clonal isolation of affinity mature memory B cells that interact with the target antigen; (ad) Culture of single B cells in a co-culture system that does not require immortalization of single B cell clones; and / or (ae) Screening of B cell culture supernatants with cell-based ELISA to identify at least one antibody that binds to a target antigen.

9. The method according to claim 7 or 8, wherein the framework regions of the non-human antibody together have the highest homology to the corresponding framework regions of the human VH1A family (SEQ ID NO: 1) and combinations of the human VL family selected from Vκ1 (SEQ ID NO: 41) and Vκ3 (SEQ ID NO: 51).

10. A method for optimizing the target parent antibody, A method comprising one or more steps of cloning nucleic acids encoding the VH CDR and VL CDR CDR1, CDR2, and CDR3 of the parent antibody to one or more nucleic acids encoding the antibody or its antigen-binding fragment according to claim 1.

11. A method for generating a diverse collection of antibodies or antigen-binding fragments thereof, A method comprising, in one or more steps, cloning one or more diverse collections of nucleic acids encoding one or more diverse collections of VH CDR and / or VL CDR CDR1, CDR2, and CDR3 to one or more nucleic acids encoding the antibody or antigen-binding fragment thereof described in claim 1.

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