Novel monoclonal antibodies to L-thyroxine and their diagnostic uses

Abstract

The present invention provides a novel monoclonal antibody that specifically binds to L-thyroxine (T4), as well as compositions and kits that include such antibodies.Furthermore, polynucleotides encoding such monoclonal antibodies, host cells expressing said antibodies, methods for producing such antibodies, and diagnostic methods using such monoclonal antibodies are provided.The monoclonal antibody of the present invention comprises a heavy chain variable domain (VH) that includes V or A at position 33; Y at position 50; W at position 52; I at position 98, G, A or V at position 99; Y at position 100; and I at position 100b, and a light chain variable domain (VL) that includes amino acids H or Y at position 28; N or K at position 29; W at position 32; G or A at position 91; Y, W or F at position 92; S or T at position 93; Y or F at position 95b; N, S, T or Q at position 95c; and H at position 96, and the positions of the amino acids in VH and VL are indicated according to the Kabat numbering scheme, respectively.

Description

[Technical field]

[0001] The present invention provides novel monoclonal antibodies against L-thyroxine (T4), as well as compositions and kits comprising such antibodies. Additionally provided are polynucleotides encoding such monoclonal antibodies, host cells expressing the antibodies, methods of producing such antibodies, and diagnostic methods using such monoclonal antibodies. [Background technology]

[0002] L-thyroxine hormone (hereafter referred to as L-T4 or T4; CAS number: 51-48-9) is the main thyroid hormone secreted by the thyroid gland into the bloodstream. Together with triiodothyronine (T3), it plays an important role in regulating the body's metabolic rate, affects the cardiovascular system, growth and bone metabolism, and is important for gonadal function and normal development of the nervous system (Kronenberg HM et al. Williams Textbook of Endocrinology. Saunders Elsevier, Philadelphia, 12th edition, 2011, chapter 10, p. 301-311). T4 circulates in the bloodstream as an equilibrium mixture of free and serum-bound hormone. Free T4 (fT4) is the unbound biologically active form and represents only 0.03% of total T4. The remaining T4 is inactive and binds to serum proteins such as thyroxine-binding globulin (TBG) (75%), prealbumin (15%), and albumin (10%) (Robbins J, Rall JE. Recent Prog Horm Res 1957; 13: 161-208; Oppenheimer JH. N Engl J Med 1968; 278 (21): 1153-1162; DeGroot LJ, Larsen PR, Hennemann G. Wiley and Sons, New York, 1984: 62-65; Ekins RP. Endocr Rev 1990; 11 (1): 5-46). Measurement of free T4 has the advantage of being independent of changes in the concentration and binding properties of these binding proteins, and therefore, additional determination of binding parameters (T uptake, TBG) is unnecessary. Free T4 is therefore a useful tool in clinical routine diagnostics for the evaluation of thyroid status.It should be measured together with TSH if thyroid disorders are suspected and also appropriate for monitoring thyroid suppression therapy (Kronenberg HM, Melmed S, Polonsky KS, et al. Williams Textbook of Endocrinology. Saunders Elsevier, Philadelphia, 12th edition, 2011, chapter 10, p. 301-311; Wu AHB. Tietz Clinical Guide To Laboratory Tests. Saunders Elsevier, Philadelphia, 4th edition, 2006, section II, p. 1076-1077; Brent GA. Thyroid Function Testing. Springer, Berlin, 1st edition, 2010, chapter 5, p. 86-88).

[0003] Various methods are available for estimating free thyroid hormone levels. Direct measurement of fT4 and fT3 by equilibrium dialysis or ultrafiltration is mainly used as a reference method to standardize immunological procedures commonly used for routine diagnostic purposes (Wu AHB. Tietz Clinical Guide To Laboratory Tests. Saunders Elsevier, Philadelphia, 4th edition, 2006, section II, p. 1076-1077; Brent GA. Thyroid Function Testing. Springer, Berlin, 1st edition, 2010, chapter 5, p. 86-88).

[0004] Some commercially available fully automated fT4 and total T4 immunoassays use polyclonal antibodies, for example produced in sheep or rabbits. However, several drawbacks arise from the use of polyclonal antibodies for routine in vitro diagnostic products: 1) Due to their origin from animal serum, the majority of the antibody preparation is initially present as non-specific IgG, so that target-specific antibodies usually have to be purified using, for example, affinity chromatography, which exposes the antibodies to unfavourable conditions, such as low pH or other denaturing conditions, during the elution step, which may result in an increase in the hydrophobicity of the purified antibodies due to partial unfolding. Even loss of high affinity antibodies may occur if they cannot be eluted at all from the target without complete denaturation of the antibodies. 2) Due to the nature of the mammalian immune system, the quality of the produced antibodies can be highly variable between different animals in terms of the distribution of affinities and specificities, and also bleeding from a single animal taken consecutively over time will inevitably vary in terms of the affinity, specificity and also the distribution of concentrations of the target-specific antibodies. Obviously, this raises the issue of lot-to-lot variability of the antibody preparation itself, but also the risk of altered behavior of immunoassays generated with different lots of purified polyclonal antibodies.

[0005] Despite the drawbacks of polyclonal antibodies, commercially available immunoassays based thereon, such as in particular Elecsys® FT4 III (material number 07976836190), show good analytical sensitivity and low cross-reactivity with respect to cross-reactants.

[0006] Even though powerful methods for generating monoclonal antibodies are available in the prior art (e.g., KOHLER, G., MILSTEIN, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497 (1975). https: / / doi.org / 10.1038 / 256495a0), it is not trivial to replace polyclonal antibodies in immunoassays without losing signal kinetics and analyte specificity. In particular, for hapten analytes such as L-T4, it is difficult to identify monoclonal antibodies that provide good kinetic characteristics without exhibiting undesirable cross-reactions with structurally closely related structures.

[0007] Thus, there is a strong need to provide new monoclonal L-T4 antibodies with excellent kinetic characteristics that can adequately discriminate L-T4 (e.g., fT4) from structurally related substances / derivatives. Furthermore, there is a need to provide L-T4 (e.g., fT4) immunoassays based on monoclonal antibodies that exhibit similar or even improved analytical performance and / or specificity with respect to cross-reactants than previously available and well-accepted assays based on polyclonal antibodies. Summary of the Invention

[0008] The above needs are addressed by the invention described herein.

[0009] In a first aspect, the present invention relates to monoclonal antibodies that specifically bind L-thyroxine (T4). Among a set of 840 monoclonal antibodies that bind to T4, the inventors surprisingly found that they have exceptionally high association rate constants (k aWe have identified a family of three closely related antibodies with very high sequence identity in their CDR sequences that share a common K ) binding constant. Such high association rate constants are important in high throughput immunoassays, such as Elecsys®-based immunoassays, where there is typically only a very short time for the formation of a usable antibody-antigen complex. Furthermore, the antibodies of the present invention have a K ) binding constant for fT4. D The family of antibodies provided herein is further characterized by high specificity for fT4, in particular being able to distinguish fT4 from closely related compounds (see below).

[0010] Together, these characteristics make the antibodies according to the first aspect of the invention a superior set of monoclonal antibodies, especially when used in competitive immunoassays to detect the level of fT4 in a sample. Due to their kinetic characteristics, the monoclonal antibodies according to the first aspect of the invention can effectively replace previously used polyclonal antibodies, thereby overcoming all the disadvantages associated with polyclonal antibodies. It is a surprising discovery that the performance of a polyclonal antibody, which comprises a mixture of different antibodies, can be very well mimicked by a single monoclonal antibody.

[0011] The crystal structure of antibody 38F8 (the member of the identified antibody family with the best performance in the immunoassay experiments of Example 4) in complex with T4 revealed all amino acid residues that directly interact with T4. Surprisingly, these amino acids are largely identical in the other two antibodies of the identified antibody family, 7D4 and 7E10. This high identity confirms that all three identified antibodies share at least largely identical interaction patterns. Some differences are noted, particularly in the specificity for T4 as well as the association rates and K for binding to T4. D As far as the present invention is concerned, it has been demonstrated that at each amino acid position, specific sequence variations do not dramatically affect antibody function.

[0012] The paratope of antibody 38F8 identified by the co-crystal structure is shown in Figure 5. Example 7 describes in silico and modeling approaches that defined possible amino acid substitutions based on sequence variations within the antibody families 38F8, 7E10 and 7D4.

[0013] As demonstrated by these analyses, the monoclonal antibodies of the present invention: i) a heavy chain variable domain (VH) comprising: a V or A at position 33; a Y at position 50; a W at position 52; an I at position 98; a G, A or V at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising amino acids H or Y at position 28; N or K at position 29; W at position 32; G or A at position 91; Y, W or F at position 92; S or T at position 93; Y or F at position 95b; N, S, T or Q at position 95c; and H at position 96.

[0014] The above defined residues were identified to directly interact with T4 in the co-crystal structure of 38F8 with L-T4. All above positions of amino acids are indicated according to the Kabat numbering scheme for VH and VL, respectively.

[0015] In embodiments, the paratope of the monoclonal antibody for binding to T4 comprises amino acids of VH at positions 33, 50, 52, 98, 99, 100 and 100b, and amino acids of VL at positions 28, 29, 32, 91, 92, 93, 95b, 95c and 96. Again, all positions are indicated according to the Kabat numbering scheme.

[0016] Methods for determining the amino acid residues belonging to the paratope are known in the art. A preferred method is the crystallization of the antibody complexed with L-T4. An exemplary embodiment for such crystallization is provided in the attached examples. The settings used and described in the attached examples can also be applied to other T4 antibodies. Based on such description, minor adaptations can be easily made by those skilled in the art.

[0017] Thus, there is provided herein a monoclonal antibody that specifically binds to L-thyroxine (T4), comprising a paratope, the paratope comprising: i) a heavy chain variable domain (VH) comprising: a V or A at position 33; a Y at position 50; a W at position 52; an I at position 98; a G, A or V at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising, or consisting of, the amino acids H or Y at position 28; N or K at position 29; W at position 32; G or A at position 91; Y, W or F at position 92; S or T at position 93; Y or F at position 95b; N, S, T or Q at position 95c; and H at position 96; wherein all positions are named according to Kabat nomenclature.

[0018] In certain embodiments, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: a V or A at position 33; a Y at position 50; a W at position 52; an I at position 98; a G, A or V at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising the amino acids H or Y at position 28; N or K at position 29; W at position 32; G or A at position 91; Y, W or F at position 92; S or T at position 93; Y or F at position 95b; N, S, or T at position 95c; and H at position 96. Including, All positions are annotated according to Kabat nomenclature. In another embodiment, the monoclonal antibody of the first aspect comprises: i) a heavy chain variable domain (VH) comprising: a V at position 33; a Y at position 50; a W at position 52; an I at position 98; a G at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising the amino acids H or Y at position 28; N or K at position 29; W at position 32; G at position 91; Y or W at position 92; S at position 93; Y at position 95b; N or S at position 95c; and H at position 96; Including, All positions are annotated according to Kabat nomenclature.

[0019] In a preferred embodiment, the monoclonal antibody of the first aspect comprises residues in antibody clone 38F8 that were found to directly interact with T4.

[0020] Thus, monoclonal antibodies are i) a heavy chain variable domain (VH) comprising: a V at position 33; a Y at position 50; a W at position 52; an I at position 98; a G at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising the amino acids H at position 28; N at position 29; W at position 32; G at position 91; Y at position 92; S at position 93; Y at position 95b; N at position 95c; and H at position 96; It may include, All positions are annotated according to Kabat nomenclature.

[0021] In another preferred embodiment, the monoclonal antibody of the first aspect comprises residues found in antibody 7D4 that correspond to the paratope residues of 38F8. Thus, in an embodiment, the first monoclonal antibody comprises: i) a heavy chain variable domain (VH) comprising: a V at position 33; a Y at position 50; a W at position 52; an I at position 98; a G at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising the amino acids Y at position 28; K at position 29; W at position 32; G at position 91; W at position 92; S at position 93; Y at position 95b; S at position 95c; and H at position 96; It may include, All positions are annotated according to Kabat nomenclature.

[0022] In another preferred embodiment, the monoclonal antibody of the first aspect comprises residues found in antibody 7E10 which correspond to the paratope residues of 38F8. i) a heavy chain variable domain (VH) comprising: a V at position 33; a Y at position 50; a W at position 52; an I at position 98; a G at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising the amino acids Y at position 28; K at position 29; W at position 32; G at position 91; W at position 92; S at position 93; Y at position 95b; N at position 95c; and H at position 96; It may include, All positions are annotated according to Kabat nomenclature.

[0023] As detailed in the appended examples, there is in silico evidence that amino acids at positions 34, 35, 51, 52a, 53, 97 and / or 100a of VH according to the Kabat numbering scheme and amino acids at positions 30, 31, 94, 95, 95a and / or 97 of VL according to the Kabat numbering scheme have some relevance in positioning amino acids that directly interact with T4. However, at least certain amino acid exchanges to other amino acids are tolerated at these positions, as assessed by variation at these amino acid positions in the closely related antibodies 38F8, 7E10 and 7D4, or in silico in the appended examples (see also Table XX).

[0024] The VH of a monoclonal antibody of the invention may comprise M, L, I or V at position 34; N, S, T or Q at position 35; I, A, L or V at position 51, T or S at position 52a; R, G, D or K at position 53; H, A, R or K at position 97; and / or N, A or Q at position 100a, where the amino acid positions are indicated according to the Kabat numbering scheme.

[0025] In one embodiment, the VH of a monoclonal antibody of the invention comprises M, L, or I at position 34; N, S, or T at position 35; I, A, or L at position 51, T or S at position 52a; R, G, D or K at position 53; H, A, R or K at position 97; and / or N, A or Q at position 100a, where the amino acid positions are indicated according to the Kabat numbering scheme.

[0026] In certain embodiments, the VH of a monoclonal antibody of the invention comprises M or L at position 34; N at position 35; I at position 51, T at position 52a; R, G or D at position 53; H or A at position 97; and / or N or A at position 100a, where the amino acid positions are indicated according to the Kabat numbering scheme.

[0027] In an even more specific embodiment, the VH of a monoclonal antibody of the invention comprises an M at position 34; an N at position 35; an I at position 51, a T at position 52a; an R at position 53; an H at position 97; and / or an N at position 100a, where the amino acid positions are indicated according to the Kabat numbering scheme.

[0028] In an even more specific embodiment, the VH of a monoclonal antibody of the invention comprises M at position 34, N at position 35, I at position 51, T at position 52a, R at position 53, H at position 97, and N at position 100a, the amino acid positions being indicated according to the Kabat numbering scheme. These amino acids can be found, for example, in the VH of antibody 38F8.

[0029] In another specific embodiment, the VH of a monoclonal antibody of the invention comprises an L at position 34, an N at position 35, an I at position 51, a T at position 52a, a G at position 53, an A at position 97, and an A at position 100a, the amino acid positions being indicated according to the Kabat numbering scheme. These amino acids can be found, for example, in the VH of antibody 7D4.

[0030] In another specific embodiment, the VH of a monoclonal antibody of the invention comprises M at position 34, N at position 35, I at position 51, T at position 52a, D at position 53, A at position 97, and A at position 100a, where the amino acid positions are indicated according to the Kabat numbering scheme. These amino acids can be found, for example, in the VH of antibody 7E10.

[0031] The VL of the monoclonal antibodies of the invention comprises N, Q, S or T at position 30; A, N or V at position 31; G, A or S at position 94; S, G, N or Q at position 95; T, S or G at position 95a; and / or V, A, I or L at position 97, where the amino acid positions are indicated according to the Kabat numbering scheme.

[0032] In one embodiment, the VL of a monoclonal antibody of the invention comprises an N at position 30; an A or N at position 31; a G, A or S at position 94; an S, G, or N at position 95; a T, S or G at position 95a; and / or a V or A at position 97, where the amino acid positions are indicated according to the Kabat numbering scheme.

[0033] In an even more specific embodiment, the VL of a monoclonal antibody of the invention comprises an N at position 30, an A at position 31, a G at position 94, an S at position 95, a T at position 95a, and / or a V at position 97, the amino acid positions being indicated according to the Kabat numbering scheme. These amino acids can be found, for example, in the VL of antibody 38F8.

[0034] In another specific embodiment, the VL of a monoclonal antibody of the invention comprises an N at position 30, an N at position 31, an A at position 94, a G at position 95, a G at position 95a, and / or an A at position 97, the amino acid positions being indicated according to the Kabat numbering scheme. These amino acids can be found, for example, in the VL of antibody 7D4.

[0035] In another specific embodiment, the VL of a monoclonal antibody of the invention comprises an N at position 30, an N at position 31, an S at position 94, an N at position 95, an S at position 95a, and / or an A at position 97, the amino acid positions being indicated according to the Kabat numbering scheme. These amino acids can be found, for example, in the VL of antibody 7E10.

[0036] As discussed in the accompanying examples, amino acids in the CDRs of novel antibodies against T4 that (i) do not interact with T4 according to the crystal structure and (ii) have not been identified as relevant for positioning interacting residues can be replaced with other amino acids. These amino acid positions include all amino acids in the CDRs (preferably defined by the Kabat numbering scheme) other than the amino acid residues at positions 33, 50, 52, 98, 99, 100, 100b, 34, 35, 51, 52a, 53, 97 and 100a of VH and 28, 29, 32, 91, 92, 93, 95b, 95c, 96, 30, 31, 94, 95, 95a and 97 of VL according to the Kabat numbering scheme. In embodiments, the following amino acid positions according to the Kabat numbering scheme can be replaced with other amino acids: i) positions 31, 32, 54 to 65, 95, 96, 100c, 101, and 102 of VH; and ii) VL positions 24, 25, 26, 27, 27a, 27b, 33, 34, 50-56, 89 and 90.

[0037] The VH of the antibody of the invention may comprise amino acids at positions 31 and 32 (according to the Kabat numbering scheme) independently selected from any amino acid other than proline (e.g., any naturally occurring amino acid other than proline), in embodiments, any amino acid other than proline or cysteine ​​(e.g., any naturally occurring amino acid other than P or C). In embodiments, the VH comprises an S, an R or a conservative substitution thereof at position 31 according to the Kabat numbering scheme, and / or an N or a conservative substitution thereof at position 32 according to the Kabat numbering scheme. For example, the amino acid at position 31 of the VH according to the Kabat numbering scheme may be selected from S or R. The amino acid at position 32 may be N. In a specific embodiment, the VH comprises an S or R at position 31 according to the Kabat numbering scheme, and / or an N at position 32 according to the Kabat numbering scheme.

[0038] In an embodiment, the CDR-H1 of the VH of the monoclonal antibody of the present invention comprises positions 31 to 35 according to the Kabat numbering scheme. In an even more preferred embodiment, the CDR-H1 of the VH consists of positions 31 to 35 according to the Kabat numbering scheme.

[0039] The VH of the monoclonal antibody of the invention preferably comprises amino acids at positions 54-65 according to the Kabat numbering scheme. These amino acids at positions 54-65 may each be individually selected from any amino acid other than proline (e.g., may be any naturally occurring amino acid other than proline), and in embodiments may be selected from any amino acid other than proline or cysteine ​​(e.g., may be any naturally occurring amino acid other than P or C). In embodiments, the amino acids at positions 54-65 according to the Kabat numbering scheme may have the amino acid sequence SGNTYYASWAKG (SEQ ID NO: 2), or a variant thereof having 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acid exchanges. Preferably, none of positions 54-65 is a proline, or in embodiments is a proline and a cysteine. In a preferred embodiment, the amino acids at positions 54 to 65 according to the Kabat numbering scheme may have the amino acid sequence SGNTYYASWAKG (SEQ ID NO: 2) or a variant thereof having no more than two amino acid exchanges, and none of positions 54 to 65 is a proline, or in an embodiment, a proline and a cysteine. In an embodiment, all amino acid exchanges in SEQ ID NO: 2 may each be individually a conservative amino acid exchange. In a specific embodiment, the amino acid sequence at positions 54 to 65 according to the Kabat numbering scheme may be selected from the group consisting of: SGNTYYASWAKG (SEQ ID NO: 1), SGNTYYATWAKG (SEQ ID NO: 2) or SGSTYYATWAKG (SEQ ID NO: 3). These are the corresponding sequences found in antibodies 38F8, 7D4 and 7E10.

[0040] In an embodiment, the CDR-H2 of the VH of the monoclonal antibody of the present invention comprises positions 50, 51, 52, 52a, and 53 to 65 (i.e., positions 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 63, and 65) according to the Kabat numbering scheme. In a preferred embodiment, the CDR-H2 of the VH of the monoclonal antibody of the present invention comprises positions 50, 51, 52, 52a, and 53 to 65 (i.e., positions 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 63, and 65) according to the Kabat numbering scheme.

[0041] In an embodiment, the VH comprises amino acids at positions 95, 96, 100c, 101 and 102 according to the Kabat numbering scheme. The amino acids at positions 95, 96, 100c, 101 and 102 are preferably individually selected from any amino acid (e.g., any naturally occurring amino acid) other than proline, or preferably other than proline and cysteine. In an embodiment, the amino acids at positions 95, 96, 100c, 101 and 102 of the VH are selected as follows: G or a conservative substitution thereof at position 95; L or a conservative substitution thereof at position 96; F or a conservative substitution thereof at position 100c; N or a conservative substitution thereof at position 101; and F or a conservative substitution thereof at position 102 (amino acid positions are indicated according to the Kabat numbering scheme). In a specific embodiment, the amino acids at positions 95, 96, 100c, 101 and 102 of VH are selected as follows: G at position 95; L at 96; F at position 100c; N at position 101; and F at position 102.

[0042] In an embodiment, the CDR-H3 of the VH of the monoclonal antibody of the present invention comprises positions 95 to 100 (i.e., positions 95, 96, 97, 98, 99, and 100), 100a, 100b, 100c, 101, and 102 according to the Kabat numbering scheme. In a preferred embodiment, the CDR-H3 of the VH of the monoclonal antibody of the present invention comprises positions 95 to 100 (i.e., positions 95, 96, 97, 98, 99, and 100), 100a, 100b, 100c, 101, and 102 according to the Kabat numbering scheme.

[0043] The VL of the monoclonal antibodies of the invention may comprise amino acids at positions 24, 25, 26, 27, 27a, 27b, 33 and 34 according to the Kabat numbering scheme. In an embodiment, the VL of the monoclonal antibodies of the invention may comprise any amino acid other than proline (e.g., any naturally occurring amino acid) at positions 24, 25, 26, 27, 27a, 27b, 33 and 34 according to the Kabat numbering scheme. In an embodiment, the VL of the monoclonal antibodies of the invention may comprise any amino acid other than proline and cysteine ​​(e.g., any naturally occurring amino acid) at positions 24, 25, 26, 27, 27a, 27b, 33 and 34 according to the Kabat numbering scheme.

[0044] In an embodiment, the VL of the monoclonal antibody of the invention comprises Q or a conservative substitution thereof at position 24; S or a conservative substitution thereof at position 25; S or a conservative substitution thereof at position 26; Q or a conservative substitution thereof at position 27; S or a conservative substitution thereof at position 27a; V or a conservative substitution thereof at position 27b; C, L or a conservative substitution thereof at position 33; and / or S or a conservative substitution thereof at position 34, with the amino acid positions indicated according to the Kabat numbering scheme. In an embodiment, the VL of the monoclonal antibody of the invention comprises Q or a conservative substitution thereof at position 24; S or a conservative substitution thereof at position 25; S or a conservative substitution thereof at position 26; Q or a conservative substitution thereof at position 27; S or a conservative substitution thereof at position 27a; V or a conservative substitution thereof at position 27b; C, L or a conservative substitution thereof at position 33; and S or a conservative substitution thereof at position 34, with the amino acid positions indicated according to the Kabat numbering scheme. In a specific embodiment, the VL of a monoclonal antibody of the invention comprises a Q at position 24, an S at position 25, an S at position 26, a Q at position 27, an S at position 27a, a V at position 27b, a C or L at position 33, and / or an S at position 34, all positions designated according to the Kabat numbering scheme. In another specific embodiment, the VL of a monoclonal antibody of the invention comprises a Q at position 24, an S at position 25, an S at position 26, a Q at position 27, an S at position 27a, a V at position 27b, a C or L at position 33, and an S at position 34, all positions designated according to the Kabat numbering scheme.

[0045] In an embodiment, the CDR-L1 of the VL of the monoclonal antibody of the present invention comprises positions 24, 25, 26, 27, 27a, 27b, 28, 29, 30, 31, 32, 33 and 34 according to the Kabat numbering scheme. In a preferred embodiment, the CDR-L1 of the VL of the monoclonal antibody of the present invention consists of positions 24, 25, 26, 27, 27a, 27b, 28, 29, 30, 31, 32, 33 and 34 according to the Kabat numbering scheme.

[0046] The VL of the monoclonal antibodies of the invention may comprise amino acids at positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme. In embodiments, the VL of the monoclonal antibodies of the invention may comprise any amino acid other than proline (e.g., any naturally occurring amino acid) at positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme. In embodiments, the VL of the monoclonal antibodies of the invention may comprise any amino acid other than proline and cysteine ​​(e.g., any naturally occurring amino acid) at positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme.

[0047] In an embodiment, the VL of the monoclonal antibody of the invention comprises G or a conservative substitution thereof at position 50; A or a conservative substitution thereof at position 51; S or a conservative substitution thereof at position 52; T or a conservative substitution thereof at position 53; L or a conservative substitution thereof at position 54; T, A or a conservative substitution thereof at position 55; and / or C, S or a conservative substitution thereof at position 56, with the amino acid positions indicated according to the Kabat numbering scheme. In an embodiment, the VL of the monoclonal antibody of the invention comprises G or a conservative substitution thereof at position 50; A or a conservative substitution thereof at position 51; S or a conservative substitution thereof at position 52; T or a conservative substitution thereof at position 53; L or a conservative substitution thereof at position 54; T, A or a conservative substitution thereof at position 55; and C, S or a conservative substitution thereof at position 56, with the amino acid positions indicated according to the Kabat numbering scheme. In a specific embodiment, the VL of the monoclonal antibody of the invention comprises GASTLTS (SEQ ID NO: 4) or GASTLAS (SEQ ID NO: 5) at positions 50 to 56 according to the Kabat numbering scheme.

[0048] In an embodiment, the CDR-L2 of the VL of the monoclonal antibody of the present invention comprises positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme. In a preferred embodiment, the CDR-L2 of the VL of the monoclonal antibody of the present invention consists of positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme.

[0049] The VL of a monoclonal antibody of the invention may comprise amino acids at positions 89 and 90 according to the Kabat numbering scheme. In embodiments, the VL of a monoclonal antibody of the invention may comprise any amino acid other than proline (e.g., any naturally occurring amino acid) at positions 89 and 90 according to the Kabat numbering scheme. In embodiments, the VL of a monoclonal antibody of the invention may comprise any amino acid other than proline and cysteine ​​(e.g., any naturally occurring amino acid) at positions 89 and 90 according to the Kabat numbering scheme.

[0050] In an embodiment, the VL of the monoclonal antibody of the invention comprises an A or a conservative substitution thereof at position 89, and / or a G or a conservative substitution thereof at position 90, and the amino acid positions are indicated according to the Kabat numbering scheme. In an embodiment, the VL of the monoclonal antibody of the invention comprises an A or a conservative substitution thereof at position 89, and a G or a conservative substitution thereof at position 90, and the amino acid positions are indicated according to the Kabat numbering scheme. In a specific embodiment, the VL of the monoclonal antibody of the invention comprises an A at position 89, and / or a G at position 90. In a specific embodiment, the VL of the monoclonal antibody of the invention comprises an A at position 89 and a G at position 90 according to the Kabat numbering scheme.

[0051] In an embodiment, the CDR-L3 of the VL of the monoclonal antibody of the present invention comprises positions 89, 90, 91, 92, 93, 94, 95, 95a, 95b, 95c, 96 and 97 according to the Kabat numbering scheme. In a preferred embodiment, the CDR-L3 of the VL of the monoclonal antibody of the present invention consists of positions 89, 90, 91, 92, 93, 94, 95, 95a, 95b, 95c, 96 and 97 according to the Kabat numbering scheme.

[0052] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 4, no more than 3, no more than 2, or no more than 1 position selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 5, no more than 6, no more than 7, no more than 3, no more than 8, no more than 9, no more than 10 ... and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 8; ii) (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a light chain variable domain (VL) comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 10.

[0053] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 8; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 10.

[0054] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at one or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at three or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at two or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 8; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at one or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 10.

[0055] In a specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 8; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at three or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 10.

[0056] In an even more specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 8; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 10.

[0057] In an embodiment, a variant of SEQ ID NO: 6 has M, L, I or V at position 4 and N, S, T or Q at position 5; a variant of SEQ ID NO: 7 has I, A, L or V at position 2, T or S at position 4 and R, G, D or K at position 5; a variant of SEQ ID NO: 8 has H, A, R or K at position 3 and N, A or Q at position 7; a variant of SEQ ID NO: 9 has N, Q, S or T at position 9 and A, N or V at position 10; and / or a variant of SEQ ID NO: 10 has G, A or S at position 6, S, G, N or Q at position 7, T, S or G at position 8 and V, A, I or L at position 12.

[0058] In specific embodiments, a variant of SEQ ID NO: 6 has an M, L or I at position 4 and an N, S or T at position 5; a variant of SEQ ID NO: 7 has an I, A or L at position 2, a T or S at position 4 and an R, G, D or K at position 5; a variant of SEQ ID NO: 8 has an H, A, R or K at position 3 and an N, A or Q at position 7; a variant of SEQ ID NO: 9 has an N, Q, S or T at position 9 and an A, N or V at position 10; and / or a variant of SEQ ID NO: 10 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and a V or A at position 12.

[0059] In even more specific embodiments, a variant of SEQ ID NO:6 has an M or L at position 4 and an N at position 5, a variant of SEQ ID NO:7 has an I at position 2, a T at position 4 and an R, G or D at position 5, a variant of SEQ ID NO:8 has an H or A at position 3 and an N or A at position 7, a variant of SEQ ID NO:9 has an N at position 9 and an A or N at position 10, and / or a variant of SEQ ID NO:10 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and / or a V or A at position 12.

[0060] In a preferred embodiment, variants of SEQ ID NOs: 6-10 have amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 12 or fewer positions compared to SEQ ID NOs: 6-10.

[0061] Also provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 4, no more than 3, no more than 2, or no more than 1 position selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than 4 selected from positions 2, and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 13; ii) (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9; and (f) a light chain variable domain (VL) comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 16.

[0062] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 13; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 16.

[0063] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at one or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at three or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at two or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 13; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at one or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 16.

[0064] In a specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 13; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at three or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 16.

[0065] In an even more specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 13; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 16.

[0066] In an embodiment, a variant of SEQ ID NO: 6 has M, L, I or V at position 4 and N, S, T or Q at position 5; a variant of SEQ ID NO: 7 has I, A, L or V at position 2, T or S at position 4 and R, G, D or K at position 5; a variant of SEQ ID NO: 13 has H, A, R or K at position 3 and N, A or Q at position 7; a variant of SEQ ID NO: 9 has N, Q, S or T at position 9 and A, N or V at position 10; and / or a variant of SEQ ID NO: 16 has G, A or S at position 6, S, G, N or Q at position 7, T, S or G at position 8 and V, A, I or L at position 12.

[0067] In specific embodiments, a variant of SEQ ID NO: 6 has an M, L or I at position 4 and an N, S or T at position 5; a variant of SEQ ID NO: 7 has an I, A or L at position 2, a T or S at position 4 and an R, G, D or K at position 5; a variant of SEQ ID NO: 13 has an H, A, R or K at position 3 and an N, A or Q at position 7; a variant of SEQ ID NO: 9 has an N, Q, S or T at position 9 and an A, N or V at position 10; and / or a variant of SEQ ID NO: 16 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and a V or A at position 12.

[0068] In even more specific embodiments, a variant of SEQ ID NO:6 has an M or L at position 4 and an N at position 5, a variant of SEQ ID NO:7 has an I at position 2, a T at position 4 and an R, G or D at position 5, a variant of SEQ ID NO:13 has an H or A at position 3 and an N or A at position 7, a variant of SEQ ID NO:9 has an N at position 9 and an A or N at position 10, and / or a variant of SEQ ID NO:16 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and / or a V or A at position 12.

[0069] In preferred embodiments, variants of SEQ ID NOs: 6, 7, 13, 9 and 16 have amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 12 or fewer positions compared to SEQ ID NOs: 6, 7, 13, 9 and 16, respectively.

[0070] Also provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 4, no more than 3, no more than 2, or no more than 1 position selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 5 ... and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9; and (f) a light chain variable domain (VL) comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 17.

[0071] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 17.

[0072] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at one or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at three or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at two or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at one or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 17.

[0073] In a specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at three or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 17.

[0074] In an even more specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 17.

[0075] In an embodiment, a variant of SEQ ID NO: 12 has M, L, I or V at position 4 and N, S, T or Q at position 5; a variant of SEQ ID NO: 7 has I, A, L or V at position 2, T or S at position 4 and R, G, D or K at position 5; a variant of SEQ ID NO: 14 has H, A, R or K at position 3 and N, A or Q at position 7; a variant of SEQ ID NO: 9 has N, Q, S or T at position 9 and A, N or V at position 10; and / or a variant of SEQ ID NO: 17 has G, A or S at position 6, S, G, N or Q at position 7, T, S or G at position 8 and V, A, I or L at position 12.

[0076] In specific embodiments, a variant of SEQ ID NO: 12 has an M, L or I at position 4 and an N, S or T at position 5; a variant of SEQ ID NO: 7 has an I, A or L at position 2, a T or S at position 4 and an R, G, D or K at position 5; a variant of SEQ ID NO: 14 has an H, A, R or K at position 3 and an N, A or Q at position 7; a variant of SEQ ID NO: 9 has an N, Q, S or T at position 9 and an A, N or V at position 10; and / or a variant of SEQ ID NO: 17 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and a V or A at position 12.

[0077] In even more specific embodiments, a variant of SEQ ID NO:12 has an M or L at position 4 and an N at position 5, a variant of SEQ ID NO:7 has an I at position 2, a T at position 4 and an R, G or D at position 5, a variant of SEQ ID NO:14 has an H or A at position 3 and an N or A at position 7, a variant of SEQ ID NO:9 has an N at position 9 and an A or N at position 10, and / or a variant of SEQ ID NO:17 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and / or a V or A at position 12.

[0078] In preferred embodiments, variants of SEQ ID NOs: 12, 7, 14, 9 and 17 have amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 12 or fewer positions compared to SEQ ID NOs: 12, 7, 14, 9 and 17, respectively.

[0079] Also provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 4, no more than 3, no more than 2, or no more than 1 position selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 5 ... and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 15; and (f) a light chain variable domain (VL) comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 18.

[0080] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 15, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at four or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 18.

[0081] In one aspect, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at one or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at three or less positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at two or less positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 15, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at one or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 18.

[0082] In a specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 2 and 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 to 3, 7, and 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at two or less positions selected from positions 1 to 6, 9, 10, 12 and 13 of SEQ ID NO: 15, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at three or less positions selected from positions 1, 2, 6 to 8 and 12 of SEQ ID NO: 18.

[0083] In an even more specific embodiment, the monoclonal antibody is i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 15, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 18.

[0084] In an embodiment, a variant of SEQ ID NO: 12 has M, L, I or V at position 4 and N, S, T or Q at position 5; a variant of SEQ ID NO: 7 has I, A, L or V at position 2, T or S at position 4 and R, G, D or K at position 5; a variant of SEQ ID NO: 14 has H, A, R or K at position 3 and N, A or Q at position 7; a variant of SEQ ID NO: 15 has N, Q, S or T at position 9 and A, N or V at position 10; and / or a variant of SEQ ID NO: 18 has G, A or S at position 6, S, G, N or Q at position 7, T, S or G at position 8 and V, A, I or L at position 12.

[0085] In specific embodiments, a variant of SEQ ID NO: 12 has an M, L or I at position 4 and an N, S or T at position 5; a variant of SEQ ID NO: 7 has an I, A or L at position 2, a T or S at position 4 and an R, G, D or K at position 5; a variant of SEQ ID NO: 14 has an H, A, R or K at position 3 and an N, A or Q at position 7; a variant of SEQ ID NO: 15 has an N, Q, S or T at position 9 and an A, N or V at position 10; and / or a variant of SEQ ID NO: 18 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and a V or A at position 12.

[0086] In even more specific embodiments, a variant of SEQ ID NO:12 has an M or L at position 4 and an N at position 5, a variant of SEQ ID NO:7 has an I at position 2, a T at position 4 and an R, G or D at position 5, a variant of SEQ ID NO:14 has an H or A at position 3 and an N or A at position 7, a variant of SEQ ID NO:15 has an N at position 9 and an A or N at position 10, and / or a variant of SEQ ID NO:18 has a G, A or S at position 6, an S, G or N at position 7, a T, S or G at position 8 and / or a V or A at position 12.

[0087] In preferred embodiments, variants of SEQ ID NOs: 12, 7, 14, 9 and 17 have amino acid substitutions (preferably conservative or highly conservative amino acid substitutions) at 12 or fewer positions compared to SEQ ID NOs: 12, 7, 14, 15 and 18, respectively.

[0088] As will be made clear in the accompanying examples, the CDR-L2 region is that of the T4-specific antibody of the invention and does not contribute to the interaction with T4. Thus, the CDR-L2 can in principle have any sequence. In a preferred embodiment, the light chain variable domain comprises (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 position of SEQ ID NO: 11. In one embodiment, the variant has no amino acid substitution at positions 1 and 2 of SEQ ID NO: 11. In a particular embodiment, the light chain variable domain of the monoclonal antibody of the invention comprises (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at no more than 1 position of SEQ ID NO: 11. For example, the light chain variable domain of the monoclonal antibody of the invention may comprise (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having an amino acid substitution (preferably a conservative or highly conservative amino acid substitution) at no more than 1 position of SEQ ID NO: 11.

[0089] In embodiments, any of the amino acid substitutions referred to herein may be conservative amino acid substitutions of the respective amino acids defined at each position in the sequence.

[0090] As used in the context of the present invention, a "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid selected from the same physicochemical group, which is as follows: a) non-polar hydrophobic amino acids consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met; b) polar neutral amino acids consisting of Ser, Thr, Asn and Gln; c) positively charged basic amino acids consisting of Arg, Lys and His; and d) negatively charged acidic amino acids consisting of Asp and Glu; When a Cys is conservatively substituted, it is replaced with Ser or Ala, and when a Pro is conservatively substituted, it is replaced with Ala.

[0091] In embodiments, any of the amino acid substitutions referred to herein may be highly conservative amino acid substitutions of the respective amino acids defined at each position in the sequence.

[0092] As used in the context of the present invention, "highly conservative amino acid substitutions" refers to the following amino acid substitutions: a) replacement of Ala with Val, Leu, Ile or Gly; b) Substitution of Arg by Lys; c) replacement of Asn with Gln; d) replacement of Asp with Glu; e) replacement of Cys with Ser; f) replacement of Gln with Asn; g) replacement of Glu with Asp; h) replacement of Gly with Ala; i) Replacement of His by Arg; j) replacement of Ile with Leu, Val or Ala; k) replacement of Leu with Ile, Val or Ala; l) replacement of Lys with Arg; m) replacement of Met by Leu, Ile, or Val; n) replacement of Phe by Tyr or Trp; o) replacement of Pro with Ala; p) replacement of Ser by Thr; q) replacement of Thr with Ser; r) replacement of Trp by Phe or Tyr; s) replacement of Tyr with Phe or Trp; and t) Replacement of Val with Leu, Ile or Ala.

[0093] In an embodiment, the heavy chain variable domain (VH) of the monoclonal antibody of the present invention comprises (a) a CDR-H1 comprising or consisting of the amino acid sequence of X1NVX2N (wherein X1 is S or R and X2 is M or L; also referred to herein as SEQ ID NO: 19 or SEQ ID NO: 19), (b) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (c) a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 21, and the light chain variable domain (VL) of the antibody of the present invention comprises (d) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and (f) a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23.

[0094] Thus, in one embodiment, a monoclonal antibody that specifically binds to T4 is provided, the monoclonal antibody comprising: i) the heavy chain variable domain (VH) of the monoclonal antibody of the present invention comprises (a) a CDR-H1 comprising or consisting of the amino acid sequence of X1NVX2N (wherein X1 is S or R and X2 is M or L), (b) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (c) a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 21; ii) The light chain variable domain (VL) of the antibody of the present invention comprises (d) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and (f) a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23.

[0095] The monoclonal antibody of the invention may further comprise in its VL a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO:24.

[0096] Thus, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: i) the heavy chain variable domain (VH) of the monoclonal antibody of the present invention comprises (a) a CDR-H1 comprising or consisting of the amino acid sequence of X1NVX2N (wherein X1 is S or R and X2 is M or L), (b) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (c) a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 21; ii) The light chain variable domain (VL) of the antibody of the present invention comprises (d) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and (f) a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 23.

[0097] In an embodiment, the heavy chain variable domain (VH) of the antibody of the present invention comprises (a) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 25 and 26, (b) a CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 27 and 28, and (c) a CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 29, and the light chain variable domain (VL) of the antibody of the present invention comprises (d) a CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 30, and (f) a CDR-L3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 31 and 32.

[0098] Thus, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: (i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 25, and 26; (b) a CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 27, and 28; and (c) a CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 29; (ii) a light chain variable domain (VL) comprising (d) a CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 30, and (f) a CDR-L3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 31 and 32.

[0099] The light chain variable domain (VL) may further comprise a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 11 or 33.

[0100] Thus, in an embodiment, there is provided herein a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising: (a) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 25 and 26; (b) a CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 27 and 28; and (c) a CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 29; (d) a light chain variable domain (VL) comprising a CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 30, a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 11 or 33, and (f) a CDR-L3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 31 and 32.

[0101] Antibodies of the invention may have different framework regions flanking the CDRs in the VH and VL domains.

[0102] The heavy chain variable domain (VH) has the formula I: FW-H1-CDR-H1-FW-H2-CDR-H2-FW-H3-CDR-H3-FW-H4 (Formula I) The VH may comprise framework regions (FW) flanking the CDRs of VH depicted in:

[0103] FW-H1 may have the amino acid sequence of SEQ ID NO:34, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO:34.

[0104] FW-H2 may have the amino acid sequence of SEQ ID NO:35, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO:35.

[0105] FW-H3 may have the amino acid sequence of SEQ ID NO:36, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO:36.

[0106] FW-H4 may have the amino acid sequence of SEQ ID NO:37, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO:37.

[0107] The light chain variable domain (VL) has the structure of formula I: FW-L1-CDR-L1-FW-L2-CDR-L2-FW-L3-CDR-L3-FW-L4 (Formula I) The VL may comprise the framework regions (FW) flanking the CDRs of the VL depicted in:

[0108] FW-L1 may have the amino acid sequence of SEQ ID NO:38, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:38.

[0109] FW-L2 may have the amino acid sequence of SEQ ID NO:39, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:39.

[0110] FW-L3 may have the amino acid sequence of SEQ ID NO:40, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO:40.

[0111] FW-L4 may have the amino acid sequence of SEQ ID NO:41, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO:41.

[0112] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 60% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 60% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 60% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 60% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 60% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 60% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 60% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 60% sequence identity thereto.

[0113] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 70% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 70% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 70% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 70% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 70% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 70% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 70% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 70% sequence identity thereto.

[0114] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 80% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 80% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 80% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 80% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 80% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 80% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 80% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 80% sequence identity thereto.

[0115] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 85% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 85% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 85% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 85% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 85% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 85% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 85% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 85% sequence identity thereto.

[0116] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 90% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 90% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 90% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 90% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 90% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 90% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 90% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 90% sequence identity thereto.

[0117] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 95% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 95% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 95% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 95% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 95% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 95% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 95% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 95% sequence identity thereto.

[0118] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least 99% sequence identity, FW-H2 may have the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 99% sequence identity, FW-H3 may have the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 99% sequence identity, FW-H4 may have the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 99% sequence identity, and / or FW-L1 may have the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 99% sequence identity thereto, FW-L2 may have the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least 99% sequence identity thereto, FW-L3 may have the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least 99% sequence identity thereto, and FW-L4 may have the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 99% sequence identity thereto.

[0119] In an embodiment, FW-H1 may have the amino acid sequence of SEQ ID NO: 34, FW-H2 may have the amino acid sequence of SEQ ID NO: 35, FW-H3 may have the amino acid sequence of SEQ ID NO: 36, and FW-H4 may have the amino acid sequence of SEQ ID NO: 37.

[0120] In an embodiment, the VH of the monoclonal antibody of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 42.

[0121] In other embodiments, the VH of the monoclonal antibody of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 43.

[0122] In yet another embodiment, the VH of the monoclonal antibody of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 44.

[0123] In an embodiment, the VL of the monoclonal antibody of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 45.

[0124] In other embodiments, the VL of the monoclonal antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 46.

[0125] In yet another alternative embodiment, the VL of the monoclonal antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 47, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 47.

[0126] For example, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising a VH comprising or consisting of the amino acid sequence of SEQ ID NO:42, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:42, and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:45, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:45.

[0127] Further, for example, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising a VH comprising or consisting of the amino acid sequence of SEQ ID NO:43, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:43, and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:46, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:46.

[0128] Further still, for example, provided herein is a monoclonal antibody that specifically binds to T4, the monoclonal antibody comprising a VH comprising or consisting of the amino acid sequence of SEQ ID NO:44, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:44, and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:47, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:47.

[0129] As demonstrated by the accompanying examples, the monoclonal antibodies of the present invention have a particularly high association rate constant (k a) This fast association rate makes the antibodies of the invention particularly suitable for high throughput immunoassay and competitive assay formats.

[0130] Thus, in embodiments, the monoclonal antibodies of the invention have an association rate constant (k a ) is at least 1.9 × 10 7 M -1 s -1 In embodiments, the association rate for binding to T4 measured at 37° C. is at least 2×10 7 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 1 × 10 8 M -1 s -1 In embodiments, the association rate for binding to T4 measured at 37° C. is at least 1×10 9 M -1 s -1 It is.

[0131] In embodiments, the monoclonal antibodies of the invention have an association rate constant (k a ) is the association rate constant (k a ), and further characterized in that the association rates correspond to at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the total association rates, and the association rates are measured under identical experimental conditions, preferably as described herein below and / or in Example 3.

[0132] In an embodiment, the monoclonal antibody of the present invention is an antibody comprising a Fab fragment, and the association rate constant (k a) is at least 1.9 × 10 7 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 2 × 10 7 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 1 × 10 8 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 1 × 10 9 M -1 s -1 be.

[0133] In an embodiment, the monoclonal antibody of the invention is a Fab antibody fragment and has an association rate constant (k a ) is at least 1.9 × 10 7 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 2 × 10 7 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 1 × 10 8 M -1 s -1 In an embodiment, the association rate constant (k a ) is at least 1 × 10 9 M -1 s -1 be.

[0134] The association rate constant (k a Methods for determining ) are known in the art. Exemplary examples are described in the accompanying Examples (especially Example 3).

[0135] In an embodiment, the association rate constant (k a The method for determining the association rate constant of 1×10 for binding to T4 measured at 37° C. for a Fab antibody fragment consisting of a heavy chain of SEQ ID NO: 48 and a light chain of SEQ ID NO: 49 was set. 9 M -1 s -1 It can be calibrated so that

[0136] In a preferred embodiment, the association rate constant (k a ) is measured by surface plasmon resonance spectroscopy (eg, BIAcore®).

[0137] As described in the attached examples, antibodies 38F8, 7E10 and 7D4 showed significant mass transport limitation (MTL) in BIAcore® experiments. Therefore, surface plasmon resonance spectroscopy measurements used to determine association rate constants must use MTL correction. MTL correction is preferably based on a two-compartment model (Myszka et.al., Biophysical Journal, Vol75, August 1998, 583-594; Biacore Insight Evaluation Software User Manual 29287248 AB, pages 214-215). In a preferred embodiment, the analysis may be performed using a GE Healthcare Biacore™ 8K instrument, and the analysis may be performed automatically using Evaluation Insight Software V3.011.15423.

[0138] k aSurface plasmon resonance measurements to determine may be performed at a temperature of 37 °C. Measurements may be performed using multi-cycle kinetics with a series of increasing L-T4 concentrations, e.g., c = 0.12 to 10 nM, with a dilution factor of 3. The system buffer may be PBS, pH 7.4, containing 11 mM PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4 + 0.05% (w / v) Tween 20 and 5% (v / v) DMSO. As sample buffer, the latter system buffer supplemented with 1 mg / mL carboxymethyl dextran (CMD) may be used.

[0139] association rate constant k a [1 / Ms], dissociation rate constant k d [s -1 ], and the dissociation equilibrium constant K D [M] can be calculated according to the Langmuir model, for example by using evaluation software corresponding to the instrument (for example as specified above).

[0140] As will be shown in the accompanying examples, the antibody of the present invention is also characterized by having a high affinity for L-T4. Thus, the monoclonal antibody of the present invention has an equilibrium dissociation constant K of binding to free L-T4 of 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, even more preferably 0.5 nM or less, even more preferably 0.3 nM or less, and most preferably 0.28 nM or less. D These values ​​are preferably determined using Biacore surface plasmon resonance spectroscopy and affinity in a solution setting. Experimental conditions are preferably as described herein below and / or in Example 3.

[0141] In embodiments, the monoclonal antibodies of the invention have a K D is the K of binding to T4 of an antibody comprising or consisting of a heavy chain of SEQ ID NO: 48 and a light chain of SEQ ID NO: 49 Dcorresponds to at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of K D are further characterized in that K is measured under the same experimental conditions. The experimental conditions are preferably as described herein below and / or in Example 3. D The preferred temperature for determining is 37°C.

[0142] In an embodiment, the monoclonal antibody of the present invention is an antibody comprising a Fab fragment, and has an equilibrium dissociation constant K D is preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, even more preferably 0.5 nM or less, most preferably 0.3 nM or less. These values ​​are preferably determined using Biacore surface plasmon resonance spectroscopy and affinity in a solution setting. Experimental conditions are preferably as described herein below and / or in Example 3. K D The preferred temperature for determining is 37°C.

[0143] In an embodiment, the monoclonal antibody of the invention is a Fab antibody fragment and has an equilibrium dissociation constant K D is preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, even more preferably 0.5 nM or less, most preferably 0.3 nM or less. These values ​​are preferably determined using Biacore surface plasmon resonance spectroscopy and affinity in a solution setting. Experimental conditions are preferably as described herein below and / or in Example 3. K D The preferred temperature for determining is 37°C.

[0144] equilibrium dissociation constant K D Methods for determining are known in the art.

[0145] In a preferred embodiment, K D can be determined by surface plasmon resonance spectroscopy (eg, BIAcore®).

[0146] K D The preferred temperature for determining is 37°C.

[0147] In a preferred embodiment, K D The method for determining the K of binding to T4 for a Fab antibody fragment consisting of a heavy chain of SEQ ID NO: 48 and a light chain of SEQ ID NO: 49 is D is 0.28 nM at 37 °C. D is calibrated.

[0148] In an embodiment, K D can be determined using kinetic measurements to determine association and dissociation rates. Thus, the method, in embodiments, includes the above k a The method may be the same surface plasmon resonance method as described for the determination of . The method preferably includes a correction for mass transport limitations.

[0149] In a preferred embodiment, K D can be determined using surface plasmon resonance spectroscopy (e.g., BIAcore®) and using the affinity in solution measurement principle. The advantage of affinity in solution analysis is that it is not subject to mass transport limitations and allows for measurements at equilibrium.

[0150] K DFor affinity in solution measurements of K, free L-T4-conjugated to a capture label (e.g., biotin) may be pre-captured (e.g., reversibly) on the sensor surface (e.g., on a CAP chip via streptavidin-on-(SA)-biotin interaction, if the capture label is biotin). A mixture of anti-T4 antibody or antigen-binding fragment thereof (e.g., Fab) and unlabeled L-T4 may be pre-incubated for several hours to reach equilibrium. The concentration of L-T4 antibody or fragment thereof (i.e., not L-T4-bound) may then be determined via binding to L-T4 displayed on the surface, using a preceding calibration for quantification. The concentration of antibody or antigen-binding fragment thereof is preferably kept constant in the mixture, while the L-T4 concentration is allowed to vary. As L-T4 increases, the "free" Fab fragment in solution decreases, resulting in a K D can be determined.

[0151] K for binding of the antibody of the present invention to free L-T4 D An exemplary setup for affinity in solution may be as follows: Following the CAP-reagent, biotinylated T4 may be reversibly captured on the sensor surface at high density, following the vendor's instructions for the CAP-Kit (Cytiva). Regeneration may be performed with a guanidinium / NaOH solution after each cycle. Pre-incubation of both interaction partners in solution may be performed as follows: the antibody or antigen-binding fragment (e.g., Fab) concentration may be kept constant at 3 nM. The L-T4 thyroid hormone concentration may be optimized for each T4 interaction. For example, 0.021 nM to 90 nM of unlabeled L-T4 may be used for pre-incubation.

[0152] The data was evaluated using a solution affinity model from Biacore Evaluation software (e.g., Biacore T200 Evaluation SW V3.2) to determine the K D can be determined.

[0153] As used herein, the phrase "specifically binds" in reference to an antibody or antibody antigen-binding fragment reactive with T4 indicates that T4 binds to the antibody or antibody antigen-binding fragment via an antigen-antibody reaction.

[0154] In embodiments, the antibodies of the invention distinguish free L-T4 from structurally related compounds such as 3-iodo-L-tyrosine (L-T3), rthyroid hormone (rT3), 3,3',5-tri-iodo-thyroacetic acid, 3,3',5,5'-tetra-iodothyroacetic acid, 3,5-di-iodo-L-tyrosine, and / or 3-iL-tyrosine. As used herein, "distinguish" or "distinguishing" refers to a binding affinity for L-T4 that is significantly higher than related compounds, i.e., a K D In a preferred embodiment, "distinguish" means that the antibody of the invention does not exhibit detectable binding to related compounds, or has a significantly lower K D (K D -XR) and L-T4 K D has a value of 4 or more, preferably 5 or more. In other words, the affinity for L-T4 compared to the affinity for related compounds is at least 4-fold, preferably at least 5-fold higher.

[0155] In an embodiment, the antibody of the present invention distinguishes L-T4 from 3-iodo-L-tyrosine (L-T3). Preferably, the K D and L-T4 K D may have a value of 4 or more, preferably 20 or more, even more preferably 30 or more, even more preferably 40 or more, and most preferably 47 or more. Alternatively or additionally, the K D and L-T4 K D may have a value of 4 or more, preferably 20 or more, even more preferably 30 or more, even more preferably 40 or more, even more preferably 50 or more, and most preferably 58 or more.

[0156] Alternatively or additionally, the antibodies of the invention distinguish L-T4 from rT3. Preferably, the K D and L-T4 K D may have a value of 4 or more, preferably 6 or more, even more preferably 8 or more, and most preferably 10 or more. Alternatively or additionally, the K D and L-T4 K D may have a value of 4 or more, preferably 8 or more, even more preferably 10 or more, even more preferably 12 or more, even more preferably 14 or more, and most preferably 18 or more.

[0157] Alternatively or additionally, the antibodies of the invention distinguish L-T4 from 3,3',5-tri-iodo-thyroacetic acid. Preferably, the K D and L-T4 K D has a value of 4 or more, preferably 10 or more, even more preferably 30 or more, even more preferably 50 or more, even more preferably 80 or more, and most preferably 92 or more.

[0158] Alternatively or additionally, the antibodies of the invention distinguish L-T4 from 3,3',5,5'-tetra-iodothyroacetic acid. Preferably, the K D and L-T4 K D has a value of 3 or more, preferably 4 or more, and most preferably 5 or more.

[0159] Alternatively or additionally, the antibodies of the invention distinguish L-T4 from 3,5-diiodo-tyrosine. Preferably, the K D and L-T4 K D has a value of 4 or more, preferably 10 or more, even more preferably 30 or more, even more preferably 50 or more, even more preferably 80, even more preferably 100 or more, even more preferably 150 or more, and most preferably 166 or more.

[0160] Alternatively or additionally, the antibody of the invention distinguishes L-T4 from 3-iL-tyrosine. Preferably, the K of 3,5-di-iodo-L-tyrosine measured at 37° C. D and L-T4 K D has a value of 4 or greater. Even more preferably, the antibodies of the invention show no interaction with 3-iL-tyrosine measurable by surface plasmon resonance spectroscopy (e.g., using the settings described herein, e.g., Example 3).

[0161] Binding of the antibodies of the present invention to L-T4 and related compounds D Methods for determining K for binding to L-T4 are state of the art. D Exemplary methods for determining K are described hereinabove and in the accompanying Examples. These methods include D This can be applied mutatis mutandis to related compounds so that the ratio of K values ​​can be calculated. D and K for L-T4 D The ratio of the K D K of antibody against L-T4 D It means to divide by.

[0162] In a preferred embodiment, K for calculating the ratio D K values ​​are determined by surface plasmon resonance spectroscopy (e.g., BIAcore®). In a preferred embodiment, multi-cycle kinetic measurements using potential cross-reactant concentrations between 0.1 nM and 900 nM may be used. Compounds structurally related to L-T4 may be injected using flow rates between 30 μL / min and 60 μL / min. The association phase may be monitored for 3 to 5 minutes and the dissociation phase for 5 to 15 minutes. The L-T4 interaction may be characterized by using additional analyte injections with a dissociation time of 30 minutes. K DSurface plasmon resonance measurements to determine the K value may be performed at a temperature of 37° C. The system buffer may be PBS, pH 7.4, containing 11 mM PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4 + 0.05% (w / v) Tween 20 and 5% (v / v) DMSO. As sample buffer, the latter system buffer supplemented with 1 mg / mL carboxymethyl dextran (CMD) may be used. The dissociation equilibrium constant K D [M] can be calculated according to the Langmuir model, for example by using evaluation software corresponding to the instrument (for example as specified above).

[0163] As demonstrated in the accompanying examples, the structurally related antibodies 38F8, 7E10 and 7D4 showed good signal-to-noise ratios in competitive immunoassays, with performance comparable to that of a polyclonal anti-L-T4 antibody.

[0164] Thus, in preferred embodiments, the T4-specific antibodies of the invention, when used in a competitive immunoassay to quantify T4 (e.g., fT4), exhibit a signal-to-noise ratio that is at least 29%, at least 30%, at least 65%, at least 66%, at least 70%, at least 80%, at least 90%, or at least 95% of that achieved with a Fab fragment comprising or consisting of the heavy chain sequence of SEQ ID NO: 48 and the light chain sequence of SEQ ID NO: 49. In these embodiments, the immunoassay setup is identical except that the antibodies are replaced.

[0165] In a preferred embodiment, the signal to noise ratio can be analyzed over an fT4 concentration range of 0 to 122.1 pmol / L.

[0166] As used herein, "signal-to-noise ratio" may also be referred to as "signal kinetic ratio" or "relative total signal span". In the case of an immunoassay format in which the measured signal decreases as the presence of T4 (e.g., fT4) in the sample increases, the signal-to-noise ratio or signal kinetic ratio refers to the ratio calculated by (i) dividing the change in signal (e.g., ECL number) between a sample without T4 and the sample with the highest measured T4 (e.g., fT4) concentration (e.g., 122.1 pmol / L) by (ii) the signal (e.g., ECL number) of the sample with the highest measured T4 concentration (e.g., 122.1 pmol / L). In the case of an immunoassay format in which the measured signal increases with increasing presence of T4 (e.g., fT4) in the sample, the signal-to-noise ratio or signal kinetic ratio relates to the ratio calculated by (i) the positive change in signal (e.g., ECL number) between a sample without T4 (e.g., fT4) and a sample with the highest measured T4 (e.g., fT4) concentration (e.g., 122.1 pmol / L) - or a defined analyte concentration in a medically relevant concentration range - divided by (ii) the signal (e.g., ECL number) of the sample without T4 (e.g., fT4).

[0167] A competitive immunoassay for assessing the signal-to-noise ratio of an antibody may have the following basic assay principle: (i) incubating a sample containing T4 (e.g., fT4) with an antibody, wherein the antibody is labeled with a detection label (e.g., a ruthenium label) for a defined period of time (e.g., 9 minutes); (ii)(i) Adding to a mixture of T4 coupled to a capture label (e.g., biotinylated T4 or T4(OSu)-bis-DADOO-biotin-hapten) and particles capable of binding to the capture label and incubating the resulting mixture for a defined time (e.g., 9 minutes); alternatively, particles capable of binding to the capture label may be added in a subsequent step while incubating the resulting mixture for a defined time (e.g., 9 minutes). (iii) detecting the labeled antibody bound to the mixture by separating the particles from the mixture and measuring the detection label signal; and (iv) determining the amount of T4 (e.g., FT4) in the sample based on the measured detection label signal.

[0168] In this competitive assay principle, the higher the amount of T4 (eg, FT4), the lower the measured signal of the detection label.

[0169] A specific and particularly preferred example of a competitive immunoassay for evaluating signal-to-noise ratio is provided in the attached Example 4. Thus, in a specific embodiment, the signal-to-noise ratio can be evaluated by a competitive immunoassay using an automated Elecsys® Immuno-Analyzer (e.g., Elecsys® cobas® e411). An assay with a total incubation time of 18 minutes can have the following settings: first incubation (9 minutes): 15 μL of T4 (e.g., fT4)-containing sample, 75 μL of ruthenium-containing L-T4 specific antibody are incubated; second incubation (9 minutes): 75 μL of T4(OSu)-bis-DADOO-biotin-hapten-conjugate solution and 35 μL of streptavidin-coated microparticles are added to the mixture of the first incubation step. The amount of T4 (e.g., fT4) in the sample can then be determined by measuring the ruthenium label signal attached to the magnetic beads. Specifically, the reaction mixture can be drawn into a measuring cell, where the microparticles can be magnetically captured on the surface of the electrode. Unbound material can then be removed with ProCell#11662988122 (Roche Diagnostics GmbH Germany). The application of voltage to the electrode can then be used to induce electrochemiluminescence-based luminescence, which is measured by a photomultiplier tube.

[0170] In embodiments, an Elecsys® Immuno-Analyzer based competitive assay may be used, as described above or in Example 4 below. In these embodiments, for an antibody of the invention, assuming a first sample has a T4 (e.g., fT4) concentration of 122.10 pmol / L and a second sample has a T4 (e.g., fT4) concentration of 0 pmol / L, the ratio of counts detected for the first sample and the second sample (i.e., counting the first sample divided by the number of counts and counting the second sample) may be 11% or less, 5% or less, or 4% or less.

[0171] The antibodies and antigen-binding fragments of the present invention can be prepared by various techniques routinely used in the art. For example, antibodies can be prepared by isolating antigen-reactive antibody-producing B cells and immunizing non-human animals (e.g., rabbits) with the subsequently isolated L-T4. To enhance immunogenicity, L-T4 for immunization can be coupled to a carrier protein (e.g., keyhole limpet hemocyanin (KLH)). Preferably, T4-NH-PEG(3)-CO-KLH is used for immunization. Screening of antibodies that bind to L-T4 can be accomplished using L-T4 as an analyte. Preferably, T4(OSu)-bis-DADOO-biotin can be coupled to a surface and antibody binding thereto can be tested. To identify antibodies that specifically bind to L-T4, counterscreening for binding to structurally related compounds such as 3-iodo-L-tyrosine (L-T3), thyroid hormone (rT3), 3,3',5-tri-iodo-thyroacetic acid, 3,3',5,5'-tetra-iodothyroacetic acid, 3,5-di-iodo-L-tyrosine and / or 3-iL-tyrosine may be performed. Preferred exemplary methods for producing antibodies according to the invention using immunization of non-human animals are provided in the accompanying examples (see especially Example 1). Selected clones for producing antibodies can be processed according to routine methods for subsequent recombinant processing.

[0172] Another suitable method for generating or isolating the antibodies and antibody antigen-binding fragments of the present invention includes, but is not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., but is not limited to, bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or yeast display libraries) using the binding activity of interest. For example, antibodies or antigen-binding fragments can be selected from such libraries by positively selecting for specific binding to L-T4, for example, by using L-T4-coupled biotin (e.g., T4(OSu)-bis-DADOO-biotin). Optionally, negative selection for binding to structurally related compounds such as 3-iodo-L-tyrosine (L-T3), thyroid hormone (rT3), 3,3',5-tri-iodo-thyroacetic acid, 3,3',5,5'-tetra-iodothyroacetic acid, 3,5-di-iodo-L-tyrosine and / or 3-iL-tyrosine can also be performed to identify antibodies that specifically bind to L-T4. Display libraries are well known in the art and are available from a variety of commercial vendors, including, for example, but not limited to, Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsried / Planegg, Del.), Biovation (Aberdeen, Scotland, UK) and Bioinvent (Lund, Sweden). Again, selected clones can be processed according to conventional methods for subsequent recombinant processing.

[0173] The antibody of the present invention can be recombinantly expressed. Thus, in certain embodiments, the monoclonal antibody of the present invention can be a recombinant antibody. Methods for producing recombinant antibodies are known in the art. An exemplary embodiment for recombinant expression and subsequent purification of the antibody according to the present invention is provided in the attached Example 4.

[0174] In a second aspect, the present invention also provides a nucleic acid molecule encoding a monoclonal antibody of the invention or any antigen-binding fragment thereof as defined hereinabove. In particular, a polynucleotide is provided encoding the heavy and / or light chain variable domain of a monoclonal antibody that specifically binds to T4 as defined hereinabove. In some embodiments, the polynucleotide may comprise additional sequences to ensure expression of not only the heavy and light chain variable domains, but also the remaining heavy and / or light chain constant regions, such that a full-length IgG antibody comprising the heavy and / or light chain variable domain of the invention is expressed. Thus, for each of the aspects and embodiments relating to a monoclonal antibody or antigen-binding fragment that specifically binds to T4 described herein, a corresponding polynucleotide is provided herein that encodes the respective antibody or antigen-binding fragment.

[0175] In a third aspect, a vector comprising the polynucleotide of the present invention is provided herein. In particular, a vector comprising a nucleic acid molecule encoding an antibody or an antibody antigen-binding fragment of the present invention is provided. As used herein, the term "vector" refers to a circular or linear nucleic acid molecule capable of autonomously replicating in a host cell into which it is introduced. Non-limiting examples of vectors suitable for use in the present invention include cosmids, plasmids (e.g., naked or encapsulated in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) and bacteriophages. However, the art provides many suitable vectors, the selection of which depends on the desired function. The development and use of suitable vectors are well documented in the art, see, for example, the techniques described in Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001) and Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989), (1994). The vectors used in connection with the present invention comprise nucleic acid sequences encoding the full-length anti-L-T4 / free L-T4 antibody antigen-binding fragments disclosed herein. Thus, for each of the aspects and embodiments relating to monoclonal antibodies or antigen-binding fragments that specifically bind to T4 described herein, vectors are provided herein that comprise the corresponding polynucleotides encoding the respective antibodies or antigen-binding fragments.

[0176] As used herein, with respect to the term "vector comprising", it is understood in the art that there are additional nucleic acid sequences present in the vector that are necessary and / or sufficient for the desired vector activity in a host cell, e.g., driving replication of the vector (and thus encoding nucleic acid sequences) and / or directing the host cell to express an antibody or antigen-binding fragment of the invention. Such additional nucleic acid sequences include, but are not limited to, sequences that control vector replication and / or expression of a desired sequence in a particular cell system. For example, a vector may comprise a nucleic acid molecule encoding an antibody or antibody antigen-binding fragment of the invention, operably linked and / or under the control of regulatory sequences. The term "regulatory sequences" refers to DNA sequences necessary to effect expression of coding sequences to which they are operably linked. The term "regulatory sequences" is intended to include at least all components whose presence may also be necessary for expression, and may further include additional advantageous components, e.g., to allow replication. As is understood in the art, the nature of such regulatory and control sequences will vary depending on the host organism. For example, in prokaryotes, control sequences generally include promoters, ribosome binding sites, and terminators. In eukaryotes, control sequences generally include promoters, terminators, and, in some cases, enhancers, transcription activators or transcription factors.

[0177] The vectors used in the present invention are preferably expression vectors. Expression vectors can direct the replication and expression of the nucleic acid molecules of the present invention in a host cell, thus providing, for example, the expression of the heavy and / or light chain variable domains of the monoclonal antibody specifically binding to T4 disclosed herein. In some embodiments, the vectors may contain additional sequences to ensure the expression of not only the heavy and light chain variable domains, but also the remaining heavy and light chain constant regions, such that a full-length IgG antibody comprising the heavy and light chain variable domains of the present invention is expressed. Suitable expression vectors are widely described in the literature, and the determination of an appropriate expression vector for a particular cell system can be easily performed by one of skill in the art using routine methods. Preferably, the vectors disclosed herein contain a recombinant polynucleotide (i.e., a nucleic acid sequence encoding the monoclonal antibody of the present invention) and a control sequence operably linked thereto. The vectors provided herein preferably further contain a promoter. The vectors described herein may also contain a selection marker gene and an origin of replication to ensure replication in the host. In addition, the vectors provided herein may also contain a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in a host cell.

[0178] The nucleic acid molecules and / or vectors of the present invention can be designed for transfection into prokaryotic or eukaryotic host cells by any means known in the art or described herein.Non-limiting examples of suitable methods include chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nucleofection), non-chemical methods (electroporation, sonoporation, phototransfection, gene electrophoresis, hydrodynamic delivery, or natural transformation when cells are contacted with the nucleic acid molecules of the present invention), particle-based methods (gene gun, magnetofection, impalfection), phage vector-based methods, and viral methods.For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, Semliki Forest viruses, or bovine papilloma viruses may be used to transfect nucleic acid molecules into target cell populations.In addition, baculovirus systems can also be used as vectors in eukaryotic expression systems of the nucleic acid molecules of the present invention.

[0179] The term "prokaryote" is meant to include all bacteria that can be transformed, transduced or transfected with DNA or DNA or RNA molecules for the expression of the protein of the present invention. Prokaryotic hosts include gram-negative as well as gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, Corynebacterium (glutamicum), Pseudomonas (fluorescens), Lactobacillus, Streptomyces, Salmonella and Bacillus subtilis. The term "eukaryote" is meant to include yeast, higher plants, insects and mammalian cells. Non-limiting examples of host cells typically used in the art include Hela, HEK293, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS / 0, SP2 / 0 and C127 cells, COS cells, such as COS1 or COS7, CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.

[0180] Thus, in a fourth aspect, the present invention relates to a host cell comprising the polynucleotide according to the present invention or the vector according to the present invention.The host cell can be a prokaryotic or eukaryotic cell.In a preferred embodiment, the host cell is a eukaryotic cell.In a particular embodiment, the cell is a HEK cell.In another particular embodiment, the host cell is a CHO cell.

[0181] When a recombinant expression vector encoding the heavy and / or light chains of the antibody of the present invention disclosed herein is introduced into a host cell, the antibody or antibody antigen-binding fragment is produced by culturing the host cell for a period of time sufficient to express the antibody or antigen-binding fragment in the host cell, or preferably to secrete the antibody or antigen-binding fragment into the medium in which the host cell is growing. The antibody and / or antigen-binding fragment can be recovered from the medium using standard protein purification methods. Methods for purifying antibodies are well known in the art. Exemplary purification methods are described in the accompanying examples.

[0182] Thus, the present invention also provides a method for the production of a monoclonal antibody that specifically binds to T4 as disclosed herein. The method comprises culturing a host cell of the present invention under suitable conditions and isolating the antibody produced. The isolated monoclonal antibody of the present invention can be obtained, for example, by a purification process as described in the accompanying examples. The present invention further provides an antibody or antigen-binding fragment obtainable by any of the methods disclosed herein.

[0183] The transformed host cells can be grown in a bioreactor and cultured by techniques known in the art to achieve optimal cell growth. The antibodies and / or antibody antigen-binding fragments of the invention can then be isolated from the cell fraction or growth medium by any conventional means, including but not limited to affinity chromatography (e.g., using a fusion tag such as Strep-tag II or His6 tag), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, or immunoprecipitation.

[0184] It will be understood that variations of the above procedure are within the scope of the present invention. For example, recombinant DNA technology can be used to remove or modify the DNA sequence encoding the antibody and / or antibody antigen-binding fragment disclosed herein, e.g., encoding the heavy and / or light chain variable domains as defined herein above. For example, recombinant DNA technology can be used to remove portions of the coding sequence that are not necessary to maintain specific and selective binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present invention. In addition, multivalent antibodies are also provided that include at least two (preferably 4, 5, 6, 7, 8) of the heavy and / or light chain variable domains of the present invention (e.g., forming an antibody Fv domain that specifically and selectively binds to T4).

[0185] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity, or to reduce, enhance or modify binding, affinity, on-rates, off-rates, avidity, specificity, half-life or any other suitable characteristic.

[0186] Also provided are humanized versions of the antibodies disclosed herein, i.e., those that contain the heavy and / or light chain CDRs as disclosed hereinabove. As is well known in the art, "humanization" (to create a humanized version of a parent antibody) refers to recombinantly engineering an antibody using CDRs derived from a non-human donor immunoglobulin in the context of human-derived framework and constant domains. During engineering, framework and / or CDR residues can be altered to preserve binding affinity and activity, e.g., specificity for T4. Methods for humanizing antibodies are known in the art and are described, for example, in Queen et al., Proc. Natl. Acad Sci USA 86 (1989), 10029-10032; Hodgson et al., Bio / Technology 9 (1991) 421.

[0187] In a fifth aspect, the present invention provides a composition comprising the antibody of the present invention, the polynucleotide of the present invention, the vector of the present invention or the host cell of the present invention. In a preferred embodiment, the composition is a diagnostic composition, i.e. a composition for use in diagnostic applications. In a preferred embodiment, the composition is for use in an in vitro diagnostic test for detecting L-T4, preferably free L-T4. In a preferred embodiment, the diagnostic composition may be a reagent for an immunoassay for detecting L-T4, preferably free L-T4. The diagnostic composition is preferably configured to allow detection of L-T4, preferably free L-T4, in a sample obtained from a subject. The sample is preferably a blood sample (e.g., whole blood, serum or plasma).

[0188] In an embodiment, the composition of the present invention is a composition for in vitro detection (preferably quantification) of T4 (preferably fT4) in a sample, preferably using immunoassay. In an embodiment, the immunoassay is a heterogeneous immunoassay. In an embodiment, the immunoassay is a competitive immunoassay.

[0189] In a sixth aspect, the present invention provides the use of the antibody according to the present invention for in vitro detection (preferably quantification) of T4 (e.g., free T4) in a sample. Detection and / or quantification of T4 (e.g., free T4) is preferably achieved by using an immunoassay. Thus, the antibody of the present invention is also provided for use in an immunoassay (e.g., heterogeneous immunoassay) for detecting T4 (e.g., free T4).

[0190] The sample may be a bodily fluid, such as, but not limited to, a blood sample, cerebrospinal fluid, semen, saliva, or urine. In an embodiment, the sample is a blood sample, such as whole blood, serum, or plasma. In an embodiment, the sample is serum or plasma.

[0191] Since T4 is a very small analyte, it is preferred that the immunoassay be competitive. In an embodiment, the assay setup is a competitive assay of the reverse titration type, for example as described herein below or in the accompanying examples. Briefly, the assay can be configured as follows: (i) incubating a sample containing T4 (e.g., free T4) with an antibody, where the antibody is labeled with a detectable label (e.g., a ruthenium label) for a defined period of time (e.g., 9 minutes); (ii)(i) adding to a mixture of T4 coupled to a capture label (e.g., T4(OSu)-bis-DADOO-biotin-hapten) and particles capable of binding to the capture label, and incubating the resulting mixture for a defined period of time (e.g., 9 minutes); (iii) detecting the labeled antibody bound to the mixture by separating the particles from the mixture and measuring the detection label signal; and (iv) determining the amount of T4 (e.g., free T4) based on the measured detectable label signal.

[0192] Optionally, where total T4 is determined in a sample in which T4 is partially bound by a binding protein, step (i) may comprise releasing the T4 from the binding protein, as described in detail below in connection with the seventh aspect of the invention.

[0193] In a seventh aspect, the present invention provides an in vitro immunoassay method for quantifying T4 (e.g., free T4) in a sample using an antibody of the present invention. The antibody may be part of a composition, particularly a diagnostic composition, of the present invention.

[0194] The sample may be a bodily fluid, such as a blood sample, cerebrospinal fluid, semen, saliva or urine. In an embodiment, the sample is a blood sample, such as whole blood, serum or plasma. In an embodiment, the sample is serum or plasma.

[0195] A method for quantifying L-T4 may include (i) incubating a sample containing L-T4 with an antibody of the present invention under conditions that allow the antibody to bind to L-T4, and (ii) quantifying the amount of L-T4 in the sample by directly or indirectly detecting the amount of L-T4 bound to the antibody of the present invention.

[0196] Direct detection means that the amount of antibody-L-T4 complex is directly detected via the detection label, and the amount of L-T4-antibody complex corresponds proportionally to the amount of L-T4 in the sample. Indirect detection means that the amount of L-T4 is indirectly correlated with the measurement signal of a detectable label (e.g., when achieved in a competitive assay format). For example, for indirect detection, the antibody of the present invention may be labeled with a detection label, and the amount of binding of the labeled antibody to L-T4 (i.e., competing with T4 for binding to the T4 antibody of the present invention) or an analog of L-T4 linked to a surface (directly or via a capture label such as biotin) is detected. Thus, in an embodiment, the amount of antibody that does not bind to L-T4 (e.g., fT4) in the sample is quantified, thereby indirectly determining the amount of L-T4 in the sample.

[0197] In embodiments, the method of the present invention may be a method for detecting total T4 (i.e., free T4 and T4 bound to binding proteins) in a sample. In these embodiments, the method may further comprise releasing T4 from the binding proteins by adding a releasing agent. Such a releasing agent may be added, for example, before or during incubation with the antibody. In embodiments, the releasing agent may be added before incubation with the antibody and may still be present during further method steps. Suitable reagents for releasing T4 from the binding proteins are known in the art (see, for example, Method Sheet for Elecsys® T4; Ref. 09007784190). In some embodiments, the releasing agent may be 8-anilino-1-naphthalenesulfonic acid (ANS). In embodiments, ANS may be added at a final concentration in the sample of 0.3 mg / ml to 0.9 mg / ml, in embodiments 0.375 mg / ml to 0.83 mg / ml. In an embodiment, ANS may be at 0.83 mg / ml during incubation with an antibody of the invention and at 0.375 mg / ml in a subsequent step in which a capture agent (eg, that competes with an antibody of the invention) is added.

[0198] The detection label linked to the antibody of the present invention can be any detection label known in the art. For example, the detection label can be selected from an enzyme or a luminescent label (e.g., fluorescent, luminescent, chemiluminescent, electrochemiluminescent or radioactive).

[0199] In an eighth aspect, a kit comprising the antibody of the present invention or a composition comprising it is provided herein. In an embodiment, the kit is a kit for detecting and / or quantifying T4 (e.g., fT4) in vitro. In an embodiment, the kit is an immunoassay kit. In an embodiment, the kit is a kit for heterogeneous immunoassay. In an embodiment, the kit is a kit for competitive immunoassay. In an embodiment, the kit may be a kit for detecting total T4 in vitro, and may further comprise a realease agent for releasing T4 from its binding protein (e.g., serum / plasma binding protein) in a sample.

[0200] The invention also provides kits comprising at least one reagent of the invention, i.e., (i) an antibody or antibody antigen-binding fragment of the invention, (ii) a nucleic acid molecule of the invention, (iii) a vector of the invention, (iv) a host cell of the invention, and / or (v) any article of manufacture (e.g., a package or container or insert) comprising one or more of the antibodies or antibody antigen-binding fragments produced or obtained by the methods of the invention. Kits may be promoted, distributed, or sold as a unit for carrying out the methods of the invention.

[0201] In embodiments, the kits of the invention may include an antibody of the invention having a detection label attached (e.g., as specified elsewhere herein). The kits may further include L-T4. The L-T4 may include a capture label. The kits may include a solid phase, such as a magnetic particle (e.g., a magnetic particle). The solid phase may be functionalized to allow for the binding of the capture label, e.g., functionalized with streptavidin. Alternatively, the solid phase may be precoated with L-T4.

[0202] Because certain detection labels require substrates or reagents to generate a detectable signal, the kits of the present invention may, in embodiments, further contain substrates and / or reagents that allow for detection of the detection label.

[0203] In a specific embodiment, the kit may contain in a first container a monoclonal antibody that specifically binds to T4 (e.g., free T4) according to the invention, and in a separate second container a mixture of a solid phase and a capture analog of L-T4 (e.g., biotinylated L-T4), where the capture analog can be bound to the solid phase. For example, the solid phase may be coated with streptavidin and the capture analog biotinylated. Alternatively, the second container may contain a solid phase immobilized with L-T4. In both embodiments, the coupling of L-T4 to the solid phase is such that L-T4 can still be recognized by the antibody of the invention.

[0204] The present invention also relates in particular to the following items:

[0205] 1. A monoclonal antibody that specifically binds to L-thyroxine (T4), said monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: a V or A at position 33; a Y at position 50; a W at position 52; an I at position 98; a G, A or V at position 99; a Y at position 100; and an I at position 100b; ii) a light chain variable domain (VL) comprising amino acids H or Y at position 28; N or K at position 29; W at position 32; G or A at position 91; Y, W or F at position 92; S or T at position 93; Y or F at position 95b; N, S, T or Q at position 95c; and H at position 96; A monoclonal antibody, wherein the amino acid positions in the VH and VL are each indicated according to the Kabat numbering scheme.

[0206] 2. The monoclonal antibody of item 1, wherein the VL comprises an N, S or T at position 95c.

[0207] 3. i) the VH comprises a V at position 33 and a G at position 99; ii) The monoclonal antibody of item 1, wherein the VL comprises G at position 91; Y or W at position 92; S at position 93; Y at position 95b and N or S at position 95c.

[0208] 4. i) the VH comprises a V at position 33 and a G at position 99; ii) The monoclonal antibody of item 1, wherein the VL comprises H at position 28; N at position 29; G at position 91; Y at position 92; S at position 93; Y at position 95b and N at position 95c.

[0209] 5. The monoclonal antibody according to any one of items 1 to 5, wherein the paratope of the monoclonal antibody for binding to T4 comprises amino acids of the VH at positions 33, 50, 52, 98, 99, 100 and 100b and amino acids of the VL at positions 28, 29, 32, 91, 92, 93, 95b, 95c and 96, wherein the positions of the amino acids in the VH and the VL, respectively, are indicated according to the Kabat numbering scheme.

[0210] 6. The monoclonal antibody according to any one of items 1 to 5, wherein the VH comprises M, L, I or V at position 34; N, S, T or Q at position 35; I, A, L or V at position 51, T or S at position 52a; R, G, D or K at position 53; H, A, R or K at position 97; and / or N, A or Q at position 100a, wherein said amino acid positions are indicated according to the Kabat numbering scheme.

[0211] 7. The monoclonal antibody of item 6, wherein the VH comprises M, L or I at position 34; N, S or T at position 35; and / or I, A or L at position 51.

[0212] 8. The monoclonal antibody of item 6, wherein the VH comprises M or L at position 34; N at position 35; I at position 51; T at position 52a; R, G or D at position 53; H or A at position 97; and / or N or A at position 100a.

[0213] 9. The monoclonal antibody of item 6, wherein the VH comprises M at position 34; N at position 35; I at position 51; T at position 52a; R at position 53; H at position 97; and / or N at position 100a.

[0214] 10. The monoclonal antibody according to any one of items 1 to 9, wherein the VL comprises N, Q, S or T at position 30; A, N or V at position 31; G, A or S at position 94; S, G, N, Q at position 95; T, S or G at position 95a; and / or V, A, I or L at position 97, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0215] 11. The monoclonal antibody of item 10, wherein the VL comprises N at position 30; A or N at position 31; S, G or N at position 95; and / or V or A at position 97.

[0216] 12. The monoclonal antibody of item 10, wherein the VL comprises N at position 30; A at position 31; G at position 94; S at position 95; T at position 95a and / or V at position 97.

[0217] 13. The monoclonal antibody according to any one of items 1 to 12, wherein the VH comprises an amino acid other than proline at positions 31 and 32, respectively, according to the Kabat numbering scheme.

[0218] 14. The monoclonal antibody according to any one of items 1 to 12, wherein the VH comprises S, R or a conservative substitution thereof at position 31 according to the Kabat numbering scheme, and / or N or a conservative substitution thereof at position 32 according to the Kabat numbering scheme.

[0219] 15. The monoclonal antibody according to any one of items 1 to 12, wherein the VH comprises S or R at position 31 according to the Kabat numbering scheme, and / or N at position 32 according to the Kabat numbering scheme.

[0220] 16. The monoclonal antibody according to any one of items 1 to 15, wherein CDR-H1 of the VH comprises or consists of positions 31 to 35 according to the Kabat numbering scheme.

[0221] 17. The monoclonal antibody according to any one of items 1 to 16, wherein the VH comprises amino acids other than proline at positions 54 to 65 according to the Kabat numbering scheme.

[0222] 18. The monoclonal antibody according to any one of items 1 to 17, wherein the VH comprises the amino acid sequence SGNTYYASWAKG (SEQ ID NO: 1), or a variant thereof having no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 amino acid substitutions at positions 54 to 65 according to the Kabat numbering scheme, wherein none of positions 54 to 65 is a proline.

[0223] 19. The monoclonal antibody of item 18, wherein no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitutions are each individually conservative amino acid substitutions.

[0224] 20. The monoclonal antibody according to any one of items 1 to 19, wherein the VH comprises an amino acid sequence from positions 54 to 65 according to the Kabat numbering scheme, selected from the group consisting of: SGNTYYASWAKG (SEQ ID NO: 1), SGNTYYATWAKG (SEQ ID NO: 2) or SGSTYYATWAKG (SEQ ID NO: 3).

[0225] 21. The monoclonal antibody according to any one of items 1 to 20, wherein CDR-H2 of said VH comprises or consists of positions 50, 51, 52, 52a and 53 to 65 according to the Kabat numbering scheme.

[0226] 22. The monoclonal antibody according to any one of items 1 to 21, wherein the VH comprises amino acids other than proline at positions 95, 96, 100c, 101 and 102 according to the Kabat numbering scheme.

[0227] 23. The monoclonal antibody according to any one of items 1 to 22, wherein the VH comprises G at position 95 or a conservative substitution thereof; L at position 96 or a conservative substitution thereof; F at position 100c or a conservative substitution thereof; N at position 101 or a conservative substitution thereof; and F at position 102 or a conservative substitution thereof, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0228] 24. The monoclonal antibody according to any one of items 1 to 23, wherein the VH comprises G at position 95; L at position 96; F at position 100c; N at position 101; and F at position 102, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0229] 25. The monoclonal antibody according to any one of items 1 to 24, wherein the CDR-H3 of said VH comprises or consists of positions 95 to 100, 100a, 100b, 100c, 101 and 102 according to the Kabat numbering scheme.

[0230] 26. The monoclonal antibody according to any one of items 1 to 25, wherein the VL comprises amino acids other than proline at positions 24, 25, 26, 27, 27a, 27b, 33 and 34 according to the Kabat numbering scheme.

[0231] 27. The monoclonal antibody according to any one of items 1 to 26, wherein the VL comprises Q or a conservative substitution thereof at position 24; S or a conservative substitution thereof at position 25; S or a conservative substitution thereof at position 26; Q or a conservative substitution thereof at position 27; S or a conservative substitution thereof at position 27a; V or a conservative substitution thereof at position 27b; C, L or a conservative substitution thereof at position 33; and / or S or a conservative substitution thereof at position 34, wherein the positions of the amino acids are indicated according to the Kabat numbering scheme.

[0232] 28. The monoclonal antibody according to any one of items 1 to 27, wherein the VL comprises a Q at position 24; an S at position 25; an S at position 26; a Q at position 27; an S at position 27a; a V at position 27b; a C or L at position 33; and / or an S at position 34, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0233] 29. The monoclonal antibody according to any one of items 1 to 31, wherein the CDR-L1 of VL comprises or consists of positions 24, 25, 26, 27, 27a, 27b, 28, 29, 30, 28, 32, 33 and 34 according to the Kabat numbering scheme.

[0234] 30. The monoclonal antibody according to any one of items 1 to 29, wherein the VL comprises amino acids other than proline at positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme.

[0235] 31. The monoclonal antibody according to any one of items 1 to 30, wherein the VL comprises G or a conservative substitution thereof at position 50; A or a conservative substitution thereof at position 51; S or a conservative substitution thereof at position 52; T or a conservative substitution thereof at position 53; L or a conservative substitution thereof at position 54; T, A or a conservative substitution thereof at position 55; and / or C, S or a conservative substitution thereof at position 56, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0236] 32. The monoclonal antibody according to any one of items 1 to 31, wherein the VL comprises GASTLTS (SEQ ID NO: 4) or GASTLAS (SEQ ID NO: 5) at positions 50 to 56 according to the Kabat numbering scheme.

[0237] 33. The monoclonal antibody according to any one of items 1 to 32, wherein the CDR-L2 of the VL comprises or consists of positions 50, 51, 52, 53, 54, 55 and 56 according to the Kabat numbering scheme.

[0238] 34. The monoclonal antibody according to any one of items 1 to 33, wherein the VL comprises amino acids other than proline at positions 89 and 90 according to the Kabat numbering scheme.

[0239] 35. The monoclonal antibody according to any one of items 1 to 34, wherein the VL comprises an A or a conservative substitution thereof at position 89 and / or a G or a conservative substitution thereof at position 90, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0240] 36. The monoclonal antibody according to any one of items 1 to 35, wherein the VL comprises an A at position 89 and / or a G at position 90, wherein the amino acid positions are indicated according to the Kabat numbering scheme.

[0241] 37. The monoclonal antibody according to any one of items 1 to 36, wherein the CDR-L3 of VL comprises or consists of positions 89, 90, 91, 92, 93, 94, 95, 95a, 95b, 95c, 96 and 97 according to the Kabat numbering scheme.

[0242] 38. A monoclonal antibody that specifically binds to L-thyroxine (T4), the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at four or less positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 8; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 10. A monoclonal antibody comprising:

[0243] 39. A monoclonal antibody that specifically binds to L-thyroxine (T4), the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at four or less positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 13; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 16. A monoclonal antibody comprising:

[0244] 40. A monoclonal antibody that specifically binds to L-thyroxine (T4), the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at four or less positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 17. A monoclonal antibody comprising:

[0245] 41. A monoclonal antibody that specifically binds to L-thyroxine (T4), the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at four or less positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 15, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions at four or less positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 18. A monoclonal antibody comprising:

[0246] 42. (a) said variant of SEQ ID NO: 6 has M, L, I or V at position 4 and N, S, T or Q at position 5; (b) said variant of SEQ ID NO: 7 has I, A, L or V at position 2, T or S at position 4 and R, G, D or K at position 5; (c) said variant of SEQ ID NO:8 has H, A, R or K at position 3 and N, A or Q at position 7; (d) said variant of SEQ ID NO: 9 has an N, Q, S or T at position 9 and an A, N or V at position 10; (e) The monoclonal antibody according to any one of items 38 to 41, wherein the variant of SEQ ID NO: 10 has G, A or S at position 6, S, G, N or Q at position 7, T, S or G at position 8 and V, A, I or L at position 12.

[0247] 43. (a) said variant of SEQ ID NO:6 has an M, L, or I at position 4 and an N, S, or T at position 5; (b) said variant of SEQ ID NO: 7 has I, A, or L at position 2, T or S at position 4, and R, G, D, or K at position 5; (c) the variant of SEQ ID NO: 13 has H, A, R or K at position 3 and N, A or Q at position 7; (d) said variant of SEQ ID NO: 9 has an N, Q, S or T at position 9 and an A, N or V at position 10; (e) The monoclonal antibody according to any one of items 38 to 41, wherein the variant of SEQ ID NO: 16 has G, A or S at position 6, S, G or N at position 7, T, S or G at position 8 and V or A at position 12.

[0248] 44. (a) said variant of SEQ ID NO: 12 has M at position 4, or L and N at position 5; (b) said variant of SEQ ID NO:7 has an I at position 2, a T at position 4, and an R, G, or D at position 5; (c) said variant of SEQ ID NO: 14 has H, or A at position 3 and N, A or Q at position 7; (d) said variant of SEQ ID NO:9 has an N at position 9 and an N or an A at position 10; (e) The monoclonal antibody according to any one of items 38 to 41, wherein the variant of SEQ ID NO: 17 has G, A or S at position 6, S, G or N at position 7, T, S or G at position 8 and V or A at position 12.

[0249] 45. (a) the variant of SEQ ID NO: 12 has an M at position 4 and an N at position 5; (b) said variant of SEQ ID NO:7 has an I at position 2, a T at position 4 and an R at position 5; (c) the variant of SEQ ID NO: 14 has an H at position 3 and an N at position 7; (d) the variant of SEQ ID NO:9 has an N at position 9 and an A at position 10; (e) The monoclonal antibody according to any one of items 38 to 41, wherein the variant of SEQ ID NO: 17 has G at position 6, S at position 7, T at position 8 and V at position 12.

[0250] 46. ​​The monoclonal antibody according to any one of items 38 to 45, wherein the light chain variable domain comprises (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having an amino acid substitution at no more than four positions of SEQ ID NO: 11.

[0251] 47. The monoclonal antibody according to any one of items 38 to 46, wherein the sum of the amino acid substitutions in the variants of CDRs H1, H2, H3, L1 and L3 is 12 or less.

[0252] 48. The monoclonal antibody according to item 46, wherein the sum of the amino acid substitutions in the variants of CDRs H1, H2, H3, L1, L2 and L3 is 13 or less.

[0253] 49. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 8; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 10. 49. The monoclonal antibody according to any one of Items 38 to 48, comprising:

[0254] 50. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 13; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 16. 49. The monoclonal antibody according to any one of Items 38 to 48, comprising:

[0255] 51. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 17. 49. The monoclonal antibody according to any one of Items 38 to 48, comprising:

[0256] 52. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 12; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 2 and positions 4 to 17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 3, 7, and positions 9 to 11 of SEQ ID NO: 14; ii) (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 15, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO: 18. 49. The monoclonal antibody according to any one of Items 38 to 48, comprising:

[0257] 53. The monoclonal antibody according to any one of items 38 to 52, wherein the light chain variable domain comprises (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having an amino acid substitution at not more than one position of SEQ ID NO: 11.

[0258] 54. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having an amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 6; (b) a CDR-H2 having an amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7; and (c) a CDR-H3 having an amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 8; ii) a light chain variable domain (VL) comprising: (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 9; and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 10. 54. The monoclonal antibody according to any one of Items 38 to 53, comprising:

[0259] 55. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 13; ii) a light chain variable domain (VL) comprising: (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 9; and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 16. 54. The monoclonal antibody according to any one of Items 38 to 53, comprising:

[0260] 56. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having an amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 6; (b) a CDR-H2 having an amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7, and (c) a CDR-H3 having an amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 14; ii) a light chain variable domain (VL) comprising: (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 9; and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 17. 54. The monoclonal antibody according to any one of Items 38 to 53, comprising:

[0261] 57. The monoclonal antibody, i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having an amino acid sequence of SEQ ID NO: 12, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 1 and 4 of SEQ ID NO: 6; (b) a CDR-H2 having an amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 5, 8 and 13 of SEQ ID NO: 7, and (c) a CDR-H3 having an amino acid sequence of SEQ ID NO: 14, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 3 and 7 of SEQ ID NO: 14; ii) a light chain variable domain (VL) comprising: (d) a CDR-L1 having an amino acid sequence of SEQ ID NO: 15, or a variant thereof having amino acid substitutions at not more than two positions selected from positions 8 and 10 of SEQ ID NO: 15; and (f) a CDR-L3 having an amino acid sequence of SEQ ID NO: 18, or a variant thereof having amino acid substitutions at not more than three positions selected from positions 4, 5 and 6 of SEQ ID NO: 18. 54. The monoclonal antibody according to any one of Items 38 to 53, comprising:

[0262] 58. The monoclonal antibody according to any one of items 38 to 57, wherein the light chain variable domain comprises (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having not more than one amino acid substitution at position 6 of SEQ ID NO: 11.

[0263] 59. The monoclonal antibody according to any one of items 38 to 58, wherein each of the amino acid substitutions is a conservative amino acid substitution, preferably a highly conservative amino acid substitution.

[0264] 60. The monoclonal antibody according to any one of items 1 to 59, wherein the heavy chain variable domain (VH) comprises (a) a CDR-H1 comprising the amino acid sequence of X1NVX2N (wherein X1 is S or R and X2 is M or L); (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and the light chain variable domain (VL) comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23.

[0265] 61. The monoclonal antibody according to any one of items 1 to 60, wherein the light chain variable domain (VL) comprises a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 24.

[0266] 62. The monoclonal antibody according to any one of items 1 to 61, wherein the heavy chain variable domain (VH) comprises (a) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 25 and 26, (b) a CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 27 and 28, and (c) a CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 29, and the light chain variable domain (VL) comprises (d) a CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 30, and (f) a CDR-L3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 31 and 32.

[0267] 63. The monoclonal antibody according to any one of items 1 to 62, wherein the light chain variable domain (VL) comprises a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11 or 33.

[0268] 64. The heavy chain variable domain (VH) has formula I: FW-H1-CDR-H1-FW-H2-CDR-H2-FW-H3-CDR-H3-FW-H4 (Formula I) and wherein the VH comprises a framework region (FW) adjacent to the CDRs of the VH represented by FW-H1 has the amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 34; FW-H2 has the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 35; FW-H3 has the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 36; 64. The monoclonal antibody according to any one of items 1 to 63, wherein FW-H4 has the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 37.

[0269] 65. The light chain variable domain (VL) has formula I: FW-L1-CDR-L1-FW-L2-CDR-L2-FW-L3-CDR-L3-FW-L4 (Formula I) and wherein the VL comprises a framework region (FW) adjacent to the CDRs of the VL represented by FW-L1 has the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 38; FW-L2 has the amino acid sequence of SEQ ID NO: 39, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 39; FW-L3 has an amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 40; 65. The monoclonal antibody according to any one of items 1 to 64, wherein FW-L4 has the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO: 41.

[0270] 66. The monoclonal antibody according to any one of items 1 to 65, wherein the VH has the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 42.

[0271] 67. The monoclonal antibody according to any one of items 1 to 66, wherein the VH has the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 43.

[0272] 68. The monoclonal antibody according to any one of items 1 to 67, wherein the VH has the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 44.

[0273] 69. The monoclonal antibody according to any one of items 1 to 68, wherein the VL has an amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 45.

[0274] 70. The monoclonal antibody according to any one of items 1 to 69, wherein the VL has an amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 46.

[0275] 71. The monoclonal antibody according to any one of items 1 to 70, wherein the VL has an amino acid sequence of SEQ ID NO: 47, or a variant thereof having at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity to SEQ ID NO: 47.

[0276] At a temperature of 72.37° C., the association rate constant of the monoclonal antibody with T4 (k a ) is 1.9 × 10 7 M -1 sec -1 In the above embodiment, 2×10 7 M -1 sec -1 In the above embodiment, 8 M -1 sec -1 In the above embodiment, 3×10 8 M -1 sec -1 or more, and in the embodiment, 1×10 9 M -1 sec -1 72. The monoclonal antibody according to any one of items 1 to 71, wherein

[0277] 73. The association rate constant (k a ) is the association rate constant (k a 73. The monoclonal antibody according to any one of items 1 to 72, wherein the association rate constant corresponds to at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the total association rate constant, wherein the association rate constant is measured under identical experimental conditions.

[0278] 74. The above k a74. The monoclonal antibody according to item 72 or 73, wherein is determined by surface plasmon resonance spectroscopy.

[0279] 75. The monoclonal antibody according to item 74, wherein the surface plasmon resonance spectroscopy is analyzed using correction for mass transport limitations.

[0280] 76. The monoclonal antibody has a K of 20 nM or less, in embodiments 10 nM or less, in embodiments 5 nM or less, in embodiments 1 nM or less, in embodiments 0.5 nM or less, in embodiments 0.3 nM or less, and in embodiments 0.28 nM or less. D 74. The monoclonal antibody according to any one of items 1 to 73, which binds to T4 at

[0281] 77. The above K D 77. The monoclonal antibody of claim 76, wherein the affinity in solution is optionally determined by surface plasmon resonance spectroscopy.

[0282] 78. The monoclonal antibody according to any one of items 1 to 77, wherein the monoclonal antibody distinguishes T4 from 3-iodo-L-tyrosine (L-T3), rThyroid hormone (rT3), 3,3',5-tri-iodo-thyroacetic acid, 3,3',5,5'-tetra-iodothyroacetic acid, 3,5-di-iodo-L-tyrosine and / or 3-iL-tyrosine.

[0283] 79. The monoclonal antibody of any one of items 1 to 78, wherein the monoclonal distinguishes T4 from L-T3 and / or rT3.

[0284] 80. Identifying T4 from each of the substances (multiple substances) is important. D are the K of each of the other substances (multiple substances are possible) D 80. The monoclonal antibody according to item 78 or 79, which means at least 4-fold lower, in an embodiment at least 5-fold lower than.

[0285] 81. The monoclonal antibody according to any one of items 1 to 80, wherein said monoclonal antibody, when used in a competitive immunoassay for quantifying T4 (e.g., fT4), exhibits a signal-to-noise ratio that is at least 29%, in an embodiment 65% and in an embodiment at least 95% of the signal-to-noise ratio achieved with a Fab fragment having the heavy chain sequence of SEQ ID NO: 48 and the light chain sequence of SEQ ID NO: 49 in an otherwise identical immunoassay setting.

[0286] 82. The monoclonal antibody according to any one of items 1 to 81, wherein the monoclonal antibody is a Fab fragment.

[0287] 83. A polynucleotide comprising: (i) the heavy chain or the heavy chain variable domain of the monoclonal antibody according to any one of items 1 to 82, and / or (ii) The light chain or the light chain variable domain of the monoclonal antibody according to any one of items 1 to 82. A polynucleotide encoding the

[0288] 84. A vector comprising the polynucleotide according to item 83.

[0289] 85. A host cell comprising a polynucleotide according to item 83 or a vector according to item 84.

[0290] 86. The host cell according to item 85, which is a prokaryotic or eukaryotic cell.

[0291] 87. The host cell according to item 85, which is a eukaryotic cell, the cell being a HEK cell or a CHO cell.

[0292] 88. A method for producing a monoclonal antibody according to any one of items 1 to 82, comprising culturing a host cell according to any one of items 85 to 87 and isolating the antibody.

[0293] 89. The antibody according to any one of items 1 to 82, obtainable by the method according to item 88.

[0294] 90. A composition comprising the antibody according to any one of items 1 to 82, the polynucleotide according to item 83, the vector according to item 84, or the host cell according to any one of items 85 to 87.

[0295] 91. A composition comprising the antibody according to any one of items 1 to 82, which is a diagnostic composition.

[0296] 92. Use of an antibody according to any one of items 1 to 82 or a composition according to item 90 or 91 for in vitro detection, preferably in vitro quantification, of T4 (e.g. fT4) in a sample, preferably using an immunoassay.

[0297] 93. The use according to item 92, wherein the immunoassay is a heterogeneous immunoassay.

[0298] 94. The use according to item 92 or 93, wherein the sample is blood, preferably plasma or serum.

[0299] 95. The use according to any one of items 92 to 94, wherein the immunoassay is a competitive immunoassay.

[0300] 96. An in vitro immunoassay method for quantifying T4 (e.g., fT4) in a sample using the antibody according to any one of items 1 to 82.

[0301] 97. The method according to item 96, wherein the sample is a body fluid.

[0302] 98. The method according to item 97, wherein the body fluid is a blood sample, cerebrospinal fluid, semen, saliva or urine.

[0303] 99. The method according to item 96 or 97, wherein the body fluid is a blood sample which is whole blood, serum or plasma.

[0304] 100. The method according to any one of items 96 to 99, wherein the method comprises (i) incubating a sample containing T4 (e.g., fT4) with an antibody according to any one of items 1 to 82 or a composition according to item 90 or 91, and (ii) quantifying the amount of T4 (e.g., fT4) in the sample by directly or indirectly detecting the antibody contained in the composition or T4 (e.g., fT4) bound to the antibody, respectively.

[0305] 101. The method according to any one of items 96 to 100, the method comprising: (i) incubating a sample containing T4 (e.g., fT4) with an antibody according to any one of items 1 to 82 or a composition according to item 90 or 91, wherein the antibody has a detection label attached thereto; and (ii) quantifying the amount of T4 (e.g., fT4) in the sample by quantifying the amount of the antibody that has not bound to T4 (e.g., fT4) in the sample via the detection label.

[0306] 102. The method according to item 101, wherein the detection label is selected from an enzymatic, luminescent, in one embodiment fluorescent, luminescent, chemiluminescent, electrochemiluminescent or radioactive emitting label.

[0307] 103. The method according to any one of items 96 to 102, wherein the method is for detecting total T4 in a sample, the method comprising treating the sample with a reagent for releasing T4 from its binding protein (e.g. a serum protein), and in an embodiment, the reagent for releasing T4 from its binding protein is 8-anilinonaphthalene-1-sulfonic acid (ANS).

[0308] 104. A kit comprising the antibody according to any one of items 1 to 82, or the composition according to item 90 or 91.

[0309] 105. The kit according to item 103, wherein the kit is a kit for detecting and / or quantifying T4 (e.g., fT4) in vitro.

[0310] 106. The kit according to item 103 or 104, which is an immunoassay kit.

[0311] The present invention provides an antibody that specifically binds to L-thyroxine (herein referred to as L-T4 or T4). Alternative names for L-thyroxine used in the art are 3,3',5,5"-tetraiodo-L-thyronine and 3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]-L-alanine. T4 has the CAS number 51-48-9. T4 circulates in the bloodstream as an equilibrium mixture of free and serum-bound hormone. Free T4 (fT4) is the unbound, biologically active form and represents only 0.03% of total T4. The remaining T4 is inactive and bound to serum proteins such as thyroxine-binding globulin (TBG) (75%), prealbumin (15%), and albumin (10%) (Robbins J, Rall JE. Recent Prog Horm Res 1957;13:161-208; Oppenheimer JH. N Engl J Med 1968;278(21):1153-1162;DeGroot LJ,Larsen PR,Hennemann G.Wiley and Sons,New York,1984:62-65;Ekins RP.Endocr Rev 1990;11(1):5-46). It is clear that the antibodies of the present invention that specifically bind to T4 / L-T4 bind T4 / L-T4 in its free form (i.e., not in its binding protein-bound form) since serum protein-bound T4 is largely inaccessible to antibody binding. Thus, in embodiments, the antibodies of the present invention may also specifically bind to fT4. In embodiments, specifically binding to fT4 does not mean that fT4 can be distinguished from serum protein-bound T4, but rather relates to distinguishing T4 from related substances / derivatives such as L-T3 and others disclosed elsewhere herein. The antibodies of the present invention may, in embodiments, be for the detection of fT4. In other embodiments, the antibodies can be used for the detection of total T4 in a sample. In the latter embodiment, the use of the antibodies typically involves pretreatment of the sample to release T4 bound to proteins (e.g., serum proteins).

[0312] As used herein, the terms "antibody", "antibodies" and similar terms refer to complete immunoglobulin molecules and include naturally occurring forms of antibodies (including but not limited to IgG, IgA, IgM, IgE) as well as recombinant antibody constructs including but not limited to single chain antibodies, chimeric antibodies, humanized antibodies, antibody fusion proteins, and multispecific antibodies; as well as all antigen-binding fragments and derivatives of the above. As used herein, the terms "antibody", "antibodies" and similar terms also refer to antigen-binding fragments thereof, which may be referred to herein as antibody antigen-binding fragments and / or simply antigen-binding fragments. These terms refer to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen, i.e., L-T4, as known in the art, and include but are not limited to antigen-binding fragments comprising paired heavy and light chain variable domains, such as Fv domains, i.e., Fab, Fab', F(ab')2 and Fv fragments, as well as recombinant constructs, e.g., single chain Fv domains known in the art as scFv. The term also includes antibody antigen-binding fragments comprising a single unpaired heavy or light chain variable domain known in the art that retain the ability to specifically and selectively bind to an antigen as defined herein, including, but not limited to, single domain antibodies based on camelid heavy chains (also referred to in the art as sdAbs, dAbs, and / or nanobodies) and V H Contains the H domain.

[0313] In certain embodiments, the monoclonal antibodies of the invention may be complete immunoglobulins, Fab, Fab', F(ab')2, Fv or scFv. In specific embodiments, the monoclonal antibodies of the invention may be Fab fragments.

[0314] Antibodies can be polyclonal or monoclonal. The antibodies of the present invention are monoclonal. As used herein with respect to antibodies or antigen-binding fragments thereof, the term "monoclonal" refers to a population of antibody polypeptides or fragments thereof produced from a single B-cell clone, which population contains only one species of antigen-binding site capable of immunoreacting with a particular epitope of an antigen. This is in contrast to "polyclonal" antibodies and compositions, which is a term that refers to a population of antibody polypeptides or antigen-binding fragments that contain multiple species of antigen-binding sites. Also included are modified forms of the monoclonal antibodies of the present invention, such as humanized or chimeric versions thereof, as well as recombinant antibody constructs, such as antibody (or antigen-binding fragment) fusion proteins, where the antibody or antigen-binding fragment contains additional domains, such as for the isolation and / or preparation of recombinantly produced antibodies / fragments / constructs.

[0315] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated using the VH or VL domain of an antibody that binds to that antigen, and a library of complementary VL or VH domains, respectively, may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0316] As used herein in connection with antibodies, the term "paratope" refers to the amino acids of VH and VL that directly interact with the antigen (e.g., L-T4). Paratope amino acids can interact with the antigen through different interaction modes. Exemplary interaction modes are hydrophobic interactions, H-bonds, H-bonds with HO coordination, and H-coordination. The interaction of each paratope residue with the antigen can be mediated individually through the amino acid side chain or the amino acid backbone.

[0317] As used herein, the term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are hypervariable in sequence and that determine antigen-binding specificity, e.g., each of the "complementarity determining regions" (CDRs).

[0318] Generally, an antibody comprises six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0319] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will appreciate that the designation of CDRs may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0320] The numbering of amino acid residues of the VH and VL of the antibody of the present invention is performed according to the Kabat nomenclature unless otherwise specified. Those skilled in the art can convert the numbering according to Kabat to other nomenclature systems such as Clothia, McCallum, etc.

[0321] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences typically occur in the following order in a VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0322] As used herein in connection with amino acids, the terms "substitution," "exchange," or "mutate" refer to the replacement of an amino acid with another amino acid. The deletion of an amino acid at a particular position and the introduction of an amino acid (or amino acids) at a different position are not expressly encompassed by the term "substitution." As stated, the present invention encompasses conservative or highly conservative amino acid substitutions as defined herein above.

[0323] In some cases, a particular amino acid is defined herein as being selected from a subset of two or more amino acids or a conservative (or highly conservative) substitution thereof. In these examples, each amino acid position may be any of the listed amino acids or a conservative (or highly conservative) amino acid substitution of any of the explicitly listed amino acids. What is meant by conservative and highly conservative substitutions as used herein is defined elsewhere herein. To give an illustrative example, if a particular amino acid is defined as "A or T or a conservative amino acid substitution thereof," the amino acid may be A or any conservative amino acid replacement thereof, or T or a conservative amino acid substitution thereof.

[0324] Amino acids are spelled out or abbreviated herein using the one-letter or three-letter code.

[0325] In the context of the present invention, it refers to variants of sequences (especially CDRs). These variants typically contain one or more amino acid substitutions. It is clear that variant CDRs are functional variants, i.e. they may differ from the reference amino acid sequence, but the different sequence has an amino acid sequence that exhibits or maintains the same functional activity as the reference sequence in the context of the described heavy and / or light chain variable domain. Specifically, as used herein, the term same functional activity refers to the fact that the antibody or antibody-binding fragment of the present invention comprising one or more variant CDRs has an exceptionally high association rate constant k a and / or affinity for binding to L-T4, specificity (especially with respect to discriminating L-T4 from structurally related compounds) and / or maintaining a high signal-to-noise ratio in immunoassays.

[0326] As used in the context of the present invention, a "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid selected from the same physicochemical group, which is as follows: a) non-polar hydrophobic amino acids consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met; b) polar neutral amino acids consisting of Ser, Thr, Asn and Gln; c) positively charged basic amino acids consisting of Arg, Lys and His; and d) negatively charged acidic amino acids consisting of Asp and Glu; When a Cys is conservatively substituted, it is replaced with Ser or Ala, and when a Pro is conservatively substituted, it is replaced with Ala.

[0327] As used in the context of the present invention, "highly conservative amino acid substitutions" refers to the following amino acid substitutions: a) replacement of Ala with Val, Leu, Ile or Gly; b) Substitution of Arg by Lys; c) replacement of Asn with Gln; d) replacement of Asp with Glu; e) replacement of Cys with Ser; f) replacement of Gln with Asn; g) replacement of Glu with Asp; h) replacement of Gly with Ala; i) Replacement of His by Arg; j) replacement of Ile with Leu, Val or Ala; k) replacement of Leu with Ile, Val or Ala; l) replacement of Lys with Arg; m) replacement of Met by Leu, Ile, or Val; n) replacement of Phe by Tyr or Trp; o) replacement of Pro with Ala; p) replacement of Ser by Thr; q) replacement of Thr with Ser; r) replacement of Trp by Phe or Tyr; s) replacement of Tyr with Phe or Trp; and t) Replacement of Val with Leu, Ile or Ala.

[0328] As used herein, the term "sequence identity percentage" in relation to amino acid sequences and / or nucleic acid sequences of polypeptides / peptides or nucleic acid molecules refers to the number of identical amino acid or nucleic acid residue matches of two or more aligned sequences compared to the number of residues that make up the entire length of the sequences being compared (or the portions being compared in their entirety). Using the alignment of two or more sequences or subsequences, the percentage of residues that are identical can be determined when comparing (sub)sequences and aligning for maximum matches over a comparison window or over a designated region measured using sequence comparison algorithms known in the art, or by manual alignment and visual inspection. Non-limiting examples of algorithms for use in determining sequence identity include, for example, those based on the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res 25 (1997), 3389-3402), the CLUSTALW computer program (Thompson, Nucl. Acids Res. 2 (1994), 4673-4680) or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 85 (1988), 2444). The FASTA algorithm typically does not take into account internal non-matching deletions or additions in the sequence, i.e., gaps, in its calculations, although this can be manually corrected to avoid overestimating the % sequence identity. However, CLUSTALW does take into account sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul et al., Nucl Acids Res., 25 (1977), 3389) are also available.

[0329] As used herein, "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" and similar terms include both genomic DNA and cDNA, as well as RNA capable of driving the expression of an antibody or antigen-binding fragment of the invention. As used herein, the term "RNA" is understood to include all forms of RNA, including mRNA, tRNA and rRNA, but also genomic RNA, such as in the case of the RNA of an RNA virus. Preferably, embodiments describing "RNA" relate to mRNA. The nucleic acid molecules / nucleic acid sequences of the invention may be of natural as well as synthetic or semi-synthetic origin. In embodiments, the nucleic acids / nucleic acid sequences of the invention may be isolated. Thus, the nucleic acid molecules may be nucleic acid molecules that have been synthesized, for example, according to conventional protocols of organic chemistry, according to recombinant methods, or that have been produced semi-synthetically, for example by combining chemical synthesis and recombinant methods. The skilled artisan is familiar with the preparation and use of such nucleic acid molecules.

[0330] As used herein, an "immunoassay" is a well-established bioanalytical method in which the detection or quantification of an analyte relies on the reaction of the analyte with at least one analyte-specific binding agent, thus forming an analyte:binder complex. In the context of the present invention, at least one of the at least one analyte-specific binding agent is an antibody of the present invention. A specific embodiment of a "sandwich" immunoassay can be used for analytes with multiple recognition epitopes. Thus, a sandwich assay requires at least two binding agents that attach to non-overlapping epitopes on the analyte. In a "heterogeneous sandwich immunoassay", one of the binding agents has the functional role of an analyte-specific capture binding agent, which is immobilized on a solid phase or will be immobilized (during the course of the assay) on a solid phase. A second analyte-specific binding agent is provided in dissolved form in the liquid phase. Once the respective analytes are bound by the first and second binding agents, a sandwich-like complex (binder-1:analyte:binder-2) is formed. The sandwich-like complex is also called a "detection complex". Within the detection complex, the analyte is sandwiched between the binding agents, ie in such a complex the analyte represents the connecting element between the first and second binding agents.

[0331] The term "heterogeneous" (as opposed to "homogeneous") denotes two essential and separate steps in the assay procedure. In the first step, a detection complex containing the label is formed and immobilized, while unbound label still surrounds the complex. Before determining the label-dependent signal, the unbound label is removed from the immobilized detection complex, thus representing the second step. In contrast, homogeneous assays generate an analyte-dependent detectable signal by a single-step incubation and do not require a washing step.

[0332] In heterogeneous immunoassays, the solid phase is functionalized such that a functional capture binding agent (first binding agent) can be attached to its surface before contacting with the analyte, or the surface of the solid phase is functionalized such that the first binding agent can be tethered after reacting with the analyte. In the latter case, the tethering process must not interfere with the ability of the binding agent to specifically capture and bind the analyte. A second binding agent present in the liquid phase is used to detect the bound analyte. Thus, in heterogeneous immunoassays, the analyte is bound to the first binding agent (capture) and the second binding agent (detector). This forms a "detection complex" in which the analyte is sandwiched between the capture binding agent and the detector binding agent. In typical embodiments, the detector binding agent is labeled before contacting with the analyte, or the label is specifically attached to the detector binding agent after analyte binding. If the detection complex is immobilized on the solid phase, the amount of label detectable on the solid phase corresponds to the amount of sandwiched analyte. After removal of unbound label, the immobilized label can be detected, indicating the presence and amount of analyte.

[0333] As used herein, a "competitive immunoassay" preferably uses a single binding agent (i.e., T4 / fT4) that directly interacts with the analyte. A "competitive heterogeneous immunoassay" typically detects a signal of a detection label that corresponds inversely to the amount of analyte in the sample. In a preferred embodiment herein, the competitive immunoassay may be a heterogeneous competitive immunoassay. In an embodiment, a sample containing the analyte is mixed with an artificially generated labeled analog of the analyte that can react with an analyte-specific binding agent (e.g., an antibody of the present invention). In the assay, the analyte and the analog compete for binding to a capture binding agent (e.g., an antibody of the present invention) that is immobilized or that is to be immobilized. The amount of binding agent is selected to be limiting in this setting. Following this binding step, the greater the amount of immobilized label, the less the amount of unlabeled analyte that was able to compete for the capture binding agent. The immobilized label is quantified after a washing step. In this setting, the amount of label detectable on the solid phase corresponds inversely to the amount of analyte initially present in the sample. In other embodiments, the competitive immunoassay may be a heterogeneous reverse titration assay. In such an assay, a sample containing an analyte is first incubated with a binder (e.g., an antibody of the present invention) to which a detection label is attached for a sufficient time to form an analyte:binder complex. The binder may be provided in excess relative to the highest analyte concentration to be measured. A capture analog (e.g., biotinylated L-T4) is then added to the mixture, which competes for binding with the binder having a detection label (e.g., competes with T4 (e.g., fT4) in the sample for the antibody of the present invention). The capture analog is immobilized to a surface (e.g., magnetic beads) during incubation with the mixture, or is immobilized (e.g., via biotin-streptavidin interaction). The amount of analyte can then be detected by removing non-surface-bound reagents and detecting the signal of the detection label. The more detection label (attached to the binder) on the surface, the less analyte was present in the sample. Therefore, also in this second setting, the amount of detectable label on the solid phase corresponds inversely to the amount of analyte initially present in the sample.

[0334] As used herein, "detectable label" refers to a label that allows detection. According to one embodiment of the present invention, the detectable label is an enzyme, or in one embodiment, a label that emits fluorescence, luminescence, chemiluminescence, electrochemiluminescence or radioactivity. In a preferred embodiment, the label is an electrochemiluminescent label, in one embodiment, a tris(2,2'-bipyridyl)ruthenium(II) complex (Ru(bpy)). All of the above labels can be used in the present invention, since interference is caused by the three-dimensional structure of the label molecule that attracts autoantibodies and similar interfering molecules, and not by the signal emission mechanism of the label, such as light or radioactivity.

[0335] As used herein, "capture label" refers to a label that can immobilize a capture agent (e.g., T4 with the capture label attached) on a surface (e.g., on a magnetic particle such as a microbead). A non-limiting example is a member of a binding pair. A non-limiting example of a capture label is biotin or a derivative thereof, which can interact with streptavidin or a derivative thereof. A variety of capture labels are well known in the art.

[0336] As used in the context of the present disclosure, a "sample" may be a liquid sample that contains or is expected to contain L_T4. The sample may be a bodily fluid, such as, but not limited to, a blood sample, cerebrospinal fluid, semen, saliva or urine. In an embodiment, the sample is a blood sample, such as whole blood, serum or plasma. In an embodiment, the sample is serum or plasma.

[0337] The word "comprise", and variations such as "comprises" and "comprising", are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps.

[0338] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0339] Concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range formats are used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as boundaries of the range, but also all of the individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. By way of illustration, a numerical range of "150 mg to 600 mg" should be interpreted not only to include the explicitly recited value of 150 mg to 600 mg, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, . . . 580, 590, 600 mg, etc., and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle also applies to ranges reciting only one numerical value. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics described.

[0340] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​in a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0341] In the above detailed description of the present invention, several individual elements, features, techniques, and / or steps are disclosed. It is easily recognized that each of these has benefits not only when considered or used alone, but also when considered and used in combination with each other. Therefore, in order to avoid excessively repetitive and redundant sections, this description has avoided repeating all possible combinations and permutations. Nevertheless, it is understood that such combinations, whether explicitly listed or not, are fully within the scope of the subject matter of this disclosure.

[0342] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or equivalent substitutions of techniques that would be apparent to those skilled in the art.

[0343] All amino acid sequences provided herein begin with the most N-terminal residue and end with the most C-terminal residue, as is conventional in the art, and the one-letter or three-letter code abbreviations used to identify amino acids throughout the invention correspond to those commonly used for amino acids.

[0344] In this specification, many documents are cited, including patent applications and manufacturer's manuals. The disclosures of these documents are not considered relevant to the patentability of this invention, but are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0345] The following diagrams are provided to aid the understanding of the present invention, the true scope of which is set forth in the claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0346] [Figure 1] Figure 1 shows the data obtained for the initial kinetic screening of the generated antibodies. The dissociation rate constant kd is plotted against the association rate constant ka, with the diagonal line showing the affinity range obtained. Shown are 840 analyzed supernatants (A) and a selected Ab selection of 50 antibodies (B). Importantly, no correction for mass transport limitations was applied for the determination of the ka and kd values ​​shown in this figure, even if such a correction was necessary. [Diagram 2] Figure 2 shows the kinetic profiles of Fab fragments 38F8, 7D4, 7E10, 3B7, 18B3 and 4H8 binding to L-T4 at 37 °C measured by Biacore. A) 38F8, B) 7D4, C) 7E10, D) 3B7, E) 18B3 and F) 4H8 binding series with increasing L-T4 concentrations c = 0.04-10 nM, dilution factor 3; replicates of 3.3 nM. Multi-cycle kinetics with measured sensorgrams (shown in black) overlaid with a Langmuir 1:1 binding model corrected for mass transport limitations (8K-insight SW). [Diagram 3] Figure 3 shows the affinity in solution (AiS) curves for Fab fragments. From left to right, 38F8, 7D4, 7E10 (top), 3B7, 18B3 and 4H8 (bottom), concentrations are held constant at 3 nM (38F8, 7D4 and 7E10), 5 nM (3B7) or 10 nM (18B3 and 4H8), respectively, and L-T4 concentrations are varied. As more L-T4 is present, there is less "free" Fab fragment in solution. The Fab concentrations determined for the competition experiments are plotted against the L-T4 competitor concentration. [Figure 4]Figure 4 shows the kinetic profiles of Fab fragments 38F8, 7D4, 7E10, 3B7, 18B3 and 4H8 binding to L-T3 at 37 °C: A) 38F8, B) 7D4, C) 7E10, D) 3B7, E) 18B3 and F) 4H8 binding series at increasing L-T3 concentrations c = 1.2 to 300 nM, dilution factor 3; replicates of 100 nM. Measured sensorgrams (left) and steady-state kinetics where the response reaches equilibrium in resonance units (RU) versus XR concentration are overlaid with the fitted binding model (right). [Diagram 5] Figure 5 shows a two-dimensional representation of the L-T4 ligand in the receptor binding pocket of Fab 38F8. The ligand is shown in stick representation and the receptor amino acids involved in binding are simplified to spheres, where the A and B letter prefixes represent the heavy and light chains, respectively. The curved dotted lines enveloping the ligand represent the main interaction areas with the ligand, while the straight dotted lines represent hydrogen bonds. The shaded spherical regions behind some of the ligand atoms indicate solvent accessibility, with larger shaded spheres indicating higher relative solvent accessibility. [Figure 6] Figure 6 shows the sequence alignment of the VH and VL domains of 38F8, 7D4, 7E10, 3B7, 18B3 and 4H8, respectively. The CDRs are highlighted in bold. Amino acid residues identified as part of the paratope that directly interacts with L-T4 according to the crystal structure of 38F8 in complex with L-T4 are underlined. [Figure 7] Balanced 3D structures of 38F8, 7D4, 4H8, and 7E10. The sequences of the heavy and light chain CDRs of all four clones are shown below. Amino acids that form the binding pocket of 38F8 and are identical in the other clones are shown in bold. Amino acids that differ in the binding pocket are shown in italics.

[0347] Array Description SEQ ID NO: 1: Amino acids (AS) 54 to 65 of VH of 38F8 according to Kabat numbering SGNTYYASWAKG SEQ ID NO: 2 AS54-65 of VH of 7D4 according to Kabat numbering SGNTYYATWAKG SEQ ID NO: 3: AS54-65 of VH of 7E10 according to Kabat numbering SGSTYYATWAKG SEQ ID NO: 4 AS50-56 of VL of 38F8 according to Kabat numbering GASTLTS SEQ ID NO: 5 AS50 to AS56 of VL of 7E10 and 7D4 according to Kabat numbering GASTLAS SEQ ID NO:6 CDR-H1 sequence of 38F8 with variations in paratope residues SNXMN, where X is V or A. SEQ ID NO: 7 CDR-H2 sequence of 38F8 YIWTRSGNTYYASWAKG SEQ ID NO: 8 CDR-H3 sequence of 38F8 with variations in paratope residues GLHIXYNIFNF, where X is G, A or V. SEQ ID NO: 9 CDR-L1 sequence of 38F8 with variations in paratope residues QSSQSVX1X2NAWCS (wherein X1 is H or Y and X2 is N or K) SEQ ID NO: 10 CDR-L3 sequence of 38F8 with variations in paratope residues AGX1X2X3GSTX4X5HV (wherein X1 is G or A, X2 is Y, W or F, X3 is S or T, X4 is Y or F, and X5 is N, S, T, Q, N) SEQ ID NO: 11 CDR-L2 sequence of 38F8 GASTLTS SEQ ID NO: 12 CDR-H1 sequence of 38F8 SNVMN SEQ ID NO: 13 CDR-H3 sequence of 38F8 with variations in paratope residues GLHIXYNIFNF, where X is G or A. SEQ ID NO: 14 CDR-H3 sequence of 38F8 GLHIGYNIFNF SEQ ID NO: 15 CDR-L1 sequence of 38F8 QSSQSVHNNAWCS SEQ ID NO: 16 CDR-L3 sequence of 38F8 with variations in paratope residues AGX1X2X3GSTX4X5HV (wherein X1 is G or A, X2 is Y, W or F, X3 is S or T, X4 is Y or F, and X5 is N, S, T) SEQ ID NO: 17 CDR-L3 sequence of 38F8 with variations in paratope residues AGGX1SGSTYX2HV (wherein X1 is Y, W, and X2 is N, S). SEQ ID NO: 18 CDR-L3 sequence of 38F8 AGGYSGSTYNHV SEQ ID NO: 19 CDR-H1 consensus sequence of 38F8, 7E10 and 7D4 X1NVX2N (wherein X1 is S or R, and X2 is M, L). SEQ ID NO: 20 CDR-H2 consensus sequence of 38F8, 7E10 and 7D4 YIWTX1SGX2TYYAX3WAKG (wherein X1 is R, G or D, X2 is N or S, and X3 is S or T). SEQ ID NO: 21 CDR-H3 consensus sequence of 38F8, 7E10 and 7D4 GLX1IGYX2IFNF (wherein X1 is H or A and X2 is N or A) SEQ ID NO: 22 CDR-L1 consensus sequence of 38F8, 7E10 and 7D4 QSSQSVX1X2NX3WX4S (wherein X1 is H or Y, X2 is N or K, X3 is A or N, and X4 is C or L). SEQ ID NO: 23 CDR-L3 consensus sequence of 38F8, 7E10 and 7D4 AGGX1SX2X3X4YX5HX6, where X1 is Y or W, X2 is G, A or S, X3 is S, G or N, X4 is T, G or S, X5 is N or S, and X6 is V or A. SEQ ID NO: 24 CDR-L2 consensus sequence of 38F8, 7E10 and 7D4 GASTLX1S (wherein X1 is T or A) SEQ ID NO: 25 CDR-H1 of 7E10 RNVMN SEQ ID NO: 26 CDR-H1 of 7D4 RNVLN CDR-H2 of SEQ ID NO: 27 7E10 YIWTDSGSTYYATWAKG SEQ ID NO: 28 CDR-H2 of 7D4 YIWTGSGNTYYATWAKQ SEQ ID NO: 29 CDR-H3 of 7D4 and 7E10 GLAIGYAIFNF SEQ ID NO: 30 CDR-L1 of 7D4 and 7E10 QSSQSVYKNNWLS CDR-L3 of SEQ ID NO: 31 7E10 AGGWSSNSYNHA SEQ ID NO: 32 CDR-L3 of 7D4 AGGWSAGGYSHA SEQ ID NO: 33 CDR-L2 of 7D4 and 7E10 GASTLAS FW-H1 of 38F8 according to SEQ ID NO: 34 Kabat LSLEESGGRLVTPGTPLTLTCTVSGIDLS FW-H2 of 38F8 according to SEQ ID NO: 35 Kabat WVRQAPGKGLEWIG FW-H3 of 38F8 according to SEQ ID NO: 36 Kabat RFTISKTSSTTVDLKMTSLTTEDTATYFCAG FW-H4 of 38F8 according to SEQ ID NO: 37 Kabat WGQGTLVTVSS FW-L1 of 38F8 according to SEQ ID NO: 38 Kabat AVLTQTPSPVSAAVGGTVTINC SEQ ID NO: 39 FW-L2 of 38F8 according to Kabat WFQKKPGQPPKQLIY FW-L3 of 38F8 according to SEQ ID NO: 40 Kabat GVPSRFKGSGSGTQFTLTISDVQCDDAATYYC SEQ ID NO: 41 FW-L4 of 38F8 according to Kabat FGGGTEVVVK SEQ ID NO: 42 VH domain of 38F8 LSLEESGGRLVTPGTPLTLTCTVSGIDLSSNVMNWVRQAPGKGLEWIGYIWTRSGNTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCAGGLHIGYNIFNFWGQGTLVTVSS SEQ ID NO: 43 VH domain of 7E10 QSVEESGGRLVTPGTPLTLTCTVSGIDLSRNVMNWVRQAPGKGLEWIGYIWTDSGSTYYATWAKGRFTISKTSSTTVELKMTSPTTEDTATYFCAGGLAIGYAIFNFWGQGTLVTVSS SEQ ID NO: 44 VH domain of 7D4 QSVEESGGRLVTPGTPLTLTCTVSGIDLSRNVLNWVRQAPGKGLEWIGYIWTGSGNTYYATWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCAGGLAIGYAIFNFWGQGTLVTVSS SEQ ID NO: 45 VL domain of 38F8 AVLTQTPSPVSAAVGGTVTINCQSSQSVHNNAWCSWFQKKPGQPPKQLIYGASTLTSGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYSGSTYNHVFGGGTEVVVK SEQ ID NO: 46 VL domain of 7E10 AVLTQTPSPVSAAVGGTVTISCQSSQSVYKNNWLSWFQQKPGQPPKLLIYGASTLASGVPSRFEGSGSGTQFTLTISDVQCDDAATYYCAGGWSSNSYNHAFGGGTGVVVT SEQ ID NO: 47 VL domain of 7D4 AAVLTQTPSPVSAAVGGTVTISCQSSQSVYKNNWLSWFQQKPGQPPKLLIYGASTLASGVPSRFEGSGSGTQFTLTISDVQCDDAATYYCAGGWSAGGYSHAFGGGTGVVVA SEQ ID NO: 48 Heavy chain of Fab 38F8 LSLEESGGRLVTPGTPLTLTCTVSGIDLSSNVMNWVRQAPGKGLEWIGYIWTRSGNTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCAGGLHIGYNIFFWG QGTLVTVSSGQPKAPSVFPLAPCCGDTPSSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCS SEQ ID NO: 49 Light chain of Fab 38F8 AVLTQTPSPVSAAVGGTVTINCQSSQSVHNNAWCSWFQKKPGQPPKQLIYGASTLTSGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYSGSTYNHVFGGGTE VVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SEQ ID NO:50 VH domain of 18B3 QSVEESGGRLVTPGTPLTLTCTLSGFSLKGYALSWVRQAPGKGLEWIGLIGNTGMTYYATWATGRFTISKTSTTVDLKMTSPTTEDTATYFCARDWFRYDTFGGTTVIYYYGMDLWGPGTLVTVSS SEQ ID NO:51 VH domain of 4H8 QSVEESGGRLVTPGTPLTLTCTASGFSLSAYYMIWVRQAPGKGLEWIGYIGGGVSASYASWANGRFTISSTSTTVDLKIPSPTTEDTATYFCARGSWNSGIDLWGQGTLVTVSS SEQ ID NO:52 VH domain of 3B7 QSLEESGGDLVKPGASLTLTCKASGIDFSGSALCWVRQAPGKGPEWIVCIYVGSFQNTYYASWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCASDASGISHYRYYFNLWGPGTLVTVSS SEQ ID NO:53 VL domain of 18B3 ALVMTQTPSPVSAAVGGTVTINCQASEEIGNNLAWFQQKPGQPPKLLIQRASTLASGVPSRFSGSGSGTDYSLTISGLQCDDAATYYCLGVLPYIGADGHAFGGGTEVVVKGDPV SEQ ID NO:54 VL domain of 4H8 AAVLTQTASPVSAAVGGTVTINCQSSQSVVNNNRLSWFQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGGYISTSDNAFGGGTEVVVKGDPV SEQ ID NO:55 VL domain of 3B7 AIEMTQTPFSVSAAVGGTVTISCQASESVYAKLGWYQQKPGQPPKLLIYDASSLASGVPSRFKGSGSGTEYSLTISDLECDDAATYYCQSAYYTRGADTWGAFGGGTEVVVKGDPV SEQ ID NO: 56 CDR-H1 of 4H8 AYYMI SEQ ID NO: 57 CDR-H2 of 4H8 YIGGGVSASYASWANG SEQ ID NO:58 CDR-H3 of 4H8 GSWNSGIDL SEQ ID NO:59 CDR-L1 of 4H8 QSSQSVVNNNRLS SEQ ID NO: 60 CDR-L2 of 4H8 KASTLAS SEQ ID NO: 61 CDR-L3 of 4H8 LGGYISTSDNA EXAMPLES

[0348] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0349] Example 1: Generation of antibodies specific to T4 Synthesis of hapten immunogens and screening reagents Synthesis of hapten T4-NH-PEG(3)-(OSu) 100 mg of L-thyroxine was dissolved in 8 mL of dry DMF, 38 μL of trimethylamine and 117 mg of NHS-PEG3-NHS were added. The reaction was stirred for 4 h. The solvent was evaporated and the product was purified by preparative HPLC reverse phase.

[0350] HPLC-ESI-MS: M+=1107.2 Da. Yield was 105 mg.

[0351] Synthesis of antigen conjugate T4-NH-PEG(3)-CO-KLH 7.9 mg of T4-NH-PEG(3)-(OSu), NHS ester, synthesized as described above, was dissolved in 1500 μL of DMSO and added to a solution of 100 mg of KLH (Keyhole Limpet Hemocyanin, Sigma H 8283). The pH was adjusted to pH=8.3 and the solution was stirred overnight. The mixture was purified in an Amicon stirred cell.

[0352] Amino group analysis: antigen-KLH ratio - approx. 200:1

[0353] The chemical structure of T4-NH-PEG(3)-CO-KLH is as follows: [ka]

[0354] Synthesis of T4(OSu)-bis-DADOO-biotin Biotin-DADOO-HS-DADOO (121 mg, see EP 451810A1) was dissolved in DMF (20 mL) containing triethylamine (36.1 μL) and T4-(Boc)-OSu (249 mg) was added. The reaction was stirred for 2 h. The solvent was removed on a rotary evaporator. The crude product was dissolved in TFA (6.0 mL) and stirred at room temperature for 30-60 min. The solvent was evaporated and the product was purified by preparative HPLC reverse phase to give 147 mg.

[0355] HPLC-ESI-MS: [M+2H + ] / 2=682.9 Da. The yield was 147 mg.

[0356] The chemical structure of T4(OSu)-bis-DADOO-biotin is as follows: [ka]

[0357] immunization New Zealand White (NZW) rabbits aged 12–16 weeks were immunized with T4-NH-PEG(3)-CO-KLH. To enhance the immunogenicity of the hapten, it was coupled to keyhole limpet hemocyanin (KLH) as a carrier protein. In the first month, animals were immunized weekly. From the second month, the immunization schedule was reduced to once a month. For the first immunization, 500 μg of T4-NH-PEG(3)-CO-KLH was dissolved in 0, 9% NaCl and emulsified in 2 ml of complete Freund's adjuvant (CFA). For all subsequent immunizations, CFA was replaced with 1 mL of incomplete Freund's adjuvant (IFA) emulsion.

[0358] Titer analysis Titer analysis was performed using an ELISA protocol. Serum titrations were performed using T4(OSu)-bis-DADOO-biotin as a positive control.

[0359] Biotinylated screening reagents were immobilized on the surface of 96-well streptavidin-coated microtiter plates by incubating 100 μl / well of a 16 ng / ml solution for 60 min at room temperature. Subsequent washes were performed using an automated instrument (Biotek) according to the manufacturer's instructions. A small amount of serum (2–3 ml per animal) from each rabbit was collected on days 45 and 105 after the start of the immunization campaign. Serum from each rabbit was diluted 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700 and 1:656100 in PBS containing 1% BSA. 100 μl of each dilution was added to the plate previously prepared with the screening peptide and incubated for 60 min at room temperature. Bound antibodies were detected with HRP-labeled F(ab')2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche). The titer of the analyzed animals was set at the 50% signal reduction of the dilution curve. [Table 1]

[0360] As demonstrated by the results in Table 1, polyclonal sera from immunized animals bound to the T4(OSu)-bis-DADOO-biotin screening peptide.

[0361] B cell cloning To enrich for antigen-reactive B cells, 100 ng / ml T4(OSu)-bis-DADOO-biotin was preincubated with peripheral blood mononuclear cell (PBMC) pools from immunized animals for 15 min at 4° C. After a washing step, antigen-reactive B cells bound to T4(OSu)-bis-DADOO-biotin were incubated with streptavidin-coated beads (Miltenyi) for 15 min at 4° C. Sorting of positive B cells using a MACS column (Miltenyi) and subsequent incubation were performed as described in Seeber et al., PLoS One 9 (2014), issue 2, e86184, with the only exception that sorting of positive B cells involved a MACS column (Miltenyi) instead of plate binding.

[0362] Subsequently, Hit-ELISA (i.e., ELISA testing binding to screening agents) was used to identify B cells with the desired binding properties, i.e. expressing antibodies that bind to T4(OSu)-bis-DADOO-biotin. T4(OSu)-bis-DADOO-biotin was immobilized on the surface of streptavidin-coated 96-well plates (Nunc) by incubation of 100 μl / well of a 100 ng / ml solution for 60 min each at room temperature. The plates were washed and 30 μl of rabbit B cell culture supernatant was transferred to each well and incubated for 1 h at room temperature. For detection of antibodies bound to the screening agents, HRP-labeled F(ab')2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche) were used according to the manufacturer's instructions. 5868 clones that bound to T4(OSu)-bis-DADOO-biotin were identified. 5868 clones that bound T4(OSu)-bis-DADOO-biotin were identified (out of 10 B cell sorting experiments with a total of 5 immunized rabbits). The V regions of 333 clones from the first B cell sorting and 421 clones from the second B cell sorting were cloned into mammalian expression vectors and subsequently expressed in 2 ml of HEK293 cells (described in Seeber et al., PLoS One 9 (2014), issue 2, e86184). After one week of expression, the supernatant of transfected HEK293 cells containing rabbit IgG was then used for an initial SPR Biacore-based selection of a subset of antibodies that met performance criteria for detailed kinetic analysis (see Examples 2 and 3) and evaluation in the Elecsys® platform-based fT4 assay (see Example 4).

[0363] Example 2: SPR Biacore kinetic screening to select antibodies The 840 recombinantly produced monoclonal antibodies identified in Example 1 as binding to T4(OSu)-bis-DADOO-biotin by ELISA were subjected to a further screening step using SPR Biacore. Specifically, 50 antibodies were pre-selected according to their kinetic characteristics, and finally a set of six antibodies was selected. In the course of selection and the following detailed evaluation of the six selected antibodies, the following characteristics of the antibodies were evaluated: kinetic parameters (K ) for binding to L-T4 and potential cross-reactivity with L-T3, D-T3, rT3, 3,3',5-triiodothyroacetic acid, 3,3',5,5'-tetraiodothyroacetic acid, 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine; D , k a , k d , velocity factor).

[0364] Kinetic screening was performed on a GE Healthcare BIAcore™ T200 instrument at 37° C. A Biacore CM5 Series S sensor was installed in the instrument and preconditioned according to the manufacturer's instructions.

[0365] The system buffer was PBS, pH 7.4, containing 11 mM PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4 + 0.05% (w / v) Tween 20 and 5% (v / v) DMSO.

[0366] System buffer supplemented with 1 mg / mL (carboxymethyl dextran (CMD)) was used as the sample buffer.

[0367] A rabbit antibody capture system was immobilized on the sensor surface. The system buffer was HBS-ET+pH 7.4 containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (w / v) Tween 20. The polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (Code No. 111-005-046, Jackson Immuno Research) was amine coupled at 25° C. using EDC / NHS chemistry according to the manufacturer's instructions.

[0368] 25 μg / mL of capture antibody was used in 10 mM sodium acetate buffer pH 5.0. The capture antibody was immobilized on flow cell Fc1-4 at a ligand density of 13000-15000 RU. Free activated carboxyl groups were saturated with 1 M ethanolamine pH 8.5.

[0369] 25 nM rabbit normal IgG (SIGMA) was used as a reference for FC1. Rabbit antibody-containing cell culture supernatants were diluted in sample buffer and captured on flow cells 2, 3 and 4. Capture was performed at 5 μl / min for 3 min. Antibody capture levels (CL) were monitored.

[0370] Kinetic screening of 840 cell culture supernatants was performed using a single concentration injection of 30 nM L-T4 (Roche) analyte. The association of the analyte was monitored for 3 min and the dissociation phase for 5 min at 60 μl / min. After each measurement cycle, the capture system was regenerated by injecting 10 mM glycine buffer pH 2.0 and pH 2.25 at 20 μL / min for 30 and 60 s.

[0371] Two reporting points, the analyte binding delay (BL), which is the signal recorded just before the end of the analyte injection, and the stability delay (SL), which is the signal just before the end of the dissociation time, were used to characterize the antibody / antigen binding stability.

[0372] association rate constant k a [M -1 s -1 ], dissociation rate constant k d [s -1 ] and the dissociation equilibrium constant K D [M] was calculated using the Langmuir model with the evaluation software. The antibody / antigen complex half-life was calculated using the formula t / 2diss=ln(2) / (k d*60) in minutes. Importantly, in this initial screening of a large number of antibodies, no mass transport correction was applied because the T200 Evaluation-Software used does not provide a correction for MTL. Therefore, if the binding between the antibody and L-T4 is diffusion limited in the Biacore setup, k a The velocity may be underestimated. Note that in Example 3, MTL correction was applied.

[0373] The molar ratio and binding stoichiometry were calculated according to the following formulas: MR=BL(antigen)*MW(antibody) / (MW(antigen)*CL(antibody)).

[0374] In this way, 840 rabbit mAbs were kinetically examined. The kinetic data of the antibody pool were statistically evaluated. The overall dissociation equilibrium constant, K D The average value of -10 M. The highest affinity found was K D 1.94×10 -12 M, and the lowest affinity in the antibody pool is K D 1.44×10 -9 (see Figure 1). The 840 antibody kinetics were first ranked according to their dissociation rate constants and a set of 50 antibodies was empirically selected from different complex stability pathways that clustered around the highest, lowest and average T4 complex stability (see Figure 1). These 50 recombinant antibodies were then kinetically examined in more detail by multi-cycle kinetics with T4 analyte concentrations between 0.04 nM and 30 nM at two different temperatures, 13 °C and 37 °C. Again, no mass transport correction was applied even if the measured signatures showed such a correction. The assay setup from the kinetic screen was used as described. The goal was to identify suitable antibodies using the principle of the rate coefficient (EP 2470563 B1). The rate coefficient as used herein is the quotient k a (37℃) / k a (13℃). The L-T4 association rate constant k a [M -1 s-1 ], and the association rate constant k a We focused on antibodies with k a Antibodies with dramatically increased binding entropy had undesirable high binding entropy and were not selected.

[0375] For example, antibody 3B7 has a a (37℃) / k a (13°C)=18 and was selected, but antibody 18C5 had a quotient k a 37℃ / K a 13℃=152 and was deselected.

[0376] From this association rate, six mAbs were selected for more detailed kinetic characterization for binding to L-T4 and the potential cross-reactants 3-iodo-L-tyrosine (L-T3), rthyroid hormone (rT3), 3,3',5-tri-iodo-thyroacetic acid, 3,3',5,5'-tetra-iodothyroacetic acid, 3,5-di-iodo-L-tyrosine, and 3-iL-tyrosine.

[0377] L-T3, D-T3, rT3, 3,3',5-triiodothyroacetic acid, 3,3',5,5'-tetraiodothyroacetic acid, 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine.

[0378] The antibodies selected were 38F8, 7D4, 7E10, 3B7, 18B3 and 4H8. These selected antibodies had rate coefficients between 2 and 35 (without applying MTL correction).

[0379] Example 3: Further SPR Biacore characterization of the six antibodies selected in Example 2 In Example 2, six antibodies were selected for more detailed evaluation. For this evaluation, their corresponding Fab fragments were used rather than the original IgG. The Fab fragments were recombinantly expressed and purified as described in Example 4A) below.

[0380] Detailed kinetic characterization of six selected antibodies The affinity of six surface-displayed Fab fragments was determined using a GE Healthcare Biacore™ 8K instrument at 37° C. Measurements were performed as described above with the following adaptations.

[0381] As a capture system, PAK<K-F(ab)2> Z-IgG(IS) (Code No. 111-005-006, Jackson Immuno Research) was used. Amine coupling was performed using 35 μg / mL PAK in 10 mM sodium acetate buffer, pH 5.0.<K-F(ab)2> The procedure was carried out as described using HBS-N buffer pH 7.4 with the concentration of Z-IgG (IS).

[0382] Multicycle kinetics was performed using a series of increasing L-T4 concentrations, c = 0.12-10 nM, with a dilution factor of 3. Buffers used were as described in Example 2. Regeneration was performed by injecting 10 mM glycine buffers pH 2.0 and pH 2.25 at 20 μL / min for 60 s.

[0383] The interactions at 37° C. are shown in FIG.

[0384] The interactions of Fab 38F8, 7D4 and 7E10 showed large mass transport limitations (MTL) when bound to L-T4, whereas Fab 3B7 showed a lower MTL. MTL corrections were applied automatically using Evaluation Insight Software V3.011.15423 from the vendor, where MTL corrections are addressed using a two-compartment model. Thus, the kinetic constants represent apparent values ​​but are corrected for the MTL.

[0385] Fabs 38F8, 7D4 and 7E10 showed significantly accelerated complex formation rates compared to Fabs 3B7, 18B3 or 4H8 at 37°C.

[0386] k of 38F8, 7D4, and 7E10 at 37°C aThe rate constant is >1.0E+09M -1 s -1 , which is close to or outside the instrument specifications. a The rate constant is 4.8E+06~1.8E+07M -1 s - The answer was 1.

[0387] The resulting affinities were determined computationally: K D = 280 pM (38F8), 125 pM (7D4), 248 pM (7E10), 37 pM (3B7), 960 pM (18B3) and 1210 pM (4H8); see Table 2 below. [Table 2]

[0388] Affinity in solution at 37℃ Complementary to the affinity obtained from the kinetic rate constant, the dissociation equilibrium constant, K D was determined by affinity in solution (AiS). The advantage of affinity in solution analysis is that it is not subject to mass transport limitations and allows measurements at equilibrium.

[0389] Biotinylated (Bi-)T4 was pre-captured on the CAP-chip sensor surface via streptavidin (SA)-biotin interaction. A mixture of anti-T4-Fab fragments and unlabeled T4 was pre-incubated for several hours to reach equilibrium. The concentration of "free" Fab fragments was determined via binding to the surface-displayed biotinylated T4 using a prior Fab calibration for quantification.

[0390] The Fab fragment concentration was held constant in the mixture while the T4 concentration was varied: as more T4 was present, there was less "free" Fab fragment in solution.

[0391] The assay method was as follows: Following the CAP-reagent, biotinylated T4 was reversibly captured at high density on the sensor surface according to the vendor's instructions for the CAP-Kit (Cytiva). Regeneration was performed after each cycle using a guanidinium / NaOH solution.

[0392] Pre-incubation of both interaction partners in solution: Fab concentrations were kept constant at 10 nM for Fabs 18B3 and 4H8, 5 nM for 3B7, and 3 nM for Fabs 38F8, 7E10 and 7D4. T4 thyroid hormone concentrations were optimized individually for each T4 interaction, i.e., c = 0.07-150 nM (18B3 and 4H8), 0.07 nM-50 nM (3B7), 0.021 nM-90 nM (38F8, 7E10 and 7D4). Data were evaluated using the affinity model in solution from the Biacore Evaluation software.

[0393] The affinity curves in solution of Fab fragments 38F8, 7D4, 7E10, 3B7, 18B3 and 4H8 are shown in FIG.

[0394] K determined by affinity in solution D The values ​​are as follows: [Table 3]

[0395] K measured for clones 38F8, 7D4, 7E10 and 3B7 D The values ​​are in a similar range to those measured in different settings in the kinetic analysis described above, confirming these results.

[0396] Fab fragment 4H8 shows a higher affinity for factor 1.7 than that measured by kinetic analysis. Both Fab fragments 18B3 and 4H8 show the weakest affinity of the six Fab fragments, confirming the ranking.

[0397] Cross-reactivity For multi-cycle cross-reactivity measurements, analyte concentrations between 0.1 nM and 900 nM were used. Potential cross-reactants were injected at 30 μL / min to 60 μL / min. The association phase was monitored for 3 min to 5 min and the dissociation phase for 5 min to 15 min. An additional analyte injection with a 30 min dissociation time was used to characterize the true T4 interaction.

[0398] The affinity of the six Fab fragments binding to the cross-reactants was determined using multiple cycle kinetics with a concentration series at 37° C. Cross-reactant L-T3 exhibits at least 49-fold weaker affinity than L-T4 at 37° C., and rT3 exhibits at least 18-fold weaker affinity than L-T4 at 37° C.

[0399] The interactions of cross reactant L-T3 at 37° C. are shown in FIG. 4. The results of all cross reactants are summarized in Table 4. For Fab 38F8, additional cross reactants were also analyzed (see Table 5). [Table 4] [Table 5]

[0400] Example 4: Characterization of antibodies selected in Example 2 in the Elecsys® competitive immunoassay A.) Purification of anti-T4-Fab Antibody candidates preselected according to their kinetic behavior by BiaCore-Analysis (see Examples 2 and 3) were expressed as His-tagged Fab fragments using transient transfection of HEK cells. Culture supernatants were concentrated using a Vivaflow 200 ultrafiltration unit (Sartorius, Germany) with a MW cutoff of 10 kDa, followed by buffer exchange via dialysis or diafiltration against 20 mM KPO4, 150 mM NaCl, 10 mM imidazole, pH 8.0. A Ni-NTA / IMAC affinity chromatography column (HisTrap, GE Healthcare, Sweden) was then equilibrated with the above dialysis buffer, and the conditioned supernatant was applied to the column at a flow rate of 60 column volumes / hour. Fab fragments were then eluted over 10-20 column volumes using a linear gradient of buffer A (20 mM KPO4, 150 mM NaCl, 10 mM imidazole, pH 8.0) and buffer B (20 mM KPO4, 150 mM NaCl, 500 mM imidazole, pH 8.0) containing 0-35% B. Fractions of eluted Fab were assessed for purity using analytical size-exclusion chromatography, and fractions with Fab purity ≥ 95% were pooled. Finally, the pooled Fab-preparation was dialyzed against a storage buffer of 20 mM KPO4, 100 mM KCl, 2% sucrose, pH 7.9, aliquoted, and stored frozen at -80 °C.

[0401] B.) Preparation of anti-T4-Fab ruthenium-labeled conjugate Purified anti-T4 Fab antibody was labeled with the N-hydroxy-succinimide activated ester of sulfobispyridyl-ruthenium according to standard laboratory procedures. Briefly, to a solution of Fab (4.4 mg, 1.03 mL in 100 mM KPO4, pH 8.4) is added 52 μL of a solution of SULFO-BPRU NHS ESTER (CAS482618-42-8: Ruthenium salt (2-), bis[[2,2'-bipyridine]-4,4'-dimethanesulfonate(2-)-κN1,κN1'][1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-κN1,κN1')-1-oxobutoxy]-2,5-pyrrolidinedione]-, sodium (1:2), (OC-6-31)-(ACI);12, 5 mg / mL in DMSO) (i.e., a stoichiometry of 6.0 moles of ruthenium-NHS-ester per mole of Fab). After stirring for 120 min at room temperature, derivatization was stopped by adding lysine to a final concentration of 10 mM. The pH was adjusted to pH 7.5 with saturated KH2PO4, and the reaction mixture was dialyzed overnight against 50 mM KPO4 / 0, 15 M KCl / 2% sucrose, pH 7.5. To remove aggregates and remaining hydrolyzed ruthenium esters, appropriate fractions were collected from size-exclusion chromatography on a Superdex 75 HR 10 / 30 column (GE Healthcare Life Sciences). After adding sucrose to a final concentration of 6.5% (w / v), the preparation was filtered through a 0.45 μm PVDF syringe filter (Acrodisk Pall Life Sciences Corp.) and stored frozen at -80 °C.

[0402] C.) Functional evaluation of anti-T4 Fab-ruthenium conjugates The assay principle of the competitive immunoassay using the automated Elecsys Immuno-Analyzer (Elecsys® cobas® e411) is summarized below. The total incubation time required for the assay is 18 min: First incubation (9 min): 15 μL of fT4-containing sample, 75 μL of ruthenium-conjugated monoclonal T4-specific Fab antibody are incubated to form a complex containing a certain proportion of ruthenium-conjugated anti-T4 Fab antibody and free T4. Second incubation (9 min): 75 μL of a solution of T4(OSu)-bis-DADOO-biotin-hapten-conjugate and 35 μL of streptavidin-coated microparticles are added to the mixture from the first incubation step. During this second incubation, the ruthenium-conjugated anti-T4 Fab antibodies that remain free after the first incubation step can bind to the biotinylated T4-hapten, and the complex of the ruthenium-conjugated antibodies bound to the T4-biotin conjugate binds to the streptavidin-coated magnetic beads / solid phase via the interaction of biotin with streptavidin. The reaction mixture is aspirated into a measuring cell, where the microparticles are magnetically captured on the surface of the electrode. Unbound material is then removed with a ProCell # 11662988122 (Roche Diagnostics GmbH Germany). The application of a voltage to the electrode then triggers the emission of light based on electrochemiluminescence, which is measured by a photomultiplier tube.

[0403] A plot of ECL signal in counts versus concentration of free T4 yields a typical hyperbolic competition curve with a falling signal with increasing T4 concentration. [Table 6]

[0404] As shown in Table 6 above, the Biacore experiments performed in Examples 2 and 3 showed exceptionally high k a The three Fab fragments 38F8, 7E10 and 7D4, which showed high kA activity against T4, were among the four best antibodies tested.a is identified as one (among other things) important factor for providing a competitive immunoassay. Furthermore, the data demonstrate that polyclonal antibodies can be successfully replaced by single monoclonal antibodies as provided herein.

[0405] Example 5: Determination of the crystal structure of Fab antibody 38F8 in complex with T4 Crystallization of Fab 38F8 with and without L-T4 hormone: The solution containing the 38F8 Fab fragment was concentrated to 18 mg / ml and subjected to crystallization screening. Crystallization drops were set up at 21° by mixing 100 nl of protein solution with 100 nl of reservoir solution (1:1 ratio) or 140 nl of protein solution with 60 nl of reservoir solution (7:3 ratio) in vapor diffusion sitting drop experiments. Crystals appeared in several conditions containing polyethylene glycol (PEG) as a precipitant. Crystals used for structure determination appeared within 2 days and grew to full size within 4 days in conditions containing the following precipitant solutions: 120 mM ethylene glycol mixture (30 mM diethylene glycol, 30 mM triethylene glycol, 30 mM tetraethylene glycol and 30 mM pentaethylene glycol), 100 mM Tris Base-BICINE pH 8.5, 20% v / v PEG 500 MME and 10% w / v PEG 20000.

[0406] Apo crystals were harvested from the 7:3 ratio droplets and crystals from the 1:1 ratio droplets were immersed for 20 h in a saturated solution of L-thyroxine hormone (L-3,5,3',5'-tetraiodothyronine or L-T4, Sigma) containing the above precipitant solution plus 10% DMSO. Crystals were harvested directly from the precipitant solution and flash cooled in liquid N2. Diffraction images were collected on an EIGER2X 16M detector at beamline X10SA at the Swiss Light Source at a temperature of 100 K and images were processed with the XDS package [Kabsch W. XDS. Acta Cryst. D66, 125-132 (2010)]. For the apo crystals, data from single crystals were merged to obtain a 1.81 Å resolution dataset in space group C121 with two molecules in the asymmetric unit (see Table 7). Similarly, for the L-T4 bound crystal, data from single crystals were merged to give a 1.66 Å resolution data set in space group C121 with two molecules in the asymmetric unit.

[0407] The structure was determined by molecular replacement in the program PHASER [McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Stroni LC, Read RJ J. Appl. Cryst. 40, 658-674 (2007)]) as part of the PHENIX [Liebschner D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Cryst. D75, 861-877 (2019)] suite. The Fab fragment from PDB-ID 6LDX was divided into constant and variable domains and used as a search model. The molecular replacement solution model was reconstructed in COOT [Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Cryst. D66, 486-501 (2010)] and refined with PHENIX Refine. [Table 7-1] [Table 7-2]

[0408] Structure of Fab 38F8 with and without L-T4 hormone To characterize the binding of Fab 38F8 to L-T4 in atomic detail, the crystal structures of the Fab in apo form and L-T4 ligand-bound form were determined. The overall conformation of the Fab is very similar in both apo and ligand-bound forms, with an RMSD of 0.11 Å2. No significant differences were observed in the side chains of the CDR loops, suggesting a fast on-rate of ligand binding. This is consistent with the Biacore data shown in Example 3.

[0409] The Fab paratope is a pocket formed at the interface between the light and heavy chains.

[0410] Analysis with the program PISA [Krissinel E and Henrick K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774--797 (2007)] revealed that 416 Å2 surface area of ​​the ligand is buried by Fab 38F8, which is 68% of the total ligand surface area. A total of 16 amino acids make up the Fab paratope, 7 from the heavy chain and 9 from the light chain (see Figure 5 and Table 8). All CDR loops except the light chain CDR2 participate in ligand binding, which is mainly governed by hydrophobic interactions with the iodine atom and the phenol ring. They expose the hydrophilic amino and carboxyl groups of the ligand to the solvent and out of the pocket. The heavy and light chain CDR3 loops of the Fab form the majority of the hydrophobic pocket, which is formed by both side chain and main chain interactions. These are summarized in Table 7.

[0411] Three hydrogen bonds serve to stabilize the ligand formed by the O4 and O4' atoms of the ligand, Tyr50 on the CRD2 of the heavy chain of the Fab, and His28 on CDR1 and His96 on CDR3 of the light chain. In addition, there are two polar-pi(arene-H bond) interactions; in one example, a water molecule coordinated by His28 and Asn29 of the Fab light chain is the hydrogen donor to one of the ligand phenol rings, and in the other, C2' is the hydrogen donor to the heterocyclic imidazole ring of His30 of the Fab light chain. All amino acid numbering follows Kabat nomenclature. [Table 8]

[0412] overview: · The Fab 38F8 paratope is a pocket composed primarily of CRD3 loop residues from both the heavy and light chains. · The conformational similarity between the apo- and ligand-bound states of Fab suggests a fast ligand-binding on-rate. · The interactions with the L-T4 ligand are mostly hydrophobic, via the iodine atom and the phenol ring, but also include three hydrogen bonds and two polar-pi interactions. 68% of the accessible surface area of ​​the ligand is filled by Fab

[0413] Example 6: Sequence comparison of screened antibodies Example 3 shows that Fab antibodies 38F8, 7E10 and 7D4 have significantly higher k aMoreover, Example 4 showed that these three clones were among the four best tested clones in a competitive immunoassay established on the Elecsys® system. To investigate whether these three antibodies and some of the other six selected antibodies (see Example 2) share sequence similarity, sequence alignment of the variable regions (VH and VL) was performed. The sequence alignment (see FIG. 6) surprisingly revealed that the antibodies 38F8, 7D4 and 7E10 have a remarkably high sequence similarity in the VH and VL regions, and especially also in the CDRs. Even more surprisingly, the conservation of the amino acid positions forming the paratope of the 38F8 antibody is extremely high. This suggests that 38F8, 7D4 and 7E10 are closely related antibodies that share high sequence similarity, especially also in the CDR sequences. As mentioned above, these antibodies have excellent functional characteristics, especially very high k a Moreover, these three Fab fragments were among the four best tested antibodies in an Elecsys®-based immunoassay for quantification of fT4 as assessed by signal-to-noise.

[0414] To quantify the observed similarity, a similarity score was obtained from the sequence alignment using a customized score matrix. This matrix is ​​constructed based on five weighted physical parameters: shape index, van der Waals volume, isoelectric point, hydrophobicity, and polarizability. These five parameters capture the most important features of the amino acids forming the paratope and allow to identify heterologous clones with similar binding motifs. Antibodies with high similarity scores have both similar binding modes as well as similar amino acid sequences in the CDR regions. The higher the similarity score, the more related the antibodies are. Similarity scores with respect to 38F8 for clones 7D4-98%, 7E10-98%, 3B7-86%, 18B3-92%, 4H8-95%. This similarity score analysis confirmed that 7E10 and 7D4 are closely related to 38F8. The paratope residues that mediate binding are highly similar (12 and 13 of the 16 amino acids are identical).

[0415] Example 7: Modeling analysis to further characterize the binding of 38F8 and related antibodies 7D4 and 7E10 to T4 The binding of Fab 38F8 to the L-T4 hormone at the atomic level was characterized using an in silico approach, aiming to define which amino acids in the CDRs are important for binding and to obtain evidence of the degree to which amino acid substitutions at specific CDR positions are tolerated.

[0416] In silico analysis showed that 7D4 and 7E10 exhibited similar kinetic properties (k a and K. D) similar to 7E10 and slightly worse Elecsys® performance. We also took advantage of the fact that 4H8 also shared significant sequence similarity with 38F8, while showing large differences in its kinetic properties as well as Elecsys® performance. Thus, a combination of known functional characteristics and sequence variations could be used as a basis to predict the impact of amino acid substitutions on the functional characteristics of 38F8 (kinetics, specificity and Elecsys® performance). In other words, this allowed us to identify important amino acid residues (i.e., cannot be substituted without compromising functional performance), potentially important amino acids (i.e., amino acids that can only be exchanged for specific amino acids) and non-important amino acids (both conservative and non-conservative amino acids should be possible) in the CDRs for maintaining 38F8 T4 binding properties (kinetics, specificity and Elecsys® performance).

[0417] As a first step, equilibrated structures of 38F8 and two highly similar antibodies, 7D4, 7E10 (similarity score >98%) and 4H8 (similarity score 95%), were generated. To obtain the equilibrated structures of 7D4, 7E10, and 4H8, the initial 3D shapes were first predicted using the MoFvAB package based on a machine learning algorithm trained on the internal Roche antibody crystal structure database. After this initial guess, the 3D structures were further optimized using molecular dynamics simulations to obtain the correct loop shapes (details of the simulation parameters are provided below). The crystal structure of 38F8 and the equilibrated structures of 7D4, 7E10, and 4H8 are shown in Figure 7. As a second step, point mutations are made in the sequence of 38F8, free energy minimization is performed, and the initial structure and the structures of the three other clones or structures are compared.

[0418] The effect of the point mutations in 38F8 on binding to the L-T4 hormone was modeled and predicted by comparing the sequences and 3D shapes of very similar clones with similar (7D4, 7E10) or different (4H8) functional characteristics (shown in Figure 7). All residues that directly contribute to the paratope are listed in Example 5 above and printed in bold in the alignment in Figure 7. Substitutions in the paratope region in the other three clones are shown in italics. With a few exceptions, all residues in the paratope are considered critical amino acids, i.e., amino acids important for maintaining functional characteristics. However, some of the interactions in the paratope are exclusively through the peptide backbone, so that at least certain amino acid substitutions must be possible at these positions (see below). In addition, other paratope amino acids form hydrophobic interactions with their side chains that can be mimicked by closely related amino acids (e.g., V33 in HC and Y95b in LC). These paratope amino acids that can be substituted for specific amino acids are grouped as potentially important in Table 9. The non-critical regions listed in Table 9 are loop fragments that are far from the binding pocket and preserve their structure even upon mutation in this region. Thus, the analysis proposes that amino acid exchanges in this "non-critical" region should be possible. Such mutations can include conservative amino acid exchanges, but also non-conservative amino acid exchanges. In addition, potential critical amino acid positions in the CDRs were found, i.e. residues that do not contribute to the paratope but can only be replaced by certain other amino acids so as not to affect the structural position of the critical paratope residues. Amino acids in the paratope region that contribute to binding only through main-chain interactions or have family variations in clones 7D4 and 7E10 are identified as potentially critical and therefore excluded from the critical regions. The critical regions include only amino acids that are involved in side-chain interactions and should not be exchanged. Table 9 summarizes the critical, potentially critical and non-critical amino acids in the CDR regions of 38F8. [Table 9]

[0419] To analyze the effect of amino acid substitutions in regions defined as potentially important, we used an energy minimization approach (details provided below). Valid point mutations are allowed if they do not affect either the hydrophobicity index, the orientation of other amino acids, or the excluded volume. As an initial guess of possible amino acid substitutions in potentially important amino acids, we considered the amino acid substitutions found in 7D4, 7E10, and 4H8 relative to 38F8. The results from modeling possible amino acid substitutions in potentially important regions are collected in Table 10 and described below. The possible mutations are divided into three groups: family variations (i.e., found in the closely related 7D4 and 7E10), amino acid substitutions approved using in silico evaluation, and other suggested substitutions with amino acids that have similar physicochemical characteristics as the in silico approved substitutions. [Table 10]

[0420] Summary of amino acid substitution analysis by CDR CDR-H1: Amino acids M34 and N35 are close to V33 (involved in the binding mode). Mutations M34L (I) and N35S (T) do not affect the conformation and orientation of V33, suggesting that these mutations do not affect paratope binding and antibody properties. Mutations to amino acids with similar physicochemical properties are possible (N35Q, M34V). V33 contributes to the interaction with L-T4 via hydrophobic side chain interactions. Substitution V33A should not affect this interaction according to a comparison of the hydrophobic index and excluded volume.

[0421] CDR-H2: Amino acids I51, T52a, R53 are close to the critical Y50 and W52. From the same clone family, the mutation R53G is possible due to the small size of G, and mutation to a different class of amino acid is important. The mutation R53D is not important due to its charged nature, and the orientation is preserved. The mutations I51A(L), R53K and T52aS do not affect the conformation and orientation of the critical Y50 and W52. Since Y50 is involved in H-bonding, exchanges at this position may be important. W52 is part of the hydrophobic interactions, and the proper orientation of the aromatic moiety is also important for the attachment point.

[0422] CDR-H3: Amino acids H97, G99, N100a are close to the critical I98, Y100, I100b. Mutations H97A and N100aA result in a reorientation of the critical IGY(98-100) fragment. Even with the altered orientation, as A is found at positions 97 and 100a in 7E10 and 7D4, antibodies with A97 and / or A100a still have superior binding affinity k a and Elecsys assay performance. The mutations H97R(K), N100aQ were found to maintain the proper orientation of the IGY(98-100) fragment and therefore may be preferred substitutions. G99 can be mutated with the mutation G99A(V), a small amino acid involved in binding through the backbone.

[0423] CDR-L1: Amino acid N30 is close to paratope amino acids H28, N29, A31, W32. Mutation N30Q(S,T) does not affect the orientation of fragment HNNAW(28-32). N30Q(S,T) is in the binding fragment HNNAW(28-32), but points in the opposite direction and does not affect binding with L-T4. Mutation H28K is important, it leads to incorrect orientation of N29 and cleavage of the aromatic bond in the binding motif. Mutation A31 slightly shifts the position of W32 (and affects pi-stacking), so this position should preferably be small and hydrophobic, e.g. V. Position W32 is important. Family variations are listed in Table 10.

[0424] CDR-L2: CDR-L2 can have various point mutations. The CDR-L2 loop is not directly involved in binding and is therefore classified as a non-critical region.

[0425] The orientation of CDR-L3: Y95b, H96 is important for the binding mode. Y95b is involved in hydrophobic interactions and can be mutated in F. Mutations Y92W(F) and S93T are not important as these amino acids are involved through main-chain interactions, but the overall orientation is shifted. Mutations G91A, G94A(S), S95G(N), T95aS, N95cS(T), V97A do not affect the conformation and orientation of the important Y95b, H96. Further mutations at certain of the above positions to amino acids with similar physicochemical properties are possible.

[0426] Computer simulation details: 1) Molecular dynamics simulation. The GROMACS simulation package was used to obtain the structures of 7E10, 7D4 and 4H8 Fab. The antibodies and environmental water were modeled with full-atom representations in the canonical (NVT) ensemble (box size: 7.0 × 7.0 × 7.0 nm3) using AMBER99SB-ILDN [Lindorff-Larsen et al., Proteins 78, 1950-58, 2010] force field parameters and the tip3p model for water [DJ Price, and CL Brooks III, J. Chem. Phys. 121, 10096, 2004] with a time step of 2 fs. The temperature was set to 300 K by the velocity rescale thermostat. Each dynamic trajectory was 300 ns long to sample the loop conformation.

[0427] 2) Structural optimization during amino acid substitution. Amino acid substitutions in the crystal structure of 38F8 are performed using the computer software platform SAMSON model [OneAngstrom, SAMSON, 2020, available at https: / / www.samson-connect.net / ]. After certain point mutations in the crystal structure, the energy of the newly obtained structure was minimized to balance the local degrees of freedom. This was done by the FIRE (Fast Inertial Relaxation Engine) optimizer for molecular structures [Bitzek et al., Physical Review Letters, 97, 170201, 2006].Physical Review Letters, 97, 170201, 2006].

Claims

1. A monoclonal antibody that specifically binds to L-thyroxine (T4), the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: V or A at position 33; Y at position 50; W at position 52; I at position 98; G, A or V at position 99; Y at position 100; and I at position 100b; ii) a light chain variable domain (VL) comprising amino acids H or Y at position 28; N or K at position 29; W at position 32; G or A at position 91; Y, W or F at position 92; S or T at position 93; Y or F at position 95b; N, S, T or Q at position 95c; and H at position 96. Including, A monoclonal antibody, wherein the amino acid positions in the VH and VL are each indicated according to the Kabat numbering scheme.

2. 2. The monoclonal antibody of claim 1, wherein the VH comprises M, L, I, or V at position 34; N, S, T, or Q at position 35; I, A, L, or V at position 51; T or S at position 52a; R, G, D, or K at position 53; H, A, R, or K at position 97; and / or N, A, or Q at position 100a, the amino acid positions being indicated according to the Kabat numbering scheme; and / or the VL comprises N, Q, S, or T at position 30; A, N, or V at position 31; G, A, or S at position 94; S, G, N, Q at position 95; T, S, or G at position 95a; and / or V, A, I, or L at position 97, the amino acid positions being indicated according to the Kabat numbering scheme.

3. A monoclonal antibody that specifically binds to L-thyroxine (T4), the monoclonal antibody comprising: i) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 1, 2, 4, and 5 of SEQ ID NO: 6; (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 2 and 4-17 of SEQ ID NO: 7; and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 1-3, 7, and 9-11 of SEQ ID NO: 8; ii) a light chain variable domain (VL) comprising: (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 1 to 6, 9, 10, 12, and 13 of SEQ ID NO: 9; and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having amino acid substitutions at not more than four positions selected from positions 1, 2, 6 to 8, and 12 of SEQ ID NO:

10. A monoclonal antibody comprising:

4. 4. The monoclonal antibody of claim 3, wherein the light chain variable domain comprises a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having amino acid substitutions at no more than four positions of SEQ ID NO:

11.

5. At a temperature of 37°C, the association rate constant (k ａ ) is 1.9*10 ７ M －１ sec －１ The monoclonal antibody according to any one of claims 1 to 4, wherein

6. The association rate constant (k ａ ) is the association rate constant (k ａ 5. The monoclonal antibody of claim 1, wherein the association rate constant corresponds to at least 10% of the association rate constant measured under identical experimental conditions.

7. 5. The monoclonal antibody of claim 1, wherein the monoclonal antibody distinguishes T4 from 3-iodo-L-tyrosine (L-T3), rthyroid hormone (rT3), 3,3',5-tri-iodo-thyroacetic acid, 3,3',5,5'-tetra-iodothyroacetic acid, 3,5-di-iodo-L-tyrosine and / or 3-i-L-tyrosine.

8. 5. The monoclonal antibody of any one of claims 1 to 4, wherein said monoclonal antibody, when used in a competitive immunoassay for quantifying T4, exhibits a signal to noise ratio that is at least 29%, in embodiments 65% and in embodiments at least 95% of the signal to noise ratio achieved with a Fab fragment having the heavy chain sequence of SEQ ID NO: 48 and the light chain sequence of SEQ ID NO: 49 in an otherwise identical immunoassay setting.

9. A polynucleotide comprising: (i) the heavy chain or heavy chain variable domain of the monoclonal antibody according to any one of claims 1 to 4, and / or (ii) 10. The light chain or light chain variable domain of the monoclonal antibody according to any one of claims 1 to 4. A polynucleotide encoding

10. A vector comprising the polynucleotide of claim 9.

11. A host cell comprising the polynucleotide of claim 9.

12. A host cell comprising the vector described in claim 10.

13. 12. A method for producing a monoclonal antibody according to any one of claims 1 to 4, said method comprising culturing a host cell according to claim 11 and isolating said antibody.

14. A composition comprising the antibody of any one of claims 1 to 4.

15. A composition comprising the polynucleotide described in claim 9.

16. A composition comprising the vector described in claim 10.

17. A composition comprising the host cell described in claim 11.

18. Use of an antibody according to any one of claims 1 to 4 for the in vitro detection or quantification of T4, in particular free T4, in a sample.

19. Use of the composition described in claim 14 for the in vitro detection or quantification of T4, particularly free T4, in a sample.

20. An in vitro immunoassay method for quantifying T4, in particular free T4, in a sample, using the antibody according to any one of claims 1 to 4.

21. A kit comprising the antibody of any one of claims 1 to 4.

22. A kit comprising the composition described in claim 14.

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