KUDIRKA ROMAS (US)
SAFINA BRIAN (US)
ZHOU MATTHEW (US)
WO2017132432A1 | 2017-08-03 |
US11033635B2 | 2021-06-15 | |||
EP0260744A2 | 1988-03-23 | |||
DE59209330C5 | 2013-10-31 |
CLAIMS: 1. An immunoconjugate comprising an antibody covalently attached to one or more STING agonist moieties by a linker, and having Formula I: or a pharmaceutically acceptable salt thereof, wherein: Ab is the antibody; p is an integer from 1 to 8; D is the STING agonist moiety having the formula: wherein Xa and Xb are independently selected from a five-membered heteroaryl, optionally substituted with R5; R1 is selected from the group consisting of H, F, Cl, Br, I, -CN, -OH, -O-(C1-C6 alkyl), and R5; R2a and R2b are independently selected from -C(=O)N(R6)2, and R5; R3 is selected from the group consisting of -(C1-C6 alkyldiyl)-, -(C1-C3 alkyldiyl)-O- (C1-C3 alkyldiyl)-, -(C1-C6 alkyldiyl)-O-, -(C1-C3 alkyldiyl)-O-(C1-C3 alkyldiyl)-O-, -(C2- C6 alkenyldiyl)-, -(C2-C6 alkenyldiyl)-O-, -(C2-C6 alkynyldiyl)-, -(C2-C6 alkynyldiyl)-O-, - (C1-C6 alkyldiyl)-N(R5)C(=O)-, -(C1-C6 alkyldiyl)-N(R5)S(O)2-, -(C1-C6 alkyldiyl)- N(R5)C(=O)-(C1-C6 alkyldiyl)-, -(C1-C6 alkyldiyl)-N(R5)S(O)2-(C1-C6 alkyldiyl)-, -(C1-C6 alkyldiyl)-N(R6)C(=O)-, -(C1-C6 alkyldiyl)-N(R6)S(O)2-, -(C1-C6 alkyldiyl)-N(R6)C(=O)- (C1-C6 alkyldiyl)-, and -(C1-C6 alkyldiyl)-N(R6)S(O)2-(C1-C6 alkyldiyl)-, where alkyldiyl, alkenyldiyl, and alkynyldiyl are optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, and R5; where one of Xa, Xb, R1, R2a, R2b and R3 is substituted with R5; R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl and C2-C6 alkynyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, - OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2; R5 is selected from the group consisting of: -(C1-C12 alkyldiyl)-*; -(C1-C12 alkyldiyl)-N(R6)-*; -(C1-C12 alkyldiyl)-O-*; -(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-*; -O-(C1-C12 alkyldiyl)-*; -O-(C1-C12 alkyldiyl)-N(R6)-*; -O-(C1-C12 alkyldiyl)-O-*; -O-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-*; -O-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-N(R6)-*; -OC(=O)N(R6)-*; -OC(=O)N(R6)-(C1-C12 alkyldiyl)-N(R6)-*; -N(R6)-*; -N(R6)-(C1-C12 alkyldiyl)-*; -N(R6)-(C1-C12 alkyldiyl)-N(R6)-*; -N(R6)-(C1-C12 alkyldiyl)-O-*; -N(R6)-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-*; -C(=O)N(R6)-*; -C(=O)N(R6)-(C1-C12 alkyldiyl)-*; -C(=O)N(R6)-(C1-C12 alkyldiyl)-N(R6)-*; -C(=O)N(R6)-(C1-C12 alkyldiyl)-O-*; -(C2-C20 heterocyclyldiyl)-*; -S(=O)2-(C2-C20 heterocyclyldiyl)-*; and -S(=O)2-(C2-C20 heterocyclyldiyl)-(C1-C12 alkyldiyl)-N(R6)-*; where the asterisk * indicates the attachment site of L; R6 is independently H or C1-C6 alkyl; L is the linker selected from the group consisting of: -C(=O)-PEG-; -C(=O)-PEG-C(=O)N(R6)-(C1-C12 alkyldiyl)-C(=O)-Gluc-; -C(=O)-PEG-O-; -C(=O)-PEG-O-C(=O)-; -C(=O)-PEG-C(=O)-; -C(=O)-PEG-C(=O)-PEP-; -C(=O)-PEG-N(R6)-; -C(=O)-PEG-N(R6)-C(=O)-; -C(=O)-PEG-N(R6)-PEG-C(=O)-PEP-; -C(=O)-PEG-N+(R6)2-PEG-C(=O)-PEP-; -C(=O)-PEG-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-; -C(=O)-PEG-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)N(R6)C(=O)-(C2-C5 monoheterocyclyldiyl)-; -C(=O)-PEG-SS-(C1-C12 alkyldiyl)-OC(=O)-; -C(=O)-PEG-SS-(C1-C12 alkyldiyl)-C(=O)-; -C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-; -C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-; -C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-N(R5)- C(=O); -C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)- N(R6)C(=O)-(C2-C5 monoheterocyclyldiyl)-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-C(=O)N(R6)-(C1-C12 alkyldiyl)-C(=O)-Gluc-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-O-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-O-C(=O)-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-C(=O)-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-N(R5)-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-N(R5)-C(=O)-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-C(=O)-PEP-; -succinimidyl-(CH2)m-C(=O)N(R6)-PEG-SS-(C1-C12 alkyldiyl)-OC(=O)-; -succinimidyl-(CH2)m-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-; -succinimidyl-(CH2)m-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)N(R6)C(=O)-; and -succinimidyl-(CH2)m-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)N(R6)C(=O)-(C2- C5 monoheterocyclyldiyl)-; PEG has the formula: -(CH2CH2O)n-(CH2)m-; m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: P where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates a point of attachment; Cyc is selected from C6-C20 aryldiyl and C1-C20 heteroaryldiyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure: ; R7 is selected from the group consisting of -CH(R8)O-, -CH2-, -CH2N(R8)-, and - CH(R8)O-C(=O)-, where R8 is selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and - C(=O)N(R9)2, where R9 is independently selected from the group consisting of H, C1-C12 alkyl, and -(CH2CH2O)n-(CH2)m-OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R9 groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, ^ CN, -CH3, -CH2CH3, -CH=CH2, -C{CH, -C{CCH3, -CH2CH2CH3, -CH(CH3)2, - CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, - C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, - CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, - CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, - N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n- (CH2)mCO2H, -O(CH2CH2O)nH, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, - SCH3, -S(O)2CH3, and -S(O)3H. 2. The immunoconjugate of claim 1 wherein the antibody is an immune checkpoint inhibitor. 3. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds PD-L1. 4. The immunoconjugate of claim 3 wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab, or a biosimilar or a biobetter thereof. 5. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds HER2. 6. The immunoconjugate of claim 5 wherein the antibody is selected from the group consisting of trastuzumab and pertuzumab, or a biosimilar or a biobetter thereof. 7. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds CEA. 8. The immunoconjugate of claim 7 wherein the antibody is labetuzumab, or a biosimilar or a biobetter thereof. 9. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds TROP2. 10. The immunoconjugate of claim 9 wherein the antibody is sacituzumab, or a biosimilar or a biobetter thereof. 11. The immunoconjugate of any one of claims 1 to 10 wherein Xa and Xb are independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl. 12. The immunoconjugate of claim 11 wherein Xa and Xb are each pyrazolyl, substituted with one or more groups selected from -CH3, -CH2CH3, -CH=CH2, -C{CH, - C{CCH3, -CH2CH2CH3, -CH(CH3)2, and -CH2CH(CH3)2. 13. The immunoconjugate of any one of claims 1 to 10 wherein one of Xa and Xb is substituted with R5. 14. The immunoconjugate of any one of claims 1 to 10 wherein R1 is selected from the group consisting of -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, and - OCH2CH2N(CH3)2. 15. The immunoconjugate of claim 14 wherein R1 is -OCH3. 16. The immunoconjugate of any one of claims 1 to 10 wherein R1 is F. 17. The immunoconjugate of any one of claims 1 to 10 wherein R2a and R2b are each -C(=O)NH2. 18. The immunoconjugate of any one of claims 1 to 10 wherein one of R2a and R2b is substituted with R5. 19. The immunoconjugate of any one of claims 1 to 10 wherein R3 is selected from - CH2CH2-, -CH=CH-, and -C{C-. 20. The immunoconjugate of any one of claims 1 to 10 wherein R3 is C2-C4 alkenyldiyl, substituted with one or more groups selected from F, -OH, and -OCH3. 21. The immunoconjugate of any one of claims 1 to 10 wherein R4 is -O-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-*. 22. The immunoconjugate of claim 21 wherein C1-C12 alkyldiyl is propyldiyl and C2- C20 heterocyclyldiyl is piperidiyl. 23. The immunoconjugate of any one of claims 1 to 10 wherein one of R1 and R4 is substituted with R5. 24. The immunoconjugate of any one of claims 1 to 10 wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-. 25. The immunoconjugate of any one of claims 1 to 10 wherein L is attached to a cysteine thiol of the antibody. 26. The immunoconjugate of any one of claims 1 to 10 wherein for the PEG, m is 1 or 2, and n is an integer from 2 to 10. 27. The immunoconjugate of claim 26 wherein n is 10. 28. The immunoconjugate of any one of claims 1 to 10 wherein L comprises PEP and PEP is a dipeptide and has the formula: . 29. The immunoconjugate of claim 28 wherein AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2; or AA1 and AA2 form a 5-membered ring proline amino acid. 30. The immunoconjugate of claim 28 wherein AA1 is -CH(CH3)2, and AA2 is -CH2CH2CH2NHC(O)NH2. 31. The immunoconjugate of claim 28 wherein AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H. 32. The immunoconjugate of claim 28 wherein PEP has the formula: wherein AA1 and AA2 are independently selected from a side chain of a naturally- occurring amino acid. 33. The immunoconjugate of any one of claims 1 to 10 wherein L comprises PEP and PEP is a tripeptide and has the formula: . 34. The immunoconjugate of any one of claims 1 to 10 wherein L comprises PEP and PEP is a tetrapeptide and has the formula: . 35. The immunoconjugate of claim 34 wherein AA1 is selected from the group consisting of Abu, Ala, and Val; AA2 is selected from the group consisting of Nle(O-Bzl), Oic and Pro; AA3 is selected from the group consisting of Ala and Met(O)2; and AA4 is selected from the group consisting of Oic, Arg(NO2), Bpa, and Nle(O-Bzl). 36. The immunoconjugate of any one of claims 1 to 10 wherein L comprises PEP and PEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala- Ala-Pro-Ala, Ala-Ala-Pro-Val, and Ala-Ala-Pro-Nva. 37. The immunoconjugate of any one of claims 1 to 10 wherein L comprises PEP and PEP is selected from the structures: . 38. The immunoconjugate of any one of claims 1 to 10 wherein L is selected from the structures: where the wavy line indicates the attachment to R5. 39. A STING agonist-linker intermediate compound having Formula II: II wherein Xa and Xb are independently selected from a five-membered heteroaryl, optionally substituted with R5; R1 is selected from the group consisting of H, F, Cl, Br, I, -CN, -OH, -O-(C1-C6 alkyl), and R5; R2a and R2b are independently selected from -C(=O)N(R6)2 and R5; R3 is selected from the group consisting of -(C1-C6 alkyldiyl)-, -(C1-C3 alkyldiyl)-O- (C1-C3 alkyldiyl)-, -(C1-C6 alkyldiyl)-O-, -(C1-C3 alkyldiyl)-O-(C1-C3 alkyldiyl)-O-, -(C2- C6 alkenyldiyl)-, -(C2-C6 alkenyldiyl)-O-, -(C2-C6 alkynyldiyl)-, -(C2-C6 alkynyldiyl)-O-, - (C1-C6 alkyldiyl)-N(R5)C(=O)-, -(C1-C6 alkyldiyl)-N(R5)S(O)2-, -(C1-C6 alkyldiyl)- N(R5)C(=O)-(C1-C6 alkyldiyl)-, -(C1-C6 alkyldiyl)-N(R5)S(O)2-(C1-C6 alkyldiyl)-, -(C1-C6 alkyldiyl)-N(R6)C(=O)-, -(C1-C6 alkyldiyl)-N(R6)S(O)2-, -(C1-C6 alkyldiyl)-N(R6)C(=O)- (C1-C6 alkyldiyl)-, and -(C1-C6 alkyldiyl)-N(R6)S(O)2-(C1-C6 alkyldiyl)-, where alkyldiyl, alkenyldiyl, and alkynyldiyl are optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, and R5; where one of Xa, Xb, R1, R2a, R2b and R3 is substituted with R5; R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl and C2-C6 alkynyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, - OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2; R5 is selected from the group consisting of: -(C1-C12 alkyldiyl)-L; -(C1-C12 alkyldiyl)-N(R6)-L; -(C1-C12 alkyldiyl)-O-L; -(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-L; -O-(C1-C12 alkyldiyl)-L; -O-(C1-C12 alkyldiyl)-N(R6)-L; -O-(C1-C12 alkyldiyl)-O-L; -O-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-L; -O-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-N(R6)-L; -OC(=O)N(R6)-L; -OC(=O)N(R6)-(C1-C12 alkyldiyl)-N(R6)-L; -N(R6)-L; -N(R6)-(C1-C12 alkyldiyl)-L; -N(R6)-(C1-C12 alkyldiyl)-N(R6)-L; -N(R6)-(C1-C12 alkyldiyl)-O-L; -N(R6)-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-L; -C(=O)N(R6)-L; -C(=O)N(R6)-(C1-C12 alkyldiyl)-L; -C(=O)N(R6)-(C1-C12 alkyldiyl)-N(R6)-L; -C(=O)N(R6)-(C1-C12 alkyldiyl)-O-L; -(C2-C20 heterocyclyldiyl)-L; -S(=O)2-(C2-C20 heterocyclyldiyl)-L; and -S(=O)2-(C2-C20 heterocyclyldiyl)-(C1-C12 alkyldiyl)-N(R6)-L; R6 is independently H or C1-C6 alkyl; L is the linker selected from the group consisting of: Q-C(=O)-PEG-; Q-C(=O)-PEG-Gluc-R7-; Q-C(=O)-PEG-O-; Q-C(=O)-PEG-O-C(=O)-; Q-C(=O)-PEG-C(=O)-; Q-C(=O)-PEG-C(=O)-PEP-; Q-C(=O)-PEG-N(R6)-; Q-C(=O)-PEG-N(R6)-C(=O)-; Q-C(=O)-PEG-N(R6)-PEG-C(=O)-PEP-; Q-C(=O)-PEG-N+(R6)2-PEG-C(=O)-PEP-; Q-C(=O)-PEG-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-; Q-C(=O)-PEG-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)N(R6)C(=O)-(C2-C5 monoheterocyclyldiyl)-; Q-C(=O)-PEG-SS-(C1-C12 alkyldiyl)-OC(=O)-; Q-C(=O)-PEG-SS-(C1-C12 alkyldiyl)-C(=O)-; Q-C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-; Q-C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-; Q-C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-N(R5)- C(=O); Q-C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)- N(R6)C(=O)-(C2-C5 monoheterocyclyldiyl)-; Q-(CH2)m-C(=O)N(R6)-PEG-; Q-(CH2)m-C(=O)N(R6)-PEG-Gluc-R7-; Q-(CH2)m-C(=O)N(R6)-PEG-O-; Q-(CH2)m-C(=O)N(R6)-PEG-O-C(=O)-; Q-(CH2)m-C(=O)N(R6)-PEG-C(=O)-; Q-(CH2)m-C(=O)N(R6)-PEG-N(R5)-; Q-(CH2)m-C(=O)N(R6)-PEG-N(R5)-C(=O)-; Q-(CH2)m-C(=O)N(R6)-PEG-C(=O)-PEP-; Q-(CH2)m-C(=O)N(R6)-PEG-SS-(C1-C12 alkyldiyl)-OC(=O)-; Q-(CH2)m-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)-; Q-(CH2)m-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)N(R6)C(=O)-; and Q-(CH2)m-C(=O)-PEP-N(R6)-(C1-C12 alkyldiyl)N(R6)C(=O)-(C2-C5 monoheterocyclyldiyl)-; PEG has the formula: -(CH2CH2O)n-(CH2)m-; m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates a point of attachment; Cyc is selected from C6-C20 aryldiyl and C1-C20 heteroaryldiyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure: ; R7 is selected from the group consisting of -CH(R8)O-, -CH2-, -CH2N(R8)-, and - CH(R8)O-C(=O)-, where R8 is selected from H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and - C(=O)N(R9)2, where R9 is independently selected from the group consisting of H, C1-C12 alkyl, and -(CH2CH2O)n-(CH2)m-OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R9 groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; Q is selected from the group consisting of N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from F, Cl, NO2, and SO3-; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, - CN, -CH3, -CH2CH3, -CH=CH2, -C{CH, -C{CCH3, -CH2CH2CH3, -CH(CH3)2, - CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, - C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, - CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, - CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, - N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n^ (CH2)mCO2H, -O(CH2CH2O)nH, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, - SCH3, -S(O)2CH3, and -S(O)3H. 40. The STING agonist-linker intermediate compound of claim 39 wherein Q is selected from: . 41. The STING agonist-linker intermediate compound of claim 39 wherein Q is phenoxy substituted with one or more groups independently selected from F, Cl, NO2, and SO3-. 42. The STING agonist-linker intermediate compound of claim 40 wherein Q is 2,3,5,6-tetrafluorophenoxy. 43. The STING agonist-linker intermediate compound of claim 40 wherein Q is 2,3,5,6-tetrafluoro-4-sulfonato-phenoxy. 44. The STING agonist-linker intermediate compound of claim 40 wherein Q is maleimide. 45. The STING agonist-linker intermediate compound of claim 39 wherein L is selected from the structures: where the wavy line indicates the attachment to R5. 46. A STING agonist-linker intermediate compound selected from Table 2. 47. An immunoconjugate prepared by conjugation of an antibody with a STING agonist-linker intermediate compound of any one of claims 39 to 46. 48. A pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate of any one of claims 1 to 10, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient. 49. A method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate according to any one of claims 1 to 10, to a patient in need thereof. 50. The method of claim 49, wherein the cancer is susceptible to a pro-inflammatory response induced by STING agonism. 51. The method of claim 49, wherein the cancer is selected from bladder cancer, salivary gland cancer, endometrial cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer. 52. Use of an immunoconjugate according to any one of claims 1 to 10 for treating cancer. 53. A method of preparing an immunoconjugate of Formula I of any one of claims 1 to 10 wherein a STING agonist-linker intermediate compound of claim 39 is conjugated with the antibody. |
Table 1. Asymmetric bis-benzimidazole compounds (BBI)
BIS-BENZIMIDAZOLE LINKER COMPOUNDS The immunoconjugates of the invention are prepared by conjugation of an antibody with an asymmetric bis-benzimidazole-linker (BBI-L) compound. The bis-benzimidazole-linker compounds comprise a STING agonist, bis-benzimidazole (BBI) moiety covalently attached to a linker unit (L). The linker units comprise functional groups and subunits which affect stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugates. The linker unit includes a reactive functional group which reacts, i.e. conjugates, with a reactive functional group of the antibody. For example, a nucleophilic group such as a lysine side chain amino of the antibody reacts with an electrophilic reactive functional group of the BBI-linker compound to form the immunoconjugate. Also, for example, a cysteine thiol of the antibody reacts with a maleimide or bromoacetamide group of the BBI-linker compound to form the immunoconjugate. Considerations for the design of the immunoconjugates of the invention include: (1) preventing the premature release of the bis-benzimidazole (BBI) moiety during in vivo circulation and (2) ensuring that a biologically active form of the BBI moiety is released at the desired site of action at an adequate rate. The complex structure of the immunoconjugate together with its functional properties requires careful design and selection of every component of the molecule including antibody, conjugation site, linker structure, and the bis-benzimidazole compound. The linker determines the mechanism and rate of adjuvant release. Generally, the linker unit (L) may be cleavable or non-cleavable. Cleavable linker units may include a peptide sequence which is a substrate for certain proteases such as Cathepsins which recognize and cleave the peptide linker unit, separating the STING agonist from the antibody (Caculitan NG, et al (2017) Cancer Res.77(24):7027-7037). Cleavable linker units may include labile functionality such as an acid-sensitive disulfide group (Kellogg, BA et al (2011) Bioconjugate Chem.22, 717−727; Ricart, A. D. et al (2011) Clin. Cancer Res.17, 6417−6427; Pillow, T., et al (2017) Chem. Sci. 8, 366-370; Zhang D, et al (2016) ACS Med Chem Lett.7(11):988-993). In some embodiments , the linker is non-cleavable under physiological conditions . As used herein , the term “physiological conditions” refers to a temperature range of 20-40 degrees Celsius , atmospheric pressure ( i.e. , 1 atm ) , a pH of about 6 to about 8 , and the one or more physiological enzymes, proteases, acids , and bases. One advantage of a non-cleavable linker between the antibody and STING agonist in an immunoconjugate is minimizing premature payload release and corresponding toxicity. STING is a broadly expressed receptor, therefore a particularly relevant consideration. In one embodiment, the invention includes a peptide linking unit, i.e. L or linker, between the cell-binding agent and the immunostimulatory moiety, comprising a peptide radical based on a linear sequence of specific amino acid residues which can be selectively cleaved by a protease such as a cathepsin, a tumor-associated elastase enzyme or an enzyme with protease- like or elastase-like activity. The peptide radical may be about two to about twelve amino acids. Enzymatic cleavage of a bond within the peptide linker releases an active form of the immunostimulatory moiety. This leads to an increase in the tissue specificity of the conjugates according to the invention and thus to an additional decrease of toxicity of the conjugates according to the invention in other tissue types. The linker provides sufficient stability of the immunoconjugate in biological media, e.g. culture medium or serum and, at the same time, the desired intracellular action within tumor tissue as a result of its specific enzymatic or hydrolytic cleavability with release of the immunostimulatory moiety, i.e. “payload”. The enzymatic activity of a protease, cathepsin, or elastase can catalyze cleavage of a covalent bond of the immunoconjugate under physiological conditions. The enzymatic activity being the expression product of cells associated with tumor tissue. The enzymatic activity on the cleavage site of the targeting peptide converts the immunoconjugate to an active immunostimulatory drug free of targeting peptide and linking group. The cleavage site may be specifically recognized by the enzyme.. Cathepsin or elastase may catalyze the cleavage of a specific peptidic bond between the C-terminal amino acid residue of the specific peptide and the immunostimulatory moiety of the immunoconjugate. In one embodiment, the invention includes a linking unit, i.e. L or linker, between the cell-binding agent and the immunostimulatory moiety, comprising a substrate for glucuronidase (Jeffrey SC, et al (2006) Bioconjug Chem.17(3):831-40), or sulfatase (Bargh JD, et al (2020) Chem Sci.11(9):2375-2380) cleavage. In particular, L may comprise a Gluc unit selected from the formulas: . Specific cleavage of the immunoconjugates of the invention takes advantage of the presence of tumor infiltrating cells of the immune system and leukocyte-secreted enzymes, to promote the activation of an anticancer drug at the tumor site. Reactive electrophilic reactive functional groups (Q in Formula II) suitable for the BBI- linker compounds include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N- hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethyl phosphines (amine reactive); maleimides (thiol reactive); halogenated acetamides such as N-iodoacetamides (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (C-H) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) and sulfotetrafluorophenyl (STP) esters (amine reactive); imidoesters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Further reagents include, but are not limited, to those described in Hermanson, Bioconjugate Techniques 2 nd Edition, Academic Press, 2008. The invention provides solutions to the limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers such as those comprising peptide units and substrates for protease may be labile in the blood stream, thereby releasing unacceptable amounts of the adjuvant/drug prior to internalization in a target cell (Khot, A. et al (2015) Bioanalysis 7(13):1633–1648). Other linkers may provide stability in the bloodstream, but intracellular release effectiveness may be negatively impacted. Linkers that provide for desired intracellular release typically have poor stability in the bloodstream. Alternatively stated, bloodstream stability and intracellular release are typically inversely related. In addition, in standard conjugation processes, the amount of adjuvant/drug moiety loaded on the antibody, i.e. drug loading, the amount of aggregate that is formed in the conjugation reaction, and the yield of final purified conjugate that can be obtained are interrelated. For example, aggregate formation is generally positively correlated to the number of equivalents of adjuvant/drug moiety and derivatives thereof conjugated to the antibody. Under high drug loading, formed aggregates must be removed for therapeutic applications. As a result, drug loading-mediated aggregate formation decreases immunoconjugate yield and can render process scale-up difficult. Exemplary embodiments include a STING agonist, bis-benzimidazole-linker compound of Formula II: wherein X a and X b are independently selected from a five-membered heteroaryl, optionally substituted with R 5 ; R 1 is selected from the group consisting of H, F, Cl, Br, I, -CN, -OH, -O-(C 1 -C 6 alkyl), and R 5 ; R 2a and R 2b are independently selected from -C(=O)N(R 6 ) 2 and R 5 ; R 3 is selected from the group consisting of -(C 1 -C 6 alkyldiyl)-, -(C 1 -C 3 alkyldiyl)-O- (C 1 -C 3 alkyldiyl)-, -(C 1 -C 6 alkyldiyl)-O-, -(C 1 -C 3 alkyldiyl)-O-(C 1 -C 3 alkyldiyl)-O-, -(C 2 - C 6 alkenyldiyl)-, -(C 2 -C 6 alkenyldiyl)-O-, -(C 2 -C 6 alkynyldiyl)-, -(C 2 -C 6 alkynyldiyl)-O-, - (C 1 -C 6 alkyldiyl)-N(R 5 )C(=O)-, -(C 1 -C 6 alkyldiyl)-N(R 5 )S(O) 2 -, -(C 1 -C 6 alkyldiyl)- N(R 5 )C(=O)-(C 1 -C 6 alkyldiyl)-, -(C 1 -C 6 alkyldiyl)-N(R 5 )S(O) 2 -(C 1 -C 6 alkyldiyl)-, -(C 1 -C 6 alkyldiyl)-N(R 6 )C(=O)-, -(C 1 -C 6 alkyldiyl)-N(R 6 )S(O) 2 -, -(C 1 -C 6 alkyldiyl)-N(R 6 )C(=O)- (C 1 -C 6 alkyldiyl)-, and -(C 1 -C 6 alkyldiyl)-N(R 6 )S(O) 2 -(C 1 -C 6 alkyldiyl)-, where alkyldiyl, alkenyldiyl, and alkynyldiyl are optionally substituted with one or more groups selected from F, Cl, -OH, -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 , and R 5 ; where one of X a , X b , R 1 , R 2a , R 2b and R 3 is substituted with R 5 ; R 4 is selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH 3 , - OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 ; R 5 is selected from the group consisting of: -(C 1 -C 12 alkyldiyl)-L; -(C 1 -C 12 alkyldiyl)-N(R 6 )-L; -(C 1 -C 12 alkyldiyl)-O-L; -(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-L; -O-(C 1 -C 12 alkyldiyl)-L; -O-(C 1 -C 12 alkyldiyl)-N(R 6 )-L; -O-(C 1 -C 12 alkyldiyl)-O-L; -O-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-L; -O-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-N(R 6 )-L; -OC(=O)N(R 6 )-L; -OC(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )-L; -N(R 6 )-L; -N(R 6 )-(C 1 -C 12 alkyldiyl)-L; -N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )-L; -N(R 6 )-(C 1 -C 12 alkyldiyl)-O-L; -N(R 6 )-(C1-C12 alkyldiyl)-(C2-C20 heterocyclyldiyl)-L; -C(=O)N(R 6 )-L; -C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-L; -C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )-L; -C(=O)N(R 6 )-(C1-C12 alkyldiyl)-O-L; -(C 2 -C 20 heterocyclyldiyl)-L; -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-L; and -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 alkyldiyl)-N(R 6 )-L; R 6 is independently H or C 1 -C 6 alkyl; L is the linker selected from the group consisting of: Q-C(=O)-PEG-; Q-C(=O)-PEG-Gluc-R 7 -; Q-C(=O)-PEG-O-; Q-C(=O)-PEG-O-C(=O)-; Q-C(=O)-PEG-C(=O)-; Q-C(=O)-PEG-C(=O)-PEP-; Q-C(=O)-PEG-N(R 6 )-; Q-C(=O)-PEG-N(R 6 )-C(=O)-; Q-C(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-; Q-C(=O)-PEG-N + (R 6 ) 2 -PEG-C(=O)-PEP-; Q-C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-; Q-C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-; Q-C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-; Q-C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-C(=O)-; Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-; Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-; Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 5 )- C(=O); Q-C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)- N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-Gluc-R 7 -; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-C(=O)-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-; Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-; Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-; and Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-; PEG has the formula: -(CH 2 CH 2 O) n -(CH 2 ) m -; m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: P where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates a point of attachment; Cyc is selected from C 6 -C 20 aryldiyl and C 1 -C 20 heteroaryldiyl, optionally substituted with one or more groups selected from F, Cl, NO 2 , -OH, -OCH 3 , and a glucuronic acid having the structure: R 7 is selected from the group consisting of -CH(R 8 )O-, -CH 2 -, -CH 2 N(R 8 )-, and - CH(R 8 )O-C(=O)-, where R 8 is selected from H, C 1 -C 6 alkyl, C(=O)-C 1 -C 6 alkyl, and - C(=O)N(R 9 ) 2 , where R 9 is independently selected from the group consisting of H, C 1 -C 12 alkyl, and -(CH 2 CH 2 O) n -(CH 2 ) m -OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R 9 groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; Q is selected from the group consisting of N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from F, Cl, NO2, and SO3 - ; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, - CN, -CH 3 , -CH 2 CH 3 , -CH=CH 2 , -C{CH, -C{CCH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , - CH 2 CH(CH 3 ) 2 , -CH 2 OH, -CH 2 OCH 3 , -CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -CH(OH)CH(CH 3 ) 2 , - C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, - CH 2 CHF 2 , -CH(CH 3 )CN, -C(CH 3 ) 2 CN, -CH 2 CN, -CH 2 NH 2 , -CH 2 NHSO 2 CH 3 , -CH 2 NHCH 3 , -CH 2 N(CH 3 ) 2 , -CO 2 H, -COCH 3 , -CO 2 CH 3 , -CO 2 C(CH 3 ) 3 , -COCH(OH)CH 3 , -CONH 2 , - CONHCH 3 , -CON(CH 3 ) 2 , -C(CH 3 ) 2 CONH 2 , -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -NHCOCH 3 , - N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, -NHC(=NH)CH 3 , -NHC(=NH)NH 2 , -NHC(=O)NH 2 , -NO 2 , =O, -OH, -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 , -O(CH 2 CH 2 O) n - (CH 2 ) m CO 2 H, -O(CH 2 CH 2 O) n H, -OCH 2 F, -OCHF 2 , -OCF 3 , -OP(O)(OH) 2 , -S(O) 2 N(CH 3 ) 2 , - SCH 3 , -S(O) 2 CH 3 , and -S(O) 3 H. An exemplary embodiment of the bis-benzimidazole-linker compound of Formula II includes wherein Q is selected from: An exemplary embodiment of the bis-benzimidazole-linker compound of Formula II includes wherein Q is phenoxy substituted with one or more groups independently selected from F, Cl, NO 2 , and SO 3 -. An exemplary embodiment of the bis-benzimidazole-linker compound of Formula II includes wherein Q is 2,3,5,6-tetrafluorophenoxy. An exemplary embodiment of the bis-benzimidazole-linker compound of Formula II includes wherein Q is 2,3,5,6-tetrafluoro-4-sulfonato-phenoxy. An exemplary embodiment of the bis-benzimidazole-linker compound of Formula II includes wherein Q is maleimide. An exemplary embodiment of the bis-benzimidazole-linker compound of Formula II includes wherein L is selected from the structures: where the wavy line indicates the attachment to R 5 . Exemplary embodiments of the asymmetric bis-benzimidazole, STING agonist-linker intermediate compound (BBI-L) are shown in Table 2. Each STING agonist-linker intermediate compound was prepared and characterized by nmr, mass spectrometry and shown to have the structure and mass indicated. The STING agonist-linker intermediate compounds of Table 2 may demonstrate the surprising and unexpected property of STING agonist selectivity which may predict useful therapeutic activity to treat cancer and other disorders when conjugated to an antibody. Table 2. Bis-benzimidazole -linker (BBI-L) Formula II compounds IMMUNOCONJUGATES The immunoconjugates of the invention induce target-specific activation of immune effector cells such as myeloid cells as well as tumor cells expressing STING themselves. Tumor targeting brings specificity to minimize off-target STING activation, and the immunoconjugate enables phagocytosis to not only increase activation of the effector cells but also immune complex uptake and subsequent tumor antigen processing and presentation. Exemplary embodiments of immunoconjugates comprise an antibody covalently attached to one or more STING agonist, asymmetric bis-benzimidazole (BBI) moieties by a linker, and having Formula I: or a pharmaceutically acceptable salt thereof, wherein: Ab is the antibody; p is an integer from 1 to 8; D is the STING agonist moiety having the formula:
wherein X a and X b are independently selected from a five-membered heteroaryl, optionally substituted R 1 is selected from the group consisting of H, F, Cl, Br, I, -CN, -OH, -O-(C 1 -C 6 alkyl), and R 5 ; R 2a and R 2b are independently selected from -C(=O)N(R 6 )2 and R 5 ; R 3 is selected from the group consisting of -(C 1 -C 6 alkyldiyl)-, -(C 1 -C 3 alkyldiyl)-O- (C 1 -C 3 alkyldiyl)-, -(C 1 -C 6 alkyldiyl)-O-, -(C 1 -C 3 alkyldiyl)-O-(C 1 -C 3 alkyldiyl)-O-, -(C 2 - C 6 alkenyldiyl)-, -(C 2 -C 6 alkenyldiyl)-O-, -(C 2 -C 6 alkynyldiyl)-, -(C 2 -C 6 alkynyldiyl)-O-, - (C1-C6 alkyldiyl)-N(R 5 )C(=O)-, -(C1-C6 alkyldiyl)-N(R 5 )S(O)2-, -(C1-C6 alkyldiyl)- N(R 5 )C(=O)-(C 1 -C 6 alkyldiyl)-, -(C 1 -C 6 alkyldiyl)-N(R 5 )S(O) 2 -(C 1 -C 6 alkyldiyl)-, -(C 1 -C 6 alkyldiyl)-N(R 6 )C(=O)-, -(C 1 -C 6 alkyldiyl)-N(R 6 )S(O) 2 -, -(C 1 -C 6 alkyldiyl)-N(R 6 )C(=O)- (C 1 -C 6 alkyldiyl)-, and -(C 1 -C 6 alkyldiyl)-N(R 6 )S(O) 2 -(C 1 -C 6 alkyldiyl)-, where alkyldiyl, alkenyldiyl, and alkynyldiyl are optionally substituted with one or more groups selected from F, Cl, -OH, -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 , and R 5 ; where one of X a , X b , R 1 , R 2a , R 2b and R 3 is substituted with R 5 ; R 4 is selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH 3 , - OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 ; R 5 is selected from the group consisting of: -(C 1 -C 12 alkyldiyl)-*; -(C 1 -C 12 alkyldiyl)-N(R 6 )-*; -(C 1 -C 12 alkyldiyl)-O-*; -(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-*; -O-(C 1 -C 12 alkyldiyl)-*; -O-(C 1 -C 12 alkyldiyl)-N(R 6 )-*; -O-(C 1 -C 12 alkyldiyl)-O-*; -O-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-*; -O-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-N(R 6 )-*; -OC(=O)N(R 6 )-*; -OC(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )-*; -N(R 6 )-*; -N(R 6 )-(C 1 -C 12 alkyldiyl)-*; -N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )-*; -N(R 6 )-(C 1 -C 12 alkyldiyl)-O-*; -N(R 6 )-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-*; -C(=O)N(R 6 )-*; -C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-*; -C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 6 )-*; -C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-O-*; -(C 2 -C 20 heterocyclyldiyl)-*; -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-*; and -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-(C 1 -C 12 alkyldiyl)-N(R 6 )-*; where the asterisk * indicates the attachment site of L; R 6 is independently H or C 1 -C 6 alkyl; L is the linker selected from the group consisting of: -C(=O)-PEG-; -C(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-C(=O)-Gluc-; -C(=O)-PEG-O-; -C(=O)-PEG-O-C(=O)-; -C(=O)-PEG-C(=O)-; -C(=O)-PEG-C(=O)-PEP-; -C(=O)-PEG-N(R 6 )-; -C(=O)-PEG-N(R 6 )-C(=O)-; -C(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-; -C(=O)-PEG-N + (R 6 ) 2 -PEG-C(=O)-PEP-; -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-; -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C1-C12 alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; -C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-; -C(=O)-PEG-SS-(C 1 -C 12 alkyldiyl)-C(=O)-; -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-; -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-; -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-N(R 5 )- C(=O); -C(=O)-(C 1 -C 12 alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)- N(R 6 )C(=O)-(C 2 -C 5 monoheterocyclyldiyl)-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 alkyldiyl)-C(=O)-Gluc-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-C(=O)-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-; -succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 alkyldiyl)-OC(=O)-; -succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)-; -succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-; and -succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 alkyldiyl)N(R 6 )C(=O)-(C 2 - C 5 monoheterocyclyldiyl)-; PEG has the formula: -(CH 2 CH 2 O) n -(CH 2 ) m -; m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: PEP has the formula: where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates a point of attachment; Cyc is selected from C 6 -C 20 aryldiyl and C 1 -C 20 heteroaryldiyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure: ; R 7 is selected from the group consisting of -CH(R 8 )O-, -CH 2 -, -CH 2 N(R 8 )-, and - CH(R 8 )O-C(=O)-, where R 8 is selected from H, C 1 -C 6 alkyl, C(=O)-C 1 -C 6 alkyl, and - C(=O)N(R 9 ) 2 , where R 9 is independently selected from the group consisting of H, C 1 -C 12 alkyl, and -(CH 2 CH 2 O) n -(CH 2 ) m -OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R 9 groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, - CN, -CH 3 , -CH 2 CH 3 , -CH=CH 2 , -C{CH, -C{CCH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , - CH 2 CH(CH 3 ) 2 , -CH 2 OH, -CH 2 OCH 3 , -CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -CH(OH)CH(CH 3 ) 2 , - C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, - CH 2 CHF 2 , -CH(CH 3 )CN, -C(CH 3 ) 2 CN, -CH 2 CN, -CH 2 NH 2 , -CH 2 NHSO 2 CH 3 , -CH 2 NHCH 3 , -CH 2 N(CH 3 ) 2 , -CO 2 H, -COCH 3 , -CO 2 CH 3 , -CO 2 C(CH 3 ) 3 , -COCH(OH)CH 3 , -CONH 2 , - CONHCH 3 , -CON(CH 3 ) 2 , -C(CH 3 ) 2 CONH 2 , -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -NHCOCH 3 , - N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, -NHC(=NH)CH 3 , -NHC(=NH)NH 2 , -NHC(=O)NH 2 , -NO 2 , =O, -OH, -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 N(CH 3 ) 2 , -O(CH 2 CH 2 O) n ^ (CH 2 ) m CO 2 H, -O(CH 2 CH 2 O) n H, -OCH 2 F, -OCHF 2 , -OCF 3 , -OP(O)(OH) 2 , -S(O) 2 N(CH 3 ) 2 , - SCH 3 , -S(O) 2 CH 3 , and -S(O) 3 H. An exemplary embodiment of the immunoconjugate of Formula I includes wherein X a and X b are independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl. An exemplary embodiment of the immunoconjugate of Formula I includes wherein X a and X b are each pyrazolyl, substituted with one or more groups selected from -CH 3 , -CH 2 CH 3 , -CH=CH 2 , -C{CH, -C{CCH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , and -CH 2 CH(CH 3 ) 2 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein one of X a and X b is substituted with R 5 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 1 is selected from the group consisting of -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 OH, and -OCH 2 CH 2 N(CH 3 ) 2 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 1 is -OCH 3 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 1 is F. An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 2a and R 2b are each -C(=O)NH 2 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein one of R 2a and R 2b is substituted with R 5 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 3 is selected from -CH 2 CH 2 -, -CH=CH-, and -C{C-. An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 3 is C2-C4 alkenyldiyl, substituted with one or more groups selected from F, -OH, and -OCH3. An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 4 is -O-(C 1 -C 12 alkyldiyl)-(C 2 -C 20 heterocyclyldiyl)-*. An exemplary embodiment of the immunoconjugate of Formula I includes wherein C 1 - C 12 alkyldiyl is propyldiyl and C 2 -C 20 heterocyclyldiyl is piperidiyl. An exemplary embodiment of the immunoconjugate of Formula I includes wherein one of R 1 and R 4 is substituted with R 5 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-. An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is attached to a cysteine thiol of the antibody. An exemplary embodiment of the immunoconjugate of Formula I includes wherein for the PEG, m is 1 or 2, and n is an integer from 2 to 10, or wherein n is 10. An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is a dipeptide and has the formula: ; and wherein AA 1 and AA 2 are independently selected from H, -CH 3 , -CH(CH 3 ) 2 , -CH 2 (C 6 H 5 ), -CH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 CH 2 CH 2 NHC(NH)NH 2 , -CHCH(CH 3 )CH 3 , -CH 2 SO 3 H, and -CH 2 CH 2 CH 2 NHC(O)NH 2 ; or AA 1 and AA 2 form a 5-membered ring proline amino acid. An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA 1 is -CH(CH3)2, and AA2 is -CH2CH2CH2NHC(O)NH2. An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA 1 and AA 2 are independently selected from GlcNAc aspartic acid, -CH 2 SO 3 H, and -CH 2 OPO 3 H. An exemplary embodiment of the immunoconjugate of Formula I includes wherein PEP has the formula: wherein AA 1 and AA 2 are independently selected from a side chain of a naturally- occurring amino acid. An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is a tripeptide and has the formula: . An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is a tetrapeptide and has the formula: and wherein: AA1 is selected from the group consisting of Abu, Ala, and Val; AA 2 is selected from the group consisting of Nle(O-Bzl), Oic and Pro; AA 3 is selected from the group consisting of Ala and Met(O) 2 ; and AA 4 is selected from the group consisting of Oic, Arg(NO 2 ), Bpa, and Nle(O-Bzl). An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala-Ala-Pro-Ala, Ala-Ala-Pro-Val, and Ala-Ala-Pro-Nva. An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is selected from the structures: ; An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is selected from the structures:
where the wavy line indicates the attachment to R 5 . The invention includes all reasonable combinations, and permutations of the features, of the Formula I embodiments. In certain embodiments, the immunoconjugate compounds of the invention include those with immunostimulatory activity. The antibody-drug conjugates of the invention selectively deliver an effective dose of a bis-benzimidazole drug to tumor tissue, whereby greater selectivity (i.e., a lower efficacious dose) may be achieved while increasing the therapeutic index (“therapeutic window”) relative to unconjugated bis-benzimidazole. Drug loading is represented by p, the number of BBI moieties per antibody in an immunoconjugate of Formula I. Drug (BBI) loading may range from 1 to about 8 drug moieties (D) per antibody. Immunoconjugates of Formula I include mixtures or collections of antibodies conjugated with a range of drug moieties, from 1 to about 8. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues such as lysine and cysteine. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. In such aspects, p may be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges thereof, such as from 1 to 8 or from 2 to 5. In any such aspect, p and n are equal (i.e., p = n = 1, 2, 3, 4, 5, 6, 7, or 8, or some range there between). Exemplary immunoconjugates of Formula I include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym.502:123-138). In some embodiments, one or more free cysteine residues are already present in an antibody forming intra-chain and inter-chain disulfide bonds (native disulfide groups), without the use of engineering, in which case the existing free, reduced cysteine residues may be used to conjugate the antibody to a drug. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues. For some immunoconjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments described herein, an antibody may have only one or a limited number of cysteine thiol groups, or may have only one or a limited number of sufficiently reactive thiol groups, to which the drug may be attached. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with a BBI-linker compound of Formula II. In certain embodiments, higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an immunoconjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug/antibody ratio) of an immunoconjugate may be controlled in different ways, and for example, by: (i) limiting the molar excess of the BBI-linker intermediate compound relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive denaturing conditions for optimized antibody reactivity. It is to be understood that where more than one nucleophilic group of the antibody reacts with a drug, then the resulting product is a mixture of immunoconjugate compounds with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual immunoconjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K.J., et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No.624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, March 27- 31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, a homogeneous immunoconjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography. An exemplary embodiment of the immunoconjugate of Formula I is selected from the Table 3 Immunoconjugates. Assessment of Immunoconjugate Activity in vitro may be conducted according to the methods of Examples 203 and 204. Table 3. BBI Immunoconjugates (IC) STING activation is canonically associated with induction of type I/III IFNs (interferons) through IRF3 (interferon regulatory factor 3) signaling, but can also induce proinflammatory cytokines such as TNFD (tumor necrosis factor alpha) through the NF-NB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway. Certain immunoconjugates may demonstrate the ability to elicit IFNO1 (interferon lambda 1) as well as TNFD, consistent with STING activation (Example 203). COMPOSITIONS OF IMMUNOCONJUGATES The invention provides a composition, e.g., a pharmaceutically or pharmacologically acceptable composition or formulation, comprising a plurality of immunoconjugates as described herein and optionally a carrier therefor, e.g., a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates can be the same or different in the composition, i.e., the composition can comprise immunoconjugates that have the same number of BBI adjuvants linked to the same positions on the antibody construct and/or immunoconjugates that have the same number of BBI adjuvants linked to different positions on the antibody construct, that have different numbers of adjuvants linked to the same positions on the antibody construct, or that have different numbers of adjuvants linked to different positions on the antibody construct. In an exemplary embodiment, a composition comprising the immunoconjugate compounds comprises a mixture of the immunoconjugate compounds, wherein the average drug (BBI) loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5. A composition of immunoconjugates of the invention can have an average adjuvant to antibody construct ratio (DAR) of about 0.4 to about 10. A skilled artisan will recognize that the number of BBI adjuvants conjugated to the antibody construct may vary from immunoconjugate to immunoconjugate in a composition comprising multiple immunoconjugates of the invention and thus the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average which may be referred to as the drug to antibody ratio (DAR). The adjuvant to antibody construct (e.g., antibody) ratio can be assessed by any suitable means, many of which are known in the art. The average number of adjuvant moieties per antibody (DAR) in preparations of immunoconjugates from conjugation reactions may be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of immunoconjugates in a composition in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous immunoconjugates where p is a certain value from immunoconjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the invention can be formulated for parenteral administration, such as IV administration or administration into a body cavity or lumen of an organ. Alternatively, the immunoconjugates can be injected into the tumor (intra-tumorally). Compositions for injection will commonly comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g., Ringer's solution. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These compositions desirably are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The composition can contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. In certain embodiments, the concentration of an immunoconjugate in a solution formulation for injection will range from about 0.1% (w/w) to about 10% (w/w). METHOD OF TREATING CANCER WITH IMMUNOCONJUGATES The invention provides a method for treating cancer. The method includes administering a therapeutically effective amount of an immunoconjugate as described herein (e.g., as a composition as described herein) to a subject in need thereof, e.g., a subject that has cancer and is in need of treatment for the cancer. The method includes administering a therapeutically effective amount of an immunoconjugate (IC) of the invention. It is contemplated that the immunoconjugate of the present invention may be used to treat various hyperproliferative diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary hyperproliferative disorders include benign or malignant solid tumors and hematological disorders such as leukemia and lymphoid malignancies. In another aspect, an immunoconjugate for use as a medicament is provided. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual comprising administering to the individual an effective amount of the immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein. In a further aspect, the invention provides for the use of an immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cancer, the method comprising administering to an individual having cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein. Carcinomas are malignancies that originate in the epithelial tissues. Epithelial cells cover the external surface of the body, line the internal cavities, and form the lining of glandular tissues. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer that begins in glandular (secretory) cells such as cancers of the breast, pancreas, lung, prostate, stomach, gastroesophageal junction, and colon) adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma; carcinoma of the breast; basal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat cell carcinoma; large cell lung carcinoma; small cell lung carcinoma; non-small cell lung carcinoma; and the like. Carcinomas may be found in prostrate, pancreas, colon, brain (usually as secondary metastases), lung, breast, and skin. Soft tissue tumors are a highly diverse group of rare tumors that are derived from connective tissue. Examples of soft tissue tumors include, but are not limited to, alveolar soft part sarcoma; angiomatoid fibrous histiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma; extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplastic small round-cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing’s sarcoma; fibromatosis (Desmoid); fibrosarcoma, infantile; gastrointestinal stromal tumor; bone giant cell tumor; tenosynovial giant cell tumor; inflammatory myofibroblastic tumor; uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindle cell or pleomorphic lipoma; atypical lipoma; chondroid lipoma; well-differentiated liposarcoma; myxoid/round cell liposarcoma; pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma; high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignant peripheral nerve sheath tumor; mesothelioma; neuroblastoma; osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolar rhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignant schwannoma; synovial sarcoma; Evan’s tumor; nodular fasciitis; desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis (PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovial sarcoma; malignant peripheral nerve sheath tumor; neurofibroma; pleomorphic adenoma of soft tissue; and neoplasias derived from fibroblasts, myofibroblasts, histiocytes, vascular cells/endothelial cells, and nerve sheath cells. A sarcoma is a rare type of cancer that arises in cells of mesenchymal origin, e.g., in bone or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissue. Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to, Askin's tumor; sarcoma botryoides; chondrosarcoma; Ewing's sarcoma; malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangiosarcoma (more commonly referred to as “angiosarcoma”); Kaposi’s sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma). A teratoma is a type of germ cell tumor that may contain several different types of tissue (e.g., can include tissues derived from any and/or all of the three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas occur most often in the ovaries in women, the testicles in men, and the tailbone in children. Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma may begin in a mole (skin melanoma), but can also begin in other pigmented tissues, such as in the eye or in the intestines. Merkel cell carcinoma is a rare type of skin cancer that usually appears as a flesh-colored or bluish-red nodule on the face, head or neck. Merkel cell carcinoma is also called neuroendocrine carcinoma of the skin. In some embodiments, methods for treating Merkel cell carcinoma include administering an immunoconjugate containing an antibody construct that is capable of binding Trop2 (e.g., sacituzumab, biosimilars thereof, or biobetters thereof). In some embodiments, the Merkel cell carcinoma has metastasized when administration occurs. Leukemias are cancers that start in blood-forming tissue, such as the bone marrow, and cause large numbers of abnormal blood cells to be produced and enter the bloodstream. For example, leukemias can originate in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for how quickly the disease develops and progresses (e.g., acute versus chronic) and for the type of white blood cell that is affected (e.g., myeloid versus lymphoid). Myeloid leukemias are also called myelogenous or myeloblastic leukemias. Lymphoid leukemias are also called lymphoblastic or lymphocytic leukemia. Lymphoid leukemia cells may collect in the lymph nodes, which can become swollen. Examples of leukemias include, but are not limited to, Acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia (CML), and Chronic lymphocytic leukemia (CLL). Lymphomas are cancers that begin in cells of the immune system. For example, lymphomas can originate in bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphomas. One category of lymphoma is Hodgkin lymphoma (HL), which is marked by the presence of a type of cell called the Reed-Sternberg cell. There are currently 6 recognized types of HL. Examples of Hodgkin lymphomas include nodular sclerosis classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte- depletion CHL, lymphocyte-rich CHL, and nodular lymphocyte predominant HL. The other category of lymphoma is non-Hodgkin lymphomas (NHL), which includes a large, diverse group of cancers of immune system cells. Non-Hodgkin lymphomas can be further divided into cancers that have an indolent (slow-growing) course and those that have an aggressive (fast-growing) course. There are currently 61 recognized types of NHL. Examples of non-Hodgkin lymphomas include, but are not limited to, AIDS-related Lymphomas, anaplastic large-cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt’s lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia/small lymphocytic lymphoma, cutaneous T-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Cell lymphoma, follicular lymphoma, hepatosplenic gamma- delta T-Cell lymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatric lymphoma, peripheral T-Cell lymphomas, primary central nervous system lymphoma, transformed lymphomas, treatment- related T-Cell lymphomas, and Waldenstrom's macroglobulinemia. Brain cancers include any cancer of the brain tissues. Examples of brain cancers include, but are not limited to, gliomas (e.g., glioblastomas, astrocytomas, oligodendrogliomas, ependymomas, and the like), meningiomas, pituitary adenomas, and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas). Immunoconjugates of the invention can be used either alone or in combination with other agents in a therapy. For instance, an immunoconjugate may be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the immunoconjugate can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Immunoconjugates can also be used in combination with radiation therapy. The immunoconjugates of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. The immunoconjugate described herein can be used to treat the same types of cancers as sacituzumab, sacituzumab govitecan, biosimilars thereof, and biobetters thereof, particularly breast cancer, especially triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma. In some embodiments, the immunoconjugates described herein may be effective in the treatment of bladder cancer, salivary gland cancer, endometrial cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer. The immunoconjugate is administered to a subject in need thereof in any therapeutically effective amount using any suitable dosing regimen, such as the dosing regimens utilized for sacituzumab, sacituzumab govitecan, biosimilars thereof, and biobetters thereof. For example, the methods can include administering the immunoconjugate to provide a dose of from about 100 ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose can range from about 5 mg/kg to about 50 mg/kg, from about 10 μg/kg to about 5 mg/kg, or from about 100 μg/kg to about 1 mg/kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 μg/kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. The immunoconjugate dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer being treated. Frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week. In another aspect, the invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to a certain cancer to be prevented. For example, the methods can include administering the immunoconjugate to provide a dose of from about 100 ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose can range from about 5 mg/kg to about 50 mg/kg, from about 10 μg/kg to about 5 mg/kg, or from about 100 μg/kg to about 1 mg/kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 μg/kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. The immunoconjugate dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer being treated. Frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week. Some embodiments of the invention provide methods for treating cancer as described above, wherein the cancer is breast cancer. Breast cancer can originate from different areas in the breast, and a number of different types of breast cancer have been characterized. For example, the immunoconjugates of the invention can be used for treating ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma; medullary carcinoma; mucinous carcinoma; papillary carcinoma; or cribriform carcinoma of the breast); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and other forms of breast cancer such as triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer. In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by STING. EXAMPLES Preparation of asymmetric bis-benzimidazole compounds and (BBI-L) Formula II compounds and intermediates Example 1 Synthesis of 1-(3-((5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5- carboxamido)-1-methyl-1H-benzo[d]imidazol-7-yl)oxy)propyl)-2 -(1-ethyl-3-methyl-1H- pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazole-5-car boxamide, BBI-1 Preparation of 4-chloro-3-methoxy-5-nitro-benzamide, 1b A solution of methyl 4-chloro-3-methoxy-5-nitro-benzoate, 1a (15 g, 61.07 mmol, 1 eq) in NH 3 •H 2 O (136.98 g, 977.13 mmol, 150.5 mL, 25% purity, 16 eq) was stirred at 50°C for 24 h. The mixture was filtered to give 1b (11.2 g, 48.57 mmol, 79.53% yield) as light yellow solid which was used into the next step without further purification. 1 H NMR (DMSO-d 6 , 400 MHz) δ8.30 (s, 1H), 8.09 (s, 1H), 7.88 (s, 1H), 7.79 (s, 1H), 4.02 (s, 3H). Preparation of 3-methoxy-4-(methylamino)-5-nitro-benzamide, 1c To a solution of 1b (3 g, 13.01 mmol, 1 eq) in DIEA (16.8 g, 130 mmol, 22.7 mL, 10 eq) was added methanamine (4.39 g, 65.0 mmol, 5 eq, HCl) and it was stirred at 120°C for 24 hrs in a 100 mL of sealed tube. The reaction mixture was quenched by addition of H2O (100 mL), filtered and washed with water (50 mL) to give a red solid. The crude product was triturated with MTBE/PE=20/80 ml at 20°C for 30 min to obtain 1c (3.2 g, crude) as red solid. 1 H NMR (DMSO-d 6 , 400 MHz) δ 8.08 (s, 1H), 7.50 (s, 1H), 3.88 (s, 3H), 2.91 (d, J = 6.8 Hz , 3H). Preparation of 3-amino-5-methoxy-4-(methylamino)benzamide, 1d To a solution of 1c (3.2 g, 14.21 mmol, 1 eq) in MeOH (25 mL) ,THF (25 mL) and H 2 O (25 mL) was added Na 2 CO 3 (6.02 g, 56.8 mmol, 4 eq) and sodium dithionite, Na 2 S 2 O 4 (17.3 g, 99.5 mmol, 21.6 mL, 7 eq). The mixture was stirred at 20 °C for 1 hr. Then it was concentrated under reduced pressure to remove MeOH and THF, the residue was diluted with H 2 O (50 mL) and filtered to give 1d (2.3 g, 11.78 mmol, 82.91% yield) as yellow solid which was used into the next step without further purification. 1 H NMR (DMSO-d 6 , 400 MHz) δ 6.94 (s, 1H), 6.90 (s, 1H), 3.85 (s, 3H), 2.70 (s, 3H) Preparation of 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)- 7-methoxy-1-methyl- 1H-benzo[d]imidazole-5-carboxamide, 1f To a solution of 1d (2.1 g, 10.7 mmol, 1 eq) in DMF (30 mL) was added 2-ethyl-5- methyl-pyrazole-3-carbonyl isothiocyanate, 1e (2.31 g, 11.8 mmol, 1.1 eq) at 0°C and the mixture was stirred for 0.5 h at the same temperature, then Et 3 N (3.27 g, 32.3 mmol, 4.5 mL, 3 eq) and EDCI (6.19 g, 32.3 mmol, 3 eq) was added and it was stirred at 25°C for another 12 h. The reaction was poured into aqueous NaHCO 3 (30 mL), filtered and washed with H 2 O (15 mL x 3), dried to give crude product. Then the crude product was triturated with CH 3 CN (50 mL) at 20°C for 20 min to obtain 1f (2.9 g, 8.14 mmol, 75.65% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ7.99 (s, 1H), 7.66 (s, 1H), 7.42-7.35 (m, 2H), 6.66 (s, 1H), 4.62-4.60 (m, 2H), 3.97 (s, 3H), 3.84 (s, 3H), 2.17 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). Preparation of 2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-hydroxy-1- methyl- benzimidazole-5- carboxamide, 1g To a solution of 1f (1.2 g, 3.37 mmol, 1 eq) in DCM (40 mL) was added dropwise BBr 3 (8.44 g, 33.7 mmol, 3.2 mL, 10 eq) at 0°C. After addition, the mixture was stirred at 20°C for 72 h. The mixture was poured into ice-water, adjusted to pH=6 with aqueous NaHCO 3 and stirred for 30 min, then it was filtered to give 1g (0.7 g, 2.04 mmol, 60.72% yield) as white solid. 1 H NMR (MeOD, 400 MHz) δ7.54 (s, 1H), 7.29 (s, 1H), 6.85 (s, 1H), 4.67 (q, J = 7.2 Hz, 2H), 4.09 (s, 3H), 2.30 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H). Preparation of tert-butyl N-[3-[6-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-3-methyl-benzimidazol-4-yl]oxypropyl]carbama te, 1h To a solution of 1g (400 mg, 1.17 mmol, 1 eq) in DMF (10 mL) was added K 2 CO 3 (226 mg, 1.64 mmol, 1.4 eq) and tert-butyl N-(3-bromopropyl)carbamate (292 mg, 1.23 mmol, 1.05 eq), and then stirred at 50°C for 2 hr. The reaction mixture was quenched by addition ice-water (20 mL) at 0°C, and then diluted with EtOAc (20 mL) and extracted with EtOAc (15 mL x 2). The combined organic layers were washed with brine 20 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude product was triturated with EtOAc/PE=5/1 at 20°C for 5 min to afford 1h (0.57 g, crude) as white solid. Preparation of 7-(3-aminopropoxy)-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-1- methyl-benzimidazole-5-carboxamide, 1i To a solution of 1h (0.47 g, 941 umol, 1 eq) in EtOAc (0.5 mL) was added HCl/EtOAc (4 M, 11.8 mL, 50 eq). The mixture was stirred at 25°C for 1 hr. Then it was concentrated to give 1i (400 mg, 917.63 umol, 97.53% yield, HCl) as a white solid which was used directly in the next step. Preparation of 7-[3-(4-carbamoyl-2-methoxy-6-nitro-anilino)propoxy]-2-[(2-e thyl-5- methyl-pyrazole-3-carbonyl)amino]-1-methyl-benzimidazole-5-c arboxamide, 1j To a solution of 1i (440 mg, 1.01 mmol, 1 eq, HCl) in butan-1-ol (7.24 g, 97.71 mmol, 8.9 mL, 96.8 eq) was added NaHCO 3 (424 mg, 5.05 mmol, 5 eq) and DIEA (652 mg, 5.05 mmol, 879 uL, 5 eq), and then stirred at 20°C for 30 min. Intermediate 1b (244 mg, 1.06 mmol, 1.05 eq) was added at 20°C under N 2 and stirred at 120 °C for another 12 hrs. After that, the mixture was concentrated to give a residue, and the residue was diluted with EtOAc (10 mL) follow by ice water (10 mL) and it was stirred for 10 min. The precipitate was filtered to give 1j (0.4 g, 674 umol, 66.76% yield) as yellow solid. Preparation of 7-[3-(2-amino-4-carbamoyl-6-methoxy-anilino)propoxy]-2-[(2-e thyl-5- methyl- pyrazole-3-carbonyl)amino]-1-methyl-benzimidazole-5-carboxam ide, 1k To a solution of 1j (100 mg, 168 umol, 1 eq) in MeOH (0.5 mL) and THF (0.5 mL) , H 2 O (0.5 mL) was added sodium dithionite (205 mg, 1.18 mmol, 7 eq) and Na 2 CO 3 (71.4 mg, 674 umol, 4 eq). The mixture was stirred at 25 °C for 2 hr. Then it was concentrated to remove MeOH and THF, the residue was diluted with H 2 O (5 mL) and stirred for 5 min. The precipitate was filtered to give 1k (80 mg, 142 umol, 84.26% yield) as yellow solid. Preparation of BBI-1 To a solution of 1k (50 mg, 89 umol, 1 eq) in DMF (1.5 mL) was added intermediate 1e (19 mg, 98 umol, 1.1 eq) at 0 °C and it was stirred for 30 min, then Et 3 N (27 mg, 266 umol, 37 uL, 3 eq) and EDCI (51 mg, 266 umol, 3 eq) was added and the mixture was stirred at 20 °C for another 16.5 hr. The reaction was added to aqueous NaHCO 3 (1 mL), filtered and washed with H2O (1 mL x 3), dried to give crude product. The residue was purified by prep-HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 25%-55%, 8 min) to afford BBI-1(14 mg, 19.32 umol, 21.77% yield) as a white solid. NMR (MeOD, 400 MHz) δ7.63 (s, 1H), 7.56 (s, 1H), 7.41 (s, 1H), 7.28 (s, 1H), 6.80 (s, 1H), 6.51 (s, 1H), 4.75-4.69 (m, 4H), 4.49-4.46 (m, 2H), 4.33-4.30 (m, 2H), 4.04 (s, 3H), 3.79 (s, 3H), 2.51-2.48 (m, 2H), 2.31 (s, 3H), 2.14 (s, 3H), 1.47 (t, J = 7.2 Hz, 3H), 1.33 (t, J = 7.2 Hz, 3H). LCMS (ESI): mass calcd. for C 35 H 40 N 12 O 6 724.32 m/z found 725.3 [M+H] + . Example 2 Synthesis of 1-[4-[6-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-3-methyl-benzimidazol-4-yl]oxybutyl]-2-[(2-e thyl-5-methyl-pyrazole-3- carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, BBI-2
Preparation of tert-butyl N-[4-[6-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-3-methyl- benzimidazol-4-yl]oxybutyl]carbamate, 2a To a solution of 2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-hydroxy-1- methyl- benzimidazole-5-carboxamide, 1g (800 mg, 2.34 mmol, 1 eq) in DMF (10 mL) was added K 2 CO 3 (355 mg, 2.57 mmol, 1.1 eq) at 50°C and it was stirred for 0.5 h, then tert-butyl N-(4- bromobutyl)carbamate (648 mg, 2.57 mmol, 527 uL, 1.1 eq) was added. The mixture was stirred at 50°C for 12 hr. The reaction mixture was quenched by addition ice water (20 mL) at 0°C, the precipitate filtered to give 2a (1.2 g, crude)as white solid which was used into the next step without further purification. 1 H NMR (MeOD, 400 MHz) δ7.63 (s, 1H), 7.41 (s, 1H), 6.73 (s, 1H), 4.77-4.67 (m, 2H), 4.32-4.22 (m, 2H), 3.98 (s, 3H), 3.17 (t, J = 7.2 Hz, 2H), 2.27 (s, 3H), 2.01-1.90 (m, 2H), 1.81-1.70 (m, 2H), 1.49-1.43 (m, 12H). Preparation 7-(4-aminobutoxy)-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)a mino]-1- methyl-benzimidazole-5-carboxamide, 2b To a solution of 2a (1.1 g, 2.14 mmol, 1 eq) in EtOAc (5 mL) was added HCl/EtOAc (4 M, 26.7 mL, 50 eq). The mixture was stirred at 25 °C for 0.5 hr. Then it was concentrated to obtain 2b (0.8 g, 1.93 mmol, 90.34% yield) as off-white solid. 1 H NMR (MeOD, 400 MHz) δ7.83 (s, 1H), 7.58 (s, 1H), 7.08 (s, 1H), 4.65 (q, J = 7.2 Hz, 2H), 4.36 (t, J = 6.0 Hz, 2H), 4.20 (s, 3H), 3.08 (t, J = 7.2 Hz, 2H), 2.37 (s, 3H), 2.09-2.01 (m, 2H), 2.00-1.90 (m, 2H), 1.49 (t, J = 7.2 Hz, 3H). Preparation of 7-[3-(4-carbamoyl-2-methoxy-6- nitro-anilino)propoxy]-2-[(2-ethyl-5- methyl-pyrazole-3-carbonyl)amino]-1-methyl-benzimidazole-5-c arboxamide, 2c To a solution of 2b (570 mg, 1.27 mmol, 1 eq, HCl) in butan-1-ol (9.39 g, 127 mmol, 11.6 mL, 100 eq) was added DIEA (818 mg, 6.33 mmol, 1.10 mL, 5 eq), NaHCO 3 (532 mg, 6.33 mmol, 5 eq) and 4-chloro-3-methoxy-5-nitro-benzamide, 1b (292 mg, 1.27 mmol, 1 eq). The mixture was stirred at 120°C for 15 hr under N 2 . The reaction mixture was quenched by addition H 2 O (50 mL) at 20°C, and then diluted with EtOAc (30 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The crude product was triturated with EtOAc/PE=10/1 at 20°C for 30 min to afford 2c (500 mg, 842 umol, 66.49% yield) as yellow solid. Preparation of 7-[4-(2-amino-4-carbamoyl-6-methoxy-anilino)butoxy] -2-[(2-ethyl-5- methyl-pyrazole-3- carbonyl)amino]-1-methyl-benzimidazole-5-carboxamide, 2d To a solution of 2c (400 mg, 658 umol, 1 eq) in MeOH (5 mL), THF (5 mL) and H 2 O (5 mL) was added Na 2 CO 3 (279 mg, 2.63 mmol, 4 eq) and disodium;BLAH (802 mg, 4.61 mmol, 7 eq). The mixture was stirred at 25 °C for 2 hr. After that, the reaction was concentrated to remove MeOH and THF, H2O (10 mL) was added and stirred for 5 min. The precipitate was filtered to give 2d (200 mg, 346 umol, 52.60% yield) as yellow solid. Preparation of BBI-2 To a solution of 2d (50 mg, 86 umol, 1 eq) in DMF (1 mL) was added 2-ethyl-5-methyl- pyrazole-3-carbonyl isothiocyanate, 1e (18 mg, 95 umol, 1.1 eq) at 0 °C and it was stirred for 30 minutes and then Et 3 N (26 mg, 259 umol, 36 uL, 3 eq) and EDCI (49 mg, 259 umol, 3 eq) was added. The mixture was stirred at 20°C for another 2 hr. The mixture was filtered and purified by prep-HPLC (column: Phenomenex Luna 80*30mm*3um;mobile phase: [water (TFA)-ACN]; B%: 25%-45%, 8 min) to obtain BBI-2 (26 mg, 35.19 umol, 40.66% yield) as a white solid. 1 H NMR (DMSO-d 6 , 400 MHz) δ8.05-7.90 (m, 2H), 7.68 (s, 1H), 7.62 (s, 1H), 7.40 (s, 1H), 7.37- 7.26 (m, 2H), 6.63 (s, 1H), 6.58 (s, 1H), 4.67-4.52 (m, 4H), 4.48-4.44 (m 2H), 4.23 (t, J = 4.8 Hz, 2H), 3.95 (s, 3H), 3.67 (s, 3H), 2.17 (s, 3H), 2.11-1.97 (m, 5H), 1.92-1.88 (m, 2H), 1.43- 1.23 (m, 6H). LCMS (ESI): mass calcd. for C 36 H 42 N 12 O 6 738.34 m/z found 739.3 [M+H] + Example 3 Synthesis of 1-(4-((5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5- carboxamido)-1-methyl-1H-benzo[d]imidazol-7-yl)oxy)butyl)-2- (1-ethyl-3-methyl-1H- pyrazole-5-carboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-ben zo[d]imidazole-5-carboxamide, BBI-3
Preparation of tert-butyl 4-[3-[5-carbamoyl-2-[4-[6-carbamoyl-2-[(2-ethyl-5-methyl- pyrazole-3-carbonyl)amino]-3-methyl-benzimidazol-4-yl]oxybut ylamino]-3-nitro- phenoxy]propyl]piperazine-1-carboxylate, 3b To a solution of 7-(4-aminobutoxy)-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)a mino]-1- methyl-benzimidazole-5-carboxamide, 2b (300 mg, 666 umol, 1.3 eq, HCl) in butan-1-ol (3.68 g, 49.65 mmol, 4.5 mL, 96.8 eq) was added NaHCO 3 (215 mg, 2.56 mmol, 99 uL, 5 eq) and DIEA (331 mg, 2.56 mmol, 446 uL, 5 eq), and then stirred at 20°C for 30 min, tert-butyl 4-[3- (5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl]piperazine-1-ca rboxylate, 3a (227 mg, 513 umol, 1 eq) was added at 20 °C under N 2 and it was stirred at 120 °C for 12 hrs. The mixture was concentrated to give a residue, then diluted with EtOAc (20 mL) and ice water (30 mL), the precipitate was filtered to give 3b (0.4 g, crude) as red solid. Preparation of tert-butyl 4-[3-[3-amino-5-carbamoyl-2-[4-[6-carbamoyl-2- [(2-ethyl-5- methyl-pyrazole-3-carbonyl) amino]-3-methyl-benzimidazol-4- yl]oxybutylamino]phenoxy]propyl]piperazine-1-carboxylate, 3c To a solution of 3b (0.4 g, 488 umol, 1 eq) in THF (5 mL), MeOH (5 mL) and H 2 O (5 mL) was added sodium dithionite (595 mg, 3.42 mmol, 743 uL, 7 eq) and Na 2 CO 3 (207 mg, 1.95 mmol, 4 eq). The mixture was stirred at 25°C for 2 hr. Water (5 mL) was added and the mixture was concentrated to remove THF and MeOH, then filtered to give 3c (0.24 g, 304 umol, 62.28% yield) as yellow solid. Preparation of tert-butyl 4-(3-((5-carbamoyl-1-(4-((5-carbamoyl-2-(1-ethyl-3-methyl- 1H-pyrazole-5-carboxamido)-1-methyl-1H-benzo[d]imidazol-7-yl )oxy)butyl)-2-(1-ethyl-3- methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)o xy)propyl)piperazine-1- carboxylate, 3d To a solution of 3c (240 mg, 304 umol, 1 eq) in DMF (5 mL) was added 2-ethyl-5- methyl-pyrazole-3-carbonyl isothiocyanate, 1e (65 mg, 334 umol, 1.1 eq) at 0 °C and it was stirred for 10 min at this temperature, then Et 3 N (92 mg, 911 umol, 127 uL, 3 eq) and EDCI (175 mg, 911 umol, 3 eq) was added. The mixture was stirred at 25 °C for another 12 hr. Then it was poured into aqueous NaHCO 3 (5 mL), filtered and the cake was washed with H 2 O (1 mL x 3), dried to afford 3d (200 mg, 210 umol, 69.21% yield) as yellow solid. Preparation of BBI-3 To a solution of 3d (0.2 g, 210 umol, 1 eq) in EtOAc (1 mL) was added HCl/EtOAc (4 M, 2.63 mL, 50 eq). The mixture was stirred at 25°C for 1 hr. The reaction was concentrated and purified by prep-HPLC (column: Phenomenex Luna 80*30mm*3um;mobile phase: [water (TFA)-ACN]; B%: 10%-40%, 8 min) to give BBI-3 (70 mg, 82.26 umol, 39.12% yield) as white solid. 1 H NMR (MeOD, 400 MHz) δ7.60 (s, 2H), 7.46 (s, 1H), 7.36 (s, 1H), 6.74 (s, 1H), 6.63 (s, 1H), 4.70 (d, J = 7.2 Hz, 2H), 4.65-4.60 (m, 2H), 4.37-4.30 (m, 4H), 3.77 (s, 3H), 3.31-3.26 (m, 4H), 2.88-2.84 (m, 4H), 2.79 (t, J = 7.2 Hz, 2H), 2.30 (s, 3H), 2.21-2.18 (m, 2H), 2.16-2.13 (m, 5H), 2.09-2.01 (m, 2H), 1.47-1.34 (m, 8H). LCMS (ESI): mass calcd. for C 42 H 54 N 14 O 6 850.44 m/z found 851.6 [M+H] + Example L-1 Synthesis of 7-[4-[5-carbamoyl-7-[3-[4-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [[2-(2,5-dioxopyrrol-1-yl)acetyl]amino] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]ethyl]piperazin-1- yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]b enzimidazol-1-yl]butoxy]-2-[(2- ethyl-5-methyl-pyrazole-3-carbonyl)amino]-1-methyl-benzimida zole-5-carboxamide, BBI-L-1
Preparation of 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2 -(2- hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]ethoxy]ethyl]aceta mide, L-1b To a solution of 2-(2,5-dioxopyrrol-1-yl)acetic acid (155 mg, 997 umol, 1 eq) in DCM (10 mL) was added HATU (398 mg, 1.05 mmol, 1.05 eq) and Et 3 N (151 mg, 1.50 mmol, 208 uL, 1.5 eq) and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethanol, L-1a (500 mg, 997 umol, 1 eq). The mixture was stirred at 0 °C for 1 hr. Then it was partitioned between ice water (20 mL) and DCM (20 mL). The organic phase was separated, washed with brine 10 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0~100% Ethyl acetate/Petroleum ether to 0-50% Methanol/Ethyl acetategradient @ 45 mL/min) to obtain L-1b (440 mg, 689 umol, 69.11% yield) as colorless oil. 1 H NMR (MeOD, 400 MHz) δ 6.77 (s, 2H), 4.21 (s, 2H), 3.75-3.71 (m, 2H), 3.70-3.59 (m, 38H), 3.59-3.55 (m, 2H), 3.48-3.44 (m, 2H). Preparation of 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- oxoethoxy)ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]aceta mide, L-1c To a solution of L-1b (240 mg, 376 umol, 1 eq) in DCM (10 mL) was added Dess-Martin periodinane, (1,1,1-Triacetoxy)-1,1-dihydro-1,2-benziodoxol-3(1H)-one, CAS Reg. No.87413- 09-0 (478 mg, 1.13 mmol, 3 eq) at 0°C. The mixture was stirred at 25°C for 1 hr. Then it was filtered and concentrated to give L-1c (0.2 g, crude) as colorless oil which was used into the next step without further purification. Preparation of BBI-L-1 To a solution of 7-[4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)a mino]-7- (3-piperazin-1-ylpropoxy)benzimidazol-1-yl]butoxy]-2-[(2-eth yl-5-methyl-pyrazole-3- carbonyl)amino]-1-methyl-benzimidazole-5-carboxamide, BBI-3 (55 mg, 51 umol, 1 eq, 2TFA) in MeOH (2 mL) was added L-1c (97.4 mg, 153 umol, 3 eq) and stirred for 20 min at 25°C, then NaBH 3 CN (9.61 mg, 153 umol, 3 eq) was added. The mixture was stirred at 25°C for 2 hr. After that, the reaction was filtered and purified by prep-HPLC (column: Phenomenex Luna 80*30mm*3um;mobile phase: [water(TFA)-ACN];B%: 20%-50%,8min) to afford BBI-L-1 (21.4 mg, 14.54 umol, 28.53% yield) as white solid. 1 H NMR (MeOD, 400 MHz) δ7.62 (s, 2H), 7.47 (s, 1H), 7.38 (s, 1H), 6.89 (s, 2H), 6.75 (s, 1H), 6.65 (s, 1H), 4.73-4.66 (m, 2H), 4.66-4.60 (m, 4H), 4.39-4.31 (m, 4H), 4.17 (s, 2H), 3.86-3.81 (m, 2H), 3.78 (s, 3H), 3.66-3.62 (m, 8H), 3.61-3.58 (m, 30H) 3.55-3.51 (m, 2H), 3.37 (t, J = 5.2 Hz, 4H), 3.07-2.74 (m, 6H), 2.30 (s, 3H), 2.25-2.13 (m, 7H), 2.10-2.02 (m, 2H), 1.48-1.28 (m, 8H). LCMS (ESI): mass calcd. for C 70 H 102 N 16 O 19 1470.75 m/z found 1471.9 [M+H] + Example 201 Preparation of Immunoconjugates (IC) In an exemplary procedure, for preparation for lysine-based conjugation, an antibody is buffer exchanged into a conjugation buffer containing 100 mM Borate, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3 using Zeba™ Spin Desalting Columns (Thermo Fisher Scientific). The concentration of the buffer-exchanged antibody was adjusted to approximately 5 – 25 mg/ml using the conjugation buffer and sterile-filtered. The bis- benzimidazole-linker (BBI-L) intermediate compound of Formula II is either dissolved in dimethylsulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5 – 20 mM. For conjugation, the antibody is mixed with 4 to about 20 molar equivalents of BBI-L. In some instances, additional DMA or DMSO up to 20% (v/v), was added to improve the solubility of BBI-L in the conjugation buffer. The reaction is allowed to proceed for approximately 30 min to 4 hours at 20 °C or 30 °C or 37 °C. The resulting conjugate is purified away from the unreacted BBI-L using two successive Zeba™ Spin Desalting Columns. The columns are pre-equilibrated with phosphate-buffered saline (PBS), pH 7.2. Adjuvant to antibody ratio (DAR) is estimated by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY TM UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO TM G2- XS TOF mass spectrometer (Waters Corporation). In an exemplary procedure, for preparation for cysteine-based conjugation, an antibody is buffer exchanged into a conjugation buffer containing PBS, pH 7.2 with 2 mM EDTA using Zeba™ Spin Desalting Columns (Thermo Fisher Scientific). The interchain disulfides are reduced using 2–4 molar excess of Tris (2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT) at 37 °C for 30 min – 2 hours. Excess TCEP or DTT was removed using a Zeba™ Spin Desalting column pre-equilibrated with the conjugation buffer. The concentration of the buffer- exchanged antibody was adjusted to approximately 5–20 mg/ml using the conjugation buffer and sterile-filtered. The BBI-L is either dissolved in dimethylsulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5–20 mM. For conjugation, the antibody is mixed with 10–20 molar equivalents of BBI-L. In some instances, additional DMA or DMSO up to 20% (v/v), was added to improve the solubility of the BBI-L in the conjugation buffer. The reaction is allowed to proceed for approximately 30 min to 4 hours at 20 °C. The resulting conjugate is purified away from the unreacted BBI-L using two successive Zeba™ Spin Desalting Columns. The columns are pre-equilibrated with phosphate-buffered saline (PBS), pH 7.2. Adjuvant to antibody ratio (DAR) is estimated by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY TM UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO TM G2-XS TOF mass spectrometer (Waters Corporation). Following conjugation, to potentially remove unreacted BBI-L and/or higher-molecular weight aggregate, the conjugates may be purified further using size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof. In another exemplary procedure, an antibody is buffer exchanged into a conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3, using G-25 SEPHADEX TM desalting columns (Sigma-Aldrich, St. Louis, MO). The eluates are then each adjusted to a concentration of about 1-10 mg/ml using the buffer and then sterile filtered. The antibody is pre-warmed to 20-30 °C and rapidly mixed with 2-20 (e.g., 7-10) molar equivalents of bis-benzimidazole-linker (BBI-L) intermediate compound of Formula II. The reaction is allowed to proceed for about 16 hours at 30 °C and the immunoconjugate (IC) is separated from reactants by running over two successive G-25 desalting columns equilibrated in phosphate buffered saline (PBS) at pH 7.2 to provide the Immunoconjugate (IC) of Table 2. Adjuvant-antibody ratio (DAR) is determined by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY TM UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO TM G2- XS TOF mass spectrometer (Waters Corporation). For conjugation, the antibody may be dissolved in a aqueous buffer system known in the art that will not adversely impact the stability or antigen-binding specificity of the antibody. Phosphate buffered saline may be used. The BBI-L is dissolved in a solvent system comprising at least one polar aprotic solvent as described elsewhere herein. In some such aspects, the BBI- L is dissolved to a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM or about 50 mM, and ranges thereof such as from about 5 mM to about 50mM or from about 10 mM to about 30 mM in pH 8 Tris buffer (e.g., 50 mM Tris). In some aspects, the BBI-L is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide) or acetonitrile, or another suitable dipolar aprotic solvent. Alternatively in the conjugation reaction, an equivalent excess of BBI-L solution may be diluted and combined with antibody solution. The BBI-L solution may suitably be diluted with at least one polar aprotic solvent and at least one polar protic solvent, examples of which include water, methanol, ethanol, n-propanol, and acetic acid. The molar equivalents of thienoazepine- linker intermediate to antibody may be about 1.5:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1, and ranges thereof, such as from about 1.5:1 to about 20:1 from about 1.5:1 to about 15:1, from about 1.5:1 to about 10:1,from about 3:1 to about 15:1, from about 3:1 to about 10:1, from about 5:1 to about 15:1 or from about 5:1 to about 10:1. The reaction may suitably be monitored for completion by methods known in the art, such as LC-MS. The conjugation reaction is typically complete in a range from about 1 hour to about 16 hours. After the reaction is complete, a reagent may be added to the reaction mixture to quench the reaction. If antibody thiol groups are reacting with a thiol-reactive group such as maleimide of the BBI-L, unreacted antibody thiol groups may be reacted with a capping reagent. An example of a suitable capping reagent is ethylmaleimide. Following conjugation, the immunoconjugates may be purified and separated from unconjugated reactants and/or conjugate aggregates by purification methods known in the art such as, for example and not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof. For instance, purification may be preceded by diluting the immunoconjugate, such in 20 mM sodium succinate, pH 5. The diluted solution is applied to a cation exchange column followed by washing with, e.g., at least 10 column volumes of 20 mM sodium succinate, pH 5. The conjugate may be suitably eluted with a buffer such as PBS. Example 202 HTRF binding assay Asymmetric bis-benzimidazole compounds (BBI) of the invention were assessed in a biochemical homogeneous time resolved fluorescence (HTRF) binding assay adapted from the human STING WT binding assay (“HTRF, A guide to Homogeneous Time Resolved Fluorescence”, (2021) PerkinElmer Cisbio; Mathis, G. Clinical Chemistry, 41(9):1391–1397). Briefly, 6His-tagged STING protein was incubated with terbium cryptate-labeled anti-6His antibody, d2-labeled 2’,3’-cGAMP, and varying concentrations of test articles in a 384-well plate format. Donor and acceptor emission signals were measured for each well by plate reader at 665 and 615 nm, respectively, and the ratio of the signals used to calculate percent inhibition of d2-labeled 2’,3’-cGAMP, cyclic dinucleotide binding. Dose-response curves generated from these data were used to calculate IC50 values. Example 203 Functional Assessment of Immunoconjugates, PBMC assay The immunoconjugates of the invention can be assessed in a co-culture assay using primary human peripheral blood mononuclear cells (PBMC) co-cultured with target antigen- expressing tumor cells. Briefly, PBMCs are freshly isolated from healthy human donor blood (Stanford Blood Center) by density centrifugation. PBMCs are then co-cultured with antigen- expressing tumor cells at a 10:1 effector to target ratio in complete medium (RPMI supplemented with 10% FBS) and incubated overnight with a range of concentrations of the indicated test articles. Activation is measured by secretion of pro-inflammatory cytokines, such as IFNO1 and TNFD, by LEGENDPLEX TM cytokine bead array (BioLegend). Example 204 Assessment of Immunoconjugate Activity In Vitro This example shows that Immunoconjugates of the invention are effective at eliciting myeloid activation, such as in dendritic cells, and therefore are useful for the treatment of cancer. Isolation of Human Conventional Dendritic Cells: Human conventional dendritic cells (cDCs) were negatively selected from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. Briefly, cells are first enriched by using a ROSETTESEP TM Human CD3 Depletion Cocktail (Stem Cell Technologies, Vancouver, Canada) to remove T cells from the cell preparation. cDCs are then further enriched via negative selection using an EASYSEP TM Human Myeloid DC Enrichment Kit (Stem Cell Technologies). c DC Activation Assay: 8 x 10 4 APCs were co-cultured with tumor cells expressing the ISAC target antigen at a 10:1 effector (cDC) to target (tumor cell) ratio. Cells were incubated in 96-well plates (Corning, Corning, NY) containing RPMI-1640 medium supplemented with 10% FBS, and where indicated, various concentrations of the indicated immunoconjugate of the invention (as prepared according to the example above). Following overnight incubation of about 18 hours, cell-free supernatants were collected and analyzed for cytokine secretion (including TNFD) using a BioLegend LEGENDPLEX cytokine bead array. Activation of myeloid cell types can be measured using various screen assays in addition to the assay described in which different myeloid populations are utilized. These may include the following: monocytes isolated from healthy donor blood, M-CSF differentiated Macrophages, GM-CSF differentiated Macrophages, GM-CSF+IL-4 monocyte-derived Dendritic Cells, conventional Dendritic Cells (cDCs) isolated from healthy donor blood, and myeloid cells polarized to an immunosuppressive state (also referred to as myeloid derived suppressor cells or MDSCs). Examples of MDSC polarized cells include monocytes differentiated toward immunosuppressive state such as M2a MΦ (IL4/IL13), M2c MΦ (IL10/TGFb), GM-CSF/IL6 MDSCs and tumor-educated monocytes (TEM). TEM differentiation can be performed using tumor-conditioned media (e.g. 786.O, MDA-MB-231, HCC1954). Primary tumor-associated myeloid cells may also include primary cells present in dissociated tumor cell suspensions (Discovery Life Sciences). Assessment of activation of the described populations of myeloid cells may be performed as a mono-culture or as a co-culture with cells expressing the antigen of interest which the immunoconjugate may bind to via the CDR region of the antibody. Following incubation for 18-48 hours, activation may be assessed by upregulation of cell surface co- stimulatory molecules using flow cytometry or by measurement of secreted proinflammatory cytokines. For cytokine measurement, cell-free supernatant is harvested and analyzed by cytokine bead array (e.g. LegendPlex from Biolegend) using flow cytometry. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.