Title of Invention

ANTILYMPHOMA TARGETING AGENTS WITH EFFECTOR AND AFFINITY FUNCTIONS LIKED BY A TRIFUNCTIONAL REAGENT

Abstract

The present invention relate to a medical agent comprising a reagent conjugated to an anti-lyrnphoma anibody is disclosed, as well as a kit containing said medical agent use of said medical agent, and a method for treatment of lymphoma. The reagent may comprise an effector, e.a. an antitumor asent or a diagnostic marker, and an affinity ligand enabling extracorporeal clearance of the agent. The three components are cound by a Afunctional linker.

Full Text

Lymphomas are usually treated by a combination of chemotherapy, radiation, surgery, and/cr bone marrow transplants. The cure rate varies greatly depending en the type of lymphoma and the progression of the disease. Because lymph tissue is found in many parts of the body, non-Hodgkin's lymphoma can start in almost any part of the body. The cancer can spread to almost any organ or tissue in the body, including the liver, bone marrow, spleen, and nose. Based on the histology, non-Hodgkin's lymphomas are divided into two groups: indolent lymphomas, which grow more'slowly and have fewer symptoms, and aggressive lymphomas, which grow more quickly. Lymphomas include follicular small cleaved cell lymphoma, adult diffuse mixed cell•lymphoma, follicular mixed cell lymphoma, adult diffuse large cell lymphoma, Treatment with standard-dose salvage chemotherapy rarely results in durable remissions and often has serious toxi-city. Although the use of high-dose chemotherapy with bone marrow transplantation has shown to be promising, not ail patients derive long-term benefits from chis type of treatment (Armitage JO. Blood 1939;73:1749-53). A curative treatment for patients with advanced low-grade lymphoma still remains to be clearly established (DeVite VT Jr., Jaffe ES, Mauch P, Longo DL. Lymphocytic lymphomas. In: DeVita VT Jr., Hellman S., Rosenberg SA. Eds. Cancer: principles and practice of oncology. 3rd ed. Vol .2. Philadelphia: J.B. Lippincott, 1989,-1741-98) . Treatment with anthracyc line-based chemotherapy regimes results in complete remission in 50-90 percent of patients with intermediate and high-grade non-Hodgkin's lymphoma and long-term disease-free survival in 3 0-60 percent. Unfortunately, few patients with low-grade lymphoma or relapses of any type of lymphoma car. be cured with conventional approaches (Armitage JO. K.Engl.J.Med. 1993; 323:1023-30). High-dose chemoradictherapy with bone The present invention encompasses a medical agent comprising a reagent conjugated to an anti-lymphoma antibody, and various methods for the treatment of lymphatic cancer, i.e. lymphoma, and NHL in particular. Summary of the Invention The object of the present invention is to solve the above discussed problem in connection with treatment of certain lymphoma diseases. This object is achieved by the present invention as specified below. The present invention relates in one aspect tc a medical agent comprising a reagent conjugated to an anti-iymphoma antibody or a variant thereof, wherein the reagent is a single molecule with at least three functional parts b) - d) wherein, a)a trifunctional cross-linking moiety is coupled to b)an affinity ligand via a linker 1, to _c)an effector agent via a covalent bond, optionally via "a linker 2, and to d} reactive moiety, optionally via a linker.3,, wherein said biomolecule reactive moiety is an anti-lymphoma antibody reactive moiety being capable of forming a bond with the anti-lymphoma antibody or a variant thereof, thereby forming a conjugate, and wherein the anti-lymphoma antibody or variants thereof is/are interacting with one or more different CD antigen(s) present on the surface of lymphoma tumour cells. ; In another aspect, the present invention relates to a composition comprising said medical agent-. I i ■ In a further aspect, the present invention "relates to a kit |for extracorporeal elimination or at least reduction of the concentration of a non-tissue-bound therapeutic or diagnostic medical agent as defined above in the plasma or whole blood of a mammalian host, wherein said medical agent previously has been introduce*! into a mammalian host and kept therein for a certain time-in .The study of human leukocyte antigens, predominantly by monoclonal antibody techniques, is a rapidly chancing field of basic research and clinical investigation. Leukocyte surface molecules defined by antibodies have been assigned cluster differentiation [CD) numbers (CD-antigens) in a series of international workshops (Paris, 1982; Boston, 1984; Oxford, 1536; Vienna, 1939, Osaka, 1996) . The CD classification of these antigens has become the standard form in published literature and provides a basis for standardization of clinical report- ■I ing. The current CD classification is presented „in the form of a list, with a brief 'summary of each antigen beside each entry. The expression "the group cf CD1 to CD247" as used herein means all the CD. molecules in the list above. In the most preferred embodiment, the anti-lymphoma antibody is directed against CD19, CD20, CD22, CD 30, ; in carticular CD 20. In the present patent application, an immunotargeting agent (immunoconjugate) is an agent carrying a cytotoxic moiety that, contrary to common cytotoxic medical agents, binds specifically to lymphatic tumor cell with a * high affinity and which could be administered parentally, preferably intravenously, to a human being. In a preferred application, the immunotargeting agents are antibodi-es, which could be of different isotypes and could originate from any species. Of particular interest are the I" monoclonal antibodies and derivatives thereof. The latter include fragments such as the F(ab')2/ F(ab') , F(ab);and the like. They also include genetically engineered chains in order to optimise the hair-life in bcdy fluid and the retention of the antibody or antibody fragments or derivatives, in the tumor tissue. In the most preferred application, the antibodies :r antibody derivatives should allow for the attachment of a sufficient number cf biotin residues to be used for extracorporeal removal through interaction with immobilized avidin, without significantly diminishing the binding properties of the targeting agent. In order to enhance the specificity, tumour specific monoclonal antibodies are used as a carrier (inmunoconjungates) of various cytotoxic moieties, such as, but net limited to, radio nuclides, chemctherapy drugs, synthetic or natural occurring toxins, immunesuppressive agents, immunostimulating agents and enzymes used in pro-drug protocols. The cytotoxic moiety is preferably a radionuclide such as a gamma-emitter e.g. iodine-131 or metal ion conjugate, where the metal is selected from a beta-particle emitter, such as yttrium or rhenium. US Patent No. 4,472,509, Gansow, et al., discloses the use of di-ethylenetriaminepentaacetic acid .(BT?A) chelating agents for the binding of radio metals to monoclonal antibodies. The patent is particularly directed co a purification technique for the removal of ncn-bcnced and adventitious-ly bonded (non-chelated) metal from radiopharmaceuticals but is illustrative of art recognized protocols for preparation of radionuclide labelled antibodies. According to such general procedures, an antibody specifically reactive with the target tissue associated antigen is reacted with a certain quantity of a selected" bifunctional chelating agent having protein binding and metal binding functionalities to produce a chelator/-antibody conjugate. In conjugating the antibodies with the chelators, an excess o.f chelating agent is reacted with the antibodies, the specific ratio being dependent upon the nature of the reagents and the desired number of chelating agents per antibody. It is a requirement that the radionuclides be bound by chelation (for metals) or covalent bonds in such a manner that they do not become separated from the biotinylation/radiolabeling compound under the conditions that the biomolecule conjugates is used (e.g, in patients) . Thus, the most stable chelates or covalent bonding arrangements are preferred. Examples of such binding/bonding moieties are: aryl halides and vinyl halides for radionuclides of halogens; N2S2 and N3S chelates for Tc and Re radionuclides; amino-carboxy derivatives such as EDTA , DTPA, derivatives of Me-DTPA and Cyclohexyl-DTPA, and cyclic amines such as NOTA, DOTA, TETA, CITC-DTPA, and triethylenetetraaminehexa-acetic acid derivatives (Yuangfang and Chuanchu, Pure & Appl. Chem. 63, 427-463, 1991) for In, Y, Pb, Bi, Cu, Sm, blood to an adsorption device and conduits for returning the processed blood or plasma to the patient. In the case plasma is processed through the adsorption device, a plasma separation device is needed as well as means of mixing the concentrated blood with processed plasma. The latter is normally achieved by leading the two components into an air-trap where the mixing occurs. In the case where whole bleed is processed/ an ordinary dialysis machine can constitute the base for such an apparatus. Dialysis machines are normally equipped with all the necessary safeguards ant monitoring devices to meet patient safety requirements as well as tc allow easy handling of the system. Hence, m a preferred embodiment whole blood is processed and a standard dialysis machine is utilised with only minor modifications of the hardware. However, such a machine requires a new program fitted to the newly intended purpose. In addition to the apparatus, special blood line tubings suitable for the intended flew and distance from the patient and the machine are needed. These line tubings could be made of any material compatible with blood or plasma and would include materials used in ordinary tubings used in dialysis. Blood access could be achieved through peripheral vein catheters or, if higher bleed flow is needed, through central vein catheters -such as, but not limited to, subclavian or femoral catheters. For affinity adsorbents, the matrix may be of various shape and chemical composition. It may, for example, constitute a column house filled with particulate polymers, the latter of natural origin or artificially made. The particles may be macrcporous or their surface may be grafted, the 'latter in order to enlarge the surface area-. The particles may be spherical or granulated and be based on polysaccharides, ceramic material, glass, silica, plastic, or any combination of these or alike materials. A combination of these could, for example, be solid particles coated with a suitable polymer of natural origin or artificially made. Artificial membranes may also be used. These may be flat sheet membranes made of cellulose, poly-" amide, polysulfone, polypropylene or other types of material which are sufficiently inert, biocompatible, nontoxic and to which'the receptor could be immobilized either directly or after chemical modification of the membrane surface. Capillary membranes like the hollow fibers made from cellulose, polypropylene or other materials suitable for this type of membranes may also be used. A preferred embodiment is a particulate material based on agarose and suitable for extracorporeal applications. In the schematically shown reagent and medical agent, respectively, the different components will be presented in more detail below. The anti-lymphoma antibody reactive moiety is chosen from a group of active esters consisting of N-hydroxy-succinimide esters, sulfo-N-hydroxysuccinimide esters, and phenolic esters; aryl and alkyl imidates; alkyl or aryl isocyanates or isothiocyanates.reacting with amino groups on "the anti-lymphoma antibody, or maleimides or alpha-haloamides reacting with sulfhydryl groups on the anti-lymphoma antibody; or aryl or alkylhydrazines or alkyl or arylhydroxylamines reacting with aldehyde or The anti-lymphoma antibody having ability to be conjugated to said anti-lymphoma antibody reactive moiety interacts with one or more different cell surface ar.ti-' * gen(s) present on the surface of lymphoma tumour ceils, said one or more cell surface ar.-i^er. (s) being cue :: more different CD antigen H;, cr variancs thereof, wherein the anti-lymphoma antibody preferably is cr.zs^v. from anti-CD20 antibodies, preferably rituximab, ibri-tumomab and tositumomab. The trifunctional cross-linking moiety is chosen from the group consisting of triamincbenzene, tricarboxy-benzene, dicarboxyanyline and diamincbenzoic acid. Linker 1 is a chemical moiety that is an attaching moiety and spacer between the trifunc~icr_al cross-linking moiety and the affinity iigand, preferably a biotin moiety, such that binding with avidin or streptavidin, or any other biotin binding species, is r.c~ diminished by steric hindrance. Linker 1 may also impart increased water solubility and biotinidase stabilization, preferably against cleavage by biotinidase by introduction of * * an alpha carboxylate or an N-methyl group. Further, it contains hydrogen bonding atoms, preferably ethers or tioethers, or ionisable groups, preferably carboxylate, sulfonates, or ammonium groups to aid in water solubili-sation of the biotin moiety. For the structural requirements of the biotin containing moiety, the following applies with reference to the following embodiment of the present invention: dase do not cleave biotin from the conjugates, otherwise the desired outcome will not be achieved. Thus, the use-ful biotin conjugate structures incorporate functional groups (Rx or R2) that block the enzymatic activity of biotinidase. While it is likely that any structure for Rx will block biotinidase, its structure is generally limited to a methyl (CH3) group, as this graup completely blocks biotinidase activity. The N-n-ethyl group decreases the binding affinity cf biotin with avidin and strept-avidin significantly, but it is still useful in.this application. Larger groups for R1 (e.g. ethyl, aryl, etc.) are not useful due to the loss of binding affinity. The alternative to having a substituent Rx is to have a substituent R2 on the atom. (e.g. methylene) adjacent to the biotinamide amine. Much larger and more varied sub-stituents can be used in this position without any significant effect on the binding affinity of biotin. Biotinidase is not completely blocked when R2 = CH3 or CH2CH3, although the rate of cleavage is slowed considerably (i.e. to 25% and 10% respectively). Complete blockage of biotinidase activity is attained when R2 = CH2OK and C02H functionalities. The important consideration is that there is no decrease in binding affinity when these groups are incorporated as R2. Larger functional groups can also.be used as R2 to block biotinidase activity, but results in a decrease in binding affinity. The larger functional groups as R2 are useful in this application if they do not cause a decrease in binding affinity greater than that obtained when Rx = CH3. The biotin affinity and water solubility of the biotin moiety in 1033 are affected by the linker moiety used. The length and nature of the linker moiety (Linker 1) will be dependent to some degree on the 'nature of the molecule that it is conjugated with. The linker moiety serves the function of providing a spacer between the biotin moiety and the rest of the conjugate such that the biotin binding is not affected by steric hindrance from where the chelating group is, but not limited to, any of the following compounds: aryl halides and vinyl halides for radionuclides of halogens; N2S2 and N3S chelates for Tc and Re radionuclides; amino-carboxy derivatives such as EDTA, DTPAf derivatives Me-DT?A and Cyclohexyl-DTPA , and cyclic amines such as NOTA,_DOTA, TETA, CITC-DTPA, and triethylenetetraaminehexaacetic acid derivatives CXuangfang and Chuanchu, Pure & Appl. Chem. 63, 427-4S3, 1991) for. In, Y, Pb, Bi, Cu, Sm, Lu.radionuclides and where the radionuclide is, but not limited, any of the following elements: Beta radiation emitters, which are useful as cytotoxic agents, include isotopes such as scandium-46, scandium-47, scandium-48, copper-67, gallium-72, gallium-73, yttrium-90, ruthenium-97, palladium-100, rhodium-101, palladium-109, samarium-153 , lutetium-177, rhenium-186, rhenium-188, rhenium-189, gold-198, radium-212 and 212 lead. The most useful gamma emitters are iodine-131 and indium-mll4. Other metal ions useful with the invention;include alpha radiation emitt ing materials such as 212-bismuth, 213-bismuth, and At- 211 as well as positron emitters such as gallium-68 and zirconium-8 9 * In the most preferred embodiment of the present invention, the medical agent is the rituximab conjugate with 1-5 groups of 3- (13'-ThioureabenzylDOTA)Trioxadi-amine-1- (13"-Biotin-Asp-OH) Trioxadiamine-5-isothiocya- 1L metal free HEPES with a minimum of 5 buffer changes over 3 days at 4#C. A solution of MitraTag-103 3 was made in water, and an appropriate volume was added to the antibody solution. After incubation overnight at room temperature, the antibody-conjugate was dialysed against 1L metal free 500 mM ammonium acetate buffer pH 5.3 with a minimum of 3 buffer changes over 3 days at 4*C. The demetalated conjugated antibody was scored au 4-8*C until used in radiolabelling experiments. 275 ixl antibody conjugate (1375 pg; 1033-Rituximab) in 500 mM ammonium acetate buffer pH 5.3 was mixed with 15 nl 111InCl3 (or 90YC13) in 50 mM HCl. The labelling -was conducted at 45*C for 16 minutes. 28 ^1 DTPA was-added to stop reaction. The quality of the radio conjugate was determined by TLC and HPLC. The number of MitraTag-103 3^ per monoclonal antibody molecule was determined by the HABA method. Example 2 - Binding of the" 1Q33-conjugated monoclonal antibody to an avidin-adsorbenr. The fraction of the 1033-rituximab radio conjugate binding to the Avidin-adsorbent utilised in the Mitradep device, was analysed utilising micro-columns. The non-bound protein fraction of a 2.4 conjugates/-IgG 1033-rituximab was 9%, and of a 4.6 conjugates/IgG 1033-rituximab 3%. This is well in line with a Poisson distribution of the conjugates. Hence, the above Rituxi-mab conjugates should contain fractions, which are not labelled with MitraTag-1033. Hence, the non-binding fraction complies with the expected fraction of non-conjugated Rituximab i.e. the non-radioactive" fraction. More than 99% of the radioactivity in a radiolabell-ed 1033-conjugate sample was bound to the micro-column with the Avidin-adsorbent. Example 3 - Depletion of 1033-rituximab conjugates during in vitro simulated treatments. The depletion kinetics of 1033-rituximab during a patient treatment was simulated in vitro utilising a Example 8 - In vivo stability cf the radiolabelled MitraTag-1033 antibody conjugates. The stability of the MitraTag:M-lQ23 moiety in vivc was determined by analysing the percentage of radioactivity in blood binding to Aviiin-microcolumns. About 0.1 ml blood was obtained from the periorbital venous plexa on following occasions: I, 3, 24, 48, and 96 hours post injection. 50 /xl bleed was applied to a microcolumn with Avidin-agarose (0.3 ml adsorbent) . After incubation for 10 minutes, the unbound radioactivity was . washed off the column- The radioactivity in the column and the collected washing fluid was measured in an automatic Nal(Tl) scintillation well counter and the bound fraction was expressed in percsr.i of the total radioactivity applied to the column. c) an effector again; covalently linked to the trifunctional cress-linking moiety, optionally via a linker 2, wherein the linker 2 provides a spacer length of 1-25 atoms and the linker contains hydrogen bonding atoms, preferably ethers or thioethers, or ioniaable groups :o aid in water solubility, and to d) a linker 3, which, covalently links the anti-CD20 antibody to the reagent, wherein the linker 3 provides a spacer length of 1-25 atoms and contains hydrogen bending atoms, preferably ethers or thioethers, or ionisable groups to.aid in water solubility, wherein the anti-CD2C antibody is selected from a grout of antibodies or variants thereof having a specific binding to CD2 0 antigens and having an affinity binding constant of at least 5x10s M-1. 2 . The medical agent according to claim 1, wherein the anti-CD20 antibody is conjugated with from 3 to 4 reagents. 3 . The medical agent according to any one of the preceding claims, wherein the affinity binding constant is at least 10a M-1. 4. The medical agent according to any one of the preceding claims, wherein the ar.ti -C32 0 antibody is ibritumomab, rituximab, or tositumomab. 5- The medical aaent according to claim 4, wherein the anti-CD20 antibody is rituximab. 6- The medical agent according to any one of the preceding claims, wherein the linkers 2 and 3 provide a spacer length of 5-18 atoms. 7. The medical agent according to any one of the preceding claims, wherein the anti-GD2C antibody variant has the same or essentially the same ability as the anfci-CD20 antibody to bind to both the anti-CD20 antibody reacting moiety and said CD antigen/antigens on the surface of a lymphoma tumour ceils, and wherein said variant is an antibody derivative, preferably the F (ah")2/ P (ab-) or ? (ab) fragment, genetically engineered hybrids or chemically synthesized peptides, preferably chimeric or humanized antibodies, and single chain antibodies, 8- The medical agent according to any OTIQ of the preceding claims, wherein the effector agent is a radionuclide bidning moiety, optionally provided with a radionuclide, a synthetic or naturally occurring toxin, an enzyme capable of converting pro-drugs, immunosuppres- sive or immunostim.ulating agents, radicsensitizers, enhancers for X-ray of MRI or ultrasound, non-radioactive elements, which can be converted to radioactive elements by means of external irradiation after the anti-CD20 antibody carrying said element has been accumulated to specific cells or tissues, or photoactive compounds or compounds used in photo-imaging or phctodynamic therapy, or any other molecule having the same or similar effect, directly or.indirectly, on lymphoma cells or lymphoma tissues. 9. The medical agent according tc claim 3, wherein the effector agent is provided. with positron-imaging radionuclides, preferably F-13, Sr-75, 3r-76 and 1-124; therapeutic radionuclides, preferably Y-90, 1-131, In- 114m, Ke-186, Re-138, Cu-67, Sm-157, Lu-177, 8i-212, si- 213, At-211, Ra-223, gamma-imaging radionuclides, prefer ably Tc99m, In-III, 1-123 and 1-125, beta-radiaticn emitters, preferably scandium-45 , scandium.-47 , scandium- 4 8, copper-67, gallium-72, gallcum-73, yttrium-3 0, ruthenium-97, palladium-10 0, rhodium-101, palladium-109, samarium-153, lutacium-177, rher.ium-13 6, rhenium-13a , rhenium-189, gold-19 3, radium-212, and lead-212, gamma emitters, preferably iodine-131 and indium-mll4 and positron emitters, preferably gallium-SS and zirconium-' 89. 10. The medical agent according to claim 9, wherein the effector agent comprises aryl halide3 and vinyl halides for radionuclides of halogens, N2S2 and U25 chelates for Tc and Re radionuclides, amino-carbo>cy derivatives, preferably EDTA and DTPA or derivatives thereof, and cyclic amines, preferably NOTA, DOTA and TETA, and derivatives thereof, for In, Y, Pb, Bi, Cu, 5m and Lu radionuclides, or any other radionuclide capable of forming a complex with said chelates. 11. The medical agent according to claim 10, '-/herein the effector agent comprises 2CTA and is provided with Y-90 or Lu-177 for therapeutic application or In-111 for diagnostic purposes. 12. The medical agent according to any one of the preceding claims, wherein the hiotin derivative is chosen from the group consisting of norbiotin, homobiotin. oxy-bio tin, imincbiotm, destibiccin, diamir.cbictm, biotin sulfoxide, and biotin sulfoner or derivatives, preferably norbiotin or bomcbiotin. * . 13. The medical agent according to any one of the preceding claims, wherein the olotinamide amine suh-stituenta are -CH-OH or -CC^H and the substituents ■adjacent to the biotin amine are -CH3 or -CH2Ch. 14. The medical agent according to any one of the preceding claims, wherein the anti-CD2 0 antibody has been covalently bound to the reagent, optionally via the linker 3, through a reaction of a group of active esters consisting of N-hydroxysuccinimide esters, sulfo-N- hydroxyauccinimide esters, and phenolic esters; aryl and alkyl imidates; alkyl or aryl isocyanatss or isothio- cyanates, with amino groups on the anti-CD20 antibody; or a reaction of maleimides or alphahaloamides with sulfhydryl groups on the anti-CD2 0 antibody? or a reaction of aryl cr alkylhydrazmes or alkyl cr aryl- ■1 hydroxylamines with -aldehyde or ketone groups naturally occurring or synthetically produced on the anti-CD2 0 antibody. 15 - The medical agent according to any one of the preceding claims, wherein the linker 2 is excluded, 16. The medical acant accordino to claims 1-15,- wherein Che anti-CD2Q antibody preferably is rituximab, wherein n is 2-4, preferably 3, c is 1-6, preferably 3, p is 1-6, preferably 3; R2 is -CH2OK cr -C02H; and RX is -CHa, -CH2OH or -K. 17. The medical agent according to claim IS, wherein it is 3-(13'-thioureabenzyl-(DOTA; trioxadiamine-1-(13' - biotin-Asp-OH) triexamine-5-isothic-cyanato- aminoisophtalate-ibritomumab, 3- (13'-thioureabenzyl- (DOTA) trioxadiamine-1- (13 ' ' -biotin-Aap-OH) trioxamine-5- isothio-cyanato-aminoisophtalate-rituximab, or 1- Isocyanato-3- ( ('IS' - (N-Biotinyl) -/3-L-Aspartyl) -4' ,7' ,10' - Trioxa-penta-Decanylamino) -1- ( (13- (Benzylthiourea-CHX- A' ') -4,7,10-Trioxatridecanediamine) -Arrdnosiophtalate-rituximab, preferably 3-(13'-thioureabenzyl-(DOTA) trioxadiamin.e-l--(13 ' ' -biotin-Asp-OH) trioxamine-5-isothio-cyanato-aminoisophtalate-rituximab. 18. The medical agent according to any one of the preceding claims, wherein it further comprises physio logically acceptable additives, preferably an ammonium 19. A medical agent: according tc any cr.s of the preceding claims, with the proviso that said reagent/- rsagents is/are ccvalently hound to the ant: -CD 2 0 antibody without the linker 3. 20. A kit for extracorporeal elimination or at least reduction of the concentration of a non-tissue hound therapeutic or diagnostic medical agent as defined in any one of claims 1-19 in the plasma or whole bleed of a mammalian host, wherein said medical agent previously has besn introduced into a mammalian host and kept therein for a certain time in order to be concentrated to the specific tissue or cells by being attached thereto, said kit comprising a) the medical agent, and b) an extracorporeal device comprising an immobilised receptor to which a bictin molecule adheres. 21. Use of a medical acent accordina to anv one of claims 1-19 or the kit according to claim 2G for the treatment of lymphoma/ preferably non-Hcdgkin's lymphoma.

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