IT202100012761A1 - Immunoadsorber containing HLA peptides as anti-HLA antibody ligands - Google Patents
Immunoadsorber containing HLA peptides as anti-HLA antibody ligands Download PDFInfo
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- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 25
- 239000003446 ligand Substances 0.000 title claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 8
- 239000000427 antigen Substances 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 238000002560 therapeutic procedure Methods 0.000 description 8
- 210000000056 organ Anatomy 0.000 description 7
- 108700028369 Alleles Proteins 0.000 description 4
- 102000004506 Blood Proteins Human genes 0.000 description 4
- 108010017384 Blood Proteins Proteins 0.000 description 4
- 206010052779 Transplant rejections Diseases 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 238000000586 desensitisation Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 3
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- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 101001100327 Homo sapiens RNA-binding protein 45 Proteins 0.000 description 1
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 102100038823 RNA-binding protein 45 Human genes 0.000 description 1
- 108010088160 Staphylococcal Protein A Proteins 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003409 anti-rejection Effects 0.000 description 1
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- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56977—HLA or MHC typing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70539—MHC-molecules, e.g. HLA-molecules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
- G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
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Description
Riassunto in italiano Summary in Italian
L?immunoadsorbimento ? una tecnica medica con cui vengono rimosse particolari proteine del sangue, responsabili di determinate patologie, attraverso il loro legame con peptidi affini (ligandi), adesi ad una matrice che si trova all?interno di un immunoadsorbitore. Le tecniche attuali non impiegano ligandi altamente specifici per la proteina da rimuovere, risultando in una efficacia parziale, ad esempio quando si tratta di anticorpi anti-HLA (rigetti umorali di trapianto d?organo o alcune circostanze pre-trapianto). La presente domanda riguarda l?ideazione di un immunoadsorbitore contenente peptidi HLA che agiscano da ligandi altamente specifici per anticorpi anti-HLA; attraverso la costruzione di un sistema matrice-ligando biocompatibile in grado di contenere combinazioni di peptidi HLA di classe I e II, l?immunoadsorbitore proposto agisce in maniera estremamente selettiva. Poich? l?anticorpo o gli anticorpi anti-HLA da rimuovere sono solitamente ben caratterizzati, l?immunoadsorbitore verr? adattato all?uso, anche in forma modulare, tramite i peptidi specifici per gli anticorpi da rimuovere. The immunoadsorption ? a medical technique with which particular blood proteins, responsible for certain pathologies, are removed through their binding with similar peptides (ligands), adherent to a matrix which is found inside an immunoadsorber. Current techniques do not employ highly specific ligands for the protein to be removed, resulting in partial efficacy, for example when dealing with anti-HLA antibodies (humoral organ transplant rejections or some pre-transplant circumstances). The present application relates to the design of an immunoadsorber containing HLA peptides which act as highly specific ligands for anti-HLA antibodies; through the construction of a biocompatible matrix-ligand system capable of containing combinations of class I and II HLA peptides, the proposed immunoadsorber acts in an extremely selective manner. because the anti-HLA antibody or antibodies to be removed are usually well characterized, the immunoadsorber will be? adapted for use, also in modular form, through the specific peptides for the antibodies to be removed.
Descrizione in italiano Description in Italian
Settore tecnico Technical field
La presente invenzione/modello di utilit? riguarda la creazione di un immunoadsorbitore contenente peptidi facenti parte della famiglia del Complesso Maggiore di Istocompatibilit?, detto anche sistema degli Human Leucocyte Antigen (HLA), di classe I e II <1>; tali peptidi, adesi ad una matrice biocompatibile, hanno il ruolo di ligandi per gli anticorpi anti-HLA specifici per gli stessi peptidi, e pertanto operano la rimozione di tali anticorpi dal plasma dei soggetti trattati con l? immunoadsorbimento grazie al presente immunoadsorbitore. Si tratta di un?invenzione/modello di utilit? nel campo della Medicina, ed in particolare nel settore della Medicina Trasfusionale ed Emaferesi. This invention/utility model? concerns the creation of an immunoadsorber containing peptides belonging to the family of the Major Histocompatibility Complex, also known as the Human Leucocyte Antigen (HLA) system, class I and II <1>; these peptides, adhered to a biocompatible matrix, have the role of ligands for the anti-HLA antibodies specific for the same peptides, and therefore remove these antibodies from the plasma of subjects treated with the? immunoadsorption thanks to the present immunoadsorber. Is it an invention/utility model? in the field of Medicine, and in particular in the field of Transfusion Medicine and Hemapheresis.
Stato dell?arte State of art
L?immunoadsorbimento (IA) ? una tecnica medica di emaferesi terapeutica con la quale ? possibile rimuovere particolari proteine del sangue quando esse sono responsabili di determinate patologie <2,3>. Nel campo della Medicina Trasfusionale ed Emaferesi, l?IA ? impiegata in circostanze particolari, quali le malattie autoimmuni o il rigetto di trapianto d?organo, laddove ? necessaria la rimozione di particolari proteine, nella fattispecie gli anticorpi, che causano la malattia o il rigetto, rispettivamente. Un?altra situazione comune di impiego dell?IA ? la terapia di desensibilizzazione nei pazienti in attesa di ricevere un trapianto d?organo o di cellule staminali emopoietiche, e che possiedono titoli alti di anticorpi anti-HLA contro l?organo o le cellule staminali, che quindi rischiano di essere rigettati <4>. Rispetto alla plasmaferesi, che consiste nella rimozione del plasma tramite emaferesi e della restituzione di plasma o albumina da donatori sani (pi? precisamente ?plasma-exchange?, PEx), l?IA, pi? recente rispetto al PEx, ? un metodo terapeutico pi? selettivo poich? rimuove le proteine ?dannose?, perlopi? tramite una sorta di filtro, l?immunoadsorbitore, contenente un sistema di tipo matrice-ligando, il quale lega le proteine plasmatiche che si vogliono rimuovere <5>, risparmiando perci? le altre proteine ?buone? del sangue, che invece la procedura di PEx rimuove comunque ed in maniera aspecifica. Infatti, mentre l?IA agisce attraverso il suddetto meccanismo, il PEx rimuove il plasma tramite centrifugazione <6>, risultando pertanto aspecifico nella rimozione. Immunoadsorption (IA) ? a medical technique of therapeutic hemapheresis with which ? It is possible to remove particular blood proteins when they are responsible for certain pathologies <2,3>. In the field of Transfusion Medicine and Hemapheresis, the AI? used in particular circumstances, such as autoimmune diseases or organ transplant rejection, where ? necessary to remove particular proteins, in this case antibodies, which cause disease or rejection, respectively. Another common AI deployment situation? desensitization therapy in patients awaiting organ or hematopoietic stem cell transplantation, and who possess high titers of anti-HLA antibodies against the organ or stem cells, which therefore risk being rejected <4>. Compared to plasmapheresis, which consists in the removal of plasma by hemapheresis and the restitution of plasma or albumin from healthy donors (more precisely ?plasma-exchange?, PEx), the AI, more? recent compared to PEx, ? a therapeutic method more? selective because? removes the ?harmful? proteins, mostly? through a sort of filter, the immunoadsorber, containing a matrix-ligand type system, which binds the plasma proteins to be removed <5>, thus saving? the other ?good? proteins blood, which instead the PEx procedure removes in any case and in a non-specific way. In fact, while AI acts through the aforementioned mechanism, PEx removes plasma by centrifugation <6>, thus resulting in non-specific removal.
Problema tecnico da risolvere Technical problem to be solved
Ad oggi, le situazioni in cui il PEx o l?IA hanno dimostrato la loro efficacia sono ben conosciute e vengono aggiornate periodicamente sulla base delle evidenze scientifiche <7 >e comprendono anche le situazioni cliniche in cui ? necessaria la rimozione degli anticorpi anti-HLA come di cui sopra; tuttavia, sebbene il PEx e/o l?IA siano considerati trattamenti standard all?interno dei protocolli terapeutici internazionalmente riconosciuti nelle terapie anti-rigetto <7 >o desensibilizzanti <8>, anche l?IA che ? pi? selettiva del PEx, comunque non impiega ligandi specifici per gli anticorpi anti-HLA, bens? per tutti i tipi di anticorpi: Proteina A stafilococcica, peptide GAM, fenilalanina e triptofano, per citare alcuni dei peptidi pi? comunemente usati, legano tutti gli anticorpi grazie ad una affinit? generica che non distingue le diversit? dei singoli tipi anticorpali <5>. In ogni caso, la rimozione degli anticorpi tramite IA, anche se aspecifica, si configura in un miglioramento dello stato della tecnica emaferetica perch? comunque evita, rispetto al PEx, di rimuovere tutto il plasma e di conseguenza anche le proteine utili quali i fattori della coagulazione in primis; tuttavia, ? ben noto il fatto che la rimozione aspecifica di tutti gli anticorpi pu? risultare in un peggioramento dell?indebolimento delle difese immunitarie in soggetti che gi? sono immunodepressi, essendo stati trapiantati o in attesa di trapianto. Degno di nota ? il fatto che il rigetto umorale del trapianto d?organo o la necessit? di realizzare una terapia desensibilizzante in individui in attesa di trapianto e che possiedono anticorpi anti-HLA sono problematiche rilevanti e rappresentano una fetta sempre pi? consistente delle complicanze che si trovano ad affrontare i medici dei Centri Trapianti. Basti pensare che le persone in attesa di trapianto d?organo solido in Italia sono oltre 8000 nel momento in cui si scrive <9 >e che oltre la met? di questi hanno uno o pi? anticorpi anti-HLA <10,11>; la presenza di tali anticorpi pu? provocare il rigetto dell?organo trapiantato o addirittura impedire la realizzazione stessa del trapianto, limitando notevolmente l?aspettativa di vita del soggetto in attesa di trapianto. Inoltre, nei casi in cui tali anticorpi anti-HLA siano presenti prima di un trapianto di cellule staminali emopoietiche e siano diretti contro il trapianto stesso, il rischio di rigetto (meglio detto ?non attecchimento? ovvero ?graft failure? in inglese) pu? arrivare al 32% <12>, causando purtroppo il decesso in molti di questi casi <12>. Il PEx o l?IA, associati o meno ad altre terapie all?interno di protocolli definiti, permettono di ridurre la quantit? di tali anticorpi anti-HLA ?dannosi? in una buona percentuale di situazioni; tuttavia, tali percentuali non arrivano mai alla totalit? dei casi <8>. Il fatto che l?efficacia non arrivi al 100% neanche con l?IA <13>, che rappresenta pur tuttavia un metodo pi? selettivo del PEx, ? dovuto al fatto che tutti i ligandi attualmente in uso (vedi sopra) non sono specifici per gli anticorpi anti-HLA, bens? lo sono per tutti gli anticorpi: infatti essi si legano ad una parte della proteina anticorpale che ? uguale, o quasi, per tutti gli anticorpi. In tal modo la rimozione non riguarda soltanto l?obiettivo selettivo della terapia che invece ? rappresentato dagli anticorpi anti-HLA specifici. To date, the situations in which PEx or AI have demonstrated their effectiveness are well known and are periodically updated on the basis of scientific evidence <7 >and also include clinical situations in which ? required removal of anti-HLA antibodies as above; however, although PEx and/or AI are considered standard treatments within internationally recognized therapeutic protocols in anti-rejection <7> or desensitizing <8> therapies, AI is also what? more selective of PEx, however it does not employ specific ligands for anti-HLA antibodies, but? for all types of antibodies: Staphylococcal Protein A, GAM peptide, phenylalanine and tryptophan, to name a few of the most? commonly used, bind all antibodies thanks to an affinity? generic that does not distinguish the diversities? of individual antibody types <5>. In any case, the removal of antibodies by AI, even if non-specific, is configured in an improvement of the state of the hemapheresis technique because? however, compared to PEx, it avoids removing all the plasma and consequently also the useful proteins such as coagulation factors in primis; However, ? well known the fact that the non-specific removal of all antibodies can? result in a worsening of the weakening of the immune defenses in subjects who already? are immunosuppressed, having received a transplant or awaiting a transplant. Noteworthy ? the fact that the humoral rejection of the organ transplant or the necessity? to carry out a desensitizing therapy in individuals waiting for a transplant and who possess anti-HLA antibodies are relevant problems and represent an increasingly large share of consisting of the complications that the doctors of the Transplant Centers are faced with. Suffice it to say that there are over 8,000 people waiting for a solid organ transplant in Italy at the time of writing <9 >, and that over half? of these have one or more? anti-HLA antibodies <10,11>; the presence of these antibodies pu? cause the rejection of the transplanted organ or even prevent the transplant itself, significantly limiting the life expectancy of the subject waiting for a transplant. Furthermore, in cases where these anti-HLA antibodies are present before a hematopoietic stem cell transplant and are directed against the transplant itself, the risk of rejection (better called ?non-engraftment? or ?graft failure? in English) can reach 32% <12>, unfortunately causing death in many of these cases <12>. PEx or AI, associated or not with other therapies within defined protocols, make it possible to reduce the amount of of these ?harmful? anti-HLA antibodies in a good percentage of situations; however, these percentages never reach the totality? of cases <8>. The fact that the effectiveness does not reach 100% even with AI <13>, which nonetheless represents a more effective method. PEx selective, ? due to the fact that all the ligands currently in use (see above) are not specific for anti-HLA antibodies, but? they are for all antibodies: in fact they bind to a part of the antibody protein that ? equal, or nearly so, for all antibodies. In this way, the removal does not only concern the selective objective of the therapy which, on the other hand, is represented by specific anti-HLA antibodies.
Descrizione dettagliata dell?invenzione Detailed description of the invention
La presente domanda riguarda l?ideazione di un immunoadsorbitore contenente peptidi HLA che agiscono da ligandi selettivi di anticorpi anti-HLA, risparmiando tutti gli altri anticorpi ed ancor pi? le altre proteine plasmatiche. Attraverso la costruzione di un sistema matrice-ligando biocompatibile contenente diverse combinazioni di peptidi HLA di classe I e II, l?immunoadsorbitore proposto sar? in grado di agire in maniera estremamente selettiva, molto di pi? rispetto ai tipi di IA attualmente in uso che di per s?, come gi? detto, sono pi? selettivi del PEx, nei casi in cui ? necessaria la rimozione di specifici anticorpi anti-HLA. La matrice per questo genere di immunoadsorbitori ? rappresentata da materiali quali sefarosio, cellulosa o altre resine insolubili in acqua: per l?immunoadsorbitore proposto un materiale come il sefarosio sembra il pi? idoneo allo scopo ma non necessariamente l?unico possibile. Le colonne per l?IA invece sono costituite da materiale plastico, come generalmente impiegato nell?ambito della emaferesi. The present application concerns the design of an immunoadsorber containing HLA peptides which act as selective ligands of anti-HLA antibodies, sparing all other antibodies and even more so. other plasma proteins. By constructing a biocompatible matrix-ligand system containing different combinations of HLA class I and class II peptides, the proposed immunoadsorber will be? able to act in an extremely selective way, much more? compared to the types of AI currently in use which in itself?, as already? said, are more selective of PEx, in cases where ? required the removal of specific anti-HLA antibodies. The matrix for this kind of immunoadsorbers? represented by materials such as sepharose, cellulose or other resins insoluble in water: for the proposed immunoadsorber, a material such as sepharose seems the most? suitable for the purpose but not necessarily the only one possible. The columns for the AI, on the other hand, are made of plastic material, as generally used in the context of hemapheresis.
L?invenzione proposta sfrutta l?affinit?, estremamente specifica e selettiva, esistente in natura tra l?antigene ed il suo anticorpo al fine di rimuovere in maniera altrettanto specifica e selettiva l?anticorpo (o gli anticorpi) anti-HLA desiderato/i. Poich? il tipo di anticorpo anti-HLA da rimuovere (o talvolta pi? di uno) ? ben caratterizzato nel soggetto da trattare con IA, l?immunoadsorbitore proposto verr? adattato, utilizzando i peptidi specifici per l?anticorpo (o gli anticorpi) da rimuovere. Il sistema HLA ? polimorfico, vale a dire che molte sono le diversit? dei peptidi e quindi dei relativi antigeni, pertanto molteplici saranno i peptidi presenti nell?immunoadsorbitore; al tal fine ? prevista la creazione di moduli, contenenti ciascuno uno o pi? peptidi in combinazione, che insieme costituiranno l?immunoadsorbitore che sar? per cos? dire ?paziente-specifico?, o quasi. Andando pi? nel dettaglio, la sequenza aminoacidica dei peptidi HLA da legare alla matrice sar? costituito da una parte costante, pertanto uguale in tutti i peptidi appartenenti allo stesso locus (vedi sotto), ed una parte variabile, che si identifica con il polimorfismo proprio al sistema HLA in natura e che riguarda i domini alfa1 ed alfa2 per i loci A, B e C (classe I) ed i domini alfa1 e beta1 per i loci DR, DQ e DP (classe II, vedi sotto). Le sequenze sono di pubblico dominio e recuperabili dalla nomenclatura HLA attuale, disponibile anche sul portale online all?indirizzo: http://hla.alleles.org/alleles/text_index.html. The proposed invention exploits the extremely specific and selective affinity existing in nature between the antigen and its antibody in order to remove the desired anti-HLA antibody (or antibodies) in an equally specific and selective manner . because the type of anti-HLA antibody to be removed (or sometimes more than one)? well characterized in the subject to be treated with AI, the proposed immunoadsorber will be? adapted, using the specific peptides for the antibody (or antibodies) to be removed. The HLA system? polymorphic, that is to say that there are many diversities? of the peptides and therefore of the relative antigens, therefore there will be many peptides present in the immunoadsorber; to that end? expected the creation of modules, each containing one or more? peptides in combination, which together will form the immunoadsorber that will be? for what? say ?patient-specific?, or almost. going more in detail, the amino acid sequence of the HLA peptides to be bound to the matrix will be? consisting of a constant part, therefore equal in all peptides belonging to the same locus (see below), and a variable part, which is identified with the polymorphism proper to the HLA system in nature and which concerns the alpha1 and alpha2 domains for loci A , B and C (class I) and the alpha1 and beta1 domains for the DR, DQ and DP loci (class II, see below). The sequences are in the public domain and can be retrieved from the current HLA nomenclature, also available on the online portal at: http://hla.alleles.org/alleles/text_index.html.
Poich? sono ben noti gli antigeni contro cui ? diretta la maggior parte degli anticorpi anti-HLA nei pazienti in attesa di trapianto, e poich? gli eventi determinanti la produzione di questi anticorpi sono le precedenti gravidanze, trasfusioni e trapianti, anche le frequenze e le specificit? anticorpali sono altrettanto note <14>. Tali antigeni vengono esposti dai peptidi adesi alla matrice in maniera univoca. Di conseguenza, i peptidi HLA da legare alla matrice saranno molteplici e si potr? procedere alla produzione di diversi kit, ciascuno contenente combinazioni dei peptidi pi? frequenti con quelli meno frequenti (vedi sotto, ?Applicazione industriale?). La distinzione tra classe I e classe II riguarda maggiormente la nomenclatura <1 >oltre che la funzione in natura e la conformazione, e descrive la presenza fisiologica delle proteine HLA nei tessuti umani, infatti le proteine di classe I sono espresse da tutte le cellule mentre quelle di classe II soltanto da alcuni sottotipi cellulari, quali le cellule del sistema immunitario. A seconda della collocazione dei geni esprimenti tali proteine, esse vengono definite ai ?loci? A, B e C (appartenenti alla classe I) e DRB1 pi? correlati, DQA1, DQB1, DPA1 e DPB1 (appartenenti alla classe II) <1>. Esistono anche altri loci ed altre classi (per es. la classe III), tuttavia il loro ruolo all?interno del presente contesto ? meno noto e pertanto non sono trattati qui. because are the antigens against which ? direct most of the anti-HLA antibodies in patients awaiting transplant, and why? the events determining the production of these antibodies are the previous pregnancies, transfusions and transplants, also the frequencies and specificities? antibodies are also known <14>. These antigens are exposed by the peptides adhered to the matrix uniquely. Consequently, the HLA peptides to be bound to the matrix will be multiple and it will be possible to proceed to the production of several kits, each containing combinations of the most common peptides? frequent with less frequent ones (see below, ?Industrial application?). The distinction between class I and class II mainly concerns the nomenclature <1 > as well as the function in nature and the conformation, and describes the physiological presence of HLA proteins in human tissues, in fact class I proteins are expressed by all cells while those of class II only from some cell subtypes, such as cells of the immune system. Depending on the location of the genes expressing these proteins, they are defined at the ?loci? A, B and C (belonging to class I) and DRB1 pi? related, DQA1, DQB1, DPA1 and DPB1 (belonging to class II) <1>. There are also other loci and other classes (eg class III), however their role within the present context? less known and therefore are not covered here.
Al fine di descrivere in dettaglio almeno un modo di attuazione dell?invenzione, si forniscono qui di seguito due esempi tipici, il primo riguardante un paziente candidato a trapianto di cellule staminali ematopoietiche da donatore familiare parzialmente compatibile (figlio) ed il secondo riguardante un paziente trapiantato di cuore con rigetto umorale del trapianto; entrambi i pazienti presentano titoli elevati di anticorpi anti-HLA la cui rimozione ? essenziale per una buona riuscita delle cure in atto. Nel primo esempio si cita una paziente, adulta, affetta da leucemia acuta e candidata a trapianto allogenico di cellule staminali ematopoietiche, la quale presenta anticorpi anti-HLA diretti contro peptidi HLA posseduti dal figlio, donatore prescelto per il trapianto. Gli anticorpi, documentati da metodica Luminex? tramite analisi di screening e successiva identificazione, sono diretti contro A32 e B7 ed i rispettivi valori di Mean Fluorescence Intensity (MFI) sono 8640 e 11450; il donatore presenta tali antigeni nel proprio assetto HLA, cos? come documentati dalla sua tipizzazione eseguita a scopo donativo: A*32:01 e B*07:02 compaiono tra gli alleli tipizzati nel donatore. In considerazione del rischio elevato di mancato attecchimento del trapianto a causa della presenza di tali anticorpi, una terapia di desensibilizzazione ? indicata prima dell?infusione delle cellule staminali, allo scopo di rimuovere gli anticorpi: a tal fine si propone l?impiego di un immunoadsorbitore contenente A32 e B7 che sar? posizionato nel circuito per IA. Le sedute di IA dipenderanno dalla discesa degli anticorpi anti-HLA diretti contro il donatore (chiamati anche ?donor-specific antibodies?, o DSA) a livelli permissivi per l?esecuzione del trapianto senza rischi aggiuntivi per l?attecchimento; tali livelli sono generalmente accettati al di sotto di 3000 MFI, sebbene la negativizzazione sia auspicabile. Nel secondo esempio si cita un paziente, adulto, affetto da rigetto cronico (umorale) di trapianto cardiaco eseguito svariati anni prima; gli anticorpi anti-HLA diretti contro il cuore trapiantato sono all?origine del fenomeno di rigetto e di conseguenza la loro rimozione ? necessaria per trattare il paziente con successo ed evitare un peggioramento della funzionalit? cardiaca con conseguente rischio di vita. Gli anticorpi, documentati da metodica Luminex? tramite analisi di screening e successiva identificazione, sono diretti contro A1 e DQ5 ed i rispettivi valori di Mean Fluorescence Intensity (MFI) sono 19500 e 5610; il cuore trapiantato presenta tali antigeni nel proprio assetto HLA, cos? come documentati dalla tipizzazione eseguita al momento del trapianto: A*01:01 e DQB1*05:02 compaiono tra gli alleli tipizzati nel donatore. In considerazione del rischio di peggioramento clinico dovuto al rigetto in corso, una terapia di desensibilizzazione ? indicata, allo scopo di rimuovere gli anticorpi: a tal fine si propone l?impiego di un immunoadsorbitore contenente A1 e DQ5 che sar? posizionato nel circuito per IA. Le sedute di IA dipenderanno dalla discesa degli anticorpi anti-HLA diretti contro il donatore (chiamati anche ?donor-specific antibodies?, o DSA) a livelli permissivi per mettere fuori pericolo il cuore trapiantato; generalmente, una terapia immunosoppressiva potenziata si accompagna al trattamento. In order to describe in detail at least one embodiment of the invention, two typical examples are provided below, the first relating to a patient candidate for hematopoietic stem cell transplantation from a partially compatible family donor (son) and the second relating to a patient heart transplant recipient with humoral graft rejection; both patients have high titers of anti-HLA antibodies whose removal? essential for successful treatment. The first example cites an adult patient affected by acute leukemia and a candidate for allogeneic hematopoietic stem cell transplantation, who has anti-HLA antibodies directed against HLA peptides possessed by her son, the donor chosen for the transplant. The antibodies, documented by Luminex method? through screening analysis and subsequent identification, they are directed against A32 and B7 and the respective values of Mean Fluorescence Intensity (MFI) are 8640 and 11450; the donor has these antigens in its HLA structure, so? as documented by his typing performed for donor purposes: A*32:01 and B*07:02 appear among the alleles typed in the donor. In view of the high risk of failure of the graft to engraft due to the presence of these antibodies, a desensitization therapy ? indicated before the infusion of stem cells, in order to remove the antibodies: for this purpose, the use of an immunoadsorber containing A32 and B7 is proposed, which will be? placed in the circuit for IA. The AI sessions will depend on the descent of anti-HLA antibodies directed against the donor (also called donor-specific antibodies, or DSA) to permissive levels for the execution of the transplant without additional risks for engraftment; such levels are generally accepted below 3000 MFI, although negativization is desirable. The second example cites an adult patient suffering from chronic (humoral) rejection of a heart transplant performed several years earlier; are the anti-HLA antibodies directed against the transplanted heart at the origin of the rejection phenomenon and consequently their removal? necessary to treat the patient successfully and avoid a deterioration of the function? heart attack with consequent risk to life. The antibodies, documented by Luminex method? through screening analysis and subsequent identification, they are directed against A1 and DQ5 and the respective values of Mean Fluorescence Intensity (MFI) are 19500 and 5610; the transplanted heart has these antigens in its HLA structure, so? as documented by typing performed at the time of transplantation: A*01:01 and DQB1*05:02 appear among the alleles typed in the donor. In view of the risk of clinical worsening due to ongoing rejection, desensitization therapy ? indicated, in order to remove the antibodies: to this end, the use of an immunoadsorber containing A1 and DQ5 is proposed, which will be? placed in the circuit for IA. AI sessions will depend on the descent of donor-directed anti-HLA antibodies (also called donor-specific antibodies, or DSA) to levels permissive to out-of-harm the transplanted heart; usually, enhanced immunosuppressive therapy accompanies treatment.
Applicazione industriale Industrial application
La presente invenzione potr? essere sviluppata su base industriale tramite la costruzione di immunoadsorbitori paziente-specifici o stereotipati (pre-definiti), anche modulari. Ogni modulo preveder? la presenza di antigeni HLA purificati di classe I e di classe II, senza limite massimo al numero di antigeni diversi presenti nello stesso modulo. Per massimizzare l?applicabilit? nella pratica clinica, i moduli verranno costruiti mettendo all?interno dello stesso modulo uno o pi? antigeni HLA maggiormente frequenti (e per i quali ? pi? frequente la presenza di anticorpi anti-HLA diretti contro) insieme ad uno o pi? antigeni meno frequenti: in tal modo si otterr? una copertura ampia dello spettro degli anticorpi anti-HLA noti mettendo insieme i moduli in combinazioni appropriate. Al fine di verificare l?efficacia dell?immunoadsorbitore ? indispensabile la sperimentazione clinica della presente invenzione, da eseguire ed eventualmente implementare di concerto tra l?inventore, l?industria e lo sperimentatore (quest?ultimo potrebbe coincidere con l?inventore). L?efficacia andr? testata in vivo e confrontata con lo standard terapeutico allo stato dell?arte. Durante l?utilizzo, l?immunoadsorbitore proposto sar? inserito nel circuito di emaferesi in maniera del tutto sovrapponibile a come viene fatto oggi con l?IA, salvo cambiamenti dello stato dell?arte intervenuti nel frattempo. The present invention could be developed on an industrial basis through the construction of patient-specific or stereotyped (pre-defined), even modular, immunoadsorbers. Each module will provide the presence of purified class I and class II HLA antigens, with no upper limit on the number of different antigens present in the same module. To maximize the? applicability? in clinical practice, the modules will be built by placing one or more? more frequent HLA antigens (and for which the presence of anti-HLA antibodies directed against is more frequent) together with one or more? less frequent antigens: in this way you will obtain? a broad spectrum coverage of known HLA antibodies by putting the modules together in appropriate combinations. In order to verify the effectiveness of the immunoadsorber ? the clinical experimentation of the present invention is essential, to be carried out and possibly implemented jointly between the inventor, the industry and the experimenter (the latter could coincide with the inventor). The efficacy will go tested in vivo and compared with the state of the art standard of care. During use, the proposed immunoadsorber will be inserted in the hemapheresis circuit in a completely superimposable way to how it is done today with the AI, except for changes in the state of the art that have occurred in the meantime.
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