Aminopeptidase antibodies

Abstract

The invention relates to antigen binding proteins and fragments thereof for binding to Insulin regulated aminopeptidase (IRAP), including soluble IRAP, and use thereof for detection of soluble IRAP.

Description

Aminopeptidase antibodiesField of the invention

[0001] The invention relates to antigen binding proteins and fragments thereof for binding to Insulin regulated aminopeptidase (IRAP), including soluble IRAP, and use thereof for detection of soluble IRAP.Cross-reference to earlier application

[0002] This application claims priority from Australian provisional application no. 2023903759, the content of which are herein incorporated by reference in their entirety.Background of the invention

[0003] Insulin regulated aminopeptidase (IRAP) is a type II transmembrane zincdependent metallopeptidase which belongs to the M1 aminopeptidase family. It has two functional domains: the extracellular / intraluminal C-terminal domain which contains the catalytic site and regulates the levels of circulating peptide hormones, and the cytosolic N-terminal domain which contains trafficking motifs that regulate the subcellular distribution of IRAP-containing vesicles.

[0004] A diverse series of names have been used for IRAP including oxytocinase, placental leucine aminopeptidase, leucyl and cystinyl aminopeptidase (LNPEP), and the angiotensin type IV receptor, reflecting the multifunctional nature of the protein and its broad tissue distribution throughout the body. For example, it has been implicated in a range of physiological processes including regulating the pregnancy hormone, oxytocin, to prevent the onset of premature labour, and trimming peptides for antigen cross presentation in dendritic cells as part of the adaptive immune response. Growing evidence suggests IRAP may also have a prominent role in disease, with global gene deletion of the enzyme in mice providing protection against ischemic stroke damage, seizures and diet-induced obesity. Similar protection is observed following inhibition of the catalytic activity of IRAP with specific inhibitors, which show promise as potential therapeutic agents for multiple disease and injured states.

[0005] Interestingly, the tissue expression of IRAP is upregulated in various pathological states including atherosclerosis and following balloon injury of carotid arteries, suggesting it may play a role in the pathogenesis or progression of thesediseases. This upregulation also extends to different forms of cancer, with high IRAP protein expression seen in cancers of the breast, colon, endometrium, lymph, prostate, skin and thyroid. Together, these findings support the notion that IRAP is a potential therapeutic target in these diseases.

[0006] Whilst IRAP is predominantly expressed in its membrane bound form, a soluble form of the enzyme can be detected in the serum of pregnant women. This soluble IRAP (sIRAP) was initially cloned from a human placental library and subsequently identified as a major enzyme present in maternal serum. It is believed to be generated by cleavage of the extracellular domain between Phe154 and Ala155 by a secretase in the ‘a disintegrin and metalloprotease domain’ (ADAM) family that has a molecular weight of -150 kDa observed in Western blots. IRAP is thought to be secreted from the apical membrane of placental syncytiotrophoblast cells following the detection of sIRAP in the conditioned media of cultured placental tissue. This secretion of sIRAP from the placenta correlates with increases in circulating oxytocin, which is a substrate of IRAP. Therefore, by regulating oxytocin levels, the soluble form of IRAP in the circulation is thought to play a role in pregnancy.

[0007] Based on the findings of increased tissue expression of IRAP in specific disease states and the presence of a soluble, secreted form of the protein in human circulation, sIRAP may serve as a potential biomarker. However, there are currently no reliable methods to measure and / or quantify secreted IRAP and validate its potential as a disease biomarker.

[0008] There is a need for reagents and methods for measuring and / or quantifying sIRAP levels, particularly in human plasma.

[0009] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0010] The invention provides an antigen binding protein for binding to soluble IRAP, the antigen binding protein having an antigen binding domain comprising:FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, andFR1 a - CDR1 a - FR2a - CDR2a - FR3a - CDR3a - FR4a, wherein:FR1 , FR2, FR3 and FR4 are each framework regions;CDR1 , CDR2 and CDR3 are each complementarity determining regions;FR1 a, FR2a, FR3a and FR4a are each framework regions;CDR1 a, CDR2a and CDR3a are each complementarity determining regions; wherein the sequence of any of the framework regions or complementarity determining regions are as described herein, preferably as described in the Table herein.

[0011] In any embodiment, CDR1 , CDR2 and CDR3 refer to complementarity determining regions from the variable heavy chain of an antibody (a VH), CDR1 a, CDR2a and CDR3a are complementarity determining regions from the variable light chain of an antibody (a VL), or where CDR1 , CDR2 and CDR3 are complementarity determining regions from the VL, CDR1 a, CDR2a and CDR3a are complementarity determining regions from VH. In such examples, the CDRs may be referred to as CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2 and CDRL3 as the case may be.

[0012] In any embodiment, the invention provides an antigen binding protein for binding to soluble IRAP, wherein the antigen binding protein competitively inhibits the binding to soluble IRAP of an antibody:- comprising a VH comprising a sequence as set forth in SEQ ID NO: 29, and a VL comprising a sequence as set forth in SEQ ID NO: 30;- comprising a VH comprising a sequence as set forth in SEQ ID NO: 59, and a VL comprising a sequence as set forth in SEQ ID NO: 60;- comprising a VH comprising a sequence as set forth in SEQ ID NO: 81 , and a VL comprising a sequence as set forth in SEQ ID NO: 82; or- comprising a VH comprising a sequence as set forth in SEQ ID NO: 96, and a VL comprising a sequence as set forth in SEQ ID NO: 97.

[0013] In any embodiment, the invention provides an antigen binding protein with a CDRH1 , a CDRH2 and / or a CDRH3 of an antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NOs: 29, 59, 81 or 96.

[0014] In any embodiment, the invention provides an antigen binding protein with a CDRL1 , a CDRL2 and / or a CDRL3 of an antigen binding domain having a variable light chain as defined in any one of SEQ ID NOs: 30, 60, 82 or 97.

[0015] In any embodiment, the invention provides an antigen binding protein for binding to soluble IRAP, the protein comprising:- a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 29, and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 30;- a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 59, and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 60;- a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 81 , and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 82; or- a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO:96, and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 97.

[0016] In any embodiment, an antigen binding protein described herein comprises:FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - linker - FR1 a - CDR1 a - FR2a - CDR2a - FR3a - CDR3a - FR4a.

[0017] As defined herein, the linker may be a chemical, one or more amino acids, or a disulphide bond formed between two cysteine residues.

[0018] In certain preferred embodiments, the invention provides an antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of (in order of N to C terminus or C to N terminus) SEQ ID NOs: 29 and 30.

[0019] In certain preferred embodiments, the invention provides an antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of (in order of N to C terminus or C to N terminus) SEQ ID NOs: 59 and 60.

[0020] In certain preferred embodiments, the invention provides an antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of (in order of N to C terminus or C to N terminus) SEQ ID NOs: 81 and 82.

[0021] In certain preferred embodiments, the invention provides an antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of (in order of N to C terminus or C to N terminus) SEQ ID NOs: 96 and 97.

[0022] In one embodiment, the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 1 (IGMT) or 15 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 2 (IGMT) or 16 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 3 (IGMT) or 17 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 29, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 4 (IMGT) or 18 (Kabat) or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 5 (IMGT) or 19 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 6 (IGMT) or 20 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 30, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence set forth in SEQ ID NO: 3; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 15, a CDR2 comprising a sequence set forth in SEQ ID NO: 16, and a CDR3 comprising a sequence set forth in SEQ ID NO: 17;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 4, a CDR2 comprising a sequence set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 6; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 18, a CDR2 comprising a sequence set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 20;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 3 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 4, a CDR2 comprising a sequence as set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 6; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO:15, a CDR2 comprising a sequence as set forth in SEQ ID NO: 16 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 17 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 18, a CDR2 comprising a sequence as set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 20; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 29, and a VL comprising a sequence set forth in SEQ ID NO: 30.

[0023] In this embodiment, when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 7, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 8, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 9, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 10, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 1 1 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 12, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 13, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 14, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0024] In this embodiment, when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 21 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 22, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 23, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 24, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 25, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 26, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 27, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence ofSEQ ID NO: 28, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0025] In another embodiment, the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 29, or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 30; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

[0026] In another embodiment, the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 31 (IGMT) or 45 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 32 (IMGT) or 46 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 33 (IMGT) or 47 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 59, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 34 (IMGT) or 48 (Kabat) or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 35 (IMGT) or 49 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 36 (IMGT) or 50 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 60, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 31 , a CDR2 comprising a sequence set forth in SEQ ID NO: 32, and a CDR3 comprising a sequence set forth in SEQ ID NO: 33; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 45, a CDR2 comprising a sequence set forth in SEQ ID NO: 46, and a CDR3 comprising a sequence set forth in SEQ ID NO: 47;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 34, a CDR2 comprising a sequence set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 36; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 48, a CDR2 comprising a sequence set forth in SEQ ID NO: 49 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 50;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 31 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 32, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 33 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 34, a CDR2 comprising a sequence as set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 36; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 45, a CDR2 comprising a sequence as set forth in SEQ ID NO: 46 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 47 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 48, a CDR2 comprising a sequence as set forth in SEQ ID NO: 49 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 50; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 59, and a VL comprising a sequence set forth in SEQ ID NO: 60.

[0027] In this embodiment, when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 37, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 38, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 39, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 40, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; andii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 41 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 42, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 43, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 44, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0028] In this embodiment, when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 51 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 52, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 53, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 54, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 55, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 56, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 57, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 58, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0029] In any embodiment, the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 59, or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 60; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

[0030] In another embodiment, the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 61 (IGMT) or 72 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 62 (IMGT) or 73 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 63 (IMGT) or 74 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 81 , or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 64 (IMGT) or 75 (Kabat) or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 35 (IMGT) or 76 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 65 (IMGT) or 77 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 82, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 61 , a CDR2 comprising a sequence set forth in SEQ ID NO: 62, and a CDR3 comprising a sequence set forth in SEQ ID NO: 63; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 71 , a CDR2 comprising a sequence set forth in SEQ ID NO: 73, and a CDR3 comprising a sequence set forth in SEQ ID NO: 74;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 64, a CDR2 comprising a sequence set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 65; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 75, a CDR2 comprising a sequence set forth in SEQ ID NO: 76 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 77;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 61 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 62, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 63 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 64, a CDR2 comprising a sequence as set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 65; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 72, a CDR2 comprising a sequence as set forth in SEQ ID NO: 73 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 74 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 75, a CDR2 comprising a sequence as set forth in SEQ ID NO: 76 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 77; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 81 , and a VL comprising a sequence set forth in SEQ ID NO: 82.

[0031] In this embodiment, when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 66, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 67, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequenceof SEQ ID NO: 68, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 69, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 41 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 42, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 70, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 71 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0032] In this embodiment, when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 78, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 52, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 79, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 54, or a sequence at least about 80%, at least 85%, at least90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 55, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 56, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 80, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 28, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0033] In any embodiment, the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 81 , or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 82; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

[0034] In another embodiment, the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 83 (IGMT) or 15 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 2 (IMGT) or 89 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 84 (IMGT) or 90 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 96, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 85 (IMGT) or 91 (Kabat) or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 5 (IMGT) or 19 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 86 (IMGT) or 92 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 97, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 83, a CDR2 comprising a sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence set forth in SEQ ID NO: 84; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 15, a CDR2 comprising a sequence set forth in SEQ ID NO: 89, and a CDR3 comprising a sequence set forth in SEQ ID NO: 90;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 85, a CDR2 comprising a sequence set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 86; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 91 , a CDR2 comprising a sequence set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 92;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 83, a CDR2 comprising a sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 84 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 85, a CDR2 comprising a sequence as set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 86; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 15, a CDR2 comprising a sequence as set forth in SEQ ID NO: 89 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 90 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 91 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 92; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 96, and a VL comprising a sequence set forth in SEQ ID NO: 97.

[0035] In this embodiment, when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 7, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 8, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 87, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 10, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 1 1 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 12, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 88, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 14, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0036] In this embodiment, when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 93, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 22, or a sequence at least about 80%, at least 85%, at least 90%, at least91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 94, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 24, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 25, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 26, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 95, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 28, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

[0037] In any embodiment, the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 96, or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 97; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12,no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

[0038] In any embodiment, an antigen binding protein of the invention can also bind full length IRAP.

[0039] In any embodiment, an antigen binding protein of the invention can be used to detect equal to or less than 0.5 ug sIRAP, equal to or less than 0.1 ug sIRAP, equal to or less than 0.05 ug sIRAP, equal to or less than 0.01 ug sIRAP, equal to or less than 0.005 ug sIRAP, equal to or less than 0.002 ug sIRAP, or equal to or less than 0.001 ug sIRAP. Preferably, the method of detection is an ELISA, such as that described herein including the Examples.

[0040] As described herein, the antigen binding protein may be in the form of:(i) a single domain antibody (sdAb);(ii) a single chain Fv fragment (scFv);(iii) a dimeric scFv (di-scFv); or(iv) one of (i) or (iii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

[0041] Further, as described herein, the antigen binding protein may be in the form of:(i) a diabody;(ii) a triabody;(iii) a tetrabody;(iv) a Fab;(v) a F(ab’)2;(vi) a Fv;(vii) a bispecific antibody or other form of multispecific antibody (including a BiTE); or(viii) one of (i) to (vii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

[0042] The foregoing antigen binding proteins can also be referred to as antigen binding domains of antibodies.

[0043] Preferably, an antigen binding protein as described herein is an antibody or antigen binding fragment thereof. Typically, the antigen binding protein is an antibody, for example, a monoclonal antibody. The antigen binding protein may be in the form of a recombinant or modified antibody (e.g., chimeric antibody, humanised antibody, human antibody, CDR-grafted antibody, primatised antibody, de-immunised antibody, synhumanised antibody, half-antibody, bispecific antibody, trispecific antibody or multispecific antibody). The antigen binding protein or antibody may further comprise a chemical modification, such as conjugation to a label.

[0044] As used herein the antigen binding protein may be a variable domain.

[0045] As used herein, the complementarity determining region sequences (CDRs) of an antigen binding protein of the invention may be defined according to the IMGT, Chothia or Kabat numbering systems, or any other CDR numbering system known to the skilled person.

[0046] The invention provides an antigen binding protein as described herein wherein an amino acid sequence forming one or more of FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4 is a human sequence.

[0047] The invention provides an anti-sIRAP binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody comprising an antigen binding protein having a sequence as described herein, or including a CDR and / or FR sequence as described herein.

[0048] An antigen binding protein as described herein may comprise a human constant region, e.g., an IgG constant region, such as an IgGi, lgG2, IgGa or lgG4 constant region or mixtures thereof. In the case of an antibody or protein comprising a VH and a Vi_, theVH can be linked to a heavy chain constant region and the VL can be linked to a light chain constant region.

[0049] In one example, an antigen binding protein comprises a VH disclosed herein linked or fused to an lgG4 constant region or stabilised lgG4 constant region (e.g., as discussed above) and the VL is linked to or fused to a kappa light chain constant region.

[0050] In any aspect of the present invention, the antibody is a naked antibody. Specifically, the antibody is in a non-conjugated form and is not adapted to form a conjugate.

[0051] The invention provides a fusion protein comprising an antigen binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody as described herein.

[0052] The invention also provides a conjugate in the form of an antigen binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody or fusion protein as described herein, conjugated to a label or moiety that facilitates detection.

[0053] The functional characteristics of an antigen binding protein of the invention will be taken to apply mutatis mutandis to an antibody of the invention.

[0054] In aspects of the invention directed to multiple polypeptide chains that form an antigen binding protein, an expression construct comprises a nucleic acid encoding a polypeptide comprising, e.g., a VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, e.g., a VL operably linked to a promoter.

[0055] In another example, the expression construct is a bicistronic expression construct, e.g., comprising the following operably linked components in 5’ to 3’ order:(i) a promoter(ii) a nucleic acid encoding a first polypeptide;(iii) an internal ribosome entry site; and(iv) a nucleic acid encoding a second polypeptide, wherein the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0056] The present invention also contemplates separate expression constructs one of which encodes a first polypeptide comprising a VH and another of which encodes a second polypeptide comprising a VL. For example, the present invention also provides a composition comprising:(i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0057] The invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified or recombinant. In one example, the cell comprises the expression construct of the invention or:(i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter, wherein the first and second polypeptides associate to form an antigen binding protein of the present invention.

[0058] Examples of cells of the present invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0059] The invention provides a nucleic acid encoding an antigen binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein. In some embodiments, the nucleic acid may comprise or consist of a sequence selected from: SEQ ID NO:98, SEQ ID NO:99; SEQ ID NO:100, SEQ ID NO:101 ; SEQ ID NO:102, SEQ ID NO:103, SEQ ID NQ:104 and SEQ ID NQ:105; or a sequence with at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. In some embodiments, the nucleic acid may comprise or consist of the sequences:SEQ ID NO:98 and SEQ ID NO:99, or sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;SEQ ID NO:100 and SEQ ID NO:101 , or sequences at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;SEQ ID NQ:102 and SEQ ID NQ:103, or sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; orSEQ ID NQ:104 and SEQ ID NQ:105, or sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0060] The invention provides a vector comprising a nucleic acid described herein.

[0061] The invention provides a cell comprising a vector or nucleic acid described herein.

[0062] The invention provides a kit or article of manufacture comprising an antigen binding protein, or including a CDR and / or FR sequence as described herein or an immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment,diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein.

[0063] The invention provides use of a sequence according to one or more of CDR1 , CDR2, CDR3, FR1 , FR2, FR3 and FR4 as described herein to produce an antigen binding protein for binding to soluble IRAP.

[0064] The invention provides use of an antigen binding protein or a CDR and / or FR sequence as described herein to produce an anti-soluble IRAP antigen binding protein having increased affinity for soluble IRAP.

[0065] The invention provides a library of nucleic acid molecules produced from the mutation of an antigen binding protein or a CDR and / or FR sequence as described herein, wherein at least one nucleic acid molecule in said library encodes an antigen binding protein for binding to soluble IRAP.

[0066] The invention provides a method for producing an antigen binding protein for binding to soluble IRAP as described herein comprising expressing a nucleic acid as described herein in a cell or animal as described herein.

[0067] An antigen binding protein as described herein may be purified, substantially purified, isolated and / or recombinant.

[0068] An antigen binding protein of the invention may be part of a supernatant taken from media in which a hybridoma expressing an antigen binding protein of the invention has been grown.

[0069] The invention also provides a method for detecting soluble IRAP in a sample, the method comprising contacting a sample with an antigen binding protein described herein, thereby detecting soluble IRAP in a sample.

[0070] In one embodiment, the method for detecting soluble IRAP in a sample comprises:- contacting a sample containing soluble IRAP with a capture antigen binding protein, wherein the capture antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the capture antigen binding protein to soluble IRAP in the sample,- detecting soluble IRAP bound to the capture antigen binding protein by contacting the bound IRAP with a detection antigen binding protein, wherein the detection antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the detection antigen binding protein to soluble IRAP bound to the capture antigen binding protein, thereby detecting soluble IRAP in a sample.

[0071] In one embodiment, the method for detecting soluble IRAP in a sample comprises:- contacting a sample containing soluble IRAP with a capture antigen binding protein, wherein the capture antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the capture antigen binding protein to soluble IRAP in the sample,- determining soluble IRAP bound to the capture antigen binding protein by contacting the bound IRAP with a detection antigen binding protein linked to a detectable label, wherein the detection antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the detection antigen binding protein to soluble IRAP bound to the capture antigen binding protein, and detecting the presence of the label, thereby detecting soluble IRAP in a sample.

[0072] In another embodiment, the invention provides a method for detecting the presence of or quantifying the level of soluble IRAP in a biological sample, the method comprises the steps of:- providing a biological sample- contacting the biological sample with a capture antigen binding protein as described herein;- incubating under conditions sufficient for a complex to form between soluble IRAP and the capture antigen binding protein;- determining the presence of or amount of the complex;wherein detection of the complex indicates the presence of soluble IRAP, and determining the amount of the complex quantifies the level of soluble IRAP.

[0073] In another embodiment, the invention provides a method for detecting the presence of or quantifying the level of soluble IRAP in a biological sample, the method comprises the steps of:- providing a biological sample- contacting the biological sample with a detection antigen binding protein as described herein;- incubating under conditions sufficient for a complex to form between soluble IRAP and the detection antigen binding protein;- determining the presence of or amount of the complex; wherein detection of the complex indicates the presence of soluble IRAP, and determining the amount of the complex quantifies the level of soluble IRAP.

[0074] In one embodiment, the invention provides a method for determining whether any soluble IRAP is present in a sample, the method comprises:(i) contacting a sample with a capture antigen binding protein, wherein the capture antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the capture antigen binding protein to soluble IRAP in the sample,(ii) subsequently to (i), providing conditions for any unbound soluble IRAP to be removed,(iii) subsequently to (ii), providing a detection antigen binding protein, wherein the detection antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the detection antigen binding protein to soluble IRAP bound to the capture antigen binding protein, and(iv) subsequently to (iii), providing conditions for any unbound detection antigen binding protein to be removed, and(v) determining the presence of any detection antigen binding protein bound to the soluble IRAP.

[0075] In any embodiment, the step of determining the presence of or amount of a complex, or determining the presence of any detection antigen binding protein bound to the soluble IRAP or detecting the label, may be any assay described herein including a competition assay, a sandwich assay, an immunofluorescence assay, a chemiluminescence immunoassay, a radio-immunoassay, an enzyme-linked immunosorbent assay, a lateral flow test, an agglutination test, a strip test, a microsphere immunoassay, or real-time surface plasmon resonance detection assay or any combination thereof.

[0076] In any embodiment, one or more or all steps of the method are automated or semi-automated.

[0077] The invention also provides a method for monitoring sIRAP levels during gestation, the method comprising determining sIRAP levels in samples taken at different times during gestation, wherein determining sIRAP levels is by any method described herein.

[0078] In one embodiment, the capture and detection antigen binding protein pairs are:- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 29 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein;- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 29 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein;- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 29 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein;- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein;- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein;- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein;- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 39 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; or- a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein.

[0079] Preferably, the detection antigen binding protein is biotinylated.

[0080] In any embodiment, the sample is any biological sample described herein. Typically, the biological sample is a body fluid, preferably the body fluid is a blood sample or a sample derived from blood. Typically, the sample derived from blood is plasma.

[0081] In one embodiment, the biological sample is derived from a pregnant woman. Preferably the biological sample derived from a pregnant woman is plasma.

[0082] In one embodiment, the sample is exposed to denaturing and / or reducing conditions. The denaturing and / or reducing conditions may be increased temperature, for example at least about 50°C, at least about 60°C, at least about 70°C, or at least about 80°C. Denaturation may also be by a detergent, such as sodium dodecyl sulfate (SDS).

[0083] In one embodiment, the capture antigen binding protein, detection antigen binding protein or both the capture antigen binding protein and detection antigen binding protein are bound to, or immobilised on, a substrate, solid phase or solid support. The substrate, solid phase or solid support may be a particle, a film, a microplate, a microtube or a test tube.

[0084] In one embodiment, the detection antigen binding protein is conjugated or linked to a detectable label.

[0085] In one embodiment, the detection antigen binding protein can be bound by another molecule which is or includes a detectable label.

[0086] In one embodiment, the method is performed in a plate, for example a 96-well plate.

[0087] In one embodiment, where the method is performed in a plate, at least about 5 pg / well, at least about 4 pg / well, at least about 3 pg / well, at least about 2 pg / well, at leastabout 1 pg / well, at least about 0.9 pg / well, at least about 0.8 pg / well, at least about 0.7 pg / well, at least about 0.6 pg / well, at least about 0.5 pg / well, at least about 0.4 pg / well, at least about 0.3 pg / well, at least about 0.2 pg / well, or at least about 0.1 pg / well of capture antibody is applied.

[0088] In one embodiment, where the method is performed in a plate, at least about 5 pg / well, at least about 4 pg / well, at least about 3 pg / well, at least about 2 pg / well, at least about 1 pg / well, at least about 0.9 pg / well, at least about 0.8 pg / well, at least about 0.7 pg / well, at least about 0.6 pg / well, at least about 0.5 pg / well, a at least bout 0.4 pg / well, at least about 0.3 pg / well, at least about 0.2 pg / well, at least about 0.1 pg / well, at least about 0.09 pg / well, at least about 0.08 pg / well, at least about 0.07 pg / well, at least about 0.06 pg / well, or at least about 0.05 pg / well of detection antibody is applied.

[0089] In one embodiment, where the method is performed in a plate, at least 5 pg / well, at least 4 pg / well, at least 3 pg / well, at least 2 pg / well, at least 1 pg / well, at least 0.9 pg / well, at least 0.8 pg / well, at least 0.7 pg / well, at least 0.6 pg / well, at least 0.5 pg / well, at least 0.4 pg / well, at least 0.3 pg / well, at least 0.2 pg / well, or at least 0.1 pg / well of capture antibody is applied.

[0090] In one embodiment, where the method is performed in a plate, at least 5 pg / well, at least 4 pg / well, at least 3 pg / well, at least 2 pg / well, at least 1 pg / well, at least 0.9 pg / well, at least 0.8 pg / well, at least 0.7 pg / well, at least 0.6 pg / well, at least 0.5 pg / well, at least 0.4 pg / well, at least 0.3 pg / well, at least 0.2 pg / well, at least 0.1 pg / well, at least 0.09 pg / well, at least 0.08 pg / well, at least 0.07 pg / well, at least 0.06 pg / well, or at least 0.05 pg / well of detection antibody is applied.

[0091] In one embodiment, at least about 0.5 pg / well of capture antibody and at least about 0.25 pg / well of detection antibody are applied.

[0092] In one embodiment, at least about 2 pg / well of capture antibody and at least about 1 pg / well of detection antibody are applied.

[0093] In one embodiment, a capture antigen binding protein is not bound to, or immobilised on, a substrate, solid phase or solid support, and instead the sample is applied to the substrate, solid phase or solid support directly. A detection antigen binding domain is then applied to facilitate indirect or direct detection of the soluble IRAP bound to, or immobilised on, a substrate, solid phase or solid support.

[0094] In one embodiment, the capture antibody is applied to a well in the presence of a buffer, preferably the buffer is 0.1 M Na2CO3 / NaHCO3, pH 9.6.

[0095] Typically, the capture antibody is applied to or coated on a well for at least about 30 minutes, at least about 45 minutes, or at least about 60 minutes.

[0096] In one embodiment, the sample is applied to a well in the presence of a buffer, preferably the buffer is 0.1 M Na2CO3 / NaHCO3, pH 9.6.

[0097] In one embodiment, the detection antibody is applied to a well in the presence of a buffer, preferably the buffer is 3% BSA in 1 x PBS.

[0098] Typically, the capture antibody is applied to a well for at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes.

[0099] In any embodiment, the method can be used to detect at least about 5 ng / ml of sIRAP, at least about 10ng / ml of sIRAP, at least about 15 ng / ml of sIRAP, at least about 20 ng / ml of sIRAP, at least about 25 ng / ml of sIRAP, at least about 50 ng / ml of sIRAP, at least about 100 ng / ml of sIRAP, at least about 200 ng / ml of sIRAP, at least about 400 ng / ml of sIRAP, at least about 500 ng / ml of sIRAP, at least about 1000 ng / ml of sIRAP, at least about 2000 ng / ml of sIRAP, at least about 3000 ng / ml of sIRAP, at least about 4000 ng / ml of sIRAP or at least about 5000 ng / ml of sIRAP.

[0100] In any embodiment, the method can be used to detect at least 5 ng / ml of sIRAP, at least 10ng / ml of sIRAP, at least 15 ng / ml of sIRAP, at least 20 ng / ml of sIRAP, at least 25 ng / ml of sIRAP, at least 50 ng / ml of sIRAP, at least 100 ng / ml of sIRAP, at least 200 ng / ml of sIRAP, at least 400 ng / ml of sIRAP, at least 500 ng / ml of sIRAP, at least 1000 ng / ml of sIRAP, at least 2000 ng / ml of sIRAP, at least about 3000 ng / ml of sIRAP, at least 4000 ng / ml of sIRAP or at least 5000 ng / ml of sIRAP.

[0101] The present invention also provides a kit for detecting the presence of or quantifying the level of soluble IRAP in a biological sample, the kit comprising; one or more detection antigen binding proteins; and optionally, one or more capture antigen binding proteins.

[0102] Preferably, the kit further comprises instructions for performing a method described herein.

[0103] Preferably, the kit further comprises a solid phase or support. The solid phase or support may be any one described herein.

[0104] In one embodiment, the invention provides a kit described herein when used in a method described herein.

[0105] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0106] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0107] Figure 1. The C-terminal domain of IRAP can be secreted into the circulation / extracellular milieu. IRAP contains two functional domains: a cytosolic N- terminal domain that contains trafficking motifs and an extracellular / intra-luminal C- terminal domain that contains the catalytic site. Following cleavage at a site near the transmembrane region, the C-terminal domain can be secreted. Current anti-IRAP antibodies that are commercially available only target the N-terminal domain.

[0108] Figure 2. Schematic summarising the method used in the generation of novel mouse monoclonal antibodies against soluble IRAP.

[0109] Figure 3. Profiles of the purified novel mouse anti-IRAP antibodies (RB9, RF7, RH3, RG4).

[0110] Figure 4. Detection of different forms of human IRAP (soluble IRAP and membrane-bound full length IRAP) by the novel mouse anti-IRAP antibodies (RB9, RF7, RH3, RG4; 1 pg / well) using indirect ELISA, as measured by absorbance at 450 nm compared to a commercially-available rabbit anti-IRAP control antibody (1 :500 concentration). Columns from left to right for each tested antibody: (i) Detection of membrane IRAP, (ii) detection of purified soluble IRAP (sIRAP) from Monash ProteinProduction Unit, (iii) detection of purified soluble IRAP from CSIRO, (iv) no antigen control.

[0111] Figure s. Detection of increasing concentrations of soluble IRAP (sIRAP) by the RB9 mouse monoclonal antibody (1 pg / well, “Ms Ab”, right columns for each tested group) compared to a commercially-available rabbit anti-IRAP control antibody (“Rb Ab”, left columns for each tested group, 1 :500 concentration), as determined by indirect ELISA. Detection of purified sIRAP was tested at the following concentrations: 0.001 pg / ml, 0.002 pg / ml, 0005 pg / ml and 0.01 pg / ml. Detection of 10 pg / ml of membrane-bound IRAP (mIRAP) was included as a control group.

[0112] Figure 6: The novel anti-IRAP antibodies can specifically detect human IRAP. Representative Western blots (uncropped 12 lanes in same gel) showing binding of the novel mouse anti-IRAP antibodies (RB9, RF7, RH3, RG4; 0.5 pg / pl) to (1 ) full length IRAP derived from membrane preparations of HEK293T cells transiently overexpressing human IRAP (0.01 pg protein / pl) or (2) purified soluble human IRAP (0.001 pg / pl). The antibodies were imaged at either different exposure times or at the same exposure time across all antibodies (n=3).

[0113] Figure 7. Specificity of the subcloned antibodies for IRAP. Western blots of the untagged and biotinylated newly subcloned monoclonal anti-IRAP antibodies RF7, RB9, RH3 and RG4 (0.0005 pg / pl) against either (a) purified soluble human IRAP (S; 0.001 pg / well) and full length human IRAP enriched membrane preparations of HEK293T cells transiently transfected with human IRAP cDNA (M; 0.1 pg proteins / well) or (b) protein lysate from cardiac tissue of 10-week old male IRAP KO (KO) or WT (WT) mice (20 pg protein / well), n=1 . All blots are imaged for the same exposure time.

[0114] Figure 8. Initial screen of newly subcloned, purified antibodies RF7, RB9, RH3, and RG4. An indirect ELISA using purified sIRAP (4 pg / ml) was conducted to compare the pre-subcloned (pre-SC) antibodies with the newly subcloned antibodies which were either untagged or biotinylated (detected using anti-Ms-HRP secondary antibody or streptavidin-HRP). All antibodies were used at 1 pg / well. The experiment was performed in duplicate (each dot is raw duplicate value, bar represents the mean, n=1 ).

[0115] Figure 9. (a) All 12 potential combinations of capture (2 pg / well) and detection (1 pg / well) antibodies were tested in a sandwich ELISA using purified sIRAP (4 pg / ml).RF7-RB9-B and RF7-RG4-B are the top performing antibody combinations, (b) For the top eight combinations (with the higher absorbance values), lower concentrations of detection antibody (0.05 & 0.2 pg / well) were used, (c) For the top 6 combinations, an even lower capture (0.2 pg / well) and detection (0.01 pg / well) antibody concentration was used. The top performing combinations were RF7-RB9-B and RF7-RG4-B. Each experiment was performed in duplicate (each dot in bar graphs show raw duplicate values, bar represents the mean, n=1 ).

[0116] Figure 10. Optimising antibody concentrations, (a) Standard curves at varying concentrations of purified sIRAP (pg / ml) were determined at high (RF7 = 2 pg / well, RB9- B = 1 pg / well), mid (RF7 = 0.5 pg / well, RB9=B = 0.25 pg / well) and low (RF7 = 0.2 pg / well, RB9-B = 0.1 pg / well) antibody concentrations, (b) The absorbance readings in sandwich ELISAs plateaued over time at high and mid antibody concentrations. Simple linear regression was conducted to generate the standard curves. Data presented is the mean ± SEM of absorbance readings from one experiment performed in duplicate (n=1 ).

[0117] Figure 11. Determining the detection range and linearity of the sandwich ELISAs. (a) A sandwich ELISA at serially diluted concentrations of purified sIRAP (0.0078 - 0.5 pg / ml) was conducted using both RF7-RB9-B (line with steepest incline) and RF7- RG4-B (bottom line extending to 0.5 pg / ml) antibody combinations at the optimised capture (0.5 pg / well) and detection (0.25 pg / well) antibody concentrations. Simple linear regression was conducted to generate the standard curve. Data presented is the mean ± SEM of absorbance readings from one representative experiment performed in duplicate (n=3). (b) The linearity of the RF7-RB9-B (bottom line) and RF7-RG4-B (top line) ELISAs at the optimised concentrations was determined by calculating the % recovery of pooled control human plasma spiked with 0.25 pg / ml purified sIRAP at serial dilutions. Data is presented as the mean ± SEM from 3 experiments performed in triplicate (n=3).

[0118] Figure 12. Validation of the sandwich ELISA on the detection of increasing sIRAP expression in human plasma at the later stages of pregnancy using Western blot analysis, (a) Representative Western blot and quantification of the optical density (OD) of sIRAP (140-150 kDa) in plasma from healthy controls and women at later stages of pregnancy (28- & 36-weeks and full term; diluted 1 :10), detected using the subcloned RF7 anti-IRAP antibody (0.0005 pg / pl). Control plasma is from three different participants and pregnant plasma is three different batches of pooled samples (n=3). Data is expressed as mean ± SEM, was corrected for total protein concentration and analysedusing a one-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01 compared to control, (b) Sandwich ELISAs using RF7-RB9-B (left column) and RF7-RG4-B (right column) antibody combinations were conducted to measure concentrations of IRAP in plasma from control and pregnant women (28- & 36-weeks & term) using a purified sIRAP standard curve (pg / ml). All samples were diluted 1 :5. Data was analysed using simple linear regression analysis to interpolate the concentrations of each sample and a oneway ANOVA with Tukey’s post-hoc test to compare groups to the control, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 vs control, n=5-6. All data was corrected for the negative control and is presented as mean ± SEM.

[0119] Figure 13. Selectivity of the subcloned antibodies for binding to IRAP but not binding to other related aminopeptidases, as determined by western blotting. Testing selectivity of the clones 1 -20 (see Table 2) in binding to (a) sIRAP hFc, (b) APN hFc, and (c) ERAP1 hFc. Four antibody clones RB9 (#8), RF7 (#16), RG4 (#17) and RH3 (#19) demonstrated selective binding to sIRAP, but no significant binding to APN or ERAP1 .

[0120] Figure 14. Comparison of an exemplary sandwich ELISA using antibodies of the invention for the detection of soluble human IRAP, compared to detection of soluble IRAP by a commercially-available ELISA from Aviva Systems Biology (OKECD09088). The “in-house” sandwich ELISA (left graph) utilised RF7 as the capture antibody and RB9-B as the detection antibody and displayed excellent detection of increasing concentrations of sIRAP (ng / ml). In contrast, the commercially available kit (“Aviva kit”, right graph) was unable to detect sIRAP at any of the tested concentrations.Sequence information

[0121] Table comprising sequence information:Table 1 : Antigen binding protein sequencesDetailed description of the embodiments

[0122] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0123] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

[0124] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0125] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the present invention.

[0126] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0127] There is growing interest in the use of the enzyme, insulin regulated aminopeptidase (IRAP), as a biomarker for conditions such as cardio-metabolic and fibrotic diseases as well as in ischemic stroke, with upregulation in its tissue expression in these conditions. However, quantification of circulating IRAP has been hampered by difficulties in detecting release of the truncated, soluble form of this enzyme into the blood stream. The present invention includes a sandwich ELISA using novel antigen binding proteins (e.g. antibodies) directed towards the soluble, secreted C-terminal portion of IRAP (sIRAP), to improve accuracy in detection and quantification of low levels of sIRAP in plasma. A series of novel anti-IRAP antigen binding proteins (e.g. antibodies) weredeveloped and found to be highly specific for sIRAP in Western blots. A sandwich ELISA was then optimised using two distinct antigen binding protein combinations to detect sIRAP in the low nanogram range (16 - 500 ng / ml) with a sensitivity of 9 ng / ml and intraassay variability <10%. Importantly, the clinical validity of the ELISA was verified by the detection of significant increases in the levels of sIRAP throughout gestation in plasma samples from pregnant women. Advantageously, the specific and sensitive immunoassay described herein can be used to detect and quantify I RAP as a biomarker for certain diseases.General

[0128] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0129] Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0130] Any example or embodiment of the present invention herein shall be taken to apply mutatis mutandis to any other example or embodiment of the invention unless specifically stated otherwise.

[0131] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology,immunohistochemistry, protein chemistry, and biochemistry). The general chemical terms used in the formulae herein have their usual meaning.

[0132] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0133] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md„ 1987 and 1991 , Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0134] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0135] As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

[0136] Reference herein to a range of, e.g., residues, will be understood to be inclusive. For example, reference to “a region comprising amino acids 56 to 65” will be understood in an inclusive manner, i.e., the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61 , 62, 63, 64 and 65 in a specified sequence.Selected Definitions

[0137] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally- associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By “substantially purified” is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

[0138] The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally- occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

[0139] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0140] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0141] As used herein, the term “antigen binding protein” is used interchangeably with “antigen binding domain” and shall be taken to mean a region of an antibody that is capable of specifically binding to an antigen, i.e., a VH or a VL or an Fv comprising both a VH and a VL. The antigen binding domain need not be in the context of an entireantibody, e.g., it can be in isolation (e.g., a domain antibody) or in another form, e.g., as described herein, such as a scFv.

[0142] For the purposes for the present disclosure, the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens by virtue of an antigen binding domain contained within a Fv. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, halfantibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (-50 to 70 kD) covalently linked and two light chains (-23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a A light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, 5, E, y, or p. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non- covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are -1 10 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH1 which is 330 to 440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. In one example, the Fc region is an lgG4 Fc region (i.e., from an lgG4 constant region), e.g., a human lgG4 Fc region. Sequences of suitable lgG4 Fc regions will be apparent to the skilled person and / or available in publically available databases (e.g., available from National Center for Biotechnology Information).

[0143] In one example, the antibody is a murine (mouse or rat) antibody, or is a primate (such as, human) antibody. In one example the antibody heavy chain is missing a C- terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.

[0144] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0145] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1 , CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1 , FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0146] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0147] “Antibodies” or “immunoglobulins” or “Igs” are gamma globulin proteins that are found in blood, or other bodily fluids of vertebrates that function in the immune system to bind antigen, hence identifying and neutralizing foreign objects.

[0148] Antibodies are generally a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to a H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0149] H and L chains define specific Ig domains. More particularly, each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and £ isotypes. Each L chain hasat the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1 ).

[0150] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated a, 5, s, y, and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0151] The constant domain includes the Fc portion which comprises the carboxyterminal portions of both H chains held together by disulfides. The effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0152] The pairing of a VH and VL together forms a "variable region" or "variable domain" including the amino -terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." The V domain contains an antigen binding protein which affects antigen binding and defines specificity of a particular antibody for its particular antigen. V regions span about 1 10 amino acid residues and consist of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3) that are each 9-12 amino acids long. The FRs largely adopt a [3-sheet configuration and the hypervariable regions form loops connecting, and in some cases forming part of, the [3-sheet structure.

[0153] "Hypervariable region", "HVR", or "HV" refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3). A number of hypervariable region delineations are in use and are encompassed herein.

[0154] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1 , CDR2 and CDR3. The CDRs of VH are also referred to herein as CDR H1 , CDR H2 and CDR H3, respectively, wherein CDR H1 corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH and CDR H3 corresponds to CDR 3 of VH. Likewise, the CDRs of VL are referred to herein as CDR L1 , CDR L2 and CDR L3, respectively, wherein CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901 - 917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and Plukthun J. Mol. Biol. 309: 657-670, 2001 ; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally-occurring amino acid. In this regard, Padlan et al., FASEB J., 9: 133-139, 1995 established that the five C- terminal amino acids of heavy chain CDR2 are not generally involved in antigen binding.

[0155] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region or CDR residues herein defined. The FRs of VH are also referred to herein as FR H1 , FR H2, FR H3 and FR H4, respectively, wherein FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH and FR H4 corresponds to FR 4 of VH. Likewise, the FRs of VL are referred to herein as FR L1 , FR L2, FR L3 and FR L4, respectively, wherein FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL and FR L4 corresponds to FR 4 of VL.

[0156] “A peptide for forming an antigen binding protein” generally refers to a peptide that may form a conformation that confers the specificity of an antibody for antigen. Examples include whole antibody or whole antibody related structures, whole antibody fragments including a variable domain, variable domains and fragments thereof, including light and heavy chains, or fragments of light and heavy chains that include some but not all of hypervariable regions or constant regions.

[0157] An "intact" or “whole” antibody is one which comprises an antigen-binding protein as well as a CL and at least heavy chain constant domains, CH1 , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.

[0158] “Whole antibody related structures” include multimerized forms of whole antibody.

[0159] “Whole antibody fragments including a variable domain” include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0160] The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigenbinding protein.

[0161] A Fab' fragment differs from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'- SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0162] A F(ab')2 fragment roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.

[0163] An "Fv" is an antibody fragment which contains a complete antigen-recognition and - binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association.

[0164] In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavychains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody.

[0165] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0166] A “single variable domain” is half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0167] "Diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). The small antibody fragments are prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that interchain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.

[0168] Diabodies may be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0169] An "isolated antibody" is one which has been identified and separated and / or recovered from a component of its pre-existing environment. Contaminant components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0170] A "human antibody" refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage -display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

[0171] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, goat or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0172] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies may be prepared by the hybridoma methodology, or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells. The "monoclonal antibodies" may also be isolated from phage antibody libraries.

[0173] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.

[0174] The term "anti-soluble IRAP antibody" or "an antibody that binds to soluble IRAP" or “soluble IRAP binding protein or antibody” refers to a protein or an antibody that is capable of binding soluble IRAP with sufficient affinity such that the antibody is useful as a diagnostic and / or detection agent in targeting proteins or cells expressing or presenting soluble IRAP. Preferably, the extent of binding of an anti-soluble IRAP antibody to an unrelated tag or protein is less than about 10% of the binding of the antibody to soluble IRAP as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to soluble IRAP has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM.

[0175] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Generally, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0176] As used herein, the term “binds” in reference to the interaction of an antigen binding protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds toepitope "A", the presence of a molecule containing epitope “A” (or free, unlabelled “A”), in a reaction containing labeled “A” and the protein, will reduce the amount of labelled “A” bound to the antibody.

[0177] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that an antigen binding protein of the invention reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells.

[0178] As used herein, the term “does not detectably bind” shall be understood to mean that an antigen binding protein, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or 8% or 6% or 5% above background. The background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control antigen. The level of binding may be detected using biosensor analysis (e.g. Biacore) in which the antigen binding protein is immobilized and contacted with an antigen.

[0179] As used herein, the term “does not significantly bind” shall be understood to mean that the level of binding of an antigen binding protein of the invention to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the antigen binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control polypeptide. The level of binding may be detected using biosensor analysis (e.g. Biacore) in which the antigen binding protein is immobilized and contacted with an antigen.

[0180] An "affinity matured" antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art.

[0181] "ADCC" refers to a process called antibody-dependent cellular cytotoxicity, which is an immune response mediated primarily by natural killer (NK) cells in humans. In ADCC, FcyRIII on the surface of an NK cell recognizes the Fe region of antibody thatis bound to antigen displayed on the surface of a target cell. This activates the NK cell, which releases perforins and granzymes, leading to lysis and apoptosis of the target cells.

[0182] "CDC" refers to a complex process called complement-dependent cytotoxicity that can lead to cell killing through the action of a cascade of proteins that can act through either of two major pathways.

[0183] "ADCP" refers to a process called antibody dependent cell-mediated phagocytosis. In this Fe receptor-mediated process, target cells to which antibodies are bound are engulfed by phagocytic cells, such as macrophage, monocytes, neutrophils, and dendritic cells. Multiple Fc receptors are involved in this process.

[0184] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0185] An "agonist antibody", as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

[0186] As meant herein, an "Fc region" is a dimer consisting of two polypeptide chains joined by one or more disulfide bonds, each chain comprising part or all of a hinge domain plus a CH2 and a CH3 domain. Each of the polypeptide chains is referred to as an "Fc polypeptide chain." To distinguish the two Fe polypeptide chains, one is referred to herein as an "A chain" and the other is referred to as a "B chain." More specifically, the Fc regions contemplated for use with the present invention are IgG Fc regions, which can be mammalian or human lgG1 , lgG2, lgG3, or lgG4 Fc regions. Among human lgG1 Fc regions, at least two allelic types are known.

[0187] An "Fc-containing protein," as meant herein, is a protein comprising an Fc region as described herein and a binding region that binds to a target molecule. The term "Fc containing protein" encompasses an antibody or an Fc fusion protein that contains an Fc region.

[0188] The present invention also provides an antigen binding protein, or a nucleic acid encoding the same, having at least 80% identity to a sequence disclosed herein. In one example, an antigen binding protein or nucleic acid of the invention comprises a sequenceat least about at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence disclosed herein.

[0189] Alternatively, or additionally, the antigen binding protein comprises a CDR (e.g., three CDRs) at least about at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to CDR(s) of a VH or VL as described herein according to any example or embodiment. In one example, the antigen binding protein comprises a FR at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to FR(s) of a VH or VL as described herein according to any example or embodiment.

[0190] In another example, a nucleic acid of the invention comprises a sequence at least about at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence encoding an antigen binding protein having a function as described herein according to any example. The present invention also encompasses nucleic acids encoding an antigen binding protein of the invention, which differs from a sequence exemplified herein as a result of degeneracy of the genetic code.

[0191] In some embodiments, the nucleic acid may comprise or consist of a sequence selected from: SEQ ID NO:98, SEQ ID NO:99; SEQ ID N0:100, SEQ ID NO:101 ; SEQ ID NO:102, SEQ ID NO:103, SEQ ID NQ:104 and SEQ ID NQ:105; or a sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. In some embodiments, the nucleic acid may comprise or consist of the sequences:SEQ ID NO:98 and SEQ ID NO:99, or sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;SEQ ID NO:100 and SEQ ID NO:101 , or sequences at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;SEQ ID NQ:102 and SEQ ID NQ:103, or sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; orSEQ ID NQ:104 and SEQ ID NQ:105, or sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0192] The present invention also contemplates a nucleic acid that hybridizes under stringent hybridization conditions to a nucleic acid encoding an antigen binding protein described herein. A “moderate stringency” is defined herein as being a hybridization and / or washing carried out in 2 x SSC buffer, 0.1 % (w / v) SDS at a temperature in the range 45°C to 65°C, or equivalent conditions. A “high stringency” is defined herein as being a hybridization and / or wash carried out in 0.1 x SSC buffer, 0.1 % (w / v) SDS, or lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using wash / hybridization solutions other than SSC known to those skilled in the art. For example, methods for calculating the temperature at which the strands of a double stranded nucleic acid will dissociate (also known as melting temperature, or Tm) are known in the art. A temperature that is similar to (e.g., within 5°C or within 10°C) or equal to the Tm of a nucleic acid is considered to be high stringency. Medium stringency is tobe considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.

[0193] The present invention also contemplates mutant forms of an antigen binding protein of the invention comprising one or more conservative amino acid substitutions compared to a sequence set forth herein. In some examples, the antigen binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity.

[0194] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), [3-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example in Kyte and Doolittle J. Mol. BioL, 157: 105-132, 1982 and hydrophilic indices are described in, e.g., US4554101.

[0195] The present invention also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids. In some examples, the antigen binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0196] In one example, the mutation(s) occur within a FR of an antigen binding domain of an antigen binding protein of the invention. In another example, the mutation(s) occur within a CDR of an antigen binding protein of the invention.

[0197] Exemplary methods for producing mutant forms of an antigen binding protein include: mutagenesis of DNA (Thie et aL, Methods Mol. Biol. 525: 309-322, 2009) or RNA (Kopsidas et aL, Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18, 2007; and WO1999 / 058661 );- introducing a nucleic acid encoding the polypeptide into a mutator cell, e.g., XL-1 Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);- DNA shuffling, e.g., as disclosed in Stemmer, Nature 370: 389-91 , 1994; and- site directed mutagenesis, e.g., as described in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0198] Exemplary methods for determining biological activity of the antigen binding proteins of the invention will be apparent to the skilled artisan and / or described herein, e.g., antigen binding. For example, methods for determining specific and selective binding to soluble IRAP are described herein.

[0199] As used herein, the properties of amino acids are defined in the following table:Assays

[0200] Detecting and / or quantifying soluble IRAP using one or more antigen binding proteins as described herein can be performed by any method suitable to identify thepresence and / or amount of soluble IRAP in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantifying may be performed by measuring the concentration of soluble IRAP in the sample or samples. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

[0201] Detecting and / or quantifying the one or more proteins may be performed using an immunological method, involving an antigen binding protein as described herein, or a fragment thereof capable of specific binding to soluble IRAP. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods may be performed, for example, in microtitre plate or strip format.

[0202] Immunological methods in accordance with the invention may be based, for example, on any of the following methods.

[0203] Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antigen binding protein has incubated with the sample and reacted with the target antigen (ie soluble IRAP) present therein to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.

[0204] In particle immunoassays, several antigen binding proteins are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the one or more proteins.

[0205] In immunonephelometry, the interaction of an antigen binding protein and target antigen results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer. The antigen concentration can be determined within minutes of the reaction.

[0206] Radioimmunoassay (RIA) methods employ radioactive isotopes such as 1125 to label either the antigen or antigen binding protein. The isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays.

[0207] Enzyme (EIA) immunoassays were developed as an alternative to radioimmunoassays (RIA). These methods use an enzyme to label either the antigen binding protein or target antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA).

[0208] ELISA is a biochemical technique used for detecting and quantifying substances such as an antibody or an antigen in a sample. One of the types of ELISA is called a “sandwich” assay because the substance to be measured is bound between two antigen binding proteins (eg antibodies) — the capture antigen binding protein (eg antibody) and the detection antigen binding protein (eg antibody). This format of ELISA is often preferred because it is sensitive and robust. When designing a sandwich ELISA it is important that the capture antigen binding protein and the detection antigen binding protein must recognise two separate non-overlapping epitopes. A capture antigen binding protein and a detection antigen binding protein that do not interfere with one another and can bind simultaneously to the antigen are considered a matched pair and are suitable for developing a sandwich ELISA. For accurate quantitative results, the signal of the unknown substance of interest should be compared against those of standards included on the same ELISA plate. The concentration range of the standards should be optimized to ensure a suitable standard curve.

[0209] In one embodiment, the level of one or more proteins is quantified using a direct or an indirect sandwich ELISA technique. In one embodiment, the capture antigen binding protein is coated onto the substrate or surface, and the sample is applied to the coated substrate. After a period of time and under conditions to allow any soluble IRAP present in the same to bind the capture antigen binding protein and the unbound soluble IRAPand remainder of the sample has been washed away, the standard ELISA protocol is followed in order to detect the amount of bound capture antigen binding protein. If the detection antigen binding protein is conjugated or linked to a detectable label, allowing direct detection of the detection antigen binding protein, this is referred to as a direct sandwich ELISA. Where the binding of the detection antigen binding protein can be indirectly detected by the detection antigen binding protein being bound by another molecule which is or includes a detectable label, this is referred to as an indirect sandwich ELISA.

[0210] In another embodiment, the level of one or more proteins is quantified using a direct or an indirect ELISA technique (ie not a sandwich ELISA). In one embodiment, the sample (ie the source of antigen) is coated onto the substrate or surface, and the detection antigen binding protein is applied to the coated substrate or surface. After a period of time and under conditions to allow any soluble IRAP present in the same to bind the detection antigen binding protein and the unbound soluble IRAP and remainder of the sample has been washed away, the standard ELISA protocol is followed in order to detect the amount of bound detection antigen binding protein. If the detection antigen binding protein is conjugated or linked to a detectable label, allowing direct detection of the detection antigen binding protein, this is referred to as a direct ELISA. Where the binding of the direct antigen binding protein can be indirectly detected by the detection antigen binding protein being bound by another molecule which is or includes a detectable label, this is referred to as an indirect ELISA.

[0211] Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

[0212] Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.

[0213] Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g., using a sensor chip) or indirect procedure may be used in the detection of soluble IRAP.

[0214] The Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention. One binding partner (eg detection antigen binding protein) is labelled with biotin and the other partner (secondary antibody) is labelled with avidin or streptavidin.

[0215] A particularly preferred form of a method of the invention is an ELISA assay as described herein. First, a complex composed of an antigen binding protein for capturing soluble IRAP in a biological sample (wherein the antigen binding protein is also referred to as a “capture antigen binding protein”), an antigen binding protein for detecting soluble IRAP (wherein the antigen binding protein is also referred to as a “detection antigen binding protein”) and soluble IRAP is formed on a solid phase (also called “solid support”). In the case where soluble IRAP is contained in the biological sample, the complex can be formed by mixing the biological sample, the capture antigen binding protein and the detection antigen binding protein together. Subsequently, a solution capture antigen binding protein to form the complex on the solid phase. Alternatively, it may be possible to use a solid phase on which the capture antigen binding protein has been immobilized in advance. That is, a solid phase on which the capture antigen binding protein has been immobilized, the biological sample and the detection antigen binding protein are brought into contact with one another to form the complex on the solid phase.

[0216] The mode of the immobilization of the capture antigen binding protein onto the solid phase may be by any means. For example, the capture antigen binding protein may be bound to the solid phase directly, or the capture antigen binding protein may be bound to the solid phase indirectly through another substance. The direct bonding is, for example, physical adsorption or the like. The indirect bonding is, for example, bonding through a combination of biotin and avidin or streptavidin. In this case, the capture antigen binding protein and the solid phase may be bound indirectly through a bond between biotin and the avidin or streptavidin by modifying the capture antigen binding protein with biotin previously and bonding the avidin or streptavidin to the solid phase previously.

[0217] The material for the solid phase can be selected from, for example, an organic polymeric compound, an inorganic compound and a biological polymer. Specific examples of the organic polymeric compound include latex, polystyrene and polypropylene. Specific examples of the inorganic compound include a magnetic material (e.g., iron oxide, chromium oxide and ferrite), silica, alumina and glass. Specific examples of the biological polymer include insoluble agarose, insoluble dextran, gelatin andcellulose. Two or more of these materials may be used in combination. The shape of the solid phase is not particularly limited, and examples of the shape include particles, a film, a microplate, a microtube and a test tube.

[0218] In this embodiment, it is possible to carry out B / F (Bound / Free) separation for removing any unreacted free component that does not contribute to the formation of the complex, between the complex formation step and the complex detection step. The term “unreacted free component” refers to a component that does not constitute the complex. Examples of the unreacted free component include an antigen binding protein that does not bind to soluble IRAP and a substance other than soluble IRAP in the biological sample (i.e., a contaminating substance). The means for the B / F separation is not particularly limited. In the case where the solid phase is composed of particles, the B / F separation can be achieved by collecting only the solid phase having the complex captured thereon by centrifugation. In the case where the solid phase is a vessel such as a microplate and a microtube, the B / F separation can be achieved by removing a solution containing the unreacted free component. In the case where the solid phase is composed of magnetic particles, the B / F separation can be achieved by removing a solution containing the unreacted free component by sucking the solution by means of a nozzle while magnetically restraining the magnetic particles with a magnet. After the removal of the unreacted free component, the solid phase having the complex captured thereon may be washed with a proper aqueous medium such as PBS.Detectable labels

[0219] The present methods of the invention involve detection of soluble IRAP, using a detection antigen binding protein linked to a detectable label.

[0220] The term "detectable" as used herein refers to an occurrence of, or a change in, a signal that is directly or indirectly detectable either by observation or by instrumentation. Typically, the detectable response is an occurrence of a signal wherein the fluorophore is inherently fluorescent. Alternatively, the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0221] The term "label," as used herein, refers to a chemical moiety or protein that is directly or indirectly detectable (e.g. due to its spectral properties, conformation or activity) when attached to antigen binding protein and used in the present methods.

[0222] Exemplary labels include a colormetric molecule, chromogenic molecule, fluorescent molecule, a luminescent molecule, a bioluminescent molecule, a radioactive molecule, and an antigenic molecule

[0223] The detection antigen binding protein may be coupled to an enzyme.Exemplary enzymes include glucose oxidase (GOx), luciferase, peptidase, protease, glycosidase, peroxidase (such as horseradish peroxidase, HRP), catalase, acetylcholinesterase, [3-galactosidase, and phosphatase (such as alkaline phosphatase, AP).

[0224] A detection label conjugated to an antigen binding protein may be a fluorochrome. Suitable fluorescent labels are known in the art and include fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor 488, Alexa fluor 594, Alexa fluor 647, Alexa fluor 710, Alexa fluor 750, Alexa fluor 405, cyanin 5 (Cy5), Cyanin 5.5 (Cy5.5), rhodamine, pacific blue (PacB), horizon violet 450 (HV450), pacific orange (PacO), horizon-V500 (HV500), Krome Orange, Brilliant Violet 421 (BV421 ), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 71 1 (BV711 ), Brilliant Violet 785 (BV785), Brilliant Ultraviolet 395 (BUV395), Brilliant Ultraviolet 496 (BUV496), Brilliant Ultraviolet 737 (BUV737), Orange Cytognos (OC)515, quantum dots and conjugates thereof coupled with PE, to APC or to PerCP (e.g. PE / Cy5, PE / Cy5.5, PE / Cy7, PerCP / Cy5.5, APC / Cy7, APC-H7, APC-Alexa750, PE-Texas Red, PE-Dazzle, PE- CF594) or any additional compatible fluorochrome or fluorochrome tandem, etc.

[0225] A suitable label may be directly or indirectly linked to the detection antigen binding protein via the use of a suitable tag. In a preferred embodiment, the detectable label is linked to streptavidin.Samples

[0226] Any biological sample that is known or suspected to contain soluble IRAP is contemplated for use in the invention.

[0227] The term sample, as used herein, shall include blood samples but may also include hematopoietic biological samples such as lymph, leukopoiesis product, bone marrow and the like; also included in the term are derivatives and fractions of such fluids. The blood sample is drawn from any site for example by venepuncture. Blood samples will usually be from about 1 to 100 ml of whole blood, i.e. from 105to 107nucleated blood cells, and may be treated with anticoagulants, e.g. heparin, EDTA, citrate, acid citrate dextrose or citrate phosphate dextrose, as known in the art.

[0228] The sample may be a bodily fluid, for example a blood sample or a sample derived from blood as discussed above. Alternatively, the same may be a tissue sample. The sample may contain a bodily fluid and a tissue sample.

[0229] The blood sample or sample derived from blood may be a whole blood, buffy coat, peripheral blood mononuclear cell (PBMC), cord blood, purified or sorted cell population, or bodily fluid. Bodily fluids include lymphatic fluid, semen, nasal secretions, bronchial secretions, alveolar fluid, cerebrospinal fluid, endolymph, synovial fluid, pleural fluid, pericardial fluid (pericardial liquor), menstrual fluid, amniotic fluid, urine, interstitial fluid, cerebrospinal fluid, peritoneal fluid, breast milk, or combinations thereof.

[0230] The tissue sample may be selected from thymus, placental, fetal membrane, uterine, ovarian, endometrial, renal, skeletal muscle, vascular endothelium, pancreas, bile duct, adrenal, cardiac, brain, bone, pituitary gland, tonsil, lymph node, bronchial, nasal or gut or skin biopsy. In one embodiment, the tissue sample may be obtained by chorionic villus sampling (CVS). Preferably, the tissue sample is treated to form a single cell suspension. Forming a single cell suspension may be through a mesh filter eg for tonsil, thymus or lymph node. Alternatively, forming a single cell suspension may be via tissue digestion and then using a mesh through filter.

[0231] Specifically, in relation to biological samples known or suspected to contain soluble IRAP, the sample may be blood, bone marrow or lymphoid tissue. The tissue may be selected from thymus, placental, fetal membrane, uterine, ovarian, endometrial, renal, skeletal muscle, vascular endothelium, pancreas, bile duct, adrenal, cardiac, brain, bone, pituitary gland, tonsil, lymph node, bronchial, nasal or gut or skin biopsy. Alternatively, the blood sample may be a whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, cord blood, purified or sorted cell population or bodily fluid. Bodily fluids include samples from the group consisting of lymphatic fluid, semen,amniotic fluid, nasal secretions, bronchial secretions, alveolar fluid, endolymph, pericardial fluid (pericardial liquor), peritoneal fluid, menstrual fluid, urine, interstitial fluid, cerebrospinal fluid, peritoneal fluid, breast milk, or combinations thereof.

[0232] The sample may be taken from any mammal including primate, in particular, human, non-human primate, murine (more particularly mouse), equine, bovine, ovine, caprine, porcine, canine, feline etc. Whole blood can be draft from the sample using any acceptable procedure. The use of whole blood allows detection of effector cells such as eosinophils and basophils. Alternatively, the blood samples may be resuspended in a solution that selectively lyses erythrocytes, e.g. ammonium chloride-potassium; ammonium oxalate, etc.

[0233] In some circumstances the sample may be subjected to treatment such as dilution in buffered medium, concentration, filtration, linearization, heat treatment, incubation in denaturing condition, or other gross treatment that will not involve the destruction of soluble IRAP. Treatments may also include removal of cells by various techniques, including centrifugation, using Ficoll-Hypaque, panning, affinity separation, using antibodies specific for one or more markers present as surface membrane proteins on the surface of cells. For example, where a sample is diluted due to its large volume the sample may require concentration or centrifugation to allow a lower amount of capture and / or detection antigen binding protein to be added.

[0234] The capture and / or detection antigen binding proteins as described above can be directly added to a whole blood sample. The amount of capture and / or detection antigen binding protein necessary to bind soluble IRAP is empirically determined by performing a test assay. The amount may vary with the affinity of the capture and / or detection antigen binding protein. The sample and capture and / or detection antigen binding proteins are incubated for a period of time sufficient for the capture and / or detection antigen binding proteins to bind to soluble IRAP. This incubation time is usually at least about 10 minutes, not more than an hour, usually not more than 30 minutes.Examples

[0235] Described in the examples below are the generation and characterisation of novel anti-IRAP antibodies which are directed towards the C-terminal domain of the enzyme. The specificity of the antibodies for IRAP was confirmed by the detection on aWestern blot of a 165 kDa band corresponding to full length, membrane-bound form of IRAP and a 150 kDa band corresponding to the secreted form of IRAP (sIRAP). Importantly, there was also a clear absence of a band in protein lysates of cardiac tissue from IRAP KO mice, validating the specificity of the antibodies. IRAP has the propensity to form homodimers, which are also detected by the antibodies.

[0236] A sandwich ELISA was then developed which involved optimisation of a range of factors including the antibody combination, antibody concentration, denaturing & reducing conditions. Parameters such as the detection range, sensitivity, intra- and interassay variability, spike recovery and linearity were also assessed. Collectively, these optimised conditions enabled the detection of nanogram levels of purified sIRAP using two different antibody combinations which is a great strength of this invention. To further validate that the optimised sandwich ELISAs can detect sIRAP, human plasma samples from women at various stages of pregnancy were tested for sIRAP expression which is known to increase in the circulation throughout gestation. Excitingly, the ELISAs with different anti-IRAP antibody combinations can detect increasing levels of sIRAP in plasma from pregnant women, which correlated with gestational period, results that were confirmed using Western blot analysis, validating its potential clinical utility in the detection and quantification of sIRAP in human plasma.Example 1 - Materials and methods

[0237] Generation of monoclonal IRAP antibodies. Antibodies directed towards the soluble C-terminal domain of human IRAP (Figure 1 ) were generated using hybridoma technology by the Monash Antibody Technologies Facility (MATF, Monash University, Australia). The antibody generation process is summarised in Figure 2. Briefly, six mice with global gene deletion of IRAP (IRAP KO) were immunised 3 times at two-week intervals (i.p.) with a combination of 16 pg of recombinant soluble human IRAP protein and immune adjuvant (Sigma #S6322) with methylated CpG until a strong serum antibody titre was reached. The spleens of the mice were fused with SP2 / 0-Ag14 myeloma cells using polyethylene glycol to generate antibody-producing hybridomas. Hybridoma colonies (adapted to 5% FB serum-free media) were grown for 10 days at which point the number of hybridoma colonies was determined and after a further 3 days incubation, an aliquot of antibody supernatant was taken for screening. The cell culture supernatants were screened for antibodies that bound to soluble human IRAP by antigen microarray and these results were confirmed by antigen ELISA. The antibody containingsupernatants were also assessed by Western blot to determine the specificity for I RAP over other related aminopeptidases including aminopeptidase N, endoplasmic reticulum aminopeptidase I and II. Based on the binding profiles, four hybridoma clones were chosen for further analysis (clones RF7, RG4, RB9 and RH3). The cells were expanded and antibody was purified from the supernatant using Protein A / G affinity chromatography by MATF, eluting bound antibody with elution / neutralisation buffer then exchanging to DPBS buffer. Purification products were assessed at 280 nm. Antibody purification profiles are shown in Figure 3. Later, the antibodies were subcloned and biotinylated by MATF.

[0238] Sources of IRAP used in experiments. Recombinant soluble human IRAP (referred to as purified si RAP) were generated by Monash Protein Production Unit (mPPU) or CSIRO (Melbourne, Australia). For the production of sIRAP from mPPU, the IRAP coding-region templates were synthesised by Genscript, codon-optimised for transcription / translation in human cells and subcloned into the pMBSV5 mammalian expression vector. The N-terminal 130 amino acids encoding the cytoplasmic and transmembrane-spanning sequences were deleted and the recombinant protein were tagged with human IgGI Fc in the C-terminus or polyhistidine tag (10xHis) in the N- terminus. For the CSIRO protein, the IRAP coding-region templates were synthesised by Genscript, codon-optimised for transcription / translation in human cells and subcloned into the mammalian expression vector, pCAGGS. The N-terminal 131 amino acids encoding the cytoplasmic and transmembrane-spanning sequences were removed and replaced by the mouse interleukin-3 (IL-3) SP, followed by four amino acids (ASIS) corresponding to the mature IL-3 N-terminus, the IRAP extracellular domain (894 amino acids) and a C- terminal with a mFlag epitope tag (DYKDDDDK). For the recombinant proteins from both sources, the plasmid was transfected into freestyle HEK 293-F cells at a ratio of 1 :3 [DNA:PEI], The cells were maintained for 7-9 days after which they were harvested and recombinant IRAP was purified using Protein G affinity / Ni-NTA affinity resin or using anion-exchange chromatography followed by gel filtration on a Superdex S200 26 / 60 column.

[0239] Protein lysates prepared from cardiac tissue of 10-week old male WT and IRAP KO mice were used to test the specificity of the novel antibodies. Briefly, the tissue was homogenised in 300-500 pl of 1 .5x Laemmli buffer (diluted from 5x Laemmli buffer (25% glycerol, 7.5% SDS, 250 mM Tris-HCI pH 8.0, 0.25 mg / ml bromophenol blue)) containing2-mercaptoethanol and then sonicated for 6 seconds, 3 times. The protein was heated in a water bath at 37°C for 10 minutes before being centrifuged at 13,000 rpm for 10 minutes at 4°C. The protein-containing supernatant was transferred to new tubes.

[0240] All experiments involving animals were approved by the Monash University Animal Ethics Committee and conducted in accordance with the National Health and Medical Research Council’s, Australian code for the care and use of animals for scientific purposes and the ARRIVE guidelines.

[0241] Membrane preparations of HEK293T cells (ATCC #CRL-3216) transiently transfected with the expression vector, pCI containing full length cDNA for human IRAP were used as an enriched source of full length IRAP (mIRAP), following the methodology previously described by Lew, R. A. et al. J. Neurochem. 86, 344-350 (2003).

[0242] The collection of plasma from healthy adults was approved by the Monash University Human Research Ethics Committee (#37732) and conducted in accordance with the NHMRC guidelines, National Statement on Ethical Conduct in Human Research 2007. Informed consent was obtained from all participants. Separate batches of pooled plasma from women at 28- and 36-weeks of gestation and full-term were kindly provided by Professor Natalie Hannan, Mercy Hospital for Women. It is worth noting that the term samples were from pregnancies where there was borderline growth restriction of the foetus.

[0243] Indirect ELISAs. All antibody and antigen concentrations used are specified in the results for each individual experiment. Nunc MaxiSorp 96-well ELISA plates were coated with the appropriate antigen diluted in coating buffer (0.1 M Na2CO3 / NaHCO3, pH 9.6) overnight (50 pl / well). Following overnight antigen coating, the plates were washed 3 times quickly with 1 x PBS. The antigen was then blocked with 5% skim milk in 1 x PBS for at least 30 minutes (100 pl / well). After washing with 1x PBS (3 quick washes), plates were incubated with the appropriate anti-IRAP antibody diluted in 5% skim milk for 1 hour (50 pl / well). Wash steps were repeated and the appropriate HRP-conjugated secondary antibody (Agilent-Dako) diluted in 5% skim milk (1 :2000) was added for 1 hour (50 pl / well). Following washing, 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (ThermoFisher) was added to each well until the desired colour was achieved (10-15 minutes; 50 pl / well), at which point the reaction was stopped with 0.2 M H2SO4 (50 pl / well). The absorbance of each well was measured at 405 nm using a CLARIOstar plate reader (BMG Labtech).

[0244] Sandwich ELISAs. All antibody and antigen concentrations used are specified in the results for each individual experiment. Nunc MaxiSorp 96-well ELISA plates were coated overnight with capture antibody diluted to varying concentrations in coating buffer (0.1 M Na2CO3 / NaHCC>3, pH 9.6; 100 pl / well). Following the overnight incubation, the plates were washed 3 times quickly with 1 x PBS. The plate coated with the antibody was then blocked with blocking buffer (3% BSA in 1x PBS) for at least one hour (200 pl / well). After washing with 1 x PBS (three quick washes), plates were incubated for 2 hours with the antigen samples diluted to varying concentrations in blocking buffer (100 pl / well). In the experiments using human plasma, the purified sIRAP standard curve was spiked with pooled healthy human plasma to mirror conditions of the diluted test plasma samples. Purified sIRAP standards / plasma samples in the experiments using RF7-RB9-B were denatured in 2.5% SDS in blocking buffer and heated at 70°C for 5 minutes. The ELISA conditions were optimised by testing varying concentrations of SDS ± 2-mercaptoethanol or dithiothreitol with heating at 70 or 95°C. After the 2-hour incubation and washing with 1 x PBS (3 quick washes), plates were incubated with the biotinylated detection antibody diluted to varying concentrations in blocking buffer for 2 hours (100 pl / well). Wash steps were repeated and streptavidin-HRP (R&D Systems) diluted in blocking buffer (1 :200) was added for 30 minutes (100 pl / well). Following washing, TMB substrate (ThermoFisher) was added to each well until the desired colour was achieved (2-15 minutes; 50 pl / well), at which point the reaction was stopped with 0.2 M H2SO4 (50 pl / well). The absorbance of each well was measured at 450 nm and corrected for optical imperfections by subtraction of the absorbance at 570 nm. In experiments using human plasma, the absorbance of the negative control containing no antigen was also subtracted from each sample reading.

[0245] Western blotting. To test the specificity of the anti-IRAP antibodies and to validate the protein expression of I RAP in various samples, standard Western blotting was conducted. All samples were diluted in 1 ,5x Laemmli buffer (diluted from 5x Laemmli buffer (25% glycerol, 7.5% SDS, 250 mM T ris-HCI pH 8.0, 0.25 mg / ml bromophenol blue)) and heated at 70°C for 5 minutes. Sources of IRAP used include mIRAP (0.01 pg protein / pl or 0.1 pg protein / well), purified sIRAP (0.001 pg / pl or 0.001 pg / well), protein lysates from hearts of male WT or IRAP KO mice (20 pg / well) and human plasma (diluted 1 :10). Before loading of samples, 7.5% or 10% gels (15 wells, 1 mm thickness) were prepared using a TGX Stain-free FastCast Acrylamide starter kit (BioRad). Precision Plus Protein Standards Dual Colour (BioRad; 5 pl) and the samples (10 pl) were loaded intothe gel and electrophoresed at 150V for -40-50 minutes in the presence of Tris / glycine / SDS running buffer. Once proteins on the gel were appropriately separated, they were transferred to a Trans-Blot Turbo Mini-size LF PVDF membrane using the Transfer-Blot Turbo transfer system (BioRad). Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1 % Tween-20 in 1 x TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk / TBS-T) for 1 hour. The rabbit anti-IRAP antibody (1 :2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 pg / ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4°C. The next day, membranes were washed with TBS-T (3x15 minutes) and then incubated with either goat anti-rabbit HRP or goat anti-mouse HRP (1 :2500; Agilent-Dako) diluted in blocking buffer on the shaker for 1 hour at room temperature. After washing with TBS-T (3x15 minutes), membranes were developed with Clarity Western ECL substrate (BioRad) for 3 minutes and imaged using the digital imager ChemiDoc MP imaging system (BioRad). Individual protein bands could be quantified by measuring the optical density (OD) per unit area using ImageLab software (BioRad).

[0246] Production of recombinant aminopeptidase N (APN) and endoplasmic reticulum aminopeptidase 1 (ERAP1). For the production of hAPN and ERAP1 from mPPU, the coding-region (2,901 bp and 2823 bp in length respectively) were synthesised by Genscript, codon-optimised for transcription / translation in human cells and subcloned into the pUC57 mammalian expression vector. The recombinant proteins were tagged with human IgG 1 Fc in the C-terminus. The plasmids were transfected into freestyle HEK 293-F cells, expressed and purified using standard methods.

[0247] Statistical analysis. Results are presented as mean ± standard error of the mean (SEM) and statistical analysis was conducted using Graph Pad Prism (Graph Pad Prism 6 Software Inc.). p<0.05 is considered statistically significant. Simple linear regression was conducted to generate standard curves at increasing sIRAP concentrations. To compare the expression of IRAP throughout gestation in human plasma in sandwich ELISAs and Western blot experiments, one-way ANOVAs with Tukey’s post-hoc test were conducted.

[0248] The formulas used to calculate the sensitivity, intra- and inter-assay variability, spike recovery and linearity of the sandwich ELISAs are summarised below.Sensitivity (ng / ml) = mean of zero standard + 2 * SD of zero standard (1 )Intra / inter assay variability (%) = fSD°frePllcatesAxIQO (OX \mean of replicates / Example 2 - Generation and characterisation of novel anti-IRAP antibodies

[0249] Antibodies that recognise the soluble C-terminal domain of human IRAP were successfully generated by Monash Antibody Technologies Facility (MATF) using hybridoma technology.

[0250] To validate the selectivity of the antibodies to bind to IRAP, and not to aminopeptidase N (APN) or endoplasmic reticulum aminopeptidase 1 (ERAP1 ), the antibody clones generated were tested for their ability to bind to sIRAPhFc, APNhFc, and ERAPI hFC using western blots (Figure 13). Four distinct clones (RF7, RB9, RH3, RG4) were selected for expansion and purification based on their binding profiles with high specificity for sIRAP over other related aminopeptidases (clearly binding to sIRAP but not to APN or ERAPI ).

[0251] Table 2: Testing selectivity of the clones in binding to IRAP, APN and ERAP1 .

[0252] To further validate the specificity of the antibodies to the different forms of I RAP, Western blotting was conducted. Recombinant soluble human IRAP, prepared by CSIRO and referred to as purified si RAP, was used as the source of si RAP in all experiments and membrane preparations of HEK293T cells transiently transfected to overexpress IRAP were used as the source of full length IRAP (mIRAP). All four mouse antibodies successfully bound to both mIRAP and sIRAP with a specific band at ~150-160 kDa (Figure 6). Dimerised forms of both mIRAP and sIRAP were also observed with sIRAP displaying a greater tendency to form dimers compared with monomers (molecular weight >250 kDa; Figure 6). Notably, differences in the binding of an antibody to mIRAP and sIRAP may be due to differences in the amount of IRAP loaded given the exact concentration of IRAP in the membrane preparation is unknown. When images were captured at the same exposure time, a difference in the intensity of the bands of each antibody can also be seen. This suggests they have different binding properties and likely bind to different epitopes of IRAP.

[0253] The antibodies were then tested in an indirect ELISA to confirm whether there were any differences in potential binding to IRAP. Each of the mouse monoclonal antibodies (RF7, RB9, RG4 and RH3) displayed an ability to preferentially bind to soluble IRAP (siRAP), compared to membrane-bound IRAP (Figure 4).

[0254] RF7 binds to ml RAP and si RAP more effectively than the other antibodies, followed by RH3 and RB9 (Figure 4). Whilst the antibodies appear to be less effective at binding to mIRAP, this is likely due to the fact that only 5-10% of the total protein in the membrane preparation used as the source of full-length IRAP is actually IRAP.

[0255] To determine the lowest detection range in an indirect ELISA of one of the antibodies, RB9 was tested against decreasing purified sIRAP concentrations. Binding of RB9 to sIRAP was detectable in the nanogram range (0.001 pg / well ; Figure 5). Notably, RB9 produced equivalent readings between purified sIRAP at 0.001 pg / well and mIRAP at 10 pg / well (Figure 5). Based on these findings, RB9 and likely the other highly specific antibodies, can detect low levels of sIRAP in the nanogram detection range.

[0256] Due to these promising results, the hybridoma cell lines were subcloned and the antibodies purified and biotinylated by MATF. The specificity of the untagged and biotinylated antibodies was then tested using Western blots. All eight antibodies produced a band at -150 kDa for purified sIRAP and at -165 kDa for mIRAP, except biotinylated RG4 which did not bind well to either form of the protein (Figure 7a). Notably, IRAP dimers are also evident in both purified sIRAP and mIRAP preparations. IRAP isoforms ranging from -120 - 165 kDa were also detected in protein lysates of cardiac tissue from male wildtype (WT), but not IRAP knockout (KO) mice with all eight antibodies (Figure 7b). Overall, the detection of a band of the correct size as purified sIRAP and the absence of specific IRAP bands in tissue from IRAP KO mice, demonstrate that all the subcloned antibodies are highly specific for IRAP.

[0257] Lastly, the ability of the highly pure and tagged antibodies to maintain their binding to purified sIRAP was tested in an indirect ELISA and the outcome was compared to the pre-subcloned antibodies (Pre-SC). All four untagged, subcloned antibodies maintained their ability to bind to sIRAP (4 pg / ml; Figure 8). However, the biotinylated antibodies which were detected using either an anti-mouse HRP secondary antibody or streptavidin-HRP, had varying decreases in their binding ability compared to their untagged counterparts (Figure 8). In particular, biotinylated RH3 displayed consistently decreased sIRAP binding compared to the untagged RH3 (Figure 9). Overall, the subcloned antibodies successfully bound specifically and selectively to IRAP, and display potential for use in an IRAP sandwich ELISA which, unlike the indirect ELISA, should have improved sensitivity and allows for quantification of the amount of IRAP in an unknown sample using an sIRAP standard curve.Example 3 - Development of sandwich ELISA

[0258] The first step in the development of a sensitive IRAP sandwich ELISA was to determine the best capture and detection antibody combination to use. All 12 possible capture (2 pg / well) and detection (1 pg / well) antibody combinations were initially screened against purified sIRAP (4 pg / ml) (Figure 9a). Eight combinations successfully detected sIRAP while the remaining four were excluded from further experimentation (Figure 9a). As referred to herein, the first antibody is a capture antibody and the second antibody is the detection antibody, for example RF7-RB9-B refers to RF7 as the capture antibody and RB9 as the detection antibody. “B” designates biotinylated. Interestingly, the combination of RF7-RH3-B, RB9-RG4-B, RH3-RF7B and RG4-RB9B consistently failed to detect sIRAP (Figure 9a). These observations suggest that these antibody pairs likely bind to epitopes in a similar region and their combined use in a sandwich ELISA is therefore not viable. The top eight antibody combinations from this initial screen were subsequently tested at lower capture (0.1 & 0.5 pg / well) and detection (0.05 & 0.2 pg / well) antibody concentrations (Figures 9b and 9c). Decreasing the capture antibody to 0.1 pg / well resulted in a loss of sIRAP detection, highlighting the importance of maintaining high capture antibody concentrations. Therefore, only data at the capture antibody concentration of 0.5 pg / well is presented. RF7-RB9-B was the top performing antibody combination (Figure 9b). The top six combinations from this experiment progressed to the next stage of testing where capture (0.2 pg / well) and detection (0.01 pg / well) antibody concentrations were further decreased. Again, RF7-RB9-B outperformed all other antibody combinations (Figure 9c). The top two combinations, RF7-RB9-B and RF7-RG4- B were both selected to progress in the development of the IRAP sandwich ELISA.

[0259] The next step in the development of the sandwich ELISA was to determine the optimal antibody concentrations that could detect the low levels of sIRAP (nanogram range) expected to be present in plasma. The RF7-RB9-B combination was used for this optimisation. As expected, the purified sIRAP detection range was highly dependent on the concentration of antibodies used. At low capture (0.2 pg / well) and detection (0.1 pg / well) antibody concentrations, purified sIRAP could only be accurately detected down to a concentration of 0.5 pg / ml (Figure 10a). Given the levels of sIRAP in plasma are expected to be in the lower nanogram range, antibody concentrations were increased to improve sensitivity of the detection. At both the mid and high capture (0.5 & 2 pg / well) and detection (0.25 & 1 pg / well) antibody concentrations tested, low nanogram levels(~10 ng / ml) of purified sIRAP could be accurately and reliably detected (Figure 10a). However, at the highest tested antibody concentrations, the standard curve quickly saturated at the relatively low antigen concentration of 125 ng / ml (Figure 10b). Therefore, the mid antibody concentrations were selected to measure sIRAP in plasma given the detection of the protein fell within a broader linear range of the standard curve.Example 4 - Validation of optimised sandwich ELISA

[0260] To determine the specific detection range for both selected antibody combinations at the optimised concentrations, sandwich ELISAs were conducted at progressively decreasing concentrations of purified sIRAP. The concentrations of sIRAP in the nanogram range could be detected with both RF7-RB9-B (16-250 ng / ml; n=3) and RF7-RG4-B (31 - 500 ng / ml; n=3; Figure 1 1 a).

[0261] The sensitivity, or lower limit of detection, of the sandwich ELISAs was also calculated by determining the lowest measurable concentration that is statistically different from the zero standard. Twelve replicates of zero standards were used in each experiment. The sensitivity of the ELISAs was calculated to be 9.1 ± 3.6 ng / ml for RF7- RB9-B (n=3) and 9.6 ± 4.4 ng / ml for RF7-RG4-B (n=3).

[0262] The precision of the ELISAs was determined by measuring the intra- and interassay variability. The coefficient of variance (%CV) within an experiment (12 replicates of purified sIRAP at 0.25 pg / ml) was calculated to be 8.32 ± 2.16% for RF7-RB9-B (n=3) and 8.10 ± 0.98% for RF7-RG4-B (n=4). The %CV between experiments conducted on separate days with 12 replicates each of 0.25 pg / ml purified sIRAP was 14.72% for RF7- RB9-B (n=3) and 26.55% for RF7-RG4-B (n=4). The inter-assay variability is slightly higher than expected, possibly due to variability in the reaction time. However, the inclusion of a standard curve on every plate will minimise the effects of variability in assay conditions when testing human samples.

[0263] The spike recovery of each ELISA was assessed by spiking a known concentration of purified sIRAP into pooled control plasma and calculating if the recovered value significantly differs from the expected concentration. This was determined to be 70.68 ± 3.04% for RF7-RB9-B (n=3) and 50.27 ± 0.96 % for RF7-RG4- B (n=3). These values indicate that there is possible interference by binding proteins in the plasma that mask the detection of purified sIRAP. To ensure consistency in theinterference between different samples, the purified sIRAP in the standard curve and human plasma samples will be prepared under identical conditions (i.e. same concentrations of buffer:plasma).

[0264] Finally, the linearity was assessed by comparing the corrected values of serially diluted pooled control plasma spiked with a known concentration of purified sIRAP. Values between 80-120% are generally accepted as linear. The % recovery of dilutions greater than 1 :2 were found to be above the accepted maximum recovery of 120% for both antibody combinations (Figure 1 1 b). Therefore, in future assays, samples will not be diluted with factors greater than 1 :2.

[0265] Given the concentration of sIRAP in diluted plasma is expected to fall within the detection range and parameters such as the intra-assay variability have been validated, the two optimised sandwich ELISAs present as a highly specific and sensitive tools to measure circulating sIRAP.Example 5 - Further validation using human plasma

[0266] To validate the optimised sandwich ELISA can detect endogenously expressed sIRAP in human plasma, samples from women at various stages of pregnancy were tested given that IRAP expression is known to increase in the circulation throughout gestation and is thought to peak at term. Plasma samples were collected from nonpregnant, young female volunteers (n=6). Plasma samples were also collected from pregnant women at 28-, 36-week gestation and at term from the Mercy Hospital for Women and these samples were pooled in separate batches (n=5-6). It is important to note that there is a possibility that the term plasma samples were from pregnancies where borderline growth restriction of the foetus had been documented.

[0267] Firstly, the ability of the RF7 monoclonal anti-IRAP antibody (ELISA capture antibody) to detect IRAP in select pregnant plasma samples was investigated by Western blot analysis. There was a significant increase (p=0.001 1 , one-way ANOVA) in IRAP expression in the third trimester of pregnancy with a specific band at the expected molecular weight for sIRAP (140-150 kDa; Figure 12a). Notably, no bands were detected using a commercially available, rabbit anti-IRAP antibody which binds to the N-terminal domain, confirming that the RF7 antibody is indeed detecting sIRAP. Overall, this preliminary finding supports previous literature and confirms the plasma samples aresuitable for use to validate the utility of the sandwich ELISAs in measuring sIRAP in human samples.

[0268] The plasma samples from pregnant women were then tested in the IRAP sandwich ELISAs. Initially, all plasma samples, including that from the later stages of pregnancy, surprisingly produced similar absorbance values which were indistinguishable from the negative control in the ELISA using the RF7-RB9-B antibody combination. Given that the Western blots using the RF7 capture antibody detected increases in sIRAP in samples from the later stages of pregnancy, the lack of positive signal in the ELISA suggests sample matrix interference with the binding of the biotinylated detection antibody (RB9-B). This may be due to the propensity of this isoform of IRAP to adopt different conformations, form homo- or hetero-dimers, or interact with binding proteins in plasma. Therefore, to disrupt the conformation of IRAP and expose the antibody binding sites, samples were tested under different denaturing & reducing conditions (see methods). For the RF7-RB9-B antibody combination, samples prepared in 2.5% SDS / 3% BSA / PBS and heated at 70°C for 5 minutes was optimal for the detection and quantification of sIRAP in pregnant plasma samples as well as the recombinant sIRAP in the standard curve. However, for the RF7-RG4-B antibody combination, heating at 70°C for 5 minutes alone was sufficient for the detection of sIRAP. Using these optimised conditions, significant increases in the levels of sIRAP were detected in plasma from women at the later stages of pregnancy with both antibody combinations (RF7-RB9-B p<0.0001 , RF7-RG4-B p=0.001 ; one-way ANOVA). A consistent level of expression around 3 pg / ml was detected in the term samples (Figure 12b). Importantly, the detection of sIRAP in pregnant plasma using the novel sandwich ELISAs mirrors the results obtained from Western blot experiments, confirming the analytical validity of the IRAP ELISAs.Example 6: Comparison to commercially-available ELISA

[0269] To further assess the ability of the novel sandwich ELISAs of the invention to detect soluble IRAP, a sandwich ELISA using RF7 as the capture antibody and RB9-B as the detection antibody was compared against a commercially-available ELISA from Aviva Systems Biology (“Aviva kit”). The Aviva kit, LNPEP ELISA Kit OKECD09088, comprises a microtiter well-plate pre-coated with an anti-IRAP antibody to bind IRAP present in the applied sample. Detection of bound LNPEP (another moniker for IRAP) is performed using an avidin-HRP conjugate and colorimetric assay following addition ofTMB substrate. The Aviva kit was used according to its provided protocols (htps: / / www.avivasysbio.eom / technical-resources / protocols-procedures / elisa#ELISA).The Aviva kit is described as having a sensitivity of 64 pg / ml of I RAP and is predicted to react with recombinant human IRAP in serum, plasma, tissue homogenates and other biological fluids. The sandwich ELISA was performed as described herein.

[0270] Both the Aviva kit and the “in-house” sandwich ELISA were tested for their ability to detect purified soluble human IRAP. The Aviva kit failed to detect soluble IRAP at any of the tested concentrations (Figure 14, right graph). In stark contrast, the novel sandwich ELISA using RF7 as the capture antibody and RB9-B as the detection antibody, advantageously showed effective detection of the increasing concentrations of sIRAP (Figure 14, left graph).Example 7 - Discussion

[0271] Biological markers or biomarkers, are defined biological characteristics that are used to indicate normal physiological processes, pathogenic processes or responses to an intervention. There is increasing awareness that the discovery of new biomarkers goes hand in hand with the identification of new therapeutic targets as they have the potential to markedly accelerate all phases of drug development and improve clinical outcomes.

[0272] IRAP is a multifaceted enzyme and is currently being pursued as a therapeutic target for conditions such as ischemic stroke as well as cardiovascular, metabolic and fibrotic disease. Excitingly, it possesses a number of unique properties which also position it as a potential biomarker for specific conditions. For example, IRAP is a type II transmembrane protein and the extracellular domain of the enzyme can be cleaved and secreted into the blood stream or interstitial space in humans and non-human primates. Changes in plasma IRAP levels may also correlate with specific physiological or pathological conditions. However, many of the commercially available ELISA kits for the detection of IRAP have not been validated nor have the antibodies been shown to bind specifically to the secreted, soluble portion of the enzyme. Therefore, the detection method with high analytical validity described herein can be used to explore the potential for IRAP to serve as a disease biomarker.

[0273] In summary, the antibodies described herein, such as RF7 that was used as the capture antibody in the ELISAs produces a single specific band representing sIRAP inhuman plasma (140-150 kDa) on a Western blot with the specificity of the antibody validated by the absence of specific bands in tissue from IRAP KO mice, and the absence of binding to other closely related members of the M1 aminopeptidases, aminopeptidase N and endoplasmic reticulum aminopeptidase 1 . Additionally, the antibodies described herein, such as RF7 can detect increasing sIRAP expression in the plasma of pregnant women which correlated with advancing stages of gestation, demonstrated by both Western blot analysis as well as the sandwich ELISAs. Therefore, the ELISAs described herein have the potential to accurately measure IRAP levels in plasma given (1 ) the high specificity of the novel antigen binding proteins for sIRAP, (2) the inclusion of appropriate positive and negative controls and (3) the confirmation of the analytical validity of the ELISA by measuring human samples where the sIRAP levels were known to be regulated.

[0274] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

CLAIMS1. An antigen binding protein for binding to soluble IRAP, wherein the antigen binding protein competitively inhibits the binding to soluble IRAP of an antibody: comprising a VH comprising a sequence as set forth in SEQ ID NO: 29, and a VL comprising a sequence as set forth in SEQ ID NO: 30; comprising a VH comprising a sequence as set forth in SEQ ID NO: 59, and a VL comprising a sequence as set forth in SEQ ID NO: 60; comprising a VH comprising a sequence as set forth in SEQ ID NO: 81 , and a VL comprising a sequence as set forth in SEQ ID NO: 82; or comprising a VH comprising a sequence as set forth in SEQ ID NO: 96, and a VL comprising a sequence as set forth in SEQ ID NO: 97.

2. The antigen binding protein according to claim 1 , wherein the antigen binding protein comprises a CDRH1 , a CDRH2 and / or a CDRH3 of an antigen binding domain having a variable heavy chain as defined in any one of SEQ ID NOs: 29, 59, 81 or 96.

3. The antigen binding protein according to claim 1 or 2, wherein the antigen binding protein comprises a CDRL1 , a CDRL2 and / or a CDRL3 of an antigen binding domain having a variable light chain as defined in any one of SEQ ID NOs: 30, 60, 82 or 97.

4. The antigen binding protein according to any one of claims 1 to 3, wherein the antigen binding protein comprises: a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 29, and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 30; a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 59, and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 60;a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 81 , and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 82; or a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO:96, and a CDR1 , a CDR2 and a CDR3 of an antigen binding domain having a variable light chain as defined in SEQ ID NO: 97.

5. The antigen binding domain according to any one of claims 1 to 4, wherein the antigen binding protein comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 1 (IGMT) or 15 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 2 or 16 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 3 or 17 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 29, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 4 (IMGT) or 18 (Kabat) or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 5 (IMGT) or 19 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 6 or 20 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 30, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence set forth in SEQ ID NO: 3; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 15, a CDR2 comprising a sequence set forth in SEQ ID NO: 16, and a CDR3 comprising a sequence set forth in SEQ ID NO: 17;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 4, a CDR2 comprising a sequence set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 6; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 18, a CDR2 comprising a sequence set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 20;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 3 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 4, a CDR2 comprising a sequence as set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 6; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO:15, a CDR2 comprising a sequence as set forth in SEQ ID NO: 16 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 17 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 18, a CDR2 comprising a sequence as set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 20; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 29, and a VL comprising a sequence set forth in SEQ ID NO: 30.

6. The antigen binding protein according to claim 5, wherein when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 7, or a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 8, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 9, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 10, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 1 1 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 12, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 13, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 14, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

7. The antigen binding protein according to claim 5, wherein when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 21 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 22, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 23, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 24, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 25, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 26, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 27, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 28, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

8. The antigen binding protein according to any one of claims 1 to 7, wherein the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 29, or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 30; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

9. The antigen binding protein according to any one of claims 1 to 4, wherein the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 31 (IGMT) or 45 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 32 (IMGT) or 46 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 33 (IMGT) or 47 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 59, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 34 (IMGT) or 48 (Kabat)or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 35 (IMGT) or 49 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 36 (IMGT) or 50 (Kabat), ora sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 60, or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 31 , a CDR2 comprising a sequence set forth in SEQ ID NO: 32, and a CDR3 comprising a sequence set forth in SEQ ID NO: 33; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 45, a CDR2 comprising a sequence set forth in SEQ ID NO: 46, and a CDR3 comprising a sequence set forth in SEQ ID NO: 47;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 34, a CDR2 comprising a sequence set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 36; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 48, a CDR2 comprising a sequence set forth in SEQ ID NO: 49 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 50;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 31 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 32, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 33 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 34, a CDR2 comprising a sequence as set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 36; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 45, a CDR2 comprising a sequence as set forth in SEQ ID NO: 46 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 47 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 48, a CDR2 comprising a sequence as setforth in SEQ ID NO: 49 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 50; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 59, and a VL comprising a sequence set forth in SEQ ID NO: 60.

10. The antigen binding protein according to claim 9, wherein when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 37, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 38, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 39, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 40, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 41 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 42, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 43, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 44, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.1 1 . The antigen binding protein according to claim 9, wherein when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 51 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 52, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 53, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 54, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 55, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 56, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 57, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acidsequence of SEQ ID NO: 58, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

12. The antigen binding protein according to any one of claims 1 to 4, or 9 to 1 1 , wherein the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 59, or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 60; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

13. The antigen binding protein according to any one of claims 1 to 4, wherein the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 61 (IGMT) or 72 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 62 (IMGT) or 73 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3comprising a sequence as set forth in SEQ ID NO: 63 (IMGT) or 74 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 81 , or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 64 (IMGT) or 75 (Kabat) or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 35 (IMGT) or 76 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 65 (IMGT) or 77 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 82, or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 61 , a CDR2 comprising a sequence set forth in SEQ ID NO: 62, and a CDR3 comprising a sequence set forth in SEQ ID NO: 63; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 71 , a CDR2 comprising a sequence set forth in SEQ ID NO: 73, and a CDR3 comprising a sequence set forth in SEQ ID NO: 74;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 64, a CDR2 comprising a sequence set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 65; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 75, a CDR2 comprising a sequence set forth in SEQ ID NO: 76 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 77;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 61 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 62, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 63 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 64, a CDR2 comprising a sequence as set forth in SEQ ID NO: 35, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 65; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 72, a CDR2 comprising a sequence as set forth in SEQ ID NO: 73 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 74 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 75, a CDR2 comprising a sequence as set forth in SEQ ID NO: 76 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 77; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 81 , and a VL comprising a sequence set forth in SEQ ID NO: 82.

14. The antigen binding protein according to claim 13, wherein when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 66, or a sequence at least about 80%, at least 85%, at least90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 67, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 68, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 69, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 41 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 42, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 70, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 71 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

15. The antigen binding protein according to claim 13, wherein when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 78, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 52, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 79, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 54, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 55, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 56, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 80, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 28, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

16. The antigen binding protein according to any one of claims 1 to 4, or 13 to 15, wherein the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 81 , or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 82; or a sequence atleast about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

17. The antigen binding protein according to any one of claims 1 to 4, wherein the antigen binding domain comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 83 (IGMT) or 15 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 2 (IMGT) or 89 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 84 (IMGT) or 90 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(ii) a VH comprising a sequence as set forth in SEQ ID NO: 96, or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iii) a VL comprising a complementarity determining region (CDR) 1 comprising a sequence as set forth in SEQ ID NO: 85 (IMGT) or 91 (Kabat) or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; a CDR2 comprising a sequence as set forth in SEQ ID NO: 5 (IMGT) or 19 (Kabat), or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto; and a CDR3 comprising a sequence as set forth in SEQ ID NO: 86 (IMGT) or 92 (Kabat), or a sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(iv) a VL comprising a sequence as set forth in SEQ ID NO: 97, or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 83, a CDR2 comprising a sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence set forth in SEQ ID NO: 84; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 15, a CDR2 comprising a sequence set forth in SEQ ID NO: 89, and a CDR3 comprising a sequence set forth in SEQ ID NO: 90;(vi) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 85, a CDR2 comprising a sequence set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 86; or comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 91 , a CDR2 comprising a sequence set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 92;(vii) a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 83, a CDR2 comprising a sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 84 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 85, a CDR2 comprising a sequence as set forth in SEQ ID NO: 5, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 86; or a VH comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 15, a CDR2 comprising a sequence as set forth in SEQ ID NO: 89 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 90 and a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 91 , a CDR2 comprising a sequence as set forth in SEQ ID NO: 19 and a CDR3 comprising a sequence as set forth in SEQ ID NO: 92; or(viii) a VH comprising a sequence as set forth in SEQ ID NO: 96, and a VL comprising a sequence set forth in SEQ ID NO: 97.

18. The antigen binding protein according to claim 17, wherein when the CDRs are determined according to the IMGT system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 7, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 8, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 87, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 10, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 1 1 , or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 12, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 88, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 14, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

19. The antigen binding protein according to claim 17, wherein when the CDRs are determined according to the Kabat system, the antigen binding domain may further comprise at least one of:(i) a VH comprising a framework region (FR) 1 comprising an amino acid sequence of SEQ ID NO: 93, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 22, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 94, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 24, or a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto; and(ii) a VL comprising a FR1 comprising an amino acid sequence of SEQ ID NO: 25, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR2 comprising an amino acid sequence of SEQ ID NO: 26, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; a FR3 comprising an amino acid sequence of SEQ ID NO: 95, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, and a FR4 comprising an amino acid sequence of SEQ ID NO: 28, or a sequence at least about 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% thereto.

20. The antigen binding protein according to any one of claims 1 to 4 or 17 to 19, wherein the antigen binding protein comprises a variable heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 96, or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; and a variable light chain comprising the amino acid sequence as set forth in SEQ ID NO: 97; or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto; wherein the variable heavy and / or light chains comprise no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 1 1 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions, or additions or combination thereof, outside the indicated CDR sequences, and wherein the antigen binding protein retains the ability to bind to soluble IRAP.

21. The antigen binding protein according to any one of claims 1 to 20, wherein the antigen binding protein can also bind full length IRAP.

22. The antigen binding protein according to any one of claims 1 to 21 , wherein the antigen binding protein can be used to detect equal to or less than 0.5 ug sIRAP, equal to or less than 0.1 ug sIRAP, equal to or less than 0.05 ug sIRAP, equal to or less than 0.01 ug sIRAP, equal to or less than 0.005 ug sIRAP, equal to or less than 0.002 ug sIRAP, or equal to or less than 0.001 ug sIRAP.

23. The antigen binding protein according to any one of claims 1 to 22, wherein the antigen binding protein is in the form of:(i) a single domain antibody (sdAb);(ii) a single chain Fv fragment (scFv);(iii) a dimeric scFv (di-scFv); or(iv) one of (i) or (iii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

24. The antigen binding protein according to any one of claims 1 to 22, wherein the antigen binding protein is in the form of:(i) a diabody;(ii) a triabody;(iii) a tetrabody;(iv) a Fab;(v) a F(ab’)2;(vi) a Fv;(vii) a bispecific antibody or other form of multispecific antibody (including a BiTE); or(viii) one of (i) to (vii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

25. The antigen binding protein according to any one of claims 1 to 24, wherein the antigen binding protein is a monoclonal antibody.

26. A fusion protein comprising the antigen binding protein according to any one of claims 1 to 25.

27. A conjugate comprising the antigen binding protein according to any one of claims 1 to 25 and a label or moiety that facilitates detection.

28. A nucleic acid encoding the antigen binding protein according to any one of claims 1 to 25.

29. A vector comprising the nucleic acid of claim 28.

30. A cell comprising the vector according to claim 29 or the nucleic acid of claim 28.31 . A method for detecting the presence of or quantifying the level of soluble IRAP in a biological sample, the method comprises the steps of:- providing a biological sample- contacting the biological sample with a detection antigen binding protein as described herein;- incubating under conditions sufficient for a complex to form between soluble IRAP and the detection antigen binding protein;- determining the presence of or amount of the complex; wherein detection of the complex indicates the presence of soluble IRAP, and determining the amount of the complex quantifies the level of soluble IRAP.

32. A method for detecting soluble IRAP in a sample comprising: contacting a sample containing soluble IRAP with a capture antigen binding protein, wherein the capture antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the capture antigen binding protein to soluble IRAP in the sample, detecting soluble IRAP bound to the capture antigen binding protein by contacting the bound IRAP with a detection antigen binding protein, wherein the detection antigen binding protein is any antigen binding protein describedherein, under conditions permitting the binding of the detection antigen binding protein to soluble IRAP bound to the capture antigen binding protein, thereby detecting soluble IRAP in a sample.

33. A method for detecting soluble IRAP in a sample comprises: contacting a sample containing soluble IRAP with a capture antigen binding protein, wherein the capture antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the capture antigen binding protein to soluble IRAP in the sample, determining soluble IRAP bound to the capture antigen binding protein by contacting the bound IRAP with a detection antigen binding protein linked to a detectable label, wherein the detection antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the detection antigen binding protein to soluble IRAP bound to the capture antigen binding protein, and detecting the presence of the label, thereby detecting soluble IRAP in a sample.

34. A method for detecting the presence of or quantifying the level of soluble IRAP in a biological sample, wherein the method comprises the steps of: providing a biological sample contacting the biological sample with a capture antigen binding protein as described herein; incubating under conditions sufficient for a complex to form between soluble IRAP and the capture antigen binding protein; determining the presence of or amount of the complex; wherein detection of the complex indicates the presence of soluble IRAP, and determining the amount of the complex quantifies the level of soluble IRAP.

35. A method for determining whether any soluble IRAP in present a sample, the method comprising:contacting a sample with a capture antigen binding protein, wherein the capture antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the capture antigen binding protein to soluble IRAP in the sample, subsequently to (i), providing conditions for any unbound soluble IRAP to be removed, subsequently to (ii), providing a detection antigen binding protein, wherein the detection antigen binding protein is any antigen binding protein described herein, under conditions permitting the binding of the detection antigen binding protein to soluble IRAP bound to the capture antigen binding protein, and subsequently to (iii), providing conditions for any unbound detection antigen binding protein to be removed, and determining the presence of any detection antigen binding protein bound to the soluble IRAP.

36. The method of any one of claims 31 to 35, wherein the step of determining the presence of or amount of a complex, or determining the presence of any detection antigen binding protein bound to the soluble IRAP or detecting the label is a competition assay, a sandwich assay, an immunofluorescence assay, a chemiluminescence immunoassay, a radio-immunoassay, an enzyme-linked immunosorbent assay, a lateral flow test, an agglutination test, a strip test, a microsphere immunoassay, or real-time surface plasmon resonance detection assay or any combination thereof.

37. The method according to any one of claims 32 to 36, wherein the capture and detection antigen binding protein pairs are: a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 29 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein;a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 29 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein; a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 29 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein; a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 59 and 60, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 59 and 60 as described herein; a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein;a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 39 and 30, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 29 and 30 as described herein; or a capture antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 81 and 82, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 81 and 82 as described herein; and a detection antigen binding protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NOs: 96 and 97, or CDR1 , CDR2 and CDR3 of SEQ ID NOs: 96 and 97 as described herein.

38. The method according to any one of claims 31 to 37, wherein the biological sample is a body fluid.

39. The method according to claim 38, wherein the body fluid is a blood sample or a sample derived from blood.

40. The method according to claim 39, wherein the sample derived from blood is plasma.

41. The method according to any one of claims 31 to 40, wherein the biological sample is derived from a pregnant woman.

42. The method according to any one of claims 31 to 41 , wherein the sample is exposed to denaturing and / or reducing conditions.

43. The method according to claim 42, wherein the denaturing and / or reducing conditions are increased temperature, for example at least about 50°C, at least about 60°C, at least about 70°C, or at least about 80°C or exposure to a detergent, such as sodium dodecyl sulfate (SDS).

44. The method according to any one of claims 32 to 43, wherein the capture antigen binding protein, detection antigen binding protein or both the capture antigen binding protein and detection antigen binding protein are bound to, or immobilised on, a substrate, solid phase or solid support.

45. The method according to claim 44, wherein the substrate, solid phase or solid support may be a particle, a film, a microplate, a microtube or a test tube.

46. The method according to any one of claims 31 to 45, wherein the detection antigen binding protein is conjugated or linked to a detectable label.

47. The method according to any one of claims 31 to 46, wherein the detection antigen binding protein can be bound by another molecule which is or includes a detectable label.

48. The method according to any one of claims 31 to 47, wherein the method is performed in a plate, for example a 96-well plate.

49. The method according to any one of claims 32 to 48, wherein the method is performed in a plate, for example a 96-well plate, wherein at least about 5 pg / well, at least about 4 pg / well, at least about 3 pg / well, at least about 2 pg / well, at least about 1 pg / well, at least about 0.9 pg / well, at least about 0.8 pg / well, at least about 0.7 pg / well, at least about 0.6 pg / well, at least about 0.5 pg / well, at least about 0.4 pg / well, at least about 0.3 pg / well, at least about 0.2 pg / well, or at least about 0.1 pg / well of capture antigen binding protein is applied.

50. The method according to any one of claims 31 to 49, wherein the method is performed in a plate, for example a 96-well plate, wherein at least about 5 pg / well, at least about 4 pg / well, at least about 3 pg / well, at least about 2 pg / well, at least about 1 pg / well, at least about 0.9 pg / well, at least about 0.8 pg / well, at least about 0.7 pg / well, at least about 0.6 pg / well, at least about 0.5 pg / well, a at least bout 0.4 pg / well, at least about 0.3 pg / well, at least about 0.2 pg / well, at least about 0.1 pg / well, at least about 0.09 pg / well, at least about 0.08 pg / well, at least about 0.07 pg / well, at least about 0.06 pg / well, or at least about 0.05 pg / well of detection antigen binding protein is applied.51 . The method according to claim 49, wherein at least 5 pg / well, at least 4 pg / well, at least 3 pg / well, at least 2 pg / well, at least 1 pg / well, at least 0.9 pg / well, at least 0.8 pg / well, at least 0.7 pg / well, at least 0.6 pg / well, at least 0.5 pg / well, at least 0.4 pg / well, at least 0.3 pg / well, at least 0.2 pg / well, or at least 0.1 pg / well of capture antigen binding protein is applied.

52. The method according to claim 50, wherein at least 5 pg / well, at least 4 pg / well, at least 3 pg / well, at least 2 pg / well, at least 1 pg / well, at least 0.9 pg / well, at least 0.8 pg / well, at least 0.7 pg / well, at least 0.6 pg / well, at least 0.5 pg / well, at least 0.4 pg / well, at least 0.3 pg / well, at least 0.2 pg / well, at least 0.1 pg / well, at least 0.09 pg / well, at least 0.08 pg / well, at least 0.07 pg / well, at least 0.06 pg / well, or at least 0.05 pg / well of detection antigen binding protein is applied.

53. The method according to any one of claims 32 to 52, wherein at least about 0.5 pg / well of capture antibody and at least about 0.25 pg / well of detection antigen binding protein are applied.

54. The method according to any one of claims 31 to 52, wherein at least about 2 pg / well of capture antigen binding protein and at least about 1 pg / well of detection antigen binding protein are applied.

55. The method according to any one of claims 49 to 54, wherein the capture antigen binding protein is applied to a well in a buffer, preferably the buffer is 0.1 M Na2CO3 / NaHCO3, pH 9.6.

56. The method according to any one of claims 49 to 55, wherein the capture antigen binding protein is applied to or coated on a well for at least about 30 minutes, at least about 45 minutes, or at least about 60 minutes.

57. The method according to any one of claims 48 to 56, the detection antigen binding protein is applied to a well in a buffer, preferably the buffer is 3% BSA in 1 x PBS.

58. The method according to any one of claims 49 to 57, wherein the capture antigen binding protein is applied to a well for at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes.

59. The method according to any one of claims 31 to 58, wherein the method can detect at least about 5 ng / ml of si RAP, at least about 10ng / ml of si RAP, at least about 15 ng / ml of sIRAP, at least about 20 ng / ml of sIRAP, at least about 25 ng / ml of sIRAP, at least about 50 ng / ml of sIRAP, at least about 100 ng / ml of sIRAP, at least about 200 ng / ml of sIRAP, at least about 400 ng / ml of sIRAP, at least about 500 ng / ml of sIRAP, at leastabout 1000 ng / ml of sIRAP, at least about 2000 ng / ml of sIRAP, at least about 3000 ng / ml of sIRAP, at least about 4000 ng / ml of sIRAP or at least about 5000 ng / ml of sIRAP.

60. The method according to any one of claims 31 to 58, wherein the method can detect at least 5 ng / ml of sIRAP, at least 10ng / ml of sIRAP, at least 15 ng / ml of sIRAP, at least 20 ng / ml of sIRAP, at least 25 ng / ml of sIRAP, at least 50 ng / ml of sIRAP, at least 100 ng / ml of sIRAP, at least 200 ng / ml of sIRAP, at least 400 ng / ml of sIRAP, at least 500 ng / ml of sIRAP, at least 1000 ng / ml of sIRAP, at least 2000 ng / ml of sIRAP, at least about 3000 ng / ml of sIRAP, at least 4000 ng / ml of sIRAP or at least 5000 ng / ml of sIRAP.61 . A kit for detecting the presence of or quantifying the level of soluble IRAP in a biological sample, the kit comprising: one or more detection antigen binding proteins; and optionally one or more capture antigen binding proteins; wherein the capture antigen binding protein is any one of the antigen binding proteins according to any one of claims 1 to 25, wherein the detection antigen binding protein is any one of the antigen binding proteins according to any one of claims 1 to 25.

62. The kit according to claim 61 , wherein the kit further comprises instructions for performing a method described herein.

63. The kit according to claim 61 or 62, wherein the kit further comprises a solid phase or support.

64. A kit according to any one of claims 61 to 63 when used in a method according to any one of claims 31 to 60.

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