Immunoconjugates and methods
Immunoconjugates of the Formula (I) include a linking group for linking an antibody targeting ligand (Ab) to a drug (D). Embodiments of such immunoconjugates are useful for delivering the drug to selected cells or tissues, e.g., for the treatment of cancer. Ab-[S-L 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I)
1 . A conjugate having Formula (III),
Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III)
wherein:
Mi is
L 2 is absent,
Z 1 and Z 2 are each individually hydrogen, halogen, NO 2 , —O—(C 1 -C 6 alkyl), or C 1 -C 6 alkyl;
L 3 is —(CH 2 )n 1 -C(═O)— or —(CH 2 CH 2 O)n 1 -(CH 2 )n 1 C(═O)—;
n 1 are independently integers of 0 to 12;
L 4 is a tetrapeptide residue;
L 5 is absent or —[NH(CH 2 )n 2 ]n 3 -;
n 2 is an integer of 0 to 6;
n 3 is an integer of 0 to 2;
L 6 is absent or
L 7 is absent
and
D is a drug moiety represented by Formula (II):
wherein:
R 1 and R 2 are each individually selected from the group consisting of hydrogen, halogen, —CN, —OR 5 , —NR 5 R 6 , a substituted or an unsubstituted C 1 -C 6 alkyl, a substituted or an unsubstituted C 1 -C 6 haloalkyl, a substituted or an unsubstituted —O—(C 1 -C 6 alkyl), a substituted or an unsubstituted —O—(C 1 -C 6 haloalkyl), —[(CY 2 ) p O(CY 2 ) q ] t CY 3 , or a substituted or an unsubstituted —O—(CR 5 R 6 ) m —O— such that R 1 and R 2 taken together form a ring;
R 3 is hydrogen or a substituted or an unsubstituted C 1 -C 6 alkyl, a substituted or an unsubstituted C 1 -C 6 haloalkyl, or —[(CY 2 ) p O(CY 2 ) q ] t CY 3 ;
R 4 is hydrogen, a substituted or an unsubstituted —(C 1 -C 6 alkyl)—X 2 , a substituted or an unsubstituted —(C 1 -C 6 haloalkyl)—X 2 , a substituted or an unsubstituted —(C 1 -C 6 alkenyl)—X 2 , a substituted or an unsubstituted —(C 1 -C 6 haloalkenyl)-X 2 , a substituted or an unsubstituted —(C 1 -C 6 alkynyl)-X 2 , or a substituted or an unsubstituted —(C 1 -C 6 haloalkynyl)-X 2 ;
X 1 is —O—;
X 2 is —OR 9 , —SR 9 , or —NHR 9 ;
R 5 and R 6 are each individually hydrogen, halogen, a substituted or an unsubstituted C 1 -C 6 alkyl, a substituted or an unsubstituted C 1 -C 6 haloalkyl, or —[(CY 2 ) p O(CY 2 ) q ] t CY 3 ;
m is 1 or 2;
n 4 and n 5 are each individually 0, 1 or 2, with the proviso that n 4 and n 5 are not both 0;
n 6 is 0, 1 or 2;
each Y is individually H or halogen;
each p is individually 1, 2, 3, 4, 5, or 6;
each q is individually 0, 1, 2, 3, 4, 5, or 6;
each t is individually 1, 2, 3, 4, 5, or 6;
R 7 is H, —COR 8 , —CO 2 R 8 , —(CO)—NHR 8 , L 4 , L 5 , L 6 , or L 7 ;
R 8 is a substituted or an unsubstituted C 1 -C 6 alkyl-X 3 , a substituted or an unsubstituted C 1 -C 6 haloalkyl-X 3 , or —[(CY 2 ) p O(CY 2 ) q ] t CY 2 —X 3 ;
R 9 is H, —COR 8 , —CO 2 R 8 , —(CO)—NHR 8 , L 4 , L 5 , L 6 , or L 7 , with the proviso that exactly one of R 7 and R 9 is L 4 , L 5 , L 6 , or L 7 ; and
each X 3 is individually —H, —OH, —SH, or —NH 2 .
2 . A process of producing an immunoconjugate, comprising: reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with a conjugate of claim 1 under reaction conditions effective to form the immunoconjugate.
3 . An antibody or antigen-binding fragment thereof, comprising:
a) a heavy chain comprising:
VHCDR 1 comprising an amino acid sequence of SEQ ID NO:15;
VHCDR 2 comprising an amino acid sequence of SEQ ID NO:16; and
VHCDR 3 comprising an amino acid sequence of SEQ ID NO:17; and
b) a light chain comprising:
VLCDR 1 comprising an amino acid sequence of SEQ ID NO:22;
VLCDR 2 comprising an amino acid sequence of DAY; and
VLCDR 3 comprising an amino acid sequence of SEQ ID NO:24;
wherein the antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase like orphan receptor 1 (ROR1).
4 . The antibody or antigen-binding fragment thereof of claim 3 , comprising an amino acid sequence of any one of SEQ ID NOs: 19, 21, 26 or 28.
5 . One or more nucleic acids encoding the antibody or antigen-binding fragment thereof of claim 3 , wherein said antibody or antigen-binding fragment thereof is encoded by one or more nucleic acids comprising a sequence of SEQ ID NOs: 18, 20, 25, or 27.
6 . A host cell comprising the one or more nucleic acids of claim 5 .
7 . An antibody or antigen-binding fragment thereof, comprising:
a) a heavy chain comprising:
VHCDR 1 comprising an amino acid sequence of SEQ ID NO:29;
VHCDR 2 comprising an amino acid sequence of SEQ ID NO:30; and
VHCDR 3 comprising an amino acid sequence of SEQ ID NO:31; and
b) a light chain comprising:
VLCDR 1 comprising an amino acid sequence of SEQ ID NO:36;
VLCDR 2 comprising an amino acid sequence of DAS; and
VLCDR 3 comprising an amino acid sequence of SEQ ID NO:38;
wherein the antibody or antigen-binding fragment specifically binds to the extracellular domain of human receptor tyrosine kinase like orphan receptor 1 (ROR1).
8 . The antibody or antigen-binding fragment thereof of claim 7 , comprising an amino acid sequence of any one of SEQ ID NOs: 33, 35, 40 or 42.
9 . One or more nucleic acids encoding the antibody or antigen-binding fragment thereof of claim 7 , wherein said antibody or antigen-binding fragment thereof is encoded by one or more nucleic acids comprising a sequence of SEQ ID NOs: 32, 34, 39, or 41.
10 . A host cell comprising the one or more nucleic acids of claim 9 .
11 . An antibody or binding fragment thereof, wherein the heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 20 and the light chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 27 or a composition comprising said antibody or binding fragment thereof.
12 . The antibody or binding fragment thereof of claim 11 or a composition comprising said antibody or binding fragment thereof, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID NO:21 and the light chain comprises the polypeptide sequence of SEQ ID NO: 28.
13 . The antibody or binding fragment thereof of claim 11 or a composition comprising said antibody or binding fragment thereof, wherein said antibody or binding fragment thereof is conjugated to a molecule.
14 . The antibody or binding fragment thereof of claim 13 or a composition comprising said antibody or binding fragment thereof, wherein the molecule is a drug, toxin, or cytokine.
15 . An antibody or binding fragment thereof, wherein the heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 34 and the light chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 41 or a composition comprising said antibody or binding fragment thereof.
16 . The antibody or binding fragment thereof of claim 15 or a composition comprising said antibody or binding fragment thereof, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID NO:35 and the light chain comprises the polypeptide sequence of SEQ ID NO: 42.
17 . The antibody or binding fragment thereof of claim 15 or a composition comprising said antibody or binding fragment thereof, wherein said antibody or binding fragment thereof is conjugated to a molecule.
18 . The antibody or binding fragment thereof of claim 17 or a composition comprising said antibody or binding fragment thereof, wherein the molecule is a drug, toxin, or cytokine.