Antibody variable domains and antibodies with reduced immunogenicity

Abstract

The present invention relates to antibody variable domains that exhibit reduced binding to pre-existing anti-drug antibodies (ADA), to antibodies comprising one or more of said antibody variable domains, and to pharmaceutical compositions comprising said antibodies. The present invention further relates to nucleic acids encoding said antibody variable domains or said antibodies, to vector(s) comprising said nucleic acids, to host cell(s) comprising said nucleic acids or said vectors, and to methods of producing said antibody variable domains or said multispecific antibodies. Furthermore, the present invention relates to methods of making said antibody variable domains and antibodies.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibody variable domains that exhibit reduced binding to pre-existing anti-drug antibodies (ADA), to antibodies comprising one or more of said antibody variable domains, and to pharmaceutical compositions comprising said antibodies. The present invention further relates to nucleic acids encoding said antibody variable domains or said antibodies, to vector(s) comprising said nucleic acids, to host cell(s) comprising said nucleic acids or said vectors, and to methods of producing said antibody variable domains or said multispecific antibodies. Furthermore, the present invention relates to methods of making said antibody variable domains and antibodies. [Background technology]

[0002] 2. Background of the Invention Over the past 40 years, since the development of the first monoclonal antibody ("mAb"; Kohler & Milstein, Nature, 1975, Vol.256, pp. 495-497), 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 antigen of interest. Although antibodies of non-human origin can be used for research and diagnostics, in therapeutic approaches, the human body typically recognizes non-human antibodies as foreign and mounts an immune response against the non-human antibody drug, reducing or eliminating its efficacy. Even when the administered antibody therapeutic has been humanized, e.g., by grafting non-human CDRs onto a human immunoglobulin scaffold to minimize the non-human component, it may still provoke an immune response, thereby compromising the efficacy and / or safety of these therapeutics.

[0003] Such immune responses usually involve the binding of anti-drug antibodies (ADAs) to the therapeutic agent, these ADAs being either antibodies already present in human serum (so-called pre-existing ADAs) and / or antibodies that form during the treatment.

[0004] The risk of ADA binding can be significantly increased for therapeutic antibodies that are composed of parts of naturally occurring human antibodies, such as Fab and Fv antibody fragments. One of the main reasons for this increased ADA binding is thought to be that in antibody fragments, a significant number of amino acids that were previously shielded by contacts with other antibody moieties or domains become exposed to the solvent and thus present to the immune system as potential epitopes.

[0005] According to the literature, a patient's antibody response depends on the presence of both B-cell and T-cell epitopes. When the B-cell receptor recognizes and binds an antigen, such as an administered therapeutic antibody, the antigen is internalized by the B cell by receptor-mediated endocytosis and undergoes proteolysis. The resulting peptides are then presented by MHC class II molecules. When the T-cell epitopes are recognized by T-helper cells, they stimulate the corresponding B cells to proliferate and differentiate into antibody-producing plasma cells.

[0006] To further reduce the response of the patient's immune system to the administered antibody, several strategies have been offered in the prior art.

[0007] For example, Zhao, L. and Li, J. (2010), BMC Struct. Biol., 10, S6, disclose a method to predict potential B-cell epitopes on protein surfaces based on structural information of antibody-antigen complexes. The authors identified common structural elements that are frequently present in B-cell epitopes. In particular, they found a significant prevalence of polar amino acids with flexible side chains, such as arginine (R), lysine (K), asparagine (N), glutamine (Q), and histidine (H), in antigen epitopes recognized by antibodies. Knowledge of these important structural elements forms the basis of strategies to avoid them.

[0008] Nataga, S. and Pastan, I. (2009), Adv Drug Deliv Rev, p. 977-985, Onda, M. et al (2008), PNAS Vol 105(32): 1 1311-11316, and Mazor, R. et al (2016), Immunol Rev. Vol. 270(1): 152-64 disclose a method to reduce the immunogenicity of foreign proteins by identifying B-cell epitopes on the protein and removing them by mutagenesis. The authors replaced bulky hydrophilic residues such as arginine (R), glutamine (Q), glutamic acid (E) or lysine (K) in solvent-exposed regions with small amino acids (such as alanine, glycine and serine).

[0009] WO2016 / 150845 discloses glycosylated immunoglobulin heavy chain variable domains (VH domains) of immunoglobulin single variable domain antibodies (ISVDs or nanobodies) that are glycosylated in such a way that the binding of said ISVD to pre-existing antibodies is reduced compared to the same ISDV in the absence of glycosylation. For example, WO2016 / 150845 discloses VH domains of ISDVs that are glycosylated or that contain a glycosylation site at one of positions 11, 12, 13, 14, 15, 46, 47, 48, 49, 101, 103, 144, 146, 148, 149 or 150 such that they are glycosylated or can be glycosylated.

[0010] US2017 / 121399A1 discloses a VH domain of a single variable domain antibody (nanobody) comprising mutations in the framework region to reduce binding to pre-existing antibodies. US2017 / 121399A1 proposes various positions for mutations, including at positions 12, 15, 48, 49, 101, 144, 146 and 148 (AHo numbering), including an extension of the C-terminus of the VH domain. Experimental data revealed that the C-terminal alanine extension and substitutions at positions 12 and 103 (AHo numbering), as well as an alanine substitution at position 101, did not further significantly reduce binding to pre-existing antibodies.

[0011] WO2011 / 075861 discloses a method for reducing the immunogenicity of an antibody variable domain, in particular an scFv, by mutating one or more amino acid residues located at the interface between the variable and constant chains of the corresponding full-length antibody. It further discloses that (i) residues present in turn regions of the secondary structure, (ii) residues with large flexible or bulky side chains, or (iii) hydrophobic residues are likely to be B-cell epitopes and thus induce an immunogenic response, and that removing such amino acid residues blocks the B-cell epitopes. It is further specified that the one or more amino acid residues to be substituted are leucine (L), valine (V), aspartic acid (D), phenylalanine (F), arginine (R) and / or glutamic acid (E). WO2011 / 075861 discloses an example of an scFv with heavy chain point mutations L12S, V103T and L144T (AHo numbering), which shows reduced binding to pre-existing ADA present in human serum compared to a version without the mutations. Thus, WO2011 / 075861 teaches how to reduce the immunogenicity of an antibody variable domain against pre-existing ADA by replacing small hydrophobic residues such as L and V located at the interface between the variable and constant chains of the corresponding full-length antibody with small, weakly hydrophilic amino acids (such as S and T), and avoiding large, bulky hydrophilic residues located at said interface.

[0012] Currently available methods provide useful suggestions on how the immunogenicity of antibody variable domains can be reduced, but the likelihood of success is case-dependent. That is, the solutions proposed to reduce the immunogenicity of antibody variable domains are not generally applicable, and antibody variable domains obtained by these methods often show significant residual immunogenicity. Thus, there remains a large unmet need for antibody variable domains that exhibit low immunogenicity and are generally applicable for the construction of antibody fragments. More specifically, it is desirable to have at hand antibody variable domains that exhibit low immunogenicity, especially with regard to reduced binding to pre-existing ADAs, and that are generally applicable for the construction of antibody fragments. Furthermore, it is desirable that these antibody variable domains exhibit high stability when incorporated into the final antibody format, making them applicable for the construction of stable antibody fragments and fragment-based multispecific antibodies suitable for therapeutic development. Summary of the Invention

[0013] The aim of the present invention is to provide antibody variable domain variants that exhibit reduced immunogenicity, more particularly that they are significantly less recognized by pre-existing ADA(s) when compared to their unmodified variants. Furthermore, these antibody variable domain variants should be highly stable to enable their application in the construction of antibody fragments and multispecific antibodies suitable for pharmaceutical development.

[0014] The inventors have surprisingly found that antibody variable domains, in particular at one or more of heavy chain framework positions 101, 146 and / or 148 (according to AHo numbering), substituted by small, moderately hydrophilic amino acids, i.e. alanine (A) or serine (S), or by hydrophilic amino acids with flexible and bulky side chains, i.e. lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), when in scFv format, show a significant reduction in binding to pre-existing anti-drug antibodies (ADA) present in human serum compared to the unsubstituted version. Furthermore, it has been found that additional substitution(s) of one or both of heavy chain framework positions 12 and 144 by alanine (A), lysine (K) or arginine (R) can further reduce binding of said antibody variable domains, when in scFv format, to pre-existing anti-drug antibodies (ADA) present in human serum.

[0015] Thus, in a first aspect, the present invention relates to a method for generating modified antibody variable domains that exhibit reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum from healthy donors when compared to the unmodified version, the reduced binding being determined by an ELISA-based pre-existing anti-drug antibody binding assay, wherein said unmodified antibody variable domains bind to a target antigen, (i) a variable heavy chain (VH) comprising from the N-terminus to the C-terminus the domains HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region; (ii) a variable light chain (VL), the variable light chain comprising, from the N-terminus to the C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region, and each LCDR representing a light chain complementarity determining region; the method comprising: - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q); - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 to obtain the modified antibody variable domain.

[0016] In a second aspect, the present invention relates to an antibody variable domain which binds to a target antigen, (i) a variable heavy chain (VH) comprising from the N-terminus to the C-terminus the domains HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region; wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, and the HFW1, HFW2, HFW3 and HFW4 are - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q); - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 (AHo numbering); a variable heavy chain (VH) having one or more substitutions selected from the group consisting of: And (ii) a variable light chain (VL), the variable light chain comprising from the N-terminus to the C-terminus the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region, and each LCDRR representing a light chain complementarity determining region; wherein said variable light chain framework regions LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks and said variable light chain framework region LFW4 is selected from Vλ frameworks, in particular Vλ framework sequences selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196. Includes.

[0017] In a third aspect, the present invention relates to an antibody comprising one or more antibody variable domains of the invention, said one or more antibody variable domains independently of each other selected from an Fv, a disulfide stabilized Fv (dsFv), an scFv, and a disulfide stabilized scFv (dsscFv).

[0018] In a fourth aspect, the present invention relates to a nucleic acid or two nucleic acids encoding an antibody variable domain or an antibody of the invention.

[0019] In a fifth aspect, the present invention relates to a vector or two vectors comprising a nucleic acid or two nucleic acids according to the invention.

[0020] In a sixth aspect, the present invention relates to a host cell(s) comprising the vector or two vectors of the invention.

[0021] In a seventh aspect, the invention relates to a method for producing an antibody variable domain of the invention or an antibody of the invention, comprising (i) providing a nucleic acid or two nucleic acids of the invention, or a vector or two vectors of the invention, expressing said nucleic acid or said two nucleic acids, or said vector or vectors and recovering said antibody variable domain or said antibody from said expression system, or (ii) providing a host cell(s) of the invention, culturing said host cell(s) and collecting said antibody variable domain or said antibody from the cell culture.

[0022] In an eighth aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharma- ceutically acceptable carrier.

[0023] In a ninth aspect, the present invention relates to a pharmaceutical composition according to the invention for use as a medicament.

[0024] The aspects, advantageous features and preferred embodiments of the present invention summarized in the following items each alone or in combination further contribute to solving the object of the present invention. 1. An antibody variable domain that binds to a target antigen: (i) a variable heavy chain (VH) comprising from the N-terminus to the C-terminus the domains HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region; wherein said variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, said HFW1, HFW2, HFW3 and HFW4 having the following structure: (AHo numbering): Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 having one or more substitutions selected from the group consisting of: and (ii) a variable light chain (VL), the variable light chain comprising from the N-terminus to the C-terminus the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region, and each LCDRR representing a light chain complementarity determining region; wherein said variable light chain framework regions LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks and said variable light chain framework region LFW4 is selected from Vλ frameworks.

[0025] 2. The HFW3 and HFW4 have (AHo numbering): - serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 2. The antibody variable domain of item 1, having one or more substitutions selected from the group consisting of:

[0026] 3. The HFW3 and HFW4 have (AHo numbering): - alanine (A), serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101; - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and - leucine (L), lysine (K) or asparagine (N) at amino acid position 148; or - serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101; - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 The antibody variable domain according to item 1, having one or more substitutions selected from the group consisting of:

[0027] 4. The HFW3 and HFW4 have (AHo numbering): - alanine (A), serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101; - Lysine (K), Aspartic acid (D), Glutamic acid (E), Arginine (R) or Glutamine (Q) at amino acid position 146; and -Lysine (K) at amino acid position 148; or - having serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101; - Lysine (K), Aspartic acid (D), Glutamic acid (E), Arginine (R) or Glutamine (Q) at amino acid position 146; and - Lysine (K) at amino acid position 148 2. The antibody variable domain of item 1, having one or more substitutions selected from the group consisting of:

[0028] 5. The HFW1 and HFW4 further comprise (AHo numbering): - alanine (A), lysine (K) or arginine (R) at amino acid position 12, in particular alanine (A) or arginine (R) at amino acid position 12; and - alanine (A), lysine (K) or arginine (R) at amino acid position 144 5. The antibody variable domain of any one of items 1 to 4, having one or two substitutions selected from the group consisting of:

[0029] 6. The variable light chain framework regions LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks and the variable light chain framework region LFW4 is selected from Vλ frameworks; The LFW3 has the following substitutions (AHo numbering): - glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101 Item 1. The antibody variable domain of any one of the preceding items, having the following structure:

[0030] 7. HFW1, HFW3 and HFW4 have the following substitutions (AHo numbering): a. alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; b. alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146; c. alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); d. alanine (A), lysine (K), or arginine (R) at amino acid position 144; and leucine (L), lysine (K), or asparagine (N) at amino acid position 148; e. alanine (A), lysine (K), or arginine (R) at amino acid position 144; at amino acid position 146, an alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); and leucine (L), lysine (K), or asparagine (N) at amino acid position 148; f. Alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; an alanine (A), lysine (K), or arginine (R) at amino acid position 144; and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); g. Alanine (A) or Arginine (R) at amino acid position 12; and at amino acid position 101, an alanine (A), a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q), or a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q); h. alanine (A) or arginine (R) at amino acid position 12; and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); i. alanine (A) or arginine (R) at amino acid position 12; at amino acid position 101, an alanine (A), a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q), or a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q); and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); j. alanine (A) or arginine (R) at amino acid position 12; at amino acid position 101, an alanine (A), a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q), or a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q); an alanine (A), lysine (K), or arginine (R) at amino acid position 144; and At amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) The antibody variable domain according to item 1, having one of the following:

[0031] 8. The HFW1, HFW3 and HFW4 have the following substitutions (AHo numbering): a. Alanine (A), Lysine (K), Arginine (R), or Asparagine (N), or Lysine (K), Arginine (R), or Asparagine (N) at amino acid position 101; b. Lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; c. serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; d. alanine (A) or lysine (K) at amino acid position 144; and leucine (L) or asparagine (N) at amino acid position 148; e. Lysine (K) at amino acid position 144; Glutamic acid (E) at amino acid position 146; and lysine (K) at amino acid position 148; f. serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; Alanine (A) or Lysine (K) at amino acid position 144; and arginine (R) or glutamine (Q) at amino acid position 146; g. Arginine (R) at amino acid position 12; and serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; h. Arginine (R) at amino acid position 12; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; i. Arginine (R) at amino acid position 12; serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; j. alanine (A) at amino acid position 12; serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; Alanine (A) or Lysine (K) at amino acid position 144; and Arginine (R) or glutamine (Q) at amino acid position 146 The antibody variable domain according to item 1, having one of the following:

[0032] 9. The HFW1, HFW3 and HFW4 are one of the substitutions indicated for position -101, and - one or more further substitutions selected from the substitutions indicated at positions 12, 144, 146 and 148, in particular one or more further substitutions selected from the substitutions indicated at positions 146 and 148, in particular one further substitution selected from the substitutions indicated at position 146 9. The antibody variable domain according to any one of items 1 to 8, comprising:

[0033] 10. The antibody variable domain according to any one of items 1 to 9, wherein the variable heavy chain framework regions HFW1, HFW2 and HFW3 are selected from the human VH framework subtypes VH1a, VH1b, VH3, VH4, VH5 and VH6.

[0034] 11. The antibody variable domain according to any one of items 1 to 9, wherein the variable heavy chain framework regions HFW1, HFW2 and HFW3 are selected from the human VH framework subtypes VH1a, VH1b, VH3 and VH4.

[0035] 12. The antibody variable domain according to any one of items 1 to 9, wherein the variable heavy chain framework regions HFW1, HFW2 and HFW3 are of the human VH framework subtype VH3.

[0036] 13. The variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are a. a combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italicized residues in Table 5) of any one of SEQ ID NOs: 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 and 175; and b. Combinations of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e. non-italicized residues in Table 5) of any one of SEQ ID NOs: 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 and 175 with 1, 2 or 3 mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering). is selected from 10. The antibody variable domain according to any one of items 1 to 9, wherein HFW1, HFW3 and HFW4 have one or more of the substitutions defined in any one of items 1 to 9.

[0037] 14. The variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are a. a combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italicized residues in Table 5) of any one of SEQ ID NOs: 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 and 170; and b. Combinations of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e. non-italicized residues in Table 5) of any one of SEQ ID NOs: 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 and 170 with 1, 2 or 3 mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering). 10. The antibody variable domain according to any one of items 1 to 9, wherein HFW1, HFW3 and HFW4 have one or more of the substitutions defined in any one of items 1 to 9.

[0038] 15. The variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are a. a combination of the framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italicized residues in Tables 1 and 3) of any one of SEQ ID NOs: 5-36 and 83-114, in particular any one of SEQ ID NOs: 8-36 and 86-114; and b. Combinations of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e. non-italicized residues in Tables 1 and 3) of any one of SEQ ID NOs: 5 to 36 and 83 to 114, in particular any one of SEQ ID NOs: 8 to 36 and 86 to 114, with 1, 2 or 3 mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering). 4. The antibody variable domain of any one of the preceding items, selected from:

[0039] 16. The variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are a. a combination of the framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italicized residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25, 28, 30-36, 86-103, 106 and 108-114, in particular any one of SEQ ID NOs: 8-25 and 86-103; and b. Combinations of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e. non-italicized residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25, 28, 30-36, 86-103, 106 and 108-114, in particular any one of SEQ ID NOs: 8-25 and 86-103, with 1, 2 or 3 mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering). 4. The antibody variable domain of any one of the preceding items, selected from:

[0040] 17. The antibody variable domain of any one of the preceding items, wherein the human antibody Vκ framework is selected from the Vκ1 framework subtype.

[0041] 18. LFW1, LFW2 and LFW3 are a. a combination of framework regions LFW1, LFW2 and LFW3 (i.e., the non-italicized residues in Table 5) of any one of SEQ ID NOs: 176, 177, 178 and 179; and b. A combination of framework regions LFW1, LFW2 and LFW3 (i.e., non-italicized residues in Table 5) of any one of SEQ ID NOs: 176, 177, 178 and 179 having 1, 2 or 3 mutations in the framework regions at a position different from position 101 (AHo numbering); wherein said LFW3 is optionally selected from the following substitutions (AHo numbering): - glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101 Item 1. The antibody variable domain of any one of the preceding items, having the following structure:

[0042] 19. The antibody variable domain of any one of the preceding items, wherein the variable light chain framework region LFW4 has a sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196.

[0043] 20. The variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are a. a combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i.e., the non-italicized residues in Table 1, Table 3 and Table 5) of any one of SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187; and b. Combinations of framework regions LFW1, LFW2, LFW3 and LFW4 (i.e. non-italicized residues in Tables 1, 3 and 5) of any one of SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187 with 1, 2 or 3 mutations in the framework region at a position other than 101 (AHo numbering). 4. The antibody variable domain of any one of the preceding items, selected from:

[0044] 21. The antibody variable domain according to any one of the preceding items, wherein the format of the antibody variable domain is selected from Fv, dsFv, scFv and dscFv, in particular Fv and scFv.

[0045] 22. The antibody variable domain of any one of the preceding items, wherein the antibody variable domain, when in scFv format, exhibits reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum, as determined in a pre-existing ADA binding assay, compared to a version of the antibody variable domain that does not comprise the substitutions defined in item 1.

[0046] 23. When the antibody variable domain is in scFv format: a. a melting temperature (Tm) when formulated in 20 mM histidine, pH 6.0, as determined by differential scanning fluorimetry (DSF), of at least 63°C, particularly at least 64°C, particularly at least 65°C; b. when formulated at a concentration of 10 mg / ml in 20 mM histidine at pH 6.0, the loss of monomer content after storage for 28 days at 4° C. is less than 5%, particularly less than 3%, particularly less than 2%; c. when formulated at a concentration of 10 mg / ml in 20 mM histidine at pH 6.0, the loss in monomer content after storage for 28 days at 40° C. is less than 15%, particularly less than 10%, particularly less than 8%; d. When formulated at a concentration of 10 mg / ml in 20 mM histidine, pH 6.0, the protein content loss is less than 15%, specifically less than 10%, after storage for 28 days at 4°C; e. when formulated at a concentration of 10 mg / ml in 20 mM histidine, pH 6.0, there is less than 10% loss in protein content, specifically less than 5%, after storage for 28 days at 40°C; f. binds its target antigen with a dissociation constant (KD) that is 3-fold lower, the same or not more than 3-fold, in particular not more than 2.5-fold, in particular not more than 2-fold, as measured by surface plasmon resonance (SPR) compared to the KD of a version of said antibody variable domain that does not contain the substitution defined in item 1; 21. The antibody variable domain according to any one of items 13 to 16 and 20, further characterized by one or more of the following features:

[0047] 24. The antibody variable domain comprises: (i) a VH sequence selected from SEQ ID NO: 204 and variants of SEQ ID NO: 204 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); and (ii) a VL sequence selected from SEQ ID NOs: 205-206 and variants of SEQ ID NOs: 205-206 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 208 to 210 and variants of SEQ ID NOs: 208 to 210 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 211-212 and variants of SEQ ID NOs: 211-212 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 213 to 215 and variants of SEQ ID NOs: 213 to 215 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 216-217 and variants of SEQ ID NOs: 216-217 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 224 to 225 and variants of SEQ ID NOs: 224 to 225 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 226-229 and variants of SEQ ID NOs: 226-229 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 230 to 233 and variants of SEQ ID NOs: 230 to 233 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 234-237, and variants of SEQ ID NOs: 234-237 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 238 and a variant of SEQ ID NO: 238 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 239 and a variant of SEQ ID NO: 239 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 240 to 242 and variants of SEQ ID NOs: 240 to 242 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 243-245, and variants of SEQ ID NOs: 236-238 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 254 and a variant of SEQ ID NO: 254 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 255 and a variant of SEQ ID NO: 255 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 256 and a variant of SEQ ID NO: 256 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 257 and a variant of SEQ ID NO: 257 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 261 to 262 and variants of SEQ ID NOs: 261 to 262 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 263-266, and variants of SEQ ID NOs: 263-266 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 267, a variant of SEQ ID NO: 267 having one, two or three mutations in the framework regions at positions different from positions 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 268 and a variant of SEQ ID NO: 268 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 269 and a variant of SEQ ID NO: 269 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 270 and a variant of SEQ ID NO: 270 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 271 to 272 and variants of SEQ ID NOs: 271 to 272 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 273 and a variant of SEQ ID NO: 273 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 274 to 275 and variants of SEQ ID NOs: 274 to 275 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 276 and a variant of SEQ ID NO: 276 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 277 to 278 and variants of SEQ ID NOs: 277 to 278 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 279-281, and variants of SEQ ID NOs: 279-281 having one, two or three mutations in the framework regions at positions (AHo numbering) different from 101; or (i) VH sequences selected from SEQ ID NOs: 282 to 283 and variants of SEQ ID NOs: 282 to 283 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 284 to 285, and variants of SEQ ID NOs: 284 to 285 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 286 and a variant of SEQ ID NO: 286 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 287 and a variant of SEQ ID NO: 287 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 288 to 291 and variants of SEQ ID NOs: 288 to 291 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 292 to 295, and variants of SEQ ID NOs: 292 to 295 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 302 to 303 and variants of SEQ ID NOs: 302 to 303 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 304 to 305, and variants of SEQ ID NOs: 304 to 305 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 309 to 310 and variants of SEQ ID NOs: 309 to 310 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 311-312 and variants of SEQ ID NOs: 311-312 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 313 to 316 and variants of SEQ ID NOs: 313 to 316 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 317-318 and variants of SEQ ID NOs: 317-318 having one, two or three mutations in the framework regions at a position (AHo numbering) different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 319 to 320 and variants of SEQ ID NOs: 319 to 320 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 321-322 and variants of SEQ ID NOs: 321-322 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 323 to 324 and variants of SEQ ID NOs: 323 to 324 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NO: 325 and a variant of SEQ ID NO: 325 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 330 to 332 and variants of SEQ ID NOs: 330 to 332 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 333 to 337, and variants of SEQ ID NOs: 333 to 337 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 338 to 340 and variants of SEQ ID NOs: 338 to 340 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 341 to 343, and variants of SEQ ID NOs: 341 to 343 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 344 to 346 and variants of SEQ ID NOs: 344 to 346 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 347-349 and variants of SEQ ID NOs: 347-349 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 350 to 352 and variants of SEQ ID NOs: 350 to 352 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 353 to 355, and variants of SEQ ID NOs: 353 to 355 having one, two or three mutations in the framework region at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 356 to 358 and variants of SEQ ID NOs: 356 to 358 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) a VL sequence selected from SEQ ID NOs: 359-361 and variants of SEQ ID NOs: 359-361 having one, two or three mutations in the framework regions at a position different from position 101 (AHo numbering); or (i) VH sequences selected from SEQ ID NOs: 362 to 364 and variants of SEQ ID NOs: 362 to 364 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering), and (ii) VL sequences selected from SEQ ID NOs: 365 to 367 and variants of SEQ ID NOs: 365 to 367 having one, two or three mutations in the framework regions at positions other than position 101 (AHo numbering). or 2. The antibody variable domain according to item 1, which is an scFv having a sequence selected from SEQ ID NOs: 207, 218 to 223, 246 to 253, 258 to 260, 296 to 301, 306 to 308, 326 to 329 and 368 to 385 and variants of SEQ ID NOs: 207, 218 to 223, 246 to 253, 258 to 260, 296 to 301, 306 to 308, 326 to 329 and 368 to 385 having 1, 2, 3, 4 or 5 mutations in the framework regions at VH positions 12, 101, 144, 146 and 148 (AHo numbering) and at positions different from VL position 101 (AHo numbering).

[0048] 25. An antibody comprising one or more antibody variable domains according to any one of items 1 to 24, wherein the one or more antibody variable domains are independently selected from Fv, dsFv, scFv and disulfide-stabilized scFv.

[0049] 26.Antibodies (i) is monospecific and is monovalent, bivalent or trivalent with respect to the target antigen; (ii) are bispecific and, independently of each other, monovalent or bivalent for each target antigen; (iii) is trispecific and monovalent for each target antigen; (iv) trispecific, being bivalent for one of the target antigens and monovalent for the other target antigen; (v) trispecific, being bivalent for two of the target antigens and monovalent for the third; (vi) is tetraspecific and monovalent for each target antigen; (vii) is tetraspecific, bivalent for one of the target antigens and monovalent for the other target antigen; or (viii) The antibody of item 25, which is tetraspecific, bivalent for two of the target antigens and monovalent for the other target antigen.

[0050] 27.Antibodies (i) a first and a second antibody variable domain as defined in any of items 1 to 24, wherein the first and the second antibody variable domain have specificity for two different target antigens; and (ii) one, two, three or four further antibody variable domains as defined in any of items 1 to 24, which independently of one another have either specificity for one of the target antigens of the first and second variable domains or specificity for a target antigen different from the target antigens of the first and second variable domains. 26. The antibody of item 25, comprising:

[0051] 28. The antibody format is selected from Fc-bearing bivalent bispecific formats, Fc-bearing trivalent bispecific formats and tetravalent bispecific formats, and trivalent bispecific IgG formats and tetravalent bispecific IgG formats, wherein the trivalent or tetravalent formats comprise one or more Fv, dsFv, scFv or disulfide-stabilized scFv; 28. The antibody according to any one of items 25 to 27, more specifically, wherein the antibody format is selected from a KiH-based format carrying an Fc; DVD-Ig; CODV-IgG and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), even more specifically DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).

[0052] 29. The antibody according to item 28, wherein the IgG is selected from the IgG subclasses IgG1 and IgG4, in particular IgG4.

[0053] 30. The antibody according to any one of items 25 to 27, wherein the antibody does not contain an immunoglobulin Fc region.

[0054] 31. The antibody according to item 30, wherein the antibody is in a format selected from the group consisting of tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, di-diabody, tandem-di-scFv, and MATCH.

[0055] 32. The antibody according to item 31, wherein the antibody does not contain a CH1 and / or CL region.

[0056] 33. The antibody according to item 32, wherein the antibody is in scDb-scFv, triabody, tetrabody or MATCH format, in particular, the antibody is in MATCH or scDb-scFv format, more particularly the antibody is in MATCH format, more particularly MATCH3 or MATCH4 format.

[0057] 34. The antibody according to any one of items 25 to 33, wherein the antibody exhibits reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum as determined in a pre-existing ADA binding assay, in particular reduced binding to pre-existing ADA when compared to a reference antibody that does not contain the substitution defined in item 1.

[0058] 35. The antibody is a single chain antibody having a sequence selected from SEQ ID NOs: 386-401, 426, 427, 440, 441 and variants of SEQ ID NOs: 386-401, 426, 427, 440, 441 with 1, 2, 3, 4 or 5 mutations in the framework regions at positions different from VH positions 12, 101, 144, 146 and 148 (AHo numbering) and VL position 101 (AHo numbering); or The antibody is selected from the group consisting of SEQ ID NOs: 402 and 403; 404 and 405; 406 and 407; 408 and 409; 410 and 411; 412 and 413; 414 and 415; 416 and 417; 418 and 419; 420 and 421; 422 and 423; 424 and 425; 428 and 429; 430 and 431; 432 and 433; 434 and 435; 436 and 437; 438 and 439; 442 and 443; 444 and 445; and 402 and 403; 404 and 405; 406 and 407; 408 and 409; 410 and 411; 412 and 413; 414 and 415; 416 and 417; 418 and 41 26. The antibody of claim 25, which is a heterodimer consisting of two chains having a pair of sequences selected from variants 9; 420 and 421; 422 and 423; 424 and 425; 428 and 429; 430 and 431; 432 and 433; 434 and 435; 436 and 437; 438 and 439; 442 and 443; 444 and 445 (wherein each sequence in the sequence pair has 1, 2, 3, 4 or 5 mutations in the framework regions at VH positions 12, 101, 144, 146 and 148 (AHo numbering) and at a position different from VL101 (AHo numbering).

[0059] 36. A nucleic acid or two nucleic acids encoding an antibody variable domain according to any one of items 1 to 24 or an antibody according to any one of items 25 to 35.

[0060] 37. A vector or two vectors comprising a nucleic acid or two nucleic acids according to item 36.

[0061] 38. A host cell or a plurality of host cells comprising a vector or two vectors according to item 37.

[0062] 39. A method for producing an antibody variable domain according to any one of items 1 to 24 or an antibody according to any one of items 25 to 35, comprising: (i) providing a nucleic acid or two nucleic acids according to item 36, or a vector or two vectors according to item 37, expressing the nucleic acid sequence or nucleic acid, or the vector or vectors, and recovering the antibody variable domain or the antibody from the expression system; or (ii) providing a host cell or a plurality of host cells according to item 38, culturing the host cell or the host cells, and recovering the antibody variable domain or the antibody from the cell culture.

[0063] 40. A pharmaceutical composition comprising the antibody according to any one of items 25 to 35 and a pharma- ceutically acceptable carrier.

[0064] 41. The antibody according to any one of items 25 to 35, or the pharmaceutical composition according to item 40, for use as a pharmaceutical.

[0065] 42. A method for generating modified antibody variable domains that exhibit reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum from healthy donors when compared to the unmodified version, the reduced binding being determined by an ELISA-based pre-existing anti-drug antibody binding assay; wherein the unmodified antibody variable domain binds to a target antigen; (i) a variable heavy chain (VH) comprising from the N-terminus to the C-terminus the domains HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region; (ii) a variable light chain (VL), the variable light chain comprising, from the N-terminus to the C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region, and each LCDR representing a light chain complementarity determining region; Including, The following substitutions (AHo numbering) in the heavy chain framework regions of the unmodified antibody variable domain: - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q); - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 to obtain the modified antibody variable domain.

[0066] 43. The method according to item 42, wherein the variable heavy chain framework regions HFW1, HFW2 and HFW3 are selected from the human VH framework subtypes VH1a, VH1b, VH3 and VH4, in particular the human VH framework subtype VH3.

[0067] 44. The method according to any one of items 42 or 43, wherein the variable light chain framework regions LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks and the variable light chain framework region LFW4 is selected from Vλ frameworks, in particular Vλ framework sequences selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196.

[0068] 45. The heavy chain framework region of the unmodified antibody variable domain comprises the following amino acids: - valine (V) or leucine (L), in particular leucine (L), at amino acid position 12; -Threonine (T) at amino acid position 101; - threonine (T) or leucine (L), in particular leucine (L), at amino acid position 144; -Threonine (T) at amino acid position 146; -Serine (S) at amino acid position 148 45. The method according to any one of items 42 to 44, comprising one or more of the following:

[0069] 46. ​​The method according to any one of items 42 to 45, wherein the modified antibody variable domain further has one or more of the characteristics defined in any one of items 1 to 24.

[0070] 47. Two or more substitutions are introduced into the heavy chain framework region of the unmodified antibody variable domain, said two or more substitutions being one of the substitutions indicated for position -101, and - one or more further substitutions selected from the substitutions indicated at positions 12, 144, 146 and 148, in particular one or more further substitutions selected from the substitutions indicated at positions 146 and 148, in particular one further substitution selected from the substitutions indicated at position 146 47. The method according to any one of items 42 to 46, selected from the group consisting of

[0071] 48. A method for producing modified antibodies that exhibit reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum from healthy donors when compared to the unmodified version, the reduced binding being determined by an ELISA-based pre-existing anti-drug antibody binding assay; The unmodified antibody is a fragment-based antibody or an antibody that comprises one or more antibody-based fragments, The method includes making the following substitutions (AHo numbering) in the VH sequence(s) of the unmodified fragment-based antibody, or in the VH sequence(s) of said antibody based fragment(s) of an unmodified antibody comprising one or more antibody based fragments: - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q); - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 to obtain the modified antibody.

[0072] 49. The method of claim 48, wherein the modified antibody comprises an antibody variable domain as defined in any one of items 1 to 24.

[0073] 50. The method according to any one of items 48 or 49, wherein the unmodified antibody comprises one or more unmodified antibody variable domains as defined in any one of items 42 to 46, wherein the one or more antibody variable domains are independently selected from Fv, dsFv, scFv and disulfide-stabilized scFv, and the method comprises introducing into at least one heavy chain framework region of the unmodified antibody variable domain one or more substitutions as defined in any one of claims 1 to 24. [Brief description of the drawings]

[0074] [Figure 1] Figure 1 shows the absorbance levels of pre-existing ADA in 20 human serum samples for PRO1922 (A) and PRO2230 (B) as determined by the ELISA-based pre-existing ADA binding assay described in Example 2. Measurements were performed with spiked and unspiked serum samples (confirmatory assay setup). To be assessed as specific, the signal reduction of spiked versus unspiked serum must be greater than 30% (>30% inhibition). Furthermore, the signal of unspiked serum must not be above the baseline to be considered significant. The baseline includes the mean value of all spiked sera of the sample plus three standard deviations (SCP, calculated in Example 2). Absorbance levels measured at 280 nm in AU. [Diagram 2]Figure 2 shows the number of positive sera of 20 human serum samples for 10 PRO2230 (α-PD-L1 and α-PD-L1 repeat) scFv variants and 10 PRO1922 (α-MSLN and α-MSLN repeat) scFv variants compared to their respective wt (PRO2230 and PRO1922) and L12R-V103T-L144Q references, as determined by an ELISA-based conventional ADA binding assay as described in Example 2. Measurements were performed using spiked and unspiked serum samples. To be assessed as specific, the signal reduction of spiked versus unspiked serum must be ≥ 30% (≥ 30% inhibition). Furthermore, the signal of unspiked serum must not be above the baseline to be considered significant. The baseline consists of the mean of all spiked sera of the sample plus three standard deviations (SCP calculated in Example 2). No bar for a variant means zero positive sera before ADA. [Diagram 3] Figure 3 shows the calculated midpoint [%] of the PEG precipitation curves as a function of 10 PRO2230 (a-PD-L1) scFv variants and 10 PRO1922 (a-MSLN) scFv variants compared to their respective PRO2230 and PRO1922 wild-type (a-PD-L1 wt and a-MSLN wt) and L12R-V103T-L144Q references. Midpoints are calculated as the half-maximal precipitating PEG concentration with 95% confidence interval (CI 95%) of the mean. Error bars represent the standard error of the fit of a sigmoidal function to the experimental data. [Figure 4] Figure 4 shows the results of chemical unfolding experiments using guanidinium hydrochloride (Gua-HCl) for several selected PRO1922 (MSLN) scFv variants and PRO2230 (PD-L1) scFv variants. Depicted is half the final guanidinium hydrochloride concentration (c1 / 2) required for complete unfolding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The inventors have surprisingly discovered that antibody variable domains substituted at one or more of heavy chain framework positions 101, 146 and 148 (according to AHo numbering) with small, moderately hydrophilic amino acids, i.e. alanine (A) or serine (S), or with hydrophilic amino acids with flexible and bulky side chains, i.e. lysine (K), arginine (R), aspartic acid (D) or glutamine (Q), when in scFv format, exhibit significantly reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum when compared to their unsubstituted versions. Furthermore, it has been found that additional substitutions at one or both of heavy chain framework positions 12 and 144 with alanine (A), lysine (K) or arginine (R) can further reduce binding of said antibody variable domains to pre-existing anti-drug antibodies (ADA) present in human serum when in scFv format.

[0076] Although many methods and strategies have been developed to reduce the immunogenicity of antibody variable domains, the reduction in immunogenicity achievable by these methods is often not as strong as desired from the perspective of drug development. Thus, there remains a great unmet need for antibody variable domains that exhibit low immunogenicity. More specifically, it is desirable to have at hand antibody variable domains that exhibit low immunogenicity, especially with respect to reduced binding to existing ADAs, and that are generally applicable to the construction of antibody fragments. Furthermore, it is desirable that these antibody variable domains exhibit high stability when incorporated into the final antibody format, and can be applied to the construction of stable antibody fragments and fragment-based multispecific antibodies suitable for therapeutic development.

[0077] The antibody variable domains of the present invention can be successfully used to construct several highly stable and functional scFvs that can be readily used in the construction of antibody fragment-based multispecific antibody formats.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0079] As used herein, the terms "comprising" and "including" are used in an open and non-limiting sense, unless otherwise indicated. With respect to such latter embodiments, the term "comprising" therefore encompasses the narrower term "consisting of."

[0080] The terms "a," "an," "the," and similar references in the context of describing the present invention (particularly in the context of the claims below) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the term "cell" includes a plurality of cells, including mixtures thereof. When the plural is used for compounds, salts, etc., this is to be construed to mean a single compound, salt, etc.

[0081] In one aspect, the invention relates to an antibody variable domain that binds to a target antigen, (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus the domains HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region, wherein said variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, and said HFW1, HFW3 and HFW4 are (AHo numbering): Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101; - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 and a variable heavy chain (VH) having one or more substitutions selected from the group consisting of: (ii) a variable light chain (VL), the variable light chain comprising from the N-terminus to the C-terminus the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region and each LCDRR representing a light chain complementarity determining region, wherein the variable light chain framework regions LFW1, LFW2 and LFW3 are selected from a human antibody Vκ framework and the variable light chain framework region LFW4 is selected from a Vλ framework; Includes.

[0082] The term "antibody" as used herein includes whole antibodies or single chains thereof; antigen-binding variable domains (i.e., "antigen-binding portions") or single chains thereof; and molecules comprising antibody CDRs, VH regions, or VL regions (including, but not limited to, multispecific antibodies). A naturally occurring "whole antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions are further subdivided into regions flanked by hypervariable regions called complementarity determining regions (CDRs) and highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0083] The term "antibody variable domain" as used herein refers to one or more portions of an intact antibody having the ability to specifically bind to a given antigen (e.g., PD-L1, CD137, ROR1, MSLN, CD3, IL-4R, IL-31 or hSA). This may be an antigen-binding fragment (i.e., "antigen-binding portion") of an intact antibody, or a single chain thereof; and a molecule comprising the antibody CDRs, VH region, or VL region. In particular, in the case of the multispecific antibodies of the invention, the term "antibody variable domain" as used herein refers in particular to Fv fragments comprising the VL and VH domains of a single arm of an antibody (Fv); disulfide stabilized Fv fragments (dsFv); single chain Fv fragments (scFv); and single chain Fv fragments (scAB) fused with an additional light chain constant domain (CL). Preferably, the antibody variable domain of the invention is selected from Fv fragments, disulfide stabilized Fv fragments (dsFv) and scFv fragments. In certain embodiments, the antibody variable domain of the invention is a single chain Fv fragment (scFv). In other particular embodiments, the VL and VH domains of the scFv fragment are stabilized by an interdomain disulfide bond, in particular said VH domain consists of a single cysteine ​​residue at position 51 (AHo numbering) and said VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0084] The term "complementarity determining region" ("CDR") refers to an amino acid sequence whose boundaries are determined using any of a number of well-known schemes. See, e.g., those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); ImMunoGeneTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)) ("IMGT" numbering scheme), and Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering). For example, in the classical format, under Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist 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 of the VH are numbered from about 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues of the VL are numbered from about 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) ("Kabat" numbering). In IMG, the CDRs of an antibody can be determined using the IMGT / DomainGap Align program.

[0085] In the context of the present invention, unless otherwise stated, the numbering system proposed by Honegger & Pluckthun ("AHo") is used (Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670).In particular, the following residues are defined as CDRs according to the AHo 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. Correspondingly, in the context of the present invention, the following residues are defined as light chain frameworks 1 to 4 (LFW1 to LFW4) and heavy chain frameworks 1 to 4 (HFW1 to HFW4), respectively, according to the AHo numbering scheme: LFW1: L1-L23; LFW2: L43-L57; LFW3: L73-L106; LFW4: L139-L149; HFW1: H1-H26; HFW2: H43-H56; HFW3: H77-H107; HFW4: H139-H149. For clarity, the numbering system according to Honegger & Pluckthun takes into account the diversity of lengths found in naturally occurring antibodies, both in the different VH and VL subfamilies and especially in the CDRs, and provides for sequence gaps. Thus, in an antibody variable domain, not all positions 1 to 149 are usually occupied by amino acid residues.

[0086] The term "binds to" as used herein refers to the ability of an individual antibody to react with an antigenic determinant. However, this does not exclude, for example, that the individual antibody may also react with homologues of the antigenic determinant (e.g., antigenic determinants from other species) or other antigenic determinants belonging to the same protein family.

[0087] The term "binding specificity" as used herein refers to the ability of an individual antibody to react with one antigenic determinant and not with a different antigenic determinant. The term "specifically binds" or "specific" as used herein refers to a measurable and reproducible interaction, such as the binding of an antibody with a target, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. However, these terms do not exclude that an individual antibody may bind with comparable affinity to the same antigenic determinant from a different species. In its most general form (and when no defining reference is cited), "specific binding" refers to the ability of an antibody to discriminate between a target of interest and unrelated molecules, for example, as determined according to a specificity assay method known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) test, peptide scan, etc. For example, a standard ELISA assay can be performed. Scoring can be performed by standard color development (e.g. tetramethylbenzidine with secondary antibodies and hydrogen peroxide with horseradish peroxide). Reactions in a given well are scored by optical density at, for example, 450 nm. A typical background (= negative reaction) would be about 0.1 OD; a typical positive reaction would be about 1 OD. That is, the ratio of positive to negative scores will be 10-fold or more. In a further example, an SPR assay can be performed, where a difference between background and signal of at least 10-fold, in particular at least 100-fold, indicates specific binding. Typically, the determination of binding specificity is not performed with a single reference molecule, but with a set of about 3-5 unrelated molecules, such as milk powder, transferrin, etc.

[0088] The antibody variable domains of the present invention bind to a target antigen, which may be any target antigen. Examples of target antigens include, but are not limited to, transmembrane molecules; receptors; ligands; growth factors; growth hormones; clotting factors; anticoagulants; plasminogen activators; serum albumin; hormone or growth factor receptors; neurotrophic factors; nerve growth factor; fibroblast growth factor; CD proteins; interferons; colony stimulating factors (CSF); interleukins (IL); T cell receptors; T cell costimulatory receptors such as CD137; surface membrane proteins; viral proteins; tumor associated antigens; integrins or interleukins; VEGF; renin; human growth hormone; bovine growth hormone. mon;growth hormone releasing factor;parathyroid hormone;thyroid stimulating hormone;lipoproteins;alpha-1-antitrypsin;insulin A chain;insulin B chain;proinsulin;follicle stimulating hormone;calcitonin;luteinizing hormone;glucagon;clotting factor VIIIC;clotting factor IX;tissue factor (TP);von Willebrand factor;protein C;atrial natriuretic factor;pulmonary surfactant;urokinase;human urine;tissue-type plasminogen activator (t-PA);bombesin;thrombin;hematopoietic growth factors;tumor necrosis factor alpha or beta;enkephalinase;RANTES (Regulated on Activation Normally T-cell Expressed and Secreted);human macrophage inflammatory protein (MlP-l) alpha;Muellerian inhibitory substance;relaxin A chain;relaxin B chain;prorelaxin;mouse gonadotropin-related peptide;microbial protein, beta-lactamase;DNase;IgE;cytotoxic T-lymphocyte-associated antigen (CTLA);CTLA-4;inhibin;activin;vascular endothelial growth factor (VEGF);protein A or D;rheumatoid factor;bone-derived neurotrophic factor (BDNF);neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6);NGF-beta;platelet-derived growth factor (PDGF);aFGF;bFGF;epidermal growth factor (EGF);TGF-alpha;TGF-beta, including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5;Insulin-like growth factor-I or -II (IGF-I or IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein, erythropoietin, bone morphogenetic factor, immunotoxin, bone morphogenetic protein (BMP), interferon-α, -β, or -γ, M-CSF, GM-CSF, or G-CSF, IL-1 to IL-10, superoxide dismutase, decay accelerating factor, AIDS envelope protein; transport proteins; homing receptors; addressrins; regulatory proteins; CD3, CD4, CD8, CD11a, CD11b, CD11c, CD18, CD19, CD20, CD34, CD40, or CD46, ICAM, VLA- 4, or VCAM; or HER2, HER3, or HER4 receptor; a member of the ErbB receptor family; EGF receptor; HER2, HER3, or HER4 receptor; cell adhesion molecule; LFA-1, Mac1, pl50.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin or αv / β3 integrin; α or β subunit of cell adhesion molecule; antibody); growth factor, VEGF; tissue factor (TF); TGF-β; α interferon (α-IFN); IL-8; IgE; blood group antigen Apo2; death receptor such as PD-1; death receptor ligand such as PD-L1; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA4 or protein C.;

[0089] The term "tumor associated antigen (TAA)" refers to an antigen expressed on the surface of a tumor cell. In certain embodiments, a TAA is an antigen that is preferentially expressed on tumor cells compared to non-tumor cells, in particular, expression of the TAA on tumor cells is at least 5-fold higher, at least 10-fold higher, at least 20-fold higher, at least 50-fold higher, or at least 100-fold higher than on non-tumor cells from the same organism or patient.

[0090] Examples of tumor-associated antigen targets include, but are not limited to, ADRB3, AFP, ALK, BCMA, beta human chorionic gonadotropin, CA-125 (MUC16), CAIX, CD123, CD133, CD135, CD135 (FLT3), CD138, CD171, CD19, CD20, CD22, CD24, CD276, CD33, CD38, CD44v6, CD79b, CD97, CDH3 (cadherin 3), CEA. , CEACAM6, CLDN6, CLEC12A (CLL1), CSPG4, CYP1B1, EGFR, EGFRvlll, EpCAM, EPHA2, ephrin B2, ERBBs (e.g., ERBB2), FAP, FGFR1, folate receptor alpha, folate receptor beta, Fos-related antigen, GA733, GD2, GD3, GFRα4, globoH, GPC3, GPR20, GPRC5D, HAVCR1, Her2 / neu (HER2), HLA-A2, HMWMAA, HPV E6 or E7, human telomerase reverse transcriptase, IL-11Ra, IL-13Ra2, intestinal carboxylesterase, KIT, Legumain, LewisY, LMP2, Ly6k, MAD-CT-1, MAD-CT-2, ML-IAP, MN-CA IX, MSLN, MUC1, mutant hsp70-2, NA-17, NCAM, neutrophil elastase, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, PANX3, PDGFR-β, PLAC1, polysialic acid, PSCA, PSMA, RAGE1, ROR1, sLe, sperm protein 17, SSEA-4, SSTR2, sTn antigen, sTn-glycopeptide, TAG72, TARP, TEM1 / CD248, TEM7R, thyroglobulin, Tn antigen, Tn-O-glycopeptide, TPBG(5T4), TRP-2, TSHR, UPK2 and VEGFR2.Preferred examples include CD138, CD79b, TPBG (5T4), HER2, MSLN, MUC1, CA-125 (MUC16), PSMA, BCMA, CD19, EpCAM, CLEC12A (CLL1), CD20, CD22, CEA, CD33, EGFR, GPC3, CD123, CD38, CD33, CD276, CDH3 (cadherin 3), FGFR1, SSTR2, CD133, EPHA2, HLA-A2, IL13RA2, ROR1, CEACAM6, CD135, GD-2, GA733, CD135 (FLT3), CSPG4, and TAG-72. Specific examples include CD138, CD79b, CD123, MSLN, PSMA, BCMA, CD19, CD20, CEA, CD38, CD33, CLEC12a, and ROR1.

[0091] In a preferred embodiment, HFW1, HFW3 and HFW4 constructed in the antibody variable domain of the present invention have lysine (K), aspartic acid (D), glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 146, and (AHo numbering): - alanine (A), serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101; and - Lysine (K) at amino acid position 148 and having one or two substitutions selected from the group consisting of:

[0092] In another preferred embodiment, the HFW1 and HFW4 constructed in the antibody variable domains of the present invention further comprise (AHo numbering): - alanine (A), lysine (K) or arginine (R) at amino acid position 12, in particular alanine (A) or arginine (R) at amino acid position 12; and - alanine (A), lysine (K) or arginine (R) at amino acid position 144 and having one or two substitutions selected from the group consisting of:

[0093] In certain embodiments, HFW1, HFW3 and HFW4 configured in the antibody variable domains of the invention have any one of the following substitutions (AHo numbering): a. alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; b. alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146; c. alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); d. alanine (A), lysine (K), or arginine (R) at amino acid position 144; and leucine (L), lysine (K), or asparagine (N) at amino acid position 148; e. alanine (A), lysine (K), or arginine (R) at amino acid position 144; at amino acid position 146, an alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); and leucine (L), lysine (K), or asparagine (N) at amino acid position 148; f. Alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; an alanine (A), lysine (K), or arginine (R) at amino acid position 144; and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); g. Alanine (A) or Arginine (R) at amino acid position 12; and at amino acid position 101, an alanine (A), a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q), or a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q); h. alanine (A) or arginine (R) at amino acid position 12; and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); i. alanine (A) or arginine (R) at amino acid position 12; at amino acid position 101, an alanine (A), a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q), or a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q); and at amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); j. alanine (A) or arginine (R) at amino acid position 12; at amino acid position 101, an alanine (A), a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q), or a serine (S), a lysine (K), an arginine (R), an asparagine (N), or a glutamine (Q); an alanine (A), lysine (K), or arginine (R) at amino acid position 144; and At amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q).

[0094] In other specific embodiments, HFW1, HFW3 and HFW4 comprised of antibody variable domains of the invention have any one of the following substitutions (AHo numbering): a. Alanine (A), Lysine (K), Arginine (R), or Asparagine (N), or Lysine (K), Arginine (R), or Asparagine (N) at amino acid position 101; b. Lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; c. serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q), or lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; d. alanine (A) or lysine (K) at amino acid position 144; and leucine (L) or asparagine (N) at amino acid position 148; e. Lysine (K) at amino acid position 144; Glutamic acid (E) at amino acid position 146; and lysine (K) at amino acid position 148; f. serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; Alanine (A) or Lysine (K) at amino acid position 144; and Arginine (R) or glutamine (Q) at amino acid position 146; or g. Arginine (R) at amino acid position 12; and serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; h. Arginine (R) at amino acid position 12; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; i. Arginine (R) at amino acid position 12; serine (S), arginine (R) or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; j. alanine (A) at amino acid position 12; serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; Alanine (A) or Lysine (K) at amino acid position 144; and Arginine (R) or glutamine (Q) at amino acid position 146;

[0095] Suitably, the framework regions HFW1, HFW2, HFW3 and HFW4 comprised in the antibody variable domain of the present invention are selected from human VH frameworks. In particular, the framework regions HFW1, HFW2 and HFW3 comprised in the antibody variable domain of the present invention are selected from human VH framework subtypes VH1a, VH1b, VH3, VH4, VH5 or VH6, in particular from human VH framework subtypes VH1a, VH1b, VH3 or VH4. In a particular embodiment, the framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH framework subtype VH3. HFW4 may be selected from the HFW4 sequence of a human germline sequence or a rearranged human antibody sequence.

[0096] In the context of the present invention, the terms "belonging to a human VHx framework subtype (or human antibody Vκ / Vλ framework)", "selected from a human VHx framework subtype (or human antibody Vκ / Vλ framework)" or "being a human VHx framework subtype" mean that the framework sequences HFW1 to HFW3 (or LF1 to LFW3) show the greatest degree of homology to the consensus sequences of said human antibody VH or VL framework subtypes, as described in Knappik et al., J. Mol. Biol. 296 (2000) 57-86 or WO2019 / 057787. In the context of the present invention, sequences of human VH domains are grouped into seven different framework subtypes, namely framework subtypes VH1a, VH1b, VH2, VH3, VH4, VH5 and VH6, based on sequence homology to sequences shown in FIG. 3 of Knappik et al., J. Mol. Biol. 296 (2000) 57-86 or WO2019 / 057787, also referred to herein as human subfamilies VH1a, VH1b, VH2, VH3, VH4, VH5 and VH6. Specific examples of VH domains belonging to the VH3 framework subtype are represented by SEQ ID NOs: 157-170 (i.e., the non-italicized residues in Table 5). Specific examples of VH domains belonging to the VH1a, VH1b, VH4, VH5 and VH6 framework subtypes are represented by SEQ ID NOs: 171-175 (i.e., the non-italicized residues in Table 5). Alternative examples of VH1a, VH1b, VH3 and VH4 sequences, as well as other VHx sequences, can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86 or WO2019 / 057787.

[0097] In a particular embodiment, the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 of the antibody variable domain of the invention are selected from a combination of framework regions (i.e. non-italicized residues in Table 1 and Table 3, i.e. all residues not marked as CDR residues) of any one of SEQ ID NOs: 5-36 and 83-114, in particular any one of SEQ ID NOs: 8-36 and 86-114; and frameworks (i.e. non-italicized residues in Table 1 and Table 3) of variants of SEQ ID NOs: 5-36 and 83-114, in particular variants of SEQ ID NOs: 8-36 and 86-114, in which no more than 5 amino acids, in particular no more than 4 amino acids, in particular no more than 3 amino acids, in particular no more than 2 amino acids, in particular no more than 1 amino acid are mutated in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering). In this context, the term "mutation" means, as various non-limiting examples, addition, substitution or deletion. The VH region further comprises a VH domain comprising positions 5 to 140 (AHo numbering), in particular at least positions 3 to 145, more particularly at least positions 2 to 147, of at least one of the sequences set forth in SEQ ID NOs: 35 to 36 and 83 to 114, with the proviso that such a VH domain exhibits the functional characteristics defined above in items 22 and 23.

[0098] In certain embodiments, the variable light chain frameworks LFW1, LFW2 and LFW3 of an antibody variable domain of the invention are selected from human antibody Vκ frameworks (e.g., Vκ1, Vκ2, Vκ3 or Vκ4 framework subtypes) or human antibody Vλ frameworks (e.g., Vλ1, Vλ2 or Vλ3 framework subtypes), in particular human antibody Vκ frameworks. In the context of the present invention, the sequences of human VL domains are classified into four different human Vκ framework subtypes, namely framework subtypes Vκ1, Vκ2, Vκ3 and Vκ4, and three different human Vλ framework subtypes, namely framework subtypes Vλ1, Vλ2 and Vλ3, also referred to herein as human Vκ subfamilies Vκ1, Vκ2, Vκ3 and Vκ4, and human Vλ subfamilies Vλ1, Vλ2 and Vλ3, based on sequence homology to sequences shown in Figure 3 of Knappik et al., J. Mol. Biol. 296 (2000) 57-86 or WO2019 / 057787. In a particular embodiment, the variable light chain frameworks LFW1, LFW2 and LFW3 of the antibody variable domains of the invention are of the Vκ1 framework subtype. Specific examples of the Vκ1 framework subtype are represented by SEQ ID NOs: 176, 177, 178 and 179 (non-italicized residues in Table 5). Alternative examples of Vκ1 sequences, and examples of Vκ2, Vκ3 or Vκ4 sequences can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86.

[0099] In another particular embodiment, the variable light chain frameworks LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks, preferably Vκ1 framework subtype, and the variable light chain framework LFW4 is selected from Vλ frameworks. In a particular embodiment, the variable light chain framework LFW4 of an antibody variable domain of the invention is selected from the group consisting of the Vλ framework 4 sequences of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196. The Vλ framework 4 sequence of SEQ ID NO: 195 is particularly applicable when it comprises a single cysteine ​​residue at variable light chain (VL) position 144 (AHo numbering) and a second single cysteine ​​is present in the corresponding variable heavy chain (VH), especially at position 51 (AHo numbering) of the VH, for the formation of an interdomain disulfide bond.

[0100] In a particular embodiment, an antibody variable domain of the invention comprises a variable light chain (VL), wherein the variable light chain framework regions LFW1, LFW2 and LFW3 are selected from a human antibody Vκ framework, in particular a Vκ1 framework subtype, and said variable light chain framework region LFW4 is selected from a Vλ framework, and wherein said LFW3 comprises the following substitutions (AHo numbering): - glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101 has.

[0101] In other particular embodiments, LFW1, LFW2 and LFW3 are selected from a combination of framework regions LFW1, LFW2 and LFW3 (i.e. non-italicized residues in Table 5) of any one of SEQ ID NOs: 176, 177, 178 and 179; and a combination of framework regions LFW1, LFW2 and LFW3 (i.e. non-italicized residues in Table 5) of any one of SEQ ID NOs: 176, 177, 178 and 179, in which no more than 5 amino acids, particularly no more than 4 amino acids, particularly no more than 3 amino acids, particularly no more than 2 amino acids, particularly no more than 1 amino acid are mutated in the framework regions at positions different from 101 (AHo numbering); and said LFW3 optionally has a glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101.

[0102] In yet other specific embodiments, the variable light chain frameworks LFW1, LFW2, LFW3 and LFW4 of the antibody variable domain of the invention are selected from the group consisting of a combination of the framework regions LFW1, LFW2, LFW3 and LFW4 (i.e. the non-italized residues of Tables 1 and 3 and Table 5) of any one of SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 182, 183, 184, 185, 186 and 187; The combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i.e. the non-italicized residues in Tables 1, 3 and 5) of any one of 16, 117, 180, 181, 182, 183, 184, 185, 186 and 187, in which no more than 5 amino acids, particularly no more than 4 amino acids, particularly no more than 3 amino acids, particularly no more than 2 amino acids, particularly no more than 1 amino acid are mutated in the framework regions at positions different from 101 (AHo numbering). In this context, the term "mutation" refers to various non-limiting examples, additions, substitutions or deletions. The VL region further comprises a VL domain comprising positions 5 to 140 (AHo numbering), in particular at least positions 3 to 145, more in particular at least positions 2 to 147, of at least one of the sequences set forth in SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187, with the proviso that such a VL domain exhibits the functional characteristics defined above in items 22 and 23.

[0103] In a preferred embodiment, the antibody variable domain of the invention is in a format selected from an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a disulfide stabilized Fv fragment (dsFv); and a single chain Fv fragment (scFv). In a specific embodiment, the antibody variable domain of the invention is selected from an Fv fragment and a single chain Fv fragment (scFv). In another specific embodiment, the VL and VH domains of the scFv fragment are stabilized by an inter-domain disulfide bond, in particular said VH domain consists of a single cysteine ​​residue at position 51 (AHo numbering) and said VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0104] The antibody variable domains according to the invention, when in scFv format, exhibit reduced immunogenicity compared to versions of said antibody variable domains that do not comprise the above defined substitutions in the VH framework regions. More specifically, the antibody variable domains according to the invention, when in scFv format, exhibit reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum, in particular reduced binding to pre-existing ADA, compared to versions of said antibody variable domains that do not comprise the above defined substitutions in the VH framework regions, as determined in pre-existing ADA binding assays, in particular pre-existing ADA binding assays as described in Example 2.

[0105] Immunogenicity, i.e., the tendency of a therapeutic protein to induce an antibody response in a patient, can be predicted, for example, by its ability to be recognized by anti-drug antibodies (ADAs) already present in healthy, untreated human serum (referred to herein as "pre-existing ADAs").

[0106] Thus, for the purposes of the present invention, the term "immunogenicity" as used herein refers to the ability of a therapeutic protein, such as an antibody, antibody fragment, or antibody binding domain, to be recognized by pre-existing ADAs in a human serum sample. Without being bound by theory, it is believed that pre-existing ADA binding as well as the induction of ADA formation during treatment is associated with the development of B-cell and / or T-cell epitopes on the therapeutic protein. The degree of such immunogenicity can be determined by ELISA assay and expressed as the percentage or number of human serum samples containing a measurable amount of pre-existing ADAs and / or ADAs formed during treatment that recognize, i.e., bind, the therapeutic protein in question (percentage or number of positive serum samples) relative to the total number of human sera tested. The reduction in immunogenicity between a therapeutic protein and a corresponding therapeutic protein that is modified to reduce immunogenicity can be measured by comparing the percentage of positive serum samples for the modified therapeutic protein with the percentage of positive serum samples for the original therapeutic protein. A lower number or percentage of positive serum samples for the modified therapeutic protein indicates reduced immunogenicity compared to the original therapeutic protein.

[0107] A serum sample is determined to contain a measurable amount of pre-existing ADA when the ELISA signal exceeds a certain threshold. This threshold is also referred to herein as the screening cut point (SCP). The SCP can be calculated as defined below or set at any value relative to the maximum ELISA signal obtained for the test serum (e.g., 20%, 15%, 10% or 5% of the maximum ELISA signal obtained for the test serum). Preferably, the SCP is calculated as defined below.

[0108] The antibody variable domains of the invention, when in scFv format, further have advantageous biophysical properties, in particular good stability. Advantageously, the antibody variable domains of the invention, when in scFv format, are further characterized by one or more of the following features: a. a melting temperature (Tm) when formulated in 20 mM histidine, pH 6.0, as determined by differential scanning fluorimetry (DSF), of at least 63°C, particularly at least 64°C, particularly at least 65°C; b. when formulated at a concentration of 10 mg / ml in 20 mM histidine at pH 6.0, the loss in monomer content after storage for 28 days at 4° C. is less than 5%, particularly less than 3%, particularly less than 2%; c) when formulated at a concentration of 10 mg / ml in 20 mM histidine at pH 6.0, the loss in monomer content after storage for 28 days at 40° C. is less than 15%, particularly less than 10%, particularly less than 8%; d. When formulated at a concentration of 10 mg / ml in 20 mM histidine, pH 6.0, the protein content loss is less than 15%, specifically less than 10%, after storage for 28 days at 4°C; e. when formulated at a concentration of 10 mg / ml in 20 mM histidine, pH 6.0, the loss of protein content is less than 10%, specifically less than 5%, after storage for 28 days at 40°C; f. binds the target antigen with a dissociation constant (KD) that is three-fold lower or no more than three-fold higher than the KD of a version of said antibody variable domain that does not contain the substitution defined in paragraph 1, as measured by surface plasmon resonance (SPR).

[0109] DSF has been previously described (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221). The midpoint of the thermal unfolding transition of scFv constructs is determined by nano-differential scanning fluorimetry as described in detail in Example 4. Briefly, 1±0.1 mg / ml and 10±1 mg / ml solutions in 20 mM histidine buffer (pH 6) are prepared and heated from 20° C. to 95° C. at 1° C. / min. The unfolding events are monitored using the change in the intrinsic fluorescence of the protein, i.e., the fluorescence emission spectrum of tryptophan (Trp). The unfolding midpoint (Tm) is defined as the inflection point (thermal midpoint) of the unfolding curve observed as a local maximum or minimum of the first derivative.

[0110] The loss of monomer content is determined by area under the curve calculation of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, as outlined in the United States Pharmacopeia (USP) Chapter 621. This method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. The separation of molecules is determined by the void volume (V0) and total permeate volume (V) of a particular column. T ) The monomer content of the scFv constructs was measured using SE-HPLC with a Shodex 402.5-4F KW column. 5 μg of scFv molecules at a given sample concentration, e.g., about 1 mg / ml or about 10 mg / ml, is injected for analysis. The monomer content is then determined by integrating the monomer peak of the scFv. A standard running buffer consisting of 50 mM sodium acetate and 250 mM sodium chloride at pH 6.0 is used for the analysis.

[0111] Furthermore, the inventors have found that the antibody variable domains of the invention maintain their binding affinity to the respective target antigens compared to the unsubstituted versions. In this respect, the expression "maintains binding affinity" means that the monovalent dissociation constant (KD) for binding of the antibody variable domains of the invention to the target antigen, as measured by surface plasmon resonance (SPR), is equal to or lower than the KD of the respective unsubstituted version (i.e., the KD of the unsubstituted version maintains the binding affinity of the antibody variable domains of the invention), i.e., equal to or lower than the KD of the respective unsubstituted version (i.e., the version of the antibody variable domain that does not contain the substitutions defined herein), or is not more than three times higher than the KD of the respective unsubstituted version (i.e., the version of the antibody variable domain that does not contain the substitutions defined herein). The determination of the individual KD values ​​by SPR is described in detail in Example 3.

[0112] As used herein, the term "affinity" refers to the strength of interaction between an antibody or antibody variable domain and an antigen at a single antigenic site. At each antigenic site, the variable region of the antibody variable domain or antibody "arm" interacts with the antigen at multiple sites through weak non-covalent forces.

[0113] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antibody variable domain) and a binding partner (e.g., an antigen or, more specifically, an epitope on an antigen). Unless otherwise indicated, "binding affinity," "bind," or "binding," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody variable domain and an antigen). The affinity of a molecule X for a partner Y is generally measured by the dissociation constant (K D) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies and antibody variable domains generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific exemplary embodiments for measuring binding affinity, i.e., binding strength, are described below.

[0114] The term “K assoc ", "K a " or "K on " as used herein is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "K dis ", "K d " or "K off " as used herein is intended to refer to the off-rate of a particular antibody-antigen interaction. In one embodiment, the term "K D " as used herein is intended to refer to the dissociation constant, which is K d and K. a (i.e., K d / K a ) and expressed as molar concentration (M). D " or "K D "value" or "KD" or "KD value" is, in one embodiment, measured using a surface plasmon resonance assay.

[0115] In another aspect, the invention relates to an antibody comprising one or more antibody variable domains of the invention, said one or more antibody variable domains independently of each other selected from Fv, dsFv, scFv and disulfide-stabilized scFv.

[0116] In certain embodiments, the antibody of the invention further comprises an antibody variable domain that is different from the antibody variable domain of the invention. More specifically, the antibody of the invention further comprises an antibody variable domain that does not have a substitution of a framework region as defined herein.

[0117] In preferred embodiments, the antibodies of the invention comprise exclusively antibody variable domains of the invention. More specifically, in these preferred embodiments, the antibodies of the invention comprise only antibody variable domains having substitutions in the framework regions as defined herein.

[0118] The term "monovalent antibody" or "an antibody that is monovalent for a target antigen" as used herein refers to an antibody that binds to a single target molecule, more specifically, to a single epitope on a target molecule, and the term "binding domain" or "monovalent binding domain" as used herein refers to a binding domain that binds to a single epitope on a target molecule.

[0119] Furthermore, the terms "binding domain" of an antibody, or "antigen-binding fragment" or "antigen-binding portion" of an antibody, etc., as used herein refer to one or more portions of an intact antibody that have the ability to bind to a given antigen, in particular to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Specifically, in the case of the antibodies of the present invention, the term "binding domain" or the term "antigen-binding fragment" or "antigen-binding portion" etc., as used herein, refers to a Fab fragment, i.e., a monovalent fragment, i.e., a monovalent fragment consisting of the VL, VH, CL and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a disulfide-stabilized Fv fragment (dsFv); and a single-chain Fv fragment (scFv). Preferably, the binding domains of the antibodies of the present invention are independently selected from Fv fragments, disulfide-stabilized Fv fragments (dsFv) and single-chain Fv fragments (scFv). In certain embodiments, the binding domains of an antibody of the invention are independently selected from an Fv fragment and a single chain Fv fragment (scFv). In other particular embodiments, the VL and VH domains of the scFv fragment are stabilized by an interdomain disulfide bond, in particular said VH domain comprises a single cysteine ​​residue at position 51 (AHo numbering) and said VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0120] As used herein, the term "bivalent antibody" or "antibody that is bivalent for a target antigen" refers to a single antibody that has two valencies, where "valency" is described as the number of antigen binding sites that bind to an epitope on a particular target molecule. Thus, a single antibody can bind to two binding sites on a target molecule and / or two target molecules due to the presence of two copies of the corresponding antigen binding site.

[0121] Similarly, the term "trivalent antibody" or "an antibody that is trivalent for a target antigen" as used herein refers to a single antibody that has three valencies. Thus, a single antibody can bind to three binding sites on a target molecule and / or can bind up to three target molecules due to the presence of three copies of the corresponding antigen-binding moiety.

[0122] When an antibody of the invention comprises two or three binding domains, said two or three binding domains bind either the same epitope or different epitopes on the target molecule. Preferably, the two or three binding domains bind to the same epitope on the target molecule.

[0123] The term "identical epitope" as used herein refers to an individual protein determinant on a protein capable of specific binding to more than one antibody, which individual protein determinant is identical, i.e., composed of the same chemically active surface groupings of molecules such as amino acids or sugar side chains having the same three-dimensional structural characteristics and the same charge characteristics for each of said antibodies.

[0124] The term "distinct epitopes" as used herein in reference to a particular protein target refers to individual protein determinants on the protein, each capable of specific binding to a different antibody, and these individual protein determinants are not identical to the different antibodies, i.e., are composed of non-identical chemically active surface groupings of molecules such as amino acids or sugar side chains that have different three-dimensional structural characteristics and different charge characteristics. These different epitopes may or may not overlap.

[0125] In one group of embodiments, the antibody format is selected from a bivalent bispecific IgG format, a trivalent bispecific IgG format and a tetravalent bispecific IgG format, said formats comprising one or more Fvs, scFvs or disulfide-stabilized scFvs. In particular, the antibody format is selected from KiH-based IgG, such as DuoBodies (Bispecific IgG prepared by Duobody Technology) (MAbs. 2017 Feb / Mar;9(2):182-212. doi: 10.1080 / 19420862.2016.1268307); DVD-Ig; IgG-scFv fusions, such as CODV-IgG, Morrison (IgG CH3-scFv fusions (Morrison-H) or IgG These include CL-scFv fusion (Morrison-L), bsAb (scFv linked to the C-terminus of the light chain), Bs1Ab (scFv linked to the N-terminus of the light chain), Bs2Ab (scFv linked to the N-terminus of the heavy chain), Bs3Ab (scFv linked to the C-terminus of the heavy chain), Ts1Ab (scFv linked to the N-terminus of both the heavy and light chains) and Ts2Ab (dsscFv linked to the C-terminus of the heavy chain). More particularly, the antibody format is selected from KiH-based IgG, e.g., DuoBodies; DVD-Ig; CODV-IgG and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), and even more particularly, DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).

[0126] In this group of embodiments, the IgG is preferably selected from the IgG subclasses IgG1 and IgG4, in particular IgG4.

[0127] In a particular embodiment, the antibody format is selected from the Morrison format, i.e., Morrison-L and Morrison-H formats. The Morrison-L and Morrison-H formats used in the present invention are tetravalent and bispecific molecular formats carrying an IgG Fc region, in particular an IgG4 Fc region. The light chain is composed of Vκ FR1-FR3 in combination with Vλ FR4, and two highly stable scFv binding domains based on the antibody variable domains of the present invention are fused to the C-terminus of the heavy chain (Morrison-H) or light chain (Morrison-L) via the linker L1.

[0128] The linker L1 is a peptide of 2 to 30 amino acids, more particularly 5 to 25 amino acids, and most particularly 10 to 20 amino acids. In a particular embodiment, the linker L1 is a peptide of 4 glycine amino acid residues and 1 serine amino acid residue (GGGGS). n (n=1, 2, 3, 4 or 5) (SEQ ID NO: 197), in particular one or more units of n=2 (SEQ ID NO: 198).

[0129] The VH and VL regions of the two scFv domains are linked by a linker L2. The linker L2 is a peptide of 10 to 40 amino acids, more particularly 15 to 30 amino acids, and most particularly 20 to 25 amino acids. In particular, the linker L2 is a peptide of 4 glycine amino acid residues and 1 serine amino acid residue (GGGGS). n (n=1, 2, 3, 4, 5, 6, 7 or 8) (SEQ ID NO: 199), in particular one or more units of n=4 (SEQ ID NO: 200).

[0130] In another group of embodiments, the antibodies of the invention do not comprise an immunoglobulin Fc region.

[0131] The term "immunoglobulin Fc region" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, i.e., the CH2 and CH3 domains of the heavy chain constant region. The term "Fc region" includes native sequence Fc regions and variant Fc regions, i.e., Fc regions that have been engineered to exhibit certain desired properties, such as, for example, altered Fc receptor binding function and / or reduced or inhibited Fab arm exchange. An example of such an engineered Fc region is the knobs-into-holes (KiH) technology (e.g., Ridgway et al., Protein Eng. 9:617-21 (1996) and Spiess et al., J Biol Chem. 288(37):26583-93 (2013)). Native sequence Fc regions include human lgG1, lgG2 (lgG2A, IgG2B), lgG3, and lgG4. "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In particular, FcR is a native sequence human FcR that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcyRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRI IB ("inhibiting receptors"), which have similar amino acid sequences that differ primarily in the cytoplasmic domain. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 5:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capet et al, Immunomethods 4: 25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. TJI (8): 6213-6 (2004); WO 2004 / 92219 (Hinton et al)). The in vivo binding to FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having mutant Fc regions. WO2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0132] In this group of embodiments, the antibody is preferably in a format selected from the group consisting of: tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, di-diabody, tandem-di-scFv, and MATCH (WO2016 / 0202457; described in Egan T., et al., MABS 9 (2017) 68-84).

[0133] In certain embodiments, the antibodies of the invention do not further comprise a CH1 and / or CL region. In these particular embodiments, the antibodies are in scDb-scFv, triabody, tetrabody or MATCH format, in particular in MATCH or scDb-scFv format. More particularly, the antibodies of the invention are in MATCH3 or MATCH4 format.

[0134] In a specific embodiment, the antibodies of the invention are trispecific and tetravalent.

[0135] In a more specific embodiment, the antibodies of the invention are trispecific and trivalent.

[0136] The antibody variable domains contained in a bi-, tri-, tetra- or penta-specific antibody of the invention can bind simultaneously to their respective antigens or receptors. The term "simultaneously" as used in this context means that one of the antibody variable domains specifically binding to, for example, MSLN and one or two of the antibody variable domains specific for, for example, CD3 and hSA bind simultaneously.

[0137] Suitably, the antibody variable domains comprised in a diabody, triabody, tetrabody or pentabody of the invention are operably linked.

[0138] The term "operably linked" as used herein indicates that two molecules (e.g., polypeptides, domains, binding domains) are linked in a manner that retains functional activity of each molecule. Two molecules may be "operably linked" whether they are directly or indirectly linked (e.g., via a linker, via a moiety, via a linker to a moiety). The term "linker" refers to a peptide or other moiety that is optionally placed between a binding domain or an antibody variable domain used in the present invention. Many strategies can be used to covalently link molecules together. These include, but are not limited to, a polypeptide linkage between the N-terminus and C-terminus of a protein or protein domain, linkage via a disulfide bond, and linkage via a chemical cross-linking reagent. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant techniques or peptide synthesis. Choosing an appropriate linker in a particular case where two polypeptide chains are to be linked depends on various parameters, including, but not limited to, the nature of the two polypeptide chains (e.g., whether they naturally oligomerize), the distance between the N-terminus and C-terminus to be linked, if known, and / or the stability of the linker against proteolysis and oxidation. Additionally, the linker may contain amino acid residues that provide flexibility.

[0139] In the context of the present invention, the term "polypeptide linker" refers to a linker consisting of a chain of amino acid residues linked by peptide bonds, which link two domains, each domain being attached to one end of the linker. The polypeptide linker should be of sufficient length to link two molecules in the correct conformation relative to each other so that they retain the desired activity. In certain embodiments, the polypeptide linker has a continuous chain of 2-30 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues). Furthermore, the amino acid residues selected for inclusion in the polypeptide linker should exhibit properties that do not significantly interfere with the activity of the polypeptide. Thus, the linker peptide, taken as a whole, should not exhibit charges that are inconsistent with the activity of the polypeptide, should not interfere with internal folding, and should not form bonds or other interactions with amino acid residues in one or more monomers that significantly impair binding of the receptor monomer domains. In certain embodiments, the polypeptide linker is an unstructured polypeptide. Useful linkers include glycine-serine, or GS linkers. A "Gly-Ser" or "GS" linker refers to a polymer of tandem glycine and serine (e.g., (Gly-Ser) n , (GSGGS) n (SEQ ID NO: 201), (GGGGS) n (SEQ ID NO: 202) and (GGGS) n(SEQ ID NO:203), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether of the Shaker potassium channel, and a wide variety of other flexible linkers as will be appreciated by those skilled in the art. Glycine-serine polymers are preferred because oligopeptides containing these amino acids are relatively unstructured and can therefore function as neutral tethers between components. Secondly, serine is hydrophilic, which can solubilize globular glycine chains. Thirdly, similar chains have been shown to be effective in linking subunits of recombinant proteins such as single chain antibodies.

[0140] Suitably, the antibody variable domain of the invention is an isolated variable domain. Similarly, the antibody of the invention is an isolated antibody. As used herein, the term "isolated variable domain" or "isolated antibody" refers to a variable domain or antibody that is substantially free of other variable domains or other antibodies with different antigen specificity (e.g., an isolated antibody variable domain that specifically binds mesothelin is substantially free of antibody variable domains that specifically bind antigens other than mesothelin). Furthermore, an isolated antibody variable domain or isolated antibody may be substantially free of other cellular material and / or chemicals.

[0141] Suitably, the antibody variable domains and antibodies of the invention are monoclonal antibody variable domains and antibodies. As used herein, the term "monoclonal antibody variable domain" or "monoclonal antibody" refers to variable domains or antibodies having substantially identical amino acid sequences or derived from the same genetic source. Monoclonal variable domains or antibodies exhibit binding specificity and affinity for a particular epitope or binding specificity and affinity for a particular epitope.

[0142] Antibody variable domains and antibodies of the present invention include, but are not limited to, chimeric antibodies, human antibodies, and humanized antibody variable domains and antibodies.

[0143] The term "chimeric antibody" or "chimeric antibody variable domain" as used herein refers to (a) an antibody molecule or antibody variable domain in which the constant region or a portion thereof has been altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species; or (b) an antibody molecule or antibody variable domain in which the variable region or a portion thereof has been altered, replaced, or exchanged with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a human immunoglobulin constant region. By replacing with a human constant region, the chimeric antibody can retain the specificity of recognizing the antigen while reducing its antigenicity in humans compared to the original mouse antibody.

[0144] The term "human antibody" or "human antibody variable domain" as used herein is intended to include antibodies or antibody variable domains having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody or antibody variable domain comprises a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence. The human antibodies and antibody variable domains of the invention may contain amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). This definition of a human antibody or antibody variable domain specifically excludes humanized antibodies or antibody variable domains that contain non-human antigen-binding residues. Human antibodies and antibody variable domains can be generated using a variety of techniques known in the art, such as phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1992); Marks et al, J. Mol. Biol, 222:581 (1991)). Human monoclonal antibodies and human monoclonal antibody variable domains can also be prepared using the methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al, J. Immunol, 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies and human antibody variable domains can be prepared by administering antigen to transgenic animals (e.g., immunized xeno-mice) that have been engineered to produce such antibodies and antibody variable domains in response to antigenic challenge, but in which the endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for ENOMOUSE™ technology).Also see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557- 3562 (2006) regarding human antibodies produced by human B cell hybridoma technology.

[0145] The term "humanized" antibody or "humanized" antibody variable domain as used herein refers to an antibody or antibody variable domain that retains the reactivity of a non-human antibody or antibody variable domain, but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody or antibody variable domain with its human counterparts (i.e., the constant regions as well as the framework portions of the variable regions). Further framework region modifications can be made not only within the human framework sequences, but also within the CDR sequences derived from the germline of other mammalian species. The humanized antibodies and antibody variable domains of the invention may contain amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or conservative substitutions to facilitate stability or manufacture). See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239: 1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31: 169-217, 1994. Other examples of human engineering techniques include, but are not limited to, the Xoma technology disclosed in US 5,766,886.

[0146] The term "recombinant humanized antibody" or "recombinant humanized antibody variable domain" as used herein includes all human antibodies and human antibody variable domains that are prepared, expressed, produced or isolated by recombinant means, such as antibodies and antibody variable domains isolated from a transfectoma, from a host cell that has been transformed to express the humanized antibody or humanized antibody variable domain, as well as antibodies and antibody variable domains that are prepared, expressed, produced or isolated by any other means that involve splicing all or part of the sequence of a human immunoglobulin gene to other DNA sequences.

[0147] Preferably, the antibody variable domains and antibodies of the invention are humanized, more preferably, the antibody variable domains and antibodies of the invention are humanized and comprise CDRs of rabbit origin.

[0148] As used herein, the terms "bispecific antibody", "trispecific antibody", "tetraspecific antibody", "pentaspecific antibody" or the more general term "multispecific antibody" refer to an antibody that binds to two or more different epitopes on at least two or more different targets, such as two different targets (bispecific), three different targets (trispecific), four different targets (tetraspecific) or five different targets (pentaspecific). Preferably, the antibodies of the invention are bispecific, trispecific or tetraspecific, particularly bispecific or trispecific, more particularly trispecific. As indicated above, the term trispecific antibody refers to an antibody that binds to at least three different epitopes on three different targets (e.g. mesothelin, CD3 and hSA, or PD-L1, CD137 and hSA).

[0149] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface substrates of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics as well as specific charge characteristics. "Conformational" epitopes are distinguished from "linear" epitopes in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0150] As used herein, the term "conformational epitope" refers to amino acid residues of an antigen that come together on the surface when the polypeptide chain folds to form the native protein.

[0151] The term "linear epitope" refers to an epitope whose points of interaction with a protein-interacting molecule (such as an antibody) all occur linearly (continuously) along the primary amino acid sequence of the protein.

[0152] As used herein, the term "recognize" refers to an antibody or antibody variable domain that finds and interacts with (eg, binds to) that conformational epitope.

[0153] The inventors have found that the antibody variable domains of the invention are successfully applied in the construction of a variety of antibody fragments, such as scFv fragments, and multispecific antibodies, such as bi- and tri-specific antibodies, exhibiting significantly reduced immunogenicity and superior stability compared to their unmodified counterparts.

[0154] The antibody variable domains and antibodies of the invention may be produced using any convenient antibody production method known in the art (for example, for production of bispecific constructs see Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14; for bispecific diabodies and tandem scFvs see Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907 (2012)713-727, and WO 99 / 57150 and WO99 / 57150). Further examples of suitable methods for the preparation of multispecific constructs include, inter alia, the Genmab technology (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150), the Merus technology (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750).

[0155] These methods typically involve the production of monoclonal antibodies or monoclonal antibody variable domains, for example, by fusing myeloma cells with spleen cells from mice immunized with the desired antigen using hybridoma technology (see, e.g., Yokoyama et al., Curr. Protoc. Immunol. Chapter 2, Unit 2.5, 2006), or by recombinant antibody engineering (repertoire cloning or phage display / yeast display) (see, e.g., Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8). Antigen binding domains or fragments or parts thereof of two or more different monoclonal antibodies can be combined to obtain bispecific or multispecific constructs using known molecular cloning techniques.

[0156] Multispecific, e.g., bispecific, trispecific, tetraspecific or pentaspecific, and / or multivalent, antibodies of the invention can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the antibodies can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Exemplary cross-linkers include protein A, carbodiimide, N-succinimidyl-5-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160: 1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229:81-83, and Glennie et al., 1987 J. Immunol. 139: 2367-2375. Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0157] Alternatively, two or more binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x Fab, mAb x scFv, mAb x dsFv or mAb x Fv fusion protein. Methods for preparing multispecific and / or multivalent antibodies and molecules are described, for example, in US 5,260,203; US 5,455,030; US 4,881,175; US 5,132,405; US 5,091,513; US 5,476,786; US 5,013,653; US 5,258,498; and US 5,482,858.

[0158] Binding of antibody variable domains and multispecific antibodies to specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0159] In a further aspect, the present invention provides a nucleic acid or two nucleic acids encoding an antibody variable domain or an antibody of the invention. Such nucleic acids may be optimized for expression in mammalian cells.

[0160] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide(s)" and refers to one or more deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses the sequence explicitly set forth, as well as its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, as described in more detail below, degenerate codon substitutions can be achieved by generating sequences in which the 3-position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0161] The present invention provides substantially purified nucleic acid molecules encoding polypeptides comprising the above-described antibody variable domains or antibody segments or domains, and when expressed from a suitable expression vector, the polypeptides encoded by these nucleic acid molecules are capable of exhibiting the antigen-binding ability of the antibody variable domains or antibodies of the present invention.

[0162] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences (e.g., sequences as described in the Examples below) encoding antibody variable domains or antibodies of the invention. Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109, 1979; the diethyl phosphoramidite method of Beaucage et al., Tetrahedron Lett., 22: 1859, 1981; and the solid support method of US 4,458,066. Mutations can be introduced into polynucleotide sequences by PCR, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0163] Also provided by the invention are expression vectors and host cells for producing the antibody variable domains or antibodies of the invention.

[0164] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having an origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0165] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which perform equivalent functions. In this particular context, the term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence. For example, a promoter sequence or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a transcriptional sequence is physically contiguous with the transcriptional sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0166] A variety of expression vectors can be used to express polynucleotides encoding antibody variable domains or antibody chain(s). Both viral-based and non-viral expression vectors can be used to produce antibodies or antibody variable domains in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors that typically carry expression cassettes for expressing proteins or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). For example, non-viral vectors useful for expressing PD-L1- or MSLN-binding polypeptides, or polynucleotides encoding such polypeptides, in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C, (Invitrogen, San Diego, Calif.), MPS V vectors, as well as numerous other vectors known in the art for expressing other proteins. Useful viral vectors include retrovirus-based vectors, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papilloma viruses, HBP Epstein-Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0167] The choice of expression vector depends on the host cell in which the vector is expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the multispecific antibody chain or variable domain. In one embodiment, an inducible promoter is employed to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population of coding sequences whose expression products are tolerated by the host cell. In addition to promoters, other regulatory elements may be necessary or desirable for efficient expression of the multispecific antibody chain or variable domain. These elements usually include an ATG initiation codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by including enhancers appropriate for the cell system used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0168] The vectors used usually encode the antibody variable domains or the antibody light and heavy chains, including the constant regions or parts thereof, if present. Such vectors express the variable regions as fusion proteins with the constant regions, thereby allowing the production of intact antibodies and their antibody variable domains. Typically, such constant regions are human.

[0169] The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in the progeny, either due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0170] Host cells for carrying and expressing the antibody variable domains or antibodies of the invention can be either prokaryotic or eukaryotic. Escherichia coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the invention. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be made, which typically contain expression control sequences (e.g., an origin of replication) compatible with the host cell. In addition, any number of different well-known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or a promoter system from phage lambda. The promoters usually control expression, optionally have operator sequences, and include ribosome binding site sequences for initiating and completing transcription and translation. Other microbes, such as yeast, can also be used to express the antibody variable domains or multispecific antibodies of the invention. Insect cells in combination with baculovirus vectors can also be used.

[0171] In one embodiment, mammalian host cells are used to express and produce the antibody variable domains or antibodies of the invention. For example, they can be either hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These include normal mortal cells, normal or abnormal immortal animal or human cells. A number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including, for example, CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally reviewed, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences such as origins of replication, promoters, enhancers (see, e.g., Queen, et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, transcription terminator sequences, etc. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific, and / or regulatable or controllable. Useful promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone inducible MMTV promoter, SV40 promoter, MRP polIII promoter, constitutive MPS V promoter, tetracycline inducible CMV promoter (such as human immediate early CMV promoter), constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0172] Methods for introducing expression vectors containing a polynucleotide sequence of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation is used for other cellular hosts (see generally Green, MR, and Sambrook, J., Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012)). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation-nucleic acid conjugates, naked DNA, artificial virions, fusion with herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced DNA uptake, and ex vivo introduction. Stable expression is often desired for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing the antibody variable domain or antibody of the invention can be prepared using an expression vector of the invention comprising a viral origin of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, the cells can be grown in enriched medium for 1-2 days before switching to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows cells that successfully express the introduced sequence to be grown in selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type. Thus, the invention provides a method of producing an antibody variable domain or an antibody of the invention, the method comprising the step of culturing a host cell comprising a nucleic acid or vector encoding the antibody variable domain or antibody of the invention, whereby the antibody variable domain or antibody of the disclosure is expressed.

[0173] In one aspect, the invention relates to a method for producing an antibody variable domain or an antibody of the invention, comprising the step of culturing a host cell expressing a nucleic acid or two nucleic acids encoding the antibody variable domain or antibody of the invention. In particular, the invention relates to a method for producing an antibody variable domain or an antibody of the invention, comprising the steps of (i) providing a nucleic acid or two nucleic acids encoding the antibody variable domain or antibody of the invention, or one or two vectors encoding the antibody variable domain or antibody of the invention, expressing said nucleic acid or said vector and recovering said antibody variable domain or said antibody from the expression system, or (ii) providing a host cell expressing a nucleic acid or two nucleic acids encoding the antibody variable domain or antibody of the invention, culturing said host cell and collecting said antibody variable domain or said multispecific antibody from the cell culture.

[0174] In a further aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a medium or diluent that does not interfere with the structure of the antibody. The pharma- ceutically acceptable carrier enhances or stabilizes the composition or facilitates the preparation of the composition. The pharma- ceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents.

[0175] Some of such carriers allow the pharmaceutical composition to be formulated for oral ingestion by a subject, for example, as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges. Some of such carriers allow the pharmaceutical composition to be formulated for injection, infusion, or topical administration. For example, a pharma- ceutically acceptable carrier can be a sterile aqueous solution.

[0176] The pharmaceutical compositions according to the present disclosure may further routinely contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, adjuvants, and supplementary immune enhancing agents such as cytokines, and optionally other therapeutic agents. The compositions may also contain antioxidants and / or preservatives. Antioxidants may include thiol derivatives (e.g., thioglycerol, cysteine, acetylcysteine, cystine, dithioerythritol, dithiothreitol, glutathione), tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, sulfites (e.g., sodium sulfate, sodium bisphenol sulfate, sodium bisulfite, sodium acetone bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate), and nordihydroguaiaretic acid. Suitable preservatives include, for example, phenol, chlorobutanol, benzyl alcohol, methylparaben, propylparaben, benzalkonium chloride, and cetylpyridinium chloride.

[0177] The pharmaceutical composition of the present invention can be administered by various methods known in the art. The route and / or mode of administration varies depending on the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or can be administered proximal to the target site. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody of the present invention, can be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0178] The pharmaceutical composition of the present invention can be prepared according to methods well known and routinely practiced in the art. For example, see Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition is preferably manufactured under GMP conditions. Typically, a therapeutically effective amount or effective amount of the antibody of the present invention is employed in the pharmaceutical composition of the present invention. The antibody of the present invention is formulated into a pharma- ceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0179] The actual dosage of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and is not toxic to the patient. The dosage selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, or its ester, salt, or amide, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors.

[0180] In one aspect, the present invention relates to an antibody of the invention or a pharmaceutical composition of the invention for use as a medicament. In a preferred embodiment, the present invention provides a multispecific antibody or a pharmaceutical composition for use in the treatment of a proliferative disease such as cancer, or a disease selected from an allergic disease, an inflammatory disease and an autoimmune disease.

[0181] In another aspect, the present invention provides a pharmaceutical composition of the invention for use in the manufacture of a medicament for the treatment of a proliferative disease such as cancer, or a disease selected from an allergic disease, an inflammatory disease and an autoimmune disease.

[0182] In another aspect, the invention relates to the use of the antibody or pharmaceutical composition of the invention for treating a proliferative disease, such as cancer, or a disease selected from an allergic disease, an inflammatory disease and an autoimmune disease in a subject in need thereof.

[0183] In another aspect, the present invention relates to a method for treating a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. In a preferred embodiment, the present invention relates to a method for treating a proliferative disease, such as cancer, or a disease selected from an allergic disease, an inflammatory disease and an autoimmune disease, in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention.

[0184] The term "subject" includes human and non-human animals.

[0185] The term "animal" includes all vertebrates, e.g., non-human mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. As used herein, the terms "patient" and "subject" are used interchangeably, unless otherwise noted.

[0186] As used herein, the terms "treatment," "treating," "treat," "treated," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of a partial or complete cure of a disease and / or side effects resulting from a disease, or a delay in the progression of a disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, e.g., a human, and includes (a) inhibiting the disease, i.e., arresting its onset; and (b) relieving the disease, i.e., causing regression of the disease.

[0187] The term "therapeutically effective amount" or "effective amount" refers to the amount of a drug that, when administered to a mammal or other subject for treating a disease, is sufficient to affect treatment for the disease. A "therapeutically effective amount" will vary depending on the drug, the disease and its severity, the age, weight, etc., of the subject to be treated.

[0188] In another aspect, the present invention relates to a method of modifying an antibody, wherein the antibody is a fragment-based or comprises one or more scFv fragments, comprising making the following substitutions (AHo numbering) in the VH sequence(s) of said fragment-based antibody or in the VH sequence(s) of an scFv fragment of said antibody: - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101, or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q); - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; - leucine (L), lysine (K) or asparagine (N) at amino acid position 148 to obtain a modified antibody, Here, the modified antibodies exhibit reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum of healthy donors when compared to the unmodified version, as determined by an ELISA-based pre-existing anti-drug antibody binding assay.

[0189] In a particular embodiment of said last aspect, said modified antibody comprises an antibody variable domain according to the invention, i.e. as defined in the claims, items 1 to 24 or in the detailed description of the invention.

[0190] Partial sequence listing (residues are designated according to the AHo numbering scheme; CDRs are defined according to Numab CDR definitions unless otherwise specified)

[0191] [Table 1-1]

[0192] [Table 1-2]

[0193] [Table 1-3]

[0194] [Table 2-1]

[0195] [Table 2-2]

[0196] [Table 2-3]

[0197] [Table 2-4]

[0198]

Table 2-5

[0199]

Table 3-1

[0200]

Table 3-2

[0201]

Table 3-3

[0202]

Table 4-1

[0203]

Table 4-2

[0204]

Table 4-3

[0205]

Table 4-4

[0206]

Table 4-5

[0207]

Table 5-1

[0208]

Table 5-2

[0209]

Table 5-3

[0210]

Table 6-1

[0211]

Table 6-2

[0212]

Table 6-3

[0213]

Table 6-4

[0214]

Table 6-5

[0215]

Table 7-1

[0216]

Table 7-2

[0217]

Table 7-3

[0218] Throughout this application, in the event of any conflict between the text of the present specification (eg, Tables 1 to 7) and the Sequence Listing, the text of the present specification shall control.

[0219] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the present invention are specifically embraced by the present invention and are disclosed herein as if each combination was individually and explicitly disclosed herein. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0220] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0221] To the extent possible under each patent law, all patents, applications, publications, test methods, literature, and other materials cited herein are incorporated herein by reference.

[0222] The following examples are illustrative of the invention described above, but are not intended to limit the scope of the invention. Other test models known to those skilled in the relevant art as such may also determine the beneficial effects of the claimed invention. EXAMPLES

[0223] Example 1: Production of scFv variants and reference scFvs according to the invention 1.1. Production of PRO1922 variants (MSLN-binding scFv) and PRO2230 variants (PD-L1-binding scFv): PRO1922, PRO2230, the variants of PRO1922 and PRO2230 of the present invention and the respective references were prepared according to the methods described in detail in patent applications WO2019 / 072868 and WO2021 / 239987.

[0224] Briefly, expression of PRO1922 and PRO2230 variants and respective references as defined herein was performed in CHO cells using the ExpiCHO Expression System (ThermoFisher). Expression was performed according to the manufacturer's instructions. Proteins were purified from the clarified harvest by affinity chromatography (Protein L and / or Protein A). If necessary, variant scFvs were purified by SE-chromatography to a final monomer content of 95% or higher. Standard analytical methods such as ESI-MS, SE-HPLC, UV280 and SDS-PAGE were applied for quality control of the produced material.

[0225] The mass of scFvs was verified by, for example, the following ESI-MS standard method. The produced scFvs were diluted 5-fold with 1% TFA. 2 μl of the sample was injected onto an ACQUITY UPLC@BioResolve-RP-mAb 2.7 μm 2.1×150 mm, 450 Å column (Waters, USA) and desalted using a gradient from 15% to 85% buffer B (0.1% formic acid, 25% propan-2-ol in acetonitrile) at 50° C. with a flow rate of 200 μl / min. MS analysis was performed on a Synapt G2 mass spectrometer directly coupled to a UPLC station. Mass spectra were acquired in positive ion mode, scanning the m / z range from 400 to 5000 da with a scan time of 1 s and an interscan delay of 0.1 s. Data were recorded with MassLynx 4.2 software (Waters, UK). When possible, single peak m / z data were deconvoluted into mass spectra applying the maximum entropy algorithm MaxEnt1 (MaxLynx).

[0226] The monomer content of the produced scFv was measured, for example, using the following standard SE-HPLC method. 5 μg of each scFv, typically present at a concentration of 0.1-10 mg / ml, was injected onto a Shodex KW402.5-4F column using a Hitachi Chromaster HPLC system at 25° C. with a flow rate of 0.35 ml / min. The mobile phase was 50 mM sodium acetate, 250 mM sodium chloride in water at pH 6.0. The elution profile was monitored at 280 nm.

[0227] PRO1922, PRO2230, the variants of PRO1922 and PRO2230 of the present invention, and the respective references generated are summarized in Table 8.

[0228] Example 2: Existing ADA Binding Assay 2.1. General measurement procedure: Numab has developed a method to detect pre-existing anti-drug antibodies in human serum using a direct assay.

[0229] 96-well half plates were coated with 100 ng / ml of test molecules (e.g., scFv molecules) for 2 hours at room temperature. The plates were blocked with PBS containing 0.2% Tween® and 1% BSA for 1 hour. Individual human sera were then added at a dilution of 1:20 (5% serum) or 1:100 (1% serum) and either unspiked (screening assay) or spiked (confirmatory assay) with the same molecules coated in the corresponding wells. The spike concentrations were 60-115 nM and the spiked samples were preincubated for 1 hour. Antibodies bound to the molecules coated on the plates were detected with 100 ng / ml of rabbit anti-human IgG-HRP for 1 hour. TMB substrate was added as a substrate and after a short incubation, the enzymatic reaction was stopped with 1 M hydrochloric acid. The optical density of each well was read at 450 nm.

[0230] All steps were performed at room temperature. Between each step, the plate was washed three times with 450 μl of wash buffer. Except for the blocking and washing steps, all assay components were added in a volume of 25 μl / well and used in duplicate. For the incubation steps, the ELISA plate was placed on a rotating mixer (40 rpm).

[0231] [Table 8-1]

[0232] [Table 8-2]

[0233] Typically, two measurements were performed for each test molecule: a first measurement was performed by applying untreated human serum, i.e., unspiked human serum (screening assay), to the plates coated with the individual test molecules, and a second measurement was performed by applying human serum preincubated with the same compound as that coated in the corresponding wells, i.e., spiked human serum (confirmatory assay), to the plates coated with the individual test molecules to determine whether the binding of the antibodies initially detected in each serum sample was specific for the test molecule. A decrease in the absorbance signal in the spiked wells indicates that the signal observed in the unspiked wells of the initial screening assay is specific for the molecule coated on the plate. The resulting inhibition percentage (% inhibition) was calculated as the decrease in the initial signal obtained for the unspiked serum (screening assay) as follows: % Inhibition = 100 x (1 - (signal spiked serum / non-signal spiked serum))

[0234] Only spiked samples with % inhibition values ​​above a certain threshold, called confirmation cut point percent (CCP%), were considered as specific binding (screening positive). The %CCP required to confirm specificity was set at 20-30% inhibition or the %CCP was calculated from the average % inhibition of all serum samples measured, as defined in section 2.2. Typically, the %CCP was set at 30% for the analysis of pre-ADA binding signals. Samples with inhibition values ​​above the %CCP were considered positive.

[0235] To exclude false positives resulting from very low or no binding of binders to ADA present in the tested serum samples (low or no binding in unspiked and spiked serum samples), a screening cut point (SCP) was calculated from the spiked serum measurements on each plate. Non-spiked samples with signals below the SCP were called "screening negatives" and were not considered. The calculation method of SCP is as described in section 2.2.

[0236] 2.2. Determination / Calculation of Screening Cut Point (SCP) and Confirmatory Cut Point (%CCP) For each test compound, 20 human serum samples from untreated healthy subjects were analyzed separately.

[0237] Screening Cut Point (SCP): The screening cut point (SCP) is the threshold at which a signal is considered positive (screening positive). The screening cut point (SCP) was calculated from the spiked serum measurements as follows: SCP=Average N+3×SDN Where: - "Average N" corresponds to the average signal from all spiked individual sera measured for a particular test compound; - "SDN" corresponds to the standard deviation calculated from all spiked individual sera measured for a particular test compound.

[0238] Confirmatory Cut Point Percentage (CCP%) Calculation: The confirmatory cut point percentage (CCP%) is the threshold at which the % inhibition is considered specific (specific inhibition).

[0239] The confirmatory cut point percentage (CCP%) is set to a specific value, for example a value in the range of 20.0-30.0%, or is calculated as follows: CCP% = average [inhibition%] + 2.33 × SDN Where: - "Average [% inhibition]" corresponds to the average inhibition value (percentage) of all spiked individual sera measured for a particular test compound; - "SDN" corresponds to the standard deviation calculated from the [% Inhibition] values ​​of all spiked individual sera measured for a particular test compound.

[0240] 2.3. Existing ADA Binding Assay Results for PRO1922 and PRO2230 Variants of the Invention PRO1922wt, PRO2230wt; 32 PRO1922 scFv variants according to the invention, 32 PRO2230 scFv variants according to the invention as well as references PRO1922-L12R-V103T-L144Q (PRO2918), PRO1922-L12S-V103T-L144T (PRO2990), PRO2230-L12R-V103T-L144Q (PRO2903) PRO2230-L12S-V103T-L144T (PRO2984) were analyzed for their ability to bind existing ADA. Measurements were performed directly in a confirmatory assay set-up with 19-20 human serum samples.

[0241] The detailed assay procedures for PRO1922-based and PRO2230-based scFvs are as follows: scFvs are diluted in PBS to a concentration of 100ng / ml, applied in a volume of 25μl to a 96-well plate (Greiner, half area, high binding) and incubated for 2h at RT. After incubation, the plate is washed 3 times and the wells are blocked for 1h at RT with 150μl per well of blocking buffer (PBS+0.1% Tween® 20+1% BSA). 20 human serum samples, 10 females and 10 males, were ordered for analysis at Dunn Labortechnik. For unspiked samples, human serum is diluted to a concentration of 5% using a commercially available low cross buffer (Candor). The dilutions were performed as follows: Primary dilution: 80 μl human serum stock + 720 μl low cross-linking buffer; 2nd dilution: 250 μl of 1st dilution + 250 μl low crosslink buffer.

[0242] For spiked samples, human serum was diluted to a concentration of 10% using a commercially available low cross-linking buffer (Candor). The scFv was also diluted to a concentration of 67 nM in low cross-linking buffer. The diluted serum and scFv were combined in a 1:1 ratio and spiked with 5% human serum. For example, 5 μl of scFv stock solution was diluted to 67 nM in low cross buffer (volume dependent on protein concentration); 50 μl of the first diluted human serum (10%) + 50 μl of the diluted scFv (67 nM).

[0243] A positive control that specifically binds to and can subsequently be detected by the human acceptor backbone of the scFv was incorporated into the assay: this control antibody was diluted to 50 μg / ml in normal human serum (Merck Millipore) and further diluted as follows for spiked and unspiked samples: For non-spiked samples: 1 μl of control antibody (50 μg / ml) + 49 μl of normal human serum to give a concentration of 1 μg / ml, and 30 μl of the diluted antibody-human serum solution was further diluted 1:20 with 570 μl of low crosslinking buffer to give a final control antibody concentration of 0.05 μg / ml. For spiked samples, 1 μl of scFv stock was diluted to 500 nM with normal human serum (volume dependent on protein concentration); 1 μl of control antibody (50 μg / ml) + 49 μl of spiked normal human serum to a concentration of 1 μg / ml; 30 μl of diluted antibody-spiked human serum solution was further diluted 1:20 with 570 μl of low crosslink buffer resulting in a final control antibody concentration of 0.05 μg / ml.

[0244] The spiked samples (spiked sera and spiked positive control) were incubated for 1 h before contacting with the immobilized scFv. After blocking, the plate was washed again 3 times and incubated for 15 min at RT with unspiked human sera and positive control in rows A-D and spiked human sera and spiked positive control in rows E-H, in a volume of 25 μl per well. The plate was then washed again 3 times and incubated for 1 h, steady state, in a volume of 25 μl per well with rabbit anti-human Fc-IgG-HRP detection antibody diluted at a concentration of 100 ng / ml. For this, the antibody was diluted 1:8000 in low cross-dilution buffer. The detection antibody binds to the Fc region of human ADA of each serum, similar to the positive control antibody. Three further washes are performed and TMB peroxidase substrate is added in a volume of 25 μl per well. For this step, the 96-well plate was stored in the dark (RT) for 15 min. The HRP conjugated to the detection antibody converts the colorless TMB into blue TMB + After incubation in the dark, 25 μl of 1 M hydrochloric acid was added to stop the enzyme reaction, and TMB was + Yellow TMB 2+ It was further oxidized to TMB. 2+ The concentration of was measured at 450 nm and was proportional to the amount of pre-existing ADA bound in the well.

[0245] The data was first analyzed by determining the number of serum samples showing 30% or more inhibition (30% CCP). These serum samples were designated "screening positives." Screening positive sera were further analyzed by calculating the SCP for each individual plate as described above, again considering the 30% CCP. The number of positive serum samples for the molecules tested are summarized in Tables 9 and 10. Some exemplary graphs of the absorbance levels of pre-existing ADA in human serum and the reduction in absorbance levels of spiked human serum for PRO1922 and PRO2230 are shown in Figure 1. Ten individual PRO2230 variants (PRO2945, PRO2942, RPO2941, PRO3349, PRO3325, PRO3346, PRO2936, PRO2931, PRO2930, PRO2938) and ten individual PRO1922 variants (PRO2947, PRO2948, PRO2951, PRO2913, PRO2915, PRO2933, PRO2934, PRO3313, PRO3340, PRO3343) compared to the wt and references PRO2230-L12R-V103T-L144Q and PRO1922-L12R-V103T-L144Q identified in two independent rounds of pre-existing ADA determination are shown in Figure 2.

[0246] [Table 9]

[0247] [Table 10]

[0248] Example 3: Determination of KD by SPR To measure the binding kinetics and affinity of PRO1922, PRO2230 and their variants, human α-PD-L1 Fc (Sino Biological, 10084-H02H, LC11NO2402) and human α-mesothelin (Acro Biosystems; MSN_H5223) were immobilized on the novel CMD200M SPR sensor prism chip (Xantec) of an SPR 24 system (Sierra Sensor-Bruker) at a surface density of 100–200 Response Units (RU). The mutant variants were then injected at concentrations of 10 nM, 2 nM, and 0.4 nM into the ligand-immobilized spots and reference spots. The wild-type variants served as controls, and the blank spots served as references.

[0249] The parameters for immobilizing the ligand on the sensor chip are as follows: Human MSLN: Concentration: 4 μg / ml; CT: 280 s; FR: 15 μl / min; Target immobilization level: 700 RU; Sodium acetate: pH 4.0; Human PD-L1: concentration: 4 μg / ml; CT: 120 s; FR: 15 μl / min; target immobilization level: 500 RU; sodium acetate pH 5.0.

[0250] After each cycle with analyte, the surface was regenerated with 3 M MgCl2 solution, leaving only free ligand available for the next analyte cycle. The binding affinity KD was calculated by the Bruker SPR software as the binding association (k on ) and dissociation (k off ) rates were calculated globally by fitting a 1:1 Langmuir model to the curves obtained by cycling with three different concentrations of analyte.

[0251] As can be seen in Table 11, the binding affinity of the anti-PD-L1 variants to PD-L1 shows little variation compared to the wild type. The binding affinity of the variants ranges from 10 to 100 pM, whereas the wild type showed an average binding affinity of 49 pM (n=4). The same is observed for variants with anti-MSLN binding specificity. The binding affinity of the variants targeting MSLN ranges from 0.5 to 3.5 nM, whereas the wild type had an average of 1.8 nM (n=2). In summary, no significant differences in binding affinity were identified between the variants and the corresponding wild type.

[0252] SPR measurements were performed at three concentrations of PRO1922 and PRO2230 scFv variants to identify possible concentration-dependent affinity effects. However, the observed differences were not significant. Furthermore, no correlation was observed between the introduced mutations and changes in affinity for PRO1922 and PRO2230 scFv variants. These results are not unexpected, as the introduced mutations are located away from the CDR regions and should have minimal impact on the binding affinity for the respective binding partners.

[0253] Example 4: Thermal stability measurement using nanoDSF The thermal stability of PRO1922, PRO2230 and their variants at a concentration of 1±0.1 mg / ml was analyzed using nanoDSF.

[0254] During nanoDSF measurements, the molecules in solution were subjected to increasing temperatures leading to unfolding of the molecules. The thermal stability of PRO1922, PRO2230, and variants at 1 ± 0.1 mg / ml was analyzed. Melting curves were generated from 20 °C to 95 °C with a temperature ramp of 1 °C / min. To monitor the unfolding events, the intrinsic fluorescence of the protein was used. The unfolding phenomenon causes a shift in the fluorescence emission spectrum of tryptophan (Trp) due to a change in the environment of the tryptophan (Trp), e.g., from the hydrophobic core to a solvent-exposed Trp residue. The spectral shift upon unfolding is recorded at two wavelengths, 330 nm and 350 nm, and the ratio of 350 nm / 330 nm is taken for data analysis. The unfolding midpoint (Tm) is defined as the inflection point (thermal midpoint) of the unfolding curve, observed as a local maximum or minimum in the first derivative. The inflection point or thermal midpoint (Tm) varies with the stability of the protein. The higher the thermal midpoint, the more stable the protein. A protein can have multiple thermal midpoints based on the number of separately unfolding domains. Thermal stability is further influenced by protein concentration and buffer conditions. Protein unfolding was performed using a Prometheus instrument (Nanotemper Technologies) and analyzed with "PR.ThermControl" v2.3.1 and "PR.Stability Analysis" v1.1 software.

[0255] Table 12 lists all measured thermal midpoints of PRO1922- and PRO2230-based scFvs measured at 1 mg / ml in 20 mM histidine, pH 6.0, as well as the onset of unfolding. PRO2230wt and some PRO2230 variants showed two thermal midpoints in the first derivative curves. The second thermal midpoint (Tm 2) could be calculated for all of these molecules, but the first thermal midpoint (Tm 1) could actually only be calculated for some of them, since the maximum could not be clearly determined for all of them.

[0256] Many PRO2230-based variants show very similar melting temperatures, Tm2, ​​within ±2.5°C, when compared to the wild type, and many PRO1922-based variants show little difference in their Tm1, within ±2.5°C, when compared to the wild type.

[0257] [Table 11]

[0258] [Table 12]

[0259] Example 5: Stability Study Ten PRO1922-based and ten PRO2230-based variants, as well as the reference molecules PRO1922-L12R-V103T-L144Q, PRO2230-L12R-V103T-L144Q and wild type, were subjected to long-term stability studies at a concentration of 10±0.5 mg / ml for 2 weeks (t2w 40°C) and 4 weeks (t4w 40°C) at 4°C and 4 weeks (t4w 4°C) at 4°C. Total proteins were analyzed after each time point for change in concentration by absorbance at 280 nm and change in monomer content by SE-HPLC.

[0260] 5.1 Protein concentration First, protein concentrations were measured for all variants in the stability study stored at 40°C and 4°C for 4 weeks. The results of the protein concentration study are summarized in Table 13. Seven of the 11 variants showed minimal concentration changes for both binding motifs compared to the initial time point t0 at 4°C and 40°C after 4 weeks. Most PRO2230 variants showed small but acceptable concentration losses after 4 weeks at 4°C. Interestingly, the corresponding PRO1922 variants did not show solubility issues at 4°C after 4 weeks. They rather showed an increase in concentration likely due to evaporation of the buffer.

[0261] 5.2 Determination of Monomer Content SE-HPLC was used to determine the monomer content of each sample. The proportion of monomers and oligomers in the samples was assessed by integration of the SE-HPLC peak areas at different time points during the study.

[0262] The results of the monomer content analysis are summarized in Table 14. In general, most of the variants tested lost less than 10% of monomer content after 4 weeks of storage at 40° C., which was considered acceptable. Most variants showed adequate monomer stability, especially when using a positively charged residue such as arginine or lysine at position T146 and a non-polar residue such as serine at position T101.

[0263] 5.3 Thermal stability measurement at a protein concentration of 10 mg / ml using nanoDSF Further nDSF measurements were performed on selected variants at a protein concentration of 10 mg / ml. The analysis results are summarized in Table 15. The melting temperatures of most variants were in a range close to their respective wild types. No significant differences in thermal stability were observed compared to the variants at mg / ml concentration.

[0264] Example 6: Solubility testing by PEG precipitation assay The apparent solubility of ten PRO1922-based variants and ten PRO2230-based variants, as well as the reference molecules PRO1922-L12R-V103T-L144Q, PRO2230-L12R-V103T-L144Q, wild-type PRO1922, and PRO2230, was analyzed by polyethylene glycol (PEG) precipitation assays.

[0265] Other methods to assess protein solubility include concentration (e.g., ultrafiltration) and lyophilization. However, these methods require excessive amounts of protein and suffer from experimental problems such as aggregation and gel formation. PEG precipitation is effective for small amounts of protein (<1 mg) and measures solubility by precipitating the protein via the excluded volume effect. At higher PEG concentrations, the protein begins to precipitate. Apparent solubility is determined at the PEG 8,000 (% w / v) concentration at which the protein concentration is halved (PEG midpoint). A 12-step gradient was created using 20 mM histidine, pH 6 and 37% (w / v) PEG 8000 in 20 mM histidine, pH 6. Then, 8 μl of protein at 10 mg / ml concentration was mixed with 72 μl of the PEG gradient solution in a 96-deep-well plate. The plates were incubated overnight and the solutions were filtered into a second 96-deep-well plate using a Pall® AcroPrep™ Advance 96-well filter plate (1.2 μm) at 2000×g for 2 min at room temperature. Protein concentrations were measured in duplicate for all proteins and all dilutions at 280 nm with a reference wavelength of 310 nm subtracted.

[0266] Data were fitted using a four-parameter sigmoidal fit (4PL, sigmoidal, least-squares fit) in Graphpad Prism 9 software to determine PEG midpoints and 95% confidence intervals. Higher PEG midpoint values ​​indicate higher apparent solubility of the protein.

[0267] [Table 13]

[0268] [Table 14]

[0269] [Table 15]

[0270] For most scFvs tested, a PEG midpoint was determined that was comparable to that observed for the unmodified scFv.

[0271] The PEG midpoint determination results for the scFvs tested are shown in Figure 3. The higher the midpoint, the more soluble the protein. PRO1922-based scFvs exhibited higher PEG midpoints on average than PRO1922-based scFvs. Overall, the scFv variants tested generally show similar solubility compared to their wild-type counterparts.

[0272] Example 7: Structural integrity by CEX-HPLC and cGE Further stability studies, namely CEX HPLC and cGE experiments, were performed on certain PRO1922 and PRO2230 variants.

[0273] CEX-HPLC analysis investigated the formation of charge variants after 4 weeks of storage at 40°C. Charge variants were evaluated during stability studies using analytical cation exchange chromatography. 30 μg of scFv was injected onto a MabPac SCX-10 (ThermoFisher Scientific) using a Hitachi Chromaster HPLC system. The main peak and charged molecules were eluted at 25°C with a flow rate of 0.85 ml / min by applying a pH gradient from pH 4 to pH 11 within 15 min. The mobile phase consisted of 15.6 mM CAPS, 9.4 mM CHES, 4.6 mM TAPS, 9.9 mM HEPPSO, 8.7 mM MOPSO, 11 mM MES, 13 mM acetic acid, 9.9 mM formic acid, and the pH was adjusted to 4 and 11 with NaOH. Species eluting before or after the main peak were grouped as acidic and basic molecules, respectively.

[0274] The starting target purity was 82.3% and 96.8% for PRO1922 and PRO2230 scFv, respectively (see Table 16). After 4 weeks at 40°C, the main peak area decreased by 0.4% for PRO1922 and 0.3% for PRO2230, indicating good chemical stability. Similar target purity as well as purity changes over time were observed for most scFvs. The largest decrease in purity was observed for the PRO1922 variants, namely L12A-T101S-L144A-T146Q (-5.7%), T101S-T146Q (-3.3%) and T101R-T146E (-2.9%). Overall, all variants showed good stability.

[0275] The selected scFv molecules and the parental scFv were further analyzed for fragmentation using reduced cGE.

[0276] Capillary gel electrophoresis (cGE) was evaluated using the Protein Express Chip on a LabChip GX Touch HT system (PerkinElmer) according to the manufacturer's protocol. scFvs were diluted to 1 mg / ml in 20 mM histidine, pH 6, denatured at 70°C for 10 min, and analyzed under non-reducing and reducing conditions. Data were integrated using LabChip GX Reviewer software (Perkin Elmer).

[0277] After 4 weeks, no significant increase in low molecular weight species (LMWS) was observed (see Table 17, LMWS formation ≦2%). The only molecule that showed an increase in LMWS formation during storage was L144K-T146E-S148K (approximately 3.7%). In summary, all variants showed excellent chemical stability.

[0278] Example 8: Chemical unfolding with guanidinium hydrochloride Chemical unfolding experiments using guanidinium hydrochloride were performed for selected PRO1922 and PRO2230 scFv variants. PRO1922 and PRO2230 scFv variants were incubated in 24 solutions ranging from 1.5 M to 5 M guanidinium hydrochloride in 20 mM histidine pH 6.0. The final protein concentration was 0.5 mg / ml. Samples were allowed to equilibrate overnight at room temperature and the intrinsic fluorescence at 330 nm and 350 nm was measured using Prometheus (Nanotemper Technologies). The change in the fluorescence ratio (350 nm / 330 nm) was used to calculate c1 / 2 and ΔG using PR.Stability Analysis v1.1 software (Nanotemper Technologies). アンフォールディング was measured.

[0279] The results of chemical unfolding using guanidinium HCl are shown in Figure 4. PRO1922 and PRO2230 show c1 / 2 of 3.9 M and 3.2 M, respectively. With the exception of the T101R variant, which shows a slightly reduced c1 / 2, all PRO1922 and PRO2230 scFv variants show similar stability in the presence of increasing concentrations of guanidinium hydrochloride.

[0280] Example 10: Hydrophobicity by Hydrophobic Interaction Chromatography (HIC) HPLC Specific PRO1922 and PRO2230 scFv variants were tested for hydrophobicity by HIC-HPLC using a Pro-Pac HIC-10 column (5 μm, 300 Å, 4.6 × 100 mm). 20 μg of protein was injected and eluted from the column using a gradient from 1.5 M to 0 M (NH4)2SO4, pH 5.5 in 20 mM histidine, at a flow rate of 1 ml / min over 12 min. Retention times were then correlated to the respective (NH4)2SO4 concentrations. A delay volume of 2.81 ml was applied to measure (NH4)2SO4 concentrations, taking into account the column volume and the dead volume of the system.

[0281] The HIC-HPLC data show only very minor changes in apparent hydrophobicity between the PRO1922 and PRO2230 scFv variants. Hydrophobic amino acids were replaced by polar or charged amino acids with low impact on the overall apparent hydrophobicity (data not shown). However, for some molecules, a slight increase in hydrophobicity was observed in both the PRO1922 and PRO2230 scFv variants. In particular for molecules with polar modifications at positions 101 and 146, namely T101Q-T146Q, T101N-T146Q, T101S-T146S. Furthermore, T101R-T146E slightly increases hydrophobicity. Interestingly, only T101Q slightly increases hydrophobicity in molecules with the corresponding single mutations. However, these changes are small and cannot be considered relevant.

[0282] [Table 16]

[0283] [Table 17]

Claims

1. A method of making modified antibody variable domains that exhibit reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum from healthy donors when compared to unmodified versions, the reduced binding being determined by an ELISA-based pre-existing anti-drug antibody binding assay; wherein the unmodified antibody variable domain binds to a target antigen, (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus the region HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region; (ii) a variable light chain (VL), the variable light chain comprising from the N-terminus to the C-terminus the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region, and each LCDR representing a light chain complementarity determining region; Including, The following substitutions (AHo numbering) were made to the heavy chain framework regions of the unmodified antibody variable domain: Alanine (A), Serine (S), Lysine (K), Arginine (R), Aspartic Acid (D), Glutamic Acid (E), Asparagine (N) or Glutamine (Q) at amino acid position 101, or Serine (S), Lysine (K), Arginine (R), Aspartic Acid (D), Glutamic Acid (E), Asparagine (N) or Glutamine (Q); Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148 to obtain said modified antibody variable domain. 。

2. 2. The method of claim 1, wherein the variable light chain framework regions LFW1, LFW2 and LFW3 are selected from a human antibody Vκ framework and the variable light chain framework region LFW4 is selected from a Vλ framework, in particular a Vλ framework sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196; and / or the variable heavy chain framework regions HFW1, HFW2 and HFW3 are selected from human VH framework subtypes VH1a, VH1b, VH3 and VH4, in particular the human VH framework subtype VH3.

3. 4. The heavy chain framework region of the unmodified antibody variable domain comprises the following amino acids: - valine (V) or leucine (L), in particular leucine (L), at amino acid position 12; - Threonine (T) at amino acid position 101; - Threonine (T) or Leucine (L), in particular Leucine (L) at amino acid position 144; - Threonine (T) at amino acid position 146; - serine (S) at amino acid position 148 The method of claim 1 or 2, comprising one or more of the following:

4. One or more substitutions to be introduced are (AHo numbering): Alanine (A), Serine (S), Lysine (K), Arginine (R), Asparagine (N) or Glutamine (Q), or Serine (S), Lysine (K), Arginine (R), Asparagine (N) or Glutamine (Q) at amino acid position 101; - Lysine (K), Aspartic acid (D), Glutamic acid (E), Arginine (R) or Glutamine (Q) at amino acid position 146; and Lysine (K) at amino acid position 148 The method according to any one of claims 1 to 3, wherein the compound is selected from the group consisting of

5. The method further comprising (AHo numbering): alanine (A), lysine (K) or arginine (R) at amino acid position 12, in particular alanine (A) or arginine (R) at amino acid position 12; and Alanine (A), Lysine (K) or Arginine (R) at amino acid position 144 The method of any one of claims 1 to 4, comprising the introduction of one or two substitutions selected from the group consisting of:

6. The following substitutions (AHo numbering) were made to the heavy chain framework regions of the unmodified antibody variable domain: a. Alanine (A), Lysine (K), Arginine (R), or Asparagine (N), or Lysine (K), Arginine (R), or Asparagine (N) at amino acid position 101; b. Lysine (K), Arginine (R), Aspartic acid (D), Glutamic acid (E), or Glutamine (Q) at amino acid position 146; c. serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; d. an alanine (A) or lysine (K) at amino acid position 144, and A leucine (L) or asparagine (N) at amino acid position 148; e. Lysine (K) at amino acid position 144; Glutamic acid (E) at amino acid position 146, and Lysine (K) at amino acid position 148; f. serine (S), arginine (R) or glutamine (Q) at amino acid position 101; alanine (A) or lysine (K) at amino acid position 144, and Arginine (R) or glutamine (Q) at amino acid position 146; g. Arginine (R) at amino acid position 12, and serine (S), arginine (R) or glutamine (Q) at amino acid position 101; h. Arginine (R) at amino acid position 12, and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; i. Arginine (R) at amino acid position 12; at amino acid position 101, serine (S), arginine (R) or glutamine (Q), and Lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146; j. alanine (A) at amino acid position 12; serine (S), arginine (R) or glutamine (Q) at amino acid position 101; alanine (A) or lysine (K) at amino acid position 144, and Arginine (R) or glutamine (Q) at amino acid position 146 The method according to any one of claims 1 to 5, further comprising introducing one of the following:

7. 1. A method of making a modified antibody that exhibits reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum from healthy donors compared to an unmodified version, the reduced binding being determined by an ELISA-based pre-existing anti-drug antibody binding assay, comprising: The unmodified antibody comprises one or more unmodified antibody variable domains as defined in any one of claims 1 to 3, wherein the one or more antibody variable domains are independently selected from Fv, disulfide stabilized Fv, scFv, and disulfide stabilized scFv; The method comprising the step of introducing one or more substitutions as defined in any one of claims 1, 4, 5 and 6 into at least one heavy chain framework region of the unmodified antibody variable domain.

8. An antibody variable domain that binds to a target antigen, (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus the region HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, where each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region; The variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, wherein HFW1, HFW3 and HFW4 are (AHo numbering): Alanine (A), Serine (S), Lysine (K), Arginine (R), Aspartic Acid (D), Glutamic Acid (E), Asparagine (N) or Glutamine (Q) at amino acid position 101, or Serine (S), Lysine (K), Arginine (R), Aspartic Acid (D), Glutamic Acid (E), Asparagine (N) or Glutamine (Q); - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146; and Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148 and a variable heavy chain (VH) having one or more substitutions selected from the group consisting of: (ii) a variable light chain (VL), comprising from the N-terminus to the C-terminus the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, each LFW representing a light chain framework region, and each LCDRR representing a light chain complementarity determining region; The variable light chain framework regions LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks, and the variable light chain framework region LFW4 is selected from a Vλ framework, in particular a Vλ framework sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196. The antibody variable domain comprising:

9. 9. The antibody variable domain of claim 8, wherein HFW1, HFW3 and HFW4 have one or more of the substitutions defined in any one of claims 4 to 6.

10. The HFW1, HFW3 and HFW4 are one of the indicated substitutions at position -101, and - one or more further substitutions selected from the substitutions indicated at positions 12, 144, 146 and 148, in particular one or more further substitutions selected from the substitutions indicated at positions 146 and 148, in particular one further substitution selected from the substitutions indicated at position 146 10. The antibody variable domain of claim 8 or 9, having the following structure:

11. the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 being a. any one of SEQ ID NOs: 8-25, 28, 30-36, 86-103, 106 and 108-114, in particular any one of SEQ ID NOs: 8-25 and 86-103 in combination with the framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italicized residues in Tables 1 and 3); and b. A combination selected from any one of SEQ ID NOs: 8-25, 28, 30-36, 86-103, 106 and 108-114, in particular a combination of the framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italicized residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25 and 86-103, with 1, 2 or 3 mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering). the variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are selected from a. a combination of the framework regions LFW1, LFW2, LFW3 and LFW4 (i.e., the non-italicized residues in Tables 1, 3 and 5) of any one of SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187; and b. The antibody variable domain of any one of claims 8-10, selected from a combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i.e. the non-italicized residues in Table 1, Table 3 and Table 5) of any one of SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187, with 1, 2 or 3 mutations in the framework regions at a position different from 101 (AHo numbering).

12. the antibody variable domain (i) a VH sequence selected from SEQ ID NO: 204 and a variant of SEQ ID NO: 204 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 205-206, and variants of SEQ ID NOs: 205-206 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 208-210, and variants of SEQ ID NOs: 208-210 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 211-212, and variants of SEQ ID NOs: 211-212 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 213-215, and variants of SEQ ID NOs: 213-215 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 216-217, and variants of SEQ ID NOs: 216-217 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 224-225, and variants of SEQ ID NOs: 224-225 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 226-229, and variants of SEQ ID NOs: 226-229 having one, two or three mutations in a framework region at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 230-233, and variants of SEQ ID NOs: 230-233 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 234-237, and variants of SEQ ID NOs: 234-237 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 238 and a variant of SEQ ID NO: 238 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 239 and a variant of SEQ ID NO: 239 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 240-242, and variants of SEQ ID NOs: 240-242 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 243-245, and a variant of SEQ ID NOs: 236-238 having one, two or three mutations in a framework region at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 254 and a variant of SEQ ID NO: 254 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 255 and a variant of SEQ ID NO: 255 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 256 and a variant of SEQ ID NO: 256 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 257 and a variant of SEQ ID NO: 257 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 261-262, and variants of SEQ ID NOs: 261-262 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 263-266, and variants of SEQ ID NOs: 263-266 having one, two or three mutations in a framework region at a position (AHo numbering) different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 267 and a variant of SEQ ID NO: 267 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 268, and a variant of SEQ ID NO: 268 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 269 and a variant of SEQ ID NO: 269 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 270 and a variant of SEQ ID NO: 270 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 271-272, and variants of SEQ ID NOs: 271-272 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 273 and a variant of SEQ ID NO: 273 having one, two or three mutations in a framework region at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 274-275, and variants of SEQ ID NOs: 274-275 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 276 and a variant of SEQ ID NO: 276 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 277-278, and variants of SEQ ID NOs: 277-278 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 279-281, and variants of SEQ ID NOs: 279-281 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 282-283, and variants of SEQ ID NOs: 282-283 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 284-285, and variants of SEQ ID NOs: 284-285 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NO: 286 and a variant of SEQ ID NO: 286 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 287 and a variant of SEQ ID NO: 287 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 288-291, and variants of SEQ ID NOs: 288-291 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 292-295, and variants of SEQ ID NOs: 292-295 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 302-303, and variants of SEQ ID NOs: 302-303 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 304-305, and variants of SEQ ID NOs: 304-305 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 309-310, and variants of SEQ ID NOs: 309-310 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 311-312, and variants of SEQ ID NOs: 311-312 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 313-316, and variants of SEQ ID NOs: 313-316 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 317-318, and variants of SEQ ID NOs: 317-318 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 319-320, and variants of SEQ ID NOs: 319-320 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 321-322, and variants of SEQ ID NOs: 321-322 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 323-324, and variants of SEQ ID NOs: 323-324 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NO: 325 and a variant of SEQ ID NO: 325 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 330-332, and variants of SEQ ID NOs: 330-332 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 333-337, and variants of SEQ ID NOs: 333-337 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 338-340, and variants of SEQ ID NOs: 338-340 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 341-343, and variants of SEQ ID NOs: 341-343 having one, two or three mutations in the framework regions at a position different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 344-346, and variants of SEQ ID NOs: 344-346 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 347-349, and variants of SEQ ID NOs: 347-349 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 350-352, and variants of SEQ ID NOs: 350-352 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 353-355, and variants of SEQ ID NOs: 353-355 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 356-358, and variants of SEQ ID NOs: 356-358 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 359-361, and variants of SEQ ID NOs: 359-361 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering); or (i) a VH sequence selected from SEQ ID NOs: 362-364, and variants of SEQ ID NOs: 362-364 having one, two or three mutations in the framework regions at positions different from 12, 101, 144, 146 and 148 (AHo numbering); (ii) a VL sequence selected from SEQ ID NOs: 365-367 and variants of SEQ ID NOs: 365-367 having one, two or three mutations in the framework regions at positions different from 101 (AHo numbering). Contains or 9. The antibody variable domain of claim 8, wherein the antibody variable domain is a scFv having a sequence selected from SEQ ID NOs: 207, 218-223, 246-253, 258-260, 296-301, 306-308, 326-329 and 368-385, and variants of SEQ ID NOs: 207, 218-223, 246-253, 258-260, 296-301, 306-308, 326-329 and 368-385 with 1, 2, 3, 4 or 5 mutations in the framework regions at positions different from VH position 12, 101, 144, 146 and 148 (AHo numbering) and VL position 101 (AHo numbering).

13. 13. An antibody comprising one or more antibody variable domains as defined in any one of claims 8 to 12, wherein said one or more antibody variable domains are independently selected from Fv, disulfide stabilized Fv, scFv, and disulfide stabilized scFv.

14. the antibody is a single chain antibody having a sequence selected from SEQ ID NOs: 386-401, 426, 427, 440, 441, and variants of SEQ ID NOs: 386-401, 426, 427, 440, 441 with 1, 2, 3, 4 or 5 mutations in the framework regions at positions different from VH position 12, 101, 144, 146 and 148 (AHo numbering) and VL position 101 (AHo numbering); or The antibodies may be selected from the group consisting of SEQ ID NOs: 402 and 403; 404 and 405; 406 and 407; 408 and 409; 410 and 411; 412 and 413; 414 and 415; 416 and 417; 418 and 419; 420 and 421; 422 and 423; 424 and 425; 428 and 429; 430 and 431; 432 and 433; 434 and 435; 436 and 437; 438 and 439; 442 and 443; 444 and 445; and SEQ ID NOs: 402 and 403; 404 and 405; 406 and 407; 408 and 409; 410 and 411; 412 and 413; 414 and 415; 416 and 417; 418 and 419; 420 and 421; 422 and 423; 424 and 425; 428 and 429; 430 and 431; 432 and 433; 434 and 435; 436 and 437; 438 and 439; 442 and 443; 444 and 445; 14. The antibody of claim 13, wherein the antibody is a dimer consisting of two chains having a sequence pair selected from variants selected from: 17; 418 and 419; 420 and 421; 422 and 423; 424 and 425; 428 and 429; 430 and 431; 432 and 433; 434 and 435; 436 and 437; 438 and 439; 442 and 443; 444 and 445, wherein each of the sequences of the sequence pair has 1, 2, 3, 4 or 5 mutations in the framework regions at positions different from VH positions 12, 101, 144, 146 and 148 (AHo numbering) and VL position 101 (AHo numbering).

15. A nucleic acid or two nucleic acids encoding an antibody variable domain according to any one of claims 8 to 12 or an antibody according to claim 13 or 14.

16. A vector or two vectors comprising the nucleic acid or two nucleic acids according to claim 15.

17. A host cell or a plurality of host cells comprising the vector or two vectors according to claim 16.

18. 17. A method for producing an antibody variable domain according to any one of claims 8 to 12 or an antibody according to claim 13 or 14, comprising (i) providing a nucleic acid or two nucleic acids according to claim 15, or a vector or two vectors according to claim 16, expressing said nucleic acid sequence or nucleic acid, or said vector or two vectors and recovering said antibody variable domain or said antibody from the expression system, or (ii) providing a host cell or a plurality of host cells according to claim 17, culturing said host cell or a plurality of host cells and recovering said antibody variable domain or said antibody from the cell culture.

19. A pharmaceutical composition comprising an antibody variable domain according to any one of claims 8 to 12, or an antibody according to claim 13 or 14, and a pharma- ceutically acceptable carrier.

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