Antibody conjugates
Abstract
Methods for covalently attaching soluble or insoluble conjugate partners (compounds, linkers or supports) to antibody molecules to form antibody conjugates useful in affinity purification, separation, diagnostic and therapeutic applications. Conjugate partners are attached to (a) carbohydrate moieties of antibody molecules, (b) sulfhydryl groups of antibody molecules or (c) amino or carboxy groups of the Fc region of antibody molecules to provide antibody conjugates which substantially retain the immunospecificity and immunoreactivity of the antibodies from which they are made. A method is described for the covalent attachment of linker groups via amide or ester bonds to compounds which contain available amino or hydroxy groups. The linkers may be designed so that they are susceptible to cleavage by any one of the serum complement enzymes.

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Expired 7 March 2003, 23.6 years ago.
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54 claims: 14 independent, 40 dependent
- 11) UCa μέθοδος παρασκευής ένός συζευκτικοϋ Αντισώματος περιλαμβάνουσαί α) ϊκθεσιν ένός Αντισώματος ή τμήματος αντισώματος κατευθυνομέναυ έναντι μιας Αντιγονικής θέσεως πρός ενα δξειδωτικό παράγοντα διά νά δημιουργηθοϋν δμάδες Αλδεϋδης είς τό καρβοϋδρικό τμήμα του Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα Αξειδωμένο αντίσωμα καί (β) Αντίδρασιν των Αλδεϋδο όμάδων τοϋ οξειδωμένου Αντισώματος μέ μία ύδράζινο,ύδράζινο ή Αμινο Αμάδα ,μιαςένώσεως διά νά σχηματισθή ένα συζευκτικό Αντισώματος έχων κυρίως τήν άνοσοαντιδραστε κδτητα καί Ανοσοειδικότητα τοϋ Αντισώματος ή τμήματος αντισώματος
- 22) Κια μέθοδος βάσει τής Αξιώσεως (I) δπου δ Αναφερθείς δξειδωτικί παράγων είναι γαλακτοσιδάση ή ύπεριωδικό.
- 33) Μία μέθοδο βάσει τής Αξιώσεως I δπου τό Αναφερθέν τμήμα Αντιβ οώματος είναι τμήμα FAB ένός Ι^Μ Αντισώματος ή ήμίσεως μορίου Αντισώματος.
- 44) Την μέθοδο βάσει τής Αξιώσεως Ι,δπου τό Αντίσωμα είναι μονοκλονικό Αντίσωμα.
- 55) Τήν μέθοδο βάσει τής Αξιώσεως Ι,δπου ή ένωσις είναι διαλυτό συνδετικό.
- 66) Τήν μέθοδο βάοει τής αξιώσεως 5 δπου τό συζευκτικό είναι προ® οκολλημένο πρός μία δεύτερη ένωσι.
- 77) Τήν μέθοδο βάσει τής Αξιώσεως 6 δπου ή δεύτερη ένωσις είναι Αδιάλυτο ύπόστρωμα. δ) Τήν μέθοδο βάσει τής Αξιώσεως I δπου ή ένωσις είναι αδιάλυτο ύϊόοτρωμα.
- 89) Τήν μέθοδο βάοει τής Αξιώσεως I δπου τό συζευκτικό αΛίτισώματοσ σταθεροποιείται διά έκθέσεως είς μία^ποτελεσματικήέύός άναγωγικοϋ παράγοντος. 1C) υ Ενα συζευκτικό Αντισώματος ,περιλαμβάνον, μία 953Λ\\πρρσκολ! · ν ' λημένη μέσω συσθενοΰς δεσμού πρός ένα καρβοϋδρικότμημα- κυρίως ,> διατηρεί τήν άνοσοαντιδραστικότητα καί άνοσοειδικότητα τοΐ άντισώματος.
- 911) “Ενα συζευκτικδ άντισώματος βάσει τής άξιώσεως 10,δπου δ συσθενής δεσμός είναι ύδραζόνη ,ίμίνη ή έναμίνη.
- 1012) Τό συζευκτικδ άντισώματος βάσει τής άξιώσεως ΙΟ,δπουή ένωσις εΐναι διαλυτό συνδετικό. Ι5)Τ0 συζευκτικδ άντισώματος βάσει τής άξιώσεως 12 δπου τό διαλυτό συνδετικό προσκολλδται είς μία δευτέρβ/ένωσι.
- 1114) Τό συζευκτικδ άντισώματος βάσει τής άξιώσεως 13 δπου ή δευτέρα ένωσις εΐναι Αδιάλυτο ύπόστρωμα.
- 1215) Τό συζευκτικδ άντισώματος βάσει τής άξιώσεως 10,δπου ή ένωσις εΐναι αδιάλυτο ύπόστρωμα.
- 1316) Τήν μέθοδο δοκιμασίας διά αντιγόνο περιλαμβάνουσα:άβάμειξιν ένός συζευκτικοΐ άντισώματος τής άξιώσεως ΙΟ μέ ενα δείγμα περί«· έχον άντιγόνο,καί άνίχνευσιν τής άλληλεπιδράσεως τοΐ άναφερθέντος συζευκτικοΐ άντισώματος καί αντιγόνου.
- 1417) Τήν μέθοδο βάσει τής άξιώσεως 16, δπου ή ένωσις τοΐ συζευκτικου άντισώματος εΐναι ένα ένζυμο ,μία ραδιενεργός ένωσις ή μία φθορίζουσα ,χημειοφωτοβολοΐσα ή δυναμικώς φθορίζουοα ή χημειοφωτοβολοΐσα ένωσις.
- 1518) Ιδία μέθοδο διαχωρισμοί μιας ένώσεως άπό ένα δείγμα,περιλαμβάνουσα:έπαφή ένός δείγματος περιέχοντος μίαένωοι μέ ένα συζευκτικόάντισώματος τής άξιώσεως 10, τό όποιο συζευκτικδ άντισώματος έχει συγγένεια πρός τήν άναφερθεΐσα ενωσιν διά νά σχηματισθή ένα σύμπλοκο ένώσεως -συζευκτικοΐ άντισώματος,καί διαχωρισμό τοΐ συμπλόκου ένώσεως-συζευκτικοΐ άντισώματος άπό τό δείγμα.
- 1619) Τήν μέθοδο βάσει τής άξιώσεως 18 ,περαιτέρω περιλαμβάνουσα άποσύνδεσιν του συμπλόκου ένώσεως-συζευκτικοΐ αντισώματος διά νά ληφθή καθαρή ένωσις.
- 1720) Ι-ία μέθοδο διαχωρισμού Ί^νός κϊΧτάρου άπό νουσα:έπαφήν ένός δείγματος περιέχοντος ένα κτικό άντισώματος τής άξιώσεως 10,τό όποιο συζευκτικ0 Χ άντισώματοσ ένα δεΐΗ&/' περιλάμ^ κύτταροσυζευ61 εχει συγγένεια πρός τό άναφερθέν κύτταρο ,διά νά σχηματισθή ένα σύμπλοκο κύττάρου-συζευκτικοΰ Αντισώματος καί διαχωρισμό τοΰ συμπλόκου κυττάρου-συζευκτικοΰ Αντισώματος Από τό δείγμα.
- 1821) Τήν μέθοδο βάσει τής Αξιώσεως 20, περιλαμβάνουσα περαιτέρω Αποσύνδεσιν τοΰ συμπλόκου κυττάρου-Αντισώματος διά νά ληφθή ένα Καθαρό κύτταρο. 32)Μία μέθοδος παρασκευής ένύς τροποποιημένου Αντισώματος περίλαμβάνουσαί (α) έκθεσιν ένός Αντισώματος ή τμήματος Αντισώματος έναντι μιας άντιγονικής θέσεως πρός ένα όξειδωτικό παράγοντα διά νά δημιουργηθοΰν δμάδες Αλδεϋδης είς τό καρβοϋδρικό τμήμα ένός Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα Αξειδωμένο Αντίσωμα, καί (β)αντίδρασιν τών Αλδέϋδο Αμάδων τοΰ Αξειδωμένου Αντισώματος μέ ένα παράγωγο ύδραζίνης ή ύδραζίνης ένός πεπτιδίου συνδέσμου , ένός συνδέσμου άμινοΑξέος ή ένός συνδέσμου τοΰ γενικοϋ τύπου w “ ( C H 2) n -Q δπου W είναι είται -φ ή -CH 2 ~* Q είναι Αμινοξύ πεπτίδιο, χηλικό ή παράγωγο χηλικοΰ ,καί η είναι Ακέραιος Από Ο-2Ο, διά νά σχηματισθή ήνα τροποποιημένο Αντίσωμα τό οποίο διατηρεί κυρίως τήν Ανοσοαντίδραστνκόιήτα καί Ανοσοειδικότητα.
- 1923)ωία μέθοδο διά τήν σύνδεαν μι“ς όμάδος πρός ένα αντίσωμα διά νά παρασκευασθή ένα συνδετικό Αντισώματος περιλαμβάνουσα! (α) έκθεσιν ένός Αντισώματος ή τμήματος Αντισώματος κατευθυνομένου έναντι μιας Αντιγονικής θέσεως πρόςένα όξειδωτικό παράγοντα διά νά δημιουργηθοΰν Αλδέϋδρ Αμάδες είς τό καρροϋδρικό τμήμα τοΰ Αντισώματοσ ήτμήυατος αντισώματος διά νά σνηυα'ίσθή έν?τ Ί οξειδωμένο Αντίσωμα, καί —' ’/f \ . (β) Αντίδρασιν τών Αμάδων Αλδεϋδής του\ΑξειδωμένουτΑντισώματος μέ ενα ύδράζινο ή ύδράζινο παράγωγο ένόΑ πέπτιδο συνδέσμου ,ένόσ συνδέσμου δμινο δξέος ή ένός συνδ'εσμου τοϋ γενικοϋ τόπου w -(CH 2 ) n -Q δπου W εΐναι είτε -^\-NH-CH 2 - ή -CHg-, Q εΐναι άμινοξύ,πεπτίδιο χηλικό ή παράγωγο χηλικοϋ ,καί η εΐναι άκέραιος άπό 0-80. , ό όποιος σύνδεσμος προσκολλαται συσθενως πρός μία θεραπευτική ή διαγνωστική ένωσι ή μία άδιάλυτο μήτρα δίά νά σχηματισθή ένα συνδετικό άντισώματος τό όποιο διατηρεΤ κυρίως τήν άνοσοαντιδραστικότητα καί άνοσοειδικότητα .
- 2024) Μία μέθοδο συνδέσεως μιας ένώσεως πρός ενα άντίσωμα διά νά παρασκευασθή ένα συζευκτικό άντισώματος , περιλαμβάνονί (α) έκθεσιν ένός άντισώματος ή τμήματος αντισώματος κατευθυνοV μένου έναντι μιας άντιγονικής θέοεως πρός ένα όξειδωτικό παράγοντα γιά νά δημιουργηθουν άλδέϋδο όμάδες είς τό καρβοϋδρικό τμήμα διά νά σχηματισθή όξειδωμένο άντίσωμα καί (β) άντίδρασιν τών δμάδων άλδεϋδης του δξειδωμένου αντισώματος μέ ένα ύδράζινο ή ύδράζινο παράγωγο ένός πέπτιδο συνδέσμου ένδς άμινοξέος ή ένός συνδέσμου του γενικοϋ τύπου W-(C H 2) n -Q δπου W εΐναι είτε φ—NH-CH 2 - ή -CH 2 ~, Q είναι αμινο δξύ, πεπτίδιο χηλικό ή παράγωγο χηλικοϋ ,καί η εΐναι άκέραιος άπό 0-20, διά νά σχηματισθή ενα τροποποιημένο άντίσωμα καί (ϊ) σχηματισμό μιας συσθενους προοκόλλήσεως ή παραγώγου χηλικού μεταξύ του μή συνδεδεμένου τελικοϋ τοϋ συνδέσμου καί μιάς θεραπευτικής ή διαγνωρτικής ένώσεως ,ή μιας άδιαλότου μήτρας, δκ νά σχηματισθή ένα συζευκτικό άντισώματος τό όποιο διατηρεί κυρίως τήν άνοσοαντιδραστικότητα καί άνοσοειδικδτητκτ, ' ν*·, ' ·'
- 2125) Μία μέθοδος βάδει τής άξιώσεως 22,23 ή 24 δποχΓδ αν’αφερθείς παράγων όξειδώσεως εΐναιχναλακτοξειδάση ή ύπ'^ριωδικό.
- 2226) Μία μέθοδο βάδει τής άξ ιώσς^)ς2\23 ή 25 δπου- τό αναφερθέν τμήμα άντισώματος εΐναι τό τμήμα FAB έ^ός 1^ Μ άντισώματος ή t>3 ήμισυ μορίου Αντισώματος.
- 2327) Μία μέθοδο παρασκευ~ης Αντισώματος τροποποιημένου περιλαμβάνουσαί (α) σύνδεσιν ένός Αντισώματος ή τμήματος Αντισώματος κατευθυνομένου έναντι μιας Αντιγονικής θέσεως πρός ένα δεύτερο Αντίσωμα Αντίσωμα κατευθυνόμενο έναντι τοΰ ϊΆΒ τμήματος τοΰ Αντισώματος ή τμήματος τοΰ Αντισώματος διά νά σχηματισθή ένα ανοσο συμπλόκου . (β) προσκόλλησιν συσθενώς ένός πέπτιδο συνδέσμου ένός συνδέσμου αμινο δξέος ή ένός συνδέσμου τοΰ γενικοΰ τύπου W-(CH 2 ) n -Q δπου%' εϊναι εϊτε -HH-CH - η -CH O -, Q εϊναι δμινο δξύ, κεπτίδιο, 2 ά χηλικό ή παράγωγο χηλικοΰ,καί η εϊναι Ακέραιος Από 0-20 πρδς τό μή συνδεδεμένο τμήμα τοΰ Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα τροποποιηβένΑντίσωμα. (γ) Αποσύνδεσιν τοΰ ανοσο συμπλόκου γιά νά Απελευθερωθή τδ τροποπο ιηβένάντ ίσωμα Από τό δεύτερο αντίσωμα, καί (δ) διαχωρισμό τοΰ τροποποιη μένου Αντισώματος από τδ δεύτερο Αντίσωμα τό όποιο διατηρεί τήν κύρια Ανοσο Αντιδραστικδτητα καί Ανοσοειδικότητα .
- 2428) Μία μέθοδο συνδέσεως μιας ένώσεως πρός ένα Αντίσωμα διά νά παρασκευασθή ένα συζευκτικό Αντισώματος,περιλαμβάνουσαί (α) σύνδεσιν τοΰ Αντισώματος ή τμήματος Αντισώματος κατευθυνομένου έναντι μιάς Αντιγονικής θέσεως πρός ένα δεύτερο Αντίσωμα κατευθυνομένου έναντι τοΰ F ft B τμήματος τοΰ Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα ανοσο σύμπλοκο. (β) προσκόλλησιν συσθενώς ένός πέπτιδο συνδέσμου ένός δμινο συνδέσμου ή ένός συνδέσμου τοΰ γενικοΰ τύπου Vi-(CH 2 )- Q . δπου W είναι εϊτε -1ΐ?-0ή 2 - ή -0 2 ~ >Q εϊναι Αμίνοξύ ,πεπτίδιο, >* ή παράγωγο χηλικοΰ,καί η εϊναι Ακέραιος Από 0-20, r δ όποιος σύνδεσμος προσκολλαταιΛσϋδ’θςνώς πρός μία θεραπευτική ή διαγνωστική ένωσι ή μία Αδιάλυτο μήτρα πρός τό μή συνδεδεμένο τμήμα τοϋ Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα συζευκτικά Αντισώματος , (γ) Αποσΰνδεσιν τοϋ ανοσο συμπλόκου γιά νά Απελευθερωτή τό συζευκτικό Αντισώματος Από τό δεύτερο Αντίσωμα ,καί (δ) διαχωρισμό τοϋ συζευκτικοϋ Αντισώματος Από τ ό δεύτερο Αντίσωμα, τό δποΤο συζευκτικά Αντισώματος διατηρεί κυρίως Ανοσοαντιδραστικότητα καί Ανοσοειδικότητα.
- 2529)ωία μέθοδο συνδέσεως μιας ενώσεως πρός ένα Αντίσωμα διά νά παρασκευασθή ένα συζευκτικό Αντισώματος περιλαμβάνουσαί (α) σύνδεσιν ένής Αντισώματος ή τμήματος Αντισώματος κατευθυνομένου έναντι Αντιγονικής θέσεως πρδς ένα δεύτερο Αντίσωμα κατευθυνόμενον έναντι τοϋ ΚΑΒ τμήματος τοϋ Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα .ανοσο σύμπλοκο (β) προσκόλλησιν συσθενώς ένός πεπτιδο συνδέσμου, ένός αμινο δξέος ’συνδέσμουή ένδς συνδέσμου τοϋ γενικοϋ τύπου W-(CH 2 ) n -Q δπου W εΐναι εΐτε -NH-CE^- η -CH 2 -,Q εΐναι δμινοδξύ ,πεπτίδιο χηλικό ή παράγωγο χηλικού ,καί η εΐναι Ακέραιος άπό 0y20. πρός τό μή συνδεδεμένο τμήμα τοϋ Αντισώματος ή τμήματος Αντισώματος διά νά σχηματισθή ένα τροποποιημένο Αντίσωμα. (ϊ) σχηματισμό ένός συσθενοϋς Αεομοϋ μεταξύ ένός μή συνδεδεμένου τελικού τού συνδέσμου καί μιας θεραπευτικής ή διαγνωστικής ένώσεις ή μιας Αδιαλύτου μήτρας γιά νά σχηματισθή ένα συζευκτικό Αντισώματος, (δ) άποούνδεσις τού ανοσο συμπλόκου γιά νά Απελευθερωθή τό συζευκτικό Αντισώματος Από τό δεύτερο Αντίσωμα,καί (ε) διαχωρισμό τού συζευκτικοϋ Αντισώματος Από σώμα ,τό όποιο συζευκτικό Αντισώματος διατηρεί αντίδραστικότητα καί τήν Ανοσοειόικδτητα. 50) Μία μέθοδο βάσει τής ας, ι'οείΰΛ 27.25 ή 29 δ:τμήμα Αντισώματος εΐναι τό FAB τμήμα ένός ήμισυ μορίου Αντισώματος. τό δεύτερο Αντίτήν κύρια Ανοσο-
- 2631) HiCa μέθοδο βάσει τής άξιώσεως 27,28 ή 29δπου τό δεύτερο Αντίσωμα είναι προσκολλημένο είς ενα Αδιάλυτο ύλικό.
- 2732) Μία μέθοδο βάσει τής Αξιώσεως 87,28 ή 29 δπου ό σύνδεσμος προσκολλαται είς τό Αντίσωμα μέσω τής καρβοδιϊμιδο Αντιδράσεως.
- 2833) Μία μέθοδο παρασκευής ένός τροποποιημένου Αντισώματος περιλαμβάνουσα! (α) εκθεσιν ένός Αντισώματος ή τού (F<£') 2 τμήματος ένός Αντισώματος είς ήπιο Αναγωγικό παράγοντα διά νά παραχθή ένα Αντίσωμα ή ένα Fap' τμήμα εχον σουλφΰδρυλ δμάδες ,τό όποιο Αντίσωμα ή Fa β'τμήμα κατευθύνεται έναντι μιας Αντιγονικής θέσεως ,καί (β) Αντίδρασιν τών Αναφερθειοών σουλφΰδρυλ όμάδων μέ ένα ίωδοάλκιλ παράγωγο ή πέπτιδο σύνδεσμο ,ένα σύνδεσμο Αμινοξέος ή ένα σύνδεσμο τού γενικού τύπου W-(CH 2 ) -Q δπου W είναι είτε -NH-CH^- ή -CH 2 -, Q είναι Αμινοξύ ,πεπτίδιο, χηλικό ή χηλικό παράγωγο ,καί η είναι ακέραιος Από 0-20. διά νά σχηματισθή συσθενής σύνδεσις μεταξύ τού Αναφερθέντος συνδέσμου και του Αναφερθέντος άναχθέντος Αντισώματος ,ή Εαβ'τμήματος φιά νά σχηματισθή ένα τροποποιημένο Αντίσωμα τό όποΤον διατηρεί κυρίως τήν Ανοσοαντιδραστικότητα καί Ανοσοειδικότητα.
- 2934) Μία μέθοδο συνδέσεως μιας ένώσεως πρός ένα Αντίσωμα διά νά παρασκευασθή ένα συζευκτικό αντισώματος περιλαμβάνουσα! (α) εκθεσιν ένός Αντισώματος ή τού (Fap') 2 τμήματος τοϋ Αντισώματος είς ήπιο Αναγωγικό παράγοντα διά νά παραχθή ένα Αντίσωμα ή Γαβ'τμήμα εχον σουλφύδρυλ όμάδες ,τό όποιο Αντίσωμα ή FaP' τμήμα κατευθύνεται, έναντι μιας Αντιγονικής θέσεως ,καί (β) Αντίδρασιν τών αναφερθειοών σουλφύδρυλ όμάδων μέ ένα ίωδοαλ»^ κυλ παράγωγο ένός πεπτιδοσυνδέσμου ένός συνδέσμου Αμινοξέως ή ένός συνδέσμου τού γενικού τύπου «'ζκίΜίΓο' W-(CE O ) -Q δπου ¥/ εί««ι είτε -1®-CK 2 “ ή εινσι · Αμινήξύ.^^επ-ΐύδΙο, χηλικό ή χηλικό παράγωγο, κατί η Ακέραιος Απδ 2-20, τό όποιο συνδετικό προσκολλαται συσθενώς είς μία θεραπευτική ή διαγνωστική ένωσι ή μία άδιάλυτο μήτρα γιά νά σχηματίση ένα ουσθενές δεσμό μεταξύ τοϋ άναφερθέντος συνδέσμου καί τοϋ άναφερθέντος άναχθέντος άντισώματος ή Ραβ'τμήματος γιά νά σχηματίσθή μία σύζευξις άντισώματος ή όποία διατηρεί τήν κύρια άνοσοαντιδραστικότητα καί άνοσοειδικότητα.
- 3035) Μία μέθοδο συνδέσεως μιας ένώσεως πρός ένα άντίσωμα γιά νά πα ρασκευασθή ένα στζευκτικό άντισώματος , περιλαμβάνουσαί έκθεσιν ένός άντισώματος ή τοϋ (FaP*)^ τμήματος ένός άντισώματος είς ϋπιο άναγωγικό παράγοντα διά νά παραχθή ένα άντίσωμα ή Ραβ' τμήμα έχο® σουλφύδρυλ όμάδες,τό όποιο άντίσωμα ή Ραβ'τμήμα κατευθύνεται έναντι μιας άντιγονικής θέσεως* (β) άντίδρασιν των άναφερθεισών σουλφύδρυλ όμάδων μέ ένα ίωδοάλκυλ παραγωγό ένός ’πέπτιδο συνδέσμου ένός συνδέσμου άμονοξέος ή ένός συνδέσμου τοϋ γενικού τόπου W-CCHg^-Q όπου W είναι είτε -NH-CHg ή -CH 2 -, Q είναι ένα άμινοξΰ ,πεπτίδιο ,χηλικου ή παράγωγο χηλικοϋ ,καί η είναι ακέραιος άπό 0-20 δ ιά νά σχηματισθή ένας συσθενής σύνδεσμος μεταξύ τοϋ άναφερθέντος συνδέσμου καί τοϋ άναχθέντος άντισώματος ή Ραβ'τμήματος,καί (γ) σχηματισμό μιας συσθενους προσκολλήσεως ή ουμπλόκου χηλικοϋ μεταξύ τοϋ μή συνδεδεμένου τελικοϋ τοϋ συνδέσμου καί μιας θεραπευτικής ή διαγνωστικής ένώσεως ή μιας άδιαλύτου μήτρας, γιά νά σχηματισθή ένα συζευκτικό άντισώματος τό όποιο διατηρεί κυρίως άνοσοαντιδραστικότητα καί άνοσοειδικότητα.
- 3136) Τήν μέθοδο τής άξιώσεως 22,23,24,27,28,29,32,34 ή 35 δπου δ άναφερθείςςύνδεσμος είναι ένα πεπτίδιο περιλαμβάνον μία σειρά αμινο όξέος προερχομένης άπό τίς σειρές άμινοξέος των πεπτιδίων τά όποια είναι υποστρώματα διά ένεργοποιημένα συστατικά oy^a,-'η α, ληρώματοε.
- 3237) Τήν μέθοδο τής άξιώσεως 23,24,28 ή 29 δπου τό άντίσωβα- δύναται νά ένεργοποιή συμπλήρωμα όρρο{3\δτζη' συνδέεται πρός αντιγόνο καί ό συσθενής δεσμός μεταξύ τοϋ συνδέσμου καί τής ένώσεωςναι. έπιδεκτικός είς διαχωρισμό διά ένεργοποιημένου συμπληρώματος ,Ινω τό προκΰπτον συζευκτικό άντισώματος διατηρεί τήν Ικανότητα νά ένεργοποιεΐ τό συμπλήρωμα.
- 3338) Τήν μέθοδο της άξιώσεως 23,24,28 ή 29 δπου τδ άντίσωμα δύναται νά ένεργοποιή συμπλήρωμα δρροϋ δταν συνδέεται πρός τό αντιγόνο καί δ συσθενής δεσμός μεταξύ τοϋ συνδέσμου καί τής ένώσεως δέν εΐναι έπιδεκτικός εΐς διαχωρισμό ύπό ένεργοποιημένου συμπληρώματος ,ένω τό προκΰπτον συζευκτικδν άντισώματος διατηρεί τήν ικανότητα νά ένεργοποιή τό συμπλήρωμα.
- 3439) Τήν μέθοδο τής άξιώσεως 23,24,28 ή 29 δπου τό άντίσωμ σ δέν δύναται νά ένεργοποιήΐή τό συμπλήρωμα δρροϋ δταν συνδέεται πρός τό άντιγόνο καί δ συσθενής δεσμός μεταξύ τοϋ συνδέσμου καί τής ένώσεως εΐναι έπιδεκτικός νά διαχωρίζεται ύπό του ένεργοποιημένου συμπληρώματος.
- 3540) Τήν μέθοδο τής άξιώσεως 23,24,28 ή 29 δπου τδ άντίσωμα δέν δύναται νά ένεργοποιή τό ’συμπλήρωμα δρροϋ δταν συνδέεται πρός τό άντιγόνο καί δ συσθενής δεσμός μεταξύ τοϋ συνδέσμου καί τής ένώσεως δέν εΐναι έπιδεκτικός νά διαχωρίζεται ύπό ένεργοποιημένου συμπληρώματος.
- 3641) Τήν μέθοδον τής άξιώσεως 23,24,28 ή 29δπου τό άντίσωμα δέν δύναται νά ένεργοποιή τό συμπλήρωμα δρροϋ δταν συνδέεται πρός άντιγόνο καί δ συσθενής δεσμός μεταξύ τοϋ συνδέσμου καί τής ένώσεως εΐναι έπιδεκτικός είς τό νά διαχωρίζεται ύπό πρωτεάσης δρροϋ.
- 3742) Τήν μέθοδον τής άξιώσεως 34, ή Β5 ,δπου δ συσθενής δεσμός μεταξύ του συνδέσμου καί τής ένώσεως εΐναι έπιδεκτικός νά διαχωρίζεται διά ένεργοποιημένου συμπληρώματος.
- 3843) Τήν μέθοδον τής άξιώσεως 34 ή 35 ,δπου δ συσθενής δεσμός <*· *' μεταξύ του συνδέσμου καί τής ένώσεως δέν εΐναι έπιδεκτι-τί0ς<νϊΓ'7? διαχωρίζεται ύπό ένεργοποιημένομ_^συμπλ.ηρώματος.
- 3944) Τήν μέθοδον τής άξιώσεως 34,fy35 οπού δ συσθενής ξεβμός μεταξύ τοϋ συνδέσμου καί τής ενώσεως είναι έπιδεκτικός νά. διαχωρί68 ζεται ύπό πρωτεασών δρροϋ.
- 4045) Τήν μέθοδο τής άξιώσεως 22,23,23,27,28,29,33,34,ή 35 ,δπου τό άντίσωμα είναι μονοκλονικό άντίσωμα.
- 4146) Τό τροποποιημένο άντίσωμα παραχθέν βάσει τής μεθόδου τής άξιώσεως 22,27 ή 33.
- 4247) Τό συζευκτικό άντισώματος παραχθέν βάσει τής μεθόδου τής άξιώσεως 23,24,28,29,34,35,37,38,39,40,41,42,43,ή 44.
- 4348) Τήν μέθοδο παραδάβιφς καί άπελευθερώσεως μιας ένώσεως είς μία άντιγονική θέσι περιλαμβάνουσαί χορήγησιν μιας άποτελεσματικής ποσότητος ένός συζευκτικοΰ άντισώματος παραχθέντος βάσει τής μεθόδου τής άξιώσεως 37 είς ένα άτομο τό όποιο έχει συναγωνιστικό σύστημα συμπληρώματος δρροϋ.
- 4449) Μία μέθοδο παραδόσεως καί άπελευθερώσεως μιας ενώσεως ρίς τήν άντιγονική θέσι περιλαμβάνουσσΐ χορήγησιν είς ένα άτομο μιας άπουελεσματικής ποσότητος ένός συζευκτικοΰ άντισώματος παραγομένου βάσει τής μεθόδου τής άξιώσεως 59 ή 42 έν μίγματι μέ άντισώματα κατευθυνό|1ένοθ έναντι τής άναφερθείσης άντιγονικής θέσεως τά δποΐα δταν συνδέονται πρός τό άντιγόνο δύνανται νά ένεργοποιοϋν τό συμπλήρωμα.
- 4550) Μία μέθοδο βάσει τής άξιώσεως 48 η 49 δπου ή άντιγονική θέσις είναι ένα βακτηριακό αντιγόνο καί ή ενωσις είναι ένας άντιβακτηριακός παράγων.
- 4651) Μία μέθοδο βάσει τής άξιώσεως 48 ή 49 δπου ή άντιγονική V θέσις είναι ένα ίϊκό αντιγόνο καί ή ενωσις είναι ένας άντι-ίϊκός W παράγων.
- 4752) Μία μέθοδος βάσει τής άξιώσεως 48 ή 49 δπουή άντιγονική θέσις είναι ένα άντιγόνο ογκου καί ή ενωσις είναι ένας παράγων έναντίε ·· τοϋ ογκου. 55) kCx μέθοδο βάσει τής άξιώσεως 48 ή Ζ-9 δπου ή άντιγονική* θέσις είναι μυκητιακό αντιγόνο καί ή ενωσις είναι άντιμύκητιοκός· παράγων.
- 4854) Μία μέθοδο βάσει τής Αξιώσεως 48 ή 49 δπου ή Αντιγονική θέσις εΐναι παράσιτικό Αντιγόνο καί ή ένωσις είναι Αντιπαρασιτικός παράγων .
- 4955) Μία μέθοδο βάσει τής Αξιώσεως 48 ή 49,δπου &£$ τό Αντιγόνο εΐναι μυκοπλαστικό Αντιγόνο καί ή ένωσις εΐναι Αντιμυκοπλασμικός παράγων.
- 5056) Μία μέθοδο βάσει τής Αξιώσεως 48 ή 49 δπου ή Αντιγονική θέσις εΐναι Αντιγόνο διάφοροποιήσεως ή ίστοσυμβατότητος καί ή ένωσις εΐναι κυτοτοξικός παράγων .
- 5157) Μία μέθοδο παραδδσεως μιδς ενώσεως είς μία αντιγονική θέσι χωρίς Απελευθέρωσιν μέσω συμπληρώματος τής άναφερθείσης ένώσεως περιλαμβάνουσα χορήγησιν είς ένα άτομο μιας αποτελεσματικής ποσδ« τητος ένός συζευκτικοΰ Αντιδώματος παραχθέντος βάσει τής μεθόδου τής άξιώσεως 38,40 ή 43.
- 5258) Τήν μέθοδο τής Αξιώσεως 57 δπου ή ένωσις εΐναι ραδιοφαρμακευτικδς, βαρύ μέταλλο,τοξίνη ή τμήμα τοξίνης.
- 5359) Μία μέθοδο παραδδσεως καί άπελευθερώσεως μιας ενώσεως είς μία άντιγονική θέσι χωρίς ένεργοποίησι συμπληρώματος περιλαμβάνουσαΖ χορήγησιν είς ένα άτομο μιας άποτελεσματικής ποσδτητος ένδς συζευκτικοΰ άντισώματος παραγομένου βάσει τής μεθόδου τής τής άξιώσεως 4ΐή 44.
- 5460) Τήν μέθοδο τής άξιώσεως 59 ,δπου ή ένωσις είναι νευρομεταφορεύς ,ορμόνη,ένζυμο ή σειρά DNA.
Independent claims54
346 paragraphs in 40 sections, as filed
I. AREA OF INVENTION * This invention relates to related antisense antibodies, including monoclonal antibodies and polyclonal antibodies (standard antiserum); diagnostic and therapeutic affinity cleansing applications.
Specifically The invention is directed to methods proskdlliseos prds THE karvoOdrika portions ends molecule Antibody text outside of the antigen coupling domain of the molecule of the antibody, prdsooulfydryl Amades molecule rum Antibody Hai prds amines or carboxy dmades OF PC tdn Antibodies al Conjugate region restrained ol THE Immune Specificity and Immunoreactivity of Antibodies Is Obtained and Further Construct the Ability to Activate Antibodies supplement.
The present invention also relates to the general region of carrier systems which can form compounds in either IN VIVO or IN VITRO target sequences. Such systems include the general region, delivery of pharmaceutical derivatives or other compounds VIVO , both fantastic IN VIVO and IN VITRO systems (e.g. Imaging systems) cell type systems and separation schemes based on Antibody-Antigen Interactions.
The present invention also comprises the attachment of a substrate ligand to an antibody so that the resulting antibody conjugates retain the ability to bind antigen and to act as an additional promoter. you are the target.
<img file="GR77424B_D0001.tif" />
<img file="GR77424B_D0002.tif" />
2) "Infrastructure of the Invention
2.1 Frequent attachment
Various Reactions may be used interchangeably <sup>n</sup>iroschollisoun covalently compound prys THE entisomata.Afto supplemented by reacting residues Amino dxeos molecule antibody comprising amino dmades lysine, free Amades carboxy lichou acid glschtamichou Hai Aschartichou dxeos, sulphydryl dmades cysteine and choichila portions tdn Aromatic amino dxeon .
solvents are severe time disadvantages of common cholecystectomy for polypeptide spine
of an Antibody molecule. These Amino acid sequences of the lightweight Immunoglobulin heavy chain contain both amino acid residues randomly dispersed throughout the molecule, including the binding region. Without any chemical modification to occur in this Antigen binding region, a change must be made to the Antibody Identification Element, such changes will have to be reported, and indeed expected, if they are altered. Antigen.
In a population of different Antibodies, such a change in the Antigen binding region results in complete inactivation of some antibodies and lower degrees of inactivation of the Other, relative to the proximity of the switch to the site.
"This deactivation may result in a change in the position of the Antigen Shift to change or conform to the binding site to become inverted, or may result in a change of the region outside the region.
V, Z / m ** '-' \
Antiserum to restrict or penetrate the Antigen with a M (. · * -4 Antigen binding region...,
<img file="GR77424B_D0003.tif" />
Probably or most commonly used non-specific method of conventional adhesions is Interaction.<img file="GR77424B_D0004.tif" />Some new carbides have been linked to the carboxyl moieties of a compound similar to the Imino moieties of the Yutismoma molecule. The spike contains a lobule or water spike thus forming a spike or spike then antithetical to the spleen of the sphincter. of SCHIFF with Electrodes of the Hydroxide Molecule. In addition, compounds with reactive sulfurhydryl moieties have conjugated yttrium moieties. The isothiocyanate ester can be re-used as a conjugating agent for adhesion of more commonly compound compounds. This method has been used for new adherent fluorescent compounds such as Antibody Medium for use in fluorescence microscopy (BRANDTZAGEy. 1973, SCAND) IMMDK. 2i 273-290) and cell sorting systems (L0KEN and HSRZENBSRG, 1975, ANMALS, NY ACAD.SU 254: 163-171).
* Disulfide bonds may be re-used as compound binding sites. * While one succeeds in re-selectively reducing only the disulfide bonds, some functional properties of the Antibody may be re-affected, such as functional affinity and affinity.
Alternative Methods of Attachment to Antibody Mutations Above the Antigen Binding Area (Outside of Beta Domains)
<img file="GR77424B_D0005.tif" />
ot attachment modes are not common to any pre-
<img file="GR77424B_D0006.tif" />
separation and cleansing schemes. 'Any offset 1'
<img file="GR77424B_D0007.tif" />
in addition to selective antiseptic release; in addition to carrier systems (see section 2.3) if abnormal bonds are to be broken prior to antibody binding, they may reach their positions.
2? 3 Carrier Systems A number of agents have been used as carrier molecules with limited success in drug delivery systems. Practically ot vectors should not be toxic and target site specific. Ideally, there must be a mechanism for releasing the active form of the compound from the vector at the target site. a wide range of pharmaceutical or cytotoxic agents as ezinex radioactive compounds (e.g. I<sup>I2</sup>\ 1<sup>1</sup>^<sup>1</sup>agents that bind DNA, e.g., alkylating agents or various antibiotics (e.g., daphnomysine, adriamycin, chlorambucil), antimetabolites such as or methotrexane, agents which inhibit cell surface expression (e.g., cell surface) ) and protein synthesis inhibitors (e.g., diphtheria toxin and toxic proteins). For observations on the fatality see BALE et al., 1980 (ANCER RESB ^ gg
<img file="GR77424B_D0008.tif" />
Some of the delivery systems which are most relevant to the present invention are discussed below.
* Liposome delivery of the pharmaceutical agents has. maximum disadvantages due to lack of target specialty. Recently, researchers have succeeded in overcoming this problem by systematically attaching whole antibodies or FAB fragments to liposomes containing a pharmaceutical agent (HEATH et al. 1981, BIOCHIH, BIOFFICIOL, 2001; CHBI.255 (17) 180158018, JANSONS AND MALLET, 1989, ANA. KEI 54-59, MARTIN ET A1, 1989, Biochem, 20 * .4229-4238).
* Others have reported the conjugation of protein A (step A protein) to liposomes to direct the preparation to multiple specific targets which have previously been linked to specific antibodies (1980s; 288: 602-604). * However, an inherent problem of particular relevance to any liposomal carrier system is that in many instances the target liposome does not selectively reach the target site of VHV. * If either liposomes are encapsulated with antibody molecules, Liposomes are easily phagocytosed by macrophages and removed from circulation before reaching other target sites. Many researchers have unknown other major problem inherent in liposome targeting systems liposomes as a pharmaceutical agent, within.
Target Target (WEINSTEIN AND PARTNERS 1978, BIOCKEM BREAKING AFTA: 272-288) Some researchers have been trying to address this problem for the past few years, using targeted liposomes. who could do that? .internally
<img file="GR77424B_D0009.tif" />
cell type stellBSSUiAK et al. 1980, PSOC. SOMETHING".
A CAP. SCI USA 77 (7) 5 4089-4195, MADIC & Companions 1900, PSOC KATL ACASD SCI USA 77- (8) 54430-4434). The problem of inserting liposomal contents is still acute but nevertheless Digestive cells are active phagocytic cells. For example, the cells of the fibroid sarcoma are much less phagocytic than the cells of the lymph nodes and leukemic cells. these liposome penetration systems lie on the capability of the stem cell itself to insert a substance or substance that will ultimately be a deadly type of cell. under its active form.
After phagocytosis, the liposome containing form is harvested form the lysosomes of the target cell. The variety of proteolytic enzymes contained in the type of lysosome makes it very difficult if not unlikely to induce binding affinities They will allow the release of the drug agent in its active form. Thus, the reliability of the enzyme content of the lysosomes of the target cell is preferable to a random system of active drug delivery.
* A number of investigations have reported 4 target systems comprising attachment of compounds or pharmaceuticals directly to typical antisense antibodies, or to FAB antibody directed / antisense sections. See unpublished articles, above, and BLY ITEMAN et al. 1981, NATURE 290, 145-146. DAVIS AND PRESTON, 1981, SCIENCE2Ig:
1385-1388, HURWITZ gL ££>, 1979, INT.J. GAICBR ££: 461-470), «US PATENT lo. 4,093,607, AND Ut PATENT). * 1446536.URBAL ANU EAK0M0RI (1980, J, BIOU.OEEIi. 255 (21): IO5O9-IO579) described a targeted antibody, a cannabinoid mediated drug, e.g. call.
Various researchers have been digesting proteins other than aphids; bodies as carriers of a verse system. For example, desoxylated glycoproteins are preferably obtained from hepatocytes. See unpublished articles above and / 60 (2): 379-384.
If both antibody carrier systems are now more specific for the target than there are liposome carrier systems there is a significant problem with the drug release agent at the target site, if mediated by the target system. The drug cell line and drug may be released by lysosomal splenome separations (see Optional Articles, Above). In addition, non-specific binding of the drug to random sites on the antibody charge may interfere with binding ability, thereby reducing the ineffectiveness of the system.
Radiopharmaceutical techniques used recently in a non -eisvolikes IN VIVO great methods based on the ability's t drganou stouchou to remove TS radiofarmakeftikd signal DPA kykloforia.Oi These techniques utilize a variety of substances by radiodraotikes to deliver compounds to the desired target, toiaites substance comprises substrate ratio substrate, ligands, ligands, radionuclides, bifunctional chemicals (including binders about one chip at one end of each)<sup>7</sup>a metal or radionuclide may be attached, and a Reactive Sample at either end may be attached to a conventional
<img file="GR77424B_D0010.tif" />
IO target) and liposomes (SPENCER, RT ED 1980. Radiopharmaceuticals Structure-Cell Relationship. STRATTON Means of Torquay. EDUCATION | EASE | LEV ANSON 1977 / NTL.J. APPL. RADIATON xaf ISOTOPER 24: 67-82) Other non-invasive techniques currently available are nuclear magnetic resonance imaging and IN VIVO spectroscopy. See BROWNELL et al. in the field of radiopharmaceuticals.
5. Summary of the Invention * The present invention relates to the systematic attachment of the compounds to the antibody molecules so that the pre-conjugated antibodies can co-exist in the ability to bind antigens and inactivate them. Such attachment methods include adhesion to the carbohydrate moieties of the antibodies to the sulfhydryl antibody groups and the amino or carboxy groups of the antibody moiety. The carbohydrate is present in the constant region of the antibody (outside of the variable domain). carbohydrate PER SE will not be introduced directly from reactive or interfering groups - with the variable field.<sup>n</sup>Thus, such an approach is an attractive means for systematically modifying antibodies without seriously affecting either immunoreactivity or immunoreactivity. By preferred embodiment the antibodies are modified by standard adhesion to the carbohydrate moiety using known reagents and<sup>-</sup>In vivo with the chemistry of the proteins of the carbohydrate reaction of carbohydrates to be modified
<img file="GR77424B_D0011.tif" />
II
<img file="GR77424B_D0012.tif" />
coprotein. First, there are many different enzymatic and non-enzymatic reactions that are primarily directed, if not exclusively, at the carbohydrate moieties.
The synthetic products of the present invention have been found to retain significant, if not complete, immunoreactivity and immunoreactivity. * When such conjugates are prepared using monoclonal antibodies, or binding constants and modifying markers are not modified. reduce the average binding constant and introduce malfunctioning heterogeneity. ' Reagents used in the new techniques of the invention are potent<sub>p</sub>It can be exclusively selective As there is no detectable reaction of the polypeptide portion of the Antibody molecule. A, I, D, I, G) and I, M, and antibodies From any source, including monoclonal antibodies.
The present invention also encompasses the use of Antibodies as a carrier molecule for targeting compound or compounds for delivery to specific cells, organ tissues or any other site IK VIVO, or IK and then any other site. alternatively, or Release may be mediated by proteasomes.
Antibodies directed against any desired target. X Antigenic targeting cells of bacterial cells, or parasites) can act as motifs in any of the vectors. * And if typical antibodies can be used *, / '5 / /' as carrier molecules, Monoclonal Antibody offers ^ -some advantages of enhanced specificity with Antigen, Improved Effect12. - ·
<img file="GR77424B_D0013.tif" />
quality of delivery system and production difficulty.
Once administered II VIVO, 6 molecule vectors · Antibody will adhere to the Antigenic determinant of the target site. Substrates are then activated (complement cascade) by the formation of certain Immunosuppressants. Some of the components of the cob complement are waterproofing agents or are specific to specific substrates or chemical bonds.
According to a method of the present invention, a compound is attached to an Antibody molecule of the Immunoglobulin class of secondary antibody or which can act as a complement. This attachment is complemented by ligands such that they are susceptible to cleave at least one or more of the activated complement fragments, and one or more different compounds may adhere to each Antibody molecule. * The resulting Antibody conjugate is administered to a subject. 1After binding to the modified VIVO Antibody and Antigen, the complement of the Atomic Drug is activated and the compounds are selectively separated and separated. Tubular conjugates may be used for IN VITRO detection and identification of the target antigen or a complement-fixation assay system.
For the exercise of the present invention it is desirable to adhere or bind to the antibody molecule without the antibody, or with the ability to activate the complement (called a complement fixation). * This invention describes the new ligands and method of attachments that may be used to access the knowledge of any Antibody capable of activating the complement.
Alternatively, techniques such as antigen imaging or separation vectors based on btC antiserum-Antibody bifurcation, where the antibodies are adhered to an insoluble matrix, require an attachment to the antigen. If the target site is not available, then either of the ligand used is insensitive to activated complement proteins or the molecule. of the Antibody is of the locus or site that does not infer the complement.
Finally, for the delivery of other compounds, such as hormones or neurotransmitters, where it is desirable to separate or unbind without activation of the carotid complement, the filament may be utilized as a binding agent of the proteasome.
the. Short description of the places
<img file="GR77424B_D0014.tif" />
* The present invention is likely to be more intelligible Referring to the following detailed description of the invention examples of specific embodiments of the invention and the appendixes thereof are shown herein. (a) and of 1 M arc (b).
* Icon 2 Represents a portion of the complementarity of the complement. CI to C9 Represents complementary proteins. * Numbers overlap indicates an active enzyme. Virus S represents a site on the cell membrane.'Figure 3 includes a generic Antibody Scheme<sub>X.</sub>- 3 for adhesion of the antineoplastic drug, ILLKERAH '(BUEROUGHS-WT5LLCOME), to the β-peptide / ly-gly- peptide.
* Icon 4 represents the stimulation spectra of two (a) unoxidized Antibody and (b) Antibody Oxidized by * of Section 6.1 * Icon 5 represents the stimulation and emission spectra of the phenylhydrazine-tricyclic compound.
Figure 6 Represents the stimulation and emission spectra of the conjugated Antibody-phenylhydrazine-Tripeptide-AMC (APTA) prepared according to section 6.3.
Figure Ί Represents SIPS patterns of fluorescence quenching data using Unmodified Antibody (.- -.) Antibody modified by the method of the invention (D - D) and Modified Antibody by Carbodiomide and Carbamide - II) * Figure 8 Represents the Results of tDV, experiments which show the specific complement mediated release of AMC together with certain controls. * The fluorescence is recorded at 460 Nm with excitation at 380 Nm. An increase in fluorescence indicates release of AMC From the Antibody-phenylhydrazine-tripeptide-AMC conjugate (APTA); (a) Represents the APTA conjugate stained with erythropoiesis luteinis (b) Represents APTA conjugate stained with rat erythrocytes fixed with glutaraldehyde and Human Supplement; (c) Represents ARTA conjugate stained with sheep erythrocyte A (E) stained with glutaraldehyde;
5; Detailed description of the invention ___
Glutoproteins are one of the few types of biologically important endotheliums that have been found to be used in elective therapeutic and / or diagnostic sites. includes immunoglobulins, a number of insect repellent drugs, Compatibility Antigens of the Cell Surface and Cell Surface Inhibitors / These S15s have carbohydrate residues that are adherent to a polypeptide backbone. Antibodies are one Tadzio TEV glykoproteinvn whose karvoOdrika portions Keynes "Tie generally in the heavy chain TDS molecule Anosoglovvylinis (See Figure I for schematic Anaparastaoi merikEn AnosoglovoullnVn) .The FAB or portions FAB any AnosoglovoulinEn containing karvoOdrika portions may be chrisimopoiithodn in the Reaction Scheme described herein The FAB fragments of IgG Immunoglobulin B obtained by separately antibody to the antibody molecule pepsin (resulting in a divalent fragment (FAB'Jg) or papain),
5.1 'Antibody Selection
According to the present invention, Antibodies may be used against any Antibody or Antibody Antibody. If typical Antibodies (Antibodies are likely to be used in monoclonal antibodies, they may have some advantages, each of them is unique. antibodies used in the present invention are likely to be directed against any predisposing agents, e.g., dog, capillary, fungal, viral, parasitic, phytoplasmic, biocompatible, differentiated and other cell membrane antigens, pathogenic and antigenic sour enzymes. , barbiturates, steroid, catecholamines or dilanine, or theophylline, or histamine, cannabinoids, and, more commonly, & one of the most prominent antigens ^^
USP 4,193,983, in particular Chapter X - Which patent description is hereby incorporated * When delivering and releasing
<img file="GR77424B_D0015.tif" />
<img file="GR77424B_D0016.tif" />
<img file="GR77424B_D0017.tif" />
such as 1 'A, I <^ D, I <| E, certain occlusions in the nasal antibody are desirable, immunoglobulins of the order M1 or Limitations of the Subclasses of G1 should be used as long as these are immunoglobulin loci as they are known. In other embodiments ot carrier immunoglobulins may be used which may not activate the complement. Such carrier immunoglobulins may comprise certain classes of antitumor antibodies.
or cp00 (1 a portion of the immunoglobulins, e.g., half of the antibody molecules (a single pair of chains; lightweight) or FAB, FAB or FAB7<sub>2 </sub>When visualizing the IN VIVO targets is to be supplemented or useful by the antibody segments as carriers it is advantageous that these segments penetrate the target sites at an increased rate. In addition, a combination of antibody reactants to different antigenic determinants may be used.
<img file="GR77424B_D0018.tif" />
TABLE I
MEDICINES FOR PARTICULAR MEDICINAL PRODUCTS
NAME / TASK TYPE 'Antibacterial Amnoglycosides
STREPTOMYCIN
NEOMYCIN
KANAMYCIN AMIKACIN GENTAMICIN TOBAMYCIN STEPTOMYCIN B
DESMOS | stor / | mydos / amido imido / amido / amido / amido
MANUFACTURED
<img file="GR77424B_D0019.tif" />
<td>SPECTINOMYCIN AMPICILLIN</td><td>ester amide</td>
<td>SULFANILAMIDE</td><td>amide</td>
<td>POLYMYXIN</td><td>amide</td>
<td>CHLORAMPHENICOL</td><td>ester</td>
<td>Anti-viral</td><td></td>
<td>ACYCLOVIR</td><td></td>
<td>VIRA A.</td><td>ester / amide</td>
<td>SYMMETREL</td><td>amide</td>
<td>Antifungal</td><td></td>
<td>NYSTATIN</td><td>ester</td>
<td>* Antineoplastic</td><td></td>
<td>ADRIAMYCIN</td><td>ester / amide</td>
DOW, LILLY, DONE, PFIPHA
RMICS
BRISTOL
BRISTOL
UPIOHM, WYETH SCHERING
LILLY
SQUIBB
UPJQHH
SQUIBB, PARKE-DAVIS, CO MER, WYETH, UPJOHN, BRI STOL.SKF
MERRELL-MATIONAL BURROUQHS-WELLCOME, DOW, PARKE-DAVIS PARKE-DAVIS
BURROUQHS-WELLCOME
PARKE-DAVIS
XNDO
SQUIBB, PRIMO .LELERLE, PFIZER.KGBIXND — RANTOS
Radiopharmaceuticals
CERUBIDINE
BLEOMYCIN
ALKERAN
VALBAN
ONCOVIN
FLUOROURACIL ^ amp> amp amp amp | amp | amp | amp | amp
ADRIA
IVES
BRISTOL
BURROUQHS-WELLCOME
LILLY
LILLY
ADRIA, ROCHE .HERBERT <sub>r</sub>I25
151
I *<sup>11</sup> (TECHNETIUM)
Heavy metals
Variety Gold Plate Antimycoplastic
TYLOSE
SPECTINOMYCIN
<img file="GR77424B_D0020.tif" />
<img file="GR77424B_D0021.tif" />
5.2 ME80D0 CONNECTIONS LIFE IN PARTS 7 ..; -. Applications for the use of standard methods or a present invention include Some novel methods for preconditioning compounds of the antibody moiety. Amino or Carboxy Teams of the TC region of the Antibody molecule whatever method used, or affinity should not be significantly altered. Antibody such as either an Immunoassay and an Immunoactive Reactor. Additional considerations include simplification of the Reaction and Stability of the Produced Conjugate.
5.2.1 Attachment to the Carbohydrate Fragment of the Antibody _______ As explained in detail below, the carbohydrate side chains of the Antibodies may selectively inhibit aldehyde generation. The resulting aldehydes will probably then be reacted with amino groups (e.g. ammonia derivatives such as hydroxylamine, odorazine, phenylhydrazine or imicarbazide) to form a base of SCHIFF, e.g.
Alternatively, the carbohydrate portion of the Antibody is likely to be modified by enzymatic techniques to potentiate binding to or reaction with other chemical groups.
5.2.II Chemical Methods of Oxidation Oxidation of the carbohydrate portion or portion of the antibody molecules leads to the formation of aldehyde groups. these hydrogen oxides and their salts are preferably secondary or undesirable side chains19
<img file="GR77424B_D0022.tif" />
action is less frequent. For general discussion, see JACKSON 1944. Slow Reaction Reactions 2 "p. 54I * BOTT0t I965. Read (donated to Organic Chemistry Volume I, (fIBSRG, exAo) 'Academic Place News,' p. 36).
Oxidation of the Antibodies with these oxidizing agents can be carried out by known methods. Oxidation is generally used in the form of an aqueous solution or the concentration is generally
0.001 to 10 nm, and preferably 1.0-50 nm.
Oxygen Carbohydrate or Salt It Is Deprived of the Type of Antibody But is generally used in excess eg twice to 10 times or the amount of Oxidized Carbohydrate. 'The ideal carbohydrate, however, can be determined experimentally.
In the method for the Antibody Oxidation Assay or Salts thereof, the (Danish ranges include a pH of about 4-8 ° C, preferably 0 ° -37 ° C and reaction period of about 15 minutes to 12 hours.
According to the Oxidation of Glycoprotein with Oxygen Shaft or Salt herein, light is preferably excluded to prevent glycoprotein peroxidation.
5.2.I.2. 'Enzymatic Methods of Oxidation' Oxidation of the carbohydrate portion of the Antibody molecules likely to occur with the Enzyme, Lactosoxidase (COOPER et al. 1959, J.BIOL CHEM 234-445-448); Antibody is used; 20 iuG / IJL. The enzyme is generally used in the 5-lOC units / ml solution, the pH ranges from about 5, about 5 to about 8.0. The pH effect , COOPER and associates, cited above, are described in colorimetric buffers and buffer concentrations of the enzyme Anti20.
5.2.1.3 Preparation of the outer antiseptic
The antisense conjugates of the invention are likely to be produced by reacting the dioxide antibody with any suitable partitioner having an amino group available. The immediately preceding prodrugs (antisense conjugates) contain a
<img file="GR77424B_D0023.tif" />
of the original additives.
The 'Antibody-OXO Ύ R ———' Antibody —CHsN — R ^ HgO
For general discussion of aldehyde reaction with aldehydes, see March 1978, Advanced Organic Chemistry, Reactions, Mechanism 22, MCGRAW-HILL co., New *, p. 674.
A solution of oxidized antibody at a concentration of about 0.5 to 20 MG / ML is mixed with the conjugate parthenol (molar ratio analogs of a reactive parthenyls with an aldehyde of antibody ranging from about 18 to about I). . Suitable temperatures are below 0 ° C, 37 ° C and pH may be about 6-8 ° C.
5.2.I.4 Antibody Stabilizer Conjunctions After which antibody conjugates have formed along with the antibody and the conjugate partner as described in section 5.2.1.3, the antibody conjugates are suitably antibacterial conjugates. sodium or sodium borohydride
<img file="GR77424B_D0024.tif" />
'Antibody - CH = NR;
<img file="GR77424B_D0025.tif" />
J.BIOL.CHEM. 254X4359.
5.1.2 Adherent Sulfhydryl Tumors of the Antibody Molecule Free sulfhydryl groups can be produced by the disulfide bonds of the immunoglobulin molecule. Those that bind the two heavy chains. The disulfide bonds close to the antigen-binding region of the antibody molecule remain. Generally unaffected (see Fig. I) Such induction results in the loss of the ability to solidify the complement other than interference with the ability of antibody-antigen binding (KRUSH et al. 1979, BIOCHEM 18: 2226-2232). The free sulfhydryl groups created in the intravitational chain region can then be reacted with iodoalkyl derivatives of any carbohydrate-containing amino groups such as amino acids This binding does not interfere with the antigen binding site of the immunoglobulin.
Antibody conjugates that are produced by adhering a compound to free sulfhydryl groups of immunoglobulin-conjugated or uninfected antibody fragments do not activate the complement. These conjugates can be separated by 1 VIVO separation or IN VIVO imaging systems where separation or release of the compound is not reminiscent. or may possibly be linked to sulfhydryl groups on the immunoblot, which may have a raised antibody through ligands that are susceptible to be separated by proteasome pathways.
<img file="GR77424B_D0026.tif" />
FAB fragments of IgG immunoglobulin are obtained by separating 22 antibody molecule with pepsin (resulting in a divalent fragment (FAB *)).<sub>2</sub>) or with papain (resulting in 2 divisional segments, 2 TABs) see image I. The TAB segments (PA> *)<sub>2 </sub>It is smaller Just because of a loose antibody odor and therefore, it penetrates into the target site or the more chaperone. , places of attack, etc.).
An additional advantage is obtained when using conjugates formed with Antibody fragments because these fragments do not cross a placonic barrier. *As a result , using the present embodiment of the invention in the in vivo position (as a ligament) can be visualized in a pregnant female without exposing the fetus to the visualized compound If adhesion of a sterile compound is present adhesion methods may be used to make Antibody conjugates for use with the Supplemental Release System. In one such embodiment a compound attached to a connective layer of I ** * of a sensitive layer, the substrate may be adhered to;
sulfhydrylates of Reduced Lyses or Antibody Parts, and to be delivered to a target with a mixture of Intact Antibody molecules that may activate the complement. The latter may act on the complement or the The use of antibody fragments as carrier molecules in the Supplemental Release System could permit the education of pregnant females and, What is the advantage of having a fast transverse coupling position? \
Based on the present invention by binding to groups of reduced antibody molecules, the ligands
<img file="GR77424B_D0027.tif" />
are modified by adhesion to an iodoalkyl group at one end of the binder. Unmodified binding of the binder may or may not adhere to a fibrin cascade. (see Table II and Table III) are modified by the addition of iodoalkyl group to form iodoalkyl conduits as described below (NB denotes an Amido or an esteric bond) 0
I-CHjC-NH-binder-lk-compound As mentioned above the binder may be a compound which is a receptor or a cleavage to be separated from an activated complement, or proteasome pathway.
* When the iodoalkyl derivatives of the ligand group react with Reduced Antibody molecules or Reduced parts of the antibody or ligand group are adherently attached to the antibody molecules or moiety.This is described below (N.B.
(Aβ or moiety) -β-αH<sub>2</sub>-0-NH-connector- #> * compound
5.2.3 Adhesion to amino or carboxyl groups of the right region of the antibody molecule.
A modification of the standard methods for binding compounds to the Antibody molecules may also be used for the purposes of the present invention. affinity of the antibody molecule / cc for> 2 g / ml. a reduced Immunosuppressive effect due to the binding of the ligands or compounds to the FAB peripheral arms of the Antigen) of the Antibody molecule, such as those used for standard binding methods;
<img file="GR77424B_D0028.tif" />
The layer could be further directed to a more Ideal site on the Antibody molecule by a new immune complex formation and separation by the complement. the carboxy moths of F<sub>Q</sub> They are reacted with a substrate binder, e.g., by soluble carbodiimide reaction.
* A ligand is adherently attached to a compound but any Reactive molecules in the compound may interfere with the binding to the antibody molecule; The ligands at the compound may be new unblocked. Probably new ligand binding or elongated by activated complement cleavage, or proteases & Ldquo; * The extent of conjugation may be further tested by restriction reagent.
BC (NB denotes an Amido or ester bond).
Aβ-COOH-V H ^-linker-carbodiimide compound X
Ab-CO - NH - Binding-Compound * The protection of FA.B brachydine is probably newly complemented in a number of ways. Anti-PAB-Antibody). The following binding reactions result in adhesion of the compound to the unbound. The Anti-FAB Antibody Probably New Immobilized on a Column To Allow Easily Separate Antibody Reactants - - - - Active Ingredients.
Such a connector is likely to be damaged
I - V., · (very well washed with buffer / From each filter <sup>G. </sup>method), adhesion of the Anti-FAB-Antibody is appropriate
<img file="GR77424B_D0029.tif" />
Substrate Matrix as a Activated SEPHAROSE Column from Cyanide Bromide. Treatment of the column with an amino-blocking agent (e.g., anhydrous oxide, carbobenzoxy chloride, etc .;) to block all free amino groups on them Exposed portions and the anti-FAB antibody and associated antibody carrier, * The column is then termed a chaotropic agent (e.g. thiocyanate, hyperchloride, iodide, etc.) or a denaturant (e.g. , formol-urine, guanidine chloride, etc. which disintegrates the carrier with anti-FAB-antibody without destroying the anti-FAE antibody (DANLICER 6H and B) 5) 11460-1467. * This treatment releases the antibody-carrying myocytes that they transfuse, leaving the immobilized anti-FAB antibody free to form acyl immune complexes.
* The column, or now consisting of immobilized anti-LAB-antibody containing blocked amino groups at non-antigenic coupling sites, is loaded with an antibody carrier. After which the antibody carrier is linked directly to an antisense type i-FAB The reaction is carried out using a substrate binder such as a compound. Thus, the only available amino groups are found at F<sub>c</sub> part of the antibody carrier or provided reaction results in an adhesion of the compound through the connecting substrate to that part of the antibody carrier. Finally, the conjugate is released from the clot by eluting with appropriate elution; agent or denaturing agent). \
5.3 Coupler Partner)
According to the methods of the invention, the antivirals can be obtained
<img file="GR77424B_D0030.tif" />
The main limiting factor is whether or not adhesion adhesion should be (I) sufficiently selective to inhibit competition, undesirable effects, or unwanted reactions (2). with the reactivity of the 'A' antibody and selectivity. For the purpose of the description, the conjugates are divided into (a) compounds of interest, which are attached directly to the antibody (b) soluble binders and (c) insoluble substrates.
5.5.1 'Compounds of interest' Partner conjugates to the antibodies are likely to be any compound or compound that retains its essential properties after reacting with the antibody, whether or not it is present.
For example, it is desirable to attach an aldehyde to the oxidized carbohydrate portion of a glycoprotein in a conjugate, or a compound to preferably contain a reactive group as a group of amino acids. enzymes, catalytic transport proteins, chemicals, receptor proteins, and immunoglobulins and fluorescent or chemo Fotsis volouses compound or potentially fluorescent or chemiluminescent compound .Dia TOD dynamically "fluorescent compound or chimeeofotovolou meaning compounds whoever fluoresce or S chimeiof otovoloun only after reaction, modified or combined with other paragonta.'Opou an interesting compound does not contain an amino group or a compound likely to be modified to make a hydrogen group available for conjugation with the resulting fibrin Preventive Use of a System of Reference (selected) for the purpose of the intended application (e.g. murder, prevention of cellulitis 27)
<img file="GR77424B_D0031.tif" />
polycarbonate, hormonal therapy, target apoptosis, or gender therapy, cell specificity eg pharmaceutical agents, toxins segments toxins, alkylating agents enzymes antibiotics, antitumor agents, antiviral agents, antiviral agents, DNA polymers radioactive isotopes fluorogenic compounds, markers, lectin compounds which alter cell membrane permeability and insoluble matrices. This is not meant to be an exhaustive list. Telkes are a combination of compounds that may be used.
5.3.2 Soluble binders
According to the invention, antibodies are likely to adhere to any interest in an insoluble substrate by means of an intermediate soluble binder, or binder having a number of anti-reactive groups, one for reacting with the antibody and one for reacting with the compound of interest or on an insoluble substrate. The binder must be chosen to act or react with the antibody (or a compound of interest or an insoluble substrate) to such as the reactivity of the antibody and the selectivity in general, These binders could include hetero-functional linkers such as mercaptoethanolamine or which could be linked to the oxidized antibody through its amino group. After disassembly of the sulfur atom, free sulfhydryl groups could be available for further replication. The binder could be of any suitable size to allow distillation or distillation. Anti-inflammatory and compound of interest or to be chosen as desired.
aromat $
5 · 3.3 Insoluble substrates
Suitable Insoluble Substrates May Adhere Directly to Antibodies or Indirectly through Soluble Binders Where One Uses Oxidized Antibody by Directly Attaching to a Substrate Containing Ammonia, Including ♦
Such exemplary substrates as any Amine or aqueous substrate, include derivatized deuterans, agarose polystyrols, polyvinyl alcohols, polyvinyl alcohols, polyacrylamides and gum glass and other suitable polymers. These substrates are normally used as beads, but they can be surface pipes and discs with special uses.
5.4 VEHICLE SYSTEMS
According to one embodiment of the present invention, a compound is likely to adhere to an Antibody directed against a wall antigen. This adhesion is complemented by chemical bonds (e.g., a ester or amido bond), and which is capable of activating the complement cascade. Binding Teams (e.g. Peptides or Amino Acids) Colonies are susceptible to be separated under one or more of the Aru complement components.
The chemical binding methods described herein allow the resulting Antibody conjugate to retain the ability to bind the Antibody and to activate the complement cascade. As a result, a conjugate is administered Av. ^
Target antigen IN VIVO activates the complement of the individual. * If the binder is specified to be partially separable or the compound will be separated, the
<img file="GR77424B_D0032.tif" />
<img file="GR77424B_D0033.tif" />
targeting one or more enzymes of the cascade complement. If the release of the compound occurs after delivery to the target site or the inefficiency of the target delivery system is greatly improved.
The method of the present invention offers another advantage over other targeting systems. It is known that all cells of a single strand do not each possess the antigenic target determinant. Thus, Delivery systems in which the insertion within the target cell will successfully influence delivery only to cells that possess antigenic determinants and which have an antagonist or antagonist complex. can be nested, escaped from treatment. According to the method of the present invention, a single antibody carrier molecule delivers the compound to the target cells. However, as soon as they are attached to the target cell, the method described in the present invention permits the release of the active compound through the activated complement-activating enzymes of the individual. As a result or compound, action will be taken on the cells that do not down-regulate the Antigenic determinants as well as on the cells that actually possess the antigens Determinant. In addition, either method or method does not depend on the conjugate insert. The targeted delivery method described herein may presently be used for a number of purposes. The selection of the antibodies binding and compounds used in the γ-DNA. From delivery for delivery, * Delivery and release of pharmaceutical compounds to specific sites) targets may result in selective killing or prevention of tumor cell proliferation, cancer cells of fungi, parasitic bacteria, or a targeted delivery of Drosophila, glomerular or neurotransmitters to selected sites that may also be complemented,<sup>X.</sup>The invention or method of the present invention is likely to have an application in parental treatment programs where one or more specific offspring are likely to be delivered in vivo or in vitro to the recipient cells, or those without this specific genus.
In an alternative embodiment, the conjugate may be so specified or delivered to the cells that it is no longer released. knobs, instruments, or places of attack. This embodiment of the invention is particularly useful in illustrating systems of cell specification techniques and separation schemes.
Specifically, in the case of imaging, a radiopharmaceutical or heavy metal is (a) highly attached to a binder or (b) not directly attached to the binder via a chelator. * Therefore, where relevant, a wide range of Antibodies to ligands and compounds of interest may be used in a variety of combinations.
5.5 Work Supplement__and Elective Selection
According to the method of the present invention, when or Release of a compound is desirable, this compound is bound to a specific site on the IgG1 antibody or specific subunits of la G (Fig. I). Antigen and activation of complement cascade.
<img file="GR77424B_D0034.tif" />
<img file="GR77424B_D0035.tif" />
The complement is the collector name for a bundle / tree that can be activated in a classical way (
E0SPTA1 PRACTICE, AUGUST 1977 * 55-45) * 0 classical by antisense antibodies of the class I or some bent G to their corresponding antigen in one way or another depending on the protein of the protein. (Reid and PORTER, 1981, ANN.REV. Biochem. 50: 455-464). The classical mode is characterized by the formation of certain antigenic antibody complexes (or immune complexes) of any of the activated proteolytic B system. IMMUNOL 95: 559-566; COHEN 1968; J. IMMUNOL 100: 407-415; COHEN AND BECKER, 1968; J. IMMUNOL 100: 405-406; ISHIZAKA et al., 1968, J. Chem. IMMUNOL IOO: 1145-1155 (These activated complement enzymes segregate and activate other components of the cascade of complement (Figure 2).
The first ingredient that is classically activated is
CL which makes a protease or which acts on both of them;
I.
<img file="GR77424B_D0036.tif" />
\ y
C2 and <4. Activated CL (cl) 2 has a specific esterase activity. Activated C4.2 (? 4β "2 <P> sometimes called C5 conversion, is a complex that separates proteolytically C1 and together with active<sub>3</sub>(c ”) separates C5. The separation of C5 is the first step in common between the classical and modes of complement activation. enzymatic actions and CL and O2?<sup>uv</sup> recently studied using models of synthetic substrates (see Table II) which are separated ύ * §<sub>X.</sub>The carboxy terminal ester or double bond in vitro substrates may be used as an antibody binding agent and a compound as described herein. <sup>7</sup> These binders will allow such separation by supplementation and subsequent release of the compound in its active form at the target site. as a binder.
So, according to the present invention, one compound is attached to one end of the substrate binding group and the other end of the binding group adheres to a specific site on the vector of the substrate molecule. or the compound likely to adhere to the carboxy end of a peptide, amino acid or other appropriately selected linker via an ester or amide bond, respectively. For example, such compounds are likely to adhere linking peptide via, a Carbodiimide Reaction. * If the compound contains functional groups which could interfere with adherence to the ligand, these functional groups may be blocked before adhering or joining and being blocked as soon as the conjugate image has been made. a general reaction scheme for attaching the Antineoplastic drug, AUCAPEN (BURROUGHS -WELLCOME) to the CBZ-gly-gly-arg peptide. The Opposite or Amino terminal of the linker group is then modified by binding to an Antibody molecule which can activate the complement. The compound may adhere to the ligand before or after the ligand adheres to the antibody molecule. ) The binder is free where a bonded compound is of particular application, or compound \ likely to<sub>;</sub>"^^: Peptide ligands are in any way intended to bind to the peptide ligands likely to vary in length. Substrate Removal from the Antibody Molecule Possible
<img file="GR77424B_D0037.tif" />
Affecting the Effectiveness of Separation or Occurring in the Amidic or Fidelity Bond between the Binding and the Joint. This binding may also include organo-compounds, e.g. specific sites on the antibody molecule. The other end of the organic polymer may be attached to an Amino acid or peptide linker. * 0 Table III Indicates Other Substrates may also be used as connecting rods to prepare the Antibody conjugates of the present invention (In the table U is probably an integer including zero) these sequences were derived from sequences of similar sequences. Acid-base additives.'0 This list is not exhaustive. When these conjugates bind to the Antigen in the presence of an Amido complement or an ester bond, the adhesion of the compound to the ligand is cleaved resulting in Release of the compound in its active form and such conjugates Release of the compound by c
Target site, and is particularly effective for IN VIVO delivery of pharmaceutical agents, Antibiotics, Antibiotics, Anti-proliferative agents and cytotoxic agents.
In an alternative embodiment the ouzets of the present invention are likely to be used to detect the Target Antigen IN VITRO.nX after the conjugate is added to a test S & T containing a Target / Supplement Target or Supplement.<sup>4</sup>A * fluorescent compound is an intron and measure of the presence of the target antigen in the test (e.g.,
TABLE II £ βNovember_Christmas_Dev_ScAssional_2: BILLING
No, Reference Ifc
Δa_c £
N-BOC-tyrosine-o-nitrophenyl ester I N-BOC-phenylalanine o-nitrophenyl ester I aN-BOC-lysine o-nitrophenyl ester I N-CBZ-tyrodine β-nitrophenyl ester 2
N-Acetyl-lys-methyl ester 3 aK-CBZ-lys-methyl ester 3 α-N-Acetyl-lys-methyl ester 3 BOC-white-gly-arg-7-5min-4-methyl coumarin 4 * I. SIM, et al., 1977, Biochem. J. 16 ^ .219-27
2. BINS, 1969, BIOCHEMISTRY 8J 4503-10.
3. COOPER, NO 1975, BIOCHEMISTRY I4I4245-5I
4. CAPORALE, ET AL., 1981, J. IMMUNOL. I28J 1963-65.
<img file="GR77424B_D0038.tif" />
TABLE III
UNITED STATES CONNECTION GROUPS
INTEREST (CI) IN PARTIAL PARTS<sup>1</sup>_______
A. Binders for the separation of t-lys **;
B. Tripeptide rows for separation by sub-x
-all-lys-white-salt-cheese-leb-lb-arg-leb-lys-leb-leb-cheese / -leb-gly-argH, N- (aa) - <-leb -gly-lys $> * <sup>the</sup> white-sweet-lyr-le-val-arg-le-val-lys-le-val-cheese-le-yl-arg -
-le-yl-chain-white-yl-styr-halo-argonH<sub>2</sub>N- (a.a.)<sub>the</sub>-
<img file="GR77424B_D0039.tif" />
-a / nala-lysal-pyrryl-aryl-lysyl-thyrlyl-argly-lyslyl-tyrval-argal-lysval-pyrryl-argil-lys-, yl-styr- /
- * -CX
<img file="GR77424B_D0040.tif" />
<img file="GR77424B_D0041.tif" />
<img file="GR77424B_D0042.tif" />
{K.
III B. Tripeptide sequences for separation from (continued) z- -glya-arg-L--gly-ala-yl-yl-ala-tyr-gly-white-Arg-gly-white-lys-gly -lee-cheese-sweet-sweet-Arg-K-sweet-sweet-lys-? - * - c! -gly-gly-cheese-gly-val-Arg-gly-val-lys-gly-val-cheese-gly-yl-Arg-gly-yl-lys- /
-gly-yl-cheese<sub>2</sub>N- (a.a.)<sub>n</sub>THE<sub>2</sub>N- (a.a.)<sub>n</sub>g- -val / -valvval -val -val -val -val -val -val -val -val -val -val -val
-Al-Argalys-Lys-Ala-Tyrol ··· Arg-White-Lys-White-Cheese-Arg-Arg-Gly-Lys-Gly-Tyr-Val-Arg-Val-Lys-Val-Tyr-yl -Arg-yl-lys-yl-styr-¼ -cl
THE<sub>?</sub>N (aa) -yl-ala-Arg-yl-alys-yl-ala-tyr-yl-white-Arg-yl-laryl-lys-yl-white-tyr-yl-argon -yl-gly-lys-yl-gly-tyr-yl-val-Arg-yl-val-lys-yl-val-tyr-yl-yl-Arg-j-yl-yl-yl-yl-yl-yl -tyr-
<img file="GR77424B_D0043.tif" />
<img file="GR77424B_D0044.tif" />
III C. Peptide sequences separated by C ^ g7 ^<sub>g</sub>~ h<sub>2</sub>n-white-gly-white-white-white-ala-white-yl-gly-sweet-sweet-white-sweet-ala-gly-val-gly-yl-ala-gly-ala-white-ala-ala- -all-val-halo-yl-val-gly-val-white-val-blM-val-val-bl-yl-yl-yl-yl-yl-yl-ala-yl-val-yl-yl
-lee-val-
<img file="GR77424B_D0045.tif" />
Tripeptide -M-CJ
1) * 0 Asterivase V ^ y represents either an amide bond (ligand
C-NH- (l) or an ester bond (Binding-CO-CI)
2) Tripeptide Represents Any Of The Tripeptides Listed In Table IIIb.
<img file="GR77424B_D0046.tif" />
V ✓
Certain Applications, or Release of Enobos, is not Desirable. According to an Alternative Embodiment of the present invention, a compound adheres to the antibody molecules via a ligand which is not susceptible to separation by an Enzyme supplement. These ligands may include amino acids, peptides, or other organo-organisms. include functional groups which may be used Hereinafter attachment to antibody molecules or antibody fragments by methods * A general pollutant for such an organic binder is
W- (CH<sub>2</sub>)<sub>n</sub>-Q donut is either -NH-OH<sub>2</sub>- or -CHgQ is an amino acid, peptide, chelate (e.g. diethylene triamino pentaacetic acid) or a chiral derivative and is an integer of 0-20, An 'envelope' compound may adhere to antibody molecules or antibody fragments which do not activate the complement. * When carrier antibodies are used which cannot activate the complement, or adhesion may be complementarily used by ligands by Enabled Supplement or by using binders that are not Properly separated by Enabled Supplement. * Non-segregated adhesion may be used to attach Antibody molecules or fragments to immobilized or insoluble matrices e.g. Agarose, polyphylamide, etc. The products are likely to be used subsequently to identify or separate specific antigenic components from complex mixtures. After washing all the non-reactive conjugates, the Antigen39 target may be eluted from the insoluble matrix by treatment with a gva denaturing or chaotropic Antigenoantibody agent.
This or non-spacer approach is particularly useful for the manufacture of antibody conjugates for use in cellular cellular assays (LOKEN and HERZENBERO, 1975, ANNALS, N.Y. ACAD, SU 254-6b-I7I) and imaging systems. infestation etc. Where or the release of the compound is not desirable. In these imaging systems, or the use of antibody fragments, it offers a distinct advantage in that such portions diffuse and penetrate the masses only. In addition, Antibody fragments do not cross placental barriers (BELLANTI, 1976; Immunology II, WB SAULERS, Philadelphia; Therefore, the Imaging of the Possible Pregnant Female In yet another Endometriosis, it may be necessary to construct the ligand in such a way as to idealize the space between the compound and the antigen. using a binder of general structure.
wherein Q is an amino acid or peptide and W and n are as described above.
In yet another embodiment of the target delivery, a release of the compound without activation of the complement is desirable if or by activation of the cascade of complement will ultimately target the target cells.
The time when delivery or release of the compound should be supplemented without killing the uterine cell. Target cells are desirable for such conjugates to be prepared by attaching the compound to a myrrh instead of - (CH<sub>2</sub>)<sub>the</sub>-
<img file="GR77424B_D0047.tif" />
body or portion of which Mg ** # was intended to activate the complement through a ligand which is moderately susceptible to being separated by proteasome or if it is administered to a subject, An Antigen-Antibody Complex Will Be Formed Rapidly After Separation Of The Compound Will Be Slowly Obtained, Thus Releasing The Compound At The Target At The First Stages Of The Activation Of The Classic Antibody Additive Complex. that a site in the FC region of the Antibody molecule should be present (Figure I). Some of the carbohydrate segments lie on the PC region thereof. Immunoglobulin Required for Nutrient CI Connection Removal of the carbohydrate moiety results in a loss of the ability of the immune complex to bind the CI component of the complement (WINIZELHAKE et al. 25). The FAB or FAB fragments of any Immunoglobulins containing carbohydrate moieties may be used in the Reaction Scheme described herein. An example of such an Immunoglobulin is the Human IgGIII, P12 and SH12, respectively. ).
According to one embodiment of the invention, the substrate binders are modified by adhesion of hydrazine or hydrazide derivatives at one end of the ligand unmodified sites on the ligand may or may not be adherently attached to a ligand; a compound via an ester or amino linkage described in paragraph 5.5 (see Table II and Table III) are trimmed by attaching a hydrazide (e.g. Phenylhydricin / '- z; at the opposite amino end of the peptide of the following structure (NB, the symbol) forms an amide or ester bond.
As Alysus, Aphusus emerges
HgN-NH- ^ C 1-6 -NH-inducible- *;
<img file="GR77424B_D0048.tif" />
* Av and the indicated structure or hydrazine present in the art could be used alternatively with the hydrazine moiety in the rectum or post position. These hydrazide derivatives of the peptide bonds which are attached to the peptide bonds The amide bond is then reacted with an Oxidized Immunoglobulin, or an Immunoglobulin moiety containing an Oxidized Carbohydrate. This results in hydrazone formation and systemic adhesion. Immunoglobulin carbohydrate side chain via a ligand or which is capable of separating the complement.
* If desired, the binder used may be separated either by activated complement or by ligated article (e.g. a binder comprising a chelating or chelating derivative for use in a Visual Element). In another embodiment, the ligand may be determined to be susceptible to articular protease separation. The resulting structure is represented schematically below (N.<sub>X.</sub>B-symbol; means amide or ester bond., Carbohydrate
Aβ-side chain -CH = N-NH-NH9 linker-compound
If this or that paragraph is primarily directed Reactions with the carbohydrate moieties of such antibodies are technically applicable to Other classes of glycoproteins as well. ~? V-<sup>/_</sup>\
5.6 Additional Uses of the Specific Antibody
Antibody conjugates of the invention are useful; for a variety of applications in medicine and industry including separation and affinity purification as well as diagnostic and therapeutic; in the ability of the antibodies to specifically distinguish between light compounds of different structure.
The novel antibody conjugates of the invention are available for use in any immunoassay or interaction between antibody and antigen provides a detectable point or a means for modifying a detectable or detectable point. specially ultraviolet or bright light, either Absorbing or emitting thermal, dichromatic, electrochemical or the like. 'Modification of the signal may be, for example, as a result of quenching or modifying fluorescence or chemiluminescence. or dynamically fluorescent or chemiluminescent).
In immunoassays, the antigen concerned is physiologically signi ή cant or marked in some way and the assay allows the distinction between, of the amount of labeled or labeled antigen the antibody binds to the Antibody and the amount of labeled or labeled antigen is not bound. , or Distinct is a Result of the Physical Separation of Bound and Non Bound Antigen.
6. Examples? Rows I y ~~,<sub>t</sub> , , , ~ ~
The following examples illustrate methods for the specific attachment of an antibody molecule to a compound of interest by means of an intermediate soluble ligand. . '
<img file="GR77424B_D0049.tif" />
6/1 'oxidation of carbohydrate portion of antibody molecule
The antibody molecule used in this example was a monoclonal IM (purified No 171) specific for an antigenic determinant on sheep erythrocytes. from the immunized rats were harvested and pooled with the SP2 / OA '14 myeloma line according to the method of MCKEARN and co-workers 1979, kvoook. * kvaoH. 47X 9ΐ-l5. Cloned cells were subsequently killed and the resulting monoclonal antibody was determined as described in KLI NAMAN and MCKEARN, 1981, J, IMMUNOL Meth.42 with lacto oxidase by modifying the method of COOPER and its associates, supra; 171 Monoclonal antibody was added to I buffer consisting of 0.135 MaNaCL 0.015 M without HCL (pH 7.0)
0.5 mM MgCl2, and 0.15 nM M CaCL<sub>2</sub>Thereafter, a 0.1 ml aliquot of a lactose & oxidase solution (WORTHINGTON BIOCHEMICAL CO. FREEHOLD, N.J) was added to a concentration of 52 enzyme / ml of the same buffer with the antibody solution. Finally, 43 µg. (WORTHINGTON BIOCHEMICAL Co., FREEHOLD, N. IT) dissolved in another 0.1 ml of the same buffers were added to the reaction mixture (or catalase was added to degrade the hydrogen peroxide which was generated during the reaction reaction time 48 hours. stored at 4 ° C. fci jwy ry 4 represents the spectra for non-oxide and oxidized (b) Antibodies.
6.2 Preparation of tripeptide-AMC for in vitro antibody molecule.
<img file="GR77424B_D0050.tif" />
trocd44
For the purpose of the present example, a synthetic taxogenic compound was used as a conjugate partner. The properties of this synthetic compound are such that the bound and free states of the fluorogenic compound are spectrophotometrically different. BIOCHEMICALS INC · GARDEN.BARK, ΐΐΐ NY (List # 51474) * This fibrin consists of a tripeptide attached via an amid linkage to the fluorescent compound of 7-amino-4-methyl-coumarin (AMC), or amino acid glycinebucyl The structure of the present compound (hereinafter called AMC tripeptide) is shown below.
! i-X<sup>5</sup>
CB <sub>g</sub>-gly-gly-Arg-N-tripeptide-AMC
The excitation and decay maxima of the free AMC (345 nm and 445 nm, respectively) differ from that of the bound AMC to the tripeptide (325 nm and 395 nm, respectively); of an AMC molecule using a fluorometric assay. The excitation and emission wavelengths of 383 and 455 LEDs may be ideally used for testing purposes, these wavelengths, the free AMC. retains 20% of its maximum fluorescence but possesses a relative fluorescence of 500-fold greater than that of an equimolar amount of bound AMC (ZIMMERMAN, et al. 1978, PROC NATL, ACAD, SU USA 75J (2) J).
750-753).
To make his or her connection special. AMC triplet to 'S' X ** '
AMC tripeptide pre-carbohydrate portion of the antibody prepared in section 6.1, a hydrazide producer was coupled to a terminal end;
The glycine of the tripeptide-AMC compound, * The presence of hydrazine is advantageous since it results in reactivity for the oxidized carbohydrate portion of the antibody molecule under very mild, unstable or unstable conditions. carbohydrate side chain of the antidote antibody molecule then with the hydrazine derivative to form a hydrazide.
To adhere a hydrazine derivative (e.g. 4-fluoro-phenylhydrazine), tripeptide-AMC was first blocked at the glycine amino end by removal of the CB group. SE LOUIS, Mo) and HB4 gas (MATHESON, EAST RUTHEPORD.NJ) gas solution through the solution for 45 minutes. was precipitated by the addition of cold diethyl ether (BAKER CHEMICAD Co PHILLIPSBURG NJ) and dissolved in absolute ethanol (PUBLICKER INDUSTRIES, Co LINFIELD, Roche). After reduction in darkness, at room temperature for 2 hours the resulting mixture was stored in darkness at 4 ° C. The byproduct (phenylhydrazine-tripeptide-AMC) has the following structure.
THE<sub>2</sub>L-L-LH-gly-gly-arg-NH-AMO
<img file="GR77424B_D0051.tif" />
* This compound was shown to be fluorescent positive by UV light and positive for the present hydrazine group. * The hydrazine bound to the tripeptide was detected by thin-layer chromatography (TLC); A blue, orange or tan brown color indicates the presence of hydrolysis, and TLC results showed the presence of a hydrolyzate opiate solution of trinitrogen sulfide dioxide solution. with the Tripeptide-AMC Migratory Strip.
Absorption and emission spectra for the phenylhydrazine-tripeptide-AMC compound as shown in Fig. 5 reveal a similarity to the "tripeptide-AMC" spectra but one movement at the most transient level. The stimulation and reduction of the phenylhydrazine-tripeptide-AMC compound are 345 Nm and 5S5 mM respectively. The product was precipitated from the solution with cold diethyl ether, washed and dissolved in dimethylsulfoxide (BAKER CHEMICAL Co., PHILLIPSBURG NJ).
6.3 Fine attachment of the phenylhydrazine-tripepto AMC to the oxidized carbohydrate portion of the Antibody molecule ._______________________________________
The preparation of the oxidative monoclonal antibody described in section 6.1 above was adjusted to pH I by adding a small amount of 0.1 M bicarbonate buffer (pH 5.0). An appreciated 10-fold excess of phenylhydrazine 6.2) Add the antibody solution which was then incubated at 37 ° C overnight (about 14 5 hours). The reaction mixture was then chromatographed on a SEPHADEX G-25 column. (PHARMACIA PINE CHEMICALS, PISCATAWAY NJ) to remove any untreated phenylhydrazine tripeptide AMC.
* Spectrophotometric analysis of the protein fractions confirmed the presence of the aminobenzyl tripeptide AMC, which is attached to the Antibody (now called the Phenylhydrazine Antibody-Ampicidal Antibody-32). , respectively (icon 6x. high peak at 285 NI, the spectrum of ouzoactivation likely due to tryptophan absorption with residual boundaries is also likely to be the result of transient transport. Resonance From the tryptophan amino acid of the antibody molecule to AMC.
Ha
7. Examples: Series II * 0 The purpose of the present series of derivatives is to further illustrate the methods for preparing antibody conjugates.
<img file="GR77424B_D0052.tif" />
<img file="GR77424B_D0053.tif" />
<img file="GR77424B_D0054.tif" />
Carbohydrate of a mouse ILM monoclonal antibody, specific for phosphorylcholine, was lysed and adhered permanently to the
A 1.6-diaminohexyl derivative of ethylene diamine-di-O-hydroxyphenylacetic acid (EBHA) was re-formed with L, β-diaminohexyl-EPOHA. For comparative purposes, I-6-diaminohexyl-EDEA as well as unmodified EDDHA were attached to identical samples of the L-monoclonal Antibody using the carbodiimide reaction. Under these conditions, or I, 6-dihydro to the available aspartic and glutamic acid residues, while the unmodified EDDHA could be further conjugated to available lysines. The binding properties of these samples were compared with the natural Antibody for a new estimated or kinship or homogeneity.
7.1 * Oxidation of mouse monoclonal I
A monoclonal IgM antibody specific for the phosphorylcholine ligand was precipitated at a concentration of 2 MG / ML phosphate buffer (PBS, 0.01 M, 0.15 M sodium chloride phosphate) pH 7.0. water bath and 56.8 µg of water. sodium bicarbonate was added (40 µl of 1.42 MG / ml solution, final concentration of hydrocarbon = 0.26 n-) M).
This reaction mixture was quenched for one hour after anyone
<img file="GR77424B_D0055.tif" />
7.2 Attachment__ to the binder to EDDHA
EDDHA (1.5 g 4.2 mmol) and triethylamine (1.2 ml, 8.4 mmol) were mixed with 40 ml of water. This superficial solution was heated to 60 ° C and stirred rapidly at 0.5 g. The solution was dried in vacuo and then dissolved in 400 mL dry N, Dimethylformamide. The solution was then cooled in an ice bath and isobutyl ester (0, 56 ML, *, 2MM) was added. The reaction mixture was reacted with 0.5 mM. . The precipitated triethylamine hydrochloride precipitate was removed by filtration and the filtrate containing EDDHA Mixed Carboxycarbonate was red.
1-Amino-6-trifluoroacetamidohexane (0.8 g, 4.1 mmol) was added to the above carboxycarbonate anhydride in EDDHA.The crude solution was stirred at 4 ° C for 0.5 hr followed by lyophilization. The oil was washed with acetone / ether (4ml) mixture to give a crude yellow product.
The solid I-amino-O-trifluoroacetamido-hexyl-EFHA was collected and hydrolyzed with 7% K 2 CO 2 and resuspended with HCl to pH δ 4 to give I, 6-diamin-hexyl-EBHA (1, 4 g). * This compound gives a positive ninhydrin test and thin-layer chromatography shows only one spot. Presence of a basic solution of equimolar amount of TpCL ^, stimulants at 20.5 Nm. Emission at 545 N. subjected to formation of the characteristic chelating energy transfer complex between EDDHI and TERBIUM ion.
7 · 3 Preparation of the 1-M-conjugate-EDDHA-Binding Antibody, oxidized by the method of paragraph 7.1 was quenched with approximately 270 full-molecular excess 1,6-diaminoxylEDBHa prepared by the method of temperature one hour. Sodium urea cyanoborohydrate was followed at a final concentration of 1 ml, M, and further reduced for 4 hours at room temperature. The mixture was then dissolved at 4 ° C for a few changes in P3S.
<img file="GR77424B_D0056.tif" />
and concentrated by ultrafiltration.
7.4 Carbodiimide Adhesion of the EDDHA Binder to L .________________________
265 microl. of antibiotic M (1.9 MG / JtL) was added IO MGI-ethyl-5 (5-dimethylaminopropyl) carbodinimide (I ML one IO MG / ML solution, pH 5.0) and PBS (pH 5.0) to 2.5 ML.T0 mixture was quenched for 2 hours at room temperature. To this was added 2.75 ML 0.01 M EDLHA (PH 5.5) and the solution quenched for 2 hours at room temperature. Ten ML ethanolamine was added and the mixture was quenched. I hour at room temperature. This was then dissolved overnight against PBS (pH 7.0). r * ·
7.6 Effects of Carbohydrate Adhesion on Antibodies.
The affinities of the unmodified mouse monoclonal antibody and antibody conjugate prepared according to paragraphs 7.3, 7.4 and 7.5 were all specifically for the phosphoryl choline group measured by fluorescence quenching using a PER spectrophotometer. (PERKIN ELMER CORPORATION, NORWALK, CONNECTICUT). The wavelengths by wave and therefore were 295 and 345 Nm respectively and the temperature was maintained at 25 ° C with a LAUDAK-L / RKouTp0 water circulation (BRINKMANTS INSTR.).
Antibody concentrations were calculated using a 6 ° C <sub>=</sub> 13.5 and ranged from 7.5XII "® to 9.7X10" ® M. All
<img file="GR77424B_D0057.tif" />
, 280 g of binding sites resulted in active and sample fluxes were 3.0 ML. The stock concentrations of the N- (2,4-dinitrophenyl) -p-aminophenylphosphorylcholine (ppro) ligand were 1,7x10 ~ 4 mM. The titrations were carried out in brine buffer at a PR of 7.4. The ligand was continuously added at a rate of 9.01 microl / l using a shake syringe and fluorescence intensity. was recorded in a single, .BM 2001 series of computers equipped with a 2040 mobile diode and a 2023 printer (COMMODORE BUSINESS MACHINES),
Ί.4 Carbodiimido preconditioning of EDDHA to LM
265 microl. Antibody (l<sub>t</sub>9 MG / ML) IOMG I-ethyl-5- (3-dimethylaminopropyl) carbodiimide (I ML of 10 MG / ML solution of pH 5.0) and PBS (pH 5.0) of up to 2.5 ML were added. two hours at room temperature. To this was added 2.75 mL of 0.01 M ELLHA (PHV5.0) and the solution was quenched for two hours at room temperature. Ten ML ethanolamine was added and the mixture was quenched for one hour at room temperature. This was then dissolved in PBS. (PH
<img file="GR77424B_D0058.tif" />
Anyone Treating a PRESET I, Analogue / Finger Converter (CONNECTICUT MICROCOM PUTERS). J. IMMUNOL I27i24O) were titrated. The binding parameters were calculated using the SIPS distribution function (SIPS, 1948, J. CHEM PIYS I6J49O) repeatedly, where the maximum or maximum damping was allowed to occur until the heterogeneity index was within 0.002 units.
* 0 calculation of Omeg. Values include a Verbal Uncertainty, possibly not less than 10% of the repetitive method used to create. This or Obverse is illustrated by the fact that any discrepancy between the actual concentration of the active sites and the calculus. From the haptic density equals the value of zmg. When the heterogeneity index criterion is used. * This Compensation results from the dependence of the concentration of the linked ligand on the Ratio of the concentration of the Antibody to the value of Q max. 'Due to this dependence or the precision of the derived values of the active constant combinations of seats.
SHIP drawings showing data for unmodified carbodiimide conjugate Antibody antibody conjugates of the invention are shown in Figure 7. Binding measurements indicate purity retention, affinity of carbohydrates; Modify® Antibody (.h *).
of the compound for conjugation of the fluorescent compound was measured to be 0.1 X 10 µm.<sup>-</sup>·<sup>1</sup>· For the unmodified Antibody: JzfcC I, IXIO<sup>6</sup> M.<sup>1</sup> for the conjugate Antibody attached to carbohydrate. a Carbodiimide Reaction (0--0) Modified Antibody Preparation Has a Majorally Diminished Link, Certainly Below Calculated I-2XIII Values *. data only if the sample is homogeneous (monoclonal) "A Test on the actual homogeneity (monoclonal taxa) of the sample is a coefficient of correlation or adjustment If the data Significantly intersect with the Report Line On the SIPS project. Research of Figures 7 shows clearly good agreement for the modified Antibody and Antibody attached to the carbohydrate and very poor agreement with that of the carbohydrate receptor. carbodiimide adhesion method, The glutamate and aspartic acid lysines are easy to find and part of the antibody molecules, including the Antigen binding sites. As a result, at least some of the Antibodies are modified at or near the binding sites by consistent effects or interactions with the antigen. The sites of attachment to the carbohydrate, however, are determined separately from each other, * While obtained for systemically modified antibodies In solution, it is believed that the conclusions of this study may be They extend to Real Antibodies alike (similar experiments with Real Antibodies cannot be done for technical reasons). * The random chemistry of the common pre-attached chemistry (Activated gels of cyanide bromide, cadmium hydride). The ability to bind when compared with a specific site of the Invention.
<img file="GR77424B_D0059.tif" />
8. Examples? Series III The purpose of this series of by-products is to provide the probability that the antibody may adhere to insoluble substrates with high ineffectiveness against carbohydrate segments and adherent antisense.
8.1 Antibody adhesion to amino acylpharosarcoma.
Antibodies and IcG antibodies were oxidized using ultraviolet as described in section 7.1 except that Sepharose CL-4B (PHARMACIA FINE CHEMICALS, INC. NEW MARKET NEW JERSEY) was used instead of ethylene glycol for quenching. After removal of sepharose CL-4B, the antibody was added with an aminohexyl-sepharose CL-4B (PHARMACIA FIBE CHEMICALS INC) which was stirred at room temperature for 30 minutes. . The resulting antisense bound resin (Aβ-CHO-SEPH) was washed extensively with PBS.<sup>3</sup>I-I G et al <sup>?</sup>Antibody I * Table IV shows that immobilization of the antibody through the carbohydrate segments of the antibody has a high degree of inefficiency.
TABLE IV 'Immunoblotting of amino acids Sepharose. Antibody M antibody% ineffective
ML binding Sepharose ^;
I
IqM · connection inefficiency
7,8
4.6 _ K-.G bonded antibody - ^^ f ^^ fcG added anti-salivary 'L, Z', Z,
8.2 Partitions
Monoclonal I / G Mouse Antibody to 2,4-Dinitrophenyl (DNP) An incomplete antigenic group was oxidized and adhered to Aminosyl Sepharose (Ab-CHOSEPH) according to the method described in section 8 above. 0NB "-3EP" Living by CUATRECASAS and Associates (1968, PROC. NATL ACAD SU USA 61: 636).
Ribonuclease bound to the DNP group (INPHnase) was prepared by mixing equimolar amounts of 2,4-dinitrofluorobenzene (ALDRICH CHEMICARS Co., INC, MIL WAUKEE, WISCONSIN), and its inhibitor 9.0 for four hours at room temperature, After a prolonged dissolution against PBS, or DNF-nase was determined to have an average of 0.8 MOLES of DNP group per mole of ribonuclease. * AKP-HNase was radiolabeled with 125-j- using chloramine-T- (ALDRICH CHEMICAL Co., INC).
The two Immobilized Anti-DNP Antibiotic Resins Ab-CHO-SEPH and Ap-CNBg-SEPH were then tested for their binding ability to t-PNE-Enase. The two antibody preparations have a very high binding affinity for DNP-Enase as it was
Obviously under the Inability of the 3M Sodium Thiocyanate to Release the Connected DNP-Enase from Any Formulation. Therefore, to modify the system so that the binding antigen was not reversible, 3 experiments were performed. Antibodies Immobilized by Carbohydrate Adhesion-
<img file="GR77424B_D0060.tif" />
TABLE V
Connection of DNR -Khasease Immobilized Antibody ____________________ resins ^ ______
MG Antibody µg) NH-Enase Binding-BNB-RKAon +
ML resin ML resins -------------- binding ___________ ____________________ MG Antibody ________ ________
Ab-ONBt-SEPH 2.0 Ab-CHO-SEPH 0.6
3,9
5,5
100
140 jjfc 40 µl of 5? 6 V / V pulp of either Aβ-CHO-SEPH or Aβ-ONBt-SEPH resin were quenched with 2 mg. I-BNP-ILase in 4M urine 0, 1.5M NaCL, 0.01M potassium phosphate, PK 7.4 (urine-PBS) at 22 ° C for 120 minutes. * 0 total test volume, 4 ML. After reduction, or resin was resuspended with PBS, * H<sup>I2</sup>DNP iNase bound to resin was determined by enumeration of LKB 1271 gamma count resins.
<img file="GR77424B_D0061.tif" />
µg of DNP-HNase bound
LNP-iNase binding <sub>=</sub>IOX -—------ 'MYP' BHP-Knase bound
MG Aβ on Aβ-ONBt-SEPH
9. Example G Series IV
The following examples illustrate a method for specifically adhering to an Antibody molecule of a peptide linked to a compound of interest (amid) via an amidic or cis-linked linker. hemolytic complement fixation assay. In addition, specific release of the compound on the surface of the Antigenic cell by enzymatic separation thereof systems:
of supplement is indicated by a non-haemolytic J-τ '
The following examples or j * vaic \ c are fluorogenic, such or release by supplementation of the fluorescent compound may be detected by a test of ability to differentiate between the bound and free morphs. <sub>G.</sub>55
The materials and methods of section 6.1 were used to describe the oxidation of the carbohydrate segments of monoclonal antibodies (No. 571) in the presence of sheep erythrocyte and serum complement; Antigen-Antibody). * Suppression of complement causes lysis of sheep erythrocytes or resulting in hemoglobin release;
The AMC tripeptide was prepared as described in section 6.2.0. The properties of the fluorogenic compound (AMC) are such that the bound and free states of the fluorogenic compound are distinguished spectrophotometrically. Conjugate Antibody. More importantly, it provides a means for quantifying the subsequent release by complementation of the compound.
The specific adhesion of phenylhydrazine tripeptide A15C to the oxidized carbohydrate segments of the antibodies was performed as described in section 6.3.
9.1 Complement fixation tests
Two types of complement fixation assays were used, both amlumaric and fluorometric. These tests determined whether the Antibody-phenylhydrazine-tripeptide-AIvC conjugate retained the complement-fixation ability of AK and complement. <sub>L.</sub>
9.1.1 Preparation of Human Supplement A 10 1.1 sample of freshly obtained human shrimp was put on ice for 17 hours. Blood was decanted by centrifugation and the resulting human serum was collected. The Human Supplement has been shown to be active in the samples following the hemolytic assay described in section 9.1.2.
9.1.2 Hemolytic assay for titration
<img file="GR77424B_D0062.tif" />
fixation of the complement
A fraction of 200 µl. sheep erythrocyte suspension specimens (GIBCO DIAGNOSTICS, MADISON, WIS) at a concentration of approximately 2X10<sup>8</sup> cells / ML were mixed with 2β microl. of the conjugate antibody mixture prepared in section 6.5 (approximately 2 µg of protein). After 15 minutes of blending and incubation at 37 ° C, 100 µl. of the human serum supplement (prepared in section 9.1.I) was added to the mixture. After 30 min to 1 h of incubation at 37 ° C, the mixture was centrifuged to pellet the cell lysates. by measuring photometric spectra of α; emoglobin released in supernatant (412 Nm);
The results of this assay showed complete hemolysis and mainly 100% binding of the antibody to the cell surface. 1 ^ 20 dilution of ovine arterial cell supernatant dissolved in distilled water has Q.1.412 '0.646, same erythrocyte dilution Sheep that were lysed by the addition of conjugate and supplement had 0.1 2 = 0.672. θ ^<sup>so</sup>The conjugate retained its ability to bind to the antigen and to solid (3rd complement).
5.1.3. Non-haemolytic assay for free delivery
G *> 'supplement ._______
The conditions for the hemolytic assay were similar. Earlier acetins above except for glutaraldehyde-fixed sheep erythrocytes (SIGMA, St-LOUIS, No) were used instead of normal sheep erythrocytes. Instead, a fluorometric assay is used to indicate release of the ADC. A non-lyticolytic system is necessary for use in the fluorometric assay, since the presence of hemoglobin interferes with the fluorescence measurements.
Prior to the use of the assay, these fixed erythrocytes showed that both the unmodified Antibody and the Antibody-Phenylhydrazine-Tripeptide-AMC Antibody were prepared as described previously.
* The non-haemolytic assay was used to demonstrate specific release by supplementation of AkC with the antibody conjugate. Similarly to the haemolytic assay, 200 microliters of glutaraldehyde-fixed red blood cells were used. at a concentration of 2X10 cells / kl; were quenched with the conjugated phenylhydrazide-tripeptide-AMC conjugate at 37 ° C for 15 min. and fluorescence at 460 nm was recorded by stimulation at 380 nm (CAP0BALE et al., I98I, J. IMMONTI 128- 1983-65), as a function of time, as controls, conjugates) were incubated with sheep erythrocytes only in the presence of sheep erythrocytes and human complement (monoclonal antibody used). Figure 8 shows the Results of these experiments, A comparator of the curve (s) or Representative of its conjugate was incubated with glutaraldehyde ovine-fixed sheep and Human Supplement (s) as control (s). release of free AkC in a sample containing the specific Target Antibody and Human Supplement. Thus, either curve (b) or any Representative of conjugates afflicted with rat erythrocytes fixed with glutaldehyde and Anthropin supplementation, or curve (C) or representing any residual or inhibitory effect of conjugates showed no release of AMC. * The invention or description contemplated herein is not intended to be limited to the purpose of the disclosed embodiments, as long as these embodiments are intended as illustrations of some embodiments of the present invention. In fact, various modifications of the invention except those indicated and described herein will be apparent to those skilled in the art. The technique From the foregoing description. These modifications are also intended to fall within the scope of the appended claims.
Contents40
70 sheets
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98 members in 13 offices
Priority claims2
| Document | Office | Kind | Date |
|---|---|---|---|
| 35631582 | United States of America | A | |
| 44205082 | United States of America | A |
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| EP0173629A1 | European Patent Office (EPO) | A1 | |
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| EP0173629B1 | European Patent Office (EPO) | B1 | |
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Numbers
- Application
- 830170696
Titles
- English
- ANTIBODY CONJUGATES
Classification
- CPC, 6
- G01N33/531
- A61K41/0042
- A61K41/0057
- G01N33/54353
- A61K47/6889
- A61K47/68
- IPC, 15
- A61K39 00
- A61K39 395
- A61K39 44
- A61K41 00
- A61K47 48
- C07K1 10
- C07K1 107
- C07K1 113
- C07K1 22
- C07K14 00
- C07K16 00
- G01N33 531
- G01N33 532
- G01N33 543
- G01N33 569