Antibody variable domains and antibodies with reduced immunogenicity

Abstract

The present invention relates to antibody variable domains that exhibit reduced binding to original anti-drug antibodies (ADAs), to antibodies comprising one or more of said antibody variable domains, and to pharmaceutical compositions comprising said antibodies. The invention also relates to a nucleic acid encoding the antibody variable domain or the antibody, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector, and a method of producing the antibody variable domain or the multispecific antibody. Furthermore, the present invention relates to a method of producing said antibody variable domains and antibodies.

Description

Technical Field

[0001] The present invention relates to antibody variable domains that exhibit reduced binding to pre-existing anti-drug antibodies (ADAs), antibodies comprising one or more of said antibody variable domains, and pharmaceutical compositions comprising said antibodies. The present invention also relates to nucleic acids encoding said antibody variable domains or said antibodies, vectors comprising said nucleic acids, host cells comprising said nucleic acids or said vectors, and methods for producing said antibody variable domains or said multispecific antibodies. In addition, the present invention relates to a method for generating said antibody variable domains and antibodies. Background Art

[0002] Since the development of the first monoclonal antibodies (“mAbs”; In the past four decades since the discovery of the human antigen antagonist (Hermann-La Roche-Posay, 1975, Vol. 256, pp. 495-497), antibodies have become an increasingly important class of biomolecules for research, diagnosis, and treatment. Initially, antibodies could only be obtained by immunizing animals with the corresponding antigen of interest. Although antibodies of non-human origin can be used for research and diagnosis, in therapeutic methods, the human body typically recognizes non-human antibodies as foreign and produces an immune response against non-human antibody drugs, reducing their effectiveness or rendering them ineffective. Even if the administered antibody therapeutics have been humanized, for example, by transplanting CDRs of non-human origin into human immunoglobulin frameworks to minimize non-human components, they can still trigger an immune response, which compromises the efficacy and / or safety of these therapeutic agents.

[0003] This immune response usually involves the binding of anti-drug antibodies (ADA) to the therapeutic agent. These ADA can be antibodies already present in human serum (so-called pre-existing ADA) and / or antibodies formed during the treatment process.

[0004] For therapeutic antibodies that contain portions of naturally occurring human antibodies, such as Fab or Fv antibody fragments, or are constructed therefrom, the risk of ADA binding can be significantly increased. It is believed that one of the main reasons for the increased ADA binding is that in antibody fragments, there are often a large number of amino acids that were previously shielded from contact with other antibody parts or domains, which now become exposed to the solvent and present themselves as potential epitopes to the immune system.

[0005] According to the literature, the patient's antibody response depends on the presence of B cell epitopes and T cell epitopes. When the B cell receptor recognizes and binds to an antigen (such as an administered therapeutic antibody), the antigen is internalized into the B cell through receptor-mediated endocytosis and undergoes proteolytic processing. The resulting peptide is then presented by MHC class II molecules. After the T cell epitope is recognized by the T helper cell, the latter stimulates the corresponding B cell to proliferate and differentiate into plasma cells that produce antibodies.

[0006] Several strategies have been proposed in the prior art to further reduce the response of the patient's immune system to the administered antibodies.

[0007] For example, Zhao, L. and Li, J. (2010), BMC Struct. Biol., 10, S6, disclose a method for predicting potential B-cell epitopes on protein surfaces based on structural information about antibody-antigen complexes. The authors identified common structural elements in B-cell epitopes. In particular, they found that polar amino acids with flexible side chains, such as arginine (R), lysine (K), asparagine (N), glutamine (Q), and histidine (H), are significantly overrepresented in epitopes recognized by antibodies. Understanding these key structural elements forms the basis for 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 for reducing the immunogenicity of foreign proteins by identifying B cell epitopes on proteins and eliminating them by mutagenesis. The authors replaced bulky hydrophilic residues such as arginine (R), glutamine (Q), glutamic acid (E) or solvent lysine (K) in solvent-exposed regions with small amino acids such as alanine, glycine and serine.

[0009] WO 2016 / 150845 discloses glycosylated immunoglobulin heavy chain variable domains (VH domains) of immunoglobulin single variable domain antibodies (ISVDs or nanobodies), wherein glycosylation reduces binding of the ISVD to the original antibody compared to the same ISDV without glycosylation. For example, WO 2016 / 150845 discloses a VH domain of an ISDV that contains a glycosylation site at one of positions 11, 12, 13, 14, 15, 46, 47, 48, 49, 101, 103, 144, 146, 148, 149 or 150, thereby being glycosylated or capable of glycosylation.

[0010] US2017 / 121399 A1 discloses the VH domain of a single variable domain antibody (Nanobody) that contains mutations in the framework region to reduce binding to the original antibody. US2017 / 121399 A1 proposes multiple mutation positions, including 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 show that in addition to the C-terminal alanine extension and substitutions at positions 12 and 103 (AHo numbering), the alanine substitution at position 101 does not further significantly reduce binding to the original antibody.

[0011] WO 2011 / 075861 discloses a method for reducing the immunogenicity of antibody variable domains (particularly scFv) by mutating one or more amino acid residues in the interface between the variable chain and the constant chain of the corresponding full-length antibody. Also disclosed are (i) residues present in the turn region of the secondary structure, (ii) residues with large flexible side chains or bulky side chains, or (iii) hydrophobic residues, which tend to become B cell epitopes and thus trigger immunogenic reactions, and removing these amino acid residues can interrupt B cell epitopes. It is also noted that the one or more amino acid residues to be replaced are leucine (L), valine (V), aspartic acid (D), phenylalanine (F), arginine (R) and / or glutamic acid (E). WO 2011 / 075861 discloses an scFv example having heavy chain point mutations L12S, V103T and L144T (AHo numbering), which show reduced binding to the original ADA present in human serum compared to the unmutated version. Therefore, WO 2011 / 075861 teaches a method for reducing the immunogenicity of antibody variable domains to pre-existing ADA, by replacing small hydrophobic residues, such as L and V, located in 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 in the interface.

[0012] Although currently available methods provide useful indications on how to reduce the immunogenicity of antibody variable domains, the chances of success depend on the specific circumstances, i.e., the solutions they propose to reduce the immunogenicity of antibody variable domains are not generally applicable, and the antibody variable domains obtained by these methods often exhibit significant residual immunogenicity. Therefore, there is still a great need to meet the needs of antibody variable domains that exhibit low immunogenicity and can be generally applied to the construction of antibody fragments. More specifically, it is hoped that there will be antibody variable domains on hand that exhibit low immunogenicity, particularly reduced binding to pre-existing ADA, and that are generally applicable to the construction of antibody fragments. In addition, it is expected that these antibody variable domains will provide high stability when integrated into the final antibody form, which will allow them to be applied to the construction of stable antibody fragments and fragment-based multispecific antibodies suitable for therapeutic agent development. Summary of the Invention

[0013] One object of the present invention is to provide antibody variable domain variants that exhibit reduced immunogenicity. More specifically, the antibody variable domain variants are significantly less recognized by native ADA than their unmodified counterparts. Furthermore, these antibody variable domain variants should be highly stable to allow their use in constructing antibody fragments and multispecific antibodies suitable for drug development.

[0014] The present inventors have now surprisingly discovered that antibody variable domains, particularly antibody variable domains in which one or more heavy chain framework positions 101, 146, and / or 148 (according to AHo numbering) are substituted with small, moderately hydrophilic amino acids (i.e., alanine (A) or serine (S)), or with hydrophilic amino acids with flexible, bulky side chains (i.e., lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q)), when in scFv format, have significantly reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum compared to their unsubstituted versions. It has also been discovered 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 the binding of the 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 a modified antibody variable domain having reduced binding to native anti-drug antibodies (ADA) present in human serum from healthy donors compared to its unmodified version, and wherein the reduced binding is determined by an ELISA-based native anti-drug antibody binding assay,

[0016] wherein the unmodified antibody variable domain binds to a target antigen and comprises:

[0017] (i) a variable heavy chain (VH), comprising from N-terminus to C-terminus the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region and each HCDR indicates a heavy chain complementarity determining region,

[0018] and

[0019] (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region,

[0020] The method comprises the following steps: introducing one or more of the following substitutions (AHo numbering) into the heavy chain framework region of the unmodified antibody variable domain:

[0021] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0022] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146;

[0023] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0024] To obtain the modified antibody variable domain.

[0025] In a second aspect, the present invention relates to an antibody variable domain that binds to a target antigen, comprising:

[0026] (i) a variable heavy chain (VH) comprising, from N-terminus to C-terminus, the region HFW1-

[0027] HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region, and each HCDR indicates a heavy chain complementarity determining region,

[0028] wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, wherein said HFW1, HFW2, HFW3 and HFW4 have one or more substitutions selected from the group consisting of:

[0029] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0030] - 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

[0031] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0032] and

[0033] (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region,

[0034] 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, a Vλ framework sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196.

[0035] In a third aspect, the present invention relates to an antibody comprising one or more antibody variable domains of the present invention, wherein the one or more antibody variable domains are independently selected from Fv, disulfide-stabilized Fv (dsFv), scFv and disulfide-stabilized scFv (dsscFv).

[0036] 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 present invention.

[0037] In a fifth aspect, the present invention relates to a vector or two vectors comprising a nucleic acid or two nucleic acids of the present invention.

[0038] In a sixth aspect, the present invention relates to a host cell or multiple host cells comprising one or two vectors of the present invention.

[0039] In a seventh aspect, the present invention relates to a method for producing an antibody variable domain of the present invention or an antibody of the present invention, comprising (i) providing one or two nucleic acids of the present invention, or one or two vectors of the present invention, expressing the one or two nucleic acids, or the one or two vectors, and collecting the antibody variable domain or the antibody from the expression system, or (ii) providing one or more host cells of the present invention, culturing the one or more host cells; and collecting the antibody variable domain or the antibody from the cell culture.

[0040] In an eighth aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

[0041] In a ninth aspect, the present invention relates to a pharmaceutical composition of the present invention for use as a medicament.

[0042] The aspects, advantageous features and preferred embodiments of the invention which are outlined in the following, individually or in combination, further contribute to solving the objects of the invention:

[0043] 1. An antibody variable domain that binds to a target antigen, comprising:

[0044] (i) a variable heavy chain (VH), comprising from N-terminus to C-terminus the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region and each HCDR indicates a heavy chain complementarity determining region,

[0045] wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, wherein said HFW1, HFW2, HFW3 and HFW4 have one or more substitutions selected from the group consisting of:

[0046] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0047] - 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

[0048] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0049] and

[0050] (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region,

[0051] 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.

[0052] 2. The antibody variable domain according to claim 1, wherein HFW3 and HFW4 have one or more substitutions selected from the group consisting of:

[0053] - serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0054] - 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

[0055] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148.

[0056] 3. The antibody variable domain according to claim 1, wherein HFW3 and HFW4 have one or more substitutions selected from the group consisting of:

[0057] - alanine (A), serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101;

[0058] - 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

[0059] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0060] or

[0061] - serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101;

[0062] - 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

[0063] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148.

[0064] 4. The antibody variable domain according to claim 1, wherein HFW3 and HFW4 have one or more substitutions selected from the group consisting of:

[0065] - alanine (A), serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101;

[0066] - lysine (K), aspartic acid (D), glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 146; and

[0067] - lysine (K) at amino acid position 148;

[0068] or

[0069] - serine (S), lysine (K), arginine (R), asparagine (N) or glutamine (Q) at amino acid position 101;

[0070] - lysine (K), aspartic acid (D), glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 146; and

[0071] - Lysine (K) at amino acid position 148.

[0072] 5. The antibody variable domain according to any one of items 1 to 4, wherein HFW1 and HFW4 further have one or two substitutions selected from the group consisting of:

[0073] - 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

[0074] - Alanine (A), Lysine (K) or Arginine (R) at amino acid position 144.

[0075] 6. The antibody variable domain according to any one of the preceding items, wherein

[0076] 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,

[0077] and wherein the LFW3 has the following substitutions (AHo numbering):

[0078] - Glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101.

[0079] 7. The antibody variable domain according to claim 1, wherein HFW1, HFW3 and HFW4 have one of the following substitutions (AHo numbering):

[0080] 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;

[0081] 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;

[0082] 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

[0083] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0084] d. Alanine (A), Lysine (K) or Arginine (R) at amino acid position 144; and

[0085] Leucine (L), lysine (K), or asparagine (N) at amino acid position 148;

[0086] e. Alanine (A), lysine (K), or arginine (R) at amino acid position 144;

[0087] 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

[0088] Leucine (L), lysine (K), or asparagine (N) at amino acid position 148;

[0089] 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;

[0090] Alanine (A), Lysine (K), or Arginine (R) at amino acid position 144; and

[0091] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0092] g. Alanine (A) or arginine (R) at amino acid position 12; and

[0093] 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;

[0094] h. Alanine (A) or Arginine (R) at amino acid position 12; and

[0095] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0096] i. Alanine (A) or arginine (R) at amino acid position 12;

[0097] 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;

[0098] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0099] j. Alanine (A) or arginine (R) at amino acid position 12;

[0100] 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;

[0101] Alanine (A), Lysine (K), or Arginine (R) at amino acid position 144; and

[0102] At amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q).

[0103] 8. The antibody variable domain according to claim 1, wherein HFW1, HFW3 and HFW4 have one of the following substitutions (AHo numbering):

[0104] a. Alanine (A), lysine (K), arginine (R) or asparagine (N), or lysine (K), arginine (R) or asparagine (N) at amino acid position 101;

[0105] b. Lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0106] 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

[0107] lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0108] d. Alanine (A) or Lysine (K) at amino acid position 144; and

[0109] Leucine (L) or asparagine (N) at amino acid position 148;

[0110] e. Lysine (K) at amino acid position 144;

[0111] Glutamic acid (E) at amino acid position 146; and

[0112] lysine (K) at amino acid position 148;

[0113] f. serine (S), arginine (R) or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101;

[0114] Alanine (A) or Lysine (K) at amino acid position 144; and

[0115] arginine (R) or glutamine (Q) at amino acid position 146;

[0116] g. Arginine (R) at amino acid position 12; and

[0117] serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101;

[0118] h. Arginine (R) at amino acid position 12; and

[0119] lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0120] i. Arginine (R) at amino acid position 12;

[0121] Serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; and

[0122] lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0123] j. Alanine (A) at amino acid position 12;

[0124] serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101;

[0125] Alanine (A) or Lysine (K) at amino acid position 144; and

[0126] Arginine (R) or glutamine (Q) at amino acid position 146.

[0127] 9. The antibody variable domain according to any one of items 1 to 8, wherein HFW1, HFW3 and HFW4 have:

[0128] - one of the substitutions shown at position 101, and

[0129] - one or more further substitutions selected from the group consisting of the substitutions shown at positions 12, 144, 146 and 148, in particular one or more further substitutions selected from the group consisting of the substitutions shown at positions 146 and 148, in particular one further substitution selected from the group consisting of the substitutions shown at position 146.

[0130] 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 human VH framework subtypes VH1a, VH1b, VH3, VH4, VH5 and VH6.

[0131] 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 human VH framework subtypes VH1a, VH1b, VH3 and VH4.

[0132] 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 human VH framework subtype VH3.

[0133] 13. The antibody variable domain according to any one of items 1 to 9, wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from

[0134] a. a combination of the framework regions HFW1, HFW2, HFW3, and HFW4 (i.e., the non-italic 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

[0135] b. a combination of framework regions HFW1, HFW2, HFW3, and HFW4 (i.e., non-italic 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, wherein there are 1, 2, or 3 mutations at positions other than 12, 101, 144, 146, and 148 (AHo numbering) within the framework region;

[0136] And wherein said HFW1, HFW3 and HFW4 have one or more substitutions as defined in any one of items 1 to 9.

[0137] 14. The antibody variable domain according to any one of items 1 to 9, wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from

[0138] a. a combination of the framework regions HFW1, HFW2, HFW3, and HFW4 (i.e., the non-italic 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

[0139] b. a combination of framework regions HFW1, HFW2, HFW3, and HFW4 (i.e., non-italic 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, wherein there are 1, 2, or 3 mutations at positions other than 12, 101, 144, 146, and 148 (AHo numbering) within the framework region;

[0140] And wherein said HFW1, HFW3 and HFW4 have one or more substitutions as defined in any one of items 1 to 9.

[0141] 15. The antibody variable domain according to any one of the preceding items, wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from

[0142] a. any one of SEQ ID NOs: 5-36 and 83-114, in particular a combination of the framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., the non-italic residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-36 and 86-114; and

[0143] b. A combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., non-italic 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, wherein there are 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework region.

[0144] 16. The antibody variable domain according to any one of the preceding items, wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from

[0145] a. 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-italic residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25 and 86-103; and

[0146] b. 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-italic residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25 and 86-103, wherein there are 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions.

[0147] 17. The antibody variable domain according to any of the preceding items, wherein the human antibody Vκ framework is selected from the Vκ1 framework subtype.

[0148] 18. The antibody variable domain according to any one of the preceding items, wherein LFW1, LFW2 and LFW3 are selected from

[0149] a. a combination of the framework regions LFW1, LFW2, and LFW3 (i.e., the non-italic residues in Table 5) of any one of SEQ ID NOs: 176, 177, 178, and 179; and

[0150] b. a combination of framework regions LFW1, LFW2, and LFW3 (i.e., non-italic residues in Table 5) of any one of SEQ ID NOs: 176, 177, 178, and 179, wherein there are 1, 2, or 3 mutations at positions different from 101 (AHo numbering) within the framework region;

[0151] and wherein said LFW3 optionally has the following substitutions (AHo numbering):

[0152] - Glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101.

[0153] 19. The antibody variable domain according to 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 NO: 188, 189, 190, 191, 192, 193, 194, 195 and 196.

[0154] 20. The antibody variable domain according to any one of the preceding items, wherein the variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are selected from

[0155] a. a combination of the framework regions LFW1, LFW2, LFW3, and LFW4 (i.e., the non-italic 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

[0156] b. A combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i.e., the non-italic 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, wherein there are 1, 2 or 3 mutations at positions different from 101 (AHo numbering) within the framework region.

[0157] 21. The antibody variable domain according to any one of the preceding items, wherein the form of the antibody variable domain is selected from Fv, dsFv, scFv and dsscFv, in particular selected from Fv and scFv.

[0158] 22. The antibody variable domain according to any of the preceding items, wherein the antibody variable domain, when in scFv format, has reduced binding to native anti-drug antibodies (ADA) present in human serum compared to a version of the antibody variable domain that does not contain the substitutions defined in item 1, as determined in a native ADA binding assay.

[0159] 23. The antibody variable domain of any one of items 13 to 16 and 20, wherein when in scFv format, the antibody variable domain is further characterized by one or more of the following features:

[0160] a. a melting temperature (Tm) of at least 63°C, particularly at least 64°C, particularly at least 65°C, as determined by differential scanning fluorimetry (DSF) when formulated in 20 mM histidine (pH 6.0);

[0161] b. When formulated in 20 mM histidine (pH 6.0) at a concentration of 10 mg / ml, the monomer content loss is less than 5%, particularly less than 3%, and particularly less than 2% after storage at 4 ° C for 28 days;

[0162] c. When formulated in 20 mM histidine (pH 6.0) at a concentration of 10 mg / ml, the monomer content loss after storage at 40°C for 28 days is less than 15%, particularly less than 10%, and particularly less than 8%;

[0163] 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%, particularly less than 10%, after storage at 4°C for 28 days;

[0164] e. When formulated in 20 mM histidine (pH 6.0) at a concentration of 10 mg / ml, the protein content loss is less than 10%, particularly less than 5%, after storage at 40°C for 28 days;

[0165] f. having a dissociation constant (KD) for binding to the target antigen that is lower or the same as or no more than 3-fold, particularly no more than 2.5-fold, and particularly no more than 2-fold, as measured by surface plasmon resonance (SPR), compared to the KD of a version of the antibody variable domain that does not comprise the substitutions defined in item 1.

[0166] 24. The antibody variable domain according to item 1, wherein the antibody variable domain comprises:

[0167] (i) a VH sequence selected from the group consisting of SEQ ID NO: 204 and a variant of SEQ ID NO: 204 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0168] and

[0169] (ii) a VL sequence selected from SEQ ID NOs: 205-206 and variants of SEQ ID NOs: 205-206 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0170] or

[0171] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 208-210 and variants of SEQ ID NOs: 208-210 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0172] and

[0173] (ii) a VL sequence selected from SEQ ID NOs: 211-212 and variants of SEQ ID NOs: 211-212 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0174] or

[0175] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 213-215 and variants of SEQ ID NOs: 213-215 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0176] and

[0177] (ii) a VL sequence selected from SEQ ID NOs: 216-217 and variants of SEQ ID NOs: 216-217 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0178] or

[0179] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 224-225 and variants of SEQ ID NOs: 224-225 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0180] and

[0181] (ii) a VL sequence selected from SEQ ID NOs: 226-229 and variants of SEQ ID NOs: 226-229 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0182] or

[0183] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 230-233 and variants of SEQ ID NOs: 230-233 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0184] and

[0185] (ii) a VL sequence selected from SEQ ID NOs: 234-237 and variants of SEQ ID NOs: 234-237 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0186] or

[0187] (i) a VH sequence selected from the group consisting of SEQ ID NO: 238 and a variant of SEQ ID NO: 238 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0188] and

[0189] (ii) a VL sequence selected from SEQ ID NO: 239 and a variant of SEQ ID NO: 239 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0190] or

[0191] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 240-242 and variants of SEQ ID NOs: 240-242 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0192] and

[0193] (ii) a VL sequence selected from variants of SEQ ID NOs: 243-245 and SEQ ID NOs: 236-238, said variants having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0194] or

[0195] (i) a VH sequence selected from the group consisting of SEQ ID NO: 254 and a variant of SEQ ID NO: 254 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0196] and

[0197] (ii) a VL sequence selected from SEQ ID NO: 255 and a variant of SEQ ID NO: 255 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0198] or

[0199] (i) a VH sequence selected from the group consisting of SEQ ID NO: 256 and a variant of SEQ ID NO: 256 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) in the framework regions,

[0200] and

[0201] (ii) a VL sequence selected from SEQ ID NO: 257 and a variant of SEQ ID NO: 257 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0202] or

[0203] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 261-262 and variants of SEQ ID NOs: 261-262 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0204] and

[0205] (ii) a VL sequence selected from SEQ ID NOs: 263-266 and variants of SEQ ID NOs: 263-266 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0206] or

[0207] (i) a VH sequence selected from the group consisting of SEQ ID NO: 267 and a variant of SEQ ID NO: 267 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) in the framework regions,

[0208] and

[0209] (ii) a VL sequence selected from SEQ ID NO: 268 and a variant of SEQ ID NO: 268 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0210] or

[0211] (i) a VH sequence selected from the group consisting of SEQ ID NO: 269 and a variant of SEQ ID NO: 269 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0212] and

[0213] (ii) a VL sequence selected from SEQ ID NO: 270 and a variant of SEQ ID NO: 270 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0214] (i) or a VH sequence selected from the group consisting of SEQ ID NOs: 271-272 and variants of SEQ ID NOs: 271-272 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework region,

[0215] and

[0216] (ii) a VL sequence selected from SEQ ID NO: 273 and a variant of SEQ ID NO: 273 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0217] or

[0218] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 274-275 and variants of SEQ ID NOs: 274-275 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0219] and

[0220] (ii) a VL sequence selected from SEQ ID NO: 276 and a variant of SEQ ID NO: 276 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region; or

[0221] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 277-278 and variants of SEQ ID NOs: 277-278 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0222] and

[0223] (ii) a VL sequence selected from SEQ ID NOs: 279-281 and variants of SEQ ID NOs: 279-281 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0224] or

[0225] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 282-283 and variants of SEQ ID NOs: 282-283 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0226] and

[0227] (ii) a VL sequence selected from SEQ ID NOs: 284-285 and variants of SEQ ID NOs: 284-285 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0228] or

[0229] (i) a VH sequence selected from the group consisting of SEQ ID NO: 286 and a variant of SEQ ID NO: 286 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) in the framework regions,

[0230] and

[0231] (ii) a VL sequence selected from SEQ ID NO: 287 and a variant of SEQ ID NO: 287 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0232] or

[0233] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 288-291 and variants of SEQ ID NOs: 288-291 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0234] and

[0235] (ii) a VL sequence selected from SEQ ID NOs: 292-295 and variants of SEQ ID NOs: 292-295 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0236] or

[0237] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 302-303 and variants of SEQ ID NOs: 302-303 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0238] and

[0239] (ii) a VL sequence selected from SEQ ID NOs: 304-305 and variants of SEQ ID NOs: 304-305 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0240] or

[0241] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 309-310 and variants of SEQ ID NOs: 309-310 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0242] and

[0243] (ii) a VL sequence selected from SEQ ID NOs: 311-312 and variants of SEQ ID NOs: 311-312 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0244] or

[0245] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 313-316 and variants of SEQ ID NOs: 313-316 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0246] and

[0247] (ii) a VL sequence selected from SEQ ID NOs: 317-318 and variants of SEQ ID NOs: 317-318 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0248] or

[0249] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 319-320 and variants of SEQ ID NOs: 319-320 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0250] and

[0251] (ii) a VL sequence selected from SEQ ID NOs: 321-322 and variants of SEQ ID NOs: 321-322 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0252] or

[0253] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 323-324 and variants of SEQ ID NOs: 323-324 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0254] and

[0255] (ii) a VL sequence selected from SEQ ID NO: 325 and a variant of SEQ ID NO: 325 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0256] or

[0257] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 330-332 and variants of SEQ ID NOs: 330-332 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0258] and

[0259] (ii) a VL sequence selected from SEQ ID NOs: 333-337 and variants of SEQ ID NOs: 333-337 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0260] or

[0261] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 338-340 and variants of SEQ ID NOs: 338-340 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0262] and

[0263] (ii) a VL sequence selected from SEQ ID NOs: 341-343 and variants of SEQ ID NOs: 341-343 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0264] or

[0265] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 344-346 and variants of SEQ ID NOs: 344-346 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0266] and

[0267] (ii) a VL sequence selected from SEQ ID NOs: 347-349 and variants of SEQ ID NOs: 347-349 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0268] or

[0269] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 350-352 and variants of SEQ ID NOs: 350-352 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0270] and

[0271] (ii) a VL sequence selected from SEQ ID NOs: 353-355 and variants of SEQ ID NOs: 353-355 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0272] or

[0273] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 356-358 and variants of SEQ ID NOs: 356-358 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0274] and

[0275] (ii) a VL sequence selected from the group consisting of SEQ ID NOs: 359-361 and variants of SEQ ID NOs: 359-361 having 1, 2 or 3 mutations at positions other than 101 (AHo numbering) within the framework region;

[0276] or

[0277] (i) a VH sequence selected from the group consisting of SEQ ID NOs: 362-364 and variants of SEQ ID NOs: 362-364 having 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) within the framework regions,

[0278] and

[0279] (ii) a VL sequence selected from SEQ ID NOs: 365-367 and variants of SEQ ID NOs: 365-367 having 1, 2 or 3 mutations at a position other than 101 (AHo numbering) within the framework region;

[0280] or

[0281] wherein the antibody variable domain is a scFv having a sequence selected from the group consisting of 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, wherein the variant has 1, 2, 3, 4 or 5 mutations at positions within the framework region that differ from VH 12, 101, 144, 146 and 148 (AHo numbering) and VL 101 (AHo numbering).

[0282] 25. An antibody comprising one or more antibody variable domains as defined in any one of items 1 to 24, wherein the one or more antibody variable domains are independently selected from the group consisting of Fv, dsFv, scFv and disulfide-stabilized scFv.

[0283] 26. The antibody according to item 25, wherein the antibody is

[0284] (i) monospecific and monovalent, bivalent, or trivalent against its target antigen;

[0285] (ii) bispecific, being monovalent or bivalent for each target antigen independently of the other;

[0286] (iii) trispecific and monovalent for each target antigen;

[0287] (iv) trispecific and bivalent against one of the target antigens and monovalent against the other target antigen;

[0288] (v) trispecific and bivalent against two of the target antigens and monovalent against the third target antigen;

[0289] (vi) tetraspecific and monovalent for each target antigen;

[0290] (vii) tetraspecific and bivalent against one of the target antigens and monovalent against the other target antigen; or

[0291] (viii) tetraspecific and bivalent against two of the target antigens and monovalent against the other target antigen.

[0292] 27. The antibody according to item 25, wherein the antibody comprises

[0293] (i) a first and a second antibody variable domain as defined in any one of items 1 to 24, wherein the first and the second antibody variable domain are specific for two different target antigens; and

[0294] (ii) one, two, three or four additional antibody variable domains as defined in any of items 1 to 24, which, independently of each other, have specificity for one of the target antigens of the first and second variable domains, or for a target antigen that is different from the target antigen of the first and second variable domains.

[0295] 28. The antibody according to any one of items 25 to 27, wherein the antibody format is selected from a bivalent bispecific format carrying an Fc, a trivalent bispecific and a tetravalent bispecific format carrying an Fc, and a trivalent bispecific IgG format and a tetravalent bispecific IgG format, wherein the trivalent or tetravalent format comprises one or more Fv, dsFv, scFv or disulfide-stabilized scFv;

[0296] More particularly, the format of the antibody is selected from Fc-bearing KiH-based formats; DVD-Ig; CODV-IgG and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), even more particularly selected from DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).

[0297] 29. The antibody according to item 28, wherein the IgG is selected from the IgG subclasses IgG1 and IgG4, in particular IgG4.

[0298] 30. The antibody according to any one of items 25 to 27, wherein the antibody does not comprise an immunoglobulin Fc region.

[0299] 31. The antibody according to claim 30, wherein the form of the antibody is selected from the group consisting of: tandem scDb (Tandab), linear dimeric scDb (LD-scDb), circular dimeric scDb (CD-scDb), tandem three-scFv, trimer (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, didiabody, tandem-di-scFv and MATCH.

[0300] 32. The antibody according to item 31, wherein the antibody does not comprise CH1 and / or CL regions.

[0301] 33. The antibody according to item 32, wherein the antibody is in the form of scDb-scFv, a trimer, a tetramer or a MATCH, in particular wherein the antibody is in the form of MATCH or scDb-scFv, more in particular wherein the antibody is in the form of MATCH, more in particular in the form of MATCH3 or MATCH4.

[0302] 34. The antibody according to any one of items 25 to 33, wherein the antibody has reduced binding to native anti-drug antibodies (ADA) present in human serum, in particular reduced binding to native ADA compared to a reference antibody not comprising the substitutions defined in item 1, as determined in a native ADA binding assay.

[0303] 35. The antibody of claim 25, wherein the antibody is a single-chain antibody having a sequence selected from the group consisting of SEQ ID NOs: 386-401, 426, 427, 440, 441 and variants of SEQ ID NOs: 386-401, 426, 427, 440, 441 having 1, 2, 3, 4 or 5 mutations at positions within the framework regions other than VH 12, 101, 144, 146 and 148 (AHo numbering) and VL 101 (AHo numbering);

[0304] or wherein the antibody is a heterodimer consisting of two chains having a pair of sequences 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: 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 variants; wherein each sequence in the sequence pair has 1, 2, 3, 4 or 5 mutations in the framework region that differ from positions VH 12, 101, 144, 146 and 148 (AHo numbering) and positions VL 101 (AHo numbering).

[0305] 36. A nucleic acid or two nucleic acids encoding the antibody variable domain of any one of items 1 to 24 or the antibody of any one of items 25 to 35.

[0306] 37. A vector or two vectors comprising the nucleic acid or the two nucleic acids of item 36.

[0307] 38. A host cell or a plurality of host cells comprising the vector or the two vectors of item 37.

[0308] 39. A method for producing the antibody variable domain of any one of items 1 to 24 or the antibody of any one of items 25 to 35, comprising (i) providing one or two nucleic acids of item 36, or one or two vectors of item 37, expressing the one or two nucleic acids, or the one or two vectors, and collecting the antibody variable domain or the antibody from the expression system, or (ii) providing one or more host cells of item 38, culturing the one or more host cells; and collecting the antibody variable domain or the antibody from the cell culture.

[0309] 40. A pharmaceutical composition comprising the antibody of any one of items 25 to 35 and a pharmaceutically acceptable carrier.

[0310] 41. The antibody according to any one of items 25 to 35, or the pharmaceutical composition according to item 40, for use as a medicament.

[0311] 42. A method for generating a modified antibody variable domain that has reduced binding to native anti-drug antibodies (ADA) present in human serum from healthy donors compared to its unmodified version, and wherein the reduced binding is determined by an ELISA-based native anti-drug antibody binding assay,

[0312] wherein the unmodified antibody variable domain binds to the target antigen and comprises

[0313] (i) a variable heavy chain (VH), comprising from N-terminus to C-terminus the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region and each HCDR indicates a heavy chain complementarity determining region,

[0314] and

[0315] (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region,

[0316] The method comprises the following steps:

[0317] Introduce one or more of the following substitutions (AHo numbering):

[0318] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0319] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146;

[0320] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0321] To obtain the modified antibody variable domain.

[0322] 43. The method according to item 42, wherein the variable heavy chain framework regions HFW1, HFW2 and HFW3 are selected from human VH framework subtypes VH1a, VH1b, VH3 and VH4, in particular are of human VH framework subtype VH3.

[0323] 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 a Vλ framework sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196.

[0324] 45. The method according to any one of items 42 to 44, wherein the heavy chain framework region of the unmodified antibody variable domain comprises one or more of the following amino acids:

[0325] - valine (V) or leucine (L), in particular leucine (L), at amino acid position 12;

[0326] - threonine (T) at amino acid position 101;

[0327] - threonine (T) or leucine (L), in particular leucine (L), at amino acid position 144;

[0328] - threonine (T) at amino acid position 146;

[0329] - Serine (S) at amino acid position 148.

[0330] 46. ​​The method according to any one of items 42 to 45, wherein the modified antibody variable domain further has one or more features as defined in any one of items 1 to 24.

[0331] 47. The method according to any one of items 42 to 46, wherein two or more substitutions are introduced in the heavy chain framework region of the unmodified antibody variable domain, the two or more substitutions being selected from:

[0332] - one of the substitutions shown at position 101, and

[0333] - one or more further substitutions selected from the group consisting of the substitutions shown at positions 12, 144, 146 and 148, in particular one or more further substitutions selected from the group consisting of the substitutions shown at positions 146 and 148, in particular one further substitution selected from the group consisting of the substitutions shown at position 146.

[0334] 48. A method for generating a modified antibody that has reduced binding to native anti-drug antibodies (ADA) present in human serum from healthy donors compared to its unmodified version, and wherein the reduced binding is determined by an ELISA-based native anti-drug antibody binding assay,

[0335] wherein the unmodified antibody is fragment-based or comprises one or more antibody-based fragments,

[0336] The method comprises the steps of introducing one or more of the following substitutions (AHo numbering) into the VH sequence of an unmodified fragment-based antibody or into the VH sequence of an unmodified antibody comprising one or more antibody-based fragments:

[0337] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0338] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146;

[0339] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0340] to obtain the modified antibody.

[0341] 49. The method according to item 48, wherein the modified antibody comprises an antibody variable domain as defined in any one of items 1 to 24.

[0342] 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, and wherein the one or more antibody variable domains are independently selected from the group consisting of Fv, dsFv, scFv and disulfide-stabilized scFv,

[0343] The method comprises the step of introducing one or more substitutions as defined in any one of claims 1 to 24 into the heavy chain framework region of at least one of the unmodified antibody variable domains. BRIEF DESCRIPTION OF THE DRAWINGS

[0344] Figure 1 Shown are the absorbance levels of native ADA against PRO1922 (A) and PRO2230 (B) in 20 human serum samples, as determined by the native ADA ELISA-based binding assay described in Example 2. Measurements were performed using spiked and unspiked serum samples (confirmation assay setup). To be considered specific, the signal reduction for spiked serum must be greater than 30% (>30% inhibition) compared to unspiked serum. In addition, the signal for unspiked serum must be above the baseline to be considered significant. The baseline is composed of the mean of all spiked sera for a sample plus three standard deviations (SCP, calculated as described in Example 2). The absorbance level is measured at 280 nm and is expressed in AU.

[0345] Figure 2The number of positive sera in 20 human serum samples for ten PRO2230 (α-PD-L1 and α-PD-L1 Repeat) scFv variants and ten PRO1922 (α-MSLN and α-MSLN Repeat) scFv variants compared to their respective wt (PRO2230 and PRO1922) and L12R-V103T-L144Q references is shown, as determined by the original ELISA-based ADA binding assay described in Example 2. Measurements were performed using spiked and unspiked serum samples. The reduction in signal for spiked serum compared to unspiked serum must be greater than 30% (>30% inhibition) to be assessed as specific. In addition, the signal for unspiked serum must be above the baseline to be considered significant. The baseline consists of the mean of all spiked sera of a sample plus three standard deviations (SCP, calculated as shown in Example 2). Variants without bar graphs mean that the original ADA-positive serum is zero.

[0346] Figure 3 Shown are the calculated midpoints [%] of the PEG precipitation curves for ten PRO2230 (a-PD-L1) scFv variants and ten 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. The midpoints are calculated as the half-maximal precipitation PEG concentration, with a 95% confidence interval (CI95%) for the mean. Error bars represent the standard error associated with the sigmoidal function fitted to the experimental data.

[0347] Figure 4 Results of chemical unfolding experiments using guanidine hydrochloride (Gua-HCl) for selected PRO1922 (MSLN) scFv variants and PRO2230 (PD-L1) scFv variants are shown. Depicted is a concentration that is half the final Gua-HCl concentration required for complete unfolding (c1 / 2). DETAILED DESCRIPTION

[0348] The present inventors surprisingly discovered that antibody variable domains substituted at one or more 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, bulky side chains (i.e., lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q)), when in scFv format, exhibit significantly reduced binding to pre-existing anti-drug antibodies (ADA) present in human serum compared to their unsubstituted versions. It was also discovered that additional substitutions at one or both of heavy chain framework positions 12 and 144 with alanine (A), lysine (K), or arginine (R) further reduced binding of the antibody variable domains (when in scFv format) to pre-existing anti-drug antibodies (ADA) present in human serum.

[0349] Although many methods and strategies have been developed to reduce the immunogenicity of antibody variable domains, from the perspective of drug development, the reduction in immunogenicity achievable by these methods is generally not as strong as desired. Therefore, there is still a large amount of unmet demand for antibody variable domains that show low immunogenicity. More specifically, it is hoped that there will be antibody variable domains on hand that exhibit low immunogenicity, particularly reduced binding to the original ADA, and that they can generally be used for the construction of antibody fragments. In addition, it is expected that these antibody variable domains provide high stability when integrated into the final antibody form, which will allow them to be applied to the construction of stable antibody fragments and fragment-based multispecific antibodies suitable for therapeutic agent development.

[0350] The antibody variable domains of the present invention can be successfully used to construct a variety of highly stable and functional scFvs, which can be easily used to construct multispecific antibody formats based on antibody fragments.

[0351] Unless defined otherwise, 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.

[0352] Unless otherwise indicated, the terms "including" and "comprising" are used herein in an open and non-limiting sense. With respect to these latter embodiments, the term "comprising" therefore encompasses the narrower term "consisting of."

[0353] Unless otherwise indicated herein or clearly contradicted by context, in the context of describing the present invention (particularly in the context of the following claims), the terms "a" and "an" and "the" and similar references should be construed to cover both the singular and the plural. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. When compounds, salts, etc. are used in the plural, this is also considered to refer to a single compound, salt, etc.

[0354] In one aspect, the present invention relates to an antibody variable domain that binds to a target antigen, comprising:

[0355] (i) a variable heavy chain (VH), comprising from N-terminus to C-terminus the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region and each HCDR indicates a heavy chain complementarity determining region,

[0356] wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, wherein said HFW1, HFW3 and HFW4 have one or more substitutions selected from the group consisting of:

[0357] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0358] - 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

[0359] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0360] and

[0361] (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region,

[0362] 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.

[0363] As used herein, the term "antibody" includes: complete antibodies or single chains thereof; any antigen-binding variable domains (i.e., "antigen-binding portions") or single chains thereof; and molecules (including but not limited to multispecific antibodies) comprising antibody CDRs, VH regions, or VL regions. A naturally occurring "complete antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), flanked by more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: 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 region of an antibody can mediate the binding of immunoglobulins 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.

[0364] As used herein, the term "antibody variable domain" refers to one or more parts of a complete antibody that have the ability to specifically bind to a given antigen (such as PD-L1, CD137, ROR1, MSLN, CD3, IL-4R, IL-31 or hSA). This can be any antigen-binding fragment (i.e., "antigen-binding portion") of a complete antibody or a single chain thereof; as well as molecules comprising antibody CDRs, VH regions or VL regions. Specifically, with respect to the multispecific antibodies of the present invention, as used herein, the term "antibody variable domain" refers in particular to an Fv fragment (Fv) consisting of the VL and VH domains of a single arm of an antibody; a disulfide-stabilized Fv fragment (dsFv); a single-chain Fv fragment (scFv); and a single-chain Fv fragment (scAB) fused with an additional light chain constant domain (CL). Preferably, the antibody variable domains of the present invention are selected from Fv fragments, disulfide-stabilized Fv fragments (dsFv) and scFv fragments. In a specific embodiment, the antibody variable domains of the present invention are single-chain Fv fragments (scFv). In other specific embodiments, the VL and VH domains of the scFv fragment are stabilized by interdomain disulfide bonds, particularly wherein the VH domain comprises a single cysteine ​​residue at position 51 (AHo numbering) and the VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0365] The term "complementarity determining region" ("CDR") refers to an amino acid sequence whose boundaries are defined using any of a number of well-known schemes, including 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 the numbering scheme described in Honegger & Plückthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering scheme). For example, for the classical form, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to Chothia, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52 -56 (HCDR2) and 95-102 (HCDR3); amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- The CDRs in the VH are numbered approximately as 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3) according to IMGT, and the CDRs in the VL are numbered approximately as 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (according to "Kabat" numbering).Under IMGT, the CDRs of an antibody can be determined using the program IMGT / DomainGap Align.

[0366] In the context of the present invention, unless otherwise specified, the numbering system suggested by Honegger & Plückthun ("AHo") is used (Honegger & Plückthun, J. Mol. Biol. 309 (2001) 657-670). Specifically, according to the AHo numbering scheme, the following residues are defined as CDRs: LCDR1 (also known as CDR-L1): L24-L42; LCDR2 (also known as CDR-L2): L58-L72; LCDR3 (also known as CDR-L3): L107-L138; HCDR1 (also known as CDR-H1): H27-H42; HCDR2 (also known as CDR-H2): H57-H76; HCDR3 (also known as CDR-H3): H108-H138. Accordingly, in the context of the present invention, the following residues are defined as light chain frameworks 1 to 4 (LFW1-LFW4) and heavy chain frameworks 1 to 4 (HFW1-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 the sake of clarity, the numbering system according to Honegger & Plückthun takes into account the length diversity found in naturally occurring antibodies, in both different VH and VL subfamilies, in particular in the CDRs, and provides for gaps in the sequence. Thus, in a given antibody variable domain, typically not all positions 1 to 149 are occupied by amino acid residues.

[0367] As used herein, the term "binding" refers to the ability of a single antibody to react with an antigenic determinant. However, this does not exclude that the single antibody may also react, for example, with homologues of the antigenic determinant (e.g., with antigenic determinants from other species) or other antigenic determinants belonging to the same protein family.

[0368] As used herein, the term "binding specificity" refers to the ability of a single antibody to react with a kind of antigenic determinant and not with different antigenic determinants. As used herein, the term "with ... specific binding" or "to ... specificity" refers to a measurable and reproducible interaction, such as the combination between a target and an antibody, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds to the target with an affinity greater than that of other targets, an avidity greater, more easily and / or longer lasting than that of other targets. However, these terms do not exclude that the single antibody can bind to the same antigenic determinant from different species with a suitable affinity. In its most general form (and when no specified standard is mentioned), "specific binding" refers to the ability of an antibody to distinguish between a target and an unrelated molecule, such as determined according to specific test methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) tests and peptide scanning. For example, standard ELISA test can be carried out. Can be scored by standard color development (for example, second antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). The reaction in some holes is scored by optical density (for example, at 450nm). Typical background (=negative reaction) may be about 0.1OD; Typical positive reaction may be about 1OD. This means that the ratio between positive and negative score can be 10 times or higher. In another example, SPR test can be carried out, wherein at least 10 times, particularly at least 100 times difference between background and signal shows specific binding. Usually, use one group of about three to five unrelated molecules such as milk powder, transferrin etc., rather than using single reference molecule, to carry out determination of binding specificity.

[0369] The antibody variable domains of the present invention bind to a target antigen, which can be any target antigen. Examples of target antigens include, but are not limited to, transmembrane molecules, receptors, ligands, growth factors, growth hormones, coagulation factors, anticoagulants, plasminogen activators, serum albumin, receptors for hormones or growth factors, neurotrophic factors, nerve growth factors, fibroblast growth factors, CD proteins, interferons, colony stimulating factors (CSF), interleukins (IL), T cell receptors, T cell co-stimulatory receptors such as CD137, surface membrane proteins, viral proteins, tumor-associated antigens, integrins or interleukins, VEGF; renin; human growth hormone; bovine growth hormone; 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; coagulation factor VIIIC; coagulation factor IX; tissue factor (TP); von Willebrand factor (von Willebrand factor) Willebrand's factor; protein C; atrial natriuretic factor; pulmonary surfactant; urokinase; human urine; tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor-α or -β; neprilysin; RANTES (regulated post-activation factor expressed and secreted by normal T cells); human macrophage inflammatory protein (MIP-1)-α; Müllerian inhibitory substance; relaxin A chain; relaxin B chain; pro-relaxin; mouse gonadotropin-related peptide; microbial protein, β-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); neurotrophins-3, -4, -5, or -6 (NT-3, NT-4, NT-5 or NT-6); NGF-β; platelet-derived growth factor (PDGF); aFGF; bFGF; epidermal growth factor (EGF); TGF-α; TGFβ, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; 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; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); 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; transporter; homing receptor; addressin; regulatory protein;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, p150.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin, or αv / β3 integrin; α or β subunit of a cell adhesion molecule; antibody); growth factor, VEGF; tissue factor (TF); TGF-β; α interferon (α-IFN); IL-8; IgE; Apo2 blood group antigen; 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.

[0370] The term "tumor-associated antigen (TAA)" refers to an antigen expressed on the surface of tumor cells. In certain embodiments, the TAA is an antigen that is preferentially expressed on tumor cells compared to non-tumor cells, particularly wherein the 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.

[0371] 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, CD33, CD38, CD44v6, CD79b, CD97, CDH3 (cadherin 3), CEA, CEACAM6, CLDN6, CLEC12A (CLL1), CSPG4, CYP1B1, EGFR, EGFRvlll, EpCAM, EPHA2, Ephrin B2, ERBB (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, mut hsp 70-2, 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-O-glycopeptide, TAG72, TARP, TEM1 / CD248, TEM7R, thyroglobulin, Tn antigen, Tn-O-glycopeptide, TPBG(5T4), TRP-2, TSHR, UPK2, and VEGFR2. Preferred examples are: 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 are: CD138, CD79b, CD123, MSLN, PSMA, BCMA, CD19, CD20, CEA, CD38, CD33, CLEC12a, and ROR1.

[0372] In a preferred embodiment, HFW1, HFW3 and HFW4 comprised in the antibody variable domains of the present invention have a lysine (K), aspartic acid (D), glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 146, and one or two substitutions selected from the group consisting of (AHo numbering):

[0373] - alanine (A), serine (S), lysine (K), arginine (R), asparagine (N), or glutamine (Q) at amino acid position 101; and

[0374] - Lysine (K) at amino acid position 148.

[0375] In other preferred embodiments, HFW1 and HFW4 comprised in the antibody variable domains of the present invention further have one or two substitutions selected from the group consisting of:

[0376] - 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

[0377] - Alanine (A), Lysine (K) or Arginine (R) at amino acid position 144.

[0378] In a specific embodiment, HFW1, HFW3 and HFW4 comprised in the variable domains of the antibodies of the invention have one of the following substitutions (AHo numbering):

[0379] 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;

[0380] 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;

[0381] 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;

[0382] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0383] d. Alanine (A), Lysine (K) or Arginine (R) at amino acid position 144; and

[0384] Leucine (L), lysine (K), or asparagine (N) at amino acid position 148;

[0385] e. Alanine (A), lysine (K), or arginine (R) at amino acid position 144;

[0386] 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

[0387] Leucine (L), lysine (K), or asparagine (N) at amino acid position 148;

[0388] 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;

[0389] Alanine (A), Lysine (K), or Arginine (R) at amino acid position 144; and

[0390] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0391] g. Alanine (A) or arginine (R) at amino acid position 12; and

[0392] 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;

[0393] h. Alanine (A) or Arginine (R) at amino acid position 12; and

[0394] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0395] i. Alanine (A) or arginine (R) at amino acid position 12;

[0396] 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;

[0397] Alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) at amino acid position 146;

[0398] j. Alanine (A) or arginine (R) at amino acid position 12;

[0399] 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;

[0400] Alanine (A), Lysine (K), or Arginine (R) at amino acid position 144; and

[0401] At amino acid position 146, alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q).

[0402] In other specific embodiments, HFW1, HFW3 and HFW4 comprised in the variable domains of the antibodies of the invention have one of the following substitutions (AHo numbering):

[0403] a. Alanine (A), lysine (K), arginine (R) or asparagine (N), or lysine (K), arginine (R) or asparagine (N) at amino acid position 101;

[0404] b. Lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0405] 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;

[0406] lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0407] d. Alanine (A) or Lysine (K) at amino acid position 144; and

[0408] Leucine (L) or asparagine (N) at amino acid position 148;

[0409] e. Lysine (K) at amino acid position 144;

[0410] Glutamic acid (E) at amino acid position 146; and

[0411] lysine (K) at amino acid position 148;

[0412] f. serine (S), arginine (R) or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101;

[0413] Alanine (A) or Lysine (K) at amino acid position 144; and

[0414] Arginine (R) or glutamine (Q) at amino acid position 146; or

[0415] g. Arginine (R) at amino acid position 12; and

[0416] serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101;

[0417] h. Arginine (R) at amino acid position 12; and

[0418] lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0419] i. Arginine (R) at amino acid position 12;

[0420] Serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101; and

[0421] lysine (K), aspartic acid (D), glutamic acid (E), or glutamine (Q) at amino acid position 146;

[0422] j. Alanine (A) at amino acid position 12;

[0423] serine (S), arginine (R), or glutamine (Q), or arginine (R) or glutamine (Q) at amino acid position 101;

[0424] Alanine (A) or Lysine (K) at amino acid position 144; and

[0425] Arginine (R) or glutamine (Q) at amino acid position 146.

[0426] Suitably, the framework regions HFW1, HFW2, HFW3, and HFW4 comprised in the variable domains of the antibodies of the invention are selected from human VH frameworks. Specifically, the framework regions HFW1, HFW2, and HFW3 comprised in the variable domains of the antibodies of the invention are selected from human VH framework subtypes VH1a, VH1b, VH3, VH4, VH5, or VH6, and in particular from human VH framework subtypes VH1a, VH1b, VH3, or VH4. In a specific embodiment, the framework regions HFW1, HFW2, HFW3, and HFW4 are selected from human VH framework subtype VH3. HFW4 can be selected from a HFW4 sequence of a human germline sequence or a rearranged human antibody sequence.

[0427] In the context of the present invention, the terms "belong to a human VHx framework subtype (or a human antibody Vκ / Vλ framework)", "selected from a human VHx framework subtype (or a human antibody Vκ / Vλ framework)" or "is of a human VHx framework subtype" mean that the framework sequences HFW1 to HFW3 (or LF1 to LFW3) show the highest degree of homology with the consensus sequence of said human antibody VH or VL framework subtype, as determined in Knappik et al., J. Mol. Biol. 296 (2000) 57-86 or WO 2019 / 057787. In the context of the present invention, based on the consensus sequence with Knappik et al., J. Mol. Biol. 296 (2000) 57-86 Figure 3 Or the sequence homology of the sequence shown in WO 2019 / 057787, the sequence of the human VH domain is divided into seven different framework subtypes, namely framework subtypes VH1a, VH1b, VH2, VH3, VH4, VH5 and VH6, also referred to herein as human subfamily 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 to 170 (i.e., the non-italic 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-italic residues in Table 5). Further examples of VH1a, VH1b, VH3 and VH4 sequences, as well as further examples of other VHx sequences can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57 / -86 or in WO 2019 / 057787.

[0428] In a specific embodiment, the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 of the antibody variable domain of the invention are selected from any one of SEQ ID NOs: 5-36 and 83-114, in particular a combination of the framework regions of any one of SEQ ID NOs: 8-36 and 86-114 (i.e., the non-italic residues in Tables 1 and 3, i.e., all residues not marked as CDR residues); and a combination of the framework regions of variants of SEQ ID NOs: 5-36 and 83-114, in particular a combination of the framework regions of variants of SEQ ID NOs: 8-36 and 86-114 (i.e., the non-italic residues in Tables 1 and 3), wherein 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 at positions other than 12, 101, 144, 146 and 148 (AHo numbering). In this regard, as various non-limiting examples, the term "mutation" refers to an addition, substitution or deletion. The VH region also includes a VH domain comprising at least positions 5 to 140 (AHo numbering), particularly at least positions 3 to 145, more particularly at least positions 2 to 147, of one of the sequences shown in SEQ ID NOs: 35-36 and 83-114, provided that the VH domain exhibits the functional characteristics defined in items 22 and 23 above.

[0429] In a specific embodiment, the variable light chain frameworks LFW1, LFW2 and LFW3 of the antibody variable domains of the present 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, based on the comparison with Knappik et al., J. Mol. Biol. 296 (2000) 57-86 Figure 3Or the sequence homology of the sequence shown in WO 2019 / 057787, the sequence of the human VL domain is divided into four different human Vκ framework subtypes, i.e. framework subtypes Vκ1, Vκ2, Vκ3 and Vκ4, and three different human Vλ framework subtypes, i.e. framework subtypes Vλ1, Vλ2 and Vλ3, also referred to herein as human Vκ subfamily Vκ1, Vκ2, Vκ3 and Vκ4, and human Vλ subfamily Vλ1, Vλ2 and Vλ3. In a specific embodiment, the variable light chain framework LFW1, LFW2 and LFW3 of the antibody variable domains of the present invention are Vκ1 framework subtypes. The specific example of Vκ1 framework subtype is represented by SEQ ID NOs: 176, 177, 178 and 179 (non-italic residues in Table 5). Further examples of Vκ1 sequences, as well as further examples of Vκ2, Vκ3 or Vκ4 sequences can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86.

[0430] In other specific embodiments, the variable light chain frameworks LFW1, LFW2, and LFW3 are selected from human antibody Vκ frameworks, preferably Vκ1 framework subtypes, and the variable light chain framework LFW4 is selected from Vλ framework subtypes. In a specific embodiment, the variable light chain framework LFW4 of the variable domain of the antibody of the present invention is selected from the group consisting of 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 comprises a single cysteine ​​residue at position 144 (AHo numbering) of the variable light chain (VL) and is particularly suitable for use in cases where a second single cysteine ​​is present in the corresponding variable heavy chain (VH), particularly at position 51 (AHo numbering) of VH, for forming interdomain disulfide bonds.

[0431] In a specific embodiment, the antibody variable domain of the invention comprises a variable light chain (VL), wherein

[0432] The variable light chain framework regions LFW1, LFW2 and LFW3 are selected from human antibody Vκ frameworks, particularly from the Vκ1 framework subtype, and the variable light chain framework region LFW4 is selected from a Vλ framework, and wherein the LFW3 has the following substitutions (AHo numbering):

[0433] - Glutamic acid (E), arginine (R) or glutamine (Q) at amino acid position 101.

[0434] In other specific embodiments, LFW1, LFW2 and LFW3 are selected from the combination of the framework regions LFW1, LFW2 and LFW3 of any one of SEQ ID NOs: 176, 177, 178 and 179 (i.e., the non-italic residues in Table 5); and the combination of the framework regions LFW1, LFW2 and LFW3 of any one of SEQ ID NOs: 176, 177, 178 and 179 (i.e., the non-italic residues in Table 5), wherein 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 at positions other than 101 (AHo numbering) in the framework region; and wherein said LFW3 optionally has glutamic acid (E), arginine (R) or a glutamine (Q) at amino acid position 101.

[0435] In other specific embodiments, the variable light chain frameworks LFW1, LFW2, LFW3 and LFW4 of the antibody variable domains of the invention are selected from the combination of the framework regions LFW1, LFW2, LFW3 and LFW4 of any one of SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187 (i.e., the non-italic residues in Tables 1, 3 and 5); and SEQ ID NOs: NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187 of any one of the framework regions LFW1, LFW2, LFW3 and LFW4 (i.e., the non-italic residues in Tables 1, 3 and 5), wherein at positions different from 101 (AHo numbering) in the framework region, 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 is mutated. In this regard, as various non-limiting examples, the term "mutation" refers to an addition, substitution or deletion. The VL region also includes a VL domain, which comprises at least positions 5 to 140 (AHo numbering) of one of the sequences shown in SEQ ID NOs: 37, 38, 39, 115, 116, 117, 180, 181, 182, 183, 184, 185, 186 and 187, in particular at least positions 3 to 145, more in particular at least positions 2 to 147, with the proviso that the VL domain exhibits the functional characteristics defined in items 22 and 23 above.

[0436] In a preferred embodiment, the form of the antibody variable domain of the present invention is selected from an Fv fragment consisting of the VL and VH domains of a single antibody arm; a disulfide bond-stabilized Fv fragment (dsFv); and a single-chain Fv fragment (scFv). In a specific embodiment, the antibody variable domain of the present invention is selected from an Fv fragment and a single-chain Fv fragment (scFv). In other specific embodiments, the VL and VH domains of the scFv fragment are stabilized by inter-domain disulfide bonds, in particular, the VH domain comprises a single cysteine ​​residue at position 51 (AHo numbering), and the VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0437] The antibody variable domains according to the present invention, when in scFv format, exhibit reduced immunogenicity compared to versions of said antibody variable domains that do not comprise the substitutions defined above in the VH framework region. More specifically, the antibody variable domains of the present invention, when in scFv format, exhibit reduced binding to native anti-drug antibodies (ADA) present in human serum, in particular reduced binding to native ADA compared to versions of said antibody variable domains that do not comprise the substitutions defined above in the VH framework region, as determined in a native ADA binding assay, in particular in the native ADA binding assay described in Example 2.

[0438] 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 (ADA) already present in the human sera of healthy and untreated individuals, referred to herein as "pre-existing ADA."

[0439] Therefore, for the purposes of the present invention, as used herein, the term "immunogenicity" refers to the ability of a therapeutic protein (e.g., an antibody, antibody fragment, or antibody binding domain) to be recognized by pre-existing ADA in a human serum sample. Without being bound by theory, it is believed that pre-existing ADA binding and the induction of ADA formation during treatment are related to the appearance of B cell and / or T cell epitopes on the therapeutic protein. The extent of this immunogenicity can be determined by ELISA assays and can be expressed as follows: the percentage or number of human serum samples containing measurable amounts of pre-existing ADA and / or ADA formed during therapeutic treatment (which recognize (i.e., bind) the therapeutic protein in question) relative to the total number of human sera tested (the percentage or number of positive serum samples). 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, the reduction in immunogenicity between the therapeutic protein and the corresponding therapeutic protein that has been modified to reduce its immunogenicity can be measured. A lower number or percentage of positive serum samples for the modified therapeutic protein indicates a reduction in immunogenicity relative to the original therapeutic protein.

[0440] When the ELISA signal exceeds a certain threshold, the serum sample is considered to contain a measurable amount of pre-existing ADA. This threshold is also referred to herein as the screening cut-point (SCP). The SCP can be calculated as defined below or set as an arbitrary 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.

[0441] When in scFv format, the antibody variable domains of the present invention also have favorable biophysical properties, especially excellent stability. Suitably, when in scFv format, the antibody variable domains of the present invention are also characterized by one or more of the following features:

[0442] a. a melting temperature (Tm) of at least 63°C, particularly at least 64°C, particularly at least 65°C, as determined by differential scanning fluorimetry (DSF) when formulated in 20 mM histidine (pH 6.0);

[0443] b. When formulated in 20 mM histidine (pH 6.0) at a concentration of 10 mg / ml, the monomer content loss is less than 5%, particularly less than 3%, and particularly less than 2% after storage at 4 ° C for 28 days;

[0444] c. When formulated in 20 mM histidine (pH 6.0) at a concentration of 10 mg / ml, the monomer content loss after storage at 40°C for 28 days is less than 15%, particularly less than 10%, and particularly less than 8%;

[0445] 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%, particularly less than 10%, after storage at 4°C for 28 days;

[0446] e. When formulated in 20 mM histidine (pH 6.0) at a concentration of 10 mg / ml, the protein content loss is less than 10%, particularly less than 5%, after storage at 40°C for 28 days;

[0447] f. It binds to the target antigen with a dissociation constant (KD) that is lower or no more than 3-fold compared to the KD of a version of the antibody variable domain that does not contain the substitution defined in item 1, as measured by surface plasmon resonance (SPR).

[0448] 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 transition midpoint of the thermal unfolding of the scFv construct was 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 were prepared in 20 mM histidine buffer (pH 6) and heated from 20°C to 95°C at a heating rate of 1°C / min. The unfolding event was monitored by changes in the intrinsic fluorescence of the protein, i.e., the tryptophan (Trp) fluorescence emission spectrum. The midpoint of unfolding (Tm) was defined as the inflection point (thermal midpoint) of the unfolding curve, observed as a local maximum or minimum of the first-order derivative.

[0449] The loss of monomer content was determined by calculating the area under the curve of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, as described in Chapter 621 of the United States Pharmacopeia (USP). This method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. The separation of the molecules occurs within the void volume (V0) and total permeate volume (V T). The monomer content of the scFv constructs was determined using SE-HPLC with a Shodex 402.5-4F KW column. 5 μg of scFv molecules at a given sample concentration (e.g., approximately 1 mg / ml or approximately 10 mg / ml) were injected for analysis. The monomer content was then determined by integrating the scFv monomer peak. A standard running buffer consisting of 50 mM sodium acetate and 250 mM sodium chloride (pH 6.0) was used for the analysis.

[0450] The inventors further found that, compared with their unsubstituted versions, the antibody variable domains of the present invention maintain their binding affinity to their respective target antigens. In this regard, the statement "maintaining their binding affinity" refers to that, as measured by surface plasmon resonance (SPR), the monovalent dissociation constant (KD) for the antibody variable domains of the present invention and its target antigen is equal to or lower than the KD of their respective unsubstituted versions (i.e., versions of the antibody variable domains not comprising the substitutions defined herein), or no more than 3 times greater than the KD of their respective unsubstituted versions (i.e., versions of the antibody variable domains not comprising the substitutions defined herein). Determining a single KD value by SPR is described in detail in Example 3.

[0451] As used herein, the term "affinity" refers to the strength of the interaction between an antibody or antibody variable domain and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody variable domain or antibody "arm" interacts with the antigen at many sites through weak non-covalent forces; the greater the interaction, the stronger the affinity.

[0452] "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 its binding partner (e.g., an antigen, or more specifically, an epitope on an antigen). Unless otherwise indicated, as used herein, "binding affinity" or "binding to" refers to 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 its partner Y can generally be expressed in terms of the dissociation constant (K D ) represents. Affinity can be measured by conventional methods known in the art, including the methods described herein. Low-affinity antibodies and antibody variable domains generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster 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 illustrative and exemplary embodiments for measuring binding affinity, i.e., binding strength, are described below.

[0453] The term "K assoc ”, “K a” or “K on ” refers to the on-rate of a particular antibody-antigen interaction, and the term “K dis ”, “K d ” or “K off " refers to the off-rate of a particular antibody-antigen interaction. In one embodiment, the term "K" as used herein D ” refers to the dissociation constant, which is given by K d With K a The ratio (K d / K a ) is obtained and expressed as molar concentration (M). In one embodiment, according to the present invention, "K D ” or “K D "KD value" or "KD value" is measured using a surface plasmon resonance assay.

[0454] In another aspect, the present invention relates to an antibody comprising one or more antibody variable domains of the present invention, wherein said one or more antibody variable domains are independently selected from the group consisting of Fv, dsFv, scFv and disulfide-stabilized scFv.

[0455] In certain embodiments, the antibodies of the present invention further comprise an antibody variable domain that is different from the antibody variable domain of the present invention. More specifically, the antibodies of the present invention further comprise an antibody variable domain that does not have a substitution in the framework region defined herein.

[0456] In preferred embodiments, the antibodies of the invention comprise only the antibody variable domains of the invention. More specifically, in these preferred embodiments, the antibodies of the invention comprise only the antibody variable domains having substitutions in the framework regions defined herein.

[0457] As used herein, the term "monovalent antibody" or "an antibody that is monovalent to its target antigen" refers to an antibody that binds to a single target molecule, more specifically, an antibody that binds to a single epitope on a target molecule. Additionally, 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.

[0458] In addition, as used herein, the term "binding domain" of an antibody or the term "antigen-binding fragment thereof" or the "antigen-binding portion" of an antibody, etc., refers to one or more parts of a complete antibody that have the ability to bind to a given antigen, especially specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by a fragment of a complete antibody. Specifically, with respect to the antibodies of the present invention, as used herein, the term "binding domain", or the term "antigen-binding fragment thereof" or "antigen-binding portion" etc. refers to a Fab fragment, i.e., a monovalent fragment consisting of 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 a specific embodiment, the binding domains of the antibodies of the present invention are independently selected from Fv fragments and single-chain Fv fragments (scFv). In other specific embodiments, the VL and VH domains of the scFv fragment are stabilized by interdomain disulfide bonds, particularly wherein the VH domain comprises a single cysteine ​​residue at position 51 (AHo numbering) and the VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0459] As used herein, the term "bivalent antibody" or "an antibody that is bivalent to its target antigen" refers to a single antibody that has two valencies, where "valency" is described as the number of antigen-binding moieties that bind to an epitope on a specific 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 moiety.

[0460] Likewise, as used herein, the term "trivalent antibody" or "an antibody that is trivalent toward its target antigen" 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 to up to three target molecules due to the presence of three copies of the corresponding antigen-binding portion.

[0461] In the case where the antibody of the present invention comprises two or three binding domains, the two or three binding domains bind to 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.

[0462] As used herein, the term "identical epitope" refers to a single protein determinant on a protein that is capable of specific binding to more than one antibody, wherein the single protein determinant is identical, i.e., consists of the same group of chemically active surface molecules such as amino acids or sugar side chains that have the same three-dimensional structural characteristics, and the same charge characteristics for each of said antibodies.

[0463] The term "different epitopes," as used herein in conjunction with a particular protein target, refers to individual protein determinants on a protein that are each capable of being specifically bound by a different antibody, wherein these individual protein determinants are distinct for the different antibodies, i.e., are composed of different chemically active surface molecular groups such as amino acids or sugar side chains, molecules having different three-dimensional structural characteristics, and molecules having different charge characteristics. These different epitopes may be overlapping or non-overlapping.

[0464] 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, wherein the format comprises one or more Fv, scFv, or disulfide-stabilized scFv. In particular, the antibody format is selected from KiH-based IgG, such as DuoBodies (bispecific IgG prepared by Duobody technology) (MAbs.2017Feb / Mar;9(2):182-212.doi:10.1080 / 19420862.2016.1268307); DVD-Ig; IgG-scFv fusion, such as CODV-IgG, Morrison (IgG CH3-scFv fusion (Morrison H) or IgG 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 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, such as DuoBodies; DVD-Ig; CODV-IgG and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), even more particularly selected from DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).

[0465] In this group of embodiments, the IgG is preferably selected from the IgG subclasses IgG1 and IgG4, in particular IgG4.

[0466] In a specific 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 molecules with an IgG Fc region, particularly an IgG4 Fc region. Two highly stable scFv binding domains (wherein the light chain comprises VκFR1 to FR3, in combination with VλFR4, and is based on the antibody variable domain of the present invention) are fused to the C-terminus of the heavy chain (Morrison-H) or light chain (Morrison-L) via a linker L1.

[0467] The linker L1 is a peptide of 2-30 amino acids, more particularly 5-25 amino acids, most particularly 10-20 amino acids. In a specific embodiment, the linker L1 comprises one or more units of four (4) glycine amino acid residues and one (1) serine amino acid residue (GGGGS) n , wherein n=1, 2, 3, 4 or 5 (SEQ ID NO: 197), in particular n=2 (SEQ ID NO: 198).

[0468] The VH region and the VL region of the two scFv domains are connected by a linker L2. The linker L2 is a peptide of 10-40 amino acids, more particularly 15-30 amino acids, and most particularly 20-25 amino acids. Specifically, the linker L2 comprises one or more units of four (4) glycine amino acid residues and one (1) serine amino acid residue (GGGGS) n , wherein n=1, 2, 3, 4, 5, 6, 7 or 8 (SEQ ID NO: 199), in particular n=4 (SEQ ID NO: 200).

[0469] In another set of embodiments, the antibodies of the invention do not comprise an immunoglobulin Fc region.

[0470] As used herein, the term "immunoglobulin Fc region" or "Fc region" is used to define the C-terminal region of the 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 altered Fc receptor binding function and / or reduced or inhibited Fab arm exchange. An example of such an engineered Fc region is knob-into-hole (KiH) technology (e.g., see 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 IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In particular, FcR is a native sequence of human FcR that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and splice forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 5: 203-234 (1997)). For an overview of FcRs, see 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). The term "FcR" herein includes other FcRs, including those to be identified in the future. 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.). In vivo binding to FcRn and the serum half-life of human FcRn high affinity binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides having variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0471] In this group of embodiments, the format of the antibody is preferably selected from the group consisting of: tandem scDb (Tandab), linear dimeric scDb (LD-scDb), circular dimeric scDb (CD-scDb), tandem three-scFv, trimer (Fab-(scFv)2), Fab-Fv2, trimer, scDb-scFv, tetramer, di-diabody, tandem-di-scFv and MATCH (described in WO 2016 / 0202457; Egan T. et al., MABS 9 (2017) 68-84).

[0472] In specific embodiments, the antibodies of the present invention further do not comprise a CH1 and / or CL region. In these specific embodiments, the antibodies are in scDb-scFv, triabody, tetrabody, or MATCH format, particularly MATCH or scDb-scFv format. More specifically, the antibodies of the present invention are in MATCH3 or MATCH4 format.

[0473] In specific embodiments, the antibodies of the invention are trispecific and tetravalent.

[0474] In a further embodiment, the antibodies of the invention are trispecific and trivalent.

[0475] The antibody variable domains contained in the bispecific, trispecific, tetraspecific or pentaspecific antibodies of the present invention are capable of binding to their respective antigens or receptors simultaneously. As used herein, the term "simultaneously" refers to the simultaneous binding of one of the antibody variable domains (e.g., specific for MSLN) and one or two additional antibody variable domains (e.g., specific for CD3 and hSA).

[0476] Suitably, the antibody variable domains comprised in the bispecific, trispecific, tetraspecific or pentaspecific antibody of the invention are operably linked.

[0477] As used herein, the term "operably connected" means that two molecules (e.g., polypeptides, domains, binding domains) are connected in a manner such that each molecule retains functional activity. Whether two molecules are directly connected or indirectly connected (e.g., through a joint, through a motif, through a joint connected to a motif), they can be "operably connected." The term "joint" refers to a peptide or other motif optionally located between the binding domain or antibody variable domains used in the present invention. A variety of strategies can be used to covalently link molecules together. These include, but are not limited to, polypeptide connections between the N-terminus and C-terminus of a protein or protein domain, connections through disulfide bonds, and connections through chemical cross-linking agents. In one aspect of this embodiment, a joint is a peptide bond produced by recombinant technology or peptide synthesis. For the specific case of connecting two polypeptide chains, selecting a suitable joint depends on various parameters, including but not limited to the properties of the two polypeptide chains (e.g., whether they are naturally oligomeric), the distance between the N-terminus and C-terminus to be connected (if known), and / or the stability of the joint to proteolysis and oxidation. In addition, the joint can include amino acid residues that provide flexibility.

[0478] In the context of the present invention, the term " polypeptide linker " refers to the joint consisting of an amino acid residue chain connected by a peptide bond, which connects two domains, and each domain is connected to one end of a joint. The length of the polypeptide linker should be enough to connect two molecules so that they have correct conformation each other, thereby maintaining required activity. In a specific embodiment, the polypeptide linker has a continuous chain of 2 to 30 amino acid residues (for example 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). In addition, the amino acid residue selected to be included in the polypeptide linker should show a characteristic that will not significantly interfere with polypeptide activity. Therefore, the linker peptide should not show an electric charge inconsistent with polypeptide activity as a whole, or interfere with internal folding, or form a key or other interactions with the amino acid residues in one or more monomers, which will seriously hinder the combination of the receptor monomer domain. In a specific embodiment, the polypeptide linker is an unstructured polypeptide. Useful linkers include glycine-serine or GS linkers. As will be understood by those skilled in the art, a "Gly-Ser" or "GS" linker refers to a polymer of glycine and serine in series (including, for example, (Gly-Ser) n 、(GSGGS) n(SEQ ID NO: 201), (GGGGS) n (SEQ ID NO: 202) and (GGGS) n (SEQ ID NO: 203) wherein n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers and other flexible linkers, such as tethers for shaker potassium channels, and a variety of other flexible peptide linkers. Glycine-serine polymers are preferred because oligopeptides comprising these amino acids are relatively unstructured and can therefore be used as neutral tethers between components. Secondly, serine is hydrophilic and therefore can dissolve substances that might be globular glycine chains. Thirdly, similar chains have been shown to effectively link subunits of recombinant proteins such as single-chain antibodies.

[0479] Suitably, the antibody variable domains of the present invention are isolated variable domains. Similarly, the antibodies of the present invention are also isolated antibodies. 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 antibodies with different antigenic specificities (e.g., an isolated antibody variable domain that specifically binds to mesothelin is substantially free of antibody variable domains that specifically bind to antigens other than mesothelin). In addition, the isolated variable domain or isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0480] Suitably, the antibody variable domains and antibodies of the present invention are monoclonal antibody variable domains and antibodies. As used herein, the term "monoclonal antibody variable domain" or "monoclonal antibody" refers to a variable domain or antibody that has substantially identical amino acid sequences or is derived from the same genetic source. A monoclonal variable domain or antibody exhibits binding specificity and affinity for a particular epitope, or exhibits binding specificity and affinity for multiple specific epitopes.

[0481] The antibody variable domains and antibodies of the present invention include, but are not limited to, chimeric, human, and humanized antibody variable domains and antibodies.

[0482] As used herein, the term "chimeric antibody" or "chimeric antibody variable domain" refers to an antibody molecule or antibody variable domain in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site (variable region) is connected to a constant region that is different or changed in class, effector function and / or species; or (b) the variable region or a portion thereof is changed, replaced or exchanged for a variable region with a different or changed antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region of a human immunoglobulin. Due to the replacement of the human constant region, the chimeric antibody can retain its specificity for recognizing the antigen while having reduced antigenicity to humans compared to the original mouse antibody.

[0483] As used herein, the term "human antibody" or "human antibody variable domain" is intended to include antibodies or antibody variable domains, the framework regions and CDR regions in their variable regions being derived from sequences of human origin. In addition, if the antibody or antibody variable domains include a constant region, the constant region is also derived from this human sequence, such as a mutant form of a human germline sequence or a human germline sequence. The human antibodies of the present invention and antibody variable domains may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). This definition of human antibodies or antibody variable domains specifically excludes humanized antibodies or antibody variable domains comprising non-human antigen-binding residues. Various techniques known in the art can be used to produce human antibodies and antibody variable domains, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227: 381 (1992); Marks et al., J. Mol. Biol, 222: 581 (1991)). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol, 147 (1): 86-95 (1991) methods described therein can also be used to prepare human monoclonal antibodies and human monoclonal antibody variable domains. 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 an antigen to a transgenic animal that has been modified to produce such antibodies and human antibody variable domains in response to an antigenic attack, but whose endogenous loci have been disabled, e.g., immune xenomice (About XENOMOUSE TM For information on human antibodies produced by human B cell hybridoma technology, see, eg, US Patent Nos. 6,075,181 and 6,150,584). See also, eg, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0484] As used herein, the term "humanized" antibody or "humanized" antibody variable domains refers to a kind of antibody or antibody variable domains, which retains the reactivity of non-human antibodies or antibody variable domains while having lower immunogenicity to the human race. For example, this can be achieved by the following method: retaining non-human CDR regions, and replacing the remainder of the antibody or antibody variable domains with human counterparts (i.e., the framework portion of constant region and variable region). Other framework region modifications can be carried out in the CDR sequence of the germline of human framework sequences and other mammalian species. The humanized antibodies of the present invention and antibody variable domains can include amino acid residues not encoded by human sequences (for example, by random or site-specific mutagenesis in vitro or by mutations introduced by somatic mutations in vivo, or conservative substitutions to promote stability or preparation). See, for example, 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 technology include, but are not limited to, the Xoma technology disclosed in US 5,766,886.

[0485] As used herein, the term "recombinant humanized antibody" or "recombinant humanized antibody variable domain" includes all human antibodies and human antibody variable domains that are prepared, expressed, created or isolated by recombinant means, such as antibodies and antibody variable domains isolated from host cells transformed to express humanized antibodies or humanized antibody variable domains, such as from a transfectoma, as well as antibodies and antibody variable domains prepared, expressed, created or isolated by any other means that involves splicing all or part of a human immunoglobulin gene sequence to other DNA sequences.

[0486] Preferably, the antibody variable domains and antibodies of the present invention are humanized. More preferably, the antibody variable domains and antibodies of the present invention are humanized and include rabbit-derived CDRs.

[0487] As used herein, the terms "bispecific antibody", "trispecific antibody", "tetraspecific antibody", "pentaspecific antibody" or the more general term "multispecific antibody" refer to antibodies that bind to two or more different epitopes on at least two or more different targets, for example, 2 different targets (bispecific), 3 different targets (trispecific), 4 different targets (tetraspecific) or 5 different targets (pentaspecific). Preferably, the antibodies of the present invention are bispecific, trispecific or tetraspecific, in particular bispecific or trispecific, more particularly trispecific. As described above, the term trispecific antibody refers to an antibody that binds to three different epitopes on at least three different targets (e.g., mesothelin, CD3 and hSA or PD-L1, CD137 and hSA).

[0488] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of chemically active surface groups of molecules (e.g., amino acids or sugar side chains) and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. "Conformational" epitopes are distinguished from "linear" epitopes in that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not lost.

[0489] As used herein, the term "conformational epitope" refers to antigenic amino acid residues that are clustered together on the surface of a polypeptide chain when it folds to form a native protein.

[0490] The term "linear epitope" refers to an epitope in which all points of interaction between a protein and an interacting molecule (eg, an antibody) occur linearly (contiguously) along the primary amino acid sequence of the protein.

[0491] As used herein, the term "recognizes" refers to an antibody or antibody variable domain finding and interacting with (eg, binding to) its conformational epitope.

[0492] The present inventors have found that the antibody variable domains of the present invention can be successfully applied to the construction of various antibody fragments (e.g., scFv fragments) and multispecific antibodies (e.g., bispecific and trispecific antibodies), which show significantly reduced immunogenicity and excellent stability when compared to their unmodified counterparts.

[0493] The antibody variable domains and multispecific antibodies of the present invention can be prepared using any convenient antibody preparation method known in the art (for the production of bispecific constructs, see, for example, Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14; for the production of bispecific diabodies and tandem scFvs, see, for example, Hornig, N. & - Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 and WO 99 / 57150). Specific examples of suitable methods for preparing multispecific constructs also include Genmab technology (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus technology (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750).

[0494] These methods generally involve the production of monoclonal antibodies or monoclonal antibody variable domains, for example, by fusing myeloma cells with spleen cells of mice that have been 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 technology (library cloning or phage display / yeast display) (see, e.g., Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8), and combining the antigen-binding domains of two or more different monoclonal antibodies, or fragments or portions thereof, using known molecular cloning techniques to obtain bispecific or multispecific constructs.

[0495] Multispecific, e.g., bispecific, trispecific, tetraspecific, or pentaspecific, and / or multivalent antibodies of the invention can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of these antibodies can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent binding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phthalimide (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 were SATA and sulfo-SMCC, both available from Pierce Chemical Co., Ltd. (Rockford, 111).

[0496] Alternatively, two or more binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This approach 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.

[0497] Binding of antibody variable domains and multispecific antibodies to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, biological assays (e.g., growth inhibition), or Western blot assays. Each of these assays typically detects the presence of a specific target protein-antibody complex by employing a labeled reagent (e.g., an antibody) specific for the target complex.

[0498] In another aspect, the invention provides a nucleic acid or two nucleic acids encoding an antibody variable domain or an antibody of the invention. Such nucleic acids can be optimized for expression in mammalian cells.

[0499] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this article, and refers to one or more deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, specific nucleic acid sequences also implicitly encompass conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences thereof, as well as sequences explicitly noted. Specifically, as described below, degenerate codon substitutions can be achieved by generating sequences in which the third 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).

[0500] The present invention provides substantially purified nucleic acid molecules encoding polypeptides comprising segments or domains of the above-mentioned antibody variable domains or antibodies. When expressed from suitable expression vectors, the polypeptides encoded by these nucleic acid molecules can exhibit the antigen-binding ability of the antibody variable domains or antibodies of the present invention.

[0501] The polynucleotide sequence can be generated from scratch by solid phase DNA synthesis or by PCR mutagenesis encoding an existing sequence of the antibody variable domains or antibodies of the present invention (e.g., the sequence described in the following examples). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as Narang et al., 1979, Meth. Enzy Mol. 68:90 phosphotriester method; Brown et al., Meth. Enzy Mol. 68:109,1979 phosphodiester method; Beaucage et al., Tetrahedron Lett., 22:1859,1981 diethylphosphoramidite method; and the solid support method of US 4,458,066. Introducing mutations into a polynucleotide sequence by PCR can be performed by methods described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (eds.), 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.

[0502] The present invention also provides expression vectors and host cells for producing the antibody variable domains or antibodies of the present invention.

[0503] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which other DNA segments can be connected. Another type of vector is a viral vector in which other DNA segments can be connected to a viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and episomal mammalian vectors with bacterial origins of replication) in the host cell they import. After importing the host cell, other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of the host cell, thereby replicating together with the host genome.

[0504] In addition, some vectors are capable of directing the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include expression vectors of this type in other forms with equivalent functions, such as viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In this specific context, the term "operably connected" refers to the functional relationship between two or more polynucleotide (e.g., DNA) fragments. Generally, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, it is operably connected to the coding sequence. Generally, the promoter transcriptional regulatory sequence operably connected to the transcribed sequence is physically continuous with the transcribed sequence, that is, they are cis. However, some transcriptional regulatory sequences, such as enhancers, do not have to be physically continuous or located near the coding sequence that enhances transcription.

[0505] Various expression vectors can be used to express polynucleotides encoding antibody variable structures or antibody chains. Both viral and non-viral expression vectors can be used to produce antibodies or antibody variable structures in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with expression cassettes for expressing proteins or RNA) and artificial chromosomes (see, for example, Harrington et al., Nat Genet. 15: 345, 1997). For example, non-viral vectors that can be used to express PD-L1- or MSLN-binding polypeptides or polynucleotides encoding such polypeptides in mammalian (eg, human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invitrogen, San Diego, Calif.), MPSV vectors and many other vectors known in the art for expressing other proteins. Useful viral vectors include those based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, those based on SV40, papillomaviruses, 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.

[0506] The selection of expression vectors depends on the expected host cell in which the vector is to be expressed. Typically, the expression vector comprises a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding a multispecific antibody chain or variable domain. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequence under non-inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. The culture of the transformed organism can be amplified under non-inducing conditions without biasing the population toward coding sequences in which the expression product is better tolerated by the host cell. In addition to the promoter, other regulatory elements may be required or desired for effective expression of the multispecific antibody chain or variable domain. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by including an enhancer suitable for the cell system in use (see, for example, 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 the CMV enhancer can be used to increase expression in mammalian host cells.

[0507] The vector to be used generally encodes the antibody variable domain or antibody light chain and heavy chain, including the constant region or part thereof, if present. Such vectors allow the variable region to be expressed as a fusion protein with the constant region, thereby resulting in the production of a complete antibody and its antibody variable domain. Typically, such constant region is human.

[0508] 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 these terms are intended to refer not only to the specific subject cell, but also to the progeny of that cell. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0509] The host cell carrying and expressing the antibody variable domains or antibodies of the present invention can be a prokaryotic organism or a eukaryotic organism. Escherichia coli is a prokaryotic host that can be used for cloning and expressing the polynucleotides of the present invention. Other applicable microbial hosts include bacillus, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia and various Pseudomonas. In these prokaryotic hosts, expression vectors can also be prepared, which generally include expression control sequences (e.g., origin of replication) compatible with the host cell. In addition, there will be any number of well-known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems or promoter systems of bacteriophage lambda. Promoters generally control expression (optionally together with operator sequences) and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express the antibody variable domains or multispecific antibodies of the present invention. Insect cells can also be used in combination with baculovirus vectors.

[0510] In one embodiment, mammalian host cells are used to express and produce antibody variable domains or antibodies of the present invention. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines with exogenous expression vectors. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including 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 described in Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89: 49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically 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 promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPS V promoters, tetracycline-inducible CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0511] The method for introducing the expression vector comprising the polynucleotide sequence of interest depends on the type of cell host. For example, calcium chloride transfection is generally used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts. (generally referring to Green, MR, and Sambrook, J., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press (2012)). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated conversion, injection and microinjection, ballistic method (ballistic method), virion, immunoliposome, polycation-nucleic acid conjugate, naked DNA, artificial virion, with the fusion of herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88 / 223, 1997), DNA absorption and in vitro transduction enhanced by reagent. In order to produce recombinant proteins for a long time and in high yield, stable expression is generally required. For example, the expression vector of the present invention can be used to prepare a cell line stably expressing the antibody variable domains of the present invention or antibody, and the expression vector comprises viral replication origin or endogenous expression element and selective marker gene. After the vector is introduced, the cells can be grown in a rich culture medium for 1 to 2 days and then switched to a selective culture 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 grow in a selective culture medium. Resistant, stably transfected cells can be propagated using tissue culture techniques suitable for the cell type. Therefore, the present invention provides a method for producing the antibody variable domains or antibodies of the present invention, wherein the method comprises the following steps: culturing a host cell, the host cell comprising a nucleic acid or vector encoding the antibody variable domains or antibodies of the present invention, thereby expressing the antibody variable domains or antibodies of the present invention.

[0512] On the one hand, the present invention relates to a method for producing an antibody variable domain or an antibody of the present invention, comprising the steps of: culturing a host cell, the host cell expressing one or two nucleic acids encoding the antibody variable domain or an antibody of the present invention. In particular, the present invention relates to a method for producing an antibody variable domain or an antibody of the present invention, the method comprising (i) providing one or two nucleic acids encoding the antibody variable domain or an antibody of the present invention, or one or two vectors encoding the antibody variable domain or an antibody of the present invention, expressing the one or two nucleic acids, or the one or two vectors, and collecting the antibody variable domain or the antibody from the expression system, or (ii) providing one or more host cells expressing one or two nucleic acids encoding the antibody variable domain or an antibody of the present invention, culturing the one or more host cells; and collecting the antibody variable domain or the multispecific antibody from the cell culture.

[0513] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to a medium or diluent that does not interfere with the structure of the antibody. A pharmaceutically acceptable carrier enhances or stabilizes the composition, or facilitates the preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0514] Some such carriers can prepare the pharmaceutical composition into, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. Some such carriers can prepare the pharmaceutical composition into, for example, injection, infusion, or topical administration. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution.

[0515] Pharmaceutical composition according to the present invention can further conventionally contain pharmaceutically acceptable concentration salt, buffer, preservative, auxiliary immunopotentiator (such as adjuvant and cytokine) and optional other therapeutic agents.Composition can also include antioxidant and / or preservative.As antioxidant, can mention thiol derivatives (such as monothioglycerol, cysteine, acetylcysteine, cystine, dithiothreitol (dithioerythreitol), dithiothreitol (dithiothreitol), glutathione), tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, sulfite (such as sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium pyrosulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiaretic acid.Suitable preservative can be such as phenol, chlorobutanol, benzyl alcohol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, benzalkonium chloride and cetylpyridinium chloride.

[0516] The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration depends on the desired result. Administration can be intravenous, intramuscular, intraperitoneal or subcutaneous administration, or administration near the target site. Pharmaceutically acceptable carriers 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 acid and other natural conditions that may inactivate the compound.

[0517] The pharmaceutical compositions of the present invention can be prepared according to methods well known and commonly practiced in the art. See, for example, 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. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or an effective dose of the antibody of the present invention is employed in the pharmaceutical compositions of the present invention. The antibody of the present invention is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus injection may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased according to the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the desired pharmaceutical carrier.

[0518] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as 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, without being toxic to the patient. The selected dosage level will depend upon 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 rate of excretion 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 prior medical history of the patient being treated, and the like.

[0519] In one aspect, the present invention relates to an antibody of the present invention or a pharmaceutical composition of the present invention for use as a medicament. In a suitable embodiment, the present invention provides a multispecific antibody or a pharmaceutical composition for use in treating a proliferative disease, such as cancer, or a disease selected from allergic, inflammatory and autoimmune diseases.

[0520] In another aspect, the present invention provides a pharmaceutical composition of the present invention for use in the preparation of a medicament for treating a proliferative disease such as cancer or a disease selected from allergic, inflammatory and autoimmune diseases.

[0521] In another aspect, the present invention relates to use of the antibody or pharmaceutical composition of the present invention for treating a proliferative disease such as cancer or a disease selected from allergic, inflammatory and autoimmune diseases in a subject in need thereof.

[0522] 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 suitable embodiment, the present invention relates to a method for treating a proliferative disease such as cancer or a disease selected from allergic, inflammatory and autoimmune diseases in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention.

[0523] The term "subject" includes human and non-human animals.

[0524] The term "animal" includes all vertebrates, such as non-human mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0525] 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 the sense of partially or completely curing a disease and / or attributing adverse effects of the disease or delaying the progression of the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, such as a human, and includes: (a) inhibiting the disease, i.e., preventing its development; and (b) relieving the disease, i.e., causing regression of the disease.

[0526] The term "therapeutically effective amount" or "effective amount" refers to the amount of an agent that, when administered to a mammal or other subject for treating a disease, is sufficient to cause such treatment of the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0527] In another aspect, the present invention relates to a method for modifying an antibody, wherein the antibody is fragment-based or comprises one or more scFv fragments, the method comprising the steps of introducing one or more of the following substitutions (AHo numbering) into the VH sequence of the fragment-based antibody or the VH sequence of the scFv fragment of the antibody:

[0528] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), or serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 101;

[0529] - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q) at amino acid position 146;

[0530] - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148;

[0531] to obtain modified antibodies;

[0532] wherein the modified antibody has reduced binding to native anti-drug antibodies (ADA) present in human serum from healthy donors compared to its unmodified version, and wherein the reduced binding is determined by an ELISA-based native anti-drug antibody binding assay.

[0533] In a specific embodiment of the last aspect, the modified antibody comprises an antibody variable domain according to the invention, as defined in the claims, items 1 to 24 or in the detailed description of the invention.

[0534] Partial sequence listing (residues are labeled according to the AHo numbering scheme, and CDRs are defined according to the Numab CDR definition, unless otherwise indicated)

[0535] Table 1. Examples of VH / VL sequences of PRO1922 variants according to the invention and VH / VL sequences of original (unmodified) PRO1922 and reference / comparative PRO1922 variants (modifications relative to original PRO1922 are shown in bold; CDR residues are shown in bold italics).

[0536]

[0537]

[0538]

[0539]

[0540] Table 2. Examples of scFv sequences of PRO1922 variants according to the invention and scFv sequences of original (unmodified) PRO1922 and reference / comparative PRO1922 variants (modifications relative to original PRO1922 and linker residues, if present, are shown in bold).

[0541]

[0542]

[0543]

[0544]

[0545]

[0546] Table 3. Examples of VH / VL sequences of PRO2230 variants according to the invention (modifications relative to the original PRO2230 are shown in bold; CDR residues are shown in bold italics).

[0547]

[0548]

[0549]

[0550] Table 4. scFv sequences of PRO2230 variants according to the invention and scFv sequences of original (unmodified) PRO2230 and reference / comparative PRO2230 variants (modifications relative to original PRO2230 and linker residues, if present, are shown in bold).

[0551]

[0552]

[0553]

[0554]

[0555] Table 5. Other sequences related to the present invention.

[0556]

[0557]

[0558] Table 6. Description of SEQ ID NOs for specific examples of VH and VL sequences of antibody variable domains and scFv sequences according to the present invention, as listed in the sequence listing.

[0559]

[0560]

[0561]

[0562]

[0563]

[0564] *) Original sequence (sequence without T101S and T146K variants) is disclosed in WO2014180577, WO2017158101, WO2017158079, WO2017158084, WO2018224443, WO2019072868, WO2018224439, WO2019072870, WO201 9072869, WO2018224441, WO2020157305, WO2020074584, WO2021089609, WO2021239 987, WO2022136669, WO2022084440, WO2022136675, WO2022136672, WO2022167460.

[0565] Table 7. Description of SEQ ID NOs of specific examples of multispecific antibody sequences according to the present invention, as listed in the sequence listing.

[0566]

[0567]

[0568]

[0569] *) Original sequence (sequence without T101S and T146K variants) is disclosed in WO2014180577, WO2017158101, WO2017158079, WO2017158084, WO2018224443, WO2019072868, WO2018224439, WO2019072870, WO201 9072869, WO2018224441, WO2020157305, WO2020074584, WO2021089609, WO2021239 987, WO2022136669, WO2022084440, WO2022136675, WO2022136672, WO2022167460.

[0570] Throughout this application, if there is a discrepancy between the text of the specification (eg, Tables 1 to 7) and the sequence listing, the text of the specification controls.

[0571] It should be 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 individual embodiments may also be provided individually or in any suitable subcombination. All combinations of embodiments relating to the invention are specifically contemplated by the invention and are disclosed herein as if each combination were individually and specifically disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically contemplated by the invention and are disclosed herein as if each such subcombination were individually and specifically disclosed herein.

[0572] The present invention is not limited to the scope of the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description. These modifications are intended to fall within the scope of the appended claims.

[0573] All patents, applications, publications, test methods, documents, and other materials cited herein are incorporated by reference to the extent permitted by their respective patent laws.

[0574] The following examples illustrate the invention described above, but are not intended to limit the scope of the invention in any way. Other test models known to those skilled in the relevant art can also determine the beneficial effects of the claimed invention.

[0575] Example

[0576] Example 1: Preparation of scFv variants according to the invention and reference scFv

[0577] 1.1. Preparation of variants of PRO1922 (MSLN binding scFv) and PRO2230 (PD-L1 binding scFv):

[0578] PRO1922, PRO2230, variants of PRO1922 and PRO2230 according to the present invention, and respective references have been produced according to the methods described in detail in patent applications WO 2019 / 072868 and WO 2021 / 239987.

[0579] Briefly, the PRO1922 and PRO2230 variants defined herein and their respective references were expressed in CHO cells using the ExpiCHO expression system (ThermoFisher). Expression was performed according to the manufacturer's instructions. Proteins (Protein L and / or Protein A) were purified from the clarified harvest by affinity chromatography. If necessary, the variant scFv was purified by SE-chromatography to a final monomer content of >95%. For quality control of the manufacturing material, standard analytical methods were used, such as ESI-MS, SE-HPLC, UV-Vis chromatography, and HPLC. 280 and SDS-PAGE.

[0580] For example, the quality of scFv has been verified by the following standard ESI-MS method. Prepared scFv was diluted 5-fold with 1% TFA. 2 μl of sample was injected into an ACQUITY UPLC@BioResolve-RP-mAb2.7μm 2.1x150 mm, The chromatographic column was loaded with 1% to 85% buffer B (0.1% formic acid, 25% propan-2-ol in acetonitrile) and desalted at 50°C at a flow rate of 200 μl / min. MS analysis was performed on a Synapt G2 mass spectrometer directly connected to a UPLC station. Mass spectra were acquired in positive ion mode by scanning the m / z range from 400 to 5000 Da with a scan duration of 1 second and an interscan delay of 0.1 second. Data were recorded using MassLynx 4.2 software (both Waters, UK). Where possible, recorded single peak m / z data were deconvoluted into mass spectra by applying the maximum entropy algorithm MaxEnt1 (MaxLynx).

[0581] The monomer content of the prepared scFvs was determined, for example, using the following standard SE-HPLC method. Using a Hitachi Chromaster HPLC system, 5 μg of the respective scFv, typically present at a concentration of 0.1 to 10 mg / ml, was injected onto a Shodex KW402.5-4F column at 25°C and a flow rate of 0.35 ml / min. The mobile phase was 50 mM sodium acetate, 250 mM sodium chloride in water, pH 6.0. The elution profile was monitored at 280 nm.

[0582] The produced PRO1922, PRO2230, variants of PRO1922 and PRO2230 according to the invention, and respective references are summarized in Table 8.

[0583] Example 2: Original ADA binding assay

[0584] 2.1. General test steps:

[0585] A method was developed at Numab to detect pre-existing anti-drug antibodies in human serum using a direct assay format.

[0586] A 96-well half-area plate is coated with 100 ng / ml of the test molecule (e.g., scFv molecule) for 2 hours at room temperature. The plate is blocked with PBS containing 0.2% Tween and 1% BSA for 1 hour. Individual human sera are then added at a dilution of 1:20 (5% serum) or 1:100 (1% serum), either unspiked (screening assay) or spiked (confirmatory assay) to the same molecule coated in the corresponding wells. The spike concentration ranges from 60 to 115 nM, and the spiked samples are pre-incubated for 1 hour. The antibodies bind to the molecules coated on the plate and are then detected with 100 ng / ml rabbit anti-human IgG-HRP for 1 hour. TMB substrate is added as a substrate, and after a brief incubation, the enzymatic reaction is stopped with 1 M HCl. The optical density of each well is read at 450 nm.

[0587] All steps were carried out 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 test components were added in a volume of 25 μl / well and used in duplicate. For the incubation step, the ELISA plate was placed on a rotary mixer (40 rpm).

[0588] Table 8: Variants of MSLN binding scFv PRO1922 and PD-L1 binding scFv PRO2230.

[0589]

[0590]

[0591] Typically, two measurements are performed for each test molecule: a first measurement in which untreated original human serum, i.e., unspiked human serum (screening assay), is applied to a plate coated with a single test molecule; and a second measurement in which human serum that has been pre-incubated with the same compound coated in the corresponding well, i.e., spiked human serum (confirming assay), is applied to a plate coated with a single test molecule to determine whether the antibody binding initially detected in each serum sample is 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 percentage of inhibition (% inhibition) is calculated as the reduction in the initial signal obtained with unspiked serum (screening assay) as follows:

[0592] % inhibition = 100*(1-(spiked serum signal / unspiked serum signal))

[0593] Only spiked samples with % inhibition values ​​above a certain threshold, called the confirmation cutoff point (% CCP) expressed as a percentage, are considered to represent specific binding (screening positive). The % CCP required to confirm specificity is set at 20-30% inhibition, or the % CCP is calculated from the average % inhibition of all serum samples, as described in Section 2.2. Typically, for analysis of native ADA binding signals, the % CCP is set at 30%. Samples with a % inhibition above the % CCP are considered positive.

[0594] To eliminate false positives caused by binders that initially showed very low or no binding to pre-existing ADA in the test serum samples (low or no binding for both unspiked and spiked serum samples), a screening cutoff point (SCP) was calculated from the spiked serum measurements for each plate. Unspiked samples with signals below the SCP were designated "screening negative" and were not considered. The SCP was calculated as described in Section 2.2.

[0595] 2.2. Determination / Calculation of Screening Cutoff Point (SCP) and Confirmation Cutoff Point (%CCP)

[0596] For each test compound, 20 individual human serum samples from healthy untreated subjects were analyzed.

[0597] Screening Cut-Off Point (SCP):

[0598] The screening cut-off point (SCP) is the threshold at which a signal is considered positive (screening positive). The screening cut-off point (SCP) is calculated from the spiked serum measurement as follows:

[0599] SCP = average value N + 3 x SDN

[0600] in:

[0601] - "Mean N" corresponds to the mean signal of all spiked individual sera measured for one specific test compound;

[0602] - "SDN" corresponds to the calculated standard deviation of all spiked individual sera measured for one specific test compound.

[0603] Calculation of the confirmation cut-off point (%CCP) expressed as a percentage:

[0604] The confirmation cut-off point (%CCP) expressed as a percentage is the threshold at which a % inhibition is considered specific (Specific Inhibition).

[0605] The confirmation cut-off point (%CCP) expressed as a percentage is either set to a specific value, such as a value in the range of 20.0% to 30.0%, or calculated as follows:

[0606] %CCP = mean [% inhibition] + 2.33 x standard deviation

[0607] in:

[0608] - "Average [% inhibition]" corresponds to the mean inhibition value (in percentage) of all spiked individual sera measured for one specific test compound;

[0609] - "SDN" corresponds to the calculated standard deviation of the [% inhibition] values ​​of all spiked individual sera measured for one specific test compound.

[0610] 2.3. Results of original ADA binding assays of PRO1922 and PRO2230 variants according to the present invention

[0611] PRO1922 wt, PRO2230 wt; 32 PRO1922 scFv variants according to the invention; 32 PRO2230 scFv variants according to the invention; and references PRO1922-L12R-V103T-L144Q (PRO2918), PRO1922-L12S-V103T-L144T (PRO2990), PRO2230-L12R-V103T-L144Q (PRO2903), and PRO2230-L12S-V103T-L144T (PRO2984) were analyzed for their ability to bind to pre-existing ADA. Measurements were performed directly in a validation assay using 19 to 20 human serum samples.

[0612] The detailed experimental procedures for PRO1922-based and PRO2230-based scFvs are as follows. The scFv was diluted to a concentration of 100 ng / ml in PBS, applied to a 96-well plate (Greiner, half area, high binding) in a volume of 25 μl, and incubated for 2 hours at room temperature. After incubation, the plate was washed three times and the wells were blocked for 1 hour at room temperature using 150 μl blocking buffer (PBS+0.1% Tween 20+1% BSA) per well. 20 human sera from 10 women and 10 men were ordered from Dunn Labortechnik for analysis. For unspiked samples, human serum was diluted to a concentration of 5% using a commercial low-cross buffer (Candor). The dilutions were as follows:

[0613] First dilution: 80 μl human serum stock solution + 720 μl low cross-linking buffer;

[0614] Second dilution: 250 μl of first dilution + 250 μl of low crossover buffer.

[0615] For spiked samples, human serum was diluted to a concentration of 10% using commercial low crossover buffer (Candor). scFv was also diluted to a concentration of 67 nM in low crossover buffer. The diluted serum and scFv were mixed in a 1:1 ratio to obtain a spiked 5% human serum. For example:

[0616] Dilute 5 μl of scFv stock to 67 nM with low-crossover buffer (volume depends on protein concentration);

[0617] 50 μl of first diluted human serum (10%) + 50 μl of diluted scFv (67 nM).

[0618] A positive control was incorporated into the assay that specifically bound to the human receptor framework of the scFv and could be tested later. This control antibody was diluted to 50 μg / ml using normal human serum (Merck Millipore) and further diluted into spiked and unspiked samples as follows.

[0619] For unspiked samples: 1 μl control antibody (50 μg / ml) + 49 μl normal human serum to obtain a concentration of 1 μg / ml; 30 μl of diluted antibody-human serum solution was further diluted 1:20 with 570 μl low-crossover buffer to obtain a final control antibody concentration of 0.05 μg / ml.

[0620] For spiked samples: dilute 1 μl of scFv stock to 500 nM using normal human serum (volume depends on protein concentration); add 1 μl of control antibody (50 μg / ml) + 49 μl of spiked normal human serum to a concentration of 1 μg / ml; further dilute 30 μl of the diluted antibody-spiked human serum solution 1:20 with 570 μl of low-crossover buffer to obtain a final control antibody concentration of 0.05 μg / ml.

[0621] The spiked samples (spiked serum and spiked positive control) were incubated for 1 hour before being allowed to add to the immobilized scFv. After blocking, the plates were washed three times again and rows AD were incubated with unspiked human serum and positive control, and rows EH were incubated with spiked human serum and spiked positive control for 15 minutes at room temperature in a volume of 25 μl per well. Afterwards, the plates were washed three times again and incubated with diluted rabbit anti-human Fc-IgG-HRP detection antibody at a concentration of 100 ng / ml for 1 hour at room temperature in a volume of 25 μl per well. Therefore, the antibody was diluted 1:8000 in low cross-dilution buffer. The detection antibody binds to the Fc region of any potential human pre-existing ADA in each serum as well as to the positive control antibody. Three more washing steps were performed and a volume of 25 μl per well of TMB peroxidase substrate was added. For this step, the 96-well plate was stored in the dark at room temperature for 15 minutes. HRP linked to the detection antibody catalyzes the oxidation of colorless TMB to blue TMB + After incubation in the dark, 25 μl of 1 M hydrochloric acid was added to terminate the enzymatic reaction, which further + TMB oxidized to yellow 2+ TMB was measured at 450 nm. 2+ The concentration of ADA is proportional to the amount of bound pre-existing ADA in the well.

[0622] The data were analyzed by first determining the number of serum samples that showed more than 30% inhibition (30% CCP). These serum samples were considered "screen positive". The screen positive sera were then further analyzed as described above by calculating the SCP for each individual plate and again taking into account a % CCP of 30%. Tables 9 and 10 summarize the number of positive serum samples for the tested molecules. Figure 1 Some example graphs for PRO1922 and PRO2230 are shown: absorbance levels of native ADA in human serum and the reduction in absorbance levels of spiked human serum. Figure 2Shown are representative comparisons of positive serum samples for 10 individual PRO2230 variants (PRO2945, PRO2942, RPO2941, PRO3349, PRO3325, PRO3346, PRO2936, PRO2931, PRO2930, PRO2938) and 10 individual PRO1922 variants (PRO2947, PRO2948, PRO2951, PRO2913, PRO2915, PRO2933, PRO2934, PRO3313, PRO3340, PRO3343) with wt and references PRO2230-L12R-V103T-L144Q and PRO1922-L12R-V103T-L144Q (identified in two independent rounds of original ADA assays).

[0623] Table 9: Number of positive serum samples for PRO1922 (MSLN binding) scFvs:

[0624]

[0625]

[0626] Table 10: Number of positive serum samples for PRO2230 (PD-L1 binding) scFvs:

[0627]

[0628] Example 3: KD determination by SPR

[0629] To determine the binding kinetics and affinity of PRO1922, PRO2230, and their mutant variants, human α-PD-L1Fc (Sino Biological, 10084-H02H, LC11NO2402) and human α-mesothelin (Acro Biosystems; MSN_H5223) were immobilized on a new CMD200M SPR sensor prism chip (Xantec) in an SPR 24 system (Sierra Sensor-Bruker) at a surface density of 100-200 response units (RU). Mutant variants at concentrations of 10 nM, 2 nM, and 0.4 nM were then injected onto the ligand-immobilized spots and reference spots. The wild-type variant was used as a control, while the blank spot was used as a reference.

[0630] The parameters for immobilizing the ligand onto the sensor chip are as follows:

[0631] Human MSLN: concentration: 4ug / ml; CT: 280s; FR: 15μl / min; target immobilization level: 700RU; sodium acetate: pH 4.0;

[0632] Human PD-L1: Concentration: 4ug / ml; CT: 120s; FR: 15μl / min; Target Immobilization Level: 500RU; Sodium Acetate pH 5.0.

[0633] After each cycle of analyte, the surface was regenerated with 3M MgCl2 solution, thus allowing the next analyte cycle to have only free ligand available. Binding affinity K D The binding association rate (k on ) and dissociation rate (k off ), which were calculated globally by fitting a 1:1 Langmuir model to the curves obtained from cycles with three different concentrations of analyte.

[0634] Compared to wt, the anti-PD-L1 variants showed little change in their binding affinity for PD-L1, as shown in Table 11. The binding affinities of the variants ranged from 10 to 100 pM, while the wild type showed an average binding affinity of 49 pM (n=4). The same situation was observed for variants with anti-MSLN binding specificity. The binding affinity of the MSLN-targeted variants ranged from 0.5 to 3.5 nM, while the wild type showed an average binding affinity of 1.8 nM (n=2). In summary, no significant differences were identified between the variants and the corresponding wild type in terms of binding affinity.

[0635] SPR measurements were performed at three concentrations of PRO1922 and PRO2230 scFv mutant variants to identify potential 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 the PRO1922 and PRO2230 scFv mutant variants. These results are not unexpected, as the introduced modifications are located far from the CDR regions and have minimal effects on the binding affinity of their respective binding partners.

[0636] Example 4: Thermal stability measurement using nanoDSF

[0637] The thermal stability of PRO1922, PRO2230 and their mutant variants at a concentration of 1 ± 0.1 mg / ml was analyzed using nanoDSF.

[0638] During nanoDSF measurements, molecules in solution are subjected to an increase in temperature, causing them to unfold. The thermal stability of PRO1922, PRO2230, and their mutants at 1±0.1 mg / ml was analyzed. Melting curves were generated by increasing the temperature from 20°C to 95°C at a rate of 1°C / min. To monitor unfolding events, the intrinsic fluorescence of the protein was used. Unfolding events cause the fluorescence emission spectrum of tryptophan (Trp) to shift due to a change in its environment, for example, from a hydrophobic core to a solvent-exposed Trp residue. The spectral shifts following unfolding were recorded at two wavelengths, 330 nm and 350 nm, and the data were analyzed using the 350 nm / 330 nm ratio. The midpoint of unfolding (Tm) was defined as the inflection point (thermal midpoint) of the unfolding curve, observed as a local maximum or minimum in the first-order 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. Proteins can have multiple thermal midpoints, depending on the number of individually unfolding domains. Protein concentration and buffer conditions further affect thermal stability. All proteins were measured in 20 mM histidine, pH 6. Protein unfolding was performed using a Prometheus apparatus (Nanotemper Technologies) and analyzed using "PR.ThermControl" v2.3.1 and "PR.Stability Analysis" v1.1 software.

[0639] Table 12 lists the thermal midpoints of all measurements of PRO1922-based and PRO2230-based scFvs measured at 1 mg / ml in 20 mM histidine, pH 6.0, as well as the onset temperature of unfolding. PRO2230wt and some PRO2230 mutant variants show two thermal midpoints in their first-order derivative curves. Although the second thermal midpoint (Tm2) can be calculated for all of these molecules, only the first thermal midpoint (Tm 1) can actually be calculated for some of these molecules because the maximum values ​​for all molecules cannot be clearly determined.

[0640] Many PRO2230-based variants showed very similar melting points, Tm2, ​​within ±2.5°C when compared to the wild type. Many PRO1922-based variants also showed very little difference, with their Tm1 within ±2.5°C when compared to the wild type.

[0641] Table 11: Summary of the binding affinities of α-PD-L1 and α-MSLN variants in the first round of selection.

[0642]

[0643] Table 12: Summary of melting temperatures and thermal unfolding onset temperatures of α-PD-L1 and α-MSLN variants in the first round of selection.

[0644]

[0645] Example 5: Stability Study

[0646] Long-term stability studies were performed at a concentration of 10 ± 0.5 mg / ml for 10 PRO1922-based variants and 10 PRO2230-based variants, as well as the reference molecules PRO1922-L12R-V103T-L144Q, PRO2230-L12R-V103T-L144Q, and wild type for two weeks (t2w 40°C) and four weeks (t4w 40°C) at 40°C, and for four weeks (t4w 4°C) at 4°C. After each time point, all proteins were analyzed for changes in concentration by absorbance at 280 nm and for monomer content by SE-HPLC.

[0647] 5.1 Protein concentration

[0648] First, the protein concentration of all stability study variants was determined after 4 weeks of storage at 40°C and 4°C. The results of the protein concentration study are summarized in Table 13. For both binding motifs, 7 of the 11 variants showed only minimal changes in concentration compared to the initial time point t0 after 4 weeks of storage at both 4°C and 40°C. Most PRO2230 variants showed a small but acceptable decrease in concentration after 4 weeks of storage at 4°C. Interestingly, the corresponding PRO1922 variants did not exhibit solubility issues after 4 weeks of storage at 4°C. Instead, they showed an increase in concentration, likely due to buffer evaporation.

[0649] 5.2 Monomer content determination

[0650] To determine the monomer content of each sample, SE-HPLC was used. The fraction of monomer and oligomer in the samples was estimated by integrating the SE-HPLC peak areas at different time points during the study.

[0651] Table 14 summarizes the results of the monomer content analysis. In general, most variants tested showed less than 10% loss of monomer content after 4 weeks of storage at 40°C, which is considered acceptable. Most variants showed suitable monomer stability, especially when a positively charged residue such as arginine or lysine was used at position T146 and a non-polar residue such as serine was used at position T101.

[0652] 5.3 Thermal stability measurement using nanoDSF at a protein concentration of 10 mg / ml

[0653] For selected variants, further nDSF measurements were performed at a protein concentration of 10 mg / ml. The results of the analysis are summarized in Table 15. The melting points determined for most mutant variants were very close to their respective wild-type ranges. No major differences in thermal stability were detected compared to the variants at a concentration of 1 mg / ml.

[0654] Example 6: Solubility testing by PEG precipitation test

[0655] Precipitation experiments using polyethylene glycol (PEG) were performed to analyze the apparent solubility of the 10 PRO1922-based variants and the 10 PRO2230-based variants, as well as the reference molecules PRO1922-L12R-V103T-L144Q, PRO2230-L12R-V103T-L144Q and wild-type PRO1922 and PRO2230.

[0656] There are other methods for assessing protein solubility, such as concentration (e.g., by ultrafiltration) or lyophilization. However, these methods require excess protein or present experimental problems, such as aggregation or gel formation. PEG precipitation is suitable for small amounts of protein (<1 mg) and measures solubility by precipitating the protein through the exclusion volume effect. As the PEG concentration increases, the protein begins to precipitate. Apparent solubility is measured at a PEG concentration of 8,000 (% w / v), at which point the protein concentration is halved (PEG 8,000). 中点 A 12-step gradient was established using 20 mM histidine (pH 6) and 37% (w / v) PEG8000 in 20 mM histidine (pH 6). Subsequently, 8 μl of protein at a concentration of 10 mg / ml was mixed with 72 μl of the PEG gradient solution using a 96-well plate. The plate was incubated overnight and the plate was washed with water. AcroPrep TM The solution was filtered into a second 96-deep-well plate using an Advance 96-well filter plate (1.2 μm) at 2000 × g for 2 minutes at room temperature. Protein concentration was measured in duplicate at 280 nm for all proteins and all dilutions, subtracting a reference wavelength of 310 nm.

[0657] The data were fitted using Graphpad Prism 9 software using a 4-parameter sigmoid fit (4PL, sigmoid, least squares fit) to determine PEG 中点 and 95% confidence interval. Higher PEG 中点 Values ​​correspond to higher apparent solubility of the protein.

[0658] Table 13: Changes in concentrations of PRO2230 and PRO1922 variants after 4 weeks of storage at 40°C and 4°C, starting from the initial time point t0.

[0659]

[0660]

[0661] Table 14: Monomer content as a function of time points t0, t2w and t4w at 40°C for stability study variants of PRO2230 and PRO1922.

[0662]

[0663] Table 15: Summary of melting temperatures and thermal unfolding onset temperatures of PRO2230 and PRO1922 variants.

[0664]

[0665] For most of the scFvs tested, PEG concentrations similar to those observed for the unmodified scFv were determined. 中点 .

[0666] The results of the PEG midpoint assay for the tested scFvs are shown in Figure 2. Figure 3 As shown. The higher the midpoint, the more soluble the protein. PRO1922-based scFvs showed, on average, a higher PEG midpoint than PRO1922-based scFvs. Overall, the tested scFv variants generally exhibited similar solubility relative to their wild-type counterparts.

[0667] Example 7: Determination of structural integrity by CEX-HPLC and cGE

[0668] For specific PRO1922 and PRO2230 mutant variants, further stability tests, namely CEXHPLC and cGE experiments, have been performed.

[0669] After four weeks of storage at 40°C, the formation of charge variants was investigated by CEX-HPLC analysis. During the stability study, charge variants were evaluated using analytical cation exchange chromatography. 30 μg of scFv were injected onto a MabPac SCX-10 (ThermoFisher Scientific) using a Hitachi Chromaster HPLC system. At 25°C, a pH gradient was run from pH 4 to pH 11 over 15 min, eluting the main peak and charged molecules at a flow rate of 0.85 ml / 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, and 9.9 mM formic acid. The pH was adjusted to 4 and 11 using NaOH. Materials eluting before or after the main peak were classified as acidic and basic molecules, respectively.

[0670] The initial target purities for PRO1922 and PRO2230 scFvs were 82.3% and 96.8%, respectively (see Table 16). After four weeks of storage at 40°C, the main peak area for PRO1922 decreased by 0.4% and that for PRO2230 by 0.3%, indicating excellent chemical stability. Similar target purities and changes in purity over time were observed for most scFvs. The largest decreases in purity were observed for PRO1922 variants, namely, L12A-T101S-L144A-T146Q (-5.7%), T101S-T146Q (-3.3%), and T101R-T146E (-2.9%). Overall, all variants demonstrated excellent stability.

[0671] Selected scFv molecules and parental scFv were further analyzed for fragmentation using reduced cGE.

[0672] Capillary gel electrophoresis (cGE) was performed using a ProteinExpress Chip on a LabChip GX Touch HT system (PerkinElmer) according to the manufacturer's protocol. scFv was diluted to 1 mg / ml with 20 mM histidine (pH 6), denatured at 70°C for 10 minutes, and analyzed under non-reducing and reducing conditions. Data were integrated using LabChip GX Reviewer Software (Perkin Elmer).

[0673] After four 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 (-3.7%). In summary, all variants exhibited excellent chemical stability.

[0674] Example 8: Chemical unfolding by guanidine hydrochloride

[0675] Chemical unfolding experiments were performed using guanidine hydrochloride for specific PRO1922 and PRO2230 scFv mutant variants. PRO1922 and PRO2230 scFv mutant variants were incubated in 20 mM histidine (pH 6.0) solutions in 24 concentrations ranging from 1.5 M to 5 M guanidine hydrochloride. The final protein concentration was 0.5 mg / ml. The samples were allowed to equilibrate overnight at room temperature, and their intrinsic fluorescence at 330 nm and 350 nm was measured using a Prometheus (Nanotemper Technologies). The c1 / 2 and ΔG values ​​were determined using the change in the fluorescence ratio (350 nm / 330 nm) using PR.Stability Analysis v1.1 software (Nanotemper Technologies). 解折叠 .

[0676] The results of chemical unfolding using guanidine hydrochloride are as follows Figure 4 The c1 / 2 values ​​for PRO1922 and PRO2230 were 3.9 M and 3.2 M, respectively. With the exception of the T101R variant, which exhibited a slightly decreased c1 / 2, all PRO1922 and PRO2230 scFv mutant variants exhibited similar stability in the presence of increasing guanidine hydrochloride concentrations.

[0677] Example 10: Determination of hydrophobicity by hydrophobic interaction chromatography (HIC)

[0678] HIC-HPLC was performed using a Pro-Pac HIC-10 column (5 μm, The hydrophobicity of specific PRO1922 and PRO2230 scFv mutant variants was determined using a 4.6x100 mm column. 20 μg of protein was injected and eluted from the column using a gradient of 1.5 M to 0 M (NH₄)₂SO₄ in 20 mM histidine (pH 5.5) over 12 minutes at a flow rate of 1 ml / min. Retention times were then correlated with corresponding (NH₄)₂SO₄ concentrations. To determine the (NH₄)₂SO₄ concentration, a 2.81 ml delay volume was used to calculate the column volume and the dead volume of the system.

[0679] HIC-HPLC data show that the apparent hydrophobicity between PRO1922 and PRO2230 scFv mutant variants has only very small changes. Although the hydrophobic amino acid is replaced by polar or charged amino acid, the impact on the overall apparent hydrophobicity is very low (data not shown). However, for some molecules, the hydrophobicity of PRO1922 and PRO2230 scFv variants is observed to increase slightly. In particular, this relates to molecules with polar modifications at positions 101 and 146, i.e., T101Q-T146Q, T101N-T146Q, and T101S-T146S. In addition, T101R-T146E also causes a slight increase in hydrophobicity. Interestingly, the only molecule with a corresponding single mutation that also causes a slight increase in hydrophobicity is T101Q. However, because these changes are very small, they are considered to be unrelated.

[0680] Table 16. Purity of PRO1922 and PRO2230 scFvs determined by CEX-HPLC after 4 weeks incubation at 40°C.

[0681]

[0682]

[0683] *Double peaks were integrated into a single target peak.

[0684] #Increase in purity caused by modification of the acidic variant and subsequent modifications.

[0685] Table 17: Purity of PRO1922 and PRO2230 scFvs determined by cGE analysis after 4 weeks incubation at 40°C.

[0686]

Claims

1. A method for generating a modified antibody variable domain having reduced binding to native anti-drug antibodies (ADA) present in human serum from healthy donors compared to its unmodified version, and wherein the reduced binding is determined by an ELISA-based native anti-drug antibody binding assay, wherein the unmodified antibody variable domain binds to a target antigen and comprises: (i) a variable heavy chain (VH), comprising from N-terminus to C-terminus the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region, and each HCDR indicates a heavy chain complementarity determining region, and (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region, The method comprises the following steps: introducing one or more of the following substitutions (AHo numbering) in the heavy chain framework region 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), or 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; - Leucine (L), Lysine (K) or Asparagine (N) at amino acid position 148; To obtain the modified antibody variable domain.

2. The method of claim 1, 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; and / or, wherein the variable heavy chain framework regions HFW1, HFW2 and HFW3 are selected from human VH framework subtypes VH1a, VH1b, VH3 and VH4, in particular human VH framework subtype VH3.

3. The method according to any one of claims 1 or 2, wherein: The heavy chain framework region of the unmodified antibody variable domain comprises one or more of 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.

4. The method according to any one of claims 1 to 3, wherein the one or more substitutions introduced are selected from (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.

5. The method according to any one of claims 1 to 4, wherein: The method further comprises introducing one or two substitutions selected from the group consisting of: - 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.

6. The method according to any one of claims 1 to 5, wherein: The heavy chain framework region of the unmodified antibody variable domain has one of the following substitutions introduced (AHo numbering): a. Alanine (A), lysine (K), arginine (R), or asparagine (N) at amino acid position 101, or Lysine (K), arginine (R), or asparagine (N); 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. 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) 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; serine (S), 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) 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.

7. A method for generating a modified antibody having reduced binding to native anti-drug antibodies (ADA) present in human serum from healthy donors compared to its unmodified version, and wherein the reduced binding is determined by an ELISA-based native anti-drug antibody binding assay, wherein the unmodified antibody comprises one or more unmodified antibody variable domains as defined in any one of claims 1 to 3, and wherein the one or more antibody variable domains are independently selected from the group consisting of Fv, disulfide-stabilized Fv, scFv and disulfide-stabilized scFv, The method comprises the following steps: One or more substitutions as defined in any one of claims 1, 4, 5 and 6 are introduced in the heavy chain framework region of at least one of the unmodified antibody variable domains.

8. An antibody variable domain that binds to a target antigen, comprising: (i) a variable heavy chain (VH), comprising from N-terminus to C-terminus the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW indicates a heavy chain framework region, and each HCDR indicates a heavy chain complementarity determining region, The variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from human VH frameworks, wherein The HFW1, HFW3 and HFW4 have one or more substitutions selected from the group consisting of (AHo numbering): - alanine (A), serine (S), lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), asparagine (N) or glutamine (Q), or 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 (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW indicates a light chain framework region, and each LCDR indicates a light chain complementarity determining region, 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, a Vλ framework sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, 192, 193, 194, 195 and 196.

9. The antibody variable domain of claim 8, wherein said HFW1, HFW3 and HFW4 have one or more substitutions as defined in any one of claims 1 to 6.

10. The antibody variable domain according to any one of claims 8 or 9, wherein the HFW1, HFW3 and HFW4 have: - one of the substitutions shown at position 101, and - one or more further substitutions selected from the group consisting of the substitutions shown at positions 12, 144, 146 and 148, in particular one or more further substitutions selected from the group consisting of the substitutions shown at positions 146 and 148, in particular one further substitution selected from the group consisting of the substitutions shown at position 146.

11. The antibody variable domain according to any one of claims 8 to 10, wherein the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from a. any one of SEQ ID NOs: 8-25, 28, 30-36, 86-103, 106 and 108-114, in particular a combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i.e., non-italic residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25 and 86-103; and b. 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-italic residues in Tables 1 and 3) of any one of SEQ ID NOs: 8-25 and 86-103, wherein there are 1, 2 or 3 mutations at positions other than 12, 101, 144, 146 and 148 (AHo numbering) in the framework regions; And among them, 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-italic 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. A combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i.e., the non-italic 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, wherein there are 1, 2 or 3 mutations at positions different from 101 (AHo numbering) in the framework region.

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

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

14. The antibody of claim 13, wherein the antibody is a single-chain antibody having a sequence selected from the group consisting of SEQ ID NOs: 386-401, 426, 427, 440, 441 and variants of SEQ ID NOs: 386-401, 426, 427, 440, 441 having 1, 2, 3, 4 or 5 mutations at positions different from VH 12, 101, 144, 146 and 148 (AHo numbering) and VL 101 (AHo numbering) within the framework region; or wherein the antibody is a heterodimer consisting of two chains having a pair of sequences 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: 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 variants; wherein each sequence in the sequence pair has 1, 2, 3, 4 or 5 mutations in the framework region at positions different from VH 12, 101, 144, 146 and 148 (AHo numbering) and VL 101 (AHo numbering).

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

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

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

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

19. A pharmaceutical composition comprising the antibody variable domain of any one of claims 8 to 12, or the antibody of claims 13 to 14, and a pharmaceutically acceptable carrier.

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