US5162505A — Proteins modified with positively charged carrier…

Claim 1

Independent Claim 272 words

A protein conjugate comprising: a pharmaceutical protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and a radionuclide attached to said pharmaceutical protein or to said positively charged agent through a bifunctional coupling agent which is bound to a site on said protein or on said positively charged agent and to said radionuclide, wherein said bifunctional coupling agent has the formula: E'--L'--Ch where: E' is a moiety bound to a site on said protein or on said positively charged agent; Ch is a moiety chelating said radionuclide; and L' is a linking radical having the formula: --X--(CH 2 l) f --(OCH 2 CH 2 ) c --(Y) q --(CH 2 l) d --(OCH 2 CH 2 ) e -- wherein f is an integer from 0 to 6; d and e are independently integers from 0 to 5 inclusive; c is an integer of from 0 to 5 inclusive; q is 0 or 1; X is selected from the group consisting of --NH--, --O-- or --S--; Y is selected from the group consisting of --CH 2 COO--, OOCH 2 --, --CH 2 CONH--, OCH 2 COO--, --NHCOCH 2 --, and --OOCCH 2 O--; provided that, when X is other than --O--, then q is 1 and y is other than --CH 2 CONH-- or --NHCOCH 2 --, and that when f is 0, then c and q are also 0.

Claim 2

Dependent Claim 272 words

A protein conjugate comprising: a pharmaceutical protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and a radionuclide attached to said pharmaceutical protein or to said positively charged agent through a bifunctional coupling agent which is bound to a site on said protein or on said positively charged agent and to said radionuclide, wherein said bifunctional coupling agent has the formula: E'--L'--Ch where: E' is a moiety bound to a site on said protein or on said positively charged agent; Ch is a moiety chelating said radionuclide; and L' is a linking radical having the formula: --X--(CH 2 l) f --(OCH 2 CH 2 ) c --(Y) q --(CH 2 l) d --(OCH 2 CH 2 ) e -- wherein f is an integer from 0 to 6; d and e are independently integers from 0 to 5 inclusive; c is an integer of from 0 to 5 inclusive; q is 0 or 1; X is selected from the group consisting of --NH--, --O-- or --S--; Y is selected from the group consisting of --CH 2 COO--, OOCH 2 --, --CH 2 CONH--, OCH 2 COO--, --NHCOCH 2 --, and --OOCCH 2 O--; provided that, when X is other than --O--, then q is 1 and y is other than --CH 2 CONH-- or --NHCOCH 2 --, and that when f is 0, then c and q are also 0.

Claim 3

Dependent Claim 52 words

The protein conjugate of claim 1 where said positively charged agent is selected from the group consisting of polyethyleneimine and polymers or copolymers of one or more amino acids having positively charged side chains selected from the group consisting of alkylamines, alkylamidines, and alkylguanidines, each optionally containing one or more alkyl substituents.

Claim 4

Dependent Claim 31 words

The protein conjugate of claim 2 where said positively charged agent is selected from the group consisting of polymers of lysine, ornithine, 2,4-diaminobutyric acid, arginine, and copolymers of said amino acids.

Claim 5

Dependent Claim 21 words

The protein conjugate of claim 3 where said polymers or copolymers comprise three to ten repeat units of said amino acids.

Claim 6

Dependent Claim 15 words

The protein conjugate of claim 3 where said positively charged agent is an L-lysine polymer.

Claim 7

Dependent Claim 18 words

The protein conjugate of claim 5 where said L-lysine polymer comprises four to eight repeat units of L-lysine.

Claim 8

Dependent Claim 21 words

The protein conjugate of claim 1 where said protein conjugate comprises a protein in combination with a therapeutic or diagnostic agent.

Claim 9

Dependent Claim 15 words

The protein conjugate of claim 7 where said protein comprises an antibody or antibody fragment.

Claim 10

Dependent Claim 13 words

The protein conjugate of claim 8 where said protein comprises a Fab' fragment.

Claim 11

Dependent Claim 18 words

The protein conjugate of claim 1 where said positively charged agent is covalently linked directly to said protein.

Claim 12

Dependent Claim 39 words

The protein conjugate of claim 1 where said positively charged agent is covalently linked to said protein through a bonding radical which is bound to a site on said protein and to a site on said positively charged agent.

Claim 13

Dependent Claim 45 words

The protein conjugate of claim 11 where said bonding radical comprises a compound of the formula E--L--A where E is bound to a site on said protein, A is bound to a site on said positively charged agent, and L is an organic linking radical.

Claim 14

Dependent Claim 13 words

The protein conjugate of claim 12 where E is a sulfhydryl selective electrophile.

Claim 15

Dependent Claim 21 words

The protein conjugate of claim 13 where E is selected from the group consisting of haloalkyl, sulfonate esters, maleimide and aziridine.

Claim 16

Dependent Claim 26 words

The protein conjugate of claim 14 where E is selected from the group consisting of ClCH 2 CONH--, BrCH 2 CONH--, ICH 2 CONH-- and maleimide.

Claim 17

Dependent Claim 22 words

The protein conjugate of claim 12 where A is selected from the group consisting of active esters, imidate esters, epoxides and aldehydes.

Claim 18

Dependent Claim 12 words

The protein conjugate of claim 16 where A is an active ester.

Claim 19

Dependent Claim 13 words

The protein conjugate of claim 17 where A is an ester of N-hydroxysuccinimide.

Claim 20

Dependent Claim 31 words

The protein conjugate of claim 12 where L is selected from the group consisting of optionally substituted alkyl, optionally substituted alkyl containing heteroatom substituents for carbon, and optionally substituted aryl groups.

Claim 21

Dependent Claim 51 words

The protein conjugate of claim 12 where L is selected from compounds of the formula --CH 2 -cyclo-C 6 H 10 --, --CONHCH 2 CH 2 --, --CH 2 C 6 H 4 , --NHCH 2 CH 2 -- and --OCH 2 CH 2 OCH 2 CH 2 OCH 2 CO--.

Claim 22

Dependent Claim 15 words

The protein conjugate of claim 11 where said bonding radical is the compound 3-sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

Claim 23

Dependent Claim 79 words

The protein conjugate of claim 1 where said positively charged agent is a polymer of L-lysine having from 4 to 8 repeat units of L-lysine; where said positively charged agent is covalently linked to said protein through a bonding radical comprising a compound of the formula E--L--A where E is selected from the group consisting of ClCH 2 CONH--, BrCH 2 CONH--, ICH 2 CONH-- and maleimide, A is an ester of N-hydroxysuccinimide, and L is a linking radical.

Claim 24

Dependent Claim 13 words

The protein conjugate of claim 22 where said protein is a Fab' fragment.

Claim 25

Dependent Claim 13 words

The protein conjugate of claim 1 where E' is a sulfhydryl selective electrophile.

Claim 26

Dependent Claim 21 words

The protein conjugate of claim 24 where E' is selected from the group consisting of haloalkyl, sulfonate ester, maleimide and aziridine.

Claim 27

Dependent Claim 26 words

The protein conjugate of claim 25 where E' is selected from the group consisting of ClCH 2 CONH--, BrCH 2 CONH--, ICH 2 CONH-- and maleimide.

Claim 28

Dependent Claim 22 words

The protein conjugate of claim 1 where E' is selected from the group consisting of active esters, imidate esters, epoxides and aldehydes.

Claim 29

Dependent Claim 12 words

The protein conjugate of claim 27 where E' is an active ester.

Claim 30

Dependent Claim 13 words

The protein conjugate of claim 28 where E' is an ester of N-hydroxysuccinimide.

Claim 31

Dependent Claim 14 words

The protein conjugate of claim 1 where L' contains at least one cleavable site.

Claim 32

Dependent Claim 29 words

The protein conjugate of claim 1 where f, d and e are each 2, c and q are each 1, X is --O-- and Y is --OCH 2 COO--.

Claim 33

Dependent Claim 133 words

The protein conjugate of claim 1 where Ch is selected from radicals of the formulas: ##STR8## wherein a, j and m are independently an integer of 1 to 3 inclusive; b is an integer from 3 to 6, inclusive; R is selected from the group consisting of R 1 CO-- or R 1 S--; R 1 is selected from the group consisting of methyl, optionally substituted lower alkyl and optionally substituted aryl; each R 2 is independently selected from the group consisting of units ##STR9## Q is selected from the group consisting of a carbonyl group and --CHR 3 ; each R 3 is independently selected from the group consisting of hydrogen, optionally substituted lower alkyl, and optionally substituted aryl; and Z designates the site of attachment of the organic linking radical L'.

Claim 34

Dependent Claim 50 words

The protein conjugate of claim 32 where a, j and m are each 1; b is 3, R is R 1 CO--, R 1 is selected from the group consisting of phenyl and phenyl substituted with a functional group; R 2 is NHCH(R 3 )CO--; and R 3 is hydrogen.

Claim 35

Dependent Claim 242 words

A protein conjugate comprising: a pharmaceutical protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and a radionuclide attached to said pharmaceutical protein or to said positively charged agent through a bifunctional coupling agent which is bound to a site on said protein or on said positively charged agent and to said radionuclide, wherein said bifunctional coupling agent has the formula: E'--L'--Ch where: E' is a moiety bound to a site on said protein or on said positively charged agent selected from the group consisting of esters of N-hydroxysuccinimide; L' is a linking radical having the formula: --X--(CH.sub.2).sub.f --(OCH.sub.2 CH.sub.2).sub.c --(Y).sub.q --(CH.sub.2).sub.d --(OCH.sub.2 CH.sub.2).sub.e where f, d and e are each 2, c and q are each 1, X is --O-- and Y is --OCH 2 COO--; and Ch is a moiety chelating said radionuclide having one of the formulas: ##STR10## where a, j and m are 1; b is 3; R is R 1 CO--; R 1 is phenyl or phenyl substituted with a functional group; R 2 is NHCH(R 3 )CO--; Q is a carbonyl group or --CHR 3 --; and R 3 is H; and Z designates the site of attachment of the organic linking radical L'.

Claim 36

Dependent Claim 242 words

A protein conjugate comprising: a pharmaceutical protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and a radionuclide attached to said pharmaceutical protein or to said positively charged agent through a bifunctional coupling agent which is bound to a site on said protein or on said positively charged agent and to said radionuclide, wherein said bifunctional coupling agent has the formula: E'--L'--Ch where: E' is a moiety bound to a site on said protein or on said positively charged agent selected from the group consisting of esters of N-hydroxysuccinimide; L' is a linking radical having the formula: --X--(CH.sub.2).sub.f --(OCH.sub.2 CH.sub.2).sub.c --(Y).sub.q --(CH.sub.2).sub.d --(OCH.sub.2 CH.sub.2).sub.e where f, d and e are each 2, c and q are each 1, X is --O-- and Y is --OCH 2 COO--; and Ch is a moiety chelating said radionuclide having one of the formulas: ##STR10## where a, j and m are 1; b is 3; R is R 1 CO--; R 1 is phenyl or phenyl substituted with a functional group; R 2 is NHCH(R 3 )CO--; Q is a carbonyl group or --CHR 3 --; and R 3 is H; and Z designates the site of attachment of the organic linking radical L'.

Claim 37

Dependent Claim 79 words

The protein conjugate of claim 34 where said positively charged agent is a polymer of L-lysine having from 4 to 8 repeat units of L-lysine; where said positively charged agent is covalently linked to said protein through a bonding radical comprising a compound of the formula E--L--A where E is selected from the group consisting of ClCH 2 CONH--, BrCH 2 CONH--, ICH 2 CONH-- and maleimide, A is an ester of N-hydroxysuccinimide and L is a linking radical.

Claim 38

Dependent Claim 107 words

A protein conjugate comprising: a pharmaceutical protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and a radionuclide attached to said pharmaceutical protein or to said positively charged agent through a bifunctional coupling agent which is bound to a site on said protein or on said positively charged agent and to said radionuclide, wherein said bifunctional coupling agent has one of the following formulas: ##STR11##

Claim 39

Dependent Claim 107 words

A protein conjugate comprising: a pharmaceutical protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and a radionuclide attached to said pharmaceutical protein or to said positively charged agent through a bifunctional coupling agent which is bound to a site on said protein or on said positively charged agent and to said radionuclide, wherein said bifunctional coupling agent has one of the following formulas: ##STR11##

Claim 40

Dependent Claim 31 words

A method of preparing the protein conjugate of claim 10 comprising contacting a protein with an excess of said positively charged agent and a limiting amount of a water soluble carbodiimide.

Claim 41

Dependent Claim 42 words

A method of preparing the protein conjugate of claim 11 comprising: (A) contacting said positively charged agent and said bonding radical under conditions effective to bond said agent and said radical: and (B) contacting the product of step (A) with said protein.

Claim 42

Dependent Claim 97 words

A radiodiagnositc or radiotherapeutic precursor comprising: A protein covalently linked to at least one positively charged agent, said protein conjugate having a positive charge in aqueous solution at physiological pH, and said positively charged agent comprising a polymer of three or more subunits selected from the group consisting of amino acids containing positively charged side chains and alkylamines; and at least one bifunctional coupling agent bound to said protein or to said positively charged agent, wherein said bifunctional coupling agent has the formula: E'--L'--Ch where E', Ch and L' have the meaning set forth at claim 1.

Claim 43

Dependent Claim 23 words

The radiodiagnositc or radiotherapeutic precursor of claim 39 where said at least one bifunctional coupling agent is bound to said positively charged agent.

Claim 44

Dependent Claim 22 words

The radiodiagnostic or radiotherapeutic precursor of claim 40 where a plurality of bifunctional coupling agents are bound to said positively charged agent.

Claim 45

Dependent Claim 29 words

A radiodiagnostic or radiotherapeutic agent comprising the radiodiagnostic or radiotherapeutic precursor of claim 39 and a metallic radionuclide bound to at least one bifunctional coupling agent of said precursor.

Claim 46

Dependent Claim 29 words

A radiodiagnostic or radiotherapeutic agent comprising the radiodiagnostic or radiotherapeutic precursor of claim 40 and a metallic radionuclide bound to at least one bifunctional coupling agent of said precursor.

Claim 47

Dependent Claim 31 words

A radiodiagnostic or radiotherapeutic agent comprising the radiodiagnostic or radiotherapeutic precursor of claim 41 and a metallic or nonmetallic radionuclide bound to at least one bifunctional coupling agent of said precursor.

Claim 48

Dependent Claim 26 words

A radiodiagnostic or radiotherapeutic agent of claim 42 where said metallic radionuclide is selected from the group consisting of Tc, In, Re, Ga, I and Y.

Claim 49

Dependent Claim 26 words

A kit for use with a metallic or nonmetallic radionuclide for preparing a radiodiagnostic or radiotherapeutic agent comprising a radiodiagnostic or radiotherapeutic precursor of claim 40.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1995-11-10	1996-05-11	1996-11-10	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1999-11-10	2000-05-11	2000-11-10	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2003-11-10	2004-05-11	2004-11-10	$7700.00	Window Closed / Lapsed

Proteins modified with positively charged carriers and compositions prepared therefrom

Abstract

Inventors

Bibliographic Details

Claims Analysis

References Cited (Backward Citations)

USPTO Annuity & Maintenance Windows

Assigned Categories & Tags

Assign Category Tag