WO2020264410A1

WO2020264410A1 - Troponin t binding agents and uses thereof

Info

Publication number: WO2020264410A1
Application number: PCT/US2020/039970
Authority: WO
Inventors: Richard A. Lerner; Jia XIE
Original assignee: Sensor-Kinesis Corporation
Priority date: 2019-06-26
Filing date: 2020-06-26
Publication date: 2020-12-30

Abstract

Presented herein, in certain embodiments, are compositions comprising binding agents that specifically bind to Troponin T and uses thereof. In some aspects, presented herein is a binding agent that specifically binds to cTn-T, or a portion thereof (sometimes referred therein as a cTn-T binding agent). In certain embodiments, a binding agent specifically binds to a variant of human cTn-T and/or to an extracellular domain of a human cTn-T comprising one or more naturally occurring variants.

Description

TROPONIN T BINDING AGENTS AND USES THEREOF

FIELD OF THE INVENTION

[0001] Embodiments of the invention relate to compositions comprising novel binding agents that specifically bind to Troponin T, as well as kits and uses thereof.

INTRODUCTION

[0002] Troponin is a protein complex found in cardiac and skeletal muscle. Troponin comprises three regulatory protein subunits named Troponin C, Troponin I and Troponin T. Cardiac Troponin (cTn) comprises Troponin C, a cardiac specific isoform of Troponin I (cardiac Troponin-I (cTn-I)) and a cardiac specific isoform of Troponin T (cardiac Troponin T (cTn-T). The presence and amounts of cTn, cTn-I, and cTn-T in peripheral body fluids, especially blood and plasma, can be sensitive and specific indicators of damage to the heart muscle.

[0003] Presented herein are novel binding agents that bind specifically to cardiac specific isoforms of Troponin T, compositions thereof and methods of using the same to detect and/or quantitate the amount of cTn and/or cTn-T in a sample.

SUMMARY

[0004] In some aspects, presented herein is a binding agent that specifically binds to cTn-T, or a portion thereof (sometimes referred to herein as a cTn-T binding agent). In certain embodiments, a binding agent specifically binds to a variant of human cTn-T and/or to an extracellular domain of a human cTn-T comprising one or more naturally occurring variants.

[0005] In some aspects, presented herein is a cTn-T binding agent comprising one or more light chain complementarity determining regions selected from a CDR-L1, a CDR-L2 and a CDR-L3, wherein the CDR-L1 comprises a polypeptide sequence having at least 80% identity to an amino acid sequence of a CDR-L1 selected from Table 1, the CDR-L2 comprises a polypeptide sequence having at least 80% identity to an amino acid sequence of a CDR-L2 selected from Table 2, and the CDR-L3 comprises a polypeptide sequence having at least 80% identity to an amino acid sequence of a CDR-L3 selected from Table 3, where the cTn-T binding agent specifically binds to a cTn-T, or a portion thereof. In some embodiments, the cTn-T binding agent comprises one or more heavy chain complementarity determining regions selected from a CDR-H1, a CDR-H2 and a CDR-H3, wherein the CDR- H1 comprises a polypeptide sequence having at least 80% identity to an amino acid sequence of a CDR-H1 selected from Table 5, the CDR-H2 comprises a polypeptide sequence having at least 80% identity to an amino acid sequence of a CDR-H2 selected from Table 6, and the CDR-H3 comprises a polypeptide sequence having at least 80% identity to an amino acid sequence of a CDR-H3 selected from Table 7, where the cTn-T binding agent specifically binds to a cTn-T, or a portion thereof.

[0006] In some aspects, presented herein is a cTn-T binding agent comprising a CDR-L1, a CDR-L2 and a CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 are selected from a CDR listed in Tables 1, 2 and 3, respectively, and the cTn-T binding agent comprises a CDR- Hl, a CDR-H2 and a CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 are selected from a CDR listed in Tables 5, 6 and 7, respectively, where the cTn-T binding agent specifically binds to a cTn-T, or a portion thereof.

[0007] Certain aspects of the technology are described further in the following description, examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The drawings illustrate embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

[0009] Fig. 1 shows an illustration of a Troponin Complex comprising Troponin I (Tnl), Troponin C (TnC), Troponin T (TnT), Actin and Tropomysin.

[0010] Fig. 2 shows a flow chart of an exemplary screening process by which cTn-T specific binding agents were isolated using a phage display methodology.

[0011] Fig. 3 shows an illustration of an exemplary immunoassay that was used to detect troponin T using a sandwich assay format where a first cTn-T-specific binding agent (e.g., Antibody- 1 (scFV Clone 1)) is immobilized on a surface (Detection sensor), cTn-T is allowed to bind to the first binding agent, and binding of a second cTn-T-specific binding agent (e.g. , Antibody-2 (scFV Clone 19)) is then detected by the sensor which indicates the presence and/or amount of cTn-T in a sample.

[0012] Fig. 4 shows a graphical results of a sandwich immunoassay utilizing the method illustrated in Fig. 3 where Antibody- 1 (scFV Clone 1) was immobilized on the surface of the detection sensor and allowed to bind a fixed amount of antigen (cTn). A second binding agent (flow Antibody 1 (scFV Clone 1), flow Antibody 2 (scFV Clone 19), flow Antibody 3 (scFV Clone 36) or flow Antibody 4 (scFV Clone 42)) was then added and binding of the second antibody was detected. Relative binding affinity of the second antibody is shown on the y-axis.

[0013] Fig. 5 shows a graphical results of sandwich immunoassay utilizing the method illustrated in Fig. 3 where Antibody-2 (scFV Clone 19) was immobilized on the surface of the detection sensor and allowed to bind a fixed amount of antigen (cTn). A second binding agent (flow Antibody 1 (clone 1), flow Antibody 2 (scFV Clone 19), flow Antibody 3 (scFV Clone 36) or flow Antibody 4 (scFV Clone 42)) was then added and binding of the second antibody was detected. Relative binding affinity of the second antibody is shown on the y- axis.

[0014] Fig. 6 shows a graphical results of sandwich immunoassay utilizing the method illustrated in Fig. 3 where Antibody-3 (scFV Clone 36) was immobilized on the surface of the detection sensor and allowed to bind a fixed amount of antigen (cTn). A second binding agent (flow antibody 1 (scFV Clone 1), flow Antibody 2 (scFV Clone 19), flow Antibody 3 (scFV Clone 36) or flow Antibody 4 (scFV Clone 42)) was then added and binding of the second antibody was detected. Relative binding affinity of the second antibody is shown on the y-axis.

[0015] Fig. 7 shows a graphical results of sandwich immunoassay utilizing the method illustrated in Fig. 3 where antibody-4 (scFV Clone 42) was immobilized on the surface of the detection sensor and allowed to bind a fixed amount of antigen (cTn). A second binding agent (flow Antibody 1 (scFV Clone 1), flow Antibody 2 (scFV Clone 19), flow Antibody 3 (scFV Clone 36) or flow Antibody 4 (scFV Clone 42)) was then added and binding of the second antibody was detected. Relative binding affinity of the second antibody is shown on the y-axis. [0016] Fig. 8 shows one possible configuration of the relative binding positions of each of the binding agents of clone 1, clone 19, clone 36 and clone 42 on cTn. As the illustration suggests, it is possible that the binding agents of clone 1 (Ab-1), clone 19 (Ab-2) and clone 36 (Ab-3) bind to overlapping epitopes while the binding agent of clone 42 appears to bind to a different epitope on cTn-T. Clone 42 may have orthogonal epitope recognition compared to the other three binding agents tested. The binding affinity (kD) of each of the binding agents Ab-1, Ab-2, Ab-3 and Ab-4 has been determined to be about 0.7 nM, 0.7 nM, 0.9 nM and 2.5 nM respectively.

[0017] Fig. 9A and 9B shows a comparison of kinetic parameters for anti-cTn-I and anti- cTn-T antibodies and anti-cTn-T scFV binding agents obtained from SPR analysis. Fig. 9A shows association constants (K_a) and dissociation constants (K_d) and Fig. 9B shows equilibrium association affinity constants (KD).

DETAILED DESCRIPTION

[0018] Presented herein, in some embodiments, are binding agents that specifically bind to cardiac Troponin T (cTn-T), or a portion thereof, as well as compositions and uses thereof.

In some embodiments cTn-T is a mammalian cTn-T. In some embodiments cTn-T is a primate cTn-T. In some embodiments cTn-T is a human cTn-T. An example of a cardiac specific isoform of human Troponin T is provided in SEQ ID NO:l herein (e.g., also see UniProtKB - P45379). Cardiac Troponin T generally comprises an immature polypeptide sequence of 310 amino acids which includes an N-terminal single sequence from amino acids 1-22, an extracellular domain from about amino acid 23-254, a transmembrane domain from about amino acid 255 to 275 and a cytoplasmic domain from about amino acid 276 to 310, numbered from the N-terminus to the C-terminus (referring to the amino acid sequence of SEQ ID NO:l). Several isoforms and variants of mammalian cTn-T have been identified and the exact positions of the various domains of cTn-T may vary among isoforms and species. Binding agents provided herein may bind to several different cardiac specific isoforms of Troponin T and may cross-react with a cTn-T derived from several different mammalian species. In some embodiments, a binding agent provided herein binds specifically to a cardiac specific isoform of human cTn-T.

[0019] Provided herein, in some embodiments, are methods and compositions for analyzing a sample. A sample (e.g., a sample comprising cTn-T, or suspected of comprising cTn-T) can be obtained from a suitable subject. A sample can be isolated or obtained directly from a subject or obtained indirectly from a subject. For example, in some embodiments, a sample is provided by, or obtained indirectly from a third party, entity or individual (e.g., a medical professional who obtained a sample directly from a patient). A sample can be any specimen that is isolated or obtained from a subject or part thereof. A sample can be any tissue or fluid that is isolated or obtained from one or more subjects. Non-limiting examples of samples include fluids or tissues obtained or derived from a subject, including, without limitation, blood or a blood product (e.g. , serum, plasma, platelets, buffy coats, lymphatic fluid or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid (CSF), spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells, neurons) or parts thereof (e.g. , mitochondrial, nucleus, extracts, lysates, or the like), urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof. A fluid or tissue sample may or may not be acellular (e.g. , cell-free). Non-limiting examples of tissues include organ tissues (e.g. , liver, kidney, lung, heart, thymus, adrenals, skin, bladder, reproductive organs, intestine, colon, spleen, brain, bone, the like or parts thereof), epithelial tissue, hair, hair follicles, ducts, canals, bone, eye, nose, mouth, throat, ear, nails, the like, parts thereof or combinations thereof. A sample may comprise cells or tissues that are normal, healthy, diseased (e.g. , infected), damaged, and/or cancerous (e.g., cancer cells). In some embodiments, a sample comprises cTn, cTn-T, mixtures thereof or portions thereof.

[0020] The term“subject” refers to a mammal. Non-limiting examples of a mammal includes humans, primates, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, and pigs) and experimental animals (e.g., mouse, rat, rabbit, and guinea pig). In some embodiments a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female.

[0021] In some embodiments, a subject has, or is suspected of having, cardiac muscle damage. In some embodiments, a subject is suffering from, or is suspected of suffering from a cardiac condition or event, non- limiting examples of which include myocardial infarction, primary or secondary ischemic myocardial injury, coronary intervention and spasm, cardiac arrhythmia, large pulmonary emboli, heart failure, acute coronary syndrome,

cardiomyopathy, atrial fibrillation, tachycardia, non-ischemic myocardial injury,

myopericarditis, cardiac trauma, cardiac toxicity, and the like. A subject suspected of suffering from a cardiac condition or event may have a family history of cardiac conditions or cardiac events or may have one or more symptoms of a cardiac condition or event. In some embodiments, a subject suspected of suffering from a cardiac condition or event may have a genetic variation that is correlated with, or predictive of, a certain cardiac condition or event. In some embodiments, a subject suspected of suffering from a cardiac condition or event may have been exposed to one or more environmental elements (e.g. , a food, toxins,

chemotherapy, or the like) that is correlated with, or suspected of causing a cardiac condition or event.

[0022] Presented herein are binding agents that specifically bind to cTn-T. In certain embodiments, a binding agent comprises or consists of one or more polypeptides or one or more proteins that bind specifically to at least one antigen (e.g., cTn-T). A binding agent often comprises at least one antigen binding portion (i.e. a binding portion). An antigen binding portion of a binding agent is that portion that binds specifically to an antigen. In certain embodiments a binding portion of a binding agent comprises or consists of a single polypeptide (e.g. , single chain antibody). In some embodiments a binding portion of a binding agent comprises or consists of two polypeptides. In some embodiments a binding portion of a binding agent comprises or consists of 2, 3, 4 or more polypeptides. In some embodiments a binding agent comprises one or more structural portions (e.g., scaffolds, linkers, structural polypeptides, constant regions and/or framework regions). In some embodiments a binding agent, or binding portion thereof is attached to a substrate (e.g. , a polymer, a non-organic material, silicon, a bead, and the like).

[0023] A binding agent may comprise one antigen binding portion or multiple antigen binding portions. For example, a binding agent that comprises one binding portion is sometimes referred to as monovalent. A binding agent that comprises two binding portions is sometimes referred as divalent. A binding agent that comprises three binding portions is sometimes referred as trivalent. A binding agent that comprises two or more binding portions is sometimes referred as multivalent. In some embodiments a binding agent comprises 1, 2,

3, 4, 5, 6, 7, 8, 9, or 10 or more binding portions hi certain embodiments, all of the binding portions of a multivalent binding agent bind to the same antigen. In certain embodiments, all of the binding portions of a multivalent binding agent comprise one or more polypeptide sequences that are at least 90%, at least 95%, at least 99% or 100% identical.

[0024] In some embodiments a binding agent comprises an antibody, or a binding portion thereof (e.g. , a binding portion thereof). In certain embodiments, a binding agent comprises or consists of an antibody, an antibody fragment and/or an antigen binding portion of an antibody (e.g., a binding fragment, i.e., a binding portion thereof). In some embodiments a binding agent is an antibody (e.g., a monoclonal antibody and/or a recombinant antibody). A binding agent or antibody can be generated, manufactured or produced by a suitable method. In some embodiments a binding agent is monoclonal. In some embodiments a binding agent is a monoclonal antibody derived from a suitable species. Certain non-limiting examples of a binding agent include monoclonal antibodies, chimeric antibodies, antibody binding fragments (e.g. , an antigen binding portion of an antibody), a CDR-grafted antibody, a humanized antibody, and a human antibody, or binding portions thereof. Human antibodies can be obtained by any suitable method. For example, human antibodies can be obtained from trans-chromosomal animals engineered to produce fully human antibodies. In certain embodiments, a binding agent is not polyclonal, and/or is not a polyclonal antibody.

[0025] In some embodiments a binding agent is derived, produced, obtained, isolated, and/or purified from a suitable species. In some embodiments a binding agent is derived, produced, obtained, isolated, and/or purified from a rabbit, goat, horse, cow, rat, mouse, fish, bird, or llama, for example. In some embodiments a binding agent is derived, produced, obtained, isolated, and/or purified from a bird (e.g., a chicken, or a bird egg). In some embodiments a binding agent is derived, produced, obtained, isolated, and/or purified from a plant (e.g. , a recombinant binding agent produced by a genetically engineered plant). In some embodiments a binding agent is derived, produced, obtained, isolated, and/or purified from a suitable mammal. In certain embodiments a suitable mammal is a genetically altered mammal (e.g., a trans-chromosomal or transgenic mammal) engineered to produce antibodies comprising human heavy chains and/or human light chains or portions thereof. In some embodiments a binding agent is produced, obtained, isolated, or purified from a prokaryotic or eukaryotic cell (e.g., a recombinant binding agent produced by a genetically engineered cell). In some embodiments a binding agent is produced, obtained, isolated, or purified from a vims or phage (e.g., a recombinant binding agent produced by a genetically engineered phage where bacteria are used to propagate the phage; e.g., phage display). In some embodiments, a binding agent is produced by a suitable process comprising phage display technology (e.g., see Antibody Phage Display: Methods and Protocols (Methods in Molecular Biology) 2^nd Edition, Editors: Robert Aitken, Humana Press, Springer Science & Business Media, 2009).

[0026] A binding agent can be expressed, isolated from and/or purified from a suitable expression system non-limiting examples of which include a suitable bacteria, phage, insect, vims, plant or mammalian expression system. For example, a nucleic acid encoding a binding agent can be introduced into a suitable mammalian cell that expresses and secretes the binding agent into the cell culture media. Any suitable mammalian cell line can be used. In certain embodiments a mammalian cell line is a Chinese hamster ovary (CHO) cell line. A method of producing a binding agent may comprise one or more of (i) introducing one or more nucleic acids into a suitable cell line wherein the nucleic acid directs the expression of a binding agent; (ii) culturing the cell line using a suitable culturing method for a period of time that allows expression of the binding agent; (iii) harvesting the cell line (e.g. , by way of generating a lysate) or harvesting conditioned media from the cell line (e.g., where the binding agent is secreted); and (iv) isolating and/or purifying the binding agent using a suitable method.

[0027] In certain embodiments, a monoclonal antibody or a monoclonal binding agent is a substantially homogeneous population of binding agents, or binding fragments thereof, where each individual binding agent in the population is substantially identical and/or binds to the same epitope, with the exception of possible variants that may arise during production of a monoclonal binding agent. In some embodiments such variants generally are absent or may be present in minor amounts. In contrast to polyclonal antibody preparations which typically include a population of different antibodies directed against different determinants (epitopes), each binding agent of a population of monoclonal binding agents often binds a single determinant on an antigen. Isolated monoclonal binding agents are often not contaminated by other immunoglobulins. Although one or more different monoclonal binding agents or polyclonal antibodies may be purposely added to a composition to form a mixture.

[0028] The modifier“monoclonal” is not to be construed as requiring production of a binding agent by any particular method. A monoclonal binding agent can be produced by any suitable method. For example, in certain embodiments, a monoclonal antibody is made by the hybridoma method (e.g., as described by Kohler et al, Nature, 256:495 (1975)), or a variation thereof. In some embodiments a monoclonal binding agent is made by a recombinant DNA method. For example, a monoclonal binding agent can be made by screening a recombinant library using a suitable expression system (e.g., a phage display expression system). In some embodiments a monoclonal binding agent is isolated from a phage library of binding agents, for example by using a technique described in Clackson et al, Nature, 352:624-628 (1991) and/or Marks et al, J. Mol Biol, 222:581-597 (1991), or a variation thereof.

[0029] In certain embodiments a binding agent comprises one or more constant regions (e.g., constant regions derived from an antibody, e.g., a mammalian antibody). A binding agent may comprise any suitable constant region of an antibody, or one or more portions thereof. In certain embodiments a binding agent comprises a constant region of an antibody light chain and/or a constant region of an antibody heavy chain. In some embodiments a binding agent comprises a lambda (l) light chain constant region, or a portion thereof. In some embodiments a binding agent comprises a kappa (K) light chain constant region, or a portion thereof. In some embodiments a binding agent comprises a polypeptide that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a polypeptide sequence of a light chain constant region of a mammalian antibody, or portion thereof. In some embodiments a binding agent comprises a polypeptide that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a polypeptide sequence of a light chain constant region of a human antibody. In some embodiments a binding agent does not include a light chain constant region.

[0030] In certain embodiments a binding agent comprises a constant region of an antibody heavy chain. A binding agent can include any suitable heavy chain constant region, or portion thereof. In mammals, an antibody can have at least five types/classes of Ig heavy chains denoted as IgA, IgD, IgE, IgG, and IgM, which are determined by the presence of distinct heavy chain constant regions, or portion thereof (e.g., CHI, CL, CH2, CH3 domains). In some embodiments a binding agent comprises one or more heavy chain constant regions of an IgM, IgD, IgA, or IgE isotype, or a portion thereof. In some embodiments a binding agent comprises a heavy chain constant region of an IgGi, IgG2, IgG3 or IgG4, or one or more portions thereof. In some embodiments a binding agent comprises a polypeptide that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to a polypeptide sequence of a heavy chain constant region of a mammalian antibody. In some embodiments a binding agent comprises a polypeptide that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical or 100% identical to a polypeptide sequence of a heavy chain constant region of a human antibody. In some embodiments a binding agent comprises one or more additions, deletions and/or modification to a constant region. A binding agent is sometimes modified to change the antibody class, or isotype of a binding agent. In some embodiments a binding agent comprises one or more additions, deletions and/or modification (one or more amino acid substitutions, deletions or additions) to modify one or more functions of a binding agent, for example to abolish, enhance or decrease serum half-life, Fc receptor binding, complement binding (e.g., Clq binding), glycosylation, sialylation, cellular toxicity, antibody-dependent cell-mediated phagocytosis (ADCP), antibody dependent cellular cytotoxicity (ADCC), and the like. In some embodiments a binding agent does not include one or more portions of a heavy chain constant region or light chain constant region. In some embodiments a binding agent does not include a heavy chain constant region.

[0031] In some embodiments a binding agent comprises or consists of one or more variable regions of an antibody, or a portion thereof. In some embodiments a binding agent comprises one or more light chain variable regions, or a portion thereof. In some embodiments a binding agent comprises one or more heavy chain variable regions, or a portion thereof. In certain embodiments a binding agent comprises at least one light chain variable region and at least one heavy chain variable region. A light chain variable region and heavy chain variable region can be on the same or different polypeptides.

[0032] In certain embodiment, a binding agent is a non-naturally occurring binding agent. Non-limiting examples of non-naturally occurring binding agents include monoclonal binding agents (e.g., monoclonal antibodies), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single-chain antibodies, Fab, Fab’, F(ab’)2, Fv fragment, single-chain Fv (scFV), scFV-Fc, (scFv)2-Fc, disulfide-linked Fvs (sdFv), VL, VH, diabody (Dab), triabody (trivalent), tetrabody (tetravalent), minibody ((scFV-CH3)2), IgGdeltaCH2, the like, combinations thereof, and antigen binding portions thereof. [0033] In some embodiments a binding agent comprises or consists of a Fab, Fab’, F(ab’)2, Fv fragment, single-chain Fv (scFv), diabody (Dab), synbody, the like and/or a combination or portion thereof (see, e.g. , U.S. Patent Nos. 6,099,842 and 5,990,296). In certain embodiments an scFV is produced as a monomer, dimer, trimer or multimer. In some embodiments, two or more scFV binding agents are attached to a suitable linker thereby producing dimeric or multimeric binding agents. In some embodiments a binding agent comprises a single-chain polypeptide comprising one or more antigen binding portions. For example, a single-chain binding agent can be constructed by joining a heavy chain variable region, or antigen binding portion thereof, and a light chain variable region, or antigen binding portion thereof, with a linker (e.g. , an amino acid, a polypeptide linker) using recombinant molecular biology processes. Such single chain binding agents often exhibit specificities and affinities for an antigen similar to a parent two-chain monoclonal binding agent. In some embodiments, a binding agent comprises engineered regions such as CDR- grafted or humanized portions.

[0034] Nucleic acids, or portions thereof, that encode a polypeptide of a binding agent may be cloned, subcloned, rearranged or modified for recombinant expression by a suitable cloning procedure and subsequently expressed using a suitable expression system by a method known to those skilled in the art (e.g., see Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; Antibody Engineering: Methods and Protocols, Vol. 248 of Methods in molecular biology, edited by Benny K. C. Lo,

Springer Science & Business Media, 2004; Antibody Engineering, Vol. 1, Roland E.

Kontermann, Stefan Diibel, Edition 2, Publisher Springer Science & Business Media, 2010; Antibody Phage Display: Methods and Protocols, Biomed Protocols, Vol. 178 of Methods in molecular biology, Editors Philippa M. O’Brien, Robert Aitken, Springer Science & Business Media, 2004).

[0035] In mammals, the heavy chain variable region and light chain variable region of an antibody each contribute three CDRs (complementarity determining regions) commonly referred to as CDR1, CDR2 and CDR3, that are separated and/or flanked by framework regions (e.g., FR1, FR2, FR3 and FR4). The term“CDR” as used herein refers to an amino acid sequence of a polypeptide identified as a complementarity determining region. In certain embodiments, definitive delineation of a CDR polypeptide sequence and

identification of residues comprising the binding site of a binding agent is accomplished by solving the structure of a binding agent and/or solving the structure of a binding agent- antigen complex. In certain embodiments, this can be accomplished by any suitable method, such as X-ray crystallography and/or computer modeling. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR sequences of a binding agent or antibody. For example, the amino acid sequence and/or location of CDRs in a polypeptide sequence of a binding agent, an antibody, a binding portion thereof or variable region thereof, can be identified using a suitable method, non-limiting examples of which include the Rabat system (e.g., see Rabat, E. A., et al, 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, as well as Johnson, G. and Wu, T. T. 2000, Nucleic Acids Research), and/or the Chothia Numbering Scheme (e.g., Chothia & Lesk, (1987) J. Mol. Biol, 196:901-917; Chothia et al, Nature, (1989) 342:878- 883; and Al-Lazikani et al, (1997) JMB 273,927-948). In some embodiments the amino sequence and/or location of CDRs of an antibody can be identified using the AbM method and/or contact method. The“AbM” definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure (see e.g., Martin et al, Proc. Natl. Acad. Sci. (USA), 86:9268-9272 (1989);“AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UR; Oxford Molecular, Ltd.). The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al,“Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl, 3: 194-198 (1999). In certain embodiments, a contact definition is based on an analysis of the available complex crystal structures (see e.g., MacCallum et ah, J. Mol. Biol, 5:732-45 (1996)).

[0036] In some embodiments a binding agent and/or an antigen binding portion of a binding agent comprises at least 2, at least 3, at least 4, at least 5 or at least 6 CDRs. In some embodiments a binding agent comprises 3 to 60 CDRs (e.g., for binding agents having multiple antigen binding portions). In some embodiments a binding agent comprises 3 to 12 CDRs. In some embodiments an antigen binding portion of a binding agent comprises 1 to 6 CDR amino acid sequences.

[0037] In certain embodiments, a binding agent and/or an antigen binding portion of a binding agent comprises one, two or three CDRs of a light chain variable region. In some embodiments a light chain variable region of a binding agent comprises one or more CDRs (e.g., one, two, three, or more CDRs). The amino acid sequences representing a CDR in a light chain variable region of an antibody or binding agent are often referred to as CDR-L1, CDR-L2, and CDR-L3 which are numbered sequentially (/._<?., LI, L2 and L3). In a mammalian antibody, the CDRs are numbered sequentially in a direction from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) of a light chain variable region. For example, in a polypeptide representing a light chain variable region of a binding agent, CDR-L1, when present, is the most N-terminal light chain CDR; CDR-L3, when present, is the most C-terminal light chain CDR; and CDR-L2, when present, is located (i) between CDR-L1 and CDR-L3, (ii) on the N-terminal side of CDR-L3 or (iii) on the C-terminal side of CDR-L1, of a light chain variable region or binding portion of a binding agent. Non limiting examples of amino acid sequences of a CDR-L1, CDR-L2 and CDR-L3 are provided in Tables 1-3, respectively. A light chain variable region or antigen binding portion of a binding agent described herein may comprise any combination of a CDR-L1, a CDR-L2, and a CDR-L3 disclosed herein, wherein the binding agent retains specific binding to cardiac Troponin T, or a portion thereof. In certain embodiments, a light chain variable region or antigen binding portion of a binding agent described herein comprises three light chain CDRs consisting of an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-L3 selected from Table 3, an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-L2 selected from Table 2 and an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-L1 of Table 1. In certain embodiments, a light chain variable region or antigen binding portion of a binding agent described herein comprises an amino acid sequence at least 70% identical to a CDR-L3 selected from Table 3, an amino acid sequence at least 70% identical to a CDR-L2 selected from Table 2 and an amino acid sequence at least 70% identical to a CDR-L1 selected from Table 1, where the binding agent retains specific binding to a human cardiac Troponin T, an isoform thereof, or a portion thereof.

[0038] In some embodiments a cTn-T binding agent comprises one or more light chain CDRs that are at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the CDR sequences listed in Tables 1, 2 or 3. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-L1 that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the sequences shown in Table 1. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-L1 of any one of the sequences shown in Table 1.

TABLE 1: CDR-L1 Sequences

[0039] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-L2 that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the sequences shown in Table 2. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-L2 of any one of the sequences shown in Table 2.

TABLE 2: CDR-L2 Sequences

[0040] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-L3 that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the sequences shown in Table 3. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-L3 of any one of the sequences shown in Table 3.

TABLE 3: CDR-L3 Sequences

[0041] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a light chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to an amino acid sequence of Table 4. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a light chain variable region sequence of Table 4.

TABLE 4: VARIABLE LIGHT CHAIN SEQUENCES

[0042] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a humanized light chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence of Table 4.

[0043] In certain embodiments, a cTn-T binding agent and/or an antigen binding portion of a cTn-T binding agent comprises one, two or three CDRs of a heavy chain variable region.

In some embodiments a heavy chain variable region comprises one or more CDRs (e.g., one, two, three, or more CDRs). The amino acid sequences representing a CDR in a heavy chain variable region of an antibody or binding agent is referred to as CDR-H1, CDR-H2, and CDR-H3, which are numbered sequentially (/._<?. , HI, H2 and H3) in the direction from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) of a heavy chain variable region. For example, in a polypeptide comprising a heavy chain variable region of a cTn-T binding agent, CDR-H1, when present, is the most N-terminal CDR; CDR-H3, when present, is the most C-terminal CDR; and CDR-H2, when present, is located (i) between CDR-H1 and CDR-H3, (ii) on the N-terminal side of CDR-H3 or (iii) on the C-terminal side of CDR-H, of a heavy chain variable region. The terms“CDR-H1”,“CDR-H2” and“CDR-H3” refer to, in part, an amino acid sequence of a polypeptide identified as, or disclosed herein as, a complementarity determining region of a cTn-T binding agent (e.g., a CDR of a heavy chain variable region of a cTn-T binding agent). Non-limiting examples of amino acid sequences of a CDR-H1, CDR-H2 and CDR-H3 are provided in Tables 5-7, respectively. A heavy chain variable region or antigen binding portion of a cTn-T binding agent described herein may comprise any combination of a CDR-H1, a CDR-H2, and a CDR-H3 disclosed herein where the cTn-T binding agent retains specific binding to cTn-T, or a portion thereof. In certain embodiments, a heavy chain variable region or antigen binding portion of a cTn-T binding agent described herein comprises three heavy chain CDRs consisting of an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-H3 selected from Table 8, an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-H2 selected from Table 7 and an amino acid sequence selected at least 70%, at least 80% or at least 90% identical to a CDR-H1 of Table 6. In certain embodiments, a heavy chain variable region or antigen binding portion of a cTn-T binding agent described herein comprises an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-H3 selected from Table 8, an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-H2 selected from Table 7 and an amino acid sequence at least 70%, at least 80% or at least 90% identical to a CDR-H1 selected from Table 6, where the cTn-T binding agent retains specific binding to cTn-T, or a portion thereof.

[0044] In some embodiments a cTn-T binding agent comprises one or more heavy chain CDRs with at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, or at least 99% identity to any one of the CDRs of Tables 5, 6 or 7. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-H1 that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the sequences shown in Table 5. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-H1 of any one of the sequences shown in Table 5. TABLE 5: CDR-H1 Sequences

[0045] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-H2 that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the sequences shown in Table 6. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-H2 of any one of the sequences shown in Table 6.

TABLE 6: CDR-H2 Sequences

[0046] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-H3 that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the sequences shown in Table 7. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a CDR-H3 of any one of the sequences shown in Table 7.

TABLE 7 : CDR-H3 Sequences

[0047] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a heavy chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence of Table 8. In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a heavy chain variable region sequence of Table 8.

TABLE 8: VARIABLE HEAVY CHAIN SEQUENCES

[0048] In some embodiments a cTn-T binding agent or the antigen binding portion of a cTn-T binding agent comprises a humanized heavy chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to a variable region sequence of Table 8.

[0049] In some embodiments a binding agent, or antigen binding portion of a binding agent comprises one or more light chain CDRs selected from a CDR of a light chain variable region of Table 4 and one or more heavy chain CDRs selected from a heavy chain variable region of Table 8. An amino acid sequence of a CDR (e.g. , a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) can be identified within a heavy chain or light chain variable region disclosed herein by any suitable method described herein or known to those skilled in the art.

[0050] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a CDR-L1 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:2; a CDR-L2 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:6; a CDR-L3 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:10; a CDR-H1 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:18; a CDR-H2 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:22; and a CDR-H3 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:26.

[0051] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a CDR-L1 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:3; a CDR-L2 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:7; a CDR-L3 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:l l; a CDR-H1 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:19; a CDR-H2 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:23; and a CDR-H3 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:27.

[0052] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a CDR-L1 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:4; a CDR-L2 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:8; a CDR-L3 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:12; a CDR-H1 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:20; a CDR-H2 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:24; and a CDR-H3 comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:28.

[0053] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a light chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14; and a heavy chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:30.

[0054] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a light chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:31.

[0055] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a light chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; and a heavy chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:32.

[0056] In some embodiments, a binding agent, or an antigen binding portion of a binding agent, comprises, or consists of, a light chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17; and a heavy chain variable region comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:33.

[0057] The term“percent identical” or“percent identity” refers to sequence identity between two amino acid sequences. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. When the equivalent site is occupied by the same or a similar amino acid residue (e.g. , similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

[0058] Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. In some

embodiments an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer.

[0059] In some embodiments a binding agent comprises one or more suitable CDR sequences or variable region sequences selected from Tables 1-8 wherein the selected polypeptide sequence comprises 0 to 5, 1 to 5, 0 to 10, 1 to 10, 0 to 15, or 1 to 12 amino acid modifications, where an amino acid modification can be an amino acid addition, an amino acid deletion and/or an amino acid substitution. Certain amino acid modifications may cause negligible effects on the binding affinity of a binding agent. In some embodiments, a binding agent disclosed herein comprises one or more amino acid analogues, non-native amino acids or amino acid derivatives.

[0060] In certain embodiments, a binding agent, or antigen binding portion of a binding agent comprises one or more framework regions (FR). Framework regions are often located between CDRs and/or flank CDR sequences of a heavy or light chain variable region of an antibody or binding agent. In mammals, a heavy chain variable region often comprises four framework regions and a light chain variable region often comprises four framework regions. Any suitable method can be used to identify one or more framework regions in an antibody, in a variable region of an antibody or in a binding agent. A binding agent may comprise synthetic or naturally occurring framework regions which are unmodified or modified (e.g. , optimized) as discussed below.

[0061] In some embodiments a binding agent, or antigen binding portion thereof is chimeric, grafted and/or humanized. Chimeric, grafted and or humanized binding agents often comprise modified or substituted constant regions and/or framework regions while maintaining binding specificity to of the binding agent to cTn-T. In some embodiments a binding agent, or antigen binding portion thereof, comprises constant regions, framework regions, or portions thereof, derived from a human antibody. In some embodiments a binding agent, or antigen binding portion thereof, comprises fully synthetic portions, one or more amino acids, or sequences of amino acids that are not found in native antibody molecules.

[0062] Naturally occurring framework regions, or portions thereof may be obtained from any suitable species. In certain embodiments the complementarity determining regions (CDRs) of the light and heavy chain variable regions of a binding agent, or an antigen binding portion thereof, is grafted into framework regions from the same, or another, species. For example, one or more framework regions of a binding agent may be derived from a rodent species (e.g., a mouse or rat) or a primate species (e.g., a human).

[0063] Methods of generating chimeric, humanized and/or optimized antibodies or binding agents, for example by modifying, substituting or deleting framework regions, or portions thereof, are known. Non-limiting examples of CDR grafting are described, e.g., in U.S.

Patent Nos. 6, 180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530, 101, and in Jones et al, Nature, 321 :522-525 (1986); Verhoeyen et al, Science, 239:1534-1536 (1988), and Winter, FEBS Letts., 430:92-94 (1998). Additional non-limiting examples of generating chimeric, grafted and/or humanized binding agents include U.S. patent No. 5,530,101; U.S. patent No. 5,707,622; U.S. patent No. 5,994,524; U.S. patent No. 6,245,894; Queen et al , (1988) PNAS 86: 10029-10033; Riechmann et al, Nature (1988) 332:323-327; Antibody Engineering: Methods and Protocols, Vol. 248 of Methods in molecular biology, edited by Benny K. C. Lo, Springer Science & Business Media, (2004); and Antibody Engineering, Vol. 1, Roland E. Kontermann, Stefan Diibel, Edition 2, Publisher Springer Science & Business Media, (2010). In some embodiments a binding agent can be humanized by exchanging one or more framework regions, or portions thereof (e.g., one or more amino acids), with one or more framework regions, or portions thereof from a human antibody. In certain embodiments, an antibody or binding agent can be humanized or grafted by transferring one or more CDRs (e.g., 1, 2, 3, 4, 5 or all 6 CDRs) from a donor binding agent (e.g., a mouse monoclonal antibody) to an acceptor binding agent (e.g., a human antibody) while retaining the binding specificity of the donor binding agent. In certain embodiments, the process of making a chimeric, grafted or humanized binding agent comprises making one or more amino acid substitutions, additions or deletions in a constant region or framework region of a binding agent. In certain embodiments, techniques such as “reshaping”,“hyperchimerization,” or“veneering/resurfacing” can be used to produce humanized binding agents (e.g., see Vaswami et al, Annals of Allergy, Asthma, & Immunol. 81 : 105 (1998); Roguska et al, Prot. Engin., 9:895-904 (1996); and U.S. Patent No.

6,072,035). In some aspects, a binding agent is modified by a method discussed above, or by another suitable method, to reduce immunogenicity (e.g., see Gilliland et al, J. Immunol, 62(6):3663-71 (1999)).

[0064] In certain embodiments, an amino acid sequence of a binding agent is modified to optimize binding affinity for a target (e.g., cTn-T), species cross-reactivity, solubility and/or function (e.g., agonist activity, or lack thereof). In some embodiments a specific combination of CDRs disclosed herein can be optimized for binding to cTn-T, and/or to optimize a function or characteristic of a binding agent disclosed herein. For example, a characterized light chain variable region disclosed herein (e.g., a light chain variable region of any one of SEQ ID NOs:35-45) can be co-expressed, using a suitable expression system, with a library of heavy chain variable regions comprising a CDR-H1 and CDR-H2 of a characterized heavy chain variable region (e.g., a heavy chain variable region selected from Tables 6 or 7), where the CDR-H3 is replaced with a library of CDR-H3 sequences, which may include one or more CDR-H3 regions of Table 8, for example. The resulting light chain/heavy chain binding agents can be screened for binding to cTn-T and/or for a specific function.

Optimized binding agents can be identified and the amino acid sequence of the CDR-H3 can be identified by a suitable method. The above screening method can be used to identify binding agents comprising specific combinations of CDRs, or specific optimized CDR sequences (e.g., CDR sequences comprising amino acid substitutions, additions or deletions) that provide a binding agent with improved binding specificity, binding affinity and/or function. Such methods of screening and optimizing binding agents are known (e.g., see Portolano et al. , (1993) Journal of Immunology 150:880-887; and Clarkson et al, (1991) Nature 352:624-628). Such references teach methods of producing antibodies that bind a specific antigen by using known variable light chain, known variable heavy chains, or portion thereof (e.g., CDRs thereof) by screening a library of complementarity variable regions.

[0065] In certain embodiments, a binding agent is modified to eliminate or add

glycosylation sites in order to optimize affinity and/or function of a binding agent (e.g. , see Co et al, Mol. Immunol, 30:1361-1367 (1993)). In some embodiments the number and/or type of glycosylation sites in a binding agent is modified or altered. An N-linked glycosylation site is often characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided in certain embodiments is a rearrangement of N-linked carbohydrate chains where one or more N- linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. In some embodiments a binding agent is modified by deleting one or more cysteine residues or substituting one or more cysteine residues for another amino acid (e.g. , serine) as compared to an unmodified binding agent. In certain embodiments cysteine variants can be useful for optimizing expression, secretion, and/or solubility. [0066] In certain embodiments a binding agent is modified to include certain amino acid additions, substitutions, or deletions designed or intended, for example, to reduce susceptibility of a binding agent to proteolysis, reduce susceptibility of a binding agent to oxidation, increase serum half-life and/or confer or modify other physicochemical, pharmacokinetic or functional properties of a binding agent.

[0067] In some embodiments a binding agent specifically binds to a mammalian cTn-T, a human cTn-T, an isoform thereof, or portion thereof. In humans, cTn-T is a protein expressed from the TNNT2 gene. In certain embodiments, a binding agent described herein specifically binds to a cTn-T with a binding affinity (KD) of 10⁵ M or less, 10⁶ M or less, 10⁷ M or less, 10 ⁸ M or less, 50 nM or less, 10 nM or less, or 1 nM or less. In certain embodiments, a binding agent described herein specifically binds to a cTn-T with a binding affinity (KD) from about lO ⁵ to 10 ¹⁵ M, lO ⁶ to 10 ¹⁵ M, lO ⁷ to 10 ¹⁵ M, lO ⁹ to 10 ¹⁵ M, lO ⁹ to 10 ¹⁴ M, 10 ⁹ to 10 ¹³ M, or 10 ⁹ to about 10 ¹² M. In some embodiments a binding agent that specifically binds to cTn-T, is a binding agent that binds cTn-T with a binding affinity (KD) equal to or less than 100 nM, equal to or less than 50 nM, equal to or less than 25 nM, equal to or less than 10 nM, equal to or less than 5 nM, equal to or less than 1 nM, equal to or less than 900 pM, equal to or less than 800 pM, equal to or less than 750 pM, equal to or less than 700 pM, equal to or less than 600 pM, equal to or less than 500 pM, equal to or less than 400 pM, equal to or less than 300 pM, equal to or less than 200 pM, or equal to or less than 100 pM. In certain aspects a binding agent specifically binds to a naturally occurring cTn-T variant or a naturally occurring cTn-T isoform. In certain aspects a binding agent specifically binds to a cTn-T comprising one or more amino acid substitutions, additions or deletions. In certain embodiments, a binding agent described herein specifically binds a cTn-T polypeptide having an amino acid sequence of SEQ ID NO: 1.

[0068] In some embodiments, a binding agent that specifically binds to cTn-T, does not specifically bind to skeletal troponin T. In some embodiments, a binding agent that specifically binds to cTn-T does not specifically bind to Troponin T1 (slow skeletal type; gene product of TNNT1 in humans), or Troponin T3 (fast skeletal type; gene product of TNNT3 in humans). In some embodiments, a binding agent that does not specifically bind to skeletal Troponin T (e.g., Troponin T1 or Troponin T3) may bind to a skeletal Troponin with a low affinity (e.g., a KD of less than 10 ⁶, less than 10 ⁵ _, less than 10⁴, or less than 10⁴). [0069] The term“specifically binds” refers to a binding agent that binds to a target peptide in preference to binding other molecules or other peptides as determined by, for example, as determined by a suitable in vitro assay (e.g. , an Elisa, Immunoblot, Flow cytometry, and the like). A specific binding interaction discriminates over non-specific binding interactions by about 2-fold or more, often about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000-fold or more, 100,000-fold or more, or 1,000,000-fold or more.

[0070] In some embodiments a binding agent comprises one or more labels. As used herein, the term“label” refers to a distinguishable identifier or detectable marker that can be detected and/or quantitated by a suitable method. In a label is often used to specifically identify and/or quantitate a binding agent, or a complex comprising a bound binding agent, among other compounds and molecule that may be present. In some embodiments, a label is a distinguishable identifier or detectable identifier that is attached to a binding agent. A label can be covalently or non-covalently attached to a binding agent. Non-limiting examples of a label include an amino acid, a peptide or polypeptide (e.g., a peptide epitope or epitope tag, a polypeptide recognized by a secondary reporter (e.g., leucine zipper, binding sites for secondary antibodies, metal binding domains, and the like)), an enzyme (e.g., horseradish peroxidase, b-galactosidase, luciferase, alkaline phosphatase), a nucleic acid (e.g., a DNA tag, barcode, a recognition sequence), a radiolabel (e.g., an isotope, radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, "Tc, ¹²⁵I, ¹³¹I), a metal or metallic label (e.g., a metallic nanoparticle, e.g., a gold nanoparticle (GNP)), a fluorescent label, a chromophore, a chemiluminescent label, an electro-chemiluminescent label (e.g., ORIGEN™), a

phosphorescent label, a quencher (e.g., a fluorophore quencher), a fluorescence resonance energy transfer (FRET) pair (e.g. , donor and acceptor), a dye, an enzyme substrate, a detectable small molecule or compound, a mass tag, quantum dots, a nanoparticle, the like or combinations thereof. In some embodiments a label is reversibly associated with a binding agent.

[0071] In certain embodiments, a label comprises a member of a binding pair. In some embodiments a binding agent comprises a member of a binding pair. In some embodiments a binding agent is attached to a label indirectly by means of a binding pair. In some embodiments a binding pair comprises at least two members (e.g., molecules) that specifically bind to (e.g., associate with) each other. Members of a binding pair often bind reversibly to each other, for example where the association of two members of a binding pair can be dissociated by a suitable method. Non- limiting examples of a binding pair includes antibody/antigen, antibody/antibody, antibody/antibody fragment, antibody/antibody receptor, antibody/protein A or protein G, hapten/anti-hapten, sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative, amine/isotriocyanate, amine/succinimidyl ester, amine/sulfonyl halides, biotin/avidin, biotin/streptavidin, folic acid/folate binding protein, receptor/ligand, vitamin B 12/intrinsic factor, analogues thereof, derivatives thereof, binding portions thereof, the like or combinations thereof. Non-limiting examples of a binding pair member include an antibody, antibody fragment, reduced antibody, chemically modified antibody, antibody receptor, an antigen, hapten, anti-hapten, a peptide, protein, nucleic acid (e.g., double- stranded DNA (dsDNA), single-stranded DNA (ssDNA), or RNA), a nucleotide, a nucleotide analog or derivative (e.g., bromodeoxyuridine (BrdU)), an alkyl moiety (e.g., methyl moiety on methylated DNA or methylated histone), an alkanoyl moiety (e.g., an acetyl group of an acetylated protein (e.g., an acetylated histone)), an alkanoic acid or alkanoate moiety (e.g., a fatty acid), a glyceryl moiety (e.g., a lipid), a phosphoryl moiety, a glycosyl moiety, a ubiquitin moiety, lectin, aptamer, receptor, ligand, metal ion, avidin, neutravidin, biotin, B12, intrinsic factor, analogues thereof, derivatives thereof, binding portions thereof, the like or combinations thereof. In some embodiments, a member of a binding pair comprises a label.

[0072] A label can be detected and/or quantitated by a variety of suitable methods, non limiting examples of which include flow cytometry, gel electrophoresis, ELISA, Western, protein-chip analysis (e.g., any chip methodology), microarray, mass spectrometry, cytofluorimetric analysis, fluorescence microscopy, confocal laser scanning microscopy, laser scanning cytometry, shear horizontal surface acoustic wave device/sensor, surface plasmon resonance, the like and combinations thereof.

[0073] In some embodiments a binding agent comprises a suitable carrier. A binding agent can be attached covalently or non-covalently to a suitable carrier. Non- limiting examples of a carrier include agents or molecules that alter the physical properties of, and/or extend the in vivo half-life of, a binding agent, non- limiting examples of which include polyethylene glycol, glycogen (e.g., by glycosylation of a binding agent), a dextran, a carrier or vehicle described in U.S. Patent No. 6,660,843, the like or combinations thereof. [0074] In some embodiments a label or carrier is bound to a binding agent by use of a suitable linker. In certain embodiments, a heavy chain variable region is attached to a light chain variable regions by use of a suitable linker. Non-limiting examples of a suitable linker include silanes, thiols, phosphonic acid, polyethylene glycol (PEG), amino acids and peptides, polymers thereof, derivatives thereof, the like and combinations thereof. In certain embodiments, a linker comprises a (G4S)3 linker with a length of 1 to 20 units (e.g., amino acid residues).

[075] In some embodiments a binding agent is used to detect or quantitate cTn and/or cTn- T, in vitro, ex vivo, or in vivo. In some embodiments a binding agent is used to detect or quantitate cTn or cTn-T in a sample or subject. In some embodiments, a method of detecting and/or quantitating cTn and/or cTn-T in a sample comprises (i) contacting a sample with a cTn-T binding agent, and (ii) detecting the presence, absence or amount of a bound complex comprising the cTn-T binding agent and cTn-T. In some embodiments, a method of detecting and/or quantitating cTn and/or cTn-T in a sample comprising one or more of (a) contacting the sample with a first cTn-T binding agent, thereby forming a first complex comprising (i) the first cTn-T binding agent binding agent and (ii) cTn-T; (b) optionally washing the first bound complex to remove unbound binding agent or unbound cTn-T, (c) contacting the first complex with a second cTn-T binding agent, thereby forming a second complex comprising (i) the first Troponin T binding agent, (ii) cTn-T and (iii) the second cTn-T binding agent; (d) optionally washing the second bound complex to remove unbound binding agent or unbound cTn-T, and (e) detecting or quantitating the presence, absence or amount of the second complex, wherein the presence, absence or amount of the second complex is often representative of the presence, absence or amount of cTn-T in the sample.

In some embodiments, a method of detecting and/or quantitating cTn and/or cTn-T in a sample comprising one or more of (a) contacting the sample with a first cTn-T binding agent, thereby forming a first complex comprising (i) the first cTn-T binding agent binding agent and (ii) cTn; (b) optionally washing the first bound complex to remove unbound binding agent, unbound cTn and/or unbound cTn-T;(c) contacting the first complex with a second cTn-T binding agent, thereby forming a second complex comprising (i) the first Troponin T binding agent, (ii) cTn and (iii) the second cTn-T binding agent; (d) optionally washing the second bound complex to remove unbound binding agent, unbound cTn-T or unbound cTn, and (e) detecting or quantitating the presence, absence or amount of the second complex, wherein the presence, absence or amount of the second complex is often representative of the presence, absence or amount of cTn or cTn-T in the sample.

[076] Any suitable method can be used to detect and/or quantitate the presence, absence and/or amount of a binding agent specifically bound to cTn, cTn-T, or a portion thereof, non limiting examples of such methods can be found in Immunology, Werner Luttmann;

Academic Press, 2006 and/or Medical Detection and Quantification of Antibodies to

Biopharmaceuticals: Practical and Applied Considerations, Michael G. Tovey; John Wiley & Sons, July 12, 2011. Additional non-limiting examples of methods that can be used to detect and/or quantitate the presence, absence and/or amount of a binding agent specifically bound to cTn, cTn-T, or a portion thereof, include use of a competitive immunoassay, a non competitive immunoassay, Western blots, a radioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a competition or sandwich ELISA, a sandwich immunoassay, an immunoprecipitation assay, an immunoradiometric assay, a fluorescent immunoassay, FRET, a protein A immunoassay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, an agglutination assay, a complement fixation assay, an

immunohistochemicai assay, an immunohistological assay, an immunocytochemical assay, a dot blot assay, a fluorescence polarization assay, a scintillation proximity assay, a homogeneous time resolved fluorescence assay, an IAsys analysis, a BIAcore analysis, protein-chip analysis (e.g. , any chip, flow cell or sensor methodology), microarray, mass spectrometry, cytofluorimetric analysis, fluorescence microscopy, confocal laser scanning microscopy, laser scanning cytometry, single molecule counting, Multiplex assays, FirePlex, Luminex, Singulex, ProQuantum High-Sensitivity Immunoassays, photoacoustic immunoassays, shear horizontal surface acoustic wave device/sensor, surface plasmon resonance, the like or a combination thereof.

Kits

[077] One or more binding agents can, if desired, be provided in a kit, pack or dispensing device, which can contain one or more cTn-T binding agents described herein. In some embodiments a kit comprises at least two cTn-T binding agents described herein, for example two different cTn-T binding agents that are suitable for using in a sandwich-type

immunoassay. [078] In some embodiments, a kit comprises an amount of a binding agent (e.g., one or two binding agents) sufficient for a user to conduct 1 to 500, 1 to 100, 1 to 50, 1 to 25, or 1 to 10 immunoassays.

[079] A kit optionally includes a product label or packaging inserts including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. Exemplary instructions include instructions to conduct a suitable immunoassay, or a detection, quantitation or diagnostic method. In certain embodiments, a kit comprises packaging material, which refers to a physical structure housing components of the kit. The packaging material can maintain the components sterilely and can be made of material commonly used for such purposes (e.g. , paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.). Product labels or inserts include“printed matter,” e.g. , paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g. , a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, optical disk such as CD, DVD, jump drive, magnetic tape, or another suitable storage media, FLASH media or memory type cards. Product labels or inserts can include identifying information of one or more components therein, e.g., concentrations, volumes, recommended storage conditions, product shelf life, and the like. Product labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, date, information and the like. In certain embodiments, a kit comprises one or more controls having a known amount of cTn, or cTn-T.

[080] In some embodiments, a kit is a diagnostic kit comprising one or more cTn-T binding agents. A binding agent comprised in a diagnostic kit can take any suitable form. In some embodiments, a diagnostic kit comprises a binding agent attached to a detectable label or member of a binding pair. In certain embodiments, for example, a diagnostic kit comprises or consists of a stick test, including necessary reagents to perform the method of the invention and to produce, for example, a colorimetric result which can be compared against a color chart or standard curve. A diagnostic kit can also comprise components necessary for detecting a binding agent that specifically binds to cTn-T, for example a secondary antibody. EXAMPLES

Example 1 - Generation of anti-cTn-T antibodies

[081] Monoclonal anti-human cTn-T scFVs were made using phage display technology and specific high-affinity scFv binding agents were selecting by multiple rounds of panning using surfaces coated with human cTn-T protein. svFV were expressed and purified from E.coli or by using a eukaryotic expression system. In some embodiments, scFV monomers were generated using a (G4S)3 linker. In some embodiments, scFY dimers were generated by adding an Fc constant region of an Ig.

[082] Surface plasmon resonance (SPR) was also used as a method to detect and quantify cTn. SPR allows real-time, label-free detection of biomolecular interactions. SPR measurements monitor the interaction between a binding agent attached to the sensor surface and a antigen in solution. Changes in the index of refraction at the surface where the binding interaction occurs are detected by the hardware and recorded as RU (resonance units).

Briefly, a cTn-T specific scFV is immobilized on a sensor chip surface followed by injection of a series of concentrations of cTn or cTn-T across the surface. The SPR sensorgram plots the binding response against time. The response typically decreases during dissociation over time. After an analysis cycle is completed, regeneration solution is passed over the sensor chip, removing bound analyte, preparing for the next analysis cycle. Curves are generated from the RU trace and are evaluated by fitting algorithms which compare the raw data to well-defined binding models. These fits allow determination of a variety of thermodynamic constants, including the apparent affinity of the binding interaction.

[083] In all measurements, the analyte used herein for both the SH-SAW system and SPR analysis was human cTn (i.e., a human troponin ITC complex, HyTest, Finland; Cat#8T62) . Fig. 9 shows histograms comparing the kinetic parameters of the anti-troponin mAbs and scFV fragments obtained from the SPR analysis. Association constants (K_a) and dissociation constants (K_d) are showing in Fig. 9A and equilibrium association constants (KD = K_d/K_a) are shown in Fig. 9B. Similarly, Table 9 summarizes the tabulated values for K_a, K_d, and KD, and epitopes (if known) of the antibodies or fragments evaluated. TABLE 9

084] SPR results showed a K_D of 1.06xl0 ⁷ M for Clone 36-hetero and 8.45xl0 ⁷ M for Clone 42-hetero. The 10 ⁷ molar affinity obtained for the anti-cTn-T scFVs was lower than anticipated but was rationalized to occur due to a heterogeneous mixture of monomer and dimerized scFv units. To improve the affinity and directionality of anti-cTn-T scFv’s Clones 36 and 42, a (G4S)3 linker with a length of 12 a.a. was added to increase the propensity of formation of monomeric scFv’s. SPR results showed a K_D of 7.08xl0 ⁷ M for Clone36- (G4S)3-mono and 2.23xl0 ⁷ M for Clone42-(G4S)3-mono.

[085] It was postulated that divalent nature antibodies (e.g. , whole IgG) would inevitably provide higher affinity compared with a monovalent scFv. To obtain a side-by-side comparison of the affinities of anti-cTn-T clones, and to demonstrate the improvement of bivalent compared with monovalent binding sites, anti-cTn-T scFv-Fc dimers were also tested by SPR. SPR results showed a K_D of 1.64xl0 ⁷ M for Clone 36-scFv-Fc and 9.43xl0 ⁸ M for Clone 42-scFv-Fc.

[086] To demonstrate the diagnostic potential of a Shear Horizontal (SH) Surface Acoustic Wave (SAW) Biosensor prototype system (SH-SAW system) developed at Sensor- Kinesis, a first anti-cTn-T scFV used for detection was conjugated to 10-nm gold nanoparticles (GNP). The GNP increased the molecular weight of the detection antibody which enhanced and improved the effective limit of detection (LOD). Briefly, the SKC SAW cTn-GNP assay consists of a sandwich type immunoassay formed by the binding of cTn-scFV-GNP complex with a surface-bound scFV. The cTn-mAb-GNP complex was created by pre-mixing a solution of cTn in a buffer with a first cTn-T specific scFV-GNP conjugate. Since the SH- SAW device measures mass/viscosity loading, adding a mass enhancer to the first scFV amplifies the detection on the output from SAW chip. The interaction of the cTn-mAb-GNP complex with a sensor surface coated with a second cTn-T forms a sandwich complex on the surface of the SAW chip. In this scheme, the GNP is linked to the sensor if and only if cTn-T is present.

[087] The limit of detection (LOD) of the SKC SAW cTn-GNP assay was approximately 4.8 ng/mL over a 20-minute sample-in-measurement-out period, with a dynamic range between 4.8 ng/mL and 4800 ng/mL of cTn.

Example 2- Representative sequences

SEQ ID NO:l - Human Cardiac Troponin T

MSDIEEVVEEYEEEEQEEAAVEEEEDWREDEDEQEEAAEEDAEAEAETEETRAEEDE

EEEEAKEAEDGPMEESKPKPRSFMPNLVPPKIPDGERVDFDDIHRKRMEKDLNELQA

LIEAHFENRKKEEEELVSLKDRIERRRAERAEQQRIRNEREKERQNRLAEERARREEE

ENRRKAEDEARKKKALSNMMHFGGYIQKQAQTERKSGKRQTEREKKKKILAERRK

VLAIDHLNEDQLREKAKELWQSIYNLEAEKFDLQEKFKQQKYEINVLRNRINDNQKV

SKTRGKAKVTGRWK

* * *

[0134] The entirety of each patent, patent application, publication or any other reference or document cited herein hereby is incorporated by reference. In case of conflict, the specification, including definitions, will control.

[0135] Citation of any patent, patent application, publication or any other document is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0137] All of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving a same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, disclosed features (e.g., antibodies) are an example of a genus of equivalent or similar features.

[0138] As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g. , 54%, 85.4%). Thus, to illustrate, reference to 80% or more identity, includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth.

[0139] Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, a reference to less than 100, includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10, includes 9, 8, 7, etc. all the way down to the number one (1).

[0140] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth.

Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

[0141] Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500- 2,000, 2,000-2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500- 6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000, includes ranges of 10-50, 50-100, 100- 1,000, 1,000-3,000, 2,000-4,000, etc.

[0142] Modifications can be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes can be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the technology.

[0143] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes

embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly excluded in the invention are nevertheless disclosed herein.

[0144] The technology illustratively described herein suitably can be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms“comprising,”“consisting essentially of,” and“consisting of’ can be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or segments thereof, and various modifications are possible within the scope of the technology claimed. The term“a” or“an” can refer to one of or a plurality of the elements it modifies (e.g.,“a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term“about” as used herein refers to a value within 10% of the underlying parameter (/._<?., plus or minus 10%), and use of the term“about” at the beginning of a string of values modifies each of the values (/._<?.,

“about 1, 2 and 3” refers to about 1, about 2 and about 3). For example, a weight of“about 100 grams” can include weights between 90 grams and 110 grams. The term,“substantially” as used herein refers to a value modifier meaning“at least 95 %”,“at least 96%”,“at least 97%”,“at least 98%”, or“at least 99%” and may include 100%. For example, a composition that is substantially free of X, may include less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of X, and/or X may be absent or undetectable in the composition. [0145] Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.

Claims

WHAT IS CLAIMED IS:

1. A Troponin T binding agent comprising:

a light chain variable region comprising a CDR-L1, a CDR-L2 and a CDR-L3, wherein

(i) the CDR-L1 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs:2-5,

(ii) the CDR-L2 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs:6-9, and

(iii) the CDR-L3 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 10-13,

and a heavy chain variable region comprising a CDR-H1, a CDR-H2 and a CDR-H3, wherein

(iv) the CDR-H1 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 18-21,

(v) the CDR-H2 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs:22-25, and

(vi) the CDR-H3 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs:26-29, wherein the Troponin T binding agent specifically binds to Troponin T, or a portion thereof.

2. The Troponin T binding agent of claim 1, wherein

the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO:2; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO:6; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 18; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO:22; and

the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO:26.

3. The Troponin T binding agent of claim 3, wherein the binding agent comprises a light chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 14 and a heavy chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:30.

4. The Troponin T binding agent of claim 1, wherein

the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO:3; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO:7; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 19; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO:23; and

the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO:27.

5. The Troponin T binding agent of claim 4, wherein the binding agent comprises a light chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:31.

6. The Troponin T binding agent of claim 1, wherein

the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO:4; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 12; the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO:20; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO:24; and

the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO:28.

7. The Troponin T binding agent of claim 6, wherein the binding agent comprises a light chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 16 and a heavy chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:32.

8. The Troponin T binding agent of claim 1, wherein the CDR-L1 comprises or consists of the amino acid sequence of SEQ ID NO:5; the CDR-L2 comprises or consists of the amino acid sequence of SEQ ID NO:9; the CDR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; the CDR-H1 comprises or consists of the amino acid sequence of SEQ ID NO:21; the CDR-H2 comprises or consists of the amino acid sequence of SEQ ID NO:25; and

the CDR-H3 comprises or consists of the amino acid sequence of SEQ ID NO:29.

9. The Troponin T binding agent of claim 8, wherein the binding agent comprises a light chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:33.

10. The Troponin T binding agent of any one of claims 1 to 9, wherein the Troponin T binding agent is an antibody, or a binding portion thereof.

11. The Troponin T binding agent of any one of claims 1 to 10, wherein the Troponin T binding agent is a monoclonal antibody, or binding portion thereof.

12. The Troponin T binding agent of any one of claims 1 to 11, wherein the Troponin T binding agent comprises a constant region of an IgG, IgD, IgE, IgA or IgM.

13. The Troponin T binding agent of claim 12, wherein the Troponin T binding agent comprises a constant region selected from an IgGi, IgG2, IgG3, and IgG4.

14. The Troponin T binding agent of any one of claims 10 to 13, wherein the Troponin T binding agent is a chimeric antibody comprising one or more human constant regions.

15. The Troponin T binding agent of any one of claims 1 to 14, wherein the Troponin T binding agent is humanized.

16. The Troponin T binding agent of claim 15, wherein the Troponin T binding agent comprises fully human framework regions 1-4.

17. The Troponin T binding agent of any one of claims 1 to 11, wherein the Troponin T binding agent comprises or consists of a Fab, Fab’, F(ab’)2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), VL, VH, diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent), tetrabody (tetravalent), minibody ((scFV-CH3)2), IgGdeltaCH2, scFv-Fc or (scFv)2-Fc, or binding portion thereof.

18. The Troponin T binding agent of claim 17, wherein the Troponin T binding agent comprises an scFV.

19. The Troponin T binding agent of claim 18, wherein the Troponin T binding agent is monomeric, dimeric or trimeric.

20. The Troponin T binding agent of any one of claims 1 to 11, and 17 to 19, wherein the Troponin T binding agent comprises a single chain polypeptide.

21. The Troponin T binding agent of any one of claims 1 to 18, wherein the binding agent binds specifically to human Troponin T.

22. The Troponin T binding agent of any one of claims 1 to 19, wherein the Troponin T binding agent binds to a human Troponin T, or a portion thereof, with a binding affinity (KD) of 50 nM or less.

23. The Troponin T binding agent of claim 22, wherein the binding agent binds to a human Troponin T with a binding affinity (KD) of 1 nM or less.

24. A kit comprising two different Troponin T binding agents selected from the Troponin T binding agent of claims 2, 4, 6 and 8.

25. The kit of claim 24, comprising (i) the Troponin T binding agent of claims 2 and 8;

(ii) the Troponin T binding agent of claims 4 and 8, (iii) the Troponin T binding agent of claims 6 and 8; or (iv) the Troponin T binding agent of claims 4 and 6.

26. A kit comprising two different Troponin T binding agents selected from the Troponin T binding agent of claims 3, 5, 7 and 9.

27. The kit of claim 26, comprising (i) the Troponin T binding agent of claims 3 and 9;

(ii) the Troponin T binding agent of claims 5 and 9, (iii) the Troponin T binding agent of claims 7 and 9; or (iv) the Troponin T binding agent of claims 5 and 7.

28. A method of detecting or quantitating an amount of cardiac Troponin (cTn) or cardiac Troponin T (cTn-T) in a sample comprising (a) contacting the sample with a first Troponin T binding agent selected from any one of claims 1-23, thereby forming a complex comprising the first Troponin T binding agent and cTn-T; and (b) detecting or quantitating a presence, absence or amount of the complex formed in (a).

29. The method of claim 28, wherein the detecting or quantitating of (b) comprises surface plasmon resonance.

30. A method of detecting or quantitating an amount of cardiac Troponin (cTn) or cardiac Troponin T (cTn-T) in a sample comprising (a) contacting the sample with a first Troponin T binding agent selected from any one of claims 1-23, thereby forming a first complex comprising (i) the first Troponin T binding agent and (ii) cTn-T; (b) contacting the complex with a second Troponin T binding agent selected from any one of claims 1-23, thereby forming a second complex comprising (i) the first Troponin T binding agent, (ii) cTn-T and

(iii) the second Troponin T binding agent; and (c) detecting or quantitating a presence, absence or amount of the second complex.

31. The method claim 30, wherein the detecting or quantitating of (c) comprises the use of a shear horizontal surface acoustic wave device.

32. The method of claim 30 or 31, wherein the first Troponin T binding agent is different than the second Troponin T binding agent.

33. The method of any one of claims 28 to 32, wherein the first Troponin binding agent is attached to a substrate.

34. The method of any one of claims 30 to 33, wherein the first Troponin T binding agent comprises the Troponin T binding agent of claim 2 or 3, and the second Troponin T binding agent comprises the binding agent of claim 4, 5, 6, 7, 8 or 9.

35. The method of any one of claims 30 to 33, wherein the first Troponin T binding agent comprises the Troponin T binding agent of claim 4 or 5, and the second Troponin T binding agent comprises the binding agent of claim 2, 3, 6, 7, 8 or 9.

36. The method of any one of claims 30 to 33, wherein the first Troponin T binding agent comprises the Troponin T binding agent of claim 6 or 7, and the second Troponin T binding agent comprises the binding agent of claim 2, 3, 4, 5, 8 or 9.

37. The method of any one of claims 30 to 33, wherein the first Troponin T binding agent comprises the Troponin T binding agent of claim 8 or 9, and the second Troponin T binding agent comprises the binding agent of claim 2, 3, 4, 5, 6 or 7.

38. The method of any one of claims 30 to 37, wherein the second troponin binding agent comprises a label.

39. The method of claim 38, wherein the label comprises a nanoparticle.

40. The method of claim 39, wherein the nanoparticle comprises a gold nanoparticle.

41. The method of any one of claims 28 to 40, further comprising one or more wash steps to reduce an amount of, or eliminate, unbound binding agent.