Binding proteins, methods and uses thereof

Abstract

This invention relates to binding proteins that bind to alanine aminotransferase (ALT). The invention also relates to bivalent, trivalent, tetravalent, or multivalent binding proteins that bind ALT. Furthermore, the invention relates to nucleic acids, vectors, and host cells for producing such binding proteins, methods for producing such binding proteins, kits comprising such binding proteins, and methods and uses of such binding proteins.

Description

Technical Field

[0001] This invention relates to binding proteins that bind to alanine aminotransferase (ALT) enzymes. The invention also relates to divalent, trivalent, tetravalent, or multivalent binding proteins that bind ALT. Furthermore, the invention relates to nucleic acids, vectors, and host cells for producing such binding proteins, methods for producing such binding proteins, kits comprising such binding proteins, and methods and uses of such binding proteins. Background Technology

[0002] In the liver, ALT enzymes play a major role in the intermediate catabolism of glucose and amino acids. Measurement of ALT enzyme levels is part of routine management in patients suspected of having liver injury. Serum ALT enzyme activity levels (considered the sum of ALT1 and ALT2 in the blood) are used as biomarkers for liver injury. For alanine and pyruvate, ALT1 and ALT2 isoform 1 enzyme activities are similar, while ALT2 isoform 2 lacks enzyme activity (Glinghammar et al., 2009). A study by Rafter et al. (2012) measured the percentage contribution of ALT1 and ALT2 enzyme activities in healthy subjects and those with liver disease. In healthy individuals, circulating activity was, on average, 92% of ALT1 and 8% of ALT2. In individuals with non-alcoholic fatty liver disease, activity was 94% of ALT1 and 6% of ALT2. In individuals with hepatitis C, 96% of ALT activity was attributed to ALT1, compared to 93% in healthy controls; ALT2 activity increased only 2.5-fold in hepatitis C patients. In individuals undergoing liver surgery, ALT levels increased from 31 IU / L to 357 IU / L. The contribution of ALT1 to this activity increased from 91% (before surgery) to 97% (after surgery), while the contribution of ALT2 activity decreased from 8% to 3%. In contrast, after strenuous exercise, the contribution of ALT1 to overall ALT activity decreased from 96% to 93%, while ALT2 increased from 4% to 7%. In conclusion, this evidence suggests that measurements of the amount of ALT1 protein in blood samples will reflect the enzyme levels detected in conventional enzyme-based assays.

[0003] When the liver is damaged, ALT levels in the blood rise, and measuring these levels is crucial for diagnosis and patient management. ALT can be elevated for a variety of reasons, including drug toxicity (e.g., due to acetaminophen overdose or prescription medications), hepatotropic viral infections (e.g., hepatitis A, B, C, D, or E), SARS-CoV-2 infection, excessive or frequent alcohol consumption, fatty liver disease, preeclampsia, inflammatory bowel disease, and the use of antibody-based therapies to treat a variety of human health conditions. Routine testing for ALT levels requires blood collection and measurement in a pathology laboratory, leading to potential treatment delays and lost follow-up, and in resource-constrained settings, pathology services may be limited or unavailable.

[0004] The ability to measure ALT levels point-of-care represents an advancement in patient management, enabling clinicians to make immediate decisions regarding further diagnostic testing and treatment pathways. A previous point-of-care test (PCT / IB2017 / 055943) developed by Anderson et al. used rabbit polyclonal immune serum targeting ALT1 to detect the amount of ALT protein present in blood samples. This invention describes how rabbit polyclonal antibodies overcome the limitations observed with mouse monoclonal antibodies and the inhibition of ALT detection in plasma samples. Specifically, interfering substances in human plasma mask epitopes recognized by mouse antibodies, thus limiting ALT1 detection. In fact, this phenomenon may be related to the presence of human anti-mouse antibodies (HAMA). Endogenous human heterophilic antibodies, targeting not only mice but also other common animal species, have been observed in 10 to 40% of people (Garcia-Gonzalez et al., 2016). Human anti-animal antibodies typically target the Fc portion of the antibody, but can also target the F(ab)2' region. These antibodies can bind to antibodies used in immunodiagnostics and affect assay performance by interfering with the binding of diagnostic antibodies to their target epitopes. In some cases, this can lead to false positives if it bridges capture and signaling antibodies, or false negatives if it blocks binding to the antigen in sandwich assays. Alternatively, in competitive assays, heterophilic antibodies bind to diagnostic antibodies and inhibit binding to the antigen, resulting in false negatives. The reasons for the generation of these HAMA antibodies are unclear, but may be related to treatment and imaging procedures using monoclonal antibody therapies, autoimmune diseases, and frequent exposure to animals. Regardless of the reason for the interference of mouse antibodies with ALT1 detection, rabbit polyclonal immune serum has been suggested as an alternative reagent for developing antigen assays to measure ALT1 in point-of-care assays (e.g., lateral flow). However, a significant limitation of using such polyclonal reagents generated in animals through vaccination is the ability to produce a consistent product and the ability to produce the quantities required for large-scale manufacturing of diagnostic tests.

[0005] Therefore, new reagents that bind to ALT1 are needed. Summary of the Invention

[0006] The inventors have developed a binding protein that binds to alanine aminotransferase (ALT).

[0007] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising an amino acid sequence selected from:

[0008] a) The amino acid sequence GPAVSNVA (SEQ ID NO: 2) as complementarity-determining region (CDR) 1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR 2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR 3;

[0009] b) The amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3; and

[0010] c) The amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, the amino acid sequence ISRSGFST (SEQ ID NO: 11) as CDR2, and the amino acid sequence AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3; and any one or more of these CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0011] In one embodiment, the binding protein binds to the epitope of alanine aminotransferase 1 (ALT1).

[0012] In one implementation, the binding protein binds to an epitope of human ALT1.

[0013] In one implementation, the binding protein is a nanobody.

[0014] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 1 (C8), or a sequence having at least 76% identity with it, or its humanized or germline form.

[0015] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or its humanized or germline form.

[0016] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 9 (C10), or a sequence having at least 76% identity with it, or its humanized or germline form.

[0017] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), the binding protein comprising an antigen-binding site of an antibody comprising an amino acid sequence selected from the following:

[0018] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0019] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0020] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0021] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and

[0022] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2 and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3;

[0023] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0024] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising a heavy chain variable region (V... H ) and light chain variable region (V L ), wherein V H and V L It includes six complementary determination regions (CDRs) selected from the following:

[0025] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0026] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0027] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0028] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and

[0029] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2 and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3;

[0030] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0031] In one implementation, the binding protein is a monoclonal antibody.

[0032] In one implementation, the binding protein is a rabbit monoclonal antibody.

[0033] In one implementation, the binding protein binds to epitopes containing residues on two monomers containing ALT1.

[0034] In one implementation, the binding protein does not bind significantly to heat-denatured ALT1.

[0035] In one implementation, the binding protein does not detectably bind to heat-denatured ALT1.

[0036] In one implementation, VL is κ1.

[0037] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site for an antibody.

[0038] In one aspect, the present invention provides a binding protein that binds to a conformational epitope of alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody.

[0039] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), wherein the binding protein does not significantly and / or detectably bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0040] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody, and said binding protein not binding significantly and / or detectably to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0041] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody comprising an amino acid sequence selected from:

[0042] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0043] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2 and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0044] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0045] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2 and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3;

[0046] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; and

[0047] f) GPAVSNVA (SEQ ID NO: 2) as CDR1, ITWSGWT (SEQ ID NO: 3) as CDR2, and NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3;

[0048] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0049] Wherein, when the amino acid sequence is a), the binding protein is a nanobody, and wherein, when the amino acid sequence is b), c), d), e), or f), the binding protein is an antibody.

[0050] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an amino acid sequence selected from:

[0051] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0052] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2 and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0053] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0054] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2 and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3;

[0055] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; and

[0056] f) GPAVSNVA (SEQ ID NO: 2) as CDR1, ITWSGWT (SEQ ID NO: 3) as CDR2, and NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3;

[0057] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0058] Wherein, when the amino acid sequence is a), the binding protein is a nanobody, and wherein, when the amino acid sequence is b), c), d), e), or f), the binding protein is an antibody.

[0059] In one aspect, the present invention provides bivalent, trivalent, tetravalent or multivalent binding proteins comprising at least one binding protein described herein or a combination thereof.

[0060] In one aspect, the present invention provides isolated nucleic acids encoding the amino acid sequence of the isolated binding protein described herein or the amino acid sequence of the divalent, trivalent, tetravalent or multivalent binding protein described herein.

[0061] In one aspect, the present invention provides a vector comprising the nucleic acid described herein.

[0062] In one aspect, the present invention provides a host cell comprising the nucleic acid or vector described herein.

[0063] In one aspect, the present invention provides a method for producing the binding protein described herein or the bivalent, trivalent, tetravalent or multivalent binding protein described herein, comprising culturing the host cells described herein in a cell culture medium and expressing the binding protein or the bivalent, trivalent, tetravalent or multivalent binding protein.

[0064] In one aspect, the present invention provides a kit or panel containing the binding protein described herein or a bivalent, trivalent, tetravalent or multivalent binding protein described herein.

[0065] In one aspect, the present invention provides a lateral flow measurement comprising: a solid support containing binding proteins described herein and / or divalent, trivalent, tetravalent or multivalent binding proteins described herein, immobilized on the solid support.

[0066] In one aspect, the present invention provides a lateral flow assay comprising: (i) a detector-binding protein coupled to a detectable label; and (ii) a capture-binding protein in a capture region on a solid support, wherein the binding protein in (i) and / or (ii) is a binding protein described herein or a divalent, trivalent, tetravalent, or multivalent binding protein described herein.

[0067] In one aspect, the present invention provides the binding protein described herein, or the bivalent, trivalent, tetravalent or multivalent binding protein described herein, or the kit or combination kit described herein, or the use of the lateral flow assay described herein for detecting ALT1.

[0068] In one aspect, the present invention provides a method for detecting ALT1, the method comprising contacting a sample with a binding protein described herein or a divalent, trivalent, tetravalent or multivalent binding protein described herein to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

[0069] In one aspect, the present invention provides the use of the binding proteins described herein, or the bivalent, trivalent, tetravalent or multivalent binding proteins described herein, or the kits or combination sets described herein, or the lateral flow assay described herein for the detection of subjects with liver injury and / or liver disease.

[0070] In one aspect, the present invention provides the use of the binding proteins described herein, or the bivalent, trivalent, tetravalent or multivalent binding proteins described herein, or the kits or combination sets described herein, or the use of the lateral flow assay described herein for detecting ALT1 in a subject.

[0071] In one aspect, the present invention provides a method for detecting ALT1, the method comprising contacting a sample with a binding protein described herein or a divalent, trivalent, tetravalent or multivalent binding protein described herein to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

[0072] In one aspect, the present invention provides the use of the binding proteins described herein, or the bivalent, trivalent, tetravalent or multivalent binding proteins described herein, or the kits or combination sets described herein, or the lateral flow assay described herein for the detection of subjects with liver injury and / or liver disease.

[0073] In one aspect, the present invention provides a method for detecting a subject suffering from liver injury and / or liver disease, the method comprising contacting a sample with a binding protein as described herein or a divalent, trivalent, tetravalent or multivalent binding protein as described herein to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

[0074] In one aspect, the present invention provides a solid or semi-solid support on which the binding protein described herein, or the divalent, trivalent, tetravalent or multivalent binding protein described herein, is immobilized.

[0075] Unless otherwise expressly stated, any embodiment described herein should be adapted to any other embodiment with the necessary modifications. For example, as those skilled in the art will understand, the examples of amino acid sequences outlined above for the binding proteins of the present invention are equally applicable to the bivalent, trivalent, tetravalent, or multivalent binding proteins of the present invention.

[0076] The scope of this invention is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this invention, as described herein.

[0077] Throughout this specification, unless otherwise expressly stated or required by the context, references to a single step, composition of matter, group of steps, or group of composition of matter shall be regarded as encompassing one or more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of composition of matter.

[0078] The invention is described below with reference to the following non-limiting embodiments and the accompanying drawings. Attached Figure Description

[0079] Figure 1. A) shows the structure of a typical human IgG molecule, whose heavy and light chains form a 150 kDa heterodimeric protein. B) shows a typical camelid antibody, which is a homodimer of two heavy chain molecules. C) shows a VHH antigen-binding fragment that can be independently expressed in bacterial cells.

[0080] Figure 2. A) shows the amino acid sequences of the VHH domains of ALT1-responsive nanobodies obtained from phage display libraries in the panning experiment. The reactivity of 94 clones to ALT1-coated ELISA plates was screened. Of these, 91 were ALT1-positive, and 86 of them possessed the full-length VHH domain. B) shows the results of screening ALT1-responsive nanobodies from the phage display library in the second panning experiment. The reactivity of 188 clones to ALT1-coated ELISA plates was screened. Of these, 60 possessed the full-length VHH domain.

[0081] Figure 3 The frequency of clones with the same complementarity-determining region 3 (CDR3) within the library isolated in Panning Experiment 1 is shown, among which two highly reactive nanobodies were identified. These are referred to below as Nb_C8 and Nb_G6. Nb_C8 is unique in this library, with only one clone having the same CDR3 region. Nb_G6 is the dominant clone isolated in this library, with 82 out of 91 clones having the same CDR3 sequence.

[0082] Figure 4The results of enzyme-linked immunosorbent assay (ELISA) for a series of nanobodies with unique complementarity-determining regions are shown. The results showed that Nb_C8 and Nb_G6 exhibited very strong binding to ALT1 (ALT1-avi), as indicated by the high absorbance at 450 nm, while Nb_C10 showed very low binding. All other nanobodies screened did not show binding to ALT1 (Figure 2 and not shown). For comparison, the binding of the nanobodies to bovine serum albumin (BSA) is shown, representing the background of the assay. WK6 represents cells only and is the background of the assay.

[0083] Figure 5. A) shows the protein-coding sequence and annotations of Nb_C8, showing the predicted boundaries of the frame region (FR) and complementarity-determining region (CDR). B) shows the protein-coding sequence of Nb_C8_FLAG. CDR domains are in bold text, while FR regions are in gray. The FLAG tag is underlined, and the polyhistidine sequence is boxed.

[0084] Figure 6. A) shows the protein-coding sequence and annotations of Nb_G6, showing the predicted boundaries of the frame (FR) and complementarity-determining region (CDR). B) shows the protein-coding sequence of Nb_G6_FLAG. CDR domains are in bold text, while FR regions are in gray. The FLAG tag is underlined, and the polyhistidine sequence is boxed.

[0085] Figure 7 The protein-coding sequence and annotation of the nanobody Nb_C10 are shown, with predicted boundaries of the frame (FR) and complementarity-determining regions (CDR). CDR domains are in bold text, while FR regions are in gray.

[0086] Figure 8 The percentage identity between nanobodies is shown (see Figures 5 to 10). Figure 7 ).

[0087] Figure 9 The use of the V, J, and D genes in Nb_G6 and Nb_C8 alpaca (Vicugna pacos) is shown. RF = Reading box.

[0088] Figure 10 The alignment between nanobodies Nb_G6 and Nb_C8 is shown, highlighting the differences in the protein-coding sequences. Complementarity-determining regions 1, 2, and 3 are indicated by gray shading. Conserved mutations (:), semi-conserved mutations (.), vacancies (-), and non-conserved mutations ().

[0089] Figure 11The alignment between nanobodies Nb_C8 and Nb_C10 is shown, highlighting the differences in the protein-coding sequences. Complementarity-determining regions 1, 2, and 3 are indicated by gray shading. Conserved mutations (:), semi-conserved mutations (.), vacancies (-), and non-conserved mutations ().

[0090] Figure 12 The alignment between nanobodies Nb_C10 and Nb_G6 is shown, highlighting the differences in the protein-coding sequences. Complementarity-determining regions 1, 2, and 3 are indicated by gray shading. Conserved mutations (:), semi-conserved mutations (.), vacancies (-), and non-conserved mutations ().

[0091] Figure 13 A) shows the Alphafold2 predicted structures of two active nanobodies, with Nb_C8 (light gray) overprinted on the predicted structure of Nb_G6 (medium gray). B) shows both active Nb_C8 and Nb_G6 overprinted on the structure of inactive Nb_C10 (black), which binds weakly to ALT1. Arrows indicate complementarity-determining regions (CDRs) 1, 2, and 3.

[0092] Figure 14 The image shows whole-cell lysates of cells expressing nanobodies Nb_C8 and Nb_G6, separated on an SDS-PAGE gel. These nanobodies have been modified by adding His, FLAG, or Avi tags. Protein sizes are indicated in kilodaltons (kDa) on the left.

[0093] Figure 15 The binding of antibodies to ALT1, ALT2, and AST in an enzyme-linked immunosorbent assay (ELISA) is shown. The absorbance at an optical density (OD) of 450 mm is shown as the antibody concentration increases. A) shows binding to Nb_G6; B) shows binding to Nb_C8; C) shows binding to a rabbit polyclonal antibody against ALT1; D) shows binding to a commercial rabbit monoclonal antibody against ALT1. A modified form of ALT2, with a 48-residue deletion at the N-terminus, is used in the ELISA to facilitate expression in *E. coli*.

[0094] Figure 16 A summary of the concentrations of each antibody required to achieve a tenfold increase in binding to ALT1, ALT2, and AST relative to the background is shown.

[0095] Figure 17 The image shows the alignment of the protein-coding sequences of ALT1 and ALT2 isotype 1. Conserved mutations (:), semi-conserved mutations (.), vacancy (-), and non-conserved mutations ().

[0096] Figure 18 The alignment of the protein-coding sequences of AST, ALT1, ALT2 isotype 1, and ALT2 isotype 2 is shown. Conserved mutations (:), semi-conserved mutations (.), vacancies (-), and non-conserved mutations ().

[0097] Figure 19 Modifications to the nanobodies are shown, in which Nb_C8 and Nb_G6 are linked together by themselves (homo-bivalent) or by each other (hetero-bivalent) through different linker sequences containing highly flexible glycine (G) and serine (S) residues. Alternative linkers used are 3 or 4 repeats of GGGGS. In the following text, bivalent nanobodies are represented by the codes shown in the table (e.g., C8-3-C8 is referred to as C3C).

[0098] Figure 20 The expression of bivalent nanobodies derived from bacterial cells was shown on an SDS-PAGE gel, and the expected increase in molecular weight from monovalent (approximately 14 kDa) to bivalent nanobodies (28 kDa) was determined. Different conditions (18 or 28 °C, growth in Terrific Broth (TB) or Autoinducible Medium (AI)) were explored to optimize nanobodies yield, and stable production of approximately 28 kDa material containing two 14 kDa Nb VHH sequences was shown. Protein sizes are indicated in kilodaltons (kDa) on the left. A) shows bivalent nanobodies C3C and C4C, and B) shows bivalent nanobodies G3G and G4G.

[0099] Figure 21 The relative binding affinity of the bivalent nanobodies to ALT1 in the enzyme-linked immunosorbent assay (ELISA) is shown. Nanobodies were added at 1000 ng / mL. Parental monovalent nanobodies Nb_C8 and Nb_G6 are shown for comparison.

[0100] Figure 22A) A biolayer interferometry (BLI) experiment measuring the binding and dissociation of receptor-ligand interactions is shown. ALT1 was immobilized on an avidin sensor, and the binding of Nb_C8 and rabbit polyclonal antibodies to the sensor was measured in real time in seconds (s). B) A BLI experiment is shown in which monovalent (C8, G6) or bivalent (G4C, C4C, C4G) nanobodies were immobilized on a nickel sensor, and the binding of five sequentially decreasing concentrations of ALT1 (1000, 333, 111, 39, 12 nM) to the sensor was measured in real time. Binding and dissociation curves for different ALT1 concentrations for each nanobody are shown. Different linkers and different nanobody combinations alter the binding and dissociation of ALT1. Such differences can provide advantages in terms of applicability to different assay systems, such as plate-based assays, biosensor-based assays, and lateral flow assays. Nb_C8 binds rapidly to ALT1 and dissociates relatively quickly, while the bivalent nanobody exhibits a slower dissociation rate while maintaining a rapid binding rate. Nb_G6 does not bind to ALT1 in this direction.

[0101] Figure 23 A) The binding (kon, or binding rate) and dissociation (kdis, or dissociation rate) of nanobodies were summarized using a 1:1 binding model. Bivalent nanobodies showed differences in their binding rates to ALT1, with G4C exhibiting a faster binding rate than other bivalent nanobodies tested. The dissociation of bivalent nanobodies also differed, with C4G exhibiting the fastest dissociation rate. D It is the equilibrium dissociation constant and R^2 represents the goodness of fit. B) describes the binding, dissociation, and K0 of the bivalent nanobody compared to monovalent Nb_C8. D The binding affinity was increased several times. Data showed that the binding of both G4C and C4C was improved, while the dissociation and K... of the bivalent nanobodies in all three tests were... D All have been improved.

[0102] Figure 24A schematic diagram of a lateral flow ALT1 detection system is shown. Nanobodies are conjugated to visible or fluorescent molecules, such as colloidal gold, gold nanoshells, europium, and other examples known to those skilled in the art. These are applied to a conjugate pad and interact with ALT present in serum, plasma, or blood samples upon contact. A run buffer is then added to the conjugate pad (shown in the upper figure), allowing the nanobodies-conjugate-ALT complexes to flow along nitrocellulose (indicated by the arrows in the lower figure), where they are captured by a second nanobodies streaked onto the nitrocellulose membrane. If nanobodies-conjugate-ALT complexes are present, these complexes are retained on the nanobodies (the lines indicated by "T" in the upper figure). The free nanobodies-conjugates are then flowed through a control line (the lines indicated by "C" in the upper figure), where ALT streaks and captures the free conjugates, confirming the validity of the test results.

[0103] Figure 25 This paper illustrates an example of how a nanobody (Nb_G6) can be used to detect ALT1 in a lateral flow assay when ALT1 is streaked onto nitrocellulose, and compares it with detection using an anti-ALT1 polyclonal antibody. The antibody was conjugated with europium and used to detect different concentrations of ALT1 protein directly streaked onto nitrocellulose.

[0104] Figure 26 An example of europium-conjugated rabbit polyclonal anti-ALT1 is shown. A practical example of using the nanobody C8_FLAG (Nb_C8_FLAG) to capture different amounts of ALT1 applied to a lateral flow assay is shown. Here, different amounts of Nb_C8_FLAG ranging from 0.1 mg / mL to 1 mg / mL are streaked onto nitrocellulose. Different amounts of ALT1 are added to the bottom of the nitrocellulose membrane, bringing it into contact with the europium-conjugated anti-ALT1 polyclonal antibody and allowing it to diffuse laterally. ALT1 is captured by the Nb_C8_FLAG band, and subsequently, the remaining europium-conjugated antibody is captured by the ALT control line.

[0105] Figure 27 An example of europium-conjugated rabbit polyclonal anti-ALT1 is shown. A practical example of using the nanobody G6_FLAG (Nb_G6_FLAG) to capture different amounts of ALT1 applied to a lateral flow assay is shown. Here, different amounts of Nb_G6_FLAG ranging from 0.1 mg / mL to 1 mg / mL are streaked onto nitrocellulose. Different amounts of ALT1 are added to the bottom of the nitrocellulose membrane, bringing it into contact with the europium-conjugated anti-ALT1 polyclonal antibody and allowing it to diffuse laterally. ALT1 is captured by the Nb_G6_FLAG band, and subsequently, the remaining europium-conjugated antibody is captured by the ALT control line.

[0106] Figure 28 A) illustrates a practical example of using nanobody C8 (Nb_C8_FLAG) to capture different amounts of ALT1 applied to a lateral flow assay and detecting it with nanobody conjugated to a gold nanoshell. A constant amount of Nb_C8_FLAG was streaked onto nitrocellulose. Different amounts of ALT1 were added to the bottom of the nitrocellulose membrane, bringing it into contact with gold nanoshells conjugated with G3G or G4C, and allowing it to diffuse laterally. ALT1 was captured by the Nb_C8_FLAG band, followed by the capture of the ALT control line by the remaining gold nanoshell-conjugated antibody. B) illustrates the quantification of the results using a lateral flow strip reader. The increased peak intensity (y-axis) reflects the higher visibility of the test line on the lateral flow strip.

[0107] Figure 29 A) An example of a lateral flow assay is shown, in which nanobody C8 (Nb_C8_FLAG) is used as the capture antibody streaked onto a nitrocellulose membrane. Different amounts of ALT1 are added to the bottom of the nitrocellulose membrane, contacting it with europium-conjugated nanobodies G6, G3G, and G4C, and allowing it to diffuse laterally. ALT1 is captured by the Nb_C8_FLAG band, and subsequently, the residual europium-conjugated antibody is captured by the ALT control line. B) Quantification of the results using a lateral flow strip reader is shown. C) Quantification of the results using a lateral flow strip reader is shown, corrected for a background of 0 g / mL ALT1 in the assay. This allows for comparison of the relative ability of each nanobody to detect ALT1. The binding ability of the bivalent nanobodies G3G or G4C to ALT1 is improved compared to the monovalent nanobody G6.

[0108] Figure 30 A) illustrates an example of a lateral flow assay where a divalent nanobody is used to capture and detect both. Here, G4C is streaked onto nitrocellulose as the capture antibody. Different amounts of ALT1 are added to the bottom of the nitrocellulose membrane, bringing it into contact with the gold nanoshell coupled with G4C, and allowing it to diffuse laterally. ALT1 is captured by the G4C strip, and subsequently, the antibody coupled to the remaining gold nanoshell is captured by the ALT control line. B) illustrates the quantification of the results using a lateral flow strip reader.

[0109] Figure 31 A) through H) show examples of different nanobodies used as capture reagents on the band with iso-divalent G4C as the detector, and their ability to detect different amounts of recombinant ALT1-his protein incorporated into the buffer solution.

[0110] Figure 32A) through H) show examples of different nanobodies used as capture reagents on the band with iso-divalent C4G as the detector, and their ability to detect different amounts of recombinant ALT1-his protein incorporated into the buffer solution.

[0111] Figure 33 A) through H) show examples of different nanobodies used as capture reagents on the band with iso-divalent C3G as the detector, and their ability to detect different amounts of recombinant ALT1-his protein incorporated into the buffer solution.

[0112] Figure 34 A) through F) show examples of different nanobodies used as capture reagents on the band with the same divalent G3G as the detector, and their ability to detect different amounts of recombinant ALT1-his protein incorporated into the buffer solution.

[0113] Figure 35 A) through C) show examples of different nanobodies used as capture reagents on the band with the same divalent G4G as the detector, and their ability to detect different amounts of recombinant ALT1-his protein incorporated into the buffer solution.

[0114] Figure 36 A) through E) show examples of different nanobodies used as capture reagents on the bands in the case of an anti-ALT1 polyclonal antibody as a detector, and their ability to detect different amounts of recombinant ALT1-his protein incorporated into a buffer solution.

[0115] Figure 37 A) An example is shown illustrating the potential impact of adding excipients on the performance of the test capture line and control line. Here, monovalent Nb_C8_FLAG (C8F) was streaked onto nitrocellulose at 0.5 mg / mL for the test line, with and without BSA and / or sucrose. ALT1 was streaked onto the control line at 50 μg / mL, with and without BSA and / or sucrose. B) Quantification of the test line results using a lateral flow strip reader is shown. C) Quantification of the control line results using a lateral flow strip reader is shown.

[0116] Figure 38A) An example is shown illustrating the potential impact of adding excipients on the performance of the test capture line and control line. Here, divalent G4C at 0.5 mg / mL was streaked onto nitrocellulose for the test line in both the presence and absence of BSA and / or sucrose. ALT1 at 50 μg / mL was streaked onto the control line in both the presence and absence of BSA and / or sucrose. B) Quantification of the test line results using a lateral flow strip reader is shown. C) Quantification of the control line results using a lateral flow strip reader is shown.

[0117] Figure 39 The evaluation of ALT1 in clinical samples with known concentrations of enzymatically catalyzed ALT is illustrated, using bivalent A)G4C as a capture antibody and B)C4C as a capture antibody. The capture antibodies were detected in a lateral flow assay using europium particle-conjugated anti-ALT polyclonal antibodies. Human plasma was obtained, and ALT levels were determined by a standard care test in a pathology laboratory that does not distinguish between ALT1 and ALT2, as previously described in the background section of the invention. Enzymatically catalyzed ALT values, in International Units (IU / L), are shown in parentheses following the sample identifier.

[0118] Figure 40 A) illustrates the correlation between enzymatically catalyzed ALT, as determined by standard pathological testing, and antigenic ALT1, obtained using a G4C capture antibody. Figure 39 Lateral flow analysis of clinical samples was used to determine ALT levels. B) shows the correlation between enzymatically catalyzed ALT determined by standard pathological testing and ALT1 measured by lateral flow assay using C4C as the capture antibody, the latter being determined by... Figure 39 The results were determined by lateral flow analysis of clinical samples.

[0119] Figure 41 The diagram illustrates nanobodies modified to increase size, affinity, and multispecificity. A) A schematic diagram of two monovalent nanobodies. B) A schematic diagram of a bivalent nanobodies consisting of two monovalent nanobodies linked by a linker. C) A schematic diagram of a bivalent nanobodies fused with the Fc domain of human IgG to generate a tetravalent molecule.

[0120] Figure 42A) SDS-PAGE gels of tetravalent Fc fusion nanobodies expressed in mammalian cells under reducing conditions are shown, and the expected molecular weight of each Fc chain fused with the divalent nanobodies is confirmed to increase from approximately 28 kDa to approximately 53 kDa. B) SDS-PAGE gels of tetravalent Fc fusion nanobodies purified from protein G sepharose and expressed in mammalian cells are shown, and the expected molecular weight of the tetravalent nanobodies Fc fusions under non-reducing and reducing conditions is confirmed to increase from approximately 53 kDa to approximately 106 kDa as expected under non-reducing conditions.

[0121] Figure 43 The binding ability of the Fc fusion nanobody to ALT1 in an enzyme-linked immunosorbent assay (ELISA) was demonstrated. The nanobody was added at 1000 ng / mL and serially diluted two-fold, with absorbance measured at 450 nm.

[0122] Figure 44 A) illustrates a lateral flow assay in which tetravalent nanobody-Fc protein (C4C-Fc, C4G-Fc, or G4C-Fc) or bivalent nanobody (G4C) protein is streaked onto a nitrocellulose membrane in the presence of excipient BSA and sucrose. Different amounts of ALT1 incorporated separately in buffer or in the presence of human plasma were added to the bottom of the nitrocellulose membrane and mixed with europium-conjugated rabbit polyclonal anti-ALT antibody, allowing it to diffuse laterally. The incorporated ALT1 was captured by the streaked protein, and subsequently, the residual europium-conjugated antibody was captured by the ALT control line. B through C) show the quantification of the results using a lateral flow strip reader. In buffer (B) and plasma (C), the tetravalent Fc fusion nanobody specifically captures and detects ALT1 in a dose-dependent manner with higher sensitivity than the bivalent G4C.

[0123] Figure 45 An illustration, including dimensions, shows an example of a lateral flow test that will be used in the assembled box.

[0124] Figure 46 An example of an assembled lateral flow test box is shown. On the left, a top view of the box is shown, and on the right, the interior of the box is shown, with the assembled lateral flow strips inside.

[0125] Figure 47 This illustrates the assembly process in a side-flow box (e.g., Figure 46In the lateral flow chamber shown, the ability of the lateral flow assay to detect different levels of ALT1 is quantified using a lateral flow strip reader. In this example, a tetravalent Fc fusion C4G-Fc is streaked onto a nitrocellulose membrane in the presence of excipient BSA and sucrose. Different amounts of ALT1 are added to the sample port, and three drops of buffer are added and allowed to diffuse laterally. ALT1 is captured by the streaked protein and detected using a europium-conjugated rabbit polyclonal antibody against ALT, which is used at two different concentrations (1 / 1000 and 1 / 2000).

[0126] Figure 48 This diagram illustrates how a lateral flow assay performed in a cassette measures ALT1 in human plasma. A tetravalent Fc-fused C4G-Fc is streaked onto a nitrocellulose membrane in the presence of excipients BSA and sucrose. A fixed volume of plasma is added to the sample port, and three drops of buffer are added to rehydrate the rabbit polyclonal anti-ALT antibody conjugated to europium particles and allow it to diffuse laterally. ALT1 in the clinical sample is captured by the streaking protein. Enzymatic ALT levels in human plasma are determined by a standard care test in a pathology laboratory. Enzymatic ALT values, in International Units (IU / L), are shown in parentheses following the sample identifier.

[0127] Figure 49 An overview of the nucleotide sequence encoding the binding protein described herein is shown. For the *E. coli* expression construct (non-Fc), the gene has an N-terminal pelB signal peptide (in bold) for periplasmic expression. The Fc fusion bivalent nanobody gene was codon-optimized for mammalian cell expression and synthesized prior to cloning into the Fc-pcDNA3 vector. These genes have an N-terminal tPA leader sequence (in bold).

[0128] Figure 50 The interaction between the Fc domain nanobody-fusion antibody and ALT1 is illustrated. A nickel sensor loaded with 5 μg / mL ALT1 protein was exposed to multiple concentrations of A) G6-Fc, B) C8-Fc, or C) C4G-Fc from 3.125 nM to 100 nM for 600 seconds to monitor binding, followed by dissociation for 600 seconds. Sensing plots for each antibody concentration are shown, with the binding and dissociation phases to the left and right of the vertical dashed line at 600 seconds, respectively.

[0129] Figure 51 The sequence of rabbit monoclonal antibody 15B5 (RmAb1), which is specific to ALT1, is shown. The variable domain sequences of the heavy chain (HC) and light chain (LC) are shown, with CDR1, 2, and 3 underlined.

[0130] Figure 52The sequence of rabbit monoclonal antibody 20C12 (RmAb2) is shown. The variable domain sequences of the heavy chain (HC) and light chain (LC) are shown, with CDR1, 2, and 3 underlined.

[0131] Figure 53 The sequence of rabbit monoclonal antibody 22G10 (RmAb3) is shown. The variable domain sequences of the heavy chain (HC) and light chain (LC) are shown, with CDR1, 2, and 3 underlined.

[0132] Figure 54 The sequence of rabbit monoclonal antibody 36D4 (RmAb4) is shown. The variable domain sequences of the heavy chain (HC) and light chain (LC) are shown, with CDR1, 2, and 3 underlined.

[0133] Figure 55 The sequence of rabbit monoclonal antibody 42F12 (RmAb5) is shown. The variable domain sequences of the heavy chain (HC) and light chain (LC) are shown, with CDR1, 2, and 3 underlined.

[0134] Figure 56 The image shows the alignment of the heavy chain sequence of the rabbit anti-ALT1 monoclonal antibody. The sequence alignment was performed using Clustal 2.1 software, and regions CDR1, 2, and 3 are outlined. Conservative mutations (:), semi-conservative mutations (.), vacancies (-), and non-conservative mutations ().

[0135] Figure 57 The alignment of the light chain sequence of the rabbit anti-ALT1 monoclonal antibody is shown. The sequence alignment was performed using Clustal 2.1 software, and regions CDR1, 2, and 3 are outlined. Conservative mutations (:), semi-conservative mutations (.), vacancies (-), and non-conservative mutations ().

[0136] Figure 58 The image shows an SDS-PAGE of purified rabbit monoclonal antibody. The antibody was expressed by co-transfecting plasmids encoding both the heavy and light chains into Expi293 cells. The protein was purified using protein A affinity chromatography, with the buffer exchanged to PBS (phosphate-buffered saline). The protein was then run on SDS-PAGE under both reducing and non-reducing conditions. Purified C4G-Fc was also run for comparison. Molecular weight markers are indicated on the left.

[0137] Figure 59 The differential scanning spectroscopy (DSS) image of the ALT-specific rabbit monoclonal antibody is shown. The protein was heat-denatured in the presence of Sypro Orange.

[0138] Figure 60A) Shows an enzyme-linked immunosorbent assay (ELISA) for binding rabbit monoclonal antibodies to ALT1. Serial dilutions of each rabbit anti-ALT monoclonal antibody and rabbit polyclonal antibody were applied to plates coated with ALT1. Data shown are mean ± standard deviation. B) Shows an enzyme-linked immunosorbent assay (ELISA) for binding rabbit monoclonal antibodies to ALT2. Serial dilutions of each rabbit anti-ALT monoclonal antibody and rabbit polyclonal antibody were applied to plates coated with ALT2. Data shown are mean ± standard deviation. C) Shows a summary of the concentrations of each antibody required to achieve a ten-fold increase in binding to ALT1 and ALT2 compared to the background.

[0139] Figure 61 The binding and dissociation of antibodies with ALT using biolayer interferometry are illustrated. A nickel sensor was used to capture the ALT1-chis protein, and different concentrations of antibody were applied and bound for 600 seconds and dissociated for 600 seconds.

[0140] Figure 62 This diagram illustrates an example of a lateral flow test suitable for detecting ALT in a self-testing device. The schematic shows the assembly of the components of the lateral flow test and the relative positions of the sample pad containing the blood capture reagent anti-blood group glycoprotein A (anti-GPA) and europium-conjugated rabbit anti-ALT1 (conjugate region), the nitrocellulose membrane sprayed with 1 mg / mL C4G-Fc (test line) and 0.1 mg / mL ALT1 (control line), the absorbent pad, and the backing card. The locations of the press-fit points, blister orifices, and sample ports on the device are shown.

[0141] Figure 63 The quantitative results of a lateral flow assay using a europium-coupled rabbit anti-ALT polyclonal antibody are shown. Different amounts of ALT1 incorporated into the buffer were applied to the cassette to visualize the quantitative detection of ALT1. After adding run buffer, the test was run for 20 minutes, followed by imaging on a fluorescence lateral flow reader. Data shown are mean ± standard deviation.

[0142] Figure 64 Images of the lateral flow device after operation are shown. 0 or 2 ng of ALT1 was incorporated into venous blood and run for 20 minutes, followed by imaging. (Using...) Figure 62 The same schematic diagram shown.

[0143] Figure 65 Images of test strips run in an AtomoRapid Pascal device after being run with venous blood containing ALT1 are shown. (Using...) Figure 62The same lateral flow configuration is shown. Blood from three different volunteers (A, B, and C) was either undoped (0 ng) or doped with 2 ng of ALT1 and ran on the lateral flow device for 20 minutes before the strips were imaged and quantified on the lateral flow reader.

[0144] Figure 66 It shows Figure 65 Quantitative results of the test line. The data shown are mean ± standard deviation.

[0145] Figure 67 An example of a modification to a lateral flow test is shown, replacing the anti-blood group glycoprotein A-coated glass fiber pad with a commercial blood retention pad. Two alternative schematic diagrams are shown, in which the length dimensions and positions of the sample pad and blood separation pad are varied to adjust blood retention capacity and test sensitivity. The region on the sample pad containing europium-coupled rabbit anti-ALT1 is shown as the coupling region. Other components of the test are... Figure 62 The same as described in [the text].

[0146] Figure 68 Images show a lateral flow test performed using venous blood with a blood retention pad. The lateral flow test uses... Figure 67 The configurations described demonstrate their ability to retain red blood cells and prevent them from entering the test window. The test is assembled in AtomoRapid. TM In the Pascal apparatus, human venous blood doped with 0, 1, or 5 ng of ALT1 was used for the test. After adding run buffer, the test was run for 20 minutes, followed by imaging on a fluorescence lateral flow reader. Single-volunteer venous blood was used, with or without varying amounts of ALT1.

[0147] Figure 69 A) shows an image of the nitrocellulose strip after running the ALT test. B) shows the quantitative data for the test strip shown in A). The test strip and... Figure 68 The same as shown.

[0148] Figure 70 A schematic diagram of a lateral flow assay for detecting ALT1 in blood using europium-conjugated rabbit polyclonal and monoclonal antibodies is shown. In this schematic, separate rehydration pads, a binding pad with europium-conjugated anti-ALT antibodies, and a separate blood sample retention pad are used. Other components of the assay include... Figure 62 As stated above.

[0149] Figure 71 It shows the use of Figure 70 Examples of images captured during lateral flow testing, as shown in the diagram. Human venous blood containing 0, 1, or 5 ng of ALT1 was used for the test at AtomoRapid. TMAssemble the test in the Pascal device. After adding the run buffer, run the test for 20 minutes, and then image on the fluorescence lateral flow reader.

[0150] Figure 72 It shows Figure 71 Quantitative analysis of the test line data. The data shown are mean ± standard deviation.

[0151] Figure 73 This paper presents quantitative data for detecting naturally occurring ALT in human plasma samples using rabbit monoclonal antibody to detect ALT1 captured by C4G-Fc on a nitrocellulose membrane. Nitrocellulose strips were used in a wet system assay, streaked with 1 mg / mL C4G-Fc and 0.1 mg / mL ALT1. Human plasma samples were added to europium-conjugated rabbit monoclonal antibody, pre-incubated in 96-well plates, and then immersed in the test strips. After 8 minutes, run buffer was added to the sample pads, and readings were taken at a fluorescence reader after another 8 minutes.

[0152] Figure 74 A visible example of the ALT1 lateral flow assay is shown. A nitrocellulose membrane streaked with 1 mg / mL LC4G-Fc and 0.1 mg / mL ALT1 was used in the wet system assay. Estapor blue and black intense particles were coupled with different amounts of RmAb5 at 50 ug / mg or 150 ug / mg. Various amounts of ALT1 were added to the run buffer and mixed with the blue or black-coupled rRmAb5 in a 96-well plate. The nitrocellulose test strip was added to the mixture of ALT1 and blue / black-coupled RmAb5 for 8 minutes, followed by the addition of run buffer. After 10 minutes, the test strip was inserted into a visible test strip reader for imaging and quantification.

[0153] Figure 75 It shows Figure 74 The quantitative analysis of the tests conducted.

[0154] Figure 76 A schematic diagram of the visible form of the ALT1 lateral flow assay is shown. In this schematic, the binding pad is sprayed with blue intensely coupled RmAb5 in the conjugate region. A longer form of binding pad is used, which also serves as the sample pad. Two blood retention pads are stacked in front of the sample port to improve blood retention. The nitrocellulose membrane is streaked with 0.05 or 0.2 mg / mL C4G-Fc and 0.1 mg / mL ALT1.

[0155] Figure 77 It shows the use of Figure 76The schematic diagram shown illustrates the results of a lateral flow test. The lateral flow device was assembled on the AtomoRapid. TM In the Pascal device, multiple amounts of ALT1 were incorporated into whole blood and added to the blood collection unit for delivery to the device. After delivery, the blister pack was popped to release the running buffer, and the test strips were imaged in a lateral flow strip reader after 20 minutes.

[0156] Figure 78 It shows Figure 77 The results are presented quantitatively. The data shown are mean ± standard deviation.

[0157] Figure 79 This paper demonstrates an example of how to modify the test to allow the detection of ALT above the ALT cutoff level. A nitrocellulose membrane was prepared by streaking with 0.5 mg / mL C4G-Fc and 0.1 mg / mL ALT1 (as a control line) and used for wet system testing. Different concentrations of RmAb5 were used to conjugate the test strips with blue-intense and black particles. The conjugates were mixed with various amounts of ALT1 in run buffer in a 96-well plate, and the nitrocellulose membrane was added. After 8 minutes, additional run buffer was added, and after another 8 minutes, the test strips were imaged using a strip reader. Images of the test strips for ALT detection using black and blue-intense particles are shown.

[0158] Figure 80 It shows Figure 79 Quantitatively.

[0159] Figure 81 This paper illustrates an example of how the test can be further modified to allow the detection of ALT above the ALT cutoff level. Nitrocellulose membranes were prepared by streaking with 0.2 and 0.05 mg / mL C4G-Fc and 0.1 mg / mL ALT1 (as a control line) and used for wet system testing. Different concentrations of RmAb5 were used to conjugate the test strips with blue-intense and black particles. The conjugates were mixed with various amounts of ALT1 in run buffer in 96-well plates, and the nitrocellulose membranes were added. After 8 minutes, additional run buffer was added, and after another 8 minutes, the test strips were imaged using a strip reader. Images of the test strips for ALT detection using black and blue intense particles are shown.

[0160] Figure 82 It shows Figure 81 The data presented are the mean ± standard deviation.

[0161] Figure 83This section illustrates an example of how rabbit anti-ALT monoclonal antibodies can be used to capture and detect ALT1. RmAb1, 3, 4, and 5, as well as polyclonal rabbit anti-ALT (poly), are conjugated with europium. Nitrocellulose is spotted with RmAb1, 3, 4, and 5, as well as polyclonal rabbit anti-ALT, to capture ALT1, and with recombinant ALT1. ALT1 diluted in run buffer is applied to test strips (+), and the bound ALT1 is detected using the europium-conjugated antibody, imaged on a fluorescent strip reader. Alternatively, no ALT is added to the run buffer as a negative control (-).

[0162] Figure 84 An example of an epitope binning experiment using biolayer interferometry (BLI) is shown. ALT-1 captured on a nickel sensor via its c-terminal histidine tag was saturated with antibody along the lines indicated by adjacent arrows (A and B) and on the x-axis (C and D). A second competing antibody, either A) C8-Fc or B) G6-Fc, was then added, and the binding and dissociation phases were monitored. For C) C8-Fc and D) G6-Fc, the percentage of inhibition was calculated as 100 - [maximum nm shift of saturated antibody / maximum nm shift of competing antibody] × 100.

[0163] Figure 85 The X-ray crystal structure of ALT1 conjugated with nanobody C8 is shown. ALT1 is a homodimer, displayed in surface mode, with each monomer shown in a distinct gray shading. In the cartoon illustration, two nanobody C8 molecules are bound to the ALT1 dimer. The ALT1-C8 crystal structure is rotated 90° (…). Figure 85 B) shows a top view of the ALT1-C8 complex. The interface between ALT1 and nanobody C8 is colored with the same shading as the interacting nanobody.

[0164] Figure 86The following diagrams show the overlap between the ALT1-C8 crystal structure and the crystal structures of ALT2 (PDB ID: 3IHJ) or the nanobody NbALFA (PDB ID: 6I2G). A) shows the overlap between ALT1 monomer A (black) and ALT2 monomer (light gray). The ALT1 monomer exhibits a similar folding pattern to ALT2, as reflected by an RMSD of 0.868 Å. B) shows the overlap between the ALT1 dimer (black) and ALT2 dimer (light gray). There are some differences in how ALT1 and ALT2 form homodimers, as reflected by a higher RMSD of 6.633 Å. C) shows the overlap between the nanobody C8 (black) and NbALFA (light gray), NbALFA being a random alpaca nanobody structure found in the RCSBPDB database. Dashed lines in the C8 structure indicate unmodeled residues. The two nanobodies show excellent alignment, with an RMSD of 0.615 Å. D) shows the overlap between the nanobody C8 and... Figure 13 The predicted overlap of the alphafold 2.0 structure is presented in the figure.

[0165] Figure 87 The residues involved at the interface between ALT1 and nanobody C8 are shown. Residues involved in salt-bridge interactions are shown in solid lines, while residues involved in hydrogen bonding are shown in dashed lines. Nanobody C8 is in contact with two monomers of the ALT1 homodimer.

[0166] Figure 88 A close-up of the bonds formed between ALT1 and nanobody C8 in the ALT1-C8 crystal structure determined by PISA analysis is shown. Salt bridges are shown as dashed lines, and hydrogen bonds as dotted lines. Residues from ALT1 are underlined.

[0167] Figure 89 A heatmap showing the ability of nanobody G6-Fc to bind to a monoalanine mutant of ALT1 is presented. Alanine residues in ALT1 were replaced at specified positions, and the protein was expressed and used to coat ELISA plates. Nanobody G6-Fc was applied in triplicate to each well, and antibody binding was detected using a species-specific antibody conjugated with horseradish peroxidase. The percentage of binding was calculated relative to wild-type ALT1. Data are presented as a heatmap of the average of two independent experiments.

[0168] Figure 90 This section shows a list of all amino acids in which the substitution of alanine leads to a decreased binding with nanobody G6. Data source: Figure 89 It lists the amino acids that cause a 75% to 100% reduction in binding, a 50% to 75% reduction in binding, or a 25% to 50% reduction in binding.

[0169] Figure 91A heatmap showing the ability of rabbit monoclonal antibodies to bind to a monoalanine mutant of ALT1 is presented. Alanine residues in ALT1 were replaced at specified positions, and the protein was expressed and used to coat ELISA plates via anti-His capture. Each rabbit monoclonal antibody was applied in triplicate to each well, and antibody binding was detected using an anti-rabbit antibody conjugated with horseradish peroxidase. The percentage of binding was calculated relative to wild-type ALT1. Data are presented as a heatmap of the average of two independent experiments.

[0170] Figure 92 The table shows a list of all amino acids whose substitution with alanine results in decreased binding to rabbit monoclonal antibody 1. Data source: Figure 91 It lists the amino acids that cause a 75% to 100% reduction in binding, a 50% to 75% reduction in binding, or a 25% to 50% reduction in binding.

[0171] Figure 93 A list of all amino acids whose substitution with alanine results in decreased binding to rabbit monoclonal antibody 3 is shown. Data source: Figure 91 It lists the amino acids that cause a 75% to 100% reduction in binding, a 50% to 75% reduction in binding, or a 25% to 50% reduction in binding.

[0172] Figure 94 A list of all amino acids whose substitution with alanine results in decreased binding to rabbit monoclonal antibody 4 is shown. Data source: Figure 91 It lists the amino acids that cause a 75% to 100% reduction in binding, a 50% to 75% reduction in binding, or a 25% to 50% reduction in binding.

[0173] Figure 95 A list of all amino acids whose substitution with alanine results in decreased binding to rabbit monoclonal antibody 5 is shown. Data source: Figure 91 It lists the amino acids that cause a 75% to 100% reduction in binding, a 50% to 75% reduction in binding, or a 25% to 50% reduction in binding.

[0174] Figure 96 Epitope binning experiments using ALT-1 and rabbit monoclonal antibodies are shown. ALT1 captured on a nickel sensor via its C-terminal histidine tag was saturated with buffer-only (KN) or RmAb1, 3, 4, or 5 (indicated by each line). A second competing antibody, RmAb1, 3, 4, or 5, was then added, and the binding and dissociation phases were monitored.

[0175] Figure 97The quantification of epitope binning experiments using ALT-1 and rabbit monoclonal antibodies is shown. For RmAb1, 3, 4, or 5, the percentage of inhibition was calculated as 100 - [maximum nm shift of saturated antibody / maximum nm shift of competing antibody] × 100.

[0176] Figure 98 The image shows dot blots of native and denatured ALT1 detected using various anti-ALT1 antibodies. Different concentrations of native (N) or denatured (D) ALT1 were spotted onto nitrocellulose membranes and detected using: G6-FLAG, C8-FLAG, C4G-Fc (A), rabbit monoclonal antibody (B), rabbit polyclonal antibody, commercial Abcam anti-ALT, commercial MyBioSource.com anti-ALT2 (C), or mouse monoclonal antibody (D).

[0177] Figure 99 Epitope binning experiments using ALT1 and mouse monoclonal antibodies are shown. ALT1 captured on a nickel sensor via its C-terminal His tag was saturated with a variety of antibodies (see figure legend). A second competitive antibody, mouse monoclonal 3H12 (A and B), 4A9 (C and D), 5H2 (E and F), or 6B5 (G and H), was then added, and binding and dissociation phases were monitored.

[0178] Figure 100 The quantification of epitope binning experiments using ALT1 and mouse monoclonal antibodies is shown. The percentage of inhibition is calculated as 100 - [maximum nm shift of saturated antibody / maximum nm shift of competing antibody] × 100, 3H12 (A), 4A9 (B), 5H2 (C), or 6B5 (D). E) is shown as a summary of (A to D) in heatmap form.

[0179] Figure 101 Embodiments of the assay of the present invention are provided. Gray boxes indicate combinations of capturing and detecting binding proteins. Light gray boxes indicate preferred combinations.

[0180] Figure 102 The differential scanning fluorometry (DSFL) image of the ALT protein is shown. The thermal stability of ALT1 was assessed by DFL. The data show that the Tm of ALT1 is 60.0 °C, as observed by the peak inflection point. The protein begins to unfold at approximately 43 °C and continues to unfold at approximately 68 °C.

[0181] Sequence List Description

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Detailed Implementation

[0189] General techniques and definitions

[0190] Unless otherwise expressly defined, all technical and scientific terms used herein should be assumed to have the same meaning as commonly understood by a person skilled in the art (e.g., amino acid sequence).

[0191] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be regarded as providing explicit support for both meanings or either meaning.

[0192] Throughout this specification, the term "including / comprises" or variations thereof will be understood to mean including the stated element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0193] As used herein, the term "object" refers to any animal. In one instance, the animal is a vertebrate. In one embodiment, the object is a mammal. In one embodiment, the mammal is a human. In some embodiments, the mammal is a human companion animal. In some embodiments, the animal is livestock or a research animal.

[0194] As used herein, the terms “binding” or “binding with” refer to the interaction between a binding protein or its antigen-binding domain and an antigen, meaning that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, a binding protein recognizes and binds to a specific protein structure, rather than a protein in general. If a binding protein binds to epitope “A”, then in a reaction containing labeled “A” and the binding protein, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) will reduce the amount of labeled “A” bound to the binding protein.

[0195] As used in this article, the term "epitope" refers to a structure on the surface of an antigen that is recognized by and can bind to a specific antibody.

[0196] As used herein, the term "conformational epitope," also known as a "discontinuous epitope" or "topographic epitope," is an epitope constructed from discontinuous portions of one or more amino acid sequences that are aggregated together by the folding of a protein in its native state. In one embodiment, the conformational epitope comprises residues on two protomers of a multimeric protein (e.g., a homodimer). In one embodiment, the conformational epitope is a conformational epitope of ALT1. In one embodiment, the conformational epitope depends on non-covalent bond formation in ALT.

[0197] As used herein, the term "complementary site" for binding proteins or their antigen-binding domains refers to a set of amino acid residues on the variable region of an antibody that directly contacts the antigen and forms the antigen-binding site of the variable region. A complementary site typically contains or consists of one or more amino acid residues from a CDR sequence. In some embodiments, the complementary site contains one or more framework regions and one or more residues from a CDR sequence.

[0198] As used herein, the terms "specific binding" or "specific to X" should be understood to mean that a binding protein of this disclosure reacts or binds to one or more specific antigens or cells expressing that antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than it reacts or binds to alternative antigens or cells. For example, a protein that specifically binds to an antigen binds to that antigen with greater affinity (e.g., 20, 40, 60, 80 to 100, 150, or 200 times greater affinity), more readily, and / or for a longer duration than it binds to other antigens. It should also be understood from this definition that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. In one embodiment, the antigen is alanine aminotransferase 1 (ALT1). In one embodiment, the antigen is alanine aminotransferase 2 (ALT2). In one embodiment, the second antigen is aspartate aminotransferase (AST). In one embodiment, the antigen is human ALT1. In one embodiment, the antigen is human ALT2. In one embodiment, the second antigen is human AST.

[0199] As used herein, "does not bind significantly to" should be understood to mean that the binding level of the binding protein to the antigen is no greater than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the binding level of the protein to antigens it is known to bind to. In one instance, binding is detected by Western blotting and / or FACS and / or ELISA and / or antibody panning (e.g., using the antibody variable region on the surface of particles (e.g., bacteriophages)). For example, the binding level of the protein of the present invention to heat-denatured ALT1 is no greater than about 20%, 15%, or 10% of its binding level to properly folded ALT1.

[0200] As used herein, “does not bind detectably to…” should be understood to mean that the binding protein does not bind to the antigen at a level significantly higher than the background level, for example, binding to ALT1 at a level less than 10%, 8%, 6%, or 5% above the background level. In the case of antibodies, the antibody binds to the antigen at a level less than 10%, 8%, 6%, or 5% higher than that of the isotype control antibody. In one instance, binding is detected by Western blotting and / or FACS and / or ELISA and / or antibody panning (e.g., using the antibody variable region on the surface of particles (e.g., phages)).

[0201] As used herein, the term “isolated” should be understood to mean that the binding protein or nucleic acid is substantially removed from its natural environment, such as being in a heterologous environment, and / or is substantially free of contaminants, such as being at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminants.

[0202] The term “recombination” as used in this article should be considered as meaning produced by combining genetic material from two or more different sources.

[0203] As used herein, the term "sample" refers to a biological sample, such as blood or its fractions, urine, tissue, cells, or saliva. In one embodiment, the sample is selected from plasma, serum, whole blood, and capillary blood. In one embodiment, the sample is plasma. In one embodiment, the sample is serum. In one embodiment, the sample is human plasma. In one embodiment, the sample is human serum. In one embodiment, the sample is whole blood. In one embodiment, the sample is capillary blood. In one embodiment, the sample is finger-prick blood. In one embodiment, the whole blood is venous blood. In one embodiment, the whole blood is capillary blood.

[0204] The term "visual" or "visible measurement" as used in this article refers to a measurement in which the results of the test can be directly interpreted by the user upon viewing, without the need for special technical equipment to detect and read the results.

[0205] The term "sensitivity" as used in this article refers to the ability of a binding protein to detect an antigen. Higher sensitivity means that the binding protein can detect lower concentrations of antigen in a sample.

[0206] As used in this article, "specificity" refers to the ability of the binding protein to detect antigens in a mixture of other antigens (which may be similar and / or different).

[0207] In this article, "protomer" refers to the basic structural unit of oligomeric proteins (such as monomers of homodimers).

[0208] As used herein, the term "antigen binding site" should be understood to refer to a structure formed by a protein that can bind to or specifically bind to an antigen. An antigen binding site need not be a series of consecutive amino acids, or even an amino acid from a single polypeptide chain. For example, in Fv, which is produced by two different polypeptide chains, the antigen binding site is formed by V... L The antigen-binding site is composed of a series of amino acids that interact with the antigen in the VH region, and it is usually, but not always, located in one or more CDRs in each variable region. In some instances, the antigen-binding site is VH. H or V L Or Fv.

[0209] As used herein, the term "affinity" refers to the strength of the interaction between the antigen and the binding protein (the overall strength of the antibody-binding protein complex). In one embodiment, the addition of an Fc region or a fragment thereof increases the affinity of the binding protein. In one embodiment, the polymeric form of the binding protein has a higher affinity than the monomeric form of the binding protein.

[0210] Alanine transaminase

[0211] As used herein, “alanine transaminase,” “ALT,” “alanine aminotransferase,” “glutamate-pyruvate transaminase,” or “serum glutamate-pyruvate transaminase” refers to an enzyme that catalyzes a reversible amination reaction between L-alanine and 2-oxoglutamate (α-ketoglutamate) by transferring an amino group from L-alanine to α-ketoglutamate to produce pyruvate and L-glutamate. Like other transaminases, ALT requires the coenzyme pyridoxal phosphate in the first stage of the reaction. Natural / naturally occurring enzymes are homodimers (comprising two protomers). Alanine transaminases play a role in gluconeogenesis and amino acid metabolism in many tissues, including skeletal muscle, kidney, and liver. In one embodiment, ALT is ALT1. In one embodiment, the monomer of the ALT1 homodimer is encoded by the amino acid sequence shown in SEQ ID NO:84. In one embodiment, ALT1 is human ALT1. In one embodiment, ALT is ALT2. In one embodiment, ALT2 is human ALT2. In one embodiment, ALT2 is ALT2 isotype 1. In one embodiment, the monomer of the ALT2 isotype 1 homodimer is encoded by the amino acid sequence shown in SEQ ID NO: 85. In one embodiment, ALT2 is ALT2 isotype 2. In one embodiment, the monomer of the ALT2 isotype 2 homodimer is encoded by the amino acid sequence shown in SEQ ID NO: 85.

[0212] As used herein, "denatured ALT1" refers to ALT1 that has lost the folded structure of the protein in its native state. In one embodiment, ALT1 is treated to break non-covalent bonds. In one embodiment, ALT1 is denatured by heating. The melting temperature of ALT1 is... Figure 102 As shown. In one embodiment, ALT1 is denatured by heating at a temperature of about 68°C. In another embodiment, ALT1 is denatured by heating at a temperature of about 68°C or higher. In yet another embodiment, ALT1 is denatured by chemical treatment.

[0213] The gene encoding ALT1 is located on human chromosome 8. The highest levels of gene transcription have been observed in the liver, followed by the colon, duodenum, fat, and kidneys, while lower levels have been detected in the skin, small intestine, and stomach. ALT1 is a cytoplasmic protein that has been reported to account for more than 90% of the activity of detectable enzymes in the blood under normal conditions (Rafter et al., 2012).

[0214] The gene encoding alanine aminotransferase 2 (ALT2) is located on human chromosome 16. ALT2 isotype 1 represents a longer transcript and encodes a longer variant. ALT2 isotype 2 is produced when a substitution of exons is used in the 5'UTR and 5' coding sequences, resulting in the use of a downstream start codon compared to variant 1. It has a shorter N-terminus than isotype 1. Unlike ALT1, several clinically significant single nucleotide polymorphisms exist in ALT2. ALT2 is a mitochondrial enzyme and has a unique tissue distribution, with its RNA transcript primarily found in fat, followed by the esophagus, pancreas, liver, stomach, brain, skin, and salivary glands, while low levels are detected in the gallbladder, testes, and thyroid gland. ALT2 accounts for a relatively small proportion of the enzymatic ALT activity found in the blood, <10% (Rafter et al., 2012). ALT2 dysregulation has been reported to promote cell survival and growth in cancer. Recessive mutations in ALT2 are associated with intellectual and developmental disabilities, postnatal microcephaly, and spastic paraplegia (Ouyang Qing et al., 2019).

[0215] binding protein

[0216] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase (ALT).

[0217] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase (ALT), said binding protein comprising an amino acid sequence selected from:

[0218] a) The amino acid sequence GPAVSNVA (SEQ ID NO: 2) as CDR1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3;

[0219] b) The amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3; and

[0220] c) The amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, ISRSGFST (SEQ ID NO: 11) as CDR2, and AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3; and

[0221] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0222] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising an amino acid sequence selected from:

[0223] a) The amino acid sequence GPAVSNVA (SEQ ID NO: 2) as CDR1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3;

[0224] b) The amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3; and

[0225] c) The amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, ISRSGFST (SEQ ID NO: 11) as CDR2, and AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3; and

[0226] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0227] In one embodiment, the present invention provides a binding protein that binds to ALT1, the binding protein comprising the amino acid sequence GPAVSNVA (SEQ ID NO: 2) as CDR1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3, wherein any one or more CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0228] In one embodiment, the present invention provides a binding protein that binds to ALT1, the binding protein comprising the amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3, wherein any one or more CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0229] In one embodiment, the present invention provides a binding protein that binds to ALT2, the binding protein comprising the amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3, wherein any one or more CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0230] In one embodiment, the present invention provides a binding protein that binds to ALT1, the binding protein comprising the amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, the amino acid sequence ISRSGFST (SEQ ID NO: 11) as CDR2, and the amino acid sequence AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3, wherein any one or more CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0231] In one embodiment, one of the CDRs has 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0232] In one embodiment, two of the CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0233] In one embodiment, the three CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0234] In one embodiment, one of CDR1 has 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0235] In one embodiment, one of CDR2 has 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0236] In one embodiment, one of CDR3 has 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0237] In one embodiment, the present invention provides a binding protein that binds to ALT1, the binding protein comprising the amino acid sequence GPAVSNVA (SEQ ID NO: 2) as CDR1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3.

[0238] In one embodiment, the present invention provides a binding protein that binds to ALT1, the binding protein comprising the amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3.

[0239] In one embodiment, the present invention provides a binding protein that binds to ALT2, the binding protein comprising the amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3.

[0240] In one embodiment, the present invention provides a binding protein that binds to ALT1, the binding protein comprising the amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, the amino acid sequence ISRSGFST (SEQ ID NO: 11) as CDR2, and the amino acid sequence AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3.

[0241] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 1 (C8), or a sequence having at least 76% identity with it, or its humanized or germline form.

[0242] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or its humanized or germline form.

[0243] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 9 (C10), or a sequence having at least 76% identity with it, or its humanized or germline form.

[0244] In one embodiment, the binding protein comprises, consists of, or is substantially composed of an amino acid sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1 (C8).

[0245] In one embodiment, the binding protein comprises, consists of, or is substantially composed of an amino acid sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 (G6).

[0246] In one embodiment, the binding protein comprises, consists of, or is substantially composed of an amino acid sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9 (C10).

[0247] In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 1 (C8). In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 5 (G6). In one embodiment, the binding protein comprises the amino acid sequence SEQ ID NO: 9 (C10).

[0248] In one embodiment, the binding protein comprises an amino acid sequence selected from SEQ ID NO: 13 to SEQ ID NO: 56, or a sequence having at least 76% identity with it, or its humanized or germline form.

[0249] In one implementation, the binding protein binds to an epitope containing residues on both protomeres of ALT1.

[0250] In one implementation, the binding protein does not bind significantly to denatured ALT1.

[0251] In one implementation, the binding protein does not bind significantly to heat-denatured ALT1.

[0252] In one implementation, the binding protein does not bind detectably to denatured ALT1.

[0253] In one embodiment, the binding protein does not detectably bind to thermally denatured ALT1. In one embodiment, ALT1 is denatured by heating to a temperature of approximately 68°C. In one embodiment, ALT1 is denatured by heating to a temperature of 68°C or higher.

[0254] In one embodiment, the binding protein binds to denatured ALT1. In another embodiment, the binding protein binds to heat-denatured ALT1.

[0255] In one embodiment, the binding protein does not significantly bind to the epitope of ALT2. In one embodiment, the binding protein does not significantly bind to the epitope of human ALT2.

[0256] In one embodiment, the binding protein does not bind significantly to AST. In another embodiment, the binding protein does not bind significantly to human AST.

[0257] In one embodiment, the binding protein does not detectably bind to the epitope of ALT2. In one embodiment, the binding protein does not detectably bind to the epitope of human ALT2.

[0258] In one embodiment, the binding protein does not detectably bind to AST. In one embodiment, the binding protein does not detectably bind to human AST.

[0259] In one implementation, the binding protein has a higher affinity for ALT1 than for ALT2.

[0260] In one implementation, the protein binds to natural ALT1 in the solution.

[0261] In one implementation, the binding protein binds to ALT1 with its conformation intact.

[0262] In one implementation, the binding protein binds to the conformational epitope of ALT1.

[0263] In one embodiment, the binding protein binds to natural ALT1 on a solid surface by streaking / coupling it onto the solid surface.

[0264] In one embodiment, the complementary site of the binding protein contains residues from two protomers of ALT (the active enzyme is a homodimer of two ALT1 protomers). In another embodiment, the complementary site of the binding protein binds residues from two promoters of ALT.

[0265] In one embodiment, the binding protein binds Asn 99 and Gln 374 of an ALT1 promerum and 93 Asp, 96 Ser and 109 Glu of a second ALT1 promerum.

[0266] In one embodiment, the complementary site of the binding protein contains residues on the two protomers of ALT (the active enzyme is a homodimer of two ALT1 monomers).

[0267] In one embodiment, the complementary site of the binding protein comprises one or more residues of the following: CDR2, FR1, and FR2. In one embodiment, the complementary site of the binding protein comprises a residue of CDR2. In one embodiment, the complementary site of the binding protein comprises a residue of FR1. In one embodiment, the complementary site of the binding protein comprises a residue of FR2.

[0268] In one embodiment, the complementary site of the binding protein contains residues in CDR2 but not residues in CDR1 or CDR3.

[0269] In one embodiment, the binding protein residues mediating contact with ALT1 include one or more residues corresponding to C8: 18 Leu, 19 Arg, 58 Ser, 68 Phe, 90 Thr, 70 Ile, 71 Ser, 76 Lys, 82 Gln, and 84 Asn.

[0270] In one embodiment, the binding protein residues mediating contact with ALT1 comprise or consist of residues corresponding to or composed of 18 Leu, 19 Arg, 58 Ser, 68 Phe, 90 Thr, 70 Ile, 71 Ser, 76 Lys, 82 Gln, and 84 Asn.

[0271] In one embodiment, the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0272] In one embodiment, the binding protein does not detectably bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0273] In one embodiment, the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 53 Arg, 61 Thr, 154 Val, 175 Glu, 178 Thr, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 225 Leu, 239 Val, 260 Arg, 266 Arg, 304 Gln, 327 Val, 358 Leu, 369 Asp, 430 Glu, 431 Leu, and 433 Leu.

[0274] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 53 Arg, 61 Thr, 154 Val, 175 Glu, 178 Thr, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 225 Leu, 239 Val, 260 Arg, 266 Arg, 304 Gln, 327 Val, 358 Leu, 369 Asp, 430 Glu, 431 Leu, and 433 Leu.

[0275] In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 53 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 154 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 175 Glu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 178 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 196 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 202 (Val). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 205 (Asp). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 212 (Arg). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 225 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 239 (Val). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 260 (Arg). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 266 (Arg). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 304 (Gln). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 327 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 369 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 430 Glu.In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 431 Leu. In another embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 433 Leu.

[0276] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 53 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 154 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 175 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 178 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 196 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 202 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 205 (Asp). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 212 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 225 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 239 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 260 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 266 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 327 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 369 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 430 Glu.In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 431 Leu. In another embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 433 Leu.

[0277] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising a heavy chain variable region (V... H ) and light chain variable region (V L ), wherein V H and V L It includes six complementary determination regions (CDRs) selected from the following:

[0278] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0279] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0280] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0281] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and

[0282] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2 and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3;

[0283] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0284] In one implementation, the binding protein binds to epitopes containing residues on two monomers containing ALT1.

[0285] In one embodiment, the binding protein does not bind significantly to human ALT2. In another embodiment, the binding protein does not bind significantly to aspartate aminotransferase (AST).

[0286] In one embodiment, the binding protein does not detectably bind to human ALT2. In another embodiment, the binding protein does not detectably bind to aspartate aminotransferase (AST).

[0287] In one embodiment, the binding protein does not bind significantly to denatured ALT1. In another embodiment, the binding protein does not bind significantly to heat-denatured ALT1.

[0288] In one embodiment, the binding protein does not detectably bind to denatured ALT1. In another embodiment, the binding protein does not detectably bind to heat-denatured ALT1.

[0289] In one implementation, the protein binds to natural ALT1 in the solution.

[0290] In one implementation, the binding protein binds to ALT1 with its conformation intact.

[0291] In one implementation, the binding protein binds to the conformational epitope of ALT1.

[0292] In one implementation, the complementary site of the binding protein contains residues on both protomers of ALT.

[0293] In one embodiment, the binding protein binds to natural ALT1 on a solid surface by streaking / coupling it onto the solid surface.

[0294] In one embodiment, the protein-binding melting temperature is from about 74°C to about 76°C. In another embodiment, the protein-binding melting temperature is from about 74.8°C to about 75.8°C.

[0295] In one embodiment, the binding rate of the binding protein is at least about 5 times higher than that of the rabbit polyclonal ALT-1 antibody. In one embodiment, the binding rate of the binding protein is at least about 5.47 times higher than that of the rabbit polyclonal ALT-1 antibody. In one embodiment, the binding rate of the binding protein is about 5.47 times to about 16.08 times higher than that of the rabbit polyclonal ALT-1 antibody.

[0296] In one embodiment, the affinity of the binding protein for ALT1 is at least about 10 times higher than that of the rabbit polyclonal anti-ALT antibody. In one embodiment, the affinity of the binding protein for ALT1 is at least about 10.56 times higher than that of the rabbit polyclonal anti-ALT antibody. In one embodiment, the affinity of the binding protein for ALT1 is at least about 10.56 times higher than that of the rabbit polyclonal anti-ALT antibody, and up to about 45.16 times higher. In one embodiment, the affinity of the binding protein for ALT1 is at least about 28.38 times higher than that of the rabbit polyclonal anti-ALT antibody, and up to about 2160 times higher. In one embodiment, the affinity of the binding protein for ALT1 is at least about 10.56 times higher than that of the rabbit polyclonal anti-ALT antibody, and up to about 2160 times higher.

[0297] In one embodiment, the binding protein can be isolated with a purity of at least about 90%. In one embodiment, the binding protein can be isolated with a purity of at least about 91%. In one embodiment, the binding protein can be isolated with a purity of at least about 92%. In one embodiment, the binding protein can be isolated with a purity of at least about 93%. In one embodiment, the binding protein can be isolated with a purity of at least about 94%. In one embodiment, the binding protein can be isolated with a purity of at least about 95%.

[0298] In one implementation, the sensitivity of the protein-bound detection of human ALT1 is at least twice that of rabbit polyclonal anti-ALT antibodies.

[0299] In one implementation, the sensitivity of the protein-bound detection of human ALT1 is at least three times higher than that of rabbit polyclonal anti-ALT antibodies.

[0300] In one implementation, the sensitivity of the protein-binding assay for detecting human ALT1 is approximately 2 to 3 times higher than that of the rabbit polyclonal anti-ALT antibody.

[0301] In one embodiment, the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO: 109. H and V containing the amino acid sequence shown in SEQ ID NO: 113 L (RmAb1), or a sequence that has at least 70% identity with it.

[0302] In one embodiment, the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO: 116. H and V containing the amino acid sequence shown in SEQ ID NO: 120 L (RmAb2), or a sequence that has at least 70% identity with it.

[0303] In one embodiment, the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO: 123. H and V containing the amino acid sequence shown in SEQ ID NO: 127 L (RmAb3), or a sequence that has at least 70% identity with it.

[0304] In one embodiment, the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO: 130. H and V containing the amino acid sequence shown in SEQ ID NO: 120 L (RmAb4), or a sequence that has at least 70% identity with it.

[0305] In one embodiment, the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO: 137. H and V containing the amino acid sequence shown in SEQ ID NO: 141 L (RmAb5), or a sequence that has at least 70% identity with it.

[0306] In one embodiment, the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 129 Ile, 131 Leu, 154 Val, 177 His, 178 Thr, 183 Leu, 202 Val, 205 Asp, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

[0307] In one embodiment, the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 183 Leu, 196 Leu, 202 Val, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 333 Asp, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

[0308] In one embodiment, the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 77 Arg, 109 Glu, 129 Ile, 131 Leu, 138 Arg, 141 Glu, 154 Val, 157 Ser, 175 Glu, 177 His, 178 Thr, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 225 Leu, 239 Val, 260 Arg, 266 Arg, 276 Gln, 304 Gln, 327 Val, 333 Asp, 340 Met, 358 Leu, 376 Gln, 408 Val, 430 Glu, 431 Leu, and 433 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 77 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 109 Glu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 131 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 138 (Arg). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 141 (Glu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 154 (Val). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 157 (Ser). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 175 (Glu).In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 177 His. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 178 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 196 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 202 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 205 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 212 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 225 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 260 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 276 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 327 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 333 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 376 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 408 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 430 Glu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 431 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 433 Leu.

[0309] In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 183 Leu, 196 Leu, 202 Val, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 333 Asp, 340 Met, 358 Leu, 376 Gln, 408 Val, 418 Val, 430 Glu, or 433 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 131 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 154 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 196 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 202 (Val). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 212 (Arg). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 222 (His). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 225 (Leu).In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 229 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 260 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 284 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 333 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 376 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 408 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 418 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 430 Glu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 433 Leu.

[0310] In one embodiment, the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 4 Ser, 7 Asp, 8 Arg, 12 Val, 16 Leu, 19 Lys, 20 Val, 24 Asp, 31 Arg, 32 Arg, 43 Gln, 44 Arg, 48 Leu, 53 Arg, 54 Gln, 56 Val, 61 Thr, 77 Arg, 102 Asp, 129 Ile, 130 Gln, 131 Leu, 154 Val, 157 Ser, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 251 Thr, 260 Arg, 266 Arg, 276 Gln, 284 Ser, 285 Gln, 303 Gln, 304 Gln, 327 Val, 333 Asp, 340 Met, 358 Leu, 408 Val, 430 Glu, 433 Leu, 461 Asp, 485 Ser, 493 Leu, or 494 Glu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 4 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 7 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 8 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 12 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 16 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 20 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 32 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 44 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 48 Leu.In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 53 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 54 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 77 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 102 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 130 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 131 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 154 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 157 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 196 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 202 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 205 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 212 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 222 His. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 225 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 229 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 251 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 260 Arg.In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 276 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 284 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 285 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 303 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 327 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 333 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 358 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 408 (Val). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 430 (Glu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 433 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 461 (Asp). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 485 (Ser). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 493 (Leu). In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 494 (Glu).

[0311] In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at one or more of the following positions: 4 Ser, 12 Val, 13 Arg, 19 Lys, 24 Asp, 31 Arg, 43 Gln, 44 Arg, 48 Leu, 53 Arg, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 177 His, 178 Thr, 183 Leu, 202 Val, 205 Asp, 212 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 340 Met, 358 Leu, 408 Val, 430 Glu, 433 Leu, 461 Asp, or 485 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 4 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 12 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 13 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 44 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 53 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 131 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 154 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 177 His.In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 178 Thr. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 202 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 205 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 212 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 260 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 284 Ser. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 408 Val. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 430 Glu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 433 Leu. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 461 Asp. In one embodiment, the binding protein does not significantly bind to ALT1 containing an alanine substitution at position 485 Ser.

[0312] In one embodiment, the binding protein does not detectably bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0313] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 129 Ile, 131 Leu, 154 Val, 177 His, 178 Thr, 183 Leu, 202 Val, 205 Asp, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

[0314] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 183 Leu, 196 Leu, 202 Val, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 333 Asp, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

[0315] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 77 Arg, 109 Glu, 129 Ile, 131 Leu, 138 Arg, 141 Glu, 154 Val, 157 Ser, 175 Glu, 177 His, 178 Thr, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 225 Leu, 239 Val, 260 Arg, 266 Arg, 276 Gln, 304 Gln, 327 Val, 333 Asp, 340 Met, 358 Leu, 376 Gln, 408 Val, 430 Glu, 431 Leu, and 433 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 77 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 109 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 131 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 138 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 141 (Glu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 154 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 157 (Ser).In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 175 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 177 His. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 178 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 196 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 202 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 205 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 212 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 225 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 239 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 260 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 266 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 276 (Gln). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 304 (Gln). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 327 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 333 (Asp). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 376 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 408 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 430 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 431 Leu.In one implementation, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 433 Leu.

[0316] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 183 Leu, 196 Leu, 202 Val, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 333 Asp, 340 Met, 358 Leu, 376 Gln, 408 Val, 418 Val, 430 Glu, or 433 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 131 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 154 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 183 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 196 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 202 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 212 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 222 (His). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 225 (Leu).In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 229 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 260 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 284 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 333 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 358 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 376 (Gln). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 408 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 418 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 430 (Glu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 433 (Leu).

[0317] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 4 Ser, 7 Asp, 8 Arg, 12 Val, 16 Leu, 19 Lys, 20 Val, 24 Asp, 31 Arg, 32 Arg, 43 Gln, 44 Arg, 48 Leu, 53 Arg, 54 Gln, 56 Val, 61 Thr, 77 Arg, 102 Asp, 129 Ile, 130 Gln, 131 Leu, 154 Val, 157 Ser, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 251 Thr, 260 Arg, 266 Arg, 276 Gln, 284 Ser, 285 Gln, 303 Gln, 304 Gln, 327 Val, 333 Asp, 340 Met, 358 Leu, 408 Val, 430 Glu, 433 Leu, 461 Asp, 485 Ser, 493 Leu, or 494 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 4 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 7 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 8 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 12 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 16 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 20 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 32 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 44 Arg.In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 53 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 54 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 77 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 102 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 130 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 131 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 154 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 157 (Ser). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 183 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 196 (Leu). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 202 (Val). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 205 (Asp). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 212 (Arg). In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 222 His. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 225 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 229 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 251 Thr.In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 260 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 276 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 284 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 285 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 303 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 327 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 333 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 408 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 430 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 433 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 461 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 485 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 493 (Leu). In another embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 494 (Glu).

[0318] In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 4 Ser, 12 Val, 13 Arg, 19 Lys, 24 Asp, 31 Arg, 43 Gln, 44 Arg, 48 Leu, 53 Arg, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 177 His, 178 Thr, 183 Leu, 202 Val, 205 Asp, 212 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 340 Met, 358 Leu, 408 Val, 430 Glu, 433 Leu, 461 Asp, or 485 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 4 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 12 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 13 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 19 Lys. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 24 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 31 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 43 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 44 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 48 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 53 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 56 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 61 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 129 Ile. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 131 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 154 Val.In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 177 His. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 178 Thr. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 183 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 202 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 205 Asp. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 212 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 239 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 260 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 266 Arg. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 284 Ser. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 304 Gln. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 340 Met. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 358 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 408 Val. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 430 Glu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 433 Leu. In one embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 461 Asp. In another embodiment, the binding protein does not detectably bind to ALT1 containing an alanine substitution at position 485 Ser.

[0319] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), the binding protein comprising an antigen-binding site of an antibody comprising an amino acid sequence selected from the following:

[0320] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0321] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0322] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0323] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and

[0324] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2 and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3;

[0325] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0326] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising a heavy chain variable region (V... H ), wherein V H Includes complementary determination regions (CDRs) selected from the following:

[0327] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2 and ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3;

[0328] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2 and ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3;

[0329] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2 and ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3;

[0330] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2 and ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3;

[0331] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2 and ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3;

[0332] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0333] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising a light chain variable region (V... L ), wherein V L Includes complementary determination regions (CDRs) selected from the following:

[0334] a) The amino acid sequence ENIYSG (SEQ ID NO: 114) as light chain CDR1, the amino acid sequence KAS as light chain CDR2, and the amino acid sequence QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0335] b) The amino acid sequence VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, the amino acid sequence GAS as light chain CDR2, and the amino acid sequence AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0336] c) The amino acid sequence QSIGNY (SEQ ID NO: 128) as light chain CDR1, the amino acid sequence RAS as light chain CDR2, and the amino acid sequence QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0337] d) The amino acid sequence QSISTA (SEQ ID NO: 135) as light chain CDR1, the amino acid sequence SAS as light chain CDR2, and the amino acid sequence QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3;

[0338] e) The amino acid sequence QSISSSY (SEQ ID NO: 142) as light chain CDR1, the amino acid sequence RVS as light chain CDR2, and the amino acid sequence QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3;

[0339] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0340] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site for an antibody.

[0341] In one aspect, the present invention provides a binding protein that binds to a conformational epitope of alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody.

[0342] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1), wherein the binding protein does not significantly and / or detectably bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0343] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody, and said binding protein not binding significantly and / or detectably to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

[0344] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody comprising an amino acid sequence selected from:

[0345] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0346] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2 and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0347] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0348] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2 and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3;

[0349] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; and

[0350] f) GPAVSNVA (SEQ ID NO: 2) as CDR1, ITWSGWT (SEQ ID NO: 3) as CDR2, and NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3;

[0351] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0352] Wherein, when the amino acid sequence is a), the binding protein is a nanobody, and wherein, when the amino acid sequence is b), c), d), e), or f), the binding protein is an antibody.

[0353] In one aspect, the present invention provides a binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an amino acid sequence selected from:

[0354] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0355] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0356] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2 and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0357] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2 and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3;

[0358] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; and

[0359] f) GPAVSNVA (SEQ ID NO: 2) as CDR1, ITWSGWT (SEQ ID NO: 3) as CDR2, and NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3;

[0360] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0361] Antibodies and their fragments

[0362] In one embodiment, the binding protein is an antibody or a fragment thereof. The term "antibody" as used herein includes monoclonal antibodies, bispecific antibodies, fusion biantibodies, triantibodies, heteroconjugated antibodies, chimeric antibodies, including intact molecules and fragments thereof (antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab').sub.2), single-chain antibody molecules (e.g., scFv), nanobodies, and other antibody-like molecules. Antibody fragments include antigen-binding fragments generated by modifying an intact antibody or antigen-binding fragments synthesized de novo using recombinant DNA methods. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a rabbit polyclonal antibody. In one embodiment, the antibody is not a polyclonal antibody. In one embodiment, the antibody is not a rabbit polyclonal antibody.

[0363] Antibodies include various forms of modification, such as, but not limited to, domain antibodies containing VH or VL domains, heavy chain variable region dimers (VHH, as described for Camelidae), light chain variable region dimers (VLL), Fv fragments containing only light chain (VL) and heavy chain (VH) variable regions that can be directly linked or linked via a linker, or Fd fragments containing heavy chain variable regions and CH1 domains.

[0364] Antibodies can consist of VHH regions, which can be single VHH domains or multiple VHH domains directly linked by linkers, and then linked to Fc regions containing the CH2 and CH3 domains of immunoglobulins.

[0365] The term "antibody" also encompasses scFvs (Bird et al., 1988; Huston et al., 1988) consisting of variable regions of heavy and light chains linked together to form a single-chain antibody, and oligomers of scFvs, such as biantibodies and triantibodies. It also encompasses fragments of antibodies, such as Fab, (Fab')2, and FabFc2 fragments, which contain variable regions and partially constant regions. It also encompasses complementarity-determining regions (CDRs)—transplanted antibody fragments and oligomers of antibody fragments. The heavy and light chain components of an Fv can be derived from the same antibody or different antibodies, resulting in chimeric Fv regions. Antibodies can be animal-derived (e.g., mouse, rabbit, or rat) or can be chimeric (Morrison et al., 1984). Antibodies can be produced by any method known in the art.

[0366] Antibodies can contain variable light chains (V L ) and variable heavy chain (VH The Fv region of the chain, where light and heavy chains can be connected directly or via a connector. In one embodiment, V L It is κ. In one implementation, V L It is κ1. In one implementation, the light chain J gene germline uses J1-2 (IGKJ1_2).

[0367] As used herein, “variable region” refers to the portion of the light and / or heavy chain of a binding protein that specifically binds to the antigen, as defined herein, and includes, for example, the amino acid sequence of CDRs (i.e., CDR1, CDR2, and CDR3) and frame regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. The term “complementarity-determining region” (i.e., CDR1, CDR2, and CDR3) as used herein refers to the amino acid residues of the variable region of the binding protein (e.g., the VHH chain) whose presence is a major contributor to specific antigen binding. For example, each VHH chain of a camel-derived binding protein typically has three CDR regions, referred to as CDR1, CDR2, and CDR3. “Frame region” refers to those variable domain residues other than the CDR residues.

[0368] There are various conventions for defining, annotating, and describing the CDR (and extrapolated FR) of binding proteins (such as VHH chain or single-domain binding proteins). Therefore, the length and sequence of a particular CDR for a binding protein can vary depending on the specific nomenclature, algorithm, etc., used to define them. Some exemplary conventions for defining CDRs include the Kabat definition (which is based on sequence variability and is the most commonly used; see, for example, Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th edition; NIH Publ. No. 91-3242(1991)), the Chothia definition (which is based on the location of structural loop regions; see, for example, Chothia, et al., (1987) J Mol. Biol. 196:901-917), the AbM definition (which is a compromise between the Kabat and Chothia definitions and is based on the OxfordMolecular AbM antibody modeling software), the IMGT definition (see, for example, https: / / www.imgt.org / IMGTindex / CDR.php), and the method described by Kontermann and Diibel (ed., Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010).

[0369] In some embodiments, the amino acid sequences of the binding proteins of this disclosure, namely CDR1, CDR2, and CDR3, are determined or defined by Lefranc unique numbering (Lefranc, 1997, Lefranc, 1999, Lefranc, 2003).

[0370] Monoclonal antibodies are preferred. The term "monoclonal antibody" or "MAb" refers to a group of homogeneous antibodies capable of binding to the same antigen, preferably to the same epitope within the antigen. This term is not intended to limit the source of the antibody or the method of its preparation. For the production of Mab, any of many known techniques can be used, such as those illustrated in US 4,196,265 or Harlow and Lane (1988) or Zola (1987).

[0371] Nanobody

[0372] In one implementation, the binding protein is a nanobody. As used herein, the terms “nanobody” or “Nb” or “single-domain binding protein” refer to a single antigen-binding domain (VHH) binding protein derived from a naturally occurring camel-based heavy chain antibody (HCAb) lacking a light chain (Muyldermans, 2013).

[0373] It should be noted that the term "nanobody" is a registered trademark of Ablynx NV and is therefore also referred to as Nanobody® and / or Nanobodies®. Nanobodies typically each have three CDRs, designated CDR1, CDR2, and CDR3. Additionally, nanobodies typically contain three or four frame regions (FRs; FR1, FR2, FR3, and optionally FR4). The nanobodies described herein may be derived from camel, dromedary camel, llama, or alpaca HCAbs. In some specific instances, the nanobodies according to this disclosure are derived from alpaca HCAbs.

[0374] In one embodiment, the binding protein described herein comprises a frame region derived from an alpaca. In one embodiment, the binding protein described herein comprises a frame region derived from a camel. In one embodiment, the binding protein described herein comprises a frame region derived from a llama. In one embodiment, the binding protein described herein comprises a frame region derived from a shark. In one embodiment, the binding protein described herein comprises a modified form of the frame region of a binding protein derived from an alpaca. In one embodiment, the binding protein described herein comprises a modified form of the frame region of a binding protein derived from a camel. In one embodiment, the binding protein described herein comprises a modified form of the frame region of a binding protein derived from a llama. In one embodiment, the binding protein described herein comprises a modified form of the frame region of a binding protein derived from a shark. In one embodiment, the binding protein described herein comprises a humanized form of the frame region of a binding protein derived from an alpaca. In one embodiment, the binding protein described herein comprises a humanized form of the frame region of a binding protein derived from a camel. In one embodiment, the binding protein described herein comprises a humanized form of the frame region of a binding protein derived from a llama. In one embodiment, the binding protein described herein comprises a humanized form of the frame region of a binding protein derived from a shark.

[0375] Humanized binding protein

[0376] As used herein, the term "humanized binding protein" or "humanized" should be understood to refer to a binding protein containing a human-like variable region comprising a CDR from a binding protein of a non-human species (e.g., camelids), which is either transplanted onto or inserted into a FR from a human binding protein (this type of binding protein is also referred to as a "CDR-transplanted binding protein"). Humanized binding proteins also include binding proteins in which one or more residues of a human protein are modified by substitution of one or more amino acids and / or one or more FR residues of a human binding protein are replaced by corresponding non-human residues. Humanized binding proteins may also contain residues that are not present in either human or non-human binding proteins. Additional regions of the binding protein (e.g., the Fc region) are typically human. Humanization can be performed using methods known in the art, such as US5225539, US6054297, US7566771, or US5585089. The term "humanized binding protein" also encompasses hyperhumanized binding proteins, such as those described in US7732578. In one embodiment, the binding protein is a humanized nanobody, such as those described in Vincke et al. 2009.

[0377] Deimmunization binding protein

[0378] As used herein, the term “deimmunizing binding protein” should be understood to mean a binding protein in which one or more epitopes (e.g., B-cell epitopes or T-cell epitopes) have been removed (i.e., mutated) to reduce the likelihood that the subject will generate an immune response against an antibody or protein (e.g., as described in WO2000 / 34317 and WO2004 / 108158). Those skilled in the art will understand that the nanobodies described herein are not “deimmunizing” because they naturally lack both B-cell and T-cell epitopes.

[0379] Germination-binding protein

[0380] As described above, a "germination-binding protein" originates from or corresponds to a sequence from a human or camelid (e.g., a germline or somatic cell), which may contain amino acid residues not encoded by the host species. For example, in some cases, mutations may be introduced, for instance, through affinity maturation or as a result of using a synthetic library not encoded by the species from which the sequence originally originated. In some embodiments, a "germination-binding protein" is a camelid-binding protein in which one or more amino acids in the chain not encoded by a camelid species are replaced or substituted with amino acids from a germline species.

[0381] Modified binding protein

[0382] For a variety of reasons, including, for example, aiding in purification and / or processing during manufacturing, direct detection, indirect detection, and modification stability, binding protein sequences can be modified by adding sequences and portions.

[0383] In one implementation, the binding protein is modified to include one or more of a tag, adapter, radioactive nucleotide, toxin, or another protein (e.g., the Fc region).

[0384] In one embodiment, the binding protein described herein comprises a tag. Those skilled in the art will understand that binding proteins are typically fused with certain tags for detection and / or purification purposes, for example, as described in Muyldermans (2013). In one embodiment, the tag is selected from one or more of the following: polyhistidine, FLAG, antibody epitope tag, c-myc, hemagglutinin, headlock, C-tag, ALFA tag, Avi, GST, maltose-binding protein (MBP). In one embodiment, the binding protein is fused with a polyhistidine tag. In one embodiment, the binding protein is fused with a FLAG tag. In one embodiment, the binding protein is fused with an antibody epitope tag. In one embodiment, the binding protein is fused with a c-myc tag. In one embodiment, the binding protein is fused with a hemagglutinin tag. In one embodiment, the binding protein is fused with a headlock tag. In one embodiment, the binding protein is fused with a C-tag. In one embodiment, the binding protein is fused with an ALFA tag. In one embodiment, the binding protein is fused with an Avi tag. In one embodiment, the binding protein is fused with a GST tag. In one embodiment, the binding protein is fused with an MBP tag. In one implementation, the tag can be cleaved from the binding protein.

[0385] In one embodiment, the binding protein comprises a detectable label, for example, as described in Muyldermans (2013). In one embodiment, the detectable label is directly detectable. In another embodiment, the detectable label is indirectly detectable. Some examples of detectable labels include metal labels, magnetic labels, beads, fluorescent labels, chemical labels, radioactive nucleotides, colored particles, quantum dots, fluorescent latex particles, carbon nanoparticles, chemiluminescence-based labels, liposome-based probes, Raman-active tags, and protein labels (Song et al., 2008; Nuntawong et al., 2022; Muyldermans, 2013). In one embodiment, the detectable label can be detected via a smartphone (Zangheri et al., 2015).

[0386] In one implementation, the detectable marker is Estapor® colored microspheres.

[0387] In one embodiment, the detectable label is selected from one or more of the following: red intense microspheres (Merck, e.g., catalog numbers FR180380637 and FR180380638), cellulose nanobeads, latex beads, alkaline phosphatase, horseradish peroxidase, colloidal gold, gold nanoshells, europium, fluorescent labels, and luminescent labels. In one embodiment, the detectable label is red intense microspheres. In one embodiment, the detectable label is cellulose nanobeads. In one embodiment, the detectable label is latex beads. In one embodiment, the detectable label is alkaline phosphatase. In one embodiment, the detectable label is horseradish peroxidase. In one embodiment, the detectable label is colloidal gold. In one embodiment, the detectable label is gold nanoshells. In one embodiment, the detectable label is europium. In one embodiment, the detectable label is red fluorescent protein. In one embodiment, the detectable label is green fluorescent protein. In one embodiment, the detectable label is estapor-blue. In one embodiment, the detectable label is an estapor-blue conjugate.

[0388] In one embodiment, the fluorescent label is selected from, but not limited to: green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), fluorescein (FITC), alexa fluorescein, 5,6-carboxymethyl fluorescein, texas red, nitrobenzene-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansanoyl chloride, rhodamine, 4'-6-diamidinyl-2-phenylindole (DAPI), and cyanine dyes Cy3, Cy3.5, Cy5, and Cy5.5 and Cy7, luciferin (5-carboxyluciferin-N-hydroxysuccinimide ester) and rhodamine (5,6-tetramethylrhodamine), Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, mNeonGreen, mUKG, AcGFP, ZsGreen, Cloverm Sapphire, T-Sapphire, Enhanced Blue Fluorescent Protein (EBFP), EBFP2, Azurite, TagBFP, mTagBFP, mKalama1, Cyan Fluorescent Protein (CFP), mCFP, Enhanced Cyan Fluorescent Protein (ECFP), mECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Super Yellow Fluorescent Protein (Super yellow fluorescent protein) protein, SYFP), Topaz, Venus, Citrine, mCitrine, YPet, TagYFP, TurboYFP, PhiYFP, ZsYellow1, mBanana, KusabiraOrange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, Red Fluorescent Protein (RFP), TurboRFP, TurboFP602, TurboFP635, Tag ref Fluorescent Protein (RFP), TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine, mKeima-Red, mRuby, mRuby2, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, mK ate2, mKate (TagFP635), HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, mNeptune, NirFP, Sirius, TagRFP657, AQ143, Kaede, KikGR1, PX-CFP2, mEos2, IrisFP, mEOS3.2. PSmOrange, PAGFP, Dronpa, allophycocyanin, GFPuv, R-phycoerythrin (RPE), peridininChlorophyll (PerCP), P3, Katusha, B-phycoerythrin (BPE), mKO, and J-Red. In one embodiment, the fluorescent protein is RFP. In another embodiment, the fluorescent protein is GFP. The maximum absorption and emission values ​​of these fluorescent agents are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm; 672 nm), Cy5.5 (682 nm; 703 nm), and Cy7 (755 nm; 778 nm).

[0389] In one embodiment, the luminescent marker is selected from: jellyfish luminescent protein, firefly luciferase, kidney luciferase, Gaussian luciferase, bacterial luciferase, and nanoluc.

[0390] In one embodiment, the magnetic marker is a magnetic or paramagnetic compound, such as iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferri-chromium-cobalt, nickel-ferri, cobalt-platinum, or strontium ferrite.

[0391] In one embodiment, the binding protein comprises one or more adapters. Examples of such adapter sequences are described in Beirnaert et al. 2017, Sabourin et al. 2007, Yang and Gruebele, 2006, Dipti et al. 2006, Anandarao et al. 2006, Wyatt et al. 1995, Bellamy-McIntyre et al. 2007, Arai et al. 2004, and Bahrami et al. 2007. The adapter can link the binding protein to another binding protein. The adapter can link the binding protein to another binding protein that detects the same antigen. The adapter can link the binding protein to different proteins. In one embodiment, the adapter comprises a sequence selected from one or more of SEQ ID NO: 65 to SEQ ID NO: 77.

[0392] In one embodiment, the binding protein is modified to contain a radionuclide. A variety of radionuclides are available. Some examples include, but are not limited to, low-energy radionuclides (e.g., suitable for diagnostic purposes), such as... 13 C 15 N、 2 H, 125 I, 123 I, 99Tc, 43 K, 52 Fe、 67 Ga、 68 Ga、 111 In, etc. For example, radionuclides are radioactive nuclides that emit gamma rays, photons, or positrons, and whose half-lives are suitable for remaining effective or detectable after the time elapsed between application and localization to the imaging site. This disclosure also covers high-energy radionuclides (e.g., for therapeutic purposes), such as... 125 I, 131 I, 123 I, 111 In、 105 Rh、 153 Sm、 67 Cu、 67 Ga、 166 Ho、 177 Lu、 186 Re and 188 Re. These isotopes typically produce high-energy alpha or beta particles with short path lengths. Such radionuclides kill cells very close to them, such as proliferative cells to which the conjugate has already attached or entered. They have little or no effect on non-localized cells and are essentially non-immunogenic. Alternatively, high-energy isotopes can be produced by thermal irradiation of other stable isotopes, for example in boron neutron capture therapy (Guan et al., 1998).

[0393] In one embodiment, the binding protein is modified to include or react with a chemiluminescent label (e.g., as described in Cabello et al., 2023). In one embodiment, the label is a horseradish peroxidase agent. In one embodiment, the chemiluminescent label is a label detectable by a smartphone (Zangheri et al., 2015).

[0394] In one embodiment, the binding protein of this disclosure is modified to include quantum dots, for example, as described in Mousavi et al., 2023. In one embodiment, the quantum dots are selected from: high-brightness multiple quantum dots (M–QD–SNs) embedded in silica-encapsulated nanoparticles, Cde / ZNS, CdSe / ZnS QDs, Qdot, CdSe / ZnS, Cu:Zu-In-S / ZnS, CdTe QDs, and CdTe. In one embodiment, the quantum dots are M-QD-SNs, for example, as described in Kim et al., 2021.

[0395] In one embodiment, the binding protein of the present invention is fused to or conjugated with a protein. Such a protein is used for one or more of the following purposes: enhancing expression, promoting purification, increasing size, solubility, enhancing binding to or retention on a solid support, and the performance characteristics of the binding protein (e.g., antigen binding).

[0396] In one embodiment, the protein is selected from: the Fc region or a fragment thereof, ferritin, maltose-binding protein (MBP), leucine zipper, glutathione S-transferase (GST), keyhole hemocyanin (KLH), albumin, cyclic protein, FKBP, calcineurin, CyPFAS, GyrB, neutral avidin, avidin, and streptavidin. In one embodiment, the protein is the Fc region. In one embodiment, the protein is a fragment of the Fc region. In one embodiment, the protein is maltose-binding protein (MBP). In one embodiment, the protein is a leucine zipper. In one embodiment, the protein is glutathione S-transferase (GST). In one embodiment, the protein is keyhole hemocyanin (KLH). In one embodiment, the protein is albumin. In one embodiment, the protein is a cyclic protein. In one embodiment, the protein is FKBP. In one embodiment, the protein is calcineurin. In one embodiment, the protein is CyPFAS. In one embodiment, the protein is GyrB. In one embodiment, the protein is neutral avidin. In one embodiment, the protein is avidin. In one implementation, the protein is streptavidin.

[0397] In one implementation, the Fc region or a fragment thereof increases the binding rate of the binding protein to ALT1.

[0398] In one implementation, the Fc region or a fragment thereof reduces the binding rate of the binding protein to ALT1.

[0399] In one implementation, the Fc region or a fragment thereof enhances the affinity of ALT1 for binding proteins.

[0400] In one embodiment, the binding protein is a nanobody fused to the Fc region or a fragment thereof.

[0401] In one embodiment, the protein forms a dimer upon expression (e.g., a dimer formed between two Fc regions (Fc region-Fc region)). In one embodiment, the protein forms a trimer upon expression. In one embodiment, the protein forms a tetramer upon expression (e.g., streptavidin).

[0402] Some examples of dimers are described in Dang et al. (2022). In one embodiment, the protein forms a homodimer upon expression. In one embodiment, the protein forms a heterodimer upon expression. In one embodiment, the protein is selected from: Fc region, ferritin, maltose-binding protein (MBP), leucine zipper, glutathione S-transferase (GST), keyhole hemocyanin (KLH), albumin, cyclic protein, FKBP, calcineurin, CyPFAS, GyrB, neutral avidin, avidin, or other such proteins. In one embodiment, the dimer is selected from: Fc region-Fc region, ferritin-ferritin, MBP-MBP, leucine zipper-leucine zipper, GST-GST, KLH-KLH, albumin-albumin, cyclic protein-cyclic protein, cyclic protein-calcineurin, FKBP-calcineurin, FKBP-CyPFas, FKBP-FKBP, and GyrB-GyrB. In one implementation, the dimer is an Fc region-Fc region.

[0403] In one embodiment, the binding protein is fused to a dimerizable protein, wherein, upon expression, the protein dimers to form a divalent, tetravalent, or multivalent binding protein. Figure 41 C provides examples of divalent binding proteins that fuse with proteins that form dimers. When dimers are formed, tetravalent binding proteins are produced.

[0404] The binding protein of this disclosure can be modified to include additional non-protein moieties known in the art and readily available. For example, suitable moieties for protein derivatization are physiologically acceptable polymers, such as water-soluble polymers. Such polymers can be used to improve stability. Some non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propylene glycol (PPG).

[0405] Bivalent, trivalent, tetravalent or multivalent binding proteins

[0406] In one aspect, the present invention provides bivalent, trivalent, tetravalent, or multivalent binding proteins comprising at least one binding protein or combination thereof as described herein. Such a form may be desirable for enhancing or otherwise modifying the effectiveness of the binding proteins of this disclosure. Advantages of such a form are described, for example, in Conrath et al., 2001 and Sparkles et al., 2018.

[0407] In some embodiments, the binding protein is a bivalent binding protein. The bivalent binding protein used herein comprises two antigen-binding domains. In one embodiment, the bivalent binding protein comprises at least one binding protein as described herein. In one embodiment, the bivalent binding protein comprises two binding proteins as described herein. In one embodiment, the bivalent binding protein is homodivalent. In one embodiment, the bivalent binding protein is heterodivalent.

[0408] In some embodiments, the binding protein is a trivalent binding protein. The trivalent binding protein used herein comprises three antigen-binding domains. In one embodiment, the trivalent binding protein comprises at least one binding protein as described herein. In one embodiment, the trivalent binding protein comprises two binding proteins as described herein. In one embodiment, the trivalent binding protein comprises three binding proteins as described herein. In one embodiment, the trivalent binding protein is homotrivalent. In one embodiment, the trivalent binding protein is heterotrivalent (comprising two or three different binding proteins).

[0409] In some embodiments, the binding protein is a tetravalent binding protein. The tetravalent binding protein used herein comprises four antigen-binding domains. In one embodiment, the tetravalent binding protein comprises at least one binding protein as described herein. In one embodiment, the tetravalent binding protein comprises two binding proteins as described herein. In one embodiment, the tetravalent binding protein comprises three binding proteins as described herein. In one embodiment, the tetravalent binding protein comprises four binding proteins as described herein. In one embodiment, the tetravalent binding protein is homotetravalent. In one embodiment, the tetravalent binding protein is heterotetravalent (comprising two, three, or four different binding proteins). In one embodiment, the tetravalent binding protein comprises an Fc region (e.g., as shown in the image). Figure 41 (As shown in C). As used herein, “Fc region” refers to a monomeric antibody tail region that interacts with a cell surface receptor called an Fc receptor. In one embodiment, the Fc region comprises the sequence shown in SEQ ID NO: 78 or a sequence having at least 70% identity with it, or a fragment thereof. In one embodiment, the Fc region comprises the sequence shown in SEQ ID NO: 78. In one embodiment, the Fc region comprises the nucleotide sequence shown in SEQ ID NO: 106 or a sequence having at least 70% identity with it, or a fragment thereof. In one embodiment, the Fc region comprises the nucleotide sequence shown in SEQ ID NO: 106. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is a human IgG Fc region.

[0410] In some embodiments, the binding protein is a multivalent binding protein. Multivalent binding proteins as used herein comprise five or more antigen-binding domains. In one embodiment, the multivalent binding protein comprises at least one binding protein as described herein. In one embodiment, the multivalent binding protein comprises two binding proteins as described herein. In one embodiment, the multivalent binding protein comprises three binding proteins as described herein. In one embodiment, the multivalent binding protein comprises four binding proteins as described herein. In one embodiment, the multivalent binding protein is a homopolyvalent binding protein. In one embodiment, the multivalent binding protein is a heteropolyvalent binding protein (comprising two, three, or four different binding proteins).

[0411] In one implementation, the bivalent, trivalent, tetravalent or multivalent binding proteins described herein have the ability to bind to two epitopes on the same molecule (bi-complementary site type) (Oliveira et al., 2013).

[0412] In one implementation, the bivalent, trivalent, tetravalent or multivalent binding proteins described herein have the ability to bind to multiple epitopes on the same molecule (multiple complementary sites) (Palomo et al., 2016).

[0413] In one implementation, the bivalent form of the binding protein has a higher affinity than the monomeric form.

[0414] In one implementation, the trivalent form of the binding protein has a higher affinity than the monomeric form.

[0415] In one implementation, the tetravalent form of the binding protein has a higher affinity than the monomeric form.

[0416] In one implementation, the multivalent form of the binding protein has a higher affinity than the monomeric form.

[0417] In one embodiment, the divalent, trivalent, tetravalent, or multivalent binding protein comprises a binding protein that contains the amino acid sequence shown in SEQ ID NO: 1 (C8), or a sequence having at least 71% identity with it, or its humanized, deimmunized, or germline form.

[0418] In one embodiment, the divalent, trivalent, tetravalent, or multivalent binding protein comprises a binding protein that contains the amino acid sequence shown in SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or its humanized, deimmunized, or germline form.

[0419] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises (i) a binding protein comprising the amino acid sequence shown in SEQ ID NO: 1 (C8), or a sequence having at least 71% identity with it, or a humanized, deimmunized, or germline form thereof; and (ii) a binding protein comprising the amino acid sequence shown in SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or a humanized, deimmunized, or germline form thereof.

[0420] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises a binding protein comprising an amino acid sequence selected from SEQ ID NO: 13 to SEQ ID NO: 56, or a sequence having at least 76% identity with it, or its humanized, deimmunized, or germline form.

[0421] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises a sequence selected from the following: C3C (SEQ ID NO: 57), C4C (SEQ ID NO: 58), G3G (SEQ ID NO: 59), G4G (SEQ ID NO: 60), C3G (SEQ ID NO: 61), C4G (SEQ ID NO: 62), G3C (SEQ ID NO: 63), and G4C (SEQ ID NO: 634) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0422] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence C3C (SEQ ID NO:57) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0423] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence C4C (SEQ ID NO:58) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0424] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence G3G (SEQ ID NO:59) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0425] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence G4G (SEQ ID NO:60) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0426] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence C3G (SEQ ID NO:61) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0427] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence C4G (SEQ ID NO:62) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0428] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence G3C (SEQ ID NO:63) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0429] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the sequence G4C (SEQ ID NO:64) or a sequence having at least 71%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0430] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is C3C (SEQ ID NO: 57). In one embodiment, the bivalent, trivalent, or multivalent binding protein is C4C (SEQ ID NO: 58). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is G3G (SEQ ID NO: 59). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is G4G (SEQ ID NO: 60). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is C3G (SEQ ID NO: 61). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is C4G (SEQ ID NO: 62). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is G3C (SEQ ID NO: 63). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein is G4C (SEQ ID NO: 64).

[0431] To generate bivalent, trivalent, tetravalent, or multivalent binding proteins as described herein, the binding proteins described herein may be linked by any method known to those skilled in the art, such as those described in Conrath et al., 2001 and Sparkles et al., 2018. In one embodiment, the binding proteins are linked by a linker (e.g., a peptide linker) or more than one linker to form bivalent, trivalent, tetravalent, or multivalent binding proteins.

[0432] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding proteins provided herein comprise at least two binding proteins as described herein linked by amino acid linker sequences of different lengths. In one embodiment, the two binding proteins are different binding proteins. In one embodiment, at least two binding proteins are the same binding protein. Examples of such linkers are as described above.

[0433] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises a linker selected from one or more of SEQ ID NO: 65 to SEQ ID NO: 77.

[0434] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGAGG (SEQ ID NO: 65). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGG (SEQ ID NO: 66). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGGGGG (SEQ ID NO: 67). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGGG (SEQ ID NO: 68). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGGGG (SEQ ID NO: 69). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGGGGGGG (SEQ ID NO: 70). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGSSG (SEQ ID NO: 71). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGG (SEQ ID NO: 72). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GAG ​​(SEQ ID NO: 73). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGAGGS (SEQ ID NO: 74). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGSGGGGSGGGGS (SEQ ID NO: 75). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 76). In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the adaptor sequence SGGSG (SEQ ID NO: 77).

[0435] In one embodiment, the tetravalent binding protein comprises an amino acid sequence selected from the following: C4C-Fc (SEQ ID NO: 79), G4G-Fc (SEQ ID NO: 80), C4G-Fc (SEQ ID NO: 81), G4C-Fc (SEQ ID NO: 82), C3G-Fc (SEQ ID NO: 159), G3C-Fc (SEQ ID NO: 161), C3C-Fc (SEQ ID NO: 155), and G3G-Fc (SEQ ID NO: 157), or a sequence having at least 76% identity with the above. In one embodiment, the tetravalent binding protein comprises the amino acid sequence C4C-Fc (SEQ ID NO: 79), or a sequence having at least 76% identity with the above. In one embodiment, the tetravalent binding protein comprises the amino acid sequence G4G-Fc (SEQ ID NO: 80), or a sequence having at least 76% identity with the above. In one embodiment, the tetravalent binding protein comprises the amino acid sequence C4G-Fc (SEQ ID NO: 81) or a sequence having at least 76% identity with it. In one embodiment, the tetravalent binding protein comprises the amino acid sequence G4C-Fc (SEQ ID NO: 82) or a sequence having at least 76% identity with it. In one embodiment, the tetravalent binding protein comprises the amino acid sequence C3G-Fc (SEQ ID NO: 159) or a sequence having at least 76% identity with it. In one embodiment, the tetravalent binding protein comprises the amino acid sequence G3C-Fc (SEQ ID NO: 161) or a sequence having at least 76% identity with it. In one embodiment, the tetravalent binding protein comprises the amino acid sequence C3C-Fc (SEQ ID NO: 155) or a sequence having at least 76% identity with it. In one embodiment, the tetravalent binding protein comprises the amino acid sequence G3G-Fc (SEQ ID NO: 157) or a sequence having at least 76% identity with it.

[0436] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises a CDR sequence selected from the following:

[0437] a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3;

[0438] b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3;

[0439] c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2, and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3;

[0440] d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and

[0441] e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3;

[0442] Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

[0443] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the following amino acid sequences: GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2, and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3; and any one or more of these CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0444] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the following amino acid sequences: GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, and ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3; VSVHYNKW (SEQ ID NO: 121) as light chain CDR1; GAS as light chain CDR2; and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3; and any one or more of these CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0445] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the following amino acid sequences: GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2, and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3; and any one or more of these CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0446] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the following amino acid sequences: GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and any one or more of these CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0447] In one embodiment, the bivalent, trivalent, tetravalent, or multivalent binding protein comprises the following amino acid sequences: GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; and any one or more of these CDRs have 0, 1, 2, or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

[0448] Production of binding proteins

[0449] Those skilled in the art will understand that the binding proteins described herein can be produced by a variety of methods, including cell-based expression systems and cell-free expression systems. In one example, the binding proteins of this disclosure are produced by culturing cells under conditions sufficient to produce the binding proteins described herein.

[0450] Those skilled in the art will understand that antibodies as described herein can be produced using recombinant genetic techniques (recombinant protein production).

[0451] In some implementations, a nucleic acid encoding a binding protein as described herein is placed in one or more expression constructs, such as expression vectors, and then transfected into host cells, such as bacterial cells, yeast cells, insect cells, or mammalian cells.

[0452] Exemplary bacterial cells include *Escherichia coli* (E. coli). Exemplary mammalian cells include simian COS cells, human embryonic kidney (HEK) cells and derivatives thereof, Chinese hamster ovary (CHO) cells, HeLa, human embryonic kidney 293 cells (HEK293), human osteosarcoma U2OS, A549, HT1080, Cath.-a differentiated cells (CAD), P19, NIH 3T3, L929, N2a, Hep G2, or myeloma cells that do not additionally produce immunoglobulins. Molecular cloning techniques for achieving these purposes are known in the art and are described, for example, in *Ausubel* or *Sambrook*. A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for generating recombinant antibodies are also known in the art. See US4816567; US7923221 and US7022500.

[0453] In some implementations, the nucleic acid is operatively linked to a promoter. The term "promoter" as used herein should be understood in its broadest context and includes transcriptional regulatory sequences of genomic genes, including TATA boxes or initiation elements required for precise transcription initiation, with or without other regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that, for example, respond to developmental and / or external stimuli or alter nucleic acid expression in a tissue-specific manner. In the context of this document, the term "promoter" is also used to describe recombinant, synthetic, or fusion nucleic acids or derivatives that confer, activate, or enhance the expression of a nucleic acid operatively linked to them. Exemplary promoters may contain one or more additional copies of specific regulatory elements for further enhancing the expression of said nucleic acid and / or altering the spatial and / or temporal expression of said nucleic acid. The term "operatively linked" as used herein means that the promoter is positioned relative to the nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0454] It should be understood that the expression of nucleic acids in a host organism or host cell can be improved by replacing the nucleotide sequence encoding a specific amino acid (i.e., a codon) with another codon that is better expressed in the host organism (i.e., codon optimization). One reason for this effect is the fact that different organisms exhibit preferences for different codons. In some embodiments, nucleic acids such as those disclosed herein are modified or optimized such that the nucleotide sequence reflects the codon preference for a specific host cell, preferably a mammalian or bacterial cell. In one embodiment, the nucleic acid is codon-optimized for a mammalian cell culture. In another embodiment, the nucleic acid is codon-optimized for a bacterial cell culture. Methods for codon optimization are apparent to those skilled in the art. For example, tools for codon optimization include, for example, GeneArtGeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).

[0455] In some implementations, the nucleic acid will contain an N-terminal sequence to aid expression in host cells. For example, for expression in bacteria such as *E. coli*, the nucleic acid may contain an N-terminal pelB signal peptide for periplasmic expression (e.g., as shown in SEQ ID NO: 107). For example, for expression in mammalian cells, the nucleic acid contains an N-terminal tPA leader sequence (e.g., as shown in SEQ ID NO: 108).

[0456] In one embodiment, the nucleic acid comprises a nucleotide sequence selected from SEQ ID NO: 87 to SEQ ID NO: 106, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 87, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 88, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 89, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 90, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 91, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 92, or an optimized form of the codon thereof, or a sequence having at least 70% identity with the nucleotide sequence. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 93 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 94 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 95 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 96 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 97 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 98 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 99 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 100 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 101 or an optimized codon form thereof, or a sequence having at least 70% identity with it. In another embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 102 or an optimized codon form thereof, or a sequence having at least 70% identity with it.In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 103 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 104 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 105 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 106 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 144 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 145 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 146 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 147 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 148 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 149 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 150 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 151 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 152 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 153 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 154 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 156 or its codon-optimized form, or a sequence having at least 70% identity with it. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 158 or its codon-optimized form, or a sequence having at least 70% identity with it.In one embodiment, the nucleic acid comprises a nucleotide sequence SEQ ID NO: 160 or an optimized codon form thereof or a sequence having at least 70% identity with it.

[0457] Isolation of binding proteins

[0458] The binding proteins of this disclosure can be isolated or purified using any method known to those skilled in the art.

[0459] When using recombinant technology, the binding proteins of this disclosure can be generated intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the protein is generated intracellularly, as a first step, particulate debris, which is host cell or lysate fragments, is removed, for example, by centrifugation or ultrafiltration. In the case of protein secretion into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentrator filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis and antibiotics can be included to prevent the growth of foreign contaminants. The supernatant can also be used directly for purification.

[0460] Binding proteins prepared from cells or supernatants can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination thereof. These methods are known in the art and are described, for example, in WO99 / 57134 or Zola (1987).

[0461] Those skilled in the art will also recognize that the binding proteins of this disclosure can be modified to include tags to facilitate purification or detection (examples of which are described above). The resulting protein is then purified using methods known in the art, such as affinity purification. For example, proteins containing a six-his tag are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) specifically bound to a six-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or further, ligands or antibodies bound to the tag are used in affinity purification methods.

[0462] Reagent test kit

[0463] In one aspect, the present invention provides a kit or combination of kits comprising binding proteins as described herein or bivalent, trivalent, tetravalent or multivalent binding proteins as described herein.

[0464] In one implementation, the kit or combination package contains strips, chips, or boxes for use on an on-the-go device.

[0465] In one implementation, the kit or combination package is selected from: lateral flow assay kits, ELISA kits, and Luminex® assay kits.

[0466] In one implementation, the kit or combination pack is designed for use by healthcare practitioners.

[0467] In one implementation, the kit or combination kit is a self-test kit designed for home use.

[0468] In one embodiment, the kit or combination pack is contained within an all-in-one device for home or healthcare practitioner use. In one embodiment, the kit or plate comprises a blister pack with a built-in lancet and running buffer. No additional consumables are provided for the test. The device is designed for home use. For certain types of treatment (medications, antibody therapy), signs of hepatotoxicity have been described, which may require monitoring of ALT levels daily, multiple times per week, or weekly.

[0469] Measurement

[0470] The methods and uses described herein are suitable for in vitro use and can be performed in assays known to those skilled in the art, including as immunoassays, chromatographic assays or homogeneous assays.

[0471] As used herein, “immunoassay” refers to an assay that uses immunoglobulins or portions thereof, capable of detecting and quantifying a desired biomarker, such as ALT. An immunoassay can be one of a range of immunoassay forms known to those skilled in the art. A wide range of immunoassay techniques are available, such as those described in Wild D. “The Immunoassay Handbook”, Nature Publishing Group, 4th edition, 2013, and subsequent innovations.

[0472] In one embodiment, an immunoassay detects total ALT (ALT1 and ALT2). In another embodiment, an immunoassay detects ALT1. In yet another embodiment, an immunoassay detects total ALT at a level comparable to the current gold standard ALT measurement (the enzymatic activity of ALT (ALT1 and ALT2) in serum / plasma (L-alanine + 2-oxoglutarate is converted to pyruvate + L-glutamate by ALT)). The enzyme assay is a coupled assay. That is, pyruvate, the product of the ALT reaction, becomes the substrate for a second reaction catalyzed by lactate dehydrogenase (LDH). This second reaction uses the cofactor NADH to convert it to NAD+. This cofactor can be monitored spectrophotometrically.

[0473] In one embodiment, the immunoassay is selected from: electrochemiluminescence (ELICA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent ELISA, fluorescent immunosorbent assay (FIA), biosensor-based assays, bead-based immunoassays, and particle-based immunoassays (e.g., mesoscale delivery platforms (MSD)). Examples of detectable groups include, but are not limited to: fluorescent dyes, enzymes, epitopes for binding a second binding reagent (e.g., when the second binding reagent / antibody is a mouse antibody, it is detected by fluorescently labeled anti-mouse antibody), members such as antigens or binding pairs, such as biotin. The surface can be a planar surface, such as in the case of a typical grid array (e.g., but not limited to, 96-well plates and planar microarrays); or a non-planar surface, such as a coated bead array technique, in which each “type” of bead is marked with, for example, a fluorescent dye (e.g., the Luminex technique described in U.S. Patent Nos. 6,599,331, 6,592,822, and 6,268,222) or a quantum dot technique (e.g., as described in U.S. Patent No. 6,306,610).

[0474] In one implementation, the immunoassay is performed on an automated platform, such as the Cobas immunoassay analyzer (Roche) or the Architect immunoassay analyzer (Abbott).

[0475] In one implementation, the immunoassay is an auxiliary / monitoring assay used in clinical trials.

[0476] Lateral flow assays, and more recently, non-lateral flow and microfluidics, provide useful settings for bioassays. Such assays can be qualitative, quantitative, or semi-quantitative. In microfluidic devices, a small amount of liquid moves through microchannels created, for example, in a chip or cartridge. A wide range of assay reagents are available, including metal nanoparticles, colored or luminescent materials. Resonance-enhanced adsorption (REA) of bioconjugated metal nanoparticles offers rapid processing times and other advantages. These devices have been combined with barcode technology to identify patients and the analytes being tested. This disclosure covers computer software and hardware for evaluating input data. Point-of-care devices and arrays, as well as high-throughput screening methods, are also considered. In one embodiment, the assay is a point-of-care device. In one embodiment, the point-of-care device includes or is accompanied by a lancet for obtaining a sample as described herein.

[0477] Qualitative assays that provide intermediate or definitive diagnoses require comprehensive thresholds, limits, or windows to allow samples to be scored as either likely to have the condition or not. Instrument readers and software are typically used to process the data and then process it through diagnostic algorithms or decision trees.

[0478] In one implementation, the bead immunoassay is selected from the Luminex LabMAP assay and the Bio-Plex multiplex immunoassay (Bio-Rad). In the bead immunoassay, the Luminex LabMAP system can be utilized. The LabMAP system comprises polystyrene microspheres internally stained with two fluorescent dyes with different spectra. Using precise ratios of these fluorescent dyes, an array of different microsphere groups with specific spectral addresses is created. Each microsphere group may have different reactants on its surface. Because microsphere groups can be distinguished by their spectral addresses, they can be combined, allowing for the simultaneous measurement of up to 100 different analytes in a single reaction vessel. A third fluorescent dye coupled to a reporter molecule quantifies the biomolecular interactions occurring on the microsphere surface. As the microspheres pass through two separate lasers in the Luminex analyzer, they are individually detected (interrogate) in a rapidly flowing fluid stream. High-speed digital signal processing classifies the microspheres based on their spectral addresses and quantifies the surface reactions of each sample within seconds.

[0479] In one embodiment, the assay is a homogeneous assay, meaning a form of assay that allows measurement via a simple mixing and reading procedure without requiring sample preparation by separation or washing. Such an assay does not include an immunosorbent solid-phase step. In one embodiment, the homogeneous assay is time-resolved Förster resonance energy transfer (FRET).

[0480] In one embodiment, the assay is based on methods such as flow cytometry, bead arrays, lateral flow, cassettes, microfluidics, or immunochromatography. In one embodiment, the assay is a point-of-care assay. In one embodiment, the point-of-care reader is an Axxin AX-2X reader, or an equivalent or modified device. For example, the device can be modified to include an LED and a filter of appropriate wavelength for the assay.

[0481] In one embodiment, the measurement is a biosensor measurement. In another embodiment, the measurement is a plasmon resonance measurement or a biolayer interferometry (BLi) measurement, for example, as described in Capelli et al. (2013) and Tokel et al. (2014). In some embodiments, the measurement allows for label-free detection of ALT1.

[0482] Capture and Detection

[0483] Those skilled in the art will understand that the assay described herein may use one binding protein as described herein, or more than one binding protein as described herein. In one embodiment, the assay described herein may include the use of two different binding proteins as described herein. In one embodiment, the assay described herein may include the use of a binding agent as described herein with a rabbit polyclonal ALT-1 antibody.

[0484] In one aspect, the invention provides a assay comprising: a solid support containing binding proteins as described herein, and / or bivalent, trivalent, tetravalent, or multivalent binding proteins as described herein, immobilized on the solid support.

[0485] In one aspect, the present invention provides an immunoassay comprising: a solid support containing binding proteins immobilized on the solid support.

[0486] In one aspect, the present invention provides an immunoassay comprising a divalent, trivalent, tetravalent, or multivalent binding protein, as described herein, immobilized on a solid support.

[0487] In one embodiment, the assay as described herein includes a binding protein for detecting ALT (detector or detector-binding protein) and a binding protein for capturing ALT (capture-binding protein). As used herein, "detector-binding protein" comprises a detectable marker as described herein that can be used to directly or indirectly measure the level of ALT in a sample. As used herein, "capture-binding protein" is used to capture ALT in a sample. In one embodiment, the capture-binding protein binds ALT before binding to the detector-binding protein. In one embodiment, the capture-binding protein binds ALT after binding to the detector-binding protein. In one embodiment, the capture-binding protein binds / immobilizes to a solid support. The term "immobilization" and variations thereof should be understood to refer to various methods and techniques for immobilizing a protein onto a particular matrix, such as those described in WO99 / 56126 or WO02 / 26292. For example, immobilization can be used to stabilize a protein so that its activity is not diminished or adversely modified by biological, chemical, or physical exposure, particularly during storage or in single-batch use. In one embodiment, the capture-binding protein binds to a solid support in the presence of one or more excipients. In one embodiment, the excipient is selected from one or more of the following: sucrose, bovine serum albumin (BSA), immunoglobulin, trehalose, casein, lactose, galactose, and detergents (e.g., Tween). In one embodiment, the excipient is sucrose. In one embodiment, the excipient is BSA. In one embodiment, the excipient is sucrose and BSA. In one embodiment, the detection and capture binding protein is a nanobody as described herein. In one embodiment, the detection and capture binding protein is a bivalent binding protein as described herein. In one embodiment, the detection and capture binding protein is a trivalent binding protein as described herein. In one embodiment, the detection and capture binding protein is a tetravalent binding protein as described herein. In one embodiment, the detection and capture binding protein is a multivalent binding protein as described herein. In one embodiment, the detection and capture binding protein is a monoclonal antibody as described herein. In one embodiment, the detection and capture binding protein is a rabbit monoclonal antibody as described herein.

[0488] In one embodiment, the detection binding protein is a nanobody as described herein. In one embodiment, the detection binding protein is a bivalent binding protein as described herein. In one embodiment, the detection binding protein is a trivalent binding protein as described herein. In one embodiment, the detection binding protein is a tetravalent binding protein as described herein. In one embodiment, the detection binding protein is a multivalent binding protein as described herein. In one embodiment, the detection binding protein is a monoclonal antibody as described herein. In one embodiment, the detection binding protein is a rabbit monoclonal antibody as described herein.

[0489] In one embodiment, the capture-binding protein is a nanobody as described herein. In one embodiment, the capture-binding protein is a bivalent binding protein as described herein. In one embodiment, the capture-binding protein is a trivalent binding protein as described herein. In one embodiment, the capture-binding protein is a tetravalent binding protein as described herein. In one embodiment, the capture binding protein is a multivalent binding protein as described herein. In one embodiment, the capture-binding protein is a monoclonal antibody as described herein. In one embodiment, the capture-binding protein is a rabbit monoclonal antibody as described herein.

[0490] In one embodiment, the detection binding protein is a rabbit polyclonal ALT antibody. In another embodiment, the capture binding protein is a rabbit polyclonal ALT antibody.

[0491] Examples of binding proteins that can be combined for capture and detection include Figure 101 As shown in the image.

[0492] In one embodiment, the capture-binding protein is a nanobody as described herein, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is a nanobody as described herein, and the detector-binding protein is a nanobody as described herein. In one embodiment, the capture-binding protein is a nanobody as described herein, and the detector-binding protein is a rabbit monoclonal antibody as described herein. In one embodiment, the capture-binding protein is a rabbit monoclonal antibody as described herein, and the detector-binding protein is a rabbit monoclonal antibody as described herein. In one embodiment, the capture-binding protein is a rabbit monoclonal antibody as described herein, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is a rabbit monoclonal antibody as described herein, and the detector-binding protein is a nanobody as described herein. In one embodiment, the capture-binding protein is a rabbit polyclonal ALT-1 antibody, and the detector-binding protein is a nanobody as described herein.

[0493] In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is selected from one or more of the following: RmAb1, RmAb2, RmAb3, RmAb4, and RmAb5. In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is RmAb2. In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is RmAb5.

[0494] In one embodiment, the capture-binding protein is RmAb1, and the detector-binding protein is selected from one or more of the following: RmAb3, RmAb4, RmAb5, and C4G-Fc. In one embodiment, the capture-binding protein is RmAb1, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is RmAb1, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is RmAb1, and the detector-binding protein is RmAb5. In one embodiment, the capture-binding protein is RmAb1, and the detector-binding protein is C4G-Fc.

[0495] In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is selected from one or more of the following: RmAb1, RmAb3, RmAb4, RmAb5, and C4G-Fc. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb5. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is C4G-Fc.

[0496] In one embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is selected from RmAb5 and C4G-Fc. In another embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is RmAb5. In yet another embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is C4G-Fc.

[0497] In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is selected from one or more of the following: RmAb3, RmAb4, RmAb5, and C4G-Fc. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb5. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is C4G-Fc.

[0498] In one embodiment, the capture-binding protein is C4C-Fc, and the detector-binding protein is selected from one or more of the following: RmAb1, RmAb4, and RmAb5. In one embodiment, the capture-binding protein is C4C-Fc, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is C4C-Fc, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is C4C-Fc, and the detector-binding protein is RmAb5.

[0499] In one embodiment, the capture-binding protein is C3G-Fc, and the detector-binding protein is selected from one or more of the following: RmAb1, RmAb4, and RmAb5. In one embodiment, the capture-binding protein is C3G-Fc, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is C3G-Fc, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is C3G-Fc, and the detector-binding protein is RmAb5.

[0500] In one embodiment, the capture-binding protein is G3C-Fc, and the detector-binding protein is selected from one or more of the following: RmAb1, RmAb4, and RmAb5. In one embodiment, the capture-binding protein is G3C-Fc, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is G3C-Fc, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is G3C-Fc, and the detector-binding protein is RmAb5.

[0501] In one embodiment, the capture-binding protein is G4C-Fc, and the detector-binding protein is selected from one or more of the following: RmAb1, RmAb4, and RmAb5. In one embodiment, the capture-binding protein is G4C-Fc, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is G4C-Fc, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is G4C-Fc, and the detector-binding protein is RmAb5.

[0502] In one embodiment, the capture-binding protein is C8, and the detector-binding protein is G6. In one embodiment, the capture-binding protein is C8, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is C8, and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is C8, and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is C8, and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is C8, and the detector-binding protein is G3G. In one embodiment, the capture-binding protein is C8, and the detector-binding protein is G4G. In one embodiment, the capture-binding protein is G6, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody.

[0503] In one embodiment, the capture-binding protein is a rabbit polyclonal ALT-1 antibody, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is a rabbit polyclonal ALT-1 antibody, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is a rabbit polyclonal ALT-1 antibody, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is a rabbit polyclonal ALT-1 antibody, and the detector-binding protein is RmAb5.

[0504] In one embodiment, the capture-binding protein is C4G-Fc, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is a C4G-Fc antibody, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is a C4G-Fc antibody, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is a C4G-Fc antibody, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is a C4G-Fc antibody, and the detector-binding protein is RmAb5. In one embodiment, the capture-binding protein is RmAb1, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody.

[0505] In one implementation, the capture-binding protein is RmAb1, and the detector-binding protein is RmAb1.

[0506] In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is RmAb3, and the detector-binding protein is RmAb5.

[0507] In one embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is RmAb4, and the detector-binding protein is RmAb5.

[0508] In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb1. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb3. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb4. In one embodiment, the capture-binding protein is RmAb5, and the detector-binding protein is RmAb5. In one embodiment, the capture-binding protein is G4C, and the detector-binding protein is RmAb1.

[0509] In one embodiment, the capture-binding protein is G4C, and the detector-binding protein is G4C. In another embodiment, the capture-binding protein is G4C, and the detector-binding protein is C3G. In yet another embodiment, the capture-binding protein is G4C, and the detector-binding protein is C4G. In yet another embodiment, the capture-binding protein is G4C, and the detector-binding protein is G3G.

[0510] In one embodiment, the capture-binding protein is C3G, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is C3G, and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is C3G, and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is C3G, and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is C3G, and the detector-binding protein is G3G. In one embodiment, the capture-binding protein is C3G, and the detector-binding protein is G4G.

[0511] In one embodiment, the capture-binding protein is C4G, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody. In one embodiment, the capture-binding protein is C4G, and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is C4G, and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is C4G, and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is C4G, and the detector-binding protein is G3G. In one embodiment, the capture-binding protein is C4G, and the detector-binding protein is G4G. In one embodiment, the capture-binding protein is C3C, and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is C3C, and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is C3C, and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is C3C, and the detector-binding protein is G3G. In one embodiment, the capture-binding protein is C4C, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody.

[0512] In one embodiment, the capture-binding protein is C4C, and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is C4C, and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is C4C, and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is C4C, and the detector-binding protein is G3G. In one embodiment, the capture-binding protein is G3G, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody.

[0513] In one embodiment, the capture-binding protein is G3G, and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is G3G, and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is G3G, and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is G3G, and the detector-binding protein is G3G. In one embodiment, the capture-binding protein is G4G, and the detector-binding protein is a rabbit polyclonal ALT-1 antibody.

[0514] In one embodiment, the capture-binding protein is G4G and the detector-binding protein is G4C. In one embodiment, the capture-binding protein is G4G and the detector-binding protein is C3G. In one embodiment, the capture-binding protein is G4G and the detector-binding protein is C4G. In one embodiment, the capture-binding protein is G4G and the detector-binding protein is G3G.

[0515] Lateral flow measurement

[0516] In one aspect, the present invention provides a lateral flow measurement comprising: a solid support containing binding proteins as described herein, and / or divalent, trivalent, tetravalent or multivalent binding proteins as described herein, immobilized on the solid support.

[0517] In one aspect, the present invention provides a lateral flow measurement comprising: a solid support containing binding proteins immobilized on the solid support.

[0518] In one aspect, the present invention provides a lateral flow measurement comprising a divalent, trivalent, tetravalent, or multivalent binding protein, as described herein, immobilized on a solid support.

[0519] In one implementation, lateral flow measurement provides the detection of ALT1.

[0520] In one aspect, the present invention provides a lateral flow assay comprising: (i) a detector-binding protein coupled to a detectable label; and (ii) a capture-binding protein in a capture region on a solid support, wherein the binding protein in (i) and / or (ii) is a binding protein as described herein, and / or a bivalent, trivalent, tetravalent, or multivalent binding protein as described herein.

[0521] In one embodiment, the lateral flow assay further includes a erythrocyte capture portion. In one embodiment, the erythrocyte capture portion is selected from erythrocyte-binding proteins and a blood retention pad. In one embodiment, the erythrocyte-binding protein is selected from binding proteins targeting the following: blood group glycoprotein B, Lewis, CD238, and CD234. In one embodiment, the erythrocyte-binding protein is blood group glycoprotein B. In one embodiment, the erythrocyte-binding protein is Lewis. In one embodiment, the erythrocyte-binding protein is CD238. In one embodiment, the erythrocyte-binding protein is CD234. In one embodiment, the blood retention pad is selected from one or more of the following: FR1, MDI, Vivid GX, CytoSep1663, CytSep 1660, CytoSep1668, Vivid GR, Vivid GX, Vivid GR, and Vivid GF. In one embodiment, the blood retention pad is FR1. In one embodiment, the blood retention pad is MDI. In one embodiment, the blood retention pad is Vivid GX. In one embodiment, the blood retention pad is CytoSep1663. In one embodiment, the blood retention pad is CytSep1660. In one embodiment, the blood retention pad is CytSep1668. In one embodiment, the blood retention pad is Vivid GR. In one embodiment, the blood retention pad is Vivid GX. In one embodiment, the blood retention pad is Vivid GR. In one embodiment, the blood retention pad is Vivid GF. In one embodiment, the lateral flow assay includes one or more of the following: a lancet, a blood collection unit, and a blister pack containing buffer. In one embodiment, the lateral flow assay includes a lancet. In one embodiment, the lateral flow assay includes a blood collection unit. In one embodiment, the lateral flow assay includes a blister pack containing buffer.

[0522] In one embodiment, the lateral flow device further includes a control line on a solid support containing ALT or ALT epitopes.

[0523] In one embodiment, the binding protein in i) and / or ii) is a binding protein as described herein. In one embodiment, the binding protein in i) and / or ii) is a nanobody as described herein. In one embodiment, the binding protein in i) and / or ii) is a nanobody fusion protein as described herein. In one embodiment, the binding protein in i) and / or ii) is a nanobody fusion protein as described herein. In one embodiment, the binding protein in i) and / or ii) is a bivalent binding protein as described herein. In one embodiment, the binding protein in i) and / or ii) is a trivalent binding protein as described herein. In one embodiment, the binding protein in i) and / or ii) is a tetravalent binding protein as described herein. In one embodiment, the binding protein in i) and / or ii) is a multivalent binding protein as described herein.

[0524] In one embodiment, the binding protein in i) is a binding protein as described herein. In one embodiment, the binding protein in i) is a nanobody as described herein. In one embodiment, the binding protein in i) is a bivalent binding protein as described herein. In one embodiment, the binding protein in i) is a trivalent binding protein as described herein. In one embodiment, the binding protein in i) is a tetravalent binding protein as described herein. In one embodiment, the binding protein in i) is a multivalent binding protein as described herein.

[0525] In one embodiment, the binding protein in ii) is a binding protein as described herein. In one embodiment, the binding protein in ii) is a nanobody as described herein. In one embodiment, the binding protein in ii) is a bivalent binding protein as described herein. In one embodiment, the binding protein in ii) is a trivalent binding protein as described herein. In one embodiment, the binding protein in ii) is a tetravalent binding protein as described herein. In one embodiment, the binding protein in ii) is a multivalent binding protein as described herein.

[0526] In one implementation, the binding protein in i) and / or ii) is selected from: C8, C6, G4C, C3G, CAG, G4G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0527] In one embodiment, the binding protein in i) and / or ii) is selected from: C8, G6, G4C, C3G, C4G, G4G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, and ALT rabbit polyclonal antibody. In one embodiment, the binding protein in i) and / or ii) is selected from: C4C-Fc, G4G-Fc, C4G-Fc, and G4C-Fc.

[0528] In one embodiment, the binding protein in i) and / or ii) is C8. In one embodiment, the binding protein in i) and / or ii) is G6. In one embodiment, the binding protein in i) and / or ii) is G4C. In one embodiment, the binding protein in i) and / or ii) is C3G. In one embodiment, the binding protein in i) and / or ii) is G4G. In one embodiment, the binding protein in i) and / or ii) is C4C-Fc. In one embodiment, the binding protein in i) and / or ii) is G4G-Fc. In one embodiment, the binding protein in i) and / or ii) is G4G-Fc. In one embodiment, the binding protein in i) and / or ii) is G4C-Fc. In one embodiment, the binding protein in i) and / or ii) is C4G-Fc. In one embodiment, the binding protein in i) and / or ii) is an ALT rabbit polyclonal antibody. In one embodiment, the ALT rabbit polyclonal antibody is the antibody described in PCT / IB2017 / 055943.

[0529] In one embodiment, the binding protein in i) is C8. In one embodiment, the binding protein in i) is G6. In one embodiment, the binding protein in i) is G4C. In one embodiment, the binding protein in i) is C3C. In one embodiment, the binding protein in i) is G4G. In one embodiment, the binding protein in i) is C4C-Fc. In one embodiment, the binding protein in i) is G4G-Fc. In one embodiment, the binding protein in i) is G4G-Fc. In one embodiment, the binding protein in i) is G4C-Fc. In one embodiment, the binding protein in i) is C4C-Fc. In one embodiment, the binding protein in i) is an ALT rabbit polyclonal antibody.

[0530] In one embodiment, the binding protein in ii) is C8. In one embodiment, the binding protein in ii) is G6. In one embodiment, the binding protein in ii) is G4C. In one embodiment, the binding protein in ii) is C36. In one embodiment, the binding protein in ii) is G4G. In one embodiment, the binding protein in ii) is C4C-Fc. In one embodiment, the binding protein in ii) is G4G-Fc. In one embodiment, the binding protein in ii) is G4G-Fc. In one embodiment, the binding protein in ii) is G4C-Fc. In one embodiment, the binding protein in ii) is G4G-Fc. In one embodiment, the binding protein in ii) is an ALT rabbit polyclonal antibody.

[0531] In one embodiment, the binding protein in i) is G4C, and the binding protein in ii) is selected from C4G, C4C, C3C, G4C, C8, G4G, G3G, and C3G. In another embodiment, the binding protein in i) is C3G, and the binding protein in ii) is selected from G4G, G3G, C8, G4C, C3C, C4C, C3G, and C4G. In another embodiment, the binding protein in i) is G4G, and the binding protein in ii) is selected from C8, C3G, and C4G. In yet another embodiment, the binding protein in i) is an anti-ALT polyclonal antibody, and the binding protein in ii) is selected from G4G, G3G, G4C, C3G, and C4G.

[0532] In one implementation, lateral flow assays can distinguish between different high (>120 IU / L) and low (<120 IU / L) levels of the enzyme ALT1 in samples as described herein.

[0533] In one implementation, when the binding protein in i) is G4C, the binding protein in ii) is selected from: C4G, C4C, C3C, G4C, C8, G4G, G3G, C3G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0534] In one implementation, when the binding protein in i) is C3G, the binding protein in ii) is selected from: G4G, G3G, C8, G4C, C3C, C4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0535] In one implementation, when the binding protein in i) is G4G, the binding protein in ii) is selected from: C8, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0536] In one implementation, when the binding protein in i) is an anti-ALT polyclonal antibody, the binding protein in ii) is selected from G4G, G3G, G4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4, and RmAb5.

[0537] In one implementation, when the binding protein in i) is RmAb1, the binding protein in ii) is selected from: G4G, G3G, C8, G4C, C3C, C4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0538] In one implementation, when the binding protein in i) is RmAb2, the binding protein in ii) is selected from: G4G, G3G, C8, G4C, C3C, C4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0539] In one implementation, when the binding protein in i) is RmAb3, the binding protein in ii) is selected from: G4G, G3G, C8, G4C, C3C, C4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0540] In one implementation, when the binding protein in i) is RmAb4, the binding protein in ii) is selected from: G4G, G3G, C8, G4C, C3C, C4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0541] In one implementation, when the binding protein in i) is RmAb5, the binding protein in ii) is selected from: G4G, G3G, C8, G4C, C3C, C4C, C3G, C4G, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

[0542] In one implementation, the binding protein in i) and / or ii) is selected from C4C-Fc, G4G-Fc, C4G-Fc and G4C-Fc.

[0543] In one embodiment, the binding protein in ii) is streaked at a concentration of about 0.05 mg / mL to about 2.5 mg / mL. In another embodiment, the binding protein in ii) is streaked at a concentration of about 0.02 mg / mL to about 0.5 mg / mL.

[0544] method

[0545] As is apparent from the description herein, this disclosure provides various methods for detecting ALT. As is apparent from the description herein, this disclosure provides various methods / uses for diagnosing / prognosticating and / or monitoring conditions / treatments associated with ALT expression. As is apparent from the description herein, this disclosure provides methods for monitoring drug induction therapy.

[0546] In one aspect, the present invention provides the use of binding proteins as described herein, or bivalent, trivalent, tetravalent or multivalent binding proteins as described herein, or kits as described herein, or lateral flow assays as described herein for the detection of ALT1.

[0547] In one aspect, the present invention provides a method for detecting ALT1, the method comprising contacting a sample with a binding protein as described herein or a divalent, trivalent, tetravalent or multivalent binding protein as described herein to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

[0548] In one aspect, the present invention provides the use of binding proteins as described herein, or bivalent, trivalent, tetravalent or multivalent binding proteins as described herein, or kits as described herein, or lateral flow assays as described herein for the detection of subjects with liver injury and / or liver disease.

[0549] In one aspect, the present invention provides a method for detecting a subject suffering from liver injury and / or liver disease, the method comprising contacting a sample with a binding protein as described herein or a bivalent, trivalent, tetravalent or multivalent binding protein as described herein to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

[0550] In one implementation, the use or method described herein is for detecting liver injury and / or liver disease in a subject.

[0551] In one implementation, the use or method described herein is for detecting, and / or monitoring, conditions or treatments related to liver injury in a subject. Examples of such conditions and treatments are described in Schaefer and John, 2023, and are provided in Table 1.

[0552] In one implementation, the condition is a pregnancy-related condition, pathogen infection, non-alcoholic fatty liver disease, fatty liver disease, or other liver injury and / or liver disease-related condition.

[0553] In one implementation, the disease is selected from: hepatotropic viruses, non-hepatotropic viruses, bacteria, fungi, parasites, toxins or substances, inflammatory diseases, metabolic or genetic diseases, pregnancy-related diseases, and ischemic or vascular diseases.

[0554] In one implementation plan, the condition is a chronic condition.

[0555] In one implementation plan, the illness is an acute illness.

[0556] In one implementation scheme, the disease is selected from: hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, Coxsackie virus, mononucleosis, adenovirus, dengue virus, coronavirus-19, fatty liver disease, acute alcoholic hepatitis or alcoholic cirrhosis, sea anemone sting, autoimmune hepatitis, biliary tract diseases such as primary biliary cholangitis or primary sclerosing cholangitis, non-alcoholic fatty liver disease, hemochromatosis, Wilson's disease, preeclampsia, acute fatty liver disease of pregnancy, HELLP syndrome, cardiogenic / distributive shock, hypotension, heatstroke, cocaine, methamphetamine, ephedrine, acute Budd-Chiari syndrome, hepatic sinusoidal obstruction syndrome, malignancy, eclampsia, Reye's syndrome, and primary graft nonfunction after liver transplantation.

[0557] In one implementation scheme, the hepatotropic virus is selected from: hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E.

[0558] In one implementation, the non-hepatotropic virus is the virus described in Spengler et al. (2019), Jafari (2023), and Gupta et al. (2021).

[0559] In one implementation, the non-hepatotropic virus is selected from viruses within the families Flaviviridae, Arenaviridae, Filoviridae, Bunyaviridae, and Togaviridae that cause viral hemorrhagic fever affecting the liver. This includes, but is not limited to, yellow fever, Lassa fever, Argentinian hemorrhagic fever, Ebola, Marburg fever, Rift Valley fever, Crimean-Congo hemorrhagic fever, and chikungunya virus.

[0560] In one embodiment, the non-hepatophilic virus is selected from: herpesviruses, including but not limited to human herpesviruses 1-8; influenza A and B viruses; adenoviruses; and coronaviruses, including but not limited to severe acute respiratory syndrome virus 1 and 2; parvoviruses, including B19; enteroviruses, including Coxsackie B virus and echovirus; paramyxoviruses, including measles; and tunica albuginea viruses, including rubella.

[0561] In one implementation, the toxin or substance-related cause is selected from: fatty liver disease, acute alcoholic hepatitis or alcoholic cirrhosis, acetaminophen, antibiotics, anticonvulsants, statins, NSAIDs, herbs / nutritional supplements, mushrooms (e.g., Amanita phalloides), herbs and dietary supplements, carbon tetrachloride, and sea anemone stings.

[0562] In one implementation, the inflammatory condition is selected from: autoimmune hepatitis, biliary tract disease (e.g., primary biliary cholangitis or primary sclerosing cholangitis).

[0563] In one implementation, the metabolic or genetic condition is selected from: non-alcoholic fatty liver disease, hemochromatosis, and Wilson's disease.

[0564] In one implementation, pregnancy-related conditions are selected from: pregnancy-related conditions, eclampsia, preeclampsia, high ALT1 levels during pregnancy in the subject, acute fatty liver of pregnancy, and HELLP syndrome.

[0565] In one implementation, ischemic or vascular conditions are selected from: cardiogenic / distributive shock, hypotension, heatstroke, cocaine, methamphetamine, ephedrine, acute Budd-Chiari syndrome, and hepatic sinusoidal obstruction syndrome.

[0566] In one implementation, the condition is selected from: malignant disease, eclampsia, Reye's syndrome, and primary graft nonfunction after liver transplantation.

[0567] In one implementation, the method or use is for monitoring for conditions in a subject as described herein.

[0568] In one implementation, the method or use is for monitoring treatment in a subject. Those skilled in the art will understand that the treatment can be any treatment in which hepatotoxicity is a known or potential problem. The method or use can serve as an early warning of elevated ALT levels (e.g., increasing over time, approaching or exceeding the upper limit of normal). The method or use can be used to determine when treatment should be adjusted, changed, or discontinued.

[0569] In one implementation, the treatment is selected from: tuberculosis treatment, neurological condition treatment, cancer treatment, and inflammatory bowel disease treatment. In one implementation, the treatment is tuberculosis treatment. In one implementation, the treatment is neurological condition treatment. In one implementation, the treatment is cancer treatment. In one implementation, the treatment is inflammatory bowel disease treatment.

[0570] In one embodiment, the treatment is selected from: anti-TNFα antibodies, infliximab, adalimumab, Bruton's tyrosine kinase inhibitors, 2MHRZ / 2MHR, 2EMRZ / 2MR, acetaminophen, antibiotics, anticonvulsants, statins, NSAIDs, and herbal / nutritional supplements. In one embodiment, the treatment is an anti-TNFα antibody. In one embodiment, the treatment is infliximab. In one embodiment, the treatment is adalimumab. In one embodiment, the treatment is a Bruton's tyrosine kinase inhibitor. In one embodiment, the treatment is 2MHRZ / 2MHR. In one embodiment, the treatment is 2EMRZ / 2MR. In one embodiment, the treatment is acetaminophen.

[0571] In one embodiment, the method or use includes retesting at later time points or at multiple time points to determine disease progression and / or response to treatment. Many treatments can negatively impact the liver and cause damage. In one embodiment, the methods and uses described herein can be used to monitor a subject's response to treatment, using an ALT1 level above the upper limit of normal as an indication of when treatment should be adjusted, changed, or discontinued. In one embodiment, testing is performed daily. In one embodiment, testing is performed weekly. In one embodiment, testing is performed twice weekly. In one embodiment, testing is performed three times weekly. In one embodiment, testing is performed monthly. In one embodiment, testing is performed every two months. In one embodiment, testing is performed three times monthly.

[0572] In one embodiment, the test is performed for at least one week. In one embodiment, the test lasts for at least two weeks. In one embodiment, the test lasts for at least three weeks. In one embodiment, the test lasts for at least four weeks. In one embodiment, the test lasts for at least five weeks. In one embodiment, the test lasts for at least six weeks. In one embodiment, the test lasts for at least seven weeks. In one embodiment, the test lasts for at least eight weeks. In one embodiment, the test lasts for at least nine weeks. In one embodiment, the test lasts for at least ten weeks. In one embodiment, the test lasts for at least eleven weeks. In one embodiment, the test lasts for at least twelve weeks. In one embodiment, the test lasts for at least eleven weeks. In one embodiment, the test lasts for at least fifteen weeks. In one embodiment, the test lasts for at least eleven weeks. In one embodiment, the test lasts for at least seventeen weeks. In one embodiment, the invention provides a concomitant assay for monitoring hepatotoxicity during drug treatment.

[0573] In one implementation, the detection of antigen-binding protein complexes above a threshold level indicates liver injury and / or liver disease in the subject. In one implementation, the threshold is the upper limit normal (ULN). Although the ULN for ALT1 or ALT1+ALT2 can vary by jurisdiction, sex, time of day, health condition, and ethnicity, it is most commonly 40 IU / L. In some jurisdictions, the ULN for ALT1 or ALT1+ALT2 is 70 IU / L.

[0574] Table 1: Example methods and uses.

[0575]

[0576]

[0577]

[0578] Example

[0579] Example 1 – Method

[0580] Production of nanobodies and polyclonal antibodies

[0581] Alpacas were immunized six times with ALT1 over 42 days. Bloodletting was performed on day 45 post-immunization. Phage display of ALT1-reactive nanobodies was performed using biopanning as previously described and with the following modifications (Pardon et al., 2014). Phages displaying ALT-specific nanobodies were enriched after two rounds of biopanning on 5 ng / mL and subsequently 10 ng / mL biotinylated ALT protein. Following the second round of biopanning, single clones were selected on microtiter plates coated with 125 nM non-biotinylated ALT1 for further analysis of the presence of ALT-reactive nanobodies by ELISA. Positive clones were sequenced and annotated using the International ImMunoGeneTics database and aligned in Geneious Prime.

[0582] To generate polyclonal antibodies against ALT, rabbits were repeatedly immunized with purified ALT1. Immunoglobulins were purified from serum using Protein G Sepharose. Further purification was performed using affinity purification of ALT1 conjugated with Sepharose. The polyclonal rabbit anti-ALT1 antibody was buffer-exchanged in PBS and stored at -80°C.

[0583] Expression and purification of nanobodies

[0584] Nanobodies were expressed in *E. coli* WK6 in Terrific broth supplemented with 0.1% glucose and 100 μg / mL ampicillin at 37 °C. Nanobody expression was induced overnight by adding 1 mM IPTG at 28 °C. Cells were harvested and resuspended in 200 mM Tris, 0.5 mM EDTA, 20% sucrose, pH 8.0, and incubated on ice for at least 1 hour. Ice-cold Milli-Q water was added, and cells were incubated on ice for at least another 1 hour. Periplasmic extracts were separated by centrifugation. Magnesium sulfate or magnesium chloride (typically 2 to 10 mM) was then added to prevent nickel ion-EDTA chelation. Nanobodies were purified by affinity chromatography using a HisTrap FF column (Cytiva) with 20 mM Tris, 300 mM NaCl, 25 mM imidazole, pH 8.0 and eluted with a 20 to 500 mM imidazole gradient. If necessary, further purify the recombinant nanobody using gel filtration chromatography with Superose 12 10 / 30 (Amersham) pre-equilibrated with PBS. Otherwise, concentrate the appropriate fraction and exchange the buffer for DPBS.

[0585] Expression and purification of ALT1 and ALT2

[0586] The amino acid sequences of human ALT1 and ALT2 were synthesized by GeneArt and subcloned into the pET expression vector using Gibson Assembly (NEB). ALT1 with a C-terminal His tag was subcloned into pET28a before the N-terminal His tag and thrombin cleavage site. ALT2 (residues 49 to 523) was subcloned into pET151 after the TEV cleavage site.

[0587] ALT1 was expressed in *E. coli* BL21 Star(DE3) cells (Invitrogen) in LB cells supplemented with 100 μg / mL ampicillin at 37 °C. ALT1 expression was induced overnight by adding 0.1 mM IPTG at 18 °C. Cells were harvested, resuspended in 20 mM Tris (pH 8.0) containing 100 μM PLP, 1 mM phenylmethylsulfonyl (PMSF), and 0.2 mg / mL lysozyme, and lysed by sonication (Branson). ALT1 was purified from the clear cell lysate by affinity chromatography using HisTrap FF (Cytiva) with 20 mM Tris, 300 mM NaCl, 25 mM imidazole, pH 8.0, eluted with a 20–500 mM imidazole gradient. Recombinant ALT1 was further purified by gel filtration chromatography using Superose 12 10 / 30 (Amersham) pre-equilibrated with 25 mM HEPES, 150 mM NaCl, pH 8.0 or PBS.

[0588] ALT2-del was expressed in *E. coli* Rosetta 2 (DE3) cells (Merck Millipore) in LB cells supplemented with 100 μg / mL ampicillin and 34 μg / mL chloramphenicol at 37 °C. ALT2-del expression was induced overnight at 18 °C by adding 0.1 mM IPTG. Cells were harvested, resuspended in 20 mM Tris (pH 8.0) containing 40 μM PLP, 1 mM phenylmethylsulfonyl (PMSF), and 0.2 mg / mL lysozyme, and lysed by sonication (Branson). ALT1 was purified from the clear cell lysate by affinity chromatography using HisTrap FF (Cytiva) with 20 mM Tris, 300 mM NaCl, 25 mM imidazole, pH 8.0, eluted with a 20–500 mM imidazole gradient. Recombinant ALT1 was further purified by gel filtration chromatography using Superose 12 10 / 30 (Amersham) pre-equilibrated with 25 mM HEPES, 150 mM NaCl, pH 8.0 or PBS.

[0589] Cloning, expression and purification of FLAG-labeled nanobodies

[0590] A nanobody comprising the pelB signal peptide and a C-terminal His tag was subcloned into the N-terminal His tag and thrombin cleavage site of pET28a. A FLAG tag was introduced into the C-terminus of the nanobody in the pET28a construct, prior to the His tag, using a Q5 site-directed mutagenesis kit (NEB, E0554S). Successful tag incorporation was verified by dideoxynucleotide sequencing (Micromon, Monash University). The FLAG-labeled nanobody was expressed overnight at 28°C in BL21 Star(DE3) self-inducing medium and purified as described above for the unlabeled form.

[0591] Cloning, expression and purification of bivalent nanobodies

[0592] Bivalent nanobodies were cloned by inserting a second nanobodies gene between the pelB sequence and the original nanobodies gene. GGGGS linkers with three or four repeats were designed between the two nanobodies using PCR overhangs, and the constructs were then assembled using Gibson Assembly (NEB). The bivalent nanobodies were expressed overnight in C41(DE3) self-inducing medium at 20°C and purified for monomeric nanobodies as described above.

[0593] Cloning, expression and purification of bivalent nanobody-Fc fusion protein

[0594] Using GenSmart TM The codon optimization tool (Genscript) optimized the bivalent nanobody gene for expression in mammalian cells and synthesized it using GenScript. The bivalent nanobody was cloned into the pcDNA3 expression vector (Invitrogen) between the tissue plasminogen activator leader sequence and the Fc region of human Fc IgG1 using NEBuilder HiFi DNA Assembly (NEB).

[0595] The tetravalent nanobody-Fc fusion protein was expressed in Expi293 cells (the expressed nucleic acid encodes a divalent monomer, which, upon expression, produces the tetravalent nanobody-Fc fusion protein) (Gibco). The cells were maintained in suspension at 37°C and 8% CO2. Cells (3 × 10⁻⁶ cells) were then... 6 ( / mL) Follow the manufacturer's instructions (Gibco) with ExpiFectamine TMReagent 293 was used for transfection at a concentration of 1 μg DNA / mL culture. One day after transfection, ExpiFectamine was added. TM 293 Transfection enhancers 1 and 2. The culture supernatant was collected by centrifugation and filtered 5 days post-transfection, then diluted with 10× PBS (Gibco). The culture supernatant was incubated with protein G sepharose (GenScript), the resin was washed with PBS, and the recombinant protein was eluted with 0.1 M glycine (pH 2.5) and immediately neutralized with 2 M Tris (pH 8.3).

[0596] Avidin captures ELISA

[0597] Maxisorp plates (96-well, Nunc) were coated overnight at 4°C or for 3 to 4 hours at ambient temperature with 5 μg / mL avidin in carbonate-bicarbonate buffer (Sigma-Aldrich, C3041) at 4°C. The wells were blocked for 1 hour with 1.5% (w / v) BSA in PBS-T (PBS containing 0.05% Tween-20), followed by incubation for 1 hour with 1 μg / mL biotinylated proteins (ALT1, ALT2, AST) in ELISA buffer (PBS-T containing 0.5% BSA (w / v)). Subsequently, the wells were incubated for 1 hour with serially diluted anti-ALT1 nanobodies, anti-ALT1 tetravalent nanobodies-Fc, anti-ALT1 rabbit polyclonal antibody, or anti-ALT1 rabbit monoclonal antibody in ELISA buffer. Wells containing anti-ALT1 nanobodies were incubated for 1 hour with mouse anti-FLAG antibody (Sigma-Aldrich, F1804, 1 / 1000), followed by incubation for another 1 hour in ELISA buffer with HRP-conjugated anti-mouse secondary antibody (Sigma-Aldrich, A3415, 1:1500). Wells containing tetravalent nanobodies-Fc were incubated for 1 hour with HRP-conjugated anti-human IgG antibody (DAKO, P0214, 1:10000). Wells containing rabbit anti-ALT1 antibody were incubated with HRP-conjugated anti-rabbit secondary antibody (Sigma-Aldrich, A1949, 1 / 50000). The bound proteins were quantified using KPL Sure Blue Reserve TMB microwell peroxidase substrate (Seracare), and the reaction was terminated by adding 0.5 M sulfuric acid. Optical densities at 620 nm and 450 nm were measured using a Multiskan SkyHigh reader (ThermoFisher), and OD (450 nm to 620 nm) was calculated. Unless otherwise specified, all incubations were performed at room temperature. After each step, the wells were washed three times with PBS-T.

[0598] To preliminarily screen the reactivity of monoclonal nanobodies to ALT1, an ELISA was performed as described above, but ALT1 was added to an avidin-coated plate at a 1 / 200 dilution for 1 hour. The nanobodies were then added at a single dilution of 15 μg / mL for 1 hour. Bivalent nanobodies against ALT1 were also screened using an avidin capture assay as described above, where ALT1 was diluted 1 / 1000 and the bivalent nanobodies were added at a single dilution of 1 μg / mL. In both assays, bound nanobodies were detected by incubation with anti-mouse anti-His (1 / 1000) and subsequently with a species-specific HRP-conjugated antibody.

[0599] Direct ELISA

[0600] Maxisorp plates (96 wells, Nunc) were coated overnight at 4°C with 1 μg / mL ALT1, ALT2, or AST in carbonate-bicarbonate buffer (Sigma-Aldrich, C3041). The wells were blocked with 1.5% (w / v) BSA in PBS-T for 1 hour, followed by incubation with serially diluted anti-ALT1 nanobodies for 1 hour. The bound anti-ALT1 nanobodies were detected using HRP-conjugated anti-lamb secondary antibody (Invitrogen, 1 / 2000). The plates were then subjected to color development and measurement as described above.

[0601] Nanobody modeling

[0602] The structure of the nanobodies was modeled using Alphafold2 (Jumper et al., 2021) via ChimeraX (Pettersen et al., 2021). Graphs were generated using the PyMOL molecular graphics system version 2.5.3, Schrödinger, LLC.

[0603] SDS-PAGE gel and Western blot

[0604] Using Bolt TMSDS-PAGE analysis was performed on Bis-Tris Plus Mini protein gels, 4% to 12% (Invitrogen), and Bolt MESSDS running buffer (Invitrogen). Western blots were transferred to nitrocellulose membranes using an iBlot 2 Western blot transfer device (Invitrogen). The membranes were blocked for 1 hour in 3% skim milk in TBS-T (TBS + 0.05% Tween-20), followed by incubation for 1 hour in blocking buffer with the primary antibody, anti-His (Rockland, 600-401-382, 1:1000). The membranes were washed with TBS-T and incubated for 1 hour in blocking buffer with anti-rabbit / HRP (Sigma, A1949, 1:15000). The membranes were washed, then ECL Prime (Amersham) was added, and the membranes were visualized on a ChemiDoc imaging system (Bio-Rad).

[0605] Production of monoclonal antibodies

[0606] To generate rabbit monoclonal antibodies, peripheral blood mononuclear cells were isolated from ALT1-immunized rabbits, and positive B cells were enriched, cultured, cloned, and sequenced (Genscript). The obtained supernatant was screened, and the top conjugate was sequenced and cloned into pcDNA3.4 for recombinant protein production. Sequence analysis of the variable domains of the heavy and light chains was performed using the IMGT / V QUEST server (Brochet et al., 2008).

[0607] Expression and purification of monoclonal antibodies

[0608] Rabbit monoclonal antibodies were expressed in Expi293 cells (Gibco) and purified using protein G Sepharose in the same manner as described for the nanobody-Fc fusion protein.

[0609] Cloning and expression of ALT1 mutants for epitope mapping

[0610] ALT1 alanine mutants were generated via Genscript using the ALT1-cHis-pET28 backbone. The ALT1 mutants were expressed in *E. coli* BL21Star(DE3) (Invitrogen) in Luria-Bertani broth (LB) supplemented with 100 μg / mL ampicillin at 37°C. ALT1 expression was induced overnight by adding 0.1 mM IPTG at 18°C. Cells were harvested and resuspended in 1 / 25 culture volume of BugBuster Master Mix (Merck, 71456-4). After 10 minutes, the lysed cells were centrifuged at 20,000 g for 10 minutes at 4°C, and the supernatant was removed. The clarified lysate was used for epitope mapping experiments.

[0611] X-ray crystallography

[0612] The ALT1-C8 complex was crystallized at 20 °C using a hanging drop vapor diffusion method, wherein the droplet contained 1 μL of protein solution (10 mg / mL, in 25 mM HEPES, 150 mM NaCl, pH 7.5) and 1 μL of storage solution (19% PEG1.5 K v / v, 20% glycerol v / v), as described in Brochet et al., 2008 and Giudicelli et al., 2011. Crystals appeared within 7 days. To collect X-ray data, the crystals were immersed in a cryoprotectant solution containing the storage solution (where PEG 1.5 K was increased to 24% v / v) and then rapidly cooled directly in liquid nitrogen.

[0613] Data for 200° were collected at 100 K using an EIGER × 16M detector at 0.954 Å on the MX2 beamline of the Australian Synchrotron (Aragao et al., 2018), with 0.1 degrees per frame and an exposure time of 0.02 seconds. The data were indexed and integrated using XDS (Kabsch, 2010) and scaled using AIMLESS (Evans & Murshudov, 2013) to a resolution of 2.7 Å, belonging to space group P41212, with cell dimensions of a = 146.846 Å, b = 146.846 Å, c = 157.369 Å, and α = 90.00°, β = 90.00°, and γ = 90.00°. Matthews coefficients predicted two ALT1-C8 complexes per asymmetric unit cell.

[0614] Using the AlphaFold2 model of ALT1 (Jumper et al., 2021) and C8 nanobodies as search models, molecular substitution was employed to resolve the structure of the ALT1-C8 complex. The generated complex models were structurally refined using REFMAC5 (Murshudov et al., 2011) and Phenix (Liebschner et al., 2019), and iterative model building was performed using COOT (Emsley et al., 2010). Structural quality was assessed using MolProbity (Williams et al., 2018). ALT1-C8 interface analysis was performed using PISA (Krissinel & Henrick, 2007). Molecular maps were generated using PyMOL (DeLano, 2002).

[0615] Biolayer Interferometry (BLI)

[0616] The affinity of nanobodies for ALT1 was measured using an Octet RED96 instrument (ForteBio). Measurements were performed using either streptavidin (SA) or Ni-NTA capture sensors (Sartorius) and kinetic buffer (20 mM HEPES, 150 mM sodium chloride, pH 7.5, supplemented with 0.1% BSA and 0.05% Tween 20). All experiments were performed at 25 °C in all-black 96-well plates (Greiner). To measure binding to ALT1, after a 180-second baseline step, ALT1 (5 μg / mL) was loaded onto the SA sensors until a 1 nm response signal was obtained from each sensor. The sensors were washed in kinetic buffer for 180 seconds, and binding of nanobodies or polyclonal anti-ALT1 antibodies was performed by immersing the sensors for 600 seconds using a 1 / 3 dilution series starting at 1000 nm. Dissociation was measured in kinetic buffer for 600 seconds after binding.

[0617] To measure the interaction between ALT1 and nanobodies, after a 180-second baseline step, the corresponding nanobodies (5 μg / mL) were loaded onto Ni-NTA sensors until each sensor acquired a response signal of 0.5 nm. The sensors were washed in kinetic buffer for 180 seconds, and ALT1 binding was performed by immersing the sensors for 600 seconds using a 1 / 3 dilution series starting at 1000 nm. Dissociation was measured for 600 seconds in kinetic buffer after binding. The sensors were regenerated by cycling for 10 seconds in either 10 mM glycine (pH 1.7) or 300 mM imidazole, followed by 10 seconds in kinetic buffer, repeated 5 times. The data were fitted using a global 1:1 model with Octet analysis software (Forte Bio) to determine the kinetics and KD values.

[0618] Tandem binning was performed using a Ni-NTA biosensor (Sartorius). ALT1-cHis was diluted to 5 μg / ml in kinetic buffer and immobilized on Ni-NTA for 600 seconds. Saturated antibody was bound at 300 nM for 600 seconds. To ensure complete self-blocking, a 150 nM competitive antibody was bound for 600 seconds and then dissociated for 600 seconds. Epitope binning analysis was performed using OctetAnalysis Studio 13.0.3.52 software.

[0619] Protein expression in ALT1 mutant lysates was quantified via BLI using an Octet RED96 instrument (ForteBio). Experiments were performed at 25 °C in all-black 96-well plates (Greiner), where the Ni-NTA capture sensor (Sartorius) was activated in kinetic buffer (1× Dulbecco phosphate-buffered saline (DPBS) supplemented with 0.1 mg / mL BSA and 0.002% Tween 20). Different concentrations (0–80 μg / mL) of purified recombinant His-labeled ALT1 were immobilized onto the sensor, and standard curves were generated using the binding rate. Next, His-labeled ALT1 mutants were immobilized in lysates diluted 1:10 in kinetic buffer, and the concentration of ALT1 mutants in the lysates was subsequently determined using the resulting maximum binding rate. Results were analyzed using Octet Analysis Studio 13.0.3.52 software (Sartorius).

[0620] In addition, to evaluate whether C4G-Fc, C8-Fc, G6-Fc, and rabbit monoclonal antibodies (RmAb1, RmAb3, RmAb4, RmAb5) have different or overlapping epitopes, BLI was used for epitope binning. 5 μg / mL of purified recombinant His-labeled ALT1 was loaded onto the Ni-NTA sensor for 300 seconds, and antibody 1 (saturated antibody) at a saturated concentration (300 nM) was allowed to bind for 600 seconds. Antibody 2 (competitive antibody) was then competed for 600 seconds at the following concentrations [C4G-Fc (90 nM); C8-Fc (90 nM); G6-Fc (200 nM); RmAb1, RmAb3, RmAb4, RmAb5 (66.7 nM); 3H12, 4A9, 5H2, 6B5 (150 nM)]. Epitope binning analysis was constructed using Octet Analysis Studio 13.0.3.52 software (Sartorius).

[0621] Differential scanning fluorescence (DSF)

[0622] Differential scanning fluorescence was used to assess the thermal stability of proteins (Niesen et al., 2007). Protein (10 μg) was diluted twice in 25 μL of 1× PBS containing 2.5 μL of 5× SYPRO Orange protein gel staining agent (Sigma-Aldrich). The samples were then heated from 25°C to 95°C in 0.5°C increments, each lasting 1 minute. Fluorescence measurements were performed at the end of each increment. Excitation was at 492 nm, and emission was at 610 nm. Tm was determined as the minimum of the negative first derivative of the melting curve.

[0623] Direct ELISA using ALT1 mutant lysate

[0624] High-binding plates (96-well half-region, Nunc, Corning 3690) were coated overnight at 4°C with 1.25 μg / mL ALT1 mutant lysate in carbonate-bicarbonate buffer (Sigma-Aldrich, C3041). The wells were blocked with 1.5% (w / v) BSA in PBS for 1 hour, followed by incubation with 0.1 μg / mL G6-Fc for 1 hour. The bound anti-ALT1 nanobody fusions were detected using HRP-conjugated anti-human IgG (Dako, 1 / 10000). The plates were developed and measured according to the overview in the ELISA section above.

[0625] Anti-His capture ELISA using ALT lysate.

[0626] High-binding plates (96-well half-region, Nunc, Corning 3690) were coated overnight at 4–8°C or for 3–4 hours at ambient temperature with 1 μg / mL mouse antihistamine tag (Bio-RAD, MCA1396) in carbonate-bicarbonate buffer (Sigma-Aldrich, C3041). The wells were blocked with 1.5% (w / v) BSA in PBS for 1 hour and then incubated with 1.25 μg / mL ALT1 mutant lysate in ELISA dilution buffer. The wells were then incubated for 1 hour with different concentrations of anti-ALT1 nanobody fusion protein or anti-ALT1 rabbit monoclonal antibody in ELISA dilution buffer. The wells containing nanobody-Fc were further incubated with HRP-conjugated anti-human IgG antibody at dilutions of 1:10000 or 1:20000 (DAKO, P0214 or Millipore, AP504P 1:20000). Incubate the wells containing rabbit anti-ALT1 antibody with HRP-conjugated anti-rabbit secondary antibody (Sigma-Aldrich, A1949, 1 / 10000). Perform color development and measurement on the plates as outlined in the ELISA section above.

[0627] Coupling of detectors for lateral flow

[0628] According to the manufacturer's instructions, nanobodies, nanobodies-Fc fusions, anti-ALT rabbit monoclonal antibodies, and anti-ALT1 rabbit polyclonal antibodies were conjugated to europium fluorescently functionalized microspheres (Merck, F1-EU 030) using the AnteoBind Nano kit (AnteoTech, A-PCKS). Nanobodies were conjugated at 75 μg / mg particles, while anti-ALT1 polyclonal or monoclonal antibodies were conjugated at 30, 50, or 100 μg / mg particles.

[0629] According to the manufacturer's instructions, nanobodies, nanobodies-Fc fusions, anti-ALT rabbit monoclonal antibodies, and anti-ALT1 rabbit polyclonal antibodies were conjugated to gold nanoshells using the BioReady High Sensitivity Gold Conjugation Kit for Lateral Flow (NanoComposix, GSZR150-10M). 30 μg of antibody was conjugated per 1 mL of nanoparticles.

[0630] According to the manufacturer's instructions, conjugate rabbit monoclonal anti-ALT antibody to Estapor BlueIntense and Black particles using the AnteoBind Nano kit (AnteoTech, A-PCKS) or the Anteobind NXT kit (Anteobind, A-LNXTK-5). Each 1 mg of nanoparticles can be conjugated with 2, 5, 20, 50, or 150 μg.

[0631] Preparation of nitrocellulose membranes for lateral flow measurement

[0632] Lateral flow lines were prepared using an IsoFlow dispenser (Imagene Technology) by spraying two lines onto a nitrocellulose membrane (NCM) (Vivid 90, Pall Corporation). The first line (test line) was streaked with monovalent, tetravalent, or Fc-fusion nanobodies (Burnet Institute) at concentrations of 0.05, 0.1, 0.2, 0.5, or 1 mg / mL. The second line (control line) was streaked with recombinant ALT1 protein at concentrations of 50 or 100 μg / mL (Burnet Institute).

[0633] Assembly and operation of lateral flow measurement in a wet system

[0634] For wet system testing, the scribe-lined NCM is laminated together with a 10 mm sample pad (1285 or 1281, Ahlstrom) and an absorbent pad (CF6 or CF5 pad, GE Healthcare) onto an adhesive backing card and cut into 4 or 5 mm test strips using a guillotine cutter (KinbioShangai Kinbio Tech).

[0635] For the assay, a total sample volume of 30 μL was prepared in the wells of a 96-well plate. Recombinant ALT1 was diluted to 0, 0.1, 0.25, 1, or 10 μg / mL, or a final total volume of 0 to 10 ng, and combined in the sample wells at various dilutions with a 6 μL gold nanoshell detector (Burnet Institute), a europium detector at a final concentration of 2.5 or 1.25 μg / mL, or a 15 μL EstaporBlue Intense or Black detector. The plasma sample volume for testing was 5 μL. Run buffer (20 mM HEPES, 150 mM NaCl, pH 7.5, 0.5% Tween-20, 0.1% BSA) was added to bring the final volume to 30 μL. The test strip was placed in each well of the 96-well plate with the sample pad at the bottom. After 8 or 10 minutes, 30 µL of run buffer was added to the wells of the 96-well plate to reduce nonspecific binding, and the assay was run again for 8 or 10 minutes. Depending on the conjugate used for detection, the intensity of the test lines can be visually assessed (gold conjugates, Estapor Blue Intense, or Black), or preferably using an automated reader such as the Axxin reader (AX-2X, Axxin Ltd Melbourne) or the Lumos Leelu reader (Lumos Diagnostics, Melbourne). This method is also used to detect europium conjugates that are not visible to the naked eye. The reader provides a digital reading for each test line, along with a photograph of the test strip.

[0636] The test results are interpreted by visually evaluating the photographed test strips and by plotting numerical readings of the test line intensity relative to the concentration of ALT1 incorporated.

[0637] Schematic diagram of lateral flow-resistant GPA assembly in the box

[0638] The europium detector was diluted 1 / 250 in BD2 conjugate drying buffer (10 mM borate, 2 mM EDTA, 0.25% Tween-20, 20% sucrose, 5% trehalose, 1% BSA, 0.35% PEG8000) or 20 mM Tris (pH 8.0), 1% BSA, 0.1% Tween-20, 10% sucrose, 5% trehalose, and sprayed onto a glass fiber pad (8951, Ahlstrom) using an IsoFlow dispenser (Imagene Technology) at 0.8 μL / mm. Anti-blood group glycoprotein A was diluted to 0.185 mg / mL in BD2 conjugate drying buffer and sprayed twice onto the same glass fiber pad at 3 μL / mm as described for the detector, and dried overnight at 37°C.

[0639] The NCM, marked as described above, is then laminated together with a glass fiber pad containing the blood-capturing reagent anti-blood group glycoprotein A (anti-GPA) and europium-conjugated rabbit anti-ALT1 at the bottom of the NCM, and an absorbent pad (CF5 absorbent pad, GE Healthcare) at the top of the NCM. All components are laminated together onto an adhesive backing card and cut into 4 mm test strips using a guillotine cutter (Kinbio Shanghai, Kinbio Tech). The strips are then assembled into a disposable plastic housing (AtomoRapid™ Diagnostics).

[0640] Schematic diagram of the lateral flow-blood retention pad assembly in the box

[0641] The europium detector (europium-coupled polyclonal rabbit anti-ALT1 or europium-coupled monoclonal rabbit anti-ALT1) was diluted (1 / 250, 1 / 500) in BD2 conjugate drying buffer or 20 mM Tris (pH 8.0), 1% BSA, 0.1% Tween 20, 10% sucrose, and 5% trehalose, and dispensed (0.8 μL / mm) onto a glass fiber pad (8951, Ahlstrom) and dried overnight at 37°C.

[0642] The NCM, as outlined above, is then laminated with a glass fiber pad containing a europium detector at the bottom of the NCM, a blood retention pad (FR1 (0.35 mm, mdi Membrane Technologies, INC.), and an absorbent pad (CF5 absorbent pad, GE Healthcare) on top of the NCM. Alternatively, the NCM is laminated with a rehydration pad (8951 glass fiber, Ahlstrom), followed by a glass fiber pad containing a europium detector at the bottom of the NCM, a blood retention pad (FR1 0.35 mm, mdi Membrane Technologies, INC.), and an absorbent pad (CF5 absorbent pad, GE Healthcare) on top of the NCM.

[0643] For test strips containing the Estapor Blue Intense detector, the detector was diluted (1 / 10, 1 / 15, or 1 / 20) in 20 mM Tris (pH 8.0), 1% BSA, 0.1% Tween 20, 10% sucrose, and 5% trehalose, and sprayed (0.8 μL / mm) onto a glass fiber pad (8951, Ahlstrom) and dried overnight at 37°C. The NCM strips, as outlined above, are laminated together with the glass fiber pad containing the Estapor Blue Intense detector at the bottom of the NCM, two overlapping blood retention pads (FR1 (0.35 mm, mdi Membrane Technologies INC)), and an absorbent pad (CF5 absorbent pad, GE Healthcare) on top of the NCM.

[0644] In each illustrative assembly, all components were laminated together onto an adhesive backing card and cut into 4 mm test strips using a guillotine cutter (Kinbio Shanghai, Kinbio Tech). The strips were then assembled into a disposable plastic housing (AtomoRapid). TM Pascal, Atomo Diagnostics).

[0645] AtomoRapid TM Lateral flow measurement in Pascal box

[0646] To run the assay using ALT-coated venous blood, different concentrations of ALT (0, 1, 2, and 5 ng / 10 μL) were added to the venous blood of healthy volunteers. Using a pipette, 10 μL or 11.5 μL of the venous blood was added to the blood collection unit (BCU) of the delivery box, and the BCU was moved to the sample area to deliver the sample. After approximately 3 to 10 seconds, the blister burst to allow the buffer to begin flowing through the test strip. The sample and buffer were allowed to diffuse laterally for 20 minutes. After 20 minutes, the test was visualized and quantified using an automated reader (e.g., a Lumos Leelu reader or a Cerberus reader (Lumos Diagnostics, Melbourne)).

[0647] To run the assay without the buffer blister pack, the assay is performed as described above, however, by adding 85 μL of running buffer (20 mM HEPES, 150 mM NaCl, pH 7.5, 0.5% Tween-20, 0.1% BSA, with or without ProClin 300) through a hole drilled in the top shell where the blister pack opening is located, instead of breaking the blister pack.

[0648] Spots and marks

[0649] Spot 12.5 ng to 100 ng of ALT1 in 1 μL onto a nitrocellulose membrane (Amersham, RPN303E) and allow it to dry. Block the blot with 3% skim milk at room temperature for 3 hours or overnight at 4°C, and incubate with antibody (1 μg / mL in blocking buffer) at room temperature for 2 hours or overnight at 4°C. Incubate the blot with anti-rabbit / HRP (1:5000, Sigma, A1949), anti-human / HRP (1:1000, DAKO, P0214), or anti-mouse / HRP (1:5000, Sigma, A2554) at room temperature for 1 hour. For FLAG-labeled nanobodies, first incubate the blot with anti-FLAG (1:5000, Sigma, F1804) for 1 hour, then incubate with anti-mouse / HRP. Wash the blot three times with PBS + 0.05% Tween-20 for 5 minutes each time between steps. The blot was visualized using Bio-Rad ChemiDoc MP with ECL (Amersham, RPN2232). The commercial rabbit anti-ALT monoclonal antibody used was Abcam EPR19616.

[0650] Example 2 – Isolation of nanobodies reacting with ALT1

[0651] In two panning experiments, nanobodies that react with ALT1 from a phage display library were screened using ELISA. In Experiment 1, the reactivity of 94 clones to ALT1-coated ELISA plates was screened. Of these, 91 were ALT1 positive, and 86 of them possessed a full-length VHH domain. Among them, 13 had unique variable domains, and 2 nanobodies possessed a unique complementarity-determining region (CDR) 3 domain. Figure 2A The two nanobodies are Nb_C8 and Nb_G6. Nb_C8 is unique, with only one clone identified possessing the precise CDR3 sequence. The CDR3 domain of Nb_G6 is the dominant nanobodies in the library. For a second experiment, 188 clones were selected, 60 of which possessed the full-length VHH sequence (…). Figure 2B When the second round of nanobodies was purified, none of them retained significant activity against ALT1.

[0652] Nb_C8 is unique in this library, with only a single clone having the same CDR3 region. Nb_G6 is the dominant clone isolated from this library, with 82 out of 91 clones having the same CDR3 sequence. Figure 3 ).

[0653] ELISA was performed on a series of nanobodies with unique CDRs. The results showed that Nb_C8 and Nb_G6 bound to ALT1 very strongly, while Nb_C10 bound very weakly. All other screened nanobodies did not show binding to ALT1. Figure 4 For comparison, the binding of the nanobody to bovine serum albumin (BSA) is shown, representing the background of the assay.

[0654] Protein-coding sequences were annotated, showing the predicted boundaries of the frame region (FR) and complementarity-determining region (CDR) of Nb_C8 and Nb_G6 (which showed strong binding to ALT1) and the nanobody Nb_C10 (which showed weak binding to ALT1). Figure 5A , 5B 6A, 6B and 7).

[0655] The percentage of sequence identity between nanobodies was analyzed. Analysis of nanobodies that strongly bind to ALT1 and those that do not showed that Nb_C8 (strong binder) and Nb_C10 (showing no / low binding to ALT1) shared only 71% identity, while Nb_G6 and Nb_C10 shared 77.0% identity. The strong ALT1 binders Nb_C8 and Nb_G6 shared 76% identity. Results are as follows... Figure 8 As shown.

[0656] The alpaca V, J, and D genes used in Nb_G6 and Nb_C8 were identified. For Nb_G6, the V gene used is IGHV3-3. 01-F shares 95.83% sequence identity with the germline sequence, while Nb_C8, using the same gene, shares 87.15% identity. This indicates that Nb_C8 has a higher degree of hypermutation than Nb_G6. This hypermutation produces a large deletion in CDR3 present in Nb_C8. Nb_G6 uses the J gene J6. 01, while Nb_C8 uses J4 01, both share 89.36% identity with the phylogenetic sequence ( Figure 9 ).

[0657] A comparison was performed between Nb_G6 and Nb_C8, highlighting differences in their protein-coding sequences. The most significant difference among these nanobodies lies in CDR3, where Nb_C8 has a 6-amino acid deletion compared to Nb_G6. Regions CDR1 and 2 are also highly different among these nanobodies, which can also lead to differences in binding properties. Point mutations are also present in FR1, 2, and 3, which can also alter their binding properties. Figure 10 ).

[0658] Alignment between Nb_C8 and Nb_C10 showed 68.2% sequence identity and 74.4% similarity, indicating that most differences exist in CDR1, 2, and 3. Nb_C8 has an 8-amino acid deletion in CDR3 compared to Nb_C10. Figure 11 ).

[0659] Alignment between Nb_C10 and Nb_G6 showed 76.0% sequence identity and 80.6% similarity, indicating that most differences exist in CDR1, 2, and 3. The CDR3 region of Nb_G6 has a 3-amino acid deletion compared to Nb_C10. Figure 12 ).

[0660] Two major differences exist in the 3D structures predicted by Alphafold2. One difference lies within CDR1, where Nb_C10 and Nb_C8 are predicted to have helical content, while Nb_G6's CDR1 is predicted to be a loop. The second major difference in the predicted structures lies in CDR3. The predicted orientation of the CDR3 loops in Nb_G6 and Nb_C8 differs significantly from the observed orientation in Nb_C10. The absence of CDR3 observed in Nb_C8 is reflected in a shorter, tighter loop structure with a short helical segment unique to this Nb at the residue LRV. Furthermore, the C-terminal segment of CDR3 in Nb_C8 does not contain a helix; both Nb_G6 and Nb_C10 contain a short β-chain at the CDR3 terminal, but they are in different orientations ( Figure 13 A and 13B).

[0661] Example 3 – Activity of Nanobodies

[0662] Nanobody sequences can be modified by adding sequences to promote site-specific changes, such as biotinylation, chemical cross-linking, conjugation with detection reagents, or the addition of an Fc domain. In this embodiment, Nb_C8 and Nb_G6 have been modified by adding His, FLAG, or Avi tags. The expression of these modified nanobodies was assessed by SDS-PAGE. Figure 14 ).

[0663] The binding activity of Nb_G6 with ALT1, ALT2, and AST was evaluated in ELISA. Nb_G6 containing the C-terminal FLAG tag bound strongly to ALT1; the amount of Nb required to achieve a 10-fold increase in background signal was 0.008 μg / mL for ALT1 and 1.15 μg / mL for ALT2. The binding of Nb_G6 to ALT1 was approximately 140-fold higher than that to ALT2. No binding to AST was detected. Figure 15 A).

[0664] The binding activity of Nb_C8 with ALT1, ALT2, and AST was evaluated in ELISA. Nb_C8 containing the C-terminal FLAG tag bound strongly to ALT1; the amount of Nb required to achieve a 10-fold increase in background signal was 0.003 μg / mL for ALT1 and 6.5 μg / mL for ALT2 and AST. The binding of Nb_C8 to ALT1 was approximately 2200 times higher than that to ALT2 and AST. Figure 15 B).

[0665] The binding activity of rabbit polyclonal antibodies against ALT1 to ALT1, ALT2, and AST was evaluated in ELISA. Rabbit polyclonal antibodies are examples of prior art polyclonal reagents against ALT1 that contain antibody-specific mixtures and do not exhibit high selec...

Claims

1. A binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising an amino acid sequence selected from the following: a) The amino acid sequence GPAVSNVA (SEQ ID NO: 2) as CDR1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3; b) The amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3; and c) The amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, ISRSGFST (SEQ ID NO: 11) as CDR2, and AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3; and Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID NO.

2. The binding protein of claim 1, wherein the binding protein is a nanobody.

3. The binding protein of claim 1 or claim 2, wherein the binding protein comprises the amino acid sequence GPAVSNVA (SEQ ID NO: 2) as CDR1, the amino acid sequence ITWSGWT (SEQ ID NO: 3) as CDR2, and the amino acid sequence NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3.

4. The binding protein of claim 1 or claim 2, wherein the binding protein comprises the amino acid sequence GRTDSFYA (SEQ ID NO: 6) as CDR1, the amino acid sequence ITWSAGST (SEQ ID NO: 7) as CDR2, and the amino acid sequence AADLSAGYESSWLEAFGS (SEQ ID NO: 8) as CDR3.

5. The binding protein of claim 1 or claim 2, wherein the binding protein comprises the amino acid sequence GRTFSSYS (SEQ ID NO: 10) as CDR1, the amino acid sequence ISRSGFST (SEQ ID NO: 11) as CDR2, and the amino acid sequence AVGRAYLPTASGTRCPREAYDY (SEQ ID NO: 12) as CDR3.

6. The binding protein of claim 1, 2 or 3, wherein the binding protein comprises amino acid SEQ ID NO: 1 (C8), or a sequence having at least 76% identity with it, or its humanized, deimmunized or germline form.

7. The binding protein of claim 1, 2 or 4, wherein the binding protein comprises amino acid SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or its humanized, deimmunized or germline form.

8. The binding protein of claim 1, 2 or 5, wherein the binding protein comprises amino acid SEQ ID NO: 9 (C10), or a sequence having at least 76% identity with it, or its humanized, deimmunized or germline form.

9. The binding protein of any one of claims 1 to 8, wherein the binding protein binds to an epitope containing residues on both protomers of ALT1.

10. The binding protein of claim 1, 3 or 6, wherein the binding protein does not bind significantly or detectably to a single monomer of ALT1.

11. The binding protein of any one of claims 1 to 10, wherein the binding protein does not bind significantly or detectably to heat-denatured ALT1.

12. The binding protein of any one of claims 1 to 11, wherein the binding protein binds to an epitope of human ALT1.

13. The binding protein of any one of claims 1 to 12, wherein the binding protein does not bind significantly or detectably to human ALT2 and / or does not bind significantly or detectably to aspartate aminotransferase (AST).

14. The binding protein of any one of claims 1 to 13, wherein the binding protein comprises a framework region derived from an alpaca, camel, llama, or shark, or a modified or humanized form thereof.

15. The binding protein of claim 14, wherein the framework region of the binding protein is derived from alpaca, or from its modified or humanized form.

16. The binding protein of any one of claims 1 to 15, wherein the binding protein comprises a tag.

17. The binding protein of claim 16, wherein the tag is selected from: polyhistidine, Flag, antibody epitope tag, c-myc, hemagglutinin, headlock, C-tag, ALFA tag, Avi, GST, MBP, Fc sequence.

18. The binding protein of claim 16 or claim 17, wherein the tag is cleavable.

19. The binding protein of any one of claims 1 to 18, wherein the binding protein comprises a detectable marker.

20. The binding protein of claim 19, wherein the detectable label is selected from: metal labels, magnetic labels, beads, colorimetric labels, radioactive labels, enzyme labels, luminescent labels, fluorescent labels, quantum dots, fluorescent latex particles, chemiluminescence-based labels, liposome-based probes, and Raman-active tags.

21. The binding protein of any one of claims 1 to 20, wherein the binding protein further comprises a linker.

22. The binding protein of claim 21, wherein the linker is selected from SEQ ID NO: 65 to SEQ ID NO:

77.

23. The binding protein of any one of claims 1 to 22, wherein the binding protein is fused to or coupled to a protein.

24. The binding protein of claim 23, wherein the protein is selected from: the Fc region or a fragment thereof, ferritin, maltose-binding protein (MBP), leucine zipper, glutathione S-transferase (GST), keyhole hemocyanin (KLH), albumin, cyclophilin, FKBP, calcineurin, CyPFAS, GyrB, neutral avidin, avidin, and streptavidin.

25. The binding protein of claim 24, wherein the protein is an Fc region or a fragment thereof.

26. The binding protein of claim 24 or claim 25, wherein the Fc region or a fragment thereof enhances the binding rate of the binding protein to ALT1.

27. The binding protein of any one of claims 24 to 26, wherein the Fc region or a fragment thereof reduces the binding rate of the binding protein to ALT1.

28. The binding protein of any one of claims 1 to 27, wherein the binding protein binds to ALT1 selected from human plasma, human serum, whole blood, and capillary blood.

29. The binding protein of claim 28, wherein the whole blood is venous blood or capillary blood.

30. The binding protein of any one of claims 1 to 29, wherein the binding protein binds Asn 99 and Gln 374 of the first ALT1 protopolymer and Asp 93, Ser 96 and Glu 109 of the second ALT1 protopolymer.

31. The binding protein of any one of claims 1 to 30, wherein the complementary site of the binding protein comprises one or more residues of CDR2, FR1 and FR2.

32. The binding protein of any one of claims 1 to 31, wherein the complementary site of the binding protein comprises residues in CDR2 but not residues in CDR1 or CDR3.

33. The binding protein of any one of claims 1 to 32, wherein the C8 residue mediating contact with ALT1 comprises one or more of the following: Leu 18, Arg 19, Ser 58, Phe 68, Thr 90, Ile 70, Ser 71, Lys 76, Gln82, and Asn 84.

34. The binding protein of any one of claims 1 to 33, wherein the binding protein does not significantly and / or detectably bind to ALT1 containing one or more of the following mutations: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu, or does not significantly and / or detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 53 Arg, 61 Thr, 154 Val, 155 Phe, 175 Glu, 178 Thr, 183 Leu, 196 Leu, 202 Val, 205 Asp, 212 Arg, 217 Asp, 225 Leu, 239 Val, 260 Arg, 263 Phe, 266 Arg, 296 Met, 299 Pro, 304 Gln, 327 Val, 330 Val, 357 Leu, 358 Leu, 369 Asp, 420Leu, 430 Glu, 431 Leu, 433 Leu, 439 Phe and 441 Leu.

35. A binding protein that binds to alanine aminotransferase 1 (ALT1), said binding protein comprising an antigen-binding site for an antibody, said antibody comprising an amino acid sequence selected from: a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3; b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2, and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3; c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2, and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3; d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2, and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; and e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2 and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No.

36. The binding protein of claim 35, wherein the binding protein is a monoclonal antibody.

37. The binding protein of claim 36, wherein the binding protein is a rabbit monoclonal antibody.

38. The binding protein of any one of claims 35 to 37, wherein the binding protein binds to an epitope containing residues on both protomers of ALT1.

39. The binding protein of any one of claims 35 to 38, wherein the binding protein does not bind significantly and / or detectably to heat-denatured ALT1.

40. The binding protein of any one of claims 35 to 39, wherein the V L It is κ1. The binding protein of 41.40, wherein the κ1 comprises Cys80, the Cys80 forming a disulfide bond with Cys 171.

42. The binding protein of claim 40 or claim 41, wherein the κ1 variable region uses the J lineage J1-2.

43. The binding protein of any one of claims 35 to 42, wherein the melting temperature of the binding protein is from about 74.8°C to about 75.8°C.

44. The binding protein of any one of claims 35 to 43, wherein the binding rate of the binding protein is at least about 5 times higher than that of the rabbit polyclonal ALT-1 antibody.

45. The binding protein of any one of claims 35 to 44, wherein the affinity of the binding protein for ALT1 is at least about 10 times higher than that of the rabbit polyclonal anti-ALT antibody.

46. ​​The binding protein of any one of claims 35 to 45, wherein the binding protein can be isolated with a purity of at least about 95%.

47. The binding protein of any one of claims 35 to 46, wherein the sensitivity of the binding protein for detecting human ALT1 is at least twice that of rabbit polyclonal anti-ALT antibody.

48. The binding protein according to any one of claims 35 to 47, wherein said binding does not significantly and / or detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 129 Ile, 131 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

49. The binding protein according to any one of claims 35 to 48, wherein said binding does not significantly and / or detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 129 Ile, 131 Leu, 154 Val, 177 His, 178 Thr, 183 Leu, 202 Val, 205 Asp, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

50. The binding protein according to any one of claims 35 to 49, wherein said binding does not significantly or detectably bind to ALT1 containing an alanine substitution at one or more of the following positions: 19 Lys, 24 Asp, 31 Arg, 43 Gln, 48 Leu, 56 Val, 61 Thr, 129 Ile, 131 Leu, 154 Val, 183 Leu, 196 Leu, 202 Val, 212 Arg, 222 His, 225 Leu, 229 Arg, 239 Val, 260 Arg, 266 Arg, 284 Ser, 304 Gln, 333 Asp, 340 Met, 358 Leu, 408 Val, 430 Glu, and 433 Leu.

51. The binding protein of any one of claims 35 to 49, wherein the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO:

109. H and V containing the amino acid sequence shown in SEQ ID NO: 113 L (RmAb1), or a sequence that has at least 70% identity with it.

52. The binding protein of any one of claims 35 to 47, wherein the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO:

116. H and V containing the amino acid sequence shown in SEQ ID NO: 120 L (RmAb2), or a sequence that has at least 70% identity with it.

53. The binding protein of any one of claims 35 to 48 or 50, wherein the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO:

123. H and V containing the amino acid sequence shown in SEQ ID NO: 127 L (RmAb3), or a sequence that has at least 70% identity with it.

54. The binding protein of any one of claims 35 to 48 or 50, wherein the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO:

130. H and V containing the amino acid sequence shown in SEQ ID NO: 134 L (RmAb4), or a sequence that has at least 70% identity with it.

55. The binding protein of any one of claims 35 to 49, wherein the binding protein comprises V containing the amino acid sequence shown in SEQ ID NO:

137. H and V containing the amino acid sequence shown in SEQ ID NO: 141 L (RmAb5), or a sequence that has at least 70% identity with it.

56. A bivalent, trivalent, tetravalent or multivalent binding protein comprising at least one binding protein or combination thereof as described in any one of claims 1 to 55.

57. The divalent, trivalent, tetravalent or multivalent binding protein of claim 56, comprising a binding protein containing the amino acid shown in SEQ ID NO: 1 (C8), or a sequence having at least 76% identity with it, or its humanized, deimmunized or germline form.

58. The divalent, trivalent, tetravalent or multivalent binding protein of claim 56 or claim 57, comprising a binding protein containing the amino acid shown in SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or a humanized, deimmunized or germline form thereof.

59. The bivalent, trivalent, tetravalent, or multivalent binding protein of any one of claims 56 to 58, comprising: (i) a binding protein comprising the amino acids shown in SEQ ID NO: 1 (C8), or a sequence having at least 76% identity with it, or its humanized, deimmunized, or germline form; and (ii) A binding protein comprising the amino acid shown in SEQ ID NO: 5 (G6), or a sequence having at least 76% identity with it, or its humanized, deimmunized or germline form.

60. The bivalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 59, wherein the binding protein is a bicomplementary site.

61. The divalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 59, wherein the binding protein is of multiple complementary morphologies.

62. The bivalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 61, wherein the binding protein comprises a linker selected from one or more of SEQ ID NO: 65 to SEQ ID NO:

77.

63. The bivalent binding protein of any one of claims 56 to 61, wherein the binding protein comprises a sequence selected from the group consisting of C3C (SEQ ID NO: 57), C4C (SEQ ID NO: 58), G3G (SEQ ID NO: 59), G4G (SEQ ID NO: 60), C3G (SEQ ID NO: 61), C4G (SEQ ID NO: 62), G3C (SEQ ID NO: 63), and G4C (SEQ ID NO: 64), or a sequence having at least 76% identity with the thereof.

64. The tetravalent binding protein of any one of claims 56 to 61, wherein the binding protein comprises a sequence selected from the group consisting of C4C-Fc (SEQ ID NO: 79), G4G-Fc (SEQ ID NO: 80), C4G-Fc (SEQ ID NO: 81), G4C-Fc (SEQ ID NO: 82), C3G-Fc (SEQ ID NO: 159), G3C-Fc (SEQ ID NO: 161), C3C-Fc (SEQ ID NO: 155), and G3G-Fc (SEQ ID NO: 157), or a sequence having at least 76% identity with the thereof.

65. The tetravalent binding protein of claim 64, wherein the binding protein comprises the sequence C4G-Fc (SEQ ID NO: 81).

66. An isolated nucleic acid encoding the amino acid sequence of the isolated binding protein of any one of claims 1 to 55 or the amino acid sequence of the divalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 65.

67. A vector comprising the nucleic acid of claim 66.

68. A host cell comprising the nucleic acid of claim 66 or the vector of claim 67.

69. The host cell of claim 68, wherein the host cell is selected from bacterial cells, mammalian cells or insect cells.

70. A method for producing a binding protein according to any one of claims 1 to 55 or a bivalent, trivalent, tetravalent or multivalent binding protein according to any one of claims 56 to 65, comprising culturing a host cell according to claim 34 or claim 35 in a cell culture medium and expressing the binding protein or the bivalent, trivalent, tetravalent or multivalent binding protein.

71. A kit or combination package comprising the binding protein of any one of claims 1 to 55 or the bivalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 65.

72. The kit or combination device of claim 71, wherein the kit or combination device comprises a strip, chip or cassette for immediate use.

73. The kit or combination device of claim 71 or claim 72, wherein the kit or combination device is selected from: lateral flow assay, ELISA, biosensor-based assay and bead assay.

74. Lateral flow measurement, which includes: solids A support, the solid support comprising a binding protein of any one of claims 1 to 55 and / or a divalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 65 fixed on the solid support.

75. Lateral flow measurement, which includes: (i) Detector-binding proteins coupled to detectable tags; (ii) Capture-binding proteins in the capture region on a solid support, The binding protein in (i) and / or (ii) is the binding protein of any one of claims 1 to 55 or the bivalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 65.

76. The lateral flow measurement of claim 74 or claim 75, further comprising a red blood cell capture portion.

77. The lateral flow assay of any one of claims 74 to 76, wherein the erythrocyte capture portion is selected from erythrocyte binding protein and blood retention pad.

78. The lateral flow assay of claim 77, wherein the blood retention pad is selected from one or more of the following: FR1, MDI, Vivid GX, CytoSep1663, CytSep 1660, CytoSep1668, Vivid GR, Vivid GX, Vivid GR and Vivid GF.

79. The lateral flow assay of claim 77, wherein the erythrocyte binding protein is selected from binding proteins targeting the following: blood group glycoprotein B, Lewis, CD238, and CD234.

80. The lateral flow measurement according to any one of claims 74 to 79, wherein the lateral flow measurement comprises one or more of a lancet, a blood collection unit, and a blister pack containing a buffer solution.

81. The lateral flow assay of any one of claims 74 to 80, wherein the binding protein binds to the capture region of the solid support in the presence of one or more excipients.

82. The lateral flow determination of claim 81, wherein the excipient is selected from one or more of the following: sucrose, bovine serum albumin (BSA), immunoglobulin, trehalose, casein, lactose, galactose, and detergents (e.g., Tween).

83. The lateral flow measurement according to any one of claims 74 to 82, further comprising a control line containing ALT or ALT epitopes on the solid support.

84. The lateral flow assay of claim 74 or 83, wherein ii) is a tetravalent binding protein.

85. The lateral flow assay according to any one of claims 74 to 84, wherein the binding protein in i) and / or ii) is a nanobody or a rabbit monoclonal antibody.

86. The lateral flow determination according to any one of claims 74 to 85, wherein the binding protein in i) and / or ii) is a divalent, trivalent, tetravalent or multivalent binding protein.

87. The lateral flow assay according to any one of claims 74 to 85, wherein the binding protein in i) is a nanobody or a rabbit monoclonal antibody, and wherein the binding protein in ii) is a rabbit polyclonal antibody; or wherein the binding protein in ii) is a nanobody or a rabbit monoclonal antibody, and wherein the binding protein in i) is a rabbit polyclonal antibody.

88. The lateral flow measurement according to any one of claims 74 to 87, wherein the measurement is capable of detecting about 0.1 ng to about 5 ng of ALT1 in the sample.

89. The lateral flow determination according to any one of claims 74 to 88, wherein the binding protein in i) is selected from: C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

90. The lateral flow determination according to any one of claims 74 to 89, wherein the binding protein in ii) is selected from: C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5.

91. The lateral flow assay of claim 89, wherein the binding protein in ii) is a rabbit polyclonal ALT-1 antibody.

92. The lateral flow assay of claim 90, wherein the binding protein in i) is a rabbit polyclonal ALT-1 antibody.

93. The lateral flow determination of claim 87, wherein the binding protein in i) is selected from G4C, C3G and G4G.

94. The lateral flow determination of claim 87, wherein the binding protein in i) and / or ii) is selected from: C4C-Fc, G4G-Fc, C4G-Fc and G4C-Fc.

95. The lateral flow measurement according to any one of claims 74 to 94, wherein: a) When the binding protein in i) is G4C, the binding proteins in ii) are C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody; b) When the binding protein in i) is C3G, the binding proteins in ii) are C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody; c) When the binding protein in i) is G4G, the binding proteins in ii) are C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody; d) When the binding protein in i) is a rabbit polyclonal ALT-1 antibody, the binding proteins in ii) are C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4 and RmAb5; e) When the binding protein in i) is RmAb1, the binding protein in ii) is selected from C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody; f) When the binding protein in i) is RmAb2, the binding protein in ii) is selected from C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody; g) When the binding protein in i) is RmAb3, the binding protein in ii) is selected from C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody; h) When the binding protein in i) is RmAb4, the binding protein in ii) is selected from C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5, and rabbit polyclonal ALT-1 antibody; and i) When the binding protein in i) is RmAb5, the binding protein in ii) is selected from C8, G6, G4C, C3G, G4G, G3C, C4G, C3C, C4C, G3G, C4C-Fc, G4G-Fc, C4G-Fc, G4C-Fc, RmAb1, RmAb2, RmAb3, RmAb4, RmAb5 and rabbit polyclonal ALT-1 antibody.

96. The lateral flow assay according to any one of claims 74 to 95, wherein the binding protein in ii) is streaked at a concentration of about 0.05 mg / mL to about 2.5 mg / mL.

97. The kit or combination set or lateral flow assay of any one of claims 74 to 96, wherein the kit or combination set or lateral flow assay is used to detect liver injury and / or liver disease.

98. The binding protein of any one of claims 1 to 55, or the bivalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 65, or the kit or combination pack of any one of claims 71 to 73, or the use of the lateral flow assay of any one of claims 74 to 96 for detecting ALT1 in a target.

99. A method for detecting ALT1, the method comprising contacting a sample with a binding protein according to any one of claims 1 to 55 or a divalent, trivalent, tetravalent or multivalent binding protein according to any one of claims 56 to 65 to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

100. The binding protein of any one of claims 1 to 55, or the bivalent, trivalent, tetravalent, or multivalent binding protein of any one of claims 56 to 65, or the kit or combination pack of any one of claims 71 to 73, or the use of the lateral flow assay of any one of claims 74 to 96 for the detection of subjects with liver injury and / or liver disease.

101. A method for detecting a subject suffering from liver injury and / or liver disease, the method comprising contacting a sample with a binding protein according to any one of claims 1 to 55 or a divalent, trivalent, tetravalent or multivalent binding protein according to any one of claims 56 to 65 to form an antigen-binding protein complex, and directly or indirectly detecting the antigen-binding protein complex.

102. The use or method of any one of claims 98 to 101, wherein the detection of antigen-binding protein complexes above a threshold level indicates liver injury and / or liver disease in the subject.

103. The use or method according to any one of claims 98 to 102, wherein the use or method is for detecting a condition selected from: pregnancy-related conditions, eclampsia, preeclampsia, high ALT1 levels during pregnancy, acute fatty liver of pregnancy, and HELLP syndrome.

104. The use or method according to any one of claims 98 to 103, wherein the use or method is for monitoring a disease in a subject.

105. The use or method of claim 104, wherein the disease is selected from: hepatotropic viruses, non-hepatotropic viruses, bacteria, fungi, parasites, toxins or substances (e.g., therapeutic treatments), inflammatory diseases, metabolic or genetic diseases, pregnancy-related diseases, and ischemic or vascular diseases.

106. The use or method of claim 104 or claim 105, wherein the disease is selected from: hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, Coxsackie virus, mononucleosis, adenovirus, dengue virus, coronavirus-19, fatty liver disease, acute alcoholic hepatitis or alcoholic cirrhosis, anemone sting, autoimmune hepatitis, biliary tract diseases such as primary biliary cholangitis or primary sclerosing cholangitis, non-alcoholic fatty liver disease, hemochromatosis, Wilson's disease, preeclampsia, acute fatty liver disease of pregnancy, HELLP syndrome, cardiogenic / distributive shock, hypotension, heatstroke, cocaine, methamphetamine, ephedrine, acute Budd-Chiari syndrome, hepatic sinusoidal obstruction syndrome, malignancy, eclampsia, Reye's syndrome, and primary graft nonfunction after liver transplantation.

107. The use or method of any one of claims 104 to 106, wherein the method or use is for monitoring treatment in a subject.

108. The use or method of claim 107, wherein the treatment is selected from: tuberculosis treatment, neurological disease treatment, cancer treatment, and inflammatory bowel disease treatment.

109. The use or method of claim 107, wherein the treatment is selected from: anti-TNFα antibodies, infliximab, adalimumab, Bruton's tyrosine kinase inhibitors, 2MHRZ / 2MHR, 2EMRZ / 2MR, acetaminophen, antibiotics, anticonvulsants, statins, NSAIDs, and herbal / nutritional supplements.

110. A binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site of an antibody comprising an amino acid sequence selected from: a) GFSLNNYN (SEQ ID NO: 110) as heavy chain CDR1, ITAGGNI (SEQ ID NO: 111) as heavy chain CDR2, ARDLAGNVYYDFDL (SEQ ID NO: 112) as heavy chain CDR3, ENIYSG (SEQ ID NO: 114) as light chain CDR1, KAS as light chain CDR2 and QGGTYSSGADIS (SEQ ID NO: 115) as light chain CDR3; b) GFDLSSYY (SEQ ID NO: 117) as heavy chain CDR1, IWLGSGNI (SEQ ID NO: 118) as heavy chain CDR2, ARGWLDDSFDP (SEQ ID NO: 119) as heavy chain CDR3, VSVHYNKW (SEQ ID NO: 121) as light chain CDR1, GAS as light chain CDR2 and AGGYSSGSDKFA (SEQ ID NO: 122) as light chain CDR3; c) GFSLITYS (SEQ ID NO: 124) as heavy chain CDR1, ISASGTA (SEQ ID NO: 125) as heavy chain CDR2, ARGSGPSGIESYKL (SEQ ID NO: 126) as heavy chain CDR3, QSIGNY (SEQ ID NO: 128) as light chain CDR1, RAS as light chain CDR2, and QGYYGIHIT (SEQ ID NO: 129) as light chain CDR3; d) GIDLSVNA (SEQ ID NO: 131) as heavy chain CDR1, IHTYDVT (SEQ ID NO: 132) as heavy chain CDR2, ARKDWTSGDSFNP (SEQ ID NO: 133) as heavy chain CDR3, QSISTA (SEQ ID NO: 135) as light chain CDR1, SAS as light chain CDR2 and QCTYHSSSTGYA (SEQ ID NO: 136) as light chain CDR3; e) GFSLSNDA (SEQ ID NO: 138) as heavy chain CDR1, ISSAGRP (SEQ ID NO: 139) as heavy chain CDR2, ARDKGYYSYHYAYDTRLDL (SEQ ID NO: 140) as heavy chain CDR3, QSISSSY (SEQ ID NO: 142) as light chain CDR1, RVS as light chain CDR2, and QGTYGSGSSSYGNA (SEQ ID NO: 143) as light chain CDR3; and f) GPAVSNVA (SEQ ID NO: 2) as CDR1, ITWSGWT (SEQ ID NO: 3) as CDR2, and NLIGLRVGPENKY (SEQ ID NO: 4) as CDR3; Any one or more of the CDRs have 0, 1, 2 or 3 amino acid changes compared to the sequence shown in the corresponding SEQ ID No. Wherein, when the amino acid sequence is a), the binding protein is a nanobody, and wherein, when the amino acid sequence is b), c), d), e), or f), the binding protein is an antibody.

111. The binding protein of claim 110, wherein the binding protein does not significantly bind to ALT1 containing alanine substitutions at one or more of the following positions: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

112. A binding protein that binds to alanine aminotransferase 1 (ALT1) and does not bind significantly and / or detectably to denatured ALT1, said binding protein comprising an antigen-binding site for an antibody, and said binding protein not binding significantly and / or detectably to ALT1 containing alanine substitutions at one or more of the following locations: 19 Lys, 24 Asp, 48 Leu, 154 Val, 183 Leu, 202 Val, 212 Arg, 239 Val, 260 Arg, 266 Arg, 304 Gln, 358 Leu, 430 Glu, and 433 Leu.

113. A solid or semi-solid support having the binding protein of any one of claims 1 to 55, or the divalent, trivalent, tetravalent or multivalent binding protein of any one of claims 56 to 65, immobilized thereon.

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