CD91 polypeptide for use in detecting hemopexin:heme complex

Abstract

The present invention relates to the use of CD91 polypeptides for the detection and quantification of hemopexin:heme complexes in biological samples, the use of CD91 polypeptides for the separation of hemopexin:heme complexes from biological samples, and the monitoring of hemolytic therapy using CD91 polypeptides.

Description

[Technical Field]

[0001] The present invention relates to the use of CD91 polypeptide for the detection and quantification of hemopexin:heme complexes in biological samples, the use of CD91 polypeptide for the isolation of hemopexin:heme complexes from biological samples, and the monitoring of hemolytic therapy using CD91 polypeptide. [Background technology]

[0002] Hemolysis is characterized by the destruction of red blood cells and is a prominent feature of anemic disorders associated with red blood cell abnormalities, such as enzyme deficiencies, abnormal hemoglobinopathy, hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria and acanthocyte anemia, as well as external factors, such as splenomegaly, autoimmune disorders (e.g., neonatal hemolytic disease), hereditary disorders (e.g., sickle cell anemia or G6PD deficiency), microangiogenic hemolysis, Gram-positive bacterial infections (e.g., Streptococcus, Enterococcus, and Staphylococcus), parasitic infections (e.g., Plasmodium), toxins, and trauma (e.g., burns) (Non-Patent Literature 1). Hemolysis is also a common disorder in blood transfusions, particularly in patients receiving massive transfusions and those using extracorporeal cardiopulmonary support devices (Non-Patent Literature 2).

[0003] The adverse effects observed in patients with hemolysis-related conditions are largely due to the release of iron and iron-containing compounds from red blood cells, such as hemoglobin (Hb) and heme (Non-Patent Literature 3). Under physiological conditions, released hemoglobin is bound by soluble proteins, such as haptoglobin, and transported to macrophages and hepatocytes (Non-Patent Literature 4 and 5). However, when the onset of hemolysis is accelerated and becomes essentially pathological, the buffering capacity of haptoglobin is overwhelmed (Non-Patent Literature 6). As a result, hemoglobin is rapidly oxidized to ferrihemoglobin, which then releases free heme (containing protoporphyrin IX and iron). Although heme plays an important role in several biological processes (e.g., as part of essential proteins such as hemoglobin and myoglobin), free heme is highly toxic. Free heme is a source of redox-active iron and generates highly toxic reactive oxygen species (ROS) that damage lipid membranes, proteins, and nucleic acids (Non-Patent Literature 7). Heme toxicity is further exacerbated by its ability to intercalate into lipid membranes, causing oxidation of membrane components and promoting cell lysis and cell death. Under physiological steady-state conditions and during mild hemolysis, the evolutionary pressure of continuous low-level extracellular Hb / heme exposure leads to compensatory mechanisms that control the harmful effects of free Hb / heme. These systems involve the release of a group of plasma proteins that bind to Hb or heme, including the Hb scavenger haptoglobin (Hp) and the heme scavenger proteins hemopexin (Hpx) and α1-microglobulin.

[0004] The main heme-binding protein in plasma and cerebrospinal fluid is hemopexin. Therefore, hemopexin is an important defense against the adverse effects of heme on cells. In a non-linear but significant relationship, when heme is bound by hemopexin in the complex, the hemopexin:heme complex is taken up into cells by various cells, including hepatocytes, macrophages, and syncytiotrophic cells, via receptor-mediated mechanisms, and the complex is degraded in lysosomes. The receptor protein for the hemopexin:heme complex is the CD91 protein (Non-Patent Literature 8).

[0005] The cluster of differentiation 91 (CD91) (Non-Patent Documents 9 and 10), also known as pro-low-density lipoprotein receptor-associated protein 1, low-density lipoprotein receptor-associated protein 1 (abbreviated as LRP1), alpha-2-macroglobulin receptor (A2MR), or apolipoprotein E receptor (APOER), was first described in Non-Patent Document 11. Human preproprotein is a 600 kDa precursor protein that is proteolytically processed by furin to form a non-covalently associated 515 kDa alpha chain and an 85 kDa beta chain (specified by SDS). The total protein molecular weight based on the primary amino acid sequence is 504,606 kDa. It is expressed in a wide range of cell types, including macrophages, hepatocytes, fibroblasts, adipocytes, neurons, vascular smooth muscle cells, and syncytiotrophic cells, most abundantly in vascular smooth muscle cells, hepatocytes, and neurons. CD91 orthologues are found in vertebrates, and their nucleotide and amino acid structures have been elucidated for many species.

[0006] As a member of the low-density lipoprotein receptor family (LDLR family), CD91 contains a cysteine-rich complement-type ligand-binding repeat, an epidermal growth factor precursor-type repeat (EGF repeat), a β-propeller domain, a transmembrane domain, and a cytoplasmic domain. The extracellular domain of CD91 is an alpha chain containing 31 cysteine-rich complement-type ligand-binding repeats arranged in four "ligand-binding domains" (numbered I, II, III, and IV), each containing 2, 8, 10, and 11 repeats, respectively. The ligand-binding domains bind a variety of ligands, including extracellular matrix proteins, growth factors, proteases, protease inhibitor complexes, and other proteins involved in lipoprotein metabolism. Of the four domains, domains II and IV bind the majority of ligands (Non-Patent Literature 12). Cysteine ​​forms intramolecular disulfide bonds, which are thought to be important for the stability of the ligand-binding domains. Twenty-two copies of the cysteine-rich EGF repeat are adjacent to the ligand-binding domains. The EGF repeat and β-propeller domain are thought to release ligands under low pH conditions, such as within endosomes, and the β-propeller domain is presumed to replace the ligand in the ligand-binding repeat. The transmembrane domain is a β-chain containing a 100-residue cytoplasmic tail. This tail contains two NPxY motifs that play a role in endocytosis and signal transduction.

[0007] CD91 plays a crucial role in intracellular signaling and endocytosis and is therefore associated with numerous intracellular and biological processes, including lipid and lipoprotein metabolism, protease degradation, regulation of platelet-derived growth factor receptors, integrin maturation and recirculation, regulation of vascular tone, regulation of blood-brain barrier permeability, cell proliferation, cell migration, inflammation, and apoptosis, as well as diseases such as neurodegenerative diseases, atherosclerosis, and cancer (Non-Patent Literature 13 and Non-Patent Literature 14).

[0008] The CD91 protein, as a receptor protein, is also involved in the endocytosis of the hemopexin:heme complex via receptor-mediated mechanisms in various cells, including hepatocytes, macrophages, and neurons, leading to heme uptake into cells and hemopexin degradation in lysosomes, thus eliminating dangerous heme from the blood. CD91 is known as the receptor for the hemopexin:heme complex with the highest affinity (Non-Patent Literature 15).

[0009] Endogenous hemopexin can control the harmful effects of free heme under physiological steady-state conditions, but under pathophysiological conditions, such as those associated with hemolysis, it has little effect in maintaining steady-state heme levels. High levels of heme lead to depletion of endogenous hemopexin, causing heme-mediated oxidative tissue damage. Studies have shown that hemopexin infusion reduces heme-induced endothelial activation, inflammation, and oxidative damage in experimental mouse models of hemolytic disorders such as sickle cell disease (SCD) and β-thalassemia. Hemopexin administration has also been shown to significantly reduce levels of pro-inflammatory cytokines and counteract heme-induced vasoconstriction in hemolytic animals (Non-Patent Literature 16 and Non-Patent Literature 17).

[0010] Because complex formation of heme released under pathophysiological conditions may require additional hemopexin administration to compensate for heme complex formation and its adverse effects, detection of the hemopexin:heme complex and determination of its amount in the subject are of paramount importance. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Schaer et al., Blood, 2013; 121(8): pp. 1276-1274. [Non-Patent Document 2] Rezogali et al., J. Cardiothorac. Vasc. Anesth., 2017;31(2):505~15 [Non-licensed Document 3] Buehler, Transfusion, 2004;44(10):1516~30 pages [Non-licensed Document 4] Schaer, Front Physiol., 2014; 5:415. [Non-licensed Document 5] Alayash, Trends Biotechnol., 2013; 31(1): 2-3 pages [Non-licensed Document 6] Muller-Eberhard, Blood, 1968; 32(5): 811~5 pages [Non-licensed Document 7] Roumenina, J. Mol. Med., 2016; 22(3): 200-13 pages [Non-licensed Document 8] Hvidberg, Blood, 2005; 106(7): 2572-9 pages [Non-licensed Document 9] Lillis, Physiol Rev., 2008; 88(3); pp. 887-918 [Non-licensed Document 10] Strickland, Arterioscler. Thromb. Vasc. Biol., 2014; 34(3): 487-98. [Non-licensed Document 11] Herz, The EMBO Journal, 1988; 7(13): 4119~27 pages [Non-licensed Document 12] Kangら, Biochem. Cell Biol., 2014; pp. 53:15~23 [Non-licensed Document 13] Lillis, J. Thromb. Haemost., 2005; 3(8): 1884-93. [Non-licensed Document 14] Smith, Exp. Cell Res., 1997; 232: 246-54 [Non-licensed Document 15] Hada et al., Biochim. Biophys. Acta, 2014;1840(7):2351-2360 [Non-Patent Document 16] Belcher et al., Blood, 2014;123(3):377-390 [Non-Patent Document 17] Belcher et al., PLoS One, 2018;13(4):e0196455 [Summary of the Invention] [Problems to be Solved by the Invention]

[0012] However, so far, no suitable detection agent has been found. CD91 is a drug suitable for the detection and quantification of hemopexin:hem complex in a sample of a subject having a condition related to hemolysis because of its high affinity for the hemopexin:hem complex. However, since the CD91 protein is large in size, it is difficult to handle. For example, recombinant production may not be possible or may only be possible under deteriorated conditions considering the large size of the protein. On the other hand, mere isolation and purification of the protein do not yield a sufficient amount for non-complex use as a detection agent. Therefore, there is a need for a detection agent for hemopexin:hem complex that enables efficient detection of the hemopexin:hem complex, and for easily, rapidly, and cost-effectively producing the efficient detection agent in high yields. [Means for Solving the Problems]

[0013] The present invention solves this problem by providing a CD91 polypeptide that binds to the hemopexin:hem complex with high affinity.

[0014] The present invention will be described in detail below. Features of the present invention will be described in individual paragraphs or sections. However, this does not mean that the features described in a paragraph or section are separate from one or more features described in other paragraphs or sections. Rather, the features described in a paragraph or section can be combined with one or more features described in other paragraphs or sections, and furthermore, the meaning of the features described in a paragraph or section is applicable even when the same feature is referred to in a different context in another paragraph or section.

[0015] As used herein, the term “comprise / es / ing” means to “include or encompass” the disclosed features and any further features not specifically described. The term “comprise / es / ing” also means “consist / s / ing of” the indicated features and therefore does not include any further features other than those indicated. Thus, the products of the present invention may also feature additional features in addition to those indicated.

[0016] In a first embodiment, the present invention provides a use of a CD91 polypeptide for the detection of a hemopexin:heme complex in a biological sample, wherein the CD91 polypeptide comprises or consists of a ligand-binding domain III of CD91 having a length of 300 to 1000 amino acids and capable of binding to a hemopexin:heme complex.

[0017] The present invention further provides an in vitro method for detecting hemopexin:heme complexes in a biological sample, comprising the steps of: incubating the biological sample with a CD91 polypeptide used in the context of the present invention; and detecting the binding of the CD91 polypeptide to the hemopexin:heme complex.

[0018] The present invention further provides a CD91 polypeptide for use in the context of the present invention for use in methods for treating hemolysis or hemolysis-related conditions.

[0019] The present invention further provides a kit comprising a CD91 polypeptide used in the context of the present invention.

[0020] The present invention further provides a solid support to which a CD91 polypeptide, used in the context of the present invention, is linked. [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows an example workflow for the dynamic characterization of the interaction between His-tagged proteins and target analytes. The assay consists of five steps: Step 1: Equilibration, Step 2: Loading (capture) of His-tagged proteins, Step 3: Baseline, Step 4: Analyte association, Step 5: Analyte dissociation. For further details, please refer to TechNote 43(6). [Figure 2] This is a schematic diagram showing the huLRP1 soluble minireceptors (5). Each of the four soluble minireceptors is illustrated in comparison to full-length endogenous hulRP1. The four putative ligand-binding domains are labeled I, II, III, and IV. [Figure 3]This figure shows the expression and purification of the huLRP1 minireceptor in ExpiCHO-S(trademark) cells. (A) Coomassie-stained reductive SDS-PAGE of huLRP1 minireceptors encoding binding domains I-IV (shown at the top of the gel) produced in transiently transfected ExpiCHO-S(trademark) cells. Co-expression of huLRPAP1(RAP)(+) resulted in a relative increase in expression levels. (B) This figure shows an anti-His Western blot of reductive SDS-PAGE of huLRP1 minireceptors encoding binding domains I-IV (shown at the top of the gel) produced in transiently transfected ExpiCHO-S(trademark) cells. (C) This figure shows Coomassie-stained reductive SDS-PAGE of huLRP1 minireceptors encoding binding domains I-IV (shown at the top of the gel) purified by tandem nickel-size exclusion chromatography. [Figure 4-1] This figure shows representative sensorgrams of each LRP1 domain analyzed for its ability to bind the heme-hpx complex. Individual LRP1 domains were immobilized on the biosensor surface. After baseline recording, four concentrations of the heme-hpx complex were tested (2.5, 1.25, 0.625, and 0.312 μM). For LRP1.3, the reference subtracted and processed kinetic dataset was fitted overall using a 1:1 binding model. The accuracy of the fitting is indicated by Chi2 and R2, shown in Table 5. Heme-hpx is shown as the red curve, with the approximation curve as the black solid line. LRP1.1, LRP1.2, LRP1.4, and LRP1.5 were not fitted. [Figure 4-2] Continuation of Figure 4-1. [Figure 4-3] Continuation of Figure 4-2. [Figure 5-1]This figure shows representative sensorgrams obtained from manual runs for all four LRP1 domains. Polyclonal anti-hemopexin was immobilized in CM5 in a subsequent heme-Hpx capture step. (A) LRP1 domain 3 and (B) domain 2 were used as analytes at 10 μM. (C) Instead of heme-Hpx, unbound Hpx was captured with LRP1 domain 3 as a negative control at 10 μM. All sensorgrams have been subtracted by reference. [Figure 5-2] Continuation of Figure 5-1. [Figure 6] This figure shows a qualitative hemopexin capture assay. (A) Schematic diagram of the assay setup. (B) Representative sensorgram of LRP1.3 binding to the heme-hx complex. Dose-dependent binding is shown. [Figure 7] This figure shows SPR sensorgrams of the interaction between heme-hpx and LRP1.3. (A) Schematic diagram of the assay setup. (B) Representative sensorgrams of the heme-hpx complex and (C) the lack of hemopexin binding to LRP1.3. (D) Steady-state 1:1 fit of heme-hx complex binding to LRP1.3, and (E) each scatcharded plot showing slight negative synergistic interaction. [Figure 8] Representative sensorgrams and steady-state fits of heme-hpx complexes from various batches. (A) Hpx batch: TO411122, (B) Hpx batch: TO341022BM ("new"), and (C) Hpx batch: TO342022B ("aged") bound to LRP1.3. [Figure 9] This graph shows that the hemopexin:heme assay is specific to hemopexin bound to heme (hemopexin:heme) and does not bind to free hemopexin (hemopexin). A 7-point standard curve was created using either the hemopexin:heme complex or free hemopexin. [Modes for carrying out the invention]

[0022] The present invention will be described below.

[0023] In the context of the present invention, it was surprisingly found that the ligand-binding domain III (LPR1) of CD91 binds to the hemopexin:heme complex but not to hemopexin alone (see Example 1). This makes it possible to use it for identifying the hemopexin:heme complex in blood and other biological samples (see Example 2).

[0024] Nucleotide and nucleic acid sequences of various CD91 proteins from various vertebrates are known in the art. For illustrative purposes only, and not limited thereto, the amino acid sequence of human CD91 disclosed herein as Sequence ID No. 1 is referred to. The corresponding nucleotide and amino acid sequences are available from NCBI (National Centre for Biotechnology Information; National Library of Medicine, Bethesda, MD20894, USA; www.ncbi.nlm.nih.gov). The amino acid sequence of isoform 1 is available under accession number Q07954.2 (LRP1_HUMAN; Pro Low-Density Lipoprotein Receptor-Related Protein 1 or Low-Density Lipoprotein Receptor-Related Protein 1; abbreviated as LRP-1). Sequence ID No. 1 is also shown below. Ligand-binding domains I, II, III, and IV are underlined and appear in the order shown, based on the reference Obermoeller-McCormick et al., J.Cell Sci., 2001, 114(5):899-908.

[0025] Sequence ID 1 1 mltpplllll pllsalvaaa idap ktcspk qfacrdqitc iskgwrcdge rdcpdgsdea 61 peicpqskaq rcqpnehncl gtelcvpmsr lcngvqdcmd gsdegphcre lqg ncsrlgc 121 qhhcvptldg ptcycnssfq lqadgktckd fdecsvygtc sqlctntdgs ficgcvegyl 181 lqpdnrscka knepvdrppv lliansqnil atylsgaqvs titptstrqt tamdfsyane 241 tvcwvhvgds aaqtqlkcar mpglkgfvde htinislslh hveqmaidwl tgnfyfvddi 301 ddrifvcnrn gdtcvtlldl elynpkgial dpamgkvfft dygqipkver cdmdgqnrtk 361 lvdskivfph gitldlvsrl vywadayldy ievvdyegkg rqtiiqgili ehlygltvfe 421 nylyatnsdn anaqqktsvi rvnrfnstey qvvtrvdkgg alhiyhqrrq prvrshacen 481 dqygkpggcs dicllanshk artcrcrsgf slgsdgksck kpehelflvy gkgrpgiirg 541 mdmgakvpde hmipienlmn praldfhaet gfiyfadtts yligrqkidg teretilkdg 601 ihnvegvavd wmgdnlywtd dgpkktisva rlekaaqtrk tliegkmthp raivvdplng 661 wmywtdweed pkdsrrgrle rawmdgshrd ifvtsktvlw pnglsldipa grlywvdafy 721 drietillng tdrkivyegp elnhafglch hgnylfwtey rsgsvyrler gvggapptvt 781 llrserppif eirmydaqqq qvgtnkcrvn nggcsslcla tpgsrqcaca edqvldadg v 841 tclampsyvp ppqcqpgefa cansrciqer wkcdgdndcl dnsdeapalc hqhtcpsdrf 901 kcennrcipn rwlcdgdndc gnsedesnat csartcppnq fscasgrcip iswtcdld dd 961 cgdrsdesas cayptcfplt qftcnnngrci ninwrcdndn dcgdnsdeag cshscsstqf 1021 kcnsgrcipe hwtcdgdndc gdysdethan ctnqatrppg gchtdefqcr ldglciplrw 1081 rcdgdtdcmd ssdekscegv thvcdpsvkf gckdsarcis kawvcdgdnd cednsdeenc 1141 eslacrppsh pcanntsvcl ppdklcdgnd dcgdgsdege lcd qcslnng gcshncsvap 1201 gegivcscpl gmelgpdnht cqiqsycakh lkcsqkcdqn kfsvkcscye gwvlepdges 1261 crsldpfkpf iifsnrheir ridlhkgdys vlvpglrnti aldfhlsqsa lywtdvvedk 1321 iyrgklldng altsfevviq yglatpegla vdwiagniyw vesnldqiev akldgtlrtt 1381 llagdiehpr aialdprdgi lfwtdwdasl prieaasmsg agrrtvhret gsggwpnglt 1441 vdylekrilw idarsdaiys arydgsghme vlrgheflsh pfavtlygge vywtdwrtnt 1501 lakankwtgh nvtvvqrtnt qpfdlqvyhp srqpmapnpc eanggqgpcs hlclinynrt 1561 vscacphlmk lhkdnttcye fkkfllyarq meirgvdlda pyynyiisft vpdidnvtvl 1621 dydareqrvy wsdvrtqaik rafingtgve tvvsadlpna hglavdwvsr nlfwtsydtn 1681 kkqinvarld gsfknavvqg leqphglvvh plrgklywtd gdnismanmd gsnrtllfsg 1741 qkgpvglaid fpesklywis sgnhtinrcn ldgsglevid amrsqlgkat alaimgdklw 1801 wadqvsekmg tcskadgsgs vvlrnsttlv mhmkvydesi qldhkgtnpc svnngdcsql 1861 clptsettrs cmctagyslr sgqqacegvg sflysvheg irgipldpnd ksdalvpvsg 1921 tslavgidfh aendtiywvd mglstisrak rdqtwredvv tngigrvegi avdwiagniy 1981 wtdqgfdvie varlngsfry vvisqgldkp raitvhpekg ylfwtewgqy priersrldg 2041 tervvlvnvs iswpngisvd yqdgklywcd artdkierid letgenrevv lssnnmdmfs 2101 vsvfedfiyw sdrthangsi krgskdnatd svplrtgigv qlkdikvfnr drqkgtnvca 2161 vanggcqqlc lyrgrgqrac acahgmlaed gascreyagy llysertilk sihlsdernl 2221 napvqpfedp ehmknviala fdyragtspg tpnriffsdi hfgniqqind dgsrritive 2281 nvgsveglay hrgwdtlywt syttstitrh tvdqtrpgaf eretvitmsg ddhprafvld 2341 ecqnlmfwtn wneqhpsimr aalsganvlt liekdirtpn gladhraek lyfsdatldk 2401 ierceydgsh ryvilksepv hpfglavyge hifwtdwvrr avqrankhvg snmkllrvdi 2461 pqqpmgiiav andtnscels pcrinnggcq dlcllthqgh vncscrggri lqddltcrav 2521 nsscraqdef ecangecinf sltcdgvphc kdksdekpsy cnsrrckktf rqcsngrcvs 2581 nmlwcngadd cgdgsdeipc nktacgvgef rcrdgtcign ssrcnqfvdc edasdemncs 2641 atdcssyfrl gvkgvlfqpc ertslcyaps wvcdgandcg dysderdcpg vkrprcplny 2701 facpsgrcip mswtcdkedd cehgedethc nkfcseaqfe cqnhrciskq wlcdgsddcg 2761 dgsdeaahce gktcgpssfs cpgthvcvpe rwlcdgdkdc adgadesiaa gclynstcdd 2821 refmcqnrqc ipkhfvcdhd rdcadgsdes peceyptcgp sefrcangrc lssrqwecdg 2881 endchdqsde apknphctsq ehkcnassqf lcssgrcvae allcngqddc gdssdergch 2941 in eclsrkls gcsqdcedlk igfkcrcrpg frlkddgrtc advdecsttf pcsqrcinth 3001 gsykclcveg yaprggdphs ckavtdeepf lifanryylr klnldgsnyt llkqglnnav 3061 aldfdyreqm iywtdvttqg smirrmhlng snvqvlhrtg lsnpdglavd wvggnlywcd 3121 kgrdtievsk lngayrtvlv ssglrepral vvdvqngyly wtdwgdhsli grigmdgssr 3181 svivdtkitw pngltldyvt eriywadare dyiefasldg snrhvvlsqd iphifaltlf 3241 edyvywtdwe tksinrahkt tgtnktllis tlhrpmdlhv fhalrqpdvp nh pckvnngg 3301 csnlcllspg gghkcacptn fylgsdgrtc vsnctasqfv ckndkcipfw wkcdteddcg 3361 dhsdeppdcp efkcrpgqfq cstgictnpa ficdgdndcq dnsdeancdi hvclpsqfkc 3421 tntnrcipgi frcngqdncg dgederdcpe vtcapnqfqc sitkrciprv wvcdrdndcv 3481 dgsdepanct qmtcgvdefr ckdsgrcipa rwkcdgeddc gdgsdepkee cdertcepyq 3541 frcknnrcvp grwqcdydnd cgdnsdeesc tprpcsesef scangrciag rwkcdgdhdc 3601 adgsdekdct prcdmdqfqc ksghciplrw rcdadadcmd gsdeeacgtg vrtcpldefq 3661 cnntlckpla wkcdgeddcg dnsdenpeec arfvcppnrp frckndrvcl wigrqcdgtd 3721 ncgdgtdeed cepptahtth ckdkkeflcr nqrclssslr cnmfddcgdg sdeedcsidp 3781 kltscatnas icgdearcvr tekaaycacr sgfhtvpgqp gcqdineclr fgtcsqlcnn 3841 tkgghlcsca rnfmkthntc kaegseyqvl yiaddneirs lfpghphsay eqafqgdesv 3901 ridamdvhvk agrvywtnwh tgtisyrslp paappttsnr hrrqidrgvt hlnisglkmp 3961 rgiaidwvag nvywtdsgrd vievaqmkge nrktlisgmi dephaivvdp lrgtmywsdw 4021 gnhpkietaa mdgtlretlv qdniqwptgl avdyhnerly wadaklsvig sirlngtdpi 4081 vaadskrgls hpfsidvfed yiygvtyinn rvfkihkfgh splvnltggl shasdvvlyh 4141 qhkqpevtnp cdrkkcewlc llspsgpvct cpngkrldng tcvpvpsptp ppdaprpgtc 4201 nlqcfnggsc flnarrqpkc rcqprytgdk celdqcwehc rnggtcaasp sgmptcrcpt 4261 gftgpkctqq vcagycanns tctvnqgnqp qcrclpgflg drcqyrqcsg ycenfgtcqm 4321 aadgsrqcrc tayfegsrce vnkcsrcleg acvvnkqsgd vtcnctdgrv apscltcvgh 4381 csnggsctmn skmmpecqcp phmtgprcee hvfsqqqpgh iasiliplll llllvlvagv 4441 vfwykrrvqg akgfqhqrmt ngamnveign ptykmyegge pddvggllda dfaldpdkpt 4501 nftnpvyatl ymgghgsrhs lastdekrel lgrgpedeig dpla

[0026] As used herein, the terms “CD91” or “CD91 protein” refer to the full-length protein in either its precursor or mature form. This protein consists of 4,544 amino acids in its precursor form and 4,525 amino acids in its mature form. Amino acids 1–19 constitute the signal sequence, amino acids 20–4419 constitute the extracellular sequence, amino acids 4420–4444 constitute the transmembrane region, and amino acids 4445–4544 constitute the cytoplasmic tail.

[0027] As used herein, the term “CD91 polypeptide” refers to any polypeptide plutide portion of a precursor or mature CD91 that binds to the hemopexin:heme complex but not to hemopexin that is not bound to heme, and does not refer to the full-length precursor or mature CD91.

[0028] According to the claims, the CD91 polypeptide has a length of 300 to 1000 amino acids.

[0029] The CD91 polypeptide used in the context of the present invention may have amino acid lengths of 400, 500, 600, 700, 800, or 900. It is also expected that it may have amino acid lengths of 400-900, 500-900, 500-800, 600-900, or 600-800.

[0030] The CD91 polypeptide used in the context of the present invention may include a sequence derived from CD91 and a further peptide sequence.

[0031] Preferably, the CD91 polypeptide contains or consists of a fragment within amino acids 2481-2942 of SEQ ID NO: 1, the starting point of this fragment is at a maximum distance of 100, 70, 50, 30, 20, 10, or 5 amino acids from amino acids 2481 and / or 2942.

[0032] The CD91 polypeptide used in the context of the present invention may comprise various portions of CD91. For example, it may comprise the amino acid sequence located at the N-terminus of CD91 and ligand-binding domain III, i.e., amino acids 2481-2942.

[0033] Unless otherwise specified, the amino acid numbering of CD91 polypeptide residues in this application refers to Sequence ID No. 1, even if the CD91 molecule does not necessarily contain all the residues of Sequence ID No. 1.

[0034] More preferably, the CD91 polypeptide comprises or consists of amino acids 2481-2942 of SEQ ID NO: 1, as shown in SEQ ID NO: 2, or the corresponding amino acid sequence of homologous CD91. SEQ ID NO: 2 shows the ligand-binding domain III of CD91. SEQ ID NO: 2 has a length of 462 amino acids. Sequence ID 2: PCRINNGGCQDLCLLTHQGHVNCSCRGGRILQDDLTCRAVNSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVSNMLWCNGADDCGDGS DEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEMNCSATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPRCPLNYFACPSGRCIPM SWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLCDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNSTCDDREFMCQ NRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWECDGENDCHDQSDEAPKNPHCTSQEHKCNASSQFLCSSGRCVAEALLCNGQDDCGDSSDERGCHIN

[0035] In a more preferred embodiment, the CD91 polypeptide used according to the present invention comprises the amino acids of SEQ ID NO: 2 and further comprises a signal sequence for secretion of the CD91 polypeptide into the culture medium, preferably the signal sequence of SEQ ID NO: 1, a linker, preferably AIDAP (SEQ ID NO: 5), a tag for purification of the CD91 polypeptide, preferably a Flag tag known in the Art (DYKDDDDKK; SEQ ID NO: 6) at the N-terminus, and a tag for purification of the CD91 polypeptide, preferably a His tag at the C-terminus. A preferred CD91 polypeptide is shown in SEQ ID NO: 3 below. This construct is used in Example 2 below. Sequence ID 3: MLTPPLLLLLPLLSALVAAAIDAPDYKDDDDKKPCRINNGGCQDLCLLTHQGHVNCSCRGGRILQDDLTCRAVNSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQ CSNGRCVSNMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEMNCSATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPRCPL NYFACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLCDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNSTCDDREFM CQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWECDGENDCHDQSDEAPKNPHCTSQEHKCNASSQFLCSSGRCVAEALLCNGQDDCGDSSDERGCHINHHHHHHHH

[0036] Those skilled in the art will understand that the CD91 polypeptide used in the context of the present invention may be even longer. For example, it may be 1100, 1200, 1300, 1400, 1600, or even 2000 or 2500 amino acid lengths.

[0037] The polypeptide of the present invention may further include a portion of the CD91 sequence adjacent to amino acids 2481-2942, i.e., adjacent to ligand-binding domain III. It may even include portions of ligand-binding domains II and IV. In this context, adjacent means that the corresponding amino acid sequence terminates at position 2480 or begins at position 2943.

[0038] The CD91 polypeptide used in the context of the present invention further comprises a ligand-binding domain III, i.e., amino acids 2481-2942, and may additionally comprise peptide stretches located between ligand-binding domains III and II and / or between ligand-binding domains III and IV. Preferably, the CD91 polypeptide may comprise a sequence of amino acids derived from CD91 that begins after the termination of ligand-binding domain II (i.e., amino acid 1183) and terminates before the termination of ligand-binding domain IV (i.e., amino acid 3293). In other words, the CD91 polypeptide comprises a sequence of amino acids derived from CD91 that begins at position 1184 and / or terminates at position 3292, comprising amino acids 2481-2942.

[0039] The polypeptide of the present invention can preferably be glycosylated.

[0040] The term "ligand-binding domain" refers to four defined regions within the CD91 protein, known as ligand-binding domains I, II, III, and IV. These domains consist of cysteine-rich complement-type ligand-binding repeats located within four ligand-binding domains: 2 repeats in ligand-binding domain I, 8 repeats in ligand-binding domain II, 10 repeats in ligand-binding domain III, and 11 repeats in ligand-binding domain IV. These repeats bind a wide range of ligands, including extracellular matrix proteins, growth factors, proteases, protease inhibitor complexes, and other proteins involved in lipoprotein metabolism. Of the four domains, domains II and VI bind the majority of ligands. To date, only a very small number of ligands have been detected in ligand-binding domain III (e.g., FVIIIa). Other ligands include apoE-rich β-VLDL and lactoferrin (Croy et al., Biochemistry, 2004;43(23):7328-35).

[0041] CD91 homologs are found in vertebrates. Therefore, the terms “CD91” or “CD91 protein” as used herein refer to any CD91 orthologue or homolog found in vertebrates. Alternatively, CD91 as used herein refers to any CD91 having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with the sequence of SEQ ID NO: 1. Alternatively, CD91 as used herein refers to any CD91 having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% amino acid homology with the sequence of SEQ ID NO: 1. Preferably, CD91 as used herein, including any CD91 found in vertebrates and any CD91 having amino acid identity or homology with the sequence of SEQ ID NO: 1 as defined above, has the same activity as CD91 according to SEQ ID NO: 1. More preferably, the CD91 referred to herein binds to the hemopexin:heme complex, preferably the human hemopexin:human heme complex, with the same affinity as CD91 of SEQ ID NO: 1 to the human hemopexin:human heme complex, or with an affinity of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the affinity of CD91 of SEQ ID NO: 1 to the human hemopexin:human heme complex. CD91 having the aforementioned definitions of relation, homology, identity, and function is also referred to herein as a “CD91 homolog”.

[0042] A CD91 polypeptide is a part of CD91. The term “CD91 polypeptide” is defined above. Therefore, as used herein, the term “CD91 polypeptide” refers to a CD91 polypeptide that is a part of any CD91 as defined above, including CD91 orthologues or homologs found in vertebrates and CD91 as defined by its amino acid identity or homology to SEQ ID NO: 1 shown above. Alternatively, a CD91 polypeptide includes an amino acid sequence that has amino acid identity with the sequence of SEQ ID NO: 2, or has at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with the sequence of SEQ ID NO: 2, or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% amino acid homology with SEQ ID NO: 2. Alternatively, the CD91 polypeptide comprises an amino acid sequence having amino acid identity with the sequence of SEQ ID NO: 3, or having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with the sequence of SEQ ID NO: 3, or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% amino acid homology with SEQ ID NO: 3. Preferably, the CD91 polypeptide referred to herein has the same activity as the CD91 polypeptide derived from SEQ ID NO: 1, or the CD91 polypeptide derived from SEQ ID NO: 2 or 3. More preferably, the CD91 polypeptide conjugates a hemopexin:heme complex, preferably a human hemopexin:human heme complex. The binding may have the same affinity as the CD91 polypeptide derived from SEQ ID NO: 1 or the CD91 polypeptide of SEQ ID NO: 2 or 3, or it may have an affinity of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the affinity of the CD91 polypeptide derived from SEQ ID NO: 1 or the CD91 polypeptide of SEQ ID NO: 2 or 3 to bind to a hemopexin:heme complex, preferably a human hemopexin:human heme complex.

[0043] The CD91 polypeptide of the present invention preferably has hemopexin:heme conjugated with a Kd of less than 100 μM, less than 50 μM, less than 10 μM, or less than 5 μM. Preferably, the Kd is less than 4 μM or less than 2 μM. The Kd may be greater than 0.1 μM or greater than 0.01 μM.

[0044] As shown in the examples, the affinity of ligand-binding domain III to the hemopexin:heme complex may be approximately 1.5 μM or approximately 2 μM.

[0045] The percentage of sequence identity refers to the percentage of amino acid residues that are identical at corresponding positions in two optimally aligned sequences. It is determined by comparing the two optimally aligned sequences across the entire comparison window. Some of the amino acid sequences in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in which identical amino acid residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman (Add.APL Math, 1981; 2:482-489), the homology alignment algorithm of Needleman and Wunsch (J.Mol.Biol., 1970; 48:443-453), the analogy method of Pearson and Lipman (PNAS, 1988; 85:2444-248), the algorithm of Karlin and Altschul (PNAS, 1990; 87:2264-228) modified by Karlin and Altschul (PNAS, 1993; 90:5873-587), or by computer implementations of these algorithms (Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575Science)). This can be done by GAP, BESTFIT, BLAST, PASTA, and TFASTA (Dr. Madison, WI) or by scrutiny. Preferably, GAP and BESTFIT are used to determine the optimal alignment. Typically, a default value of 5.00 is used for the gap weight and a default value of 0.30 is used for the gap weight length.

[0046] The percentage of sequence homology refers to the percentage of amino acid residues that are homologous at corresponding positions in two optimally aligned sequences. The "percentage of homology" between two sequences is determined in substantially the same way as the method described above for determining the "percentage of identity," except that the calculation takes into account not only homologous positions but also identical positions. Two homologous amino acids can be either identical or homologous amino acids. Homologous amino acid residues have similar chemical-physical properties; for example, amino acids belong to the same group: aromatic amino acids (Phe, Trp, Tyr), acidic amino acids (Glu, Asp), polar amino acids (Gin, Asn), basic amino acids (Lys, Arg, His), aliphatic amino acids (Ala, Leu, Ile, Val), amino acids with a hydroxyl group (Ser, Thr), or amino acids with a short side chain (Gly, Ala, Ser, Thr, Met). Substitutions between such homologous amino acids are expected not to alter the protein's phenotype (conservative substitutions).

[0047] Furthermore, as used herein, the CD91 polypeptide may refer to one homolog of the CD91 polypeptide, or a mixture of CD91 polypeptide homologs derived from two or more individuals of the same species and / or from two or more individuals of different species, insofar as the CD91 polypeptide binds to the hemopexin:heme complex.

[0048] When attempting to detect the hemopexin-heme complex in humans, it is generally preferable that the CD91 polypeptide be derived from human CD91. However, it should be understood that CD91 polypeptides derived from non-human homologs of CD91 can be used for the detection of the hemopexin-heme complex in humans, as long as the non-human homolog of CD91 has the ability to bind to the human hemopexin-heme complex. In other cases, CD91 polypeptides derived from human CD91 or non-human homologs of CD91 can be used for the detection of the hemopexin-heme complex in non-humans. Thus, as long as the human or non-human homolog of the CD91 polypeptide has the ability to bind to the hemopexin-heme complex in non-humans, the non-human homolog of the CD91 polypeptide may be from the same species or a different species. As above, when a CD91 polypeptide is intended for the detection of the hemopexin-heme complex in non-humans, it is generally preferable that the CD91 polypeptide be derived from the same species as the species in which the hemopexin-heme complex is to be detected. Exemplary examples of non-human homologs of CD91, including those originating from rabbits, mice, dogs, cats, cattle, horses, pigs, sheep, goats, or birds, are well known to those skilled in the art.

[0049] When referring to a specific amino acid sequence identified by a sequence number, or a specific amino acid region within a specific amino acid sequence identified by a sequence number, it is understood that the corresponding amino acid sequences or amino acid regions of different CD91 homologs or different CD91 polypeptide homologs are each included in that specific amino acid sequence or amino acid region. For example, when referring to the fragment between amino acids 2481 and 2942 in sequence number 1, the corresponding amino acid regions of the CD91 polypeptide homolog are covered. When referring to amino acids 2481 to 2942 by sequence number 2, the corresponding amino acid regions of the CD91 polypeptide homolog are covered.

[0050] As used herein, the terms “fragment of the ligand-binding domain III of CD91,” “the fragment,” or “fragment,” or corresponding expressions, mean any fragment of the ligand-binding domain III of CD91 that can bind to a hemopexin:heme complex, preferably a human hemopexin:human heme complex. Binding may be with the same affinity as the CD91 polypeptide derived from SEQ ID NO: 1 or the CD91 polypeptide of SEQ ID NO: 2 or 3, or with an affinity of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the affinity with which the CD91 polypeptide derived from SEQ ID NO: 1 or the CD91 polypeptide of SEQ ID NO: 2 binds to a hemopexin:heme complex, preferably a human hemopexin:human heme complex. Importantly, the fragment does not bind to hemopexin that is not bound to heme.

[0051] The term “CD91 polypeptide having length ~” as used herein should be understood to mean that the CD91 polypeptide is precisely the length indicated and is not longer by the inclusion of additional amino acids. However, a CD91 polypeptide defined by its length may contain additional amino acids at the N and / or C terminus that are longer but not included in the indicated length. Such additional amino acids may be derived from non-CD91 protein sequences, e.g., signal peptides and / or fusion tags, or from CD91 protein subsequences removed from the CD91 polypeptide sequence within the CD91 protein, including signal sequences, one or more growth factor repeats, and / or transmembrane domains.

[0052] As used herein, the term “derived from ~” means that a CD91 polypeptide has the same amino acid sequence as the corresponding amino acid sequence of CD91 from the subject or species from which it originates, or is substantially identical thereto (i.e., at least 90%, 94%, 95%, 96%, 97%, 98%, or 99%). The term “derived from ~” is also used in connection with hemopexin to mean that hemopexin has the same amino acid sequence as the corresponding amino acid sequence of hemopexin from the subject or species from which hemopexin originates, or is substantially identical thereto (i.e., at least 90%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0053] CD91 polypeptides as referred to herein may exist in “at least partially purified,” “isolated,” or “purified” forms. Terms such as “at least partially purified,” “isolated,” or “purified” are used herein to mean that the CD91 polypeptide is provided in an isolated and / or purified form, meaning that it has been separated, isolated, or purified from its natural environment or the environment in which it is produced. Therefore, the CD91 polypeptide is preferably free from or substantially free from naturally associated substances, such as other polypeptides or nucleic acids, or substances that naturally associate with it during preparation (e.g., cellular substances in cell cultures). For example, at least partially purified CD91 polypeptide may contain impurities of 50% or less (of total protein), preferably 45% or less, preferably 40% or less, preferably 35% or less, preferably 30% or less, preferably 25% or less, and preferably 20% or less (of total protein). The isolated or purified CD91 polypeptide may contain impurities of 15% or less, preferably 10% or less, preferably 5% or less, or preferably 1% or less (of total protein), and most preferably is impurity-free. Impurities do not refer to components present in the assay solution for performing the assay.

[0054] CD91 polypeptide can be produced by any method known in the art that can produce CD91 polypeptide. For example, CD91 polypeptide can be produced by recombination or synthesis using routine methods and reagents well known in the art. Preferably, CD91 polypeptide is produced by recombination, i.e., genetic modification by genetic recombination, in a suitable host cell (e.g., bacteria, yeast, insect cells, mammalian cells) according to a method known in the art (see, for example, Ausubel et al., Curr. Protoc. Mol. Biol., 2nd edition, 1992 and Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press).

[0055] Methods for producing polypeptides by recombination are well known in the art. For example, a nucleic acid molecule containing a nucleotide sequence encoding the CD91 polypeptide can be introduced and expressed in a suitable host cell (e.g., Escherichia coli), and the expressed CD91 polypeptide can be isolated / purified from the host cell (e.g., in an inclusion body) using routine methods and readily available reagents. Methods for introducing a DNA construct encoding the CD91 polypeptide into a host cell are well known in the art, including, for example, standard transformation and transfection techniques (e.g., electroporation, chemical transformation). Those skilled in the art can easily select an appropriate method for introducing the DNA construct into a host cell. Various methods for expressing proteins in host cells are well known in the art (e.g., IPTG-induced expression in Escherichia coli). Those skilled in the art can easily select an appropriate method for expressing the CD91 polypeptide in a host cell. The expressed CD91 polypeptide can be isolated from the host cell using known methods and reagents, including, for example, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. Alternatively, the expressed CD91 polypeptide can be secreted into the culture medium by a signal peptide. It can then be recovered from the culture medium by methods known in the art. For example, the CD91 polypeptide can be purified from host cells or cell solubilizes of host cells or from the culture medium by conjugating it to a support using standard techniques and reagents. For example, the CD91 polypeptide can be isolated by conjugating it to a solid support via a specific receptor, such as an antibody or fragment thereof that is specific to the CD91 polypeptide. Alternatively, the CD91 polypeptide can be isolated by a tag that enables purification by methods known in the art.

[0056] To produce recombinant CD91 polypeptide, a construct containing a nucleotide sequence encoding the CD91 polypeptide is produced, for example, by cloning the nucleotide sequence into a vector cloning site. Suitable vectors are known in the art and include plasmids, cosmids, artificial chromosomes (e.g., bacterial, yeast, or human), bacteriophages, viral vectors (retrovirus, lentivirus, adenovirus, adeno-associated virus), or nanomodified materials (e.g., ormosyl). The vector preferably contains one or more suitable sequences that can be expressed in a suitable host. Such sequences may be promoter sequences recognized by one or more proteins endogenous to the host cell that can direct the transcription of nucleic acids operably ligated to the promoter in the host cell. In addition to promoter sequences, other sequences that can be operably ligated to the CD91 polypeptide nucleotide sequence include, but are not limited to, sequences encoding fusion tags, sequences encoding signal peptides, start sequences, terminator sequences, transcription and translation stop signals, and selectable marker sequences, such as antibiotic resistance genes, e.g., kanamycin resistance genes. Various selectable markers are known in the art and can be used in the present invention. Furthermore, the construct may further include one or more sequences encoding an amino acid sequence for a desired purpose, e.g., a protein tag, or another type of tag, e.g., a chemical molecule, e.g., biotin, a linker sequence for attaching it to the CD91 polypeptide. For example, if a protein tag is present, the coding sequence of the protein tag can be inserted into the coding DNA sequence of the CD91 polypeptide, e.g., after the start codon or before the stop codon while maintaining the reading frame. This generates an N-terminal or C-terminal protein tag of the CD91 polypeptide.

[0057] Hemopexin, at least in part, serves as the first line of defense against heme toxicity due to its ability to bind heme with high affinity (Kd < 1 pM) and function as a heme-specific carrier from the bloodstream to the liver. Hemopexin has also been reported to possess serine protease activity, as well as several other functions, such as anti-inflammatory and pro-inflammatory activity, cell adhesion, and the ability to inhibit the binding of certain divalent metal ions (Mauk, MR, 2011. An alternative view of the proposed alternative activities of hemopexin. Protein Science, Vol. 20, pp. 791-805).

[0058] Hemopexin is a 61±2 kDa plasma β-1B glycoprotein composed of a single 439-amino acid long peptide chain with a carbohydrate content of 20-22%, formed by two 4-blade β-propeller domains, which resemble two thick disks fixed together at a 90° angle and linked by interdomain linker peptides. Heme is released into the bloodstream as a result of intravascular and extravascular hemolysis and binds between the two 4-blade β-propeller domains in a highly hydrophobic pocket formed by interdomain linker peptides. Residues His213 and His266 coordinate the heme iron atom, forming a stable bis-histidyl complex similar to that of hemoglobin.

[0059] Hemopexin contains 20-22% carbohydrates, including sialic acid, mannose, galactose, and glucosamine. Twelve cysteine ​​residues were found in the protein sequence, which likely constitute six disulfide crosslinks. Hemopexin has high affinity (K) for heme. dDue to its ability to bind at <1 pM and function as a heme-specific carrier from the bloodstream to the liver, it serves as the first line of defense against heme toxicity. It binds heme in equimolar ratios, but there is no evidence that the heme is covalently bound to the protein. Further characteristics include: a UV extinction coefficient of 1.97 [mL(mg×cm)] at 280 nm; a theoretical pI of 6.55; and a mean hydrophobicity of -0.43 (no high-scoring hydrophobic segments).

[0060] As used herein, the term “hemopexin” is intended to mean a hemopexin protein as naturally found in vertebrates, such as humans, rabbits, mice, dogs, cats, cattle, horses, pigs, sheep, goats, or birds, preferably in the blood of humans. Preferably, the hemopexin is a mature form of human hemopexin consisting of amino acid residues 24-462 of the NCBI reference sequence NP_000604.1, shown as SEQ ID NO: 4, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4, or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% homology with SEQ ID NO: 4. Sequence ID 4 1 MARVLGAPVA LGLWSLCWSL AIATPLPPTS AHGNVAEGET KPPDDVTERC SDGWSFDATT 61 LDDNGTMLFF KGEFVWKSHK WDRELISERW KNFPSPVDAA FRQGHNSVFL IKGDKVWVYP 121 PEKKEKGYPK LLQDEFPGIP SPLDAAVECH RGECQAEGVL FFQGDREWFW DLATGTMKER 181 SWPAVGNCSS ALRWLGRYYC FQGNQFLRFD PVRGEVPPRY PRDVRDYFMP CPGRGHGHRN 241 GTGHGNSTHH GPEYMRCSPH LVLSALTSDN HGATYAFSGT HYWRLDTSRD GWHSWPIAHQ 301 WPQGPSAVDA AFSWEEKLYL VQGTQVYVFL TKGGYTLVSG YPKRLEKEVG TPHGIILDSV 361 DAAFICPGSS RLHIMAGRRL WWLDLKSGAQ ATWTELPWPH EKVDGALCME KSLGPNSCSA 421 NGPGLYLIHG PNLYCYSDVE KLNAAKALPQ PQNVTSLLGC TH

[0061] Detection of the hemopexin:heme complex by the CD91 polypeptide can be carried out by any method known in the art that can detect ligand binding to the receptor. Such methods include immunoassays, high-performance liquid chromatography (HPLC) assays, or typical ligand-binding assays, such as labeled, unlabeled, thermodynamic, or structure-based assays (see Yakimchuk K., Materials and Methods, 2011; 1:199 (last modified March 26, 2020)).

[0062] Detection can be performed in a solid phase or in solution. Preferably, the method is a solid phase method. Therefore, the CD91 polypeptide can be or may be preferably linked to a solid support. Alternatively, the hemopexin:human heme complex can be linked to a solid support.

[0063] Preferably, the CD91 polypeptide is linked to a solid support, or is suitable for linking to a solid support. Any suitable polymer, preferably a synthetic polymer, can be used as the solid support. Linking to the solid support may be direct, meaning that the CD91 polypeptide is linked without an intermediate, especially if the CD91 polypeptide has a high affinity for the surface of the solid support. Thus, this linking may be nonspecific via adsorption. An example of a suitable surface for linking the CD91 polypeptide is a charged surface in which the charged region of the CD91 polypeptide has a high affinity. Preferably, the surface is a highly charged polystyrene surface, such as that present on a MaxiSorp® plate. According to one embodiment, the solid support may be a resin material or bead-like particles, both of which may be suitable for chromatographic methods. Alternatively, linking to the solid support may be indirect, meaning that the CD91 polypeptide is linked via a linking compound. The linking compound is an intermediate compound that is bonded to the solid support, between the solid support surface and the CD91 polypeptide. An example of indirect linkage is antigen-antibody linkage, in which an antibody specific to the CD91 polypeptide (which is the antigen) is bound to a solid support, and the CD91 polypeptide is linked to the solid support by the antibody. Alternatively, a linking compound other than an antibody for the CD91 polypeptide may be bound to the solid support. Further alternatives may include a tag that links the CD91 polypeptide to the solid support via an affinity linking partner bound to the solid support. Such tags include affinity tags, e.g., chicken-binding protein (CBP), maltose-binding protein (MBP), Strep tag, glutathione S-transferase (GST), or poly(His) tag, or linking partners, e.g., biotin, which are known in the art and are discussed herein. Linking the CD91 polypeptide to a solid support does not interfere with the binding of the hemopexin:heme complex to the CD91 polypeptide.

[0064] Preferably, the detection method is an immunoassay, which preferably measures the presence or concentration of the hemopexin:heme complex in a biological sample using a detection antibody that specifically binds to hemopexin. Most preferably, the method is an enzyme-linked immunosorbent assay (ELISA), which may be a sandwich ELISA assay.

[0065] In a particularly preferred embodiment, the amount of hemopexin:heme complex in a biological sample is determined.

[0066] To carry out this method, the CD91 polypeptide is preferably ligated to a solid support. The biological sample is then added to the solid support. A detection antibody is applied to the surface of the solid support so that the detection antibody can bind to the hemopexin in the hemopexin:heme complex bound to the CD91 polypeptide. The antibody may be ligated to an enzyme, or detected by a secondary antibody ligated to an enzyme. In the final step, a reading component containing a reaction solution with an enzyme substrate is added. The enzymatic reaction generates a detectable signal. The detectable signal may be a color, fluorescence, or electrochemical signal. Types of enzymes and substrates for generating color, fluorescence, or electrochemical signals are known in the art. Between each step, i.e., between the ligation step, the biological sample addition step, the detection antibody addition step, and the substrate addition step, the solid support is typically washed with a neutral detergent solution to remove any unbound or nonspecifically bound components, such as proteins or antibodies. After the final washing step, the solid support is expressed by adding the enzyme substrate to generate a signal. The presence and / or amount of hemopexin:heme complex in a biological sample is determined by measuring absorbance, fluorescence, or electrochemical signals.

[0067] During experimental investigations of conditions for detecting hemopexin:heme complexes using CD91 polypeptide, it was found that a classical plate-based format is highly effective for the detection and quantification of hemopexin:heme complexes. Therefore, CD91 polypeptide is nonspecifically conjugated as a coating to MaxiSorp® high-binding plates, which have high affinity for molecules with polar or hydrophilic groups. The hemopexin:heme complex of a sample is conjugated to the CD91 polypeptide bound to the plate and detected using rabbit anti-hemopexin antibody and HRP-conjugated anti-rabbit antibody against the anti-hemopexin antibody. The detection antibody and secondary antibody are known in the art and commercially available. For example, the detection antibody is an anti-hemopexin antibody, e.g., rabbit polyclonal antibody (Ab48135) from Abcam. The secondary antibody is an anti-detection antibody, such as a nonspecific goat anti-rabbit antibody, such as one linked to horseradish peroxidase (HRP), an enzyme that catalyzes the oxidation of various organic substrates with hydrogen peroxide, e.g., from Seracare (5220-0336). TMB (3,3',5,5'-tetramethylbenzidine) is used as the peroxidase substrate to generate a detectable signal. The reaction between the TMB substrate and HRP produces a measurable color change (deep blue, readable directly at 650 nm, or deep yellow, readable at 450 nm after stopping with an acid solution) that correlates with the level of the hemopexin:heme complex in the sample. In this way, the amount of the hemopexin:heme complex can be determined. Generally, the conditions described in Example 2 below can be used.

[0068] Hemopexin: For the detection of the hemopexin:hem complex, a detection antibody against hemopexin in the hemopexin:hem complex can be used. The "detection antibody" is an antibody for its detection that is specific to hemopexin and binds to hemopexin in the hemopexin:hem complex. The detection antibody does not interfere with the binding of hemopexin to the CD91 polypeptide and to heme. Those skilled in the art know how to prepare antibodies against specific proteins. Furthermore, antibodies specific to hemopexin that can be used in the present invention are commercially available. For example, there is a rabbit polyclonal anti-hemopexin antibody (Ab48135) from Abcam.

[0069] The detection of the hemopexin:hem complex in a biological sample via a detection antibody can be performed via a "secondary antibody" that can bind to the detection antibody but does not bind to the antigens present in the reaction solution. The secondary antibody is usually a non-specific antibody that discriminates antibodies of different species by binding to the heavy chain of the detection antibody and does not interfere with the binding of the detection antibody that binds to hemopexin. Therefore, the detection antibody usually originates from one species, for example, the rabbit species, and the secondary antibody is directed against the species-specific sequence of the detection antibody. The secondary antibody is commercially available, for example, from Seracare (5220-0336).

[0070] The terms "specific binding", "specific binding of an antibody", "specific antibody" or corresponding terms refer to a binding reaction in which a compound or antibody binds to a specific binding partner, which is an antigen in the case of an antibody, but does not bind to other molecules present in the reaction mixture in such a large amount (less than 10% of the binding to a specific protein). Generally, an antibody of a binding pair that "specifically binds" to its corresponding binding partner, such as an antigen in the case of an antibody, has an equilibrium affinity constant of more than about 10 5 mol / liter (e.g., 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 mol / liter or more).

[0071] As used herein, the term “antibody” includes full-length antibodies known in the art, or molecules containing at least one antibody variable domain but not the entire structure of an antibody, as defined herein, and “antibody derivatives.” Antibody derivatives can still bind to target molecules. Derivatives may be antibody fragments, e.g., Fab, Fab2, scFv, Fv or a portion thereof, or other derivatives or combinations of immunoglobulins, e.g., nanobodies, diabodies, minibodies, single-domain antibodies of camelids, single domains or Fab fragments, heavy and light chain domains of the variable region (e.g., VL, VH, VHH including Fd, V-lambda and V-kappa), and minidomains consisting of two beta-strands of an immunoglobulin domain connected by at least two structural loops.

[0072] The ligation of CD91 polypeptides or the detection of hemopexin:heme complexes can also be performed by “antibody mimics.” “Antibody mimics” are defined herein as organic compounds that can specifically bind CD91 polypeptides or hemopexin:heme complexes like antibodies, but are not structurally related to antibodies. Antibody mimics are typically artificial peptides or proteins with a molecular weight of approximately 3–20 kDa. Non-limiting examples of antibody mimics include afibody, affilin, affimer, afitin, antikalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, Z domain of protein A, gamma B crystallin, ubiquitin, cystatin, and Sulfolobus acidocardarius. Sac7D derived from acidocaldarius, lipocalin, the A domain of the membrane receptor, the ankyrin repeat motif, the SH3 domain of Fyn, the Kunits domain of the protease inhibitor, the 10th type III domain of fibronectin, and synthetic heterobivalent or heteropolyvalent ligands (Josan et al., Bioconjug. Chem., 2011; 22: pp. 1270-1278; Shallal et al., Bioconjug. Chem., 2014; 25: pp. 393-405; and Xu et al., PNAS, 2012; 109: pp. 21295-300).

[0073] The binding of the CD91 polypeptide or the detection of the hemopexin:heme complex can also be performed by any other compound that can specifically bind to the CD91 polypeptide or hemopexin, respectively, but does not interfere with the binding of other components necessary for the detection of the hemopexin:heme complex in the biological sample. "Specific binding" is meant in the sense described above, i.e., not binding to other molecules present in the reaction mixture in significant amounts (less than 10% of the binding to the CD91 polypeptide or hemopexin:heme complex, respectively).

[0074] The detection method of the present invention can also be carried out by linking the hemopexin:heme complex of a biological sample to a solid support via a specific compound such as an antibody, then adding a CD91 polypeptide, and detecting the hemopexin:heme complex by detecting the bound CD91 polypeptide.

[0075] Detection of hemopexin:heme complexes in biological samples can also be performed in solution. The biological sample may be a biological sample containing hemopexin:heme complexes, or a mixture of the biological sample and a solution containing buffers or salts to facilitate the completion of the detection method. Therefore, the binding of CD91 polypeptide to the hemopexin:heme complex is performed in solution. For detection of the complex of CD91 polypeptide and hemopexin:heme complex, a detection antibody is added, similar to detection using a solid phase. The entire complex is then ligated to a solid support and read.

[0076] A biological sample can be any fluid in which hemopexin:heme complexes may be present.

[0077] A biological sample can be any biological fluid from a subject in which a hemopexin:heme complex is present or expected to be present. The hemopexin may be endogenous hemopexin and / or exogenous hemopexin administered to the subject, preferably to treat a hemolytic or hemolytic condition. Examples of biological samples may be blood, cerebrospinal fluid (CSF), amniotic fluid, urine, saliva, mucus, sweat, bronchoalveolar lavage fluid, sputum, semen, tears, feces, and / or bile. The blood may be untreated (and whole blood) or treated blood. Preferably, the blood used in the use and methods of the present invention is treated blood. Examples of treated blood include plasma or serum.

[0078] Biological samples may be derived from patients with hemolytic conditions. Preferably, the hemolytic conditions are acute and / or chronic hemolytic conditions, and more preferably selected from the group consisting of: sickle cell anemia, hemolytic anemia, bone marrow aplastic crisis, hyperhemolytic crisis, transfusion-induced hemolysis, hemolytic uremic syndrome, myocardial infarction, acute chest syndrome, pulmonary hypertension, lower leg ulcer, growth retardation, osteoinfarction, eclampsia of pregnancy, renal failure, acute kidney injury, acute respiratory distress syndrome (ARDS), stroke including hemorrhagic stroke, subarachnoid hemorrhage, intracranial hemorrhage. ICH), splenic hemocytosis, splenic infarction, autoimmune diseases including autoimmune hemolytic anemia, microbial infection, increased susceptibility to infection, malaria infection, trauma, transplant-related conditions, open cardiac surgery using cardiopulmonary bypass, burns, e.g., hemoglobinemia or hemoglobinemia with post-burn hemolysis, hereditary spherocytosis, hereditary ellipticosis, thalassemia, congenital dyserythropoiesis, paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus, and chronic lymphocytic leukemia. More preferably, the hemolytic condition is sickle cell anemia.

[0079] A biological sample can be any fluid resulting from a manufacturing process that may contain hemopexin:heme complexes. Examples of such fluids may include intermediate or final products of protein manufacturing processes. Such processes may produce proteins derived from human plasma or recombinantly expressed human proteins. For example, the term “biological sample” also encompasses cell culture supernatants and mixtures of cell culture supernatants with other components such as heme.

[0080] In a preferred embodiment of the present invention, the CD91 polypeptide used may further comprise a signal peptide and / or one or more tags.

[0081] The CD91 polypeptide may have additional components added that are useful for isolating the CD91 polypeptide and / or for performing the method of the present invention, or additional components may be added. The additional components do not participate in binding to the hemopexin:heme complex. If they are derived from the CD91 protein, they are not directly adjacent to the CD91 polypeptide sequence within the CD91 protein and do not participate in binding to the hemopexin:heme complex. Preferred additional components may be short peptide sequences (usually 16-30 amino acids long) present at the N-terminus of the newly synthesized CD91 polypeptide, such as signal peptides, which are used to direct the CD91 polypeptide into the secretory pathway. Signal peptides for use in the present invention are known in the art. Preferably, the signal peptide linked to the CD91 polypeptide is derived from the same CD91 protein. The signal peptide is cleaved from the CD91 polypeptide during secretion and does not become part of the CD91 polypeptide used in the assay of the present invention.

[0082] Other additional components may be one or more tags. A tag is a component that is fused to or bound to the CD91 polypeptide to provide the CD91 polypeptide with desirable properties. A tag may also be a protein tag. Since tags are typically fused to the CD91 polypeptide at the N-terminus and / or C-terminus, they are also referred to as fusion tags. Fusion tags are affinity tags that enable protein purification by affinity to a binding partner. Purification is performed via binding to the binding partner by methods known in the art, e.g., chromatography. Affinity partners, purification methods via affinity partners, and systems and kits are known in the art and / or commercially available (see, e.g., Kimple et al., Curr Protoc Protein Sci., 2013; 73:9.9.1-9.9.23). Tags may also be useful for detection, such as detection via the presence of a binding partner, fluorescence, or a recognition motif; to increase expression; to increase solubility; to improve protein folding; or to facilitate separation in HPLC, for example, by increasing polarity. Examples of fusion tags are known in the art and include affinity tags that enable isolation by affinity techniques. Examples include chitin-binding protein (CBP), maltose-binding protein (MBP), Strep tags, glutathione S-transferase (GST), or poly(His) tags that enable isolation by affinity techniques (affinity tags). For example, poly(His) tags bind to metal matrices. Stabilization tags are used, in particular, for recombinant proteins expressed in chaperone-deficient species such as E. coli, to support proper folding in the protein and prevent protein precipitation. Examples are thioredoxin and poly(NANP). Some affinity tags, such as MBP and GST, play a dual role as solubilizers. Chromatographic tags are used to alter the chromatographic properties of a protein to provide different resolutions across specific separation techniques. Examples include polyanionic amino acids, e.g., FLAG tags. Epitope tags are short peptide sequences that have high affinity for antibodies.Examples include V5 tags, Myc tags, HA tags, Spot tags, T7 tags, and NE tags. These tags are particularly useful in Western blotting, immunofluorescence, and immunoprecipitation experiments. Fluorescent tags allow for visual reading. One example is GFP, and its variants are the most commonly used fluorescent tags. More advanced applications of GFP include its use as a folding receptor (fluorescent when folded, colorless when unfolded). Alternatively, the tag could be an enzyme that enables the detection of CD91 polypeptides, such as horseradish peroxidase, which enables chemiluminescent detection. Alternatively, the tag could be a binding partner of a binding pair, enabling the detection of CD91 polypeptides via the binding of the tag to its binding partner. One example is the biotin and avidin / streptavidin system, where, as a tag, biotin can be bound to CD91 polypeptides, and avidin / streptavidin can be bound to a solid support for capturing CD91 polypeptides. The tag may be removable by chemical agents or enzymes, and thus the CD91 polypeptide can be separated from the tag, for example, after binding to its affinity partner in the fusion tag during isolation.

[0083] In a preferred embodiment of the present invention, the CD91 polypeptide is bonded to a solid support, preferably a solid support having a highly charged polystyrene surface, more preferably a MaxiSorp® plate.

[0084] The solid support used herein is any solid support that enables the binding of CD91 polypeptides or the binding of hemopexin:heme complexes of a biological sample for the detection of hemopexin:heme complexes. Preferably, the CD91 polypeptide is ligated to the solid support. Alternatively, the solid support is any solid support that enables the binding of CD91 polypeptides by a binding tag via an affinity partner of a tag already bound to the solid support for isolation, purification, or detection. Examples of solid supports are plates, preferably well plates, beads, electromagnetic beads, resins, and columns, where beads or resins may be part of a column or constitute a column, filter, or membrane. Plate-based methods are preferred in the ligation of CD91 polypeptides for the detection of hemopexin:heme complexes because they have advantages over non-plate-based methods, such as higher throughput, greater sensitivity, and easier execution. The plates are more preferably well plates, more preferably multi-well plates, e.g., 6, 12, 24, 48, or 96 plates. More preferably, the surface of the plate is a highly charged polystyrene surface, such as that present on a MaxiSorp® plate.

[0085] Detection of hemopexin:heme complexes in biological samples using CD91 polypeptides can be useful for monitoring hemolysis or the treatment of hemolysis or hemolysis-related conditions in a subject, preferably for monitoring hemolysis or hemolysis-related conditions caused by hemopexin.

[0086] The formation of heme complexes by hemopexin directly protects cells from heme toxicity. In subjects suffering from hemolysis, hemopexin expression increases, forming complexes with toxic heme to detoxify it. Subsequently, the hemopexin:heme complex is removed from the body. However, even if a subject is suffering from hemolysis, endogenous hemopexin is produced only in limited quantities in the body, so the hemopexin produced may not be sufficient to form complexes with large amounts of heme. Therefore, additional treatment for hemolysis is necessary. Such treatment may involve the administration of exogenous hemopexin to form complexes with large amounts of heme and detoxify it.

[0087] Hemopexin helps detoxify biological fluids by forming a complex with heme. Elevated heme levels during hemolysis lead to a limited increase in hemopexin expression. Therefore, endogenous hemopexin:heme complex levels indicate hemolysis or the severity of hemolysis. Thus, higher hemopexin:heme complex levels indicate greater hemolysis severity. However, this correlation is limited because hemopexin is produced only to a limited extent. Therefore, detecting the concentration of hemopexin:heme complex in body fluids indicates the appearance and severity of hemolysis in a subject.

[0088] In addition, the detection and quantification of hemopexin:heme complexes in subjects can be useful in monitoring whether hemolytic treatment is effective in those subjects. Therefore, if the level of hemopexin:heme complexes in subjects with hemolysis decreases with treatment, this may indicate effective hemolytic treatment. Thus, determining the level of hemopexin:heme complexes in subjects helps monitor the effectiveness of hemolytic treatment and allows for the application of treatment based on the amount of hemopexin:heme complexes.

[0089] Furthermore, the detection and quantification of hemopexin:heme complexes in subjects can be useful in monitoring whether hemopexin treatment is effective for hemolysis in those subjects. If high levels of heme are produced during hemolysis and endogenous hemopexin is not produced in sufficient quantities to fully complex with the heme, administration of exogenous hemopexin can raise the hemopexin:heme complex level above the endogenous level. Thus, such high hemopexin:heme complex levels indicate efficient complex formation with a large amount of heme by exogenous hemopexin, but may also indicate that high levels of heme are being produced in the subject, which suggests that hemolysis is still progressing and is a serious condition. Therefore, determining the level of hemopexin:heme complexes in subjects can help monitor the effectiveness of hemopexin treatment and allow for the indication of hemopexin treatment according to the level of hemopexin:heme complexes in the subjects.

[0090] Alternatively, the detection of hemopexin:heme complexes may be useful in monitoring the efficacy of hemopexin as a pharmaceutical drug in subjects with hemolysis during clinical trials involving hemopexin. Quantifying hemopexin:heme complex levels above a certain level may indicate that therapeutically administered hemopexin is effective in complexing with heme and therefore effective as a pharmaceutical drug.

[0091] Therefore, in a preferred embodiment, the use of the present invention is intended for the diagnosis or monitoring of hemolysis in a subject, or for monitoring the treatment of hemolysis or a hemolysis-related condition in a subject.

[0092] In a further embodiment, the present invention relates to an in vitro method for detecting a hemopexin:heme complex in a biological sample, comprising the steps of: incubating the biological sample with the CD91 polypeptide as defined above; and detecting the binding of the CD91 polypeptide to the hemopexin:heme complex.

[0093] All embodiments defined above are also applicable to this method of the present invention.

[0094] According to one embodiment, an in vitro method for detecting the hemopexin:heme complex in a biological sample comprises at least the following method steps: - A step of incubating a biological sample with the CD91 polypeptide defined above, - A step to detect the binding of the CD91 polypeptide to the hemopexin:heme complex. Includes.

[0095] According to one embodiment, an in vitro method for detecting the hemopexin:heme complex in a biological sample comprises at least the following method steps: - A step of preparing a solid support to which the CD91 polypeptide defined above is linked, - A step of incubating a biological sample with CD91 polypeptide, - Depending on the circumstances, the process may include a step of performing a cleaning process, - A step to detect the binding of the CD91 polypeptide to the hemopexin:heme complex, - A step to determine the amount of hemopexin:heme complex in a biological sample. Includes.

[0096] In a more preferred embodiment, the method of the present invention is used to determine the amount of heme in a biological sample, preferably blood or serum, that is capable of binding to hemopexin.

[0097] It is well known that not all heme present in serum is reachable by hemopecin, as is the heme bound to hemoglobin. The method of the present invention can also be used to determine the amount of reachable heme in a biological sample, i.e., the amount of heme reachable to form a complex with hemopecin. For this purpose, an excess of hemopecin is added to the biological sample to generate an additional hemopecin:heme complex. The amount of hemopecin:heme complex is then determined in the sample with and without the excess hemopecin. The difference in molar concentration between the two samples is the concentration of heme reachable by hemopecin (because hemopecin:heme binds in a 1:1 ratio).

[0098] According to one embodiment, a method for determining the amount of reachable heme in a biological sample includes at least the following steps: - Additional hemopexin: The process involves adding excess hemopexin to a biological sample to generate a heme complex (with spikes), and preparing a biological sample without the added excess hemopexin (without spikes). - A step of determining the amount of hemopexin:heme complex in a biological sample with excess hemopexin added (with spikes) and a biological sample without excess hemopexin added (without spikes) by preferably applying an in vitro method for detecting hemopexin:heme complex in a biological sample as described herein, - Based on the assumption that hemopexin:heme binds in a 1:1 ratio, the process involves calculating the difference in the molar concentrations of the determined hemopexin:heme complex between two samples: one with an excess of hemopexin added (with spikes) and one without an excess of hemopexin added (without spikes), in order to obtain a heme concentration that can reach hemopexin. Includes.

[0099] According to one embodiment, a method for determining the relative concentration (C) of achievable heme in a biological sample includes at least the following steps: - Additional hemopexin: The process involves adding excess hemopexin to a biological sample to generate a heme complex (with spikes), and preparing a biological sample without the added excess hemopexin (without spikes). - A step of determining the concentration of hemopexin:heme complex (B) in a biological sample to which excess hemopexin has been added (with spikes) and the concentration of hemopexin:heme complex (A) in a biological sample without excess hemopexin has been added (without spikes), preferably by applying an in vitro method for detecting hemopexin:heme complex in a biological sample as described herein, - A step to determine the concentration of heme that can reach hemopexin by subtracting the concentration of the hemopexin:heme complex present in a sample without spikes (A) from the concentration of the hemopexin:heme complex present in a sample with spikes (B), and assuming that heme binds to hemopexin in a 1:1 ratio, dividing the resulting delta-hemopexin:heme complex concentration (BA) by the molecular weight of hemopexin. - A step to obtain the relative concentration (C) of heme that can reach hemopexin in the sample by dividing the delta value from the previous step by the molecular weight of hemopexin. Includes.

[0100] Therefore, the achievable relative concentration of heme can be calculated as (C) = ([with spike (B) - without spike (A)] / 63000) * 1000 (μM).

[0101] The present invention also relates to a method for separating hemopexin:heme complexes in vitro or in vitro from a biological sample, comprising the steps of: incubating the CD91 polypeptide defined above with the biological sample; and separating the CD91 polypeptide-hemopexin:heme complexes from the biological sample.

[0102] The separation of hemopexin:heme complexes from a biological sample means removing or depleting the hemopexin:heme complex from a biological sample by binding the hemopexin:heme complex to a CD91 polypeptide and removing the resulting complex of the CD91 polypeptide and the hemopexin:heme complex from the biological sample. The separation of hemopexin:heme complexes from a biological sample can be carried out by any method known to those skilled in the art. Primarily, the separation corresponds to the first part of the procedure for detecting hemopexin:heme complexes in the present invention, where the CD91 polypeptide and the hemopexin:heme complex are bound together. Therefore, the above description applies to embodiments of the separation of hemopexin:heme complexes from a biological sample. Preferably, as described above, the CD91 polypeptide is bound to a solid support, the solid support to which the CD91 polypeptide is bound is brought into contact with the biological sample, and the solid support to which the CD91 polypeptide is bound and the bound hemopexin:heme complex are removed from the biological sample. Therefore, effective removal can be achieved when a biological sample is passed, for example, through a column, resin, filter, or membrane, or subsequently brought into contact with beads that can be removed by, for example, centrifugation or magnetic separation.

[0103] The separation of the hemopexin:heme complex from a biological sample may be performed in vitro or extracorporeally. This may be useful in removing the hemopexin:heme complex from the biological sample. "Extracorporeal" separation means an operation performed outside the subject using a blood sample obtained from the subject. Thus, blood is collected from the subject and, if applicable, processed as described above and contacted with the CD91 polypeptide. The resulting complex of the CD91 polypeptide and the hemopexin:heme complex is separated from the blood, and the removed blood is returned to the subject. The extracorporeal operation can be performed in vitro, i.e., blood is collected from the subject, contacted with the CD91 polypeptide in vitro, the resulting complex of the CD91 polypeptide and the hemopexin:heme complex is separated, and the blood is returned to the body. Alternatively, the extracorporeal operation may be performed in a circuit operation in which the hemopexin:heme complex is separated and the blood is circulated from subject to subject before returning the blood to the subject's circulation. All operations for transporting blood outside the body are performed in an extracorporeal circuit. Preferably, the procedure is apheresis, in which the blood of the subject is passed through a device that separates and removes the hemopexin:heme complex using CD91 polypeptide and returns the remainder to circulation.

[0104] A further aspect of the present invention is a CD91 polypeptide as defined above for use in a method of treating a condition, given that it is desirable to block or reduce the interaction of the hemopexin:heme complex with its endogenous CD91 receptor with respect to the condition. In such a situation, the polypeptide of the present invention can be administered to a subject in need as a decoy of the hemopexin:heme complex to block or reduce CD91 receptor-mediated endocytosis and / or further downstream signaling.

[0105] Preferably, the hemolytic condition is an acute hemolytic state and / or a chronic hemolytic state, more preferably selected from the group consisting of: sickle cell anemia, hemolytic anemia, bone marrow aplastic crisis, hyperhemolytic crisis, transfusion-induced hemolysis, hemolytic uremic syndrome, myocardial infarction, acute chest syndrome, pulmonary hypertension, lower leg ulcer, growth retardation, osteoinfarction, eclampsia of pregnancy, renal failure, acute kidney injury, acute respiratory distress syndrome (ARDS), stroke including hemorrhagic stroke, subarachnoid hemorrhage, intracranial hemorrhage ( ICH), splenic hemocytosis, splenic infarction, autoimmune diseases including autoimmune hemolytic anemia, microbial infections, increased susceptibility to infection, malaria infection, trauma, transplant-related conditions, open cardiac surgery using cardiopulmonary bypass, burns, e.g., hemoglobinemia or hemoglobinemia with post-burn hemolysis, hereditary spherocytosis, hereditary ellipticosis, thalassemia, congenital dyserythropoiesis-associated anemia, paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus, and chronic lymphocytic leukemia.

[0106] The term "hemolysis-related condition" refers to any condition in an object in which hemolysis occurs, including any condition in which hemolysis is the cause of the condition or any condition that triggers hemolysis.

[0107] Treatment of hemolysis or hemolysis-related conditions involves the formation of hemopexin:heme complexes in the target's blood. Therefore, CD91 polypeptide can be administered to the target to form hemopexin:heme complexes in body fluids.

[0108] For therapeutic use of CD91 polypeptide, the CD91 polypeptide is provided as a pharmaceutical composition comprising the CD91 polypeptide and a pharmaceutically acceptable carrier. For the manufacture of the pharmaceutical composition, the CD91 polypeptide must be in a pharmaceutical dosage form comprising the CD91 polypeptide and a mixture of components such as a pharmaceutically acceptable carrier that provides the desired properties. The pharmaceutical composition comprises one or more pharmaceutically acceptable carriers known to those skilled in the art.

[0109] Pharmaceutical compositions can be formulated for systemic, nasal, parenteral, enteral, or topical administration. Parenteral administration includes subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal administration.

[0110] Pharmaceutical compositions can be formulated in various dosage forms, including solid dosage forms for oral administration, such as capsules, tablets, pills, powders, and granules; liquid dosage forms for oral administration, such as pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs; injectable preparations, such as sterile aqueous or oily suspensions for injection; and dosage forms for topical or transdermal administration.

[0111] The specific therapeutically effective dose level for any given subject varies depending on a variety of factors, including dosage form, age, weight, and sex of the subject, duration of treatment, and similar factors well known in the medical field.

[0112] In a preferred embodiment, a method for treating hemolysis or a hemolysis-related condition includes the use of a CD91 polypeptide to separate the hemopexin:heme complex from the blood. All embodiments described above in the context of separating the hemopexin:heme complex from the blood also apply to this embodiment.

[0113] In particular, the separation can be performed outside the body by apheresis, or it can be performed in vivo in the blood.

[0114] Regarding the removal of the CD91 polypeptide-hemopexin:heme complex from body fluids, the complex is removed by the subject and therefore removed from the subject by a natural process. Alternatively, the CD91 polypeptide-hemopexin:heme complex is separated extracorporeally, either in vitro or in an extracorporeal circuit from the subject's blood. This removes the blood from the subject, separates the CD91 polypeptide-hemopexin:heme complex from the blood, and then returns the blood to the subject. Separation can be achieved by binding the CD91 polypeptide-hemopexin:heme complex to a solid support via the CD91 polypeptide or hemopexin:heme complex, and then removing the solid support from the blood. Binding of the CD91 polypeptide or hemopexin:heme complex to a solid support is described herein.

[0115] The present invention also discloses a method for treating a subject by administering a pharmaceutically effective amount of CD91 polypeptide. All the embodiments described above also apply to this method of the present invention.

[0116] The target of treatment with CD91 polypeptide is animals, such as vertebrates, such as humans, rabbits, mice, dogs, cats, cattle, horses, pigs, sheep, goats, or birds, preferably humans.

[0117] A further aspect of the present invention refers to a kit comprising the CD91 polypeptide. The kit is an assembly of separate units comprising the CD91 polypeptide and further components necessary to carry out the method of the present invention. The CD91 polypeptide may exist in a soluble and free form, as defined herein, or in a solid form such as a powder for reconstitution in solution, or it may be bound to a solid support. If not already bound to a solid support, a solid support may be additionally present in the kit to which the CD91 polypeptide is bound during the execution of the method. The CD91 polypeptide can be bound to a support covalently or noncovalently. The CD91 polypeptide can be bound to a support directly or indirectly. The CD91 polypeptide may have binding tags as described herein. Further reagents or solutions may be present as applicable. Such solutions may be a solution adapted to link a CD91 polypeptide to a solid support, a solution adapted to conjugate a hemopexin:heme complex in a biological sample to a CD91 polypeptide, a solution containing an anti-hemopexin detection antibody adapted to conjugate an anti-hemopexin:heme complex, a solution containing a secondary antibody adapted to conjugate a secondary antibody to a detection antibody, and / or a solution containing a substrate adapted to an enzymatic reaction of the substrate by an enzyme linked to a secondary antibody. Instead of an adapted solution, the anti-hemopexin detection antibody, secondary antibody, or substrate may also exist in a solid form such as a powder, or in a storage solution. The solid form or storage solution is for use in the solution for carrying out each reaction. The kit may also include instructions for carrying out the methods of the present invention.

[0118] Further aspects of the present invention relate to a solid support to which a CD91 polypeptide is ligated. The solid support is as defined herein. The CD91 polypeptide may have tags as defined herein ligated to it.

[0119] The sequences referred to herein are outlined in the table below:

[0120] [Table 1]

[0121] The present invention will be further described here with reference to the following embodiments and drawings, but these embodiments and drawings are intended to be illustrative and not to limit the present invention. [Examples]

[0122] 1. Introduction Hemopexin (HPX) is a plasma glycoprotein that binds to free heme with high affinity. Heme released from heme-binding proteins due to internal bleeding, hemolysis, myolysis, or other cell damage is highly toxic due to its oxidative and pro-inflammatory effects. Hemopexin scavenges free heme, and this heme-hemopexin (hemu-hpx) is taken up by the liver via CD91 / LRP1 (low-density lipoprotein receptor-associated protein 1) receptor-mediated endocytosis. Plasma hemopexin plays a role in the metabolic processing of heme and inhibits the toxicity caused by the oxidative catalytic activity of heme.

[0123] CD91, also known as low-density lipoprotein receptor-associated protein 1 (LRP1, synonyms alpha-2 macroglobulin receptor and apolipoprotein E receptor), was first described by Herz et al. in The EMBO Journal, 1988; 7(13): 4119-27. The human protein is a precursor protein of approximately 600 kDa, which is proteolytically processed by furin to form a non-covalently associated 515 kDa alpha chain and an 85 kDa beta chain. It is expressed in a wide range of cell types, including macrophages, hepatocytes, fibroblasts, adipocytes, neurons, vascular smooth muscle cells, and syncytial trophoblasts, most abundantly in vascular smooth muscle cells, hepatocytes, and neurons. CD91 orthologs are found in vertebrates, and their nucleotide and amino acid structures have been elucidated for many species.

[0124] The analysis of the biogenesis and ligand-binding properties of LRP1 has been hindered by the difficulty in recombinant expression of the full-length receptor. Based on previous studies (Bu et al., J Biol Chem., 1996; 271(36):222 pp. 18-24; Neels et al., J Biol Chem., 1999; 274(44):313 pp. 5-11; and Willnow et al., J Biol Chem. 1994; 269(22):158 pp. 27-32), four recombinant soluble ligand-binding domains (I-IV) of LRP1 were constructed and used to test their binding to the heme-hx complex.

[0125] 2. Research purpose The primary objective was to investigate and determine whether it was possible to express and purify recombinant variants of the four ligand-binding repeat clusters contained within CD91 / LRP1.

[0126] The second objective was to investigate which of the four domains could bind the heme-hpx complex on two different platforms (BLI and SPR).

[0127] 3. Materials and Methods 3.1 Cell culture ExpiCHO-S® cells and mammalian expression vector pcDNA3.1 were obtained from Invitrogen®, Thermo Fisher Scientific (R790-07, V790-20). Cells were cultured in GIBCO® ExpiCHO Expression Medium (Invitrogen®, Thermo Fisher Scientific). Cells were maintained at 37°C in an incubator with an 8% CO2 atmosphere.

[0128] 3.2 Creation of cDNA expression plasmids cDNA encoding a soluble human (hu)LRP1 minireceptor, each containing four ligand-binding domains I-IV (Genbank acceptance number NP_002323.2) and huhLRPAP1 (Genbank acceptance number NP_002328.1), was codon-optimized for human expression and synthesized using GeneArt (Invitrogen®, ThermoFisher Scientific) with the Kozak consensus sequence (Kozak M., Nucleic Acids Res., 1987; 15(20): pp. 8125-48) (GCCACC) immediately upstream of the initiating methionine (+1). The huLRP1 ligand-binding domain was based on the boundary previously described by Obermoeller-McCormick et al., J Cell Sci, 2001; 114(5): pp. 899-908. This is shown in Figure 2. Each huLRP1 soluble minireceptor encoded a huLRP1 signal peptide, an N-terminal FLAG tag, and a C-terminal 8×His tag fused within the frame. These are specified as follows: huLRP1 binding domain I (amino acids 25-113) [huLRP1(1-24)-FLAG-huLRP1(25-113)-8His]; huLRP1 binding domain II (amino acids 806-1183) [huLRP1(1-24)-FLAG-huLRP1(806-1183)-8His]; huLRP1 binding domain III (amino acids 2481-2942) [huLRP1(1-24)-FLAG-huLRP1(2481-2942)-8His]; HuLRP1 binding domain IV (amino acids 3293-3783) [huLRP1(1-24)-FLAG-huLRP1(3293-3783)-8His]. Once each cDNA was completed, it was digested with NheI and XhoI and ligated to pcDNA3.1 (Invitrogen®, Thermo Fisher Scientific). Large-scale plasmid DNA preparation was performed using QIAGEN Plasmid Giga Kits (12191) according to the manufacturer's instructions.The nucleotide sequences of the plasmid constructs were validated by sequencing both strands using BigDye® Terminator Version 3.1 Ready Reaction Cycle Sequencing (Invitrogen®, Thermo Fisher Scientific. 4337455) and Applied Biosystems' 3130xl Genetic Analyzer.

[0129] 3.3 Transient transfection for recombinant protein production Transient transfection of an expression plasmid encoding hulRP1 soluble minireceptor-binding domains I-IV (90%) together with human LDL receptor protein-related protein 1 (huLRPAP1, RAP, 10%) was performed using ExpiCHO-S® cells with Expifectamine® transfection reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. Cells were 6 × 10⁶ viable cells. 6 The cells were transfected at a final concentration of cells / mL and incubated in a shaking incubator (Infors) at 37°C in 8% CO2 for 20 hours. After 20 hours, Enhancer® and Feed® were added to the culture. The culture was then incubated for a further 5 days at 32°C, 5% CO2, and 70% humidity. Five days after transfection, a second dose of Feed® was added to the culture, and the cultures were returned to an incubator at 32°C, 5% CO2, and 70% humidity. The cell culture supernatant was collected by centrifugation at 2500 rpm and then filtered through a 0.45 μm filter (Nalgene) before purification. The expression of recombinant huLRP1 soluble minireceptor in the culture supernatant was confirmed by SDS-PAGE (NuPAGE system, Thermo Fisher Scientific, MA, USA) and by Western blot analysis using an anti-His antibody (His-tagged antibody [FITC], GenScript, Cat#A01620).

[0130] 3.4 Purification of HULRP1 soluble minireceptors HuLRP1 soluble minireceptors were purified by tandem nickel-size exclusion chromatography using an AKTA Pure Protein Purification System (GE Healthcare Life Sciences). The samples were applied to a HisPrep® FF 16 / 10 Ni+ Sepharose 20 mL column in the presence of 2.5 mM NiCl2. Column chromatography was performed according to the manufacturer's instructions. The eluted samples were applied directly to a Hi Load 26 / 600 Superdex 200 pg column (GE Healthcare Bio-Sciences, PA, USA) and eluted in 10 mM Hepes, 150 mM NaCl, pH 7.3. The HuLRP1 soluble minireceptor-containing fraction was pooled and concentrated using an Amicon Ultra-15 centrifugal filter unit 10 kDa (Merck-Millipore, MS, USA) according to the manufacturer's protocol. The concentration of the purified recombinant protein was measured at A280nm using a Trinean DropSense96 system (Trinean, Gentbrugge, Belgium), and the purity of the concentrate was verified by SDS-PAGE (NuPAGE system, Thermo Fisher Scientific, MA, USA).

[0131] 3.5 LRP1 domain used in binding studies (BLI and SPR) The proteins listed in Table 1 below were used in the binding studies:

[0132] [Table 2]

[0133] 3.6 Hemopexin batches used in binding studies (BLI and SPR) The hemopexin batches listed in Table 2 below were used in the binding study:

[0134] [Table 3]

[0135] 3.7 Anti-PentaHis (HIS1K) Biosensor Real-time binding analysis of heme-hpx complexes to BLI-immobilized LRP1 soluble minireceptors was performed using anti-pentaHis biosensors (Figure 1). These were pre-immobilized with highly specific penta-His antibodies. Protein immobilization was achieved by directly capturing His-tagged proteins for subsequent kinetic analysis.

[0136] 3.8 Mini LRP1 binding study (BLI) conducted in Octet As previously mentioned, an anti-PentaHis antibody pre-coated biosensor (catalog number: 18-5120, ForteBio, TechNote 43: Anti-Penta-HIS (HIS1K) biosensors for label-free analysis of HIS-tagged protein, 2019) was used. Various LRP1 domains were immobilized at a concentration of 15 μg / mL in 10× kinetic buffer (PBS, 0.1% BSA, 0.02% Tween20). The heme-Hpx complex was diluted in 10× kinetic buffer (PBS, 0.1% BSA, 0.02% Tween20). The association and dissociation kinetics of the heme-hemopexin complex were performed at concentrations of 2.5, 1.25, 0.625, and 0.3125 μM. The settings for each binding step were selected as shown in Table 3 below. A reference control was included in all experiments (ligand loaded into the sensor without analytes). The data was acquired using OctetRED96 (ForteBio) at 30°C with the following settings:

[0137] [Table 4]

[0138] The data was analyzed using data analysis software (ForteBio, version 9.0). The data was processed by baseline alignment to the y-axis, inter-process correction, reference sensor subtraction, and curve smoothing using Savitzky-Golay filtering. The processed dynamic dataset was then fitted to the overall model using a 1:1 coupled model. The fitting accuracy was measured by the Chi parameter, which represents how similar the measurement results are to those derived from the model used to analyze the data. 2 and R 2 It was written by [author's name].

[0139] 3.9 Mini LRP1 coupling research conducted with Biacore (SPR) 3.9.1 Qualitative hemopexin capture assay 3.9.1.1 LRP1 domain that binds the heme-Hpx complex Using a standard NHS / EDC chemical reaction, rabbit anti-hemopexin polyclonal antibody was directly immobilized to approximately 18,000 RU on the carboxymethyl dextran surface of a CM5 sensor chip. Heme-hpx conjugates from CSL, Kankakee were captured to approximately 350 RU in flow cells 2 and 4, respectively, at the beginning of each cycle. The reference surface (flow cells 1 and 3) consisted solely of anti-hemopexin polyclonal antibody. 10 μM of each fraction (domains 1, 2, and 4) of LRP1 (see Table 1 for details) was injected onto the heme-hpx capture antibody surface for 120 seconds.

[0140] 3.9.1.2 LRP1.3 binding to heme-Hpx complex LRP1.3 (Fraction 3, Pool 1, Sample 4204) was injected into the heme-hpx complex capture surface in a 2-fold dilution series at concentrations ranging from 0.312 to 20 μM, in double repeats over 30 seconds. Dissociation was monitored for 60 seconds. The antibody surface was preconditioned by injecting 10 mM glycine pH 1.7 four times for 20 seconds each time. Regeneration was performed at the end of each cycle using 10 mM glycine pH 1.7 for 20 seconds. Raw data from the active surface (flow cells 2 and 4) were double-referenced by subtracting signals from the reference surface (flow cells 1 and 3) and blank injections.

[0141] The assay was performed in triple replicates at 37°C in HBS-N supplemented with 5 mM CaCl2 at pH 7.3. All buffers and solutions were filtered (0.22 μm) before use.

[0142] 3.9.2 NTA Capture and Crosslinking Assay Heme-hpx complex-bound LRP1.3 was also analyzed using a capture-crosslinking method with an NTA chip. His-tagged LRP1.3 ((Fraction 3, Pool-1, Sample 4204)) was anchored to flow cell 4 of an NTA chip using an EDC / NHS chemical reaction. LRP1.3 anchoring on the NTA chip was performed by forming a His-tagged nickel-NTA complex on a surface pre-activated using EDC / NHS. Flow cell 3, which was treated similarly but without LRP1.3 anchoring, was used as a reference cell. Immobilization was performed at a neutral pH. This method reduces heterogeneity of the chip surface by anchoring His-tagged LRP1.3 to approximately 800 RU by injecting 1 μg / ml for 1 minute and orienting the ligand.

[0143] The capture-and-crosslinking protocol method was performed in manual mode according to the following steps:

[0144] [Table 5]

[0145] The residual amine activity on the surface after mooring was deactivated by running a buffer over it overnight. This was avoided because a chemical deactivation step using ethanolamine has been found to affect ligand activity.

[0146] The heme-hpx complex was injected twice in a 2-fold dilution series at concentrations ranging from 0.62 to 20 μM, in double repeats over 30 seconds. Dissociation was monitored for 60 seconds. Since the complex completely dissociated, no regeneration step was required between cycles. Sensorgram data from the active surface (flow cell 4) was double-referenced by subtracting signals from the reference surface (flow cell 3) and blank injection. The sensorgram data were fitted to a 1:1 steady-state model.

[0147] 4. Results and Findings 4.1 Expression and purification of human LRP1 soluble minireceptors To investigate the hemopexin:heme binding properties of hulRP1 binding domains I, II, III, and IV, the inventors transiently expressed plasmids encoding each of the soluble minireceptors in ExpiCHO-S® cells (huLRP1 binding domain I (amino acids 25-113), HuLRP1 binding domain II (amino acids 806-1183), HuLRP1 binding domain III (amino acids 2481-2942), and HuLRP1 binding domain IV (amino acids 3293-3783)) (Figure 2). Expression of the huLRP1 soluble minireceptors was performed with or without co-expression of huLRPAP-1 (RAP) (Figure 3A). SDS-PAGE analysis demonstrated that co-expression of RAP is not essential for huLRP1 minireceptor secretion, but minireceptor expression with RAP co-expression was approximately twice as high (Figure 3A). This suggests that co-expression of RAP may promote the secretion of the huLRP1 minireceptor (5). SDS-PAGE analysis demonstrated that all proteins moved to higher molecular weights than their predicted molecular weights (huLRP1-binding domain I - 12.3 kDa, huLRP1-binding domain II - 43.6 kDa, huLRP1-binding domain III - 53.4 kDa, huLRP1-binding domain IV - 57.2 kDa). This is most likely due to glycosylation (Figure 3A-C).

[0148] 4.2 Identification of LRp1.3 as a binding domain for the heme-hpx complex To investigate and determine which of the LRP1 domains binds to the heme-hpx complex, each domain was anchored to a biosensor (HIS1K; anti-pentaHis) via an expression His tag. As shown in Figure 4, all four ligand-binding domains of LRP1 were tested for their binding to the heme-hpx complex. Only LRP1.3 showed clear binding to the complex. LRP1.3 bound to the heme-hpx complex in a dose-dependent manner with an affinity of approximately 1.5 μM, which is explained by a simple 1:1 kinetic model.

[0149] [Table 6]

[0150] 4.3 Confirmation of IRP1.3 by qualitative hemopexin capture SPR assay All four domains of LRP1 were tested for their binding to the heme-hpx complex by capturing and immobilizing the complex via an immobilized anti-hpx antibody on the chip surface. The assay strategy is outlined in Figure 6A. Only LRP1.3 showed clear binding to the complex, as observed in the BLI binding experiment and shown in Figure 5A. Binding to LRP1.2 was not shown. Hemopexin (10 μM) alone did not show significant binding to the LRP1 fraction tested (Figure 5C). Furthermore, LRP1.3 bound to the heme-hpx complex was tested dose-dependently at an affinity of approximately 2 μM. Concentration-dependent secondary components were also observed, and the data could not be adequately explained by a simple 1:1 kinetic model (Figure 6B). Therefore, an alternative assay setup was used to estimate the kinetic parameters.

[0151] 4.4 Estimation of kinetic parameters based on NTA capture crosslinking SPR assay In the next step, we developed an alternative assay setup as shown in Figure 7A, as described above. As shown in Figure 7B, the heme-hpx complex dose-dependently bound LRP1.3 and, with a steady-state affinity of approximately 2 μM, summarized in Table 6, produced reproducible K D We were able to calculate it.

[0152] [Table 7]

[0153] As determined, the affinity of heme-hpx complex-bound LRP1.3 was reported to be approximately 2.6 μM. Additional complexes were tested with this assay setup, and all showed similar steady-state affinities of approximately 1.4 μM (Table 7 and Figure 8).

[0154] [Table 8]

[0155] 5. Discussion and Conclusion The ability to express different LRP1 immune receptors (domains) facilitated the identification of the heme-hemopexin binding domain in vitro. In summary, we were able to demonstrate that CD91 / LRP1 domain III is involved in the binding of the heme-hemopexin complex using different methodologies (BLI and SPR) and different capture strategies. Since hemopexin that has not formed a complex (apoHpx) does not bind, binding becomes highly specific when hemopexin forms a complex with a heme molecule. This was again confirmed in both platforms used in this study. It was further found that none of the CD91 / LRP1 domains I, II, and IV bind the heme-hemopexin complex.

[0156] Furthermore, as summarized for LRP1.3 in Table 8, the inventors were able to demonstrate similar binding kinetics independently of the platform and hemopexin batch used.

[0157] [Table 9]

[0158] 6. Preparation of heme-hemopexin complex Preparation of heme-hemopexin (heme-hpx) complex 1. Dilute hemopexin (100 mg / mL) with PBS pH 7.4 to approximately 28-31 mg / mL (approximately 460-509 μM) (final volume depends on batch size). Note: Hemopexin concentrate is already approximately 30 mg / mL in PBS pH 7.4, so dilution is not necessary. 2. While in a laminar flow hood, filter the PBS-diluted hemopexin through a 0.2 μm filter into a sterile bottle or a heat-shocked bottle equipped with a stirring rod. 3. Take out 10 mL and place it in a sterile vial. Set this aside to be used as a control that does not contain hemin. 4. Determine the μM concentration of PBS-diluted hemopexin using a molecular weight of 60,888. 5. Prepare hemin (Frontier Scientific, Cat no: H651-9, mw=651.94) in 100% DMSO (Sigma-Aldrich, Cat no: D2438-10mL, sterile filtered, bioperformance certified, EP, USP test specifications) at a concentration (typically about 15-17 mg / mL) that leads to the same μM concentration determined for hemopexin when diluted 1 / 50. Prepare immediately before use. The final volume depends on the hemopexin batch size. 6. Add the volume of hemin required to dilute the hemin to 1 / 50 in hemopexin. This should yield equimuth hemopexin and heme (e.g., 500 μM + 500 μM) relative to a 100% saturated target. This should still be done under a laminar flow hood. 7. Immediately close the lid and mix by inverting and rotating. 8. Dilute DMSO 1 / 50 in a vial containing 10 mL of hemopexin from step c). Use this as a control and blank to determine the saturation percentage. 9. Incubate both the complex and the control overnight at 37°C in an incubator equipped with a stirring plate, gently stirring to promote the formation of the heme-hpx complex. 10. Take out 1 mL of the sample for saturation analysis. 11. Based on the initial hemopexin concentration of 28–31 mg / mL, prepare two dilutions of the 10% DMSO complex and control in PBS to approximately 0.45 mg / mL and 0.25 mg / mL. This should yield 414 nm absorbance values ​​of approximately 0.83 and 0.45, respectively. 12. Use a control as a blank to determine the saturation percentage at 414 nm. 13. Calculate the μM of the heme-hemopexin complex at each concentration using the extinction coefficient from 123.1. 14. Divide the μM of the complex in the reactant by the μM of hemopexin and multiply by 100 to obtain the saturation percentage. Average the two readings of the final saturation percentage.

[0159] Ultrafiltration and diafiltration of heme-hemopexin complexes 1. Filter the heme-hx complex through a 0.2 μM solution. 2. Ultrafiltration is performed to the target concentration / volume using a BioMax 10kd mwco Millipore Pellicon cassette. 3. Diafiltration is performed with 10x volume of hemopexin FIN buffer (0.9 mM citrate, 14.1 mM S. phosphate, 150 mM NaCl, pH 7.2). 4. Ultrafiltration is performed to restore the final target volume. 5. Remove the retention solution for analysis. 6. Dilute to approximately 0.45 mg / mL and 0.25 mg / mL to determine the μM and mg / mL of the heme-hx complex. This can then be used to back-calculate the hemopexin mg / mL using the saturation percentage. 7. Divide the mixture and label the tubes. 8. Freeze at -65°C or below. [Examples]

[0160] Hemopexin: Assay design for heme complexes and heme reachable to hemopexin 1. Introduction The objective of the following experiment is to determine whether 1-24-FLAG-HuLRP1-domain III (CD91 domain III) can be used as a coating reagent for measuring hemopexin:heme complexes in blood samples. For this purpose, this construct, which specifically binds to hemopexin:heme complexes, is applied as a coating reagent to Maxisorp ELISA plates. After incubation, the LPR1 coated plates are washed and then blocked with 1% casein in PBS to reduce nonspecific binding. After blocking, the plates are washed, and each sample is added to the plates in two formats: 1. Dilute the serum sample 50-fold and perform an assay to measure the hemopexin:heme complex. 2. Hemopexin, which forms complexes with any free heme in the sample, either does not spike (to quantify the endogenous complex concentration) or spikes (to measure heme that can reach hemopexin).

[0161] Sample type 2 may be omitted if only the hemopexin:heme complex is being quantified.

[0162] The samples referred to in this second embodiment are serum samples obtained from either healthy individuals or patients with sickle cell disease.

[0163] After incubation of the sample, the plate is washed again and incubated with the primary detection antibody, anti-hemopexin rabbit polyclonal antibody (Abcam, Ab48135). After washing the plate again, the secondary detection antibody, goat anti-rabbit horseradish peroxidase (HRP) conjugate antibody, is added. After another incubation, tetramethylbenzidine (TMB) is applied to each well to reduce hydrogen peroxidase in HRP, causing a blue color change. The reaction is stopped by the addition of sulfuric acid, causing the reduced hydrogen peroxidase to turn yellow. The intensity of this color change is proportional to the concentration of hemopexin:heme complex present in the sample.

[0164] Using a plate reader, read the plate at 450 nm (620 nm reference). Interpolate the hemopexin:heme complex concentration from each sample from the standard curve. Determine the relative quasi-quantitative concentration of heme reachable to hemopexin by subtracting the hemopexin:heme complex concentration present in the sample with hemopexin spikes (B) from the hemopexin:heme complex concentration present in the sample with hemopexin spikes (A). Divide the resulting delta hemopexin:heme complex concentration (BA) by the molecular weight of hemopexin. Since heme binds to hemopexin in a 1:1 ratio, the concentration obtained after dividing the delta value by the molecular weight of hemopexin is the relative quasi-molar concentration of heme reachable to hemopexin in the sample (C).

[0165] The reachable relative concentration of heme (C) = ([with spikes (B) - without spikes (A)] / 63000) * 1000 (μM)

[0166] 2. Equipment and Software • Calibrated variable-volume single-channel pipette capable of dispensing 1 μL to 1 mL. • Calibrated variable-volume multichannel pipette capable of dispensing 1 μL to 300 μL • A plate reader capable of reading at 450nm (Tecan M200 or equivalent) • Magellan Software V7.2 • Microsoft Excel • Plate shaker (Micromix 5 or equivalent) Vortex Mixer · timer • 96-well plate washer (must be able to dispense 300 μL per well and perform 5 washes)

[0167] 3.Material • 96F Maxisorp NUNC ELISA plate (Thermo Fisher Cat No. 439454)

[0168] 4. Reagents • 1-24-FLAG-HuLRP1 Domain III (CD91 Domain III) • Hemopexin: Heme complex Hemopexin Abcam anti-hemopexin rabbit polyclonal antibody primary detection (1.056 mg / mL) Cat No. Ab48135. • Secondary detection of goat anti-rabbit horseradish peroxidase (HRP) antibody (Seracare Cat No. 5220-0336) • Blocking buffer / assay diluent: 1% casein in PBS (Thermo Fisher Cat No. 37528) • Dulbecco phosphate-buffered saline (DPBS) (Sigma, Cat.#D8537) • Washing buffer: 1 x PBS / Tween • Substrate: TMB A and B (KPL Cat No. 5120-0049 and 5120-0038) · Stop liquid: Sulfuric acid (0.5M) (Thermo fisher Cat No.124240010)

[0169] 5. Operation / Procedure 5.1 Preparation of Reagents • 1-24-FLAG-HuLRP1 Domain III (CD91 Domain III) ○ 5 μg / mL required; diluted with D-PBS. ○ 11 mL is required per plate. • Primary detection antibody: Anti-hemopexin rabbit polyclonal ○ Requires 1 μg / mL; diluted with blocking buffer / assay diluent (1% casein in PBS). ○ 11 mL is required per plate. • Secondary detection antibody: Goat anti-rabbit HRP ○ The stock reagent needs to be diluted to 1 / 2000, and then diluted with blocking buffer / assay diluent (1% casein in PBS). ○That is, 5.5 μL of HRP in 10994.5 μL of blocking buffer / assay diluent. • Substrates: TMB A and B ○ Mix TMB solutions A and B in equal volumes immediately before use. ○ 12 mL is required per plate. ○That is, 6 mL of TMB A + 6 mL of TMB B

[0170] 5.2 Preparation of Standards A standard curve is created from the hemopexin:heme complex. The following pre-dilution (PD) steps are performed in blocking buffer / assay diluent (1% casein in PBS).

[0171] [Table 10]

[0172] Next, as shown in Table 2, a standard curve is created by serially diluting PD1 twice with blocking buffer / assay diluent (1x casein in PBS) on the day of the assay.

[0173] [Table 11]

[0174] 5.3 Preparation of controls The positive control (PC) is the hemopexin:heme complex. The PC is prepared using 100% pooled human serum according to Table 3. The negative control (NC) is the same 100% pooled human serum used to prepare the PC.

[0175] [Table 12]

[0176] • Before plating, apply a 50-fold dilution to the PC and NC. That is, 5 μL of control + 245 μL of blocking buffer / assay buffer.

[0177] 5.4 Preparation of Test Samples · Test samples should be thawed at room temperature before assay. As determined during validation, samples are stable at room temperature (RT) for up to 24 hours. Test samples can be assayed as only hemopexin:hem complex (sample type 1) or both hemopexin:hem complex (1) and hemopexin:accessible heme (sample type 2). · Reading of hemopexin:hem complex (sample type 1): ○ Dilute serum samples 50-fold with blocking buffer / assay diluent. That is, add 5 μL of control to 245 μL of blocking buffer / assay diluent. · Reading of heme accessible to hemopexin (sample type 2): ○ Spike each test sample with 500 μg / mL of hemopexin according to Table 4. Incubate at room temperature for 30 minutes. ○ Samples without spike must be diluted with DPBS according to the final hemopexin spike. ○ That is, add 2.5 μL of DPBS to 47.5 μL of serum. Note: This must be done to adjust the endogenous complex concentration of samples without spike at the same dilution factor as samples with spike. ○ Apply a 50-fold dilution to samples with spike. That is, add 5 μL of control to 245 μL of blocking buffer / assay diluent.

[0178]

Table 13

[0179] 5.5 Assay Procedure 1. Coat a Maxisorp NUNC 96-well plate with 100 μL per well of capture coating solution. Cover the plate with a plate sealer and incubate at RT for 1 hour (±5 minutes) with shaking. 2. Wash the plate five times with at least 200 μL of 1×PBS / Tween wash buffer per well (using a plate washer or manually). 3. Block the plate with 200 μL of blocking buffer / assay diluent (1% casein in PBS) per well. Cover the plate with a plate sealer and incubate at RT for 1 hour (±5 minutes) with shaking. 4. Wash the plate five times with 1x PBS / Tween washing buffer (using a plate washer or manually). 5. Add 100 μL of standard / control / sample to a suitable well. Cover the plate with a plate sealer and incubate with shaking at RT for 1 hour (±5 minutes). 6. Wash the plate five times with 1x PBS / Tween washing buffer (using a plate washer or manually). 7. Add 100 μL of anti-hemopexin rabbit polyclonal antibody primary detection solution to each well. Cover the plate with a plate sealer and incubate with shaking at RT for 1 hour (±5 minutes). 8. Wash the plate five times with 1x PBS / Tween washing buffer (using a plate washer or manually). 9. Add 100 μL of goat anti-rabbit HRP secondary detection per well. Cover the plate with a plate sealer and incubate in RT for 1 hour (±5 minutes) with shaking, protected from light. 10. Wash the plate five times with 1x PBS / Tween washing buffer (using a plate washer or manually). 11. Add 100 μL of substrate per well and incubate with shaking at RT for 3.5 minutes or until the optimal color change occurs. Add 50 μL of stop solution (0.5 M sulfuric acid) per well using the same pipetting motion as when the TMB was added. 12. Add the stop solution and read the plate with a plate reader at 450 nm (620 nm reference) within 10 minutes.

[0180] 6. Hemopexin: Validation data for heme assays and assays of heme reachable to hemopexin. 6.1 System Compatibility 1. Ten separate assays were plated using double replicates and performed using a 10-point standard curve. The standard curve was fitted using Magellan software with a 5-parameter logistic curve fit to generate R values, and the CV and RE were calculated in Excel. 2. As shown below, eight out of ten validation plates produced acceptable %CV and %RE within 25%. Therefore, the assay standard curve was considered to fit the qualified assay.

[0181] [Table 14]

[0182] [Table 15]

[0183] 7. Specificity To evaluate the cross-reactivity between free hemopexin (apohemopexin) and the hemopexin:heme complex, assay specificity was tested. A 7-point standard curve was created using either the hemopexin:heme complex or free hemopexin. As shown in Figure 9, the assay is specific to heme-bound hemopexin and does not bind to free hemopexin.

[0184] 8. Intermediate accuracy / simultaneous repeatability Five positive controls (UHPC, HPC, MPC, LPC, ULPC) and negative controls (NC) derived from hemopexin:heme complexes were evaluated for simultaneous reproducibility (intra-assay precision (three sets of triple replicates in one assay run), inter-assay precision (three independent assay runs by one operator), and inter-operator precision (two operators each performing two assay runs)).

[0185] As shown in Tables 6, 7, and 8, the negative controls had a CV < 25% for all parameters. As shown in Tables 6, 7, and 8, HPC, MPC, LPC, and ULPC showed acceptable precision as the %CV was less than 25%.

[0186]

Table 16

[0187]

Table 17

[0188]

Table 18

[0189] 9. Accuracy Accuracy was evaluated using the same data created for precision (Section 7.3). The accuracy of the controls was calculated after subtracting the NC (background) for each set. As shown in Tables 6, 7, and 8, HPC, MPC, LPC, and ULPC showed acceptable accuracy (RE± ≤ 25% for HPC, MPC, LPC, and RE± ≤ 30% for ULPC). UHPC failed its accuracy criteria (RE± > 30%). This reduces the upper limit of assay quantification to the HPC concentration (20 μg / mL on the plate), as further discussed in the context (Section 7.5).

[0190] 10. Range and Quantification Limit The assay range was determined by evaluating the highest and lowest concentrations that conformed to CV ≤ 25% and RE ± ≤ 30% using pooled healthy serum controls spiked with a known amount of hemopexin:heme spike control, in combination with a standard curve. The effective range of assay quantification is based on the precision and accuracy of the spike control. The upper limit of assay quantification was determined to be 1000 μg / mL (20 μg / mL on plate), and the lower limit to assay quantification was determined to be 50 μg / mL (1 μg / mL on plate). To determine the minimum detection limit of the assay, the hemopexin:heme complex was spiked at 500 μg / mL in pooled human serum at a 50-fold dilution of the sample. After 50-fold dilution, the spiked samples were serially diluted in parallel with similarly diluted unspiked serum samples before assay.

[0191] [Table 19]

[0192] As shown above, an acceptable RE (≤30%) was achieved at a plate concentration of 0.15 μg / mL. Therefore, the quantification range of the assay is determined to be 50–1000 μg / mL (1–20 μg / mL on plate) in 100% human serum, based on the performance of the control. However, based on the range data, an acceptable detection was achieved at a plate concentration of 0.15 μg / mL after background subtraction. Therefore, the assay detection limit for the hemopexin:heme complex is 0.15 μg / mL at the plate concentration after background subtraction. Concentrations of 0.15–1 μg / mL can be reported with a certain degree of uncertainty.

[0193] 11. Selectivity of heme assays that can reach hpx The selectivity of heme reachable by hemopexin was evaluated by first spiking hemopexin in the individual to generate additional complexes with free heme in each sample. The relative concentration of heme reachable by hemopexin was then determined. The relative concentration of heme reachable by hemopexin (μM) (C) = ([with spike (B) - without spike (A)] / 63000) * 1000 It was calculated as follows.

[0194] The results indicated that while individuals have basal levels of hemopexin:heme complex in their serum, free heme is relatively rare in the tested samples.

[0195] [Table 20]

[0196] When hemopexin alone was spiked (B), an increase exceeding the endogenous hemopexin:heme complex concentration (A) was detected in all individuals (delta complex concentration was greater than 0 μg / mL). The results indicated that the endogenous concentration of the hemopexin:heme complex was low in SCD serum and plasma individuals, as well as in hemolyzed individuals (with the exception of one donor). However, the addition of hemopexin resulted in slightly positive delta complex concentrations in all samples, leading to achievable positive heme measurements.

[0197] 12. Conclusion Qualified validation of the determination of hemopexin:heme complexes and heme reachable to hemopexin in human serum and plasma demonstrated that the assay is suitable for use as a quasi-quantitative assay for the complex relative trends of reachable heme in serum samples. The plate-based quantification range for hemopexin:heme complexes was defined as 50–1000 g / mL in 100% human serum (1–20 μg / mL on plate), and the detection limit was reduced to 0.15 μg / mL (plate-based concentration, divided by background) with some uncertainty. This assay is selective for hemopexin complexed with heme, as opposed to free apohemopexin. The assay demonstrated acceptable inter-assay, intra-assay, and operator-to-operator precision and accuracy. The determination of heme reachable to hemopexin follows the same results as the complex quantification aspect of this assay.

[0198] This ELISA-based assay is a qualified assay for 1) quasi-quantification of hemopexin:heme complexes, and 2) determining relative hemopexin-reachable heme from hemopexin-spiked serum samples. This assay can be performed as a single-component assay measuring only hemopexin:heme complex concentration, or as a two-part assay measuring both hemopexin:heme complex concentration and hemopexin-reachable heme.

[0199] 13. Definitions / Abbreviations Conc concentration CV (Coefficient of Variation) DF Dilution Factor Hpx Hemopexin Hpx: Heme Hemopexin: Heme complex μg / mL: micrograms per milliliter OD optical density SCD (Sickle Cell Disease) UHPC ultra-high positive control HPC High Positive Control MPC Intermediate Positive Control LPC has a relatively low positive control ULPC very low-positive control

Claims

1. Use of a CD91 polypeptide for the detection of hemopexin-heme complexes in biological samples, wherein the CD91 polypeptide has a length of 300 to 1000 amino acids and includes or comprises a ligand-binding domain III of CD91 that can bind to hemopexin-heme complexes.

2. The use according to claim 1, wherein the CD91 polypeptide has a length of 400-900 amino acids, 500-900 amino acids, 500-800 amino acids, 600-900 amino acids, or 600-800 amino acids.

3. The use according to claim 1 or 2, wherein the CD91 polypeptide comprises the amino acid sequence according to SEQ ID NO: 2 or the corresponding homologous CD91 amino acid sequence, or comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with the sequence of SEQ ID NO: 2, or having at least 85%, 90%, 95%, or 100% amino acid homology with SEQ ID NO:

2.

4. The use according to any one of claims 1 to 3, wherein the polypeptide further comprises a signal peptide and / or one or more tags.

5. The use according to any one of claims 1 to 4, wherein the CD91 polypeptide is linked to a solid support.

6. The use according to claim 5, wherein the solid support is a well plate.

7. The use according to any one of claims 1 to 6, wherein detection is performed by using an immunoassay.

8. The use according to claim 7, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

9. The use according to any one of claims 1 to 8, wherein the amount of hemopexin-heme complex in a biological sample is determined.

10. The use according to any one of claims 1 to 9 for diagnosing or monitoring hemolysis in a subject, or for monitoring treatment with hemopexin for monitoring treatment of hemolysis or a hemolysis-related condition in a subject.

11. Use of CD91 polypeptide as used in any one of claims 1 to 6 for separating hemopexin-heme complexes in vitro or in vitro from a biological sample.

12. The use according to claim 11, comprising depleting the hemopexin-heme complex from a biological sample.

13. An in vitro method for detecting a hemopexin-heme complex in a biological sample, comprising the steps of: incubating the biological sample with a CD91 polypeptide used in the use described in any one of claims 1 to 6; and detecting the binding between the CD91 polypeptide and the hemopexin-heme complex.

14. The method according to claim 13, wherein detection is performed using an immunoassay.

15. The method according to claim 13 or 14, further comprising determining the amount of hemopexin-heme complex in a biological sample.

16. The method according to any one of claims 13 to 15, for monitoring hemolysis in a subject, or for monitoring the treatment of hemolysis or a hemolysis-related condition.

17. A kit comprising a CD91 polypeptide used in the use described in any one of claims 1 to 6.

18. A solid support to which a CD91 polypeptide used in the use described in any one of claims 1 to 6 is linked.

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