PYRROLOBENZODIAZEPINE RESISTANCE
20220347309 · 2022-11-03
Inventors
Cpc classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
A61K31/5517
HUMAN NECESSITIES
C12Q2600/112
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K47/68
HUMAN NECESSITIES
A61K31/5517
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to methods of determining if a proliferative disorder such as cancer is resistant to treatment with a pyrrolobenzodiazepine (PBD) agent, such as a therapeutic antibody-drug conjugate (ADC) comprising a PBD warhead conjugated to an antibody (PBD-ADC). The present disclosure also describes methods of selecting subjects suitable for treatment with a PBD agent, and methods of reducing the resistance of a proliferative disorder to a PBD agent.
Claims
1-101. (canceled)
102. A method of treating a proliferative disease in a subject, wherein the proliferative disease is resistant to a PBD agent, the method comprising administering to the subject an antagonist of one or more PBD-resistance genes in combination with a therapeutically effective amount of the PBD agent.
103. The method of claim 102, wherein the subject is selected for treatment by a method comprising the steps of: (a) determining whether one or more PBD-resistance genes are overexpressed in a sample from the subject, and (b) selecting the subject for treatment with the PBD agent if overexpression of the one or more PBD-resistance genes is not detected in the sample.
104. A method of reducing the resistance of a proliferative cell to a PBD-agent, the method comprising contacting the proliferative cell with an antagonist of one or more PBD-resistance genes.
105. A method for determining whether a proliferative disease in a subject is resistant to treatment with a pyrrolobenzodiazepine (PBD) agent, the method comprising determining whether one or more PBD-resistance genes are overexpressed in a sample from the subject, wherein overexpression of the one or more PBD-resistance genes indicates that the proliferative disease is resistant to treatment with the PBD agent.
106. The method of claim 102, wherein the antagonist is an siRNA, or an miRNA, such as miR-200c, miR-212, miR-328, miR-519c, miR-520h, miR-297, or miR-379.
107. The method of claim 102, wherein the antagonist is selected from the group consisting of: MK-571, Biricodar, Probenecid, Reversan, Fumitremorgin C, and Ko143.
108. The method of claim 102, wherein the antagonist is an ABCC2 inhibitor selected from the group consisting of: probenecid, furosemide, ritonavir, saquinavir, lamivudine, abacavir, emtricitabine, efavirenz, delavirdine, nevirapine, cidofovir, adefovir, tenofovir, cyclosporine, PSC833, and MK571.
109. The method of claim 102, wherein the antagonist is an ABCG2 inhibitor selected from the group consisting of: febuxostat, Fumitremorgin C, elacridar, tariquidar, and Ko143.
110. The method of claim 102, wherein the one or more PBD-resistance genes are selected from the group consisting of: (i) ABCG2, ABCC2, SLCO2B1, SLC7A7, SLC22A3, SLCO2A1, ABCC12, ATP7A, AQP7, SLC5A1, SLC16A2, SLC7A9, ABCB4, ABCC11, ABCF1, SLC28A3, and ABCB6; (ii) ABCG2, ABCC2, SLCO2B1, SLC7A7, SLC22A3, SLCO2A1, ABCC12, ATP7A, AQP7, and SLC5A1; (iii) ABCG2, ABCC2, SLCO2B1, SLC7A7, and SLC22A3; (iv) ABCG2, ABCC2, SLCO2B1, and SLC7A7; (v) ABCG2, ABCC2, and SLCO2B1; (vi) ABCG2, ABCC2, and SLC7A7; (vii) ABCG2, ABCC2, and SLC22A3; (viii) ABCG2 and ABCC2; (ix) ABCG2 and SLCO2B1; (x) ABCG2; and (xi) ABCC2.
111. The method of claim 103, wherein the method comprises determining whether two, three, four, or five or more PBD-resistance genes are overexpressed in the sample.
112. The method of claim 103, wherein determining PBD-resistance gene overexpression comprises measuring the level of mRNA transcription from the one or more PBD-resistance genes and/or measuring the level of PBD-resistance polypeptide expression.
113. The method of claim 103, wherein overexpression is indicated by an at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold increase relative to a control sample.
114. The method of claim 103, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001.
115. The method of claim 102, wherein the proliferative disease is cancer.
116. The method of claim 115, wherein the cancer is: (i) a benign, pre malignant, or malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis; (ii) a solid tumour; (iii) a solid tumour associated with CD25+ve infiltrating cells; (iv) a solid tumour associated with CD25+ve infiltrating cells, wherein the solid tumour is selected from the group consisting of pancreatic cancer, breast cancer (including triple negative breast cancer), colorectal cancer, gastric and oesophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, bladder, and head and neck cancer; (iv) lymphoma or leukaemia; or (v) selected from: Hodgkin's Lymphoma; non-Hodgkin's, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL) Marginal Zone B-cell lymphoma (MZBL); and leukemias, including Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukaemia (AML), and Acute Lymphoblastic Leukaemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome-negative ALL (Ph−ALL).
117. The method of claim 102, wherein the PBD agent is a conjugate of formula L-(D.sup.L).sub.p, where D.sup.L is of formula I or II: ##STR00117## wherein: L is a cell binding agent (CBA) such as an antibody; when there is a double bond present between C2′ and C3′, R.sup.12 is selected from the group consisting of: (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (ib) C.sub.1-5 saturated aliphatic alkyl; (ic) C.sub.3-6 saturated cycloalkyl; ##STR00118## wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5; ##STR00119## wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00120## where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2′ and C3′, R.sup.12 is ##STR00121## where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups; R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ are selected from O, S, or NH; R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively; R.sup.L1′ is a linker for connection to the cell binding agent (CBA); R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R.sup.20 and R.sup.21 either together form a double bond between the nitrogen and carbon atoms to which they are bound or; R.sup.20 is selected from H and R.sup.C, where R.sup.C is a capping group; R.sup.21 is selected from OH, OR.sup.A and SO.sub.zM; when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (ib) C.sub.1-5 saturated aliphatic alkyl; (ic) C.sub.3-6 saturated cycloalkyl; ##STR00122## wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5; ##STR00123## wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00124## where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and C3, R.sup.2 is ##STR00125## where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; R.sup.22 is of formula IIIa, formula IIIb or formula IIIc: ##STR00126## where A is a C.sub.5-7 aryl group, and either (i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a single bond and —Z—(CH.sub.2).sub.n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or (ii) Q.sup.1 is —CH═CH—, and Q.sup.2 is a single bond; ##STR00127## where; R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and unsubstituted C.sub.1-2 alkyl; ##STR00128## where Q is selected from O—R.sup.L2′, S—R.sup.L2′ and NR.sup.N—R.sup.L2′, and R.sup.N is selected from H, methyl and ethyl X is selected from the group comprising: O—R.sup.L2′, S—R.sup.L2′, CO.sub.2—R.sup.L2′, CO—R.sup.L2′, NH—C(═O)—R.sup.L2′, NHNH—R.sup.L2′, CONHNH—R.sup.L2′, ##STR00129## NR.sup.NR.sup.L2′, wherein R.sup.N is selected from the group comprising H and C.sub.1-4 alkyl; R.sup.L2′ is a linker for connection to the cell binding agent (CBA); R.sup.10 and R.sup.11 either together form a double bond between the nitrogen and carbon atoms to which they are bound or; R.sup.10 is H and R.sup.11 is selected from OH, OR.sup.A and SO.sub.zM; R.sup.30 and R.sup.31 either together form a double bond between the nitrogen and carbon atoms to which they are bound or; R.sup.30 is H and R.sup.31 is selected from OH, OR.sup.A and SO.sub.zM.
118. The method of claim 117, wherein the PBD agent comprises a compound of the formula: ##STR00130## ##STR00131## wherein CBA is a cell binding agent such as an antibody.
119. The method of claim 102, wherein the PBD agent is a compound of the formula (III):
L-(DL).sub.p (III) wherein: L is a cell binding agent (CBA) such as an antibody; DL is ##STR00132## wherein: X is selected from the group comprising: a single bond, —CH.sub.2— and —C.sub.2H.sub.4—; n is from 1 to 8; m is 0 or 1; R.sup.7 is either methyl or phenyl; when there is a double bond between C2 and C3, R.sup.2 is selected the group consisting of: (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (ib) C.sub.1-5 saturated aliphatic alkyl; (ic) C.sub.3-6 saturated cycloalkyl; ##STR00133## wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5; ##STR00134## wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00135## where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond between C2 and C3, R.sup.2 is ##STR00136## where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; when there is a double bond between C2′ and C3′, R.sup.12 is selected the group consisting of: (iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (iib) C.sub.1-5 saturated aliphatic alkyl; (iic) C.sub.3-6 saturated cycloalkyl; ##STR00137## wherein each of R.sup.31, R.sup.32 and R.sup.33 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5; ##STR00138## wherein one of R.sup.35a and R.sup.35b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00139## where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond between C2′ and C3′, R.sup.12 is ##STR00140## where R.sup.36a and R.sup.36b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.36a and R.sup.36b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; and p is from 1 to 8.
120. The method of claim 119, wherein DL is: ##STR00141##
121. The method of claim 102, wherein the PBD agent is selected from ADCT-301, ADCT-401, ADCT-402, ADCT-602, ADCT-601, or ADCT-701.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0443] Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:
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STATEMENTS OF INVENTION
[0491] 1. A method for determining whether a proliferative disease in a subject is resistant to treatment with a pyrrolobenzodiazepine (PBD) agent, [0492] the method comprising determining whether one or more PBD-resistance genes are overexpressed in a sample from the subject, [0493] wherein overexpression of the one or more PBD-resistance genes indicates that the proliferative disease is resistant to treatment with the PBD agent.
[0494] 2. A method for selecting a subject for treatment with a pyrrolobenzodiazepine (PBD) agent, the method comprising the steps of: [0495] (a) determining whether one or more PBD-resistance genes are overexpressed in a sample from the subject, and [0496] (b) selecting the subject for treatment with the PBD agent if overexpression of the one or more PBD-resistance genes is not detected in the sample.
[0497] 3. The method of statement 2, wherein the subject has, is suspected of having, has been diagnosed with, or is at risk of, a proliferative disease.
[0498] 4. A method of reducing the resistance of a proliferative cell to a PBD-agent, the method comprising contacting the proliferative cell with an antagonist of one or more PBD-resistance genes.
[0499] 5. A method of treating a proliferative disease in a subject, wherein the proliferative disease is resistant to a PBD agent, the method comprising administering to the subject an antagonist of one or more PBD-resistance genes in combination with a therapeutically effective amount of the PBD agent.
[0500] 6. The method of either one of statements 4 or 5, wherein the antagonist is administered before the PBD agent, simultaneous with the PBD agent, or after the PBD agent.
[0501] 7. The method of any one of statements 4 to 6, wherein the antagonist reduces the level of mRNA transcription from the one or more PBD-resistance genes.
[0502] 8. The method of any one of statements 4 to 7, wherein the antagonist reduces the level of one or more PBD-resistance polypeptide expression.
[0503] 9. The method of any one of statements 4 to 8, wherein the antagonist reduces the activity of one or more PBD-resistance polypeptide.
[0504] 10. The method of any one of statements 4 to 9, wherein the antagonist is selected from the group consisting of: [0505] (a) an anti-PBD-resistance antibody; [0506] (b) an RNA agent that reduces the expression of one or more PBD-resistance gene; [0507] (c) a mimetic or analog of (b); [0508] and [0509] (d) an agent that increased the expression of the RNA agent of (b).
[0510] 11. The method of statement 10, wherein the antagonist is miR-200c, miR-212, miR-328, miR-519c, miR-520h, miR-297, or miR-379.
[0511] 12. The method of any one of statements 4 to 9, wherein the antagonist is selected from the group consisting of: MK-571, Biricodar, Probenecid, Reversan, Fumitremorgin C, and Ko143.
[0512] 13. The method of any one of statements 4 to 10, wherein the one or more PBD-resistance genes comprises ABCC2 and the antagonist reduces ABCC2 activity.
[0513] 14. The method of statement 13, wherein the antagonist is an ABCC2 inhibitor selected from the group consisting of: probenecid, furosemide, ritonavir, saquinavir, lamivudine, abacavir, emtricitabine, efavirenz, delavirdine, nevirapine, cidofovir, adefovir, tenofovir, cyclosporine, PSC833, and MK571.
[0514] 15. The method of any one of statements 4 to 10, wherein the antagonist reduces ABCC2 expression.
[0515] 16. The method of statement 15, wherein the antagonist is an siRNA, or an miRNA, such as miR-297 or miR-379.
[0516] 17. The method of any one of statements 4 to 10, wherein the one or more PBD-resistance genes comprises ABCG2 and the antagonist reduces ABCG2 activity.
[0517] 18. The method of statement 17, wherein the antagonist is an ABCG2 inhibitor selected from the group consisting of: febuxostat, Fumitremorgin C, elacridar, tariquidar, and Ko143.
[0518] 19. The method of any one of statements 4 to 10, wherein the antagonist reduces ABCG2 expression.
[0519] 20. The method of statement 19, wherein the antagonist is an siRNA, or an miRNA, such as miR-200c, miR-212, miR-328, miR-519c, or miR-520h.
[0520] 21. A method of treating a proliferative disease in a subject, the method comprising [0521] a) selecting a subject for treatment with a pyrrolobenzodiazepine (PBD) agent according to either one of statements 2 or 3, and [0522] b) administering to the subject a therapeutically effective amount of the PBD agent, optionally wherein the PBD agent is administered according to the method of any one of statements 5 to 20.
[0523] 22. The method of any one of statements 1 to 21, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, SLCO2B1, SLC7A7, SLC22A3, SLCO2A1, ABCC12, ATP7A, AQP7, SLC5A1, SLC16A2, SLC7A9, ABCB4, ABCC11, ABCF1, SLC28A3, and ABCB6.
[0524] 23. The method of any one of statements 1 to 22, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, SLCO2B1, SLC7A7, SLC22A3, SLCO2A1, ABCC12, ATP7A, AQP7, and SLC5A1.
[0525] 24. The method of any one of statements 1 to 23, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, SLCO2B1, SLC7A7, and SLC22A3.
[0526] 25. The method of any one of statements 1 to 24, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, SLCO2B1, and SLC7A7.
[0527] 26. The method of any one of statements 1 to 25, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, and SLCO2B1.
[0528] 27. The method of any one of statements 1 to 25, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, and SLC7A7.
[0529] 28. The method of any one of statements 1 to 25, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2, ABCC2, and SLC22A3.
[0530] 29. The method of any one of statements 1 to 28, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2 and ABCC2.
[0531] 30. The method of any one of statements 1 to 26, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2 and SLCO2B1.
[0532] 31. The method of any one of statements 1 to 30, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCG2.
[0533] 32. The method of any one of statements 1 to 29, wherein the one or more PBD-resistance genes are selected from the group consisting of: ABCC2.
[0534] 33. The method of any one of statements 1 to 30, wherein two or more PBD-resistance genes are selected.
[0535] 34. The method of any one of statements 1 to 28, wherein three or more PBD-resistance genes are selected.
[0536] 35. The method of any one of statements 1 to 25, wherein four or more PBD-resistance genes are selected.
[0537] 36. The method of any one of statements 1 to 24, wherein five or more PBD-resistance genes are selected.
[0538] 37. The method of any one of statements 1 to 36, determining PBD-resistance gene overexpression comprises measuring the level of mRNA transcription from the one or more PBD-resistance genes.
[0539] 38. The method of statement 37, wherein the level of mRNA transcription is determined by cDNA PCR array, RT-PCR, fluorescence in situ hybridization (FISH), Southern Blot, immunohistochemisty (IHC), polymerase chain reaction (PCR), quantitative PCR (qPCR), quantitative real-time PCR (qRT-PCR), comparative genomic hybridization, microarray based comparative genomic hybridization, or ligase chain reaction (LCR).
[0540] 39. The method of any one of statements 1 to 36, wherein determining PBD-resistance gene overexpression comprises measuring the level of PBD-resistance polypeptide expression.
[0541] 40. The method of statement 39, wherein determining the level of PBD-resistance polypeptide expression comprises contacting the sample with an anti-PBD-resistance antibody and detecting binding of the anti-PBD-resistance antibody to PBD-resistance polypeptide.
[0542] 41. The method of any one of statements 1 to 36, wherein determining PBD-resistance gene overexpression comprises measuring PBD-resistance polypeptide activity.
[0543] 42. The method of any one of statements 1 to 41, wherein overexpression is indicated by an at least 2-fold increase relative to a control sample.
[0544] 43. The method of statement 42, wherein overexpression is indicated by an at least 5-fold increase relative to a control sample.
[0545] 44. The method of statement 43, wherein overexpression is indicated by an at least 10-fold increase relative to a control sample.
[0546] The method of statement 44, wherein overexpression is indicated by an at least 20-fold increase relative to a control sample.
[0547] 46. The method of statement 45, wherein overexpression is indicated by an at least 50-fold increase relative to a control sample.
[0548] 47. The method of statement 46 wherein overexpression is indicated by an at least 100-fold increase relative to a control sample.
[0549] 48. The method of any one of statements 1 to 47, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.05.
[0550] 49. The method of any one of statements 1 to 48, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.01.
[0551] 50. The method of any one of statements 1 to 49, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.005.
[0552] 51. The method of any one of statements 1 to 50, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.001.
[0553] 52. The method of any one of statements 1 to 51, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.0005.
[0554] 53. The method of any one of statements 1 to 52, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.0001.
[0555] 54. The method of any one of statements 1 to 53, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.00005.
[0556] 55. The method of any one of statements 1 to 54, wherein overexpression is indicated by an increase relative to a control sample that has a p-value no greater than 0.00001.
[0557] 56. The method of any one of statements 1 to 55, the proliferative disease is cancer.
[0558] 57. The method of any one of statements 1 to 56, wherein the proliferative disorder or cancer is a benign, pre malignant, or malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.
[0559] 58. The method of any one of statements 1 to 56, wherein the proliferative disorder or cancer is a solid tumour.
[0560] 59. The method of statement 58, wherein the solid tumour is associated with CD25+ve infiltrating cells; [0561] optionally wherein the solid tumour is associated with high levels of CD25+ve infiltrating cells.
[0562] 60. The method of statement 59, wherein the solid tumour is selected from the group consisting of pancreatic cancer, breast cancer (including triple negative breast cancer), colorectal cancer, gastric and oesophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, bladder, and head and neck cancer.
[0563] 61. The method of any one of statements 1 to 56, wherein the proliferative disorder or cancer is lymphoma or leukaemia.
[0564] 62. The method of statement 61, wherein the proliferative disorder or cancer is selected from: [0565] Hodgkin's Lymphoma; [0566] non-Hodgkin's, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL) Marginal Zone B-cell lymphoma (MZBL); and [0567] leukemias, including Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukaemia (AML), and Acute Lymphoblastic Leukaemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome-negative ALL (Ph−ALL).
[0568] 63. The method of any one of statements 1 to 62, wherein the sample is a neoplasm sample.
[0569] 64. The method of any one of statements 1 to 63, wherein the sample is a tumour sample.
[0570] 65. The method of any one of statements 1 to 64, wherein the sample is a circulating fluid such as blood, plasma, serum or lymph.
[0571] 66. The method of any one of statements 1 to 65, wherein the control is the expression level of the one or more PBD-resistance genes in a healthy sample from the subject.
[0572] 67. The method of any one of statements 1 to 66, wherein the control is the expression level of the one or more PBD-resistance genes in a healthy subject.
[0573] 68. The method of any one of statements 1 to 65, wherein the control is the expression level of the one or more PBD-resistance genes in a subject having a disorder known not to be PBD-resistant.
[0574] 69. The method of statement 68, wherein the subject having a disorder that is known not to be PBD-resistant has previously been successfully treated with a PBD agent.
[0575] 70. The method of any one of statements 1 to 65, wherein the control is the average expression level of the one or more PBD-resistance genes in a control population.
[0576] 71. The method of statement 70, wherein the control population is a population of healthy subjects.
[0577] 72. The method of any one of statements 1 to 71, wherein the sample and control are taken from the same tissue.
[0578] 73. The method of any one of statements 1 to 72, wherein the PBD agent comprises a compound of the formula:
##STR00090## [0579] wherein positions 1-3 and 6-9 are optionally substituted; [0580] wherein the B-ring has either an imine (N═C), a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position; and, optionally [0581] wherein the chiral C11a position has a (S)-configuration.
[0582] 74. The method of any one of statements 1 to 73, wherein the PBD agent is, comprises, or releases a compound of the formula:
##STR00091##
[0583] 75. The method of any one of statements 1 to 74, wherein the PBD agent is a conjugate of formula L-(D.sup.L).sub.p, where D.sup.L is of formula I or II:
##STR00092##
wherein:
L is a cell binding agent (CBA); [0584] when there is a double bond present between C2′ and C3′, R.sup.12 is selected from the group consisting of:
(ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(ib) C.sub.1-5 saturated aliphatic alkyl;
(ic) C.sub.3-6 saturated cycloalkyl;
##STR00093##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5;
##STR00094##
wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
##STR00095##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,
R.SUP.12 .is
[0585] ##STR00096##
where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;
R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo;
where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups;
R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo;
R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from 0, S, or NH;
R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively;
[Formula I]
[0586] R.sup.L1′ is a linker for connection to the cell binding agent (CBA);
R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R.sup.20 and R.sup.21 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;
R.sup.20 is selected from H and R.sup.C, where R.sup.C is a capping group;
R.sup.21 is selected from OH, OR.sup.A and SO.sub.zM;
when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of:
(ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(ib) C.sub.1-5 saturated aliphatic alkyl;
(ic) C.sub.3-6 saturated cycloalkyl;
##STR00097##
wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;
##STR00098##
wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
##STR00099##
where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2 and C3,
R.SUP.2 .is
[0587] ##STR00100##
where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;
[Formula II]
[0588] R.sup.22 is of formula IIIa, formula IIIb or formula IIIc:
##STR00101##
where A is a C.sub.5-7 aryl group, and either
(i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a single bond and —Z—(CH.sub.2).sub.n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q.sup.1 is —CH═CH—, and Q.sup.2 is a single bond;
##STR00102##
where;
R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and unsubstituted C.sub.1-2 alkyl;
##STR00103##
where Q is selected from O—R.sup.L2′, S—R.sup.L2′ and NR.sup.N—R.sup.L2′, and R.sup.N is selected from H, methyl and ethyl
X is selected from the group comprising: O—R.sup.L2′, S—R.sup.L2′, CO.sub.2—R.sup.L2′, NH—C(═O)—R.sup.L2′, NHNH—R.sup.L2′, CONHNH—R.sup.L2′,
##STR00104##
NR.sup.NR.sup.L2′, wherein R.sup.N is selected from the group comprising H and C.sub.1-4 alkyl;
R.sup.L2′ is a linker for connection to the cell binding agent (CBA);
R.sup.10 and R.sup.11 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;
R.sup.10 is H and R.sup.11 is selected from OH, OR.sup.A and SO.sub.zM;
R.sup.30 and R.sup.31 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;
R.sup.30 is H and R.sup.31 is selected from OH, OR.sup.A and SO.sub.zM.
[0589] 76. The method of statement 75, wherein the PBD agent comprises a compound of the formula:
##STR00105## ##STR00106##
[0590] 77. The method of any one of statements 1 to 73, wherein the PBD agent is a compound of the formula (III):
L-(DL).sub.p (Ill)
wherein:
L is a cell binding agent (CBA);
DL is
[0591] ##STR00107##
wherein:
X is selected from the group comprising: a single bond, —CH.sub.2— and —C.sub.2H.sub.4—;
n is from 1 to 8;
m is 0 or 1;
R.sup.7 is either methyl or phenyl;
when there is a double bond between C2 and C3, R.sup.2 is selected the group consisting of:
(ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(ib) C.sub.1-5 saturated aliphatic alkyl;
(ic) C3-6 saturated cycloalkyl;
##STR00108##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;
##STR00109##
wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
##STR00110##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond between C2 and C3, R.sup.2 is
##STR00111##
where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;
when there is a double bond between C2′ and C3′, R.sup.12 is selected the group consisting of:
(iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(iib) C.sub.1-5 saturated aliphatic alkyl;
(iic) C.sub.3-6 saturated cycloalkyl;
##STR00112##
wherein each of R.sup.31, R.sup.32 and R.sup.33 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5;
##STR00113##
wherein one of R.sup.35a and R.sup.35b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
##STR00114##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond between C2′ and C3′, R.sup.12 is
##STR00115##
where R.sup.36a and R.sup.36b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.36a and R.sup.36b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;
and p is from 1 to 8.
[0592] 78. The method of statement 77, wherein DL is:
##STR00116##
[0593] 79. The method of any one of statements 1 to 75, wherein the PBD agent comprises a PBD conjugated to a cell binding agent.
[0594] 80. The method of any one of statements 1 to 76, wherein the cell-binding moiety is an antibody.
[0595] 81. The method of statement 80, wherein the antibody binds CD25 and comprises a VH domain having the sequence of SEQ ID NO.1 and a VL domain having the sequence of SEQ ID NO.2.
[0596] 82. The method of statement 80, wherein the antibody binds CD19 and comprises a VH domain having the sequence of SEQ ID NO.12 and a VL domain having the sequence of SEQ ID NO.14.
[0597] 83. The method of statement 80, wherein the antibody binds CD22 and comprises a VH domain having the sequence of SEQ ID NO.15 and a VL domain having the sequence of SEQ ID NO.16.
[0598] 84. The method of statement 80, wherein the antibody binds PSMA and comprises a VH domain having the sequence of SEQ ID NO.23 and a VL domain having the sequence of SEQ ID NO.24.
[0599] 85. The method of statement 80, wherein the antibody binds AXL and comprises a VH domain having the sequence of SEQ ID NO.31 and a VL domain having the sequence of SEQ ID NO.32.
[0600] 86. The method of statement 80, wherein the antibody binds DLK1 and comprises a VH domain having the sequence of SEQ ID NO.41 and a VL domain having the sequence of SEQ ID NO.42.
[0601] 87. The method of statement 80, wherein the antibody binds KAAG1 and comprises a VH domain having the sequence of SEQ ID NO.61 and a VL domain having the sequence of SEQ ID NO.62, SEQ ID NO.73, or SEQ ID NO.75.
[0602] 88. The method of statement 80, wherein the antibody binds Mesothelin and comprises: [0603] (a) a VH having the sequence of SEQ ID NO. 81 a VL having the sequence of SEQ ID NO. 82; [0604] (b) a VH having the sequence of SEQ ID NO. 92 a VL having the sequence of SEQ ID NO. 93; [0605] (c) a VH having the sequence of SEQ ID NO. 103 a VL having the sequence of SEQ ID NO. 104; or [0606] (d) a VH having the sequence of SEQ ID NO. 114 a VL having the sequence of SEQ ID NO. 115.
[0607] 89. The method of any one of statements 1 to 73, wherein the PBD agent is ADCx25.
[0608] 90. The method of any one of statements 1 to 73, wherein the PBD agent is ADCx19.
[0609] 91. The method of any one of statements 1 to 73, wherein the PBD agent is ADCx22.
[0610] 92. The method of any one of statements 1 to 73, wherein the PBD agent is ADCxPSMA.
[0611] 93. The method of any one of statements 1 to 73 wherein the PBD agent is ADCxAXL.
[0612] 94. The method of any one of statements 1 to 73, wherein the PBD agent is ADCxDLK1.
[0613] 95. The method of any one of statements 1 to 73, wherein the PBD agent is ADCxKAAG1.
[0614] 96. The method of any one of statements 1 to 73, wherein the PBD agent is ADCxMesothelin.
[0615] 97. The method of any one of statements 1 to 73, wherein the PBD agent is selected from ADCT-301, ADCT-401, ADCT-402, ADCT-602, ADCT-601, or ADCT-701.
[0616] 98. A PBD agent as defined in any one of statements 73 to 97 for use in a method of any one of statements 5 to 72.
[0617] 99. Use of a PBD agent as defined in any one of statements 73 to 97 in the preparation of a medicament for use in a method of any one of statements 5 to 72.
[0618] 100. An antagonist of one or more PBD-resistance genes as defined in any one of statements 4 to 20 for use in a method of any one of statements 4 to 97.
[0619] 101. Use of an antagonist of one or more PBD-resistance genes as defined in any one of statements 4 to 20 in the preparation of a medicament for use in a method of any one of statements 4 to 97.
EXAMPLES
Example 1: ADC and PBD Acquired Resistance Cell Line Generation
Generation of Acquired Resistance in Human Haematological Karpas-299 Cell Line
[0620] To generate a resistant population to either ADCT-301 or SG3199, human anaplastic large cell lymphoma Karpas-299 cells (expressing CD25) were incubated with an approximate IC50 dose of the drug for 96 hours. The cells were then washed and returned to fresh medium until normal cell growth was recovered. This process was repeated until a measurable and stable loss in cytotoxic efficacy was established using the MTS assay.
[0621] To measure the in vitro cytotoxicity with the MTS assay, cells were incubated with a 10-fold dilution range of ADCT-301 or SG3199, and incubated with continuous exposure for 96 hours (at least three cell doubling times under normal cell culture conditions). After incubation the CellTiter 960 AQueous One Solution Cell Proliferation Assay (MTS) was used to measure growth inhibition, by adding 20 μl reagent to the cells and incubating for 3-4 hours, then reading the absorbance at 492 nm. Mean % growth of the ADC or PBD treated cells was calculated relative to an untreated control. The growth inhibition curves for the wildtype and ADCT-301 acquired resistant cells are shown in
Generation of Acquired Resistance in Human Solid Tumour NCI-N87 Cell Line
[0622] To generate a resistant population to either ADCT-502 or SG3199, human gastric cancer NCI-N87 cells (expressing HER2) were incubated with an approximate IC50 dose of the drug for 144 hours. The cells were then washed and returned to fresh medium until normal cell growth was recovered. This process was repeated until a measurable and stable loss in cytotoxic efficacy was established using the MTS assay.
[0623] To measure the in vitro cytotoxicity with the MTS assay, cells were incubated with a 10-fold dilution range of ADCT-502 or SG3199, and incubated with continuous exposure for 144 hours (at least three cell doubling times under normal cell culture conditions). After incubation the CellTiter 960 AQueous One Solution Cell Proliferation Assay (MTS) was used to measure growth inhibition, by adding 20 μl reagent to the cells and incubating for 3-4 hours, then reading the absorbance at 492 nm. Mean % growth of the ADC or PBD treated cells was calculated relative to an untreated control. The growth inhibition curves for the wildtype and ADCT-502 acquired resistant cells are shown in
Example 2: Cross Resistance
Determination of Cross-Resistance in Karpas-299 Acquired Resistant Cell Lines
[0624] To measure the cross-resistance of the Karpas ADCR cells to the SG3199 PBD warhead, and the PBDR cells to ADCT-301, the in vitro cytotoxicity assay was carried out as described above, treating the cells with the opposite drug molecule to the one with which they were generated. The growth inhibition curves for the wildtype and ADCT-301 acquired resistant cells are shown in
[0625] The mean IC50 values and fold resistance compared to the wt cell line are detailed in the table below.
TABLE-US-00003 ADCT-301 IC.sub.50 SG3199 IC.sub.50 pM ng/mL (fold (fold resistance resistance compared compared to Cell line to wildtype) wildtype) Karpas wt 1.5 1.5 Karpas ADCr >1000 (>667) 306.5 (4.4) Karpas PBDr 72.8 (48.5) 204.5 (2.9)
[0626] To further investigate the cross-resistance of the Karpas ADCR and PBDR cells to other PBD dimer-containing ADCs, cytotoxic sensitivity of both resistant cell lines was compared to the wt cells treated with the C2-linked PBD-ADC HuMax-TAC-SG3560, using the same MTS assay protocol previously described (
Determination of Cross-Resistance in NCI-N87 Acquired Resistant Cell Lines
[0627] To measure the cross-resistance of the NCI-N87 ADCR cells to the SG3199 PBD warhead, and the PBDR cells to ADCT-502, the in vitro cytotoxicity assay was carried out as described above, treating the cells with the opposite drug molecule to the one with which they were generated. The mean IC50 values and fold resistance compared to the wt cell line are detailed in the table below.
[0628] The mean IC50 values and fold resistance compared to the wt cell line are detailed in the table below.
TABLE-US-00004 ADCT-502 IC.sub.50 SG3199 IC.sub.50 pM ng/mL (fold (fold resistance resistance compared compared Cell line to wildtype) to wildtype) NCI-N87 wt 1.3 15.9 NCI-N87 ADCr 9.9 (7.6) 60.4 (3.8) NCI-N87 PBDr 7.2 (5.5) 58.6 (3.7)
Example 3: DNA Interstrand Cross-Linking
Determination of DNA Interstrand Cross-Linking in Karpas-299 Wild Type and Acquired Resistant Cell Lines
[0629] The formation of inter-strand cross-links (ICLs) by either ADCT-301 or SG3199 was measured using a modification of the single cell gel electrophoresis (comet) assay (Spanswick V J, Hartley J M, Hartley J A. Measurement of DNA interstrand crosslinking in individual cells using the Single Cell Gel Electrophoresis (Comet) assay. Methods Mol Biol. 613, 267-82 (2010)). Cells were treated with 130 μM ADCT-301 or 280 μM SG3199 for 2 hours, then washed and incubated for 24 hours under normal cell culture conditions. All cells were irradiated with 18 Gy (5 Gy/min for 3.6 min). Comet slides were reviewed under a 20× objective on an epifluorescence microscope equipped with: Hg arc lamp; 580 nm dichroic mirror; and 535 nm excitation and 645 nm emission filters suitable for visualising of propidium iodide staining with a minimum of 50 Comet images acquired per treatment condition. The Olive Tail Moment (OTM) was determined as the product of the tail length and the fraction of total DNA in the tail as recorded by Komet 6 software (Andor Technology, Belfast, UK) and the percentage reduction in OTM calculated according to the formula:
% decrease in tail moment=[1−(TMdi−TMcu)/(TMci−TMcu)]*100 [0630] [TMdi=Tail Moment drug irradiated; TMcu=TM control un-irradiated; TMci=TM control irradiated]
[0631] The level of DNA interstrand cross-linking for ADCT-301 in Karpas-ADCR and for SG3199 in Karpas-PBDR compared to wildtype cells are shown in
Determination of DNA Interstrand Cross-Linking in NCI-N87 Wild Type and Acquired Resistant Cell Lines
[0632] The formation of inter-strand cross-links (ICLs) by either ADCT-502 or SG3199 was measured using the modification of the single cell gel electrophoresis (comet) assay. Cells were treated with 1 nM ADCT-502 or 1.7 nM SG3199 for 2 hours, then washed and incubated for 24 hours under normal cell culture conditions. All cells were irradiated with 18 Gy (5 Gy/min for 3.6 min). ICL formation was quantitated by measuring Olive tail moment (OTM) using the Komet 4 software, and the percentage reduction in OTM compared to an untreated irradiated control.
[0633] The level of DNA interstrand cross-linking for ADCT-502 in NCI-N87-ADCR and for SG3199 in NCI-N87-PBDR compared to wildtype cells are shown in
Karpas-299 2-Hour Exposure Cytotoxicity
[0634] To investigate growth inhibition under the same conditions under which DNA ICL formation was measured, Karpas-ADCR cells were treated with 130 pM ADCT-301, and Karpas-PBDR cells treated with 280 pM SG3199 for 2 hours, then washed and incubated for 96 hours under normal cell culture conditions. The MTS assay and calculation of % growth compared to the untreated control was carried out as previously described.
[0635] The resulting growth inhibition values for ADCT-301 in Karpas-ADCR versus wildtype and for SG3199 in Karpas-PBDR versus wildtype are shown in
NCI-N87 2-Hour Exposure Cytotoxicity
[0636] To investigate growth inhibition under the same conditions under which DNA ICL formation was measured, NCI-N87-ADCR cells were treated with 1 nM ADCT-502, and PBDR cells treated with 1.7 nM SG3199 for 2 hours, then washed and incubated for 144 hours under normal cell culture conditions. The MTS assay and calculation of % growth compared to the untreated control was carried out as previously described.
[0637] The resulting growth inhibition values for ADCT-502 in NCI-N87-ADCR versus wildtype and for SG3199 in NCI-N87-PBDR versus wildtype are shown in
Karpas-R Cross-Resistant ICL Formation
[0638] To investigate if the cross-resistance of the Karpas ADCR cells to the SG3199 PBD warhead, and the PBDR cells to ADCT-301, also shows a change in ICL formation, the comet assay was carried out as described above, treating the cells with the opposite drug molecule to the one with which they were generated. The results are shown in
[0639] The mean % reduction in OTM and p-value of the t-tests comparing the resistant line with the wt cell line are detailed in the table below.
TABLE-US-00005 ADCT-301 % red. SG3199 % red. Cell line OTM (p-value) OTM (p-value) Karpas wt 53.5 55.5 Karpas ADCr 15 (0.0004) 7.2 (0.0006) Karpas PBDr 34.3 (0.01) 25.7 (0.003)
NCI-N87 Cross-Resistant ICL Formation
[0640] To investigate if the cross-resistance of the NCI-N87 ADCR cells to the SG3199 PBD warhead, and the PBDR cells to ADCT-502, also shows a change in ICL formation, the comet assay was carried out as described above, treating the cells with the opposite drug molecule to the one with which they were generated. The results are shown in
[0641] The mean % reduction in OTM and p-value of the t-tests comparing the resistant line with the wt cell line are detailed in the table below.
TABLE-US-00006 ADCT-502 % red. SG3199 % red. Cell line OTM (p-value) OTM (p-value) NCI-N87 wt 54.7 51.0 NCI-N87 ADCr 29 (0.004) 24 (0.04) NCI-N87 PBDr 26 (0.0005) 30.7 (0.004)
Karpas Cross-Resistance Single Dose 2-Hour Exposure Cytotox
[0642] To investigate whether the cross-resistance in ICL formation observed in the Karpas resistant cell lines also correlates with growth inhibition at this dose, ADCR cells were treated with 280 μM SG3199, and PBDR cells treated with 130 μM ADCT-301 for 2 hours, then washed and incubated for 96 hours under normal cell culture conditions. The MTS assay and calculation of % growth compared to the untreated control was carried out as previously described. The results are shown in
[0643] The mean % growth values and p-values from the t-test comparing the resistant cells to the wt cell line are detailed in the table below.
TABLE-US-00007 ADCT-301 2 SG3199 2 hr mean % hr mean % Cell line growth (p-value) growth (p-value) Karpas wt 33.8 33.8 Karpas ADCr 85.9 (0.0004) 86.3 (0.008) Karpas PBDr 74.7 (0.01) 78.5 (0.035)
NCI-N87 Cross-Resistant Single-Dose Cytotox
[0644] To investigate whether the cross-resistance in ICL formation observed in the NCI-N87 resistant cell lines also correlates with growth inhibition at this dose, ADCR cells were treated with 1 nM SG3199, and PBDR cells treated with 1.7 nM ADCT-502 for 2 hours, then washed and incubated for 144 hours under normal cell culture conditions. The MTS assay and calculation of % growth compared to the untreated control was carried out as previously described. The results are shown in
[0645] The mean % growth values and p-values from the t-test comparing the resistant cells to the wt cell line are detailed in the table below.
TABLE-US-00008 ADCT-502 2 SG3199 2 hr mean % hr mean % Cell line growth (p-value) growth (p-value) NCI-N87 wt 23.5 29.0 NCI-N87 ADCr 61.5 (0.0008) 57.2 (0.03) NCI-N87 PBDr 51.9 (0.02) 76.6 (0.004)
Cross-Resistance to Other Chemotherapeutic Drugs
[0646] Cross-resistance to three conventional DNA-interacting chemotherapeutic drugs, cisplatin, doxorubicin and melphalan was also measured using the MTS assay in Karpas and NCI-N87 parental, ADC and PBD resistant cell lines. No cross-resistance was seen between any of these drugs in either the Karpas-299 ADCr or PBDr cell lines. In contrast, there was a decrease in growth inhibition in both NCI-N87 ADCr and PBDr cells treated with all three of these drugs compared to the parental cell line.
Example 4: Antigen Expression
Karpas Resistant Line CD25 Antibody Binding
[0647] Karpas wt and acquired resistant cells were blocked at 4° C. for 30 mins before being incubated with a serial dilution series of HuMax-TAC mAb for 1 hour on ice. The cells were then washed and incubated with a F(ab′)2-Goat anti-Human IgG Fc Secondary Antibody conjugated to Alexa Fluor 488. After incubation for 1 hour on ice in the dark the cells were washed and run on a Fortessa X20 flow cytometer, and the mean fluorescence intensity (MFI) determined using the B530/30 laser/filter. The resulting MFI curves are shown in
Example 5: Human Drug Transporter Expression—PCR Array
Karpas-R Drug Transporters PCR Array
[0648] The real-time RT2 Profiler PCR Array for human drug-transporters (Qiagen) was used to probe cDNA generated from Karpas acquired resistant and wt cell line lysates. CT values were normalized based on a panel of housekeeping genes (HKG). The Qiagen data analysis web portal calculates fold change/regulation using ΔΔCT method, in which ΔCT was calculated between gene of interest (GOI) and an average of HKGs, followed by DD CT calculations (ΔCT(Test Group)−ΔCT(Control Group)). Fold Change was then calculated using 2{circumflex over ( )}(−ΔΔCT) formula.
[0649] The volcano plot of Karpas wildtype versus Karpas-ADCR is shown in
TABLE-US-00009 Gene Fold upregulation (p-value) ABCG2 168.1 0.0002 SLCO2A1 4.6 0.009 ABCC2 3.5 0.004
[0650] The volcano plot of Karpas wildtype versus Karpas-PBDR is shown in
TABLE-US-00010 Gene Fold upregulation (p-value) SLCO2B1 15.5 0.0006 ABCC12 6.4 0.01 ATP7A 4.6 0.0001 SLC16A2 2.8 0.002 ABCG2 2.8 0.05 SLC7A9 2.5 0.0004 ABCB4 2.3 0.008 ABCC11 2.1 0.01
NCI-N87-R Drug Transporters PCR Array
[0651] The real-time RT2 Profiler PCR Array for human drug-transporters (Qiagen) was used to probe cDNA generated from NCI-N87 resistant and wt cell line lysates. CT values were normalized based on a panel of housekeeping genes (HKG). The Qiagen data analysis web portal calculates fold change/regulation using ΔΔCT method, in which ΔCT was calculated between gene of interest (GOI) and an average of HKGs, followed by DD CT calculations (ΔCT(Test Group)−ΔCT(Control Group)). Fold Change was then calculated using 2{circumflex over ( )}(−ΔΔCT) formula.
[0652] The volcano plot of NCI-N87 wildtype versus NCI-N87-ADCR is shown in
TABLE-US-00011 Gene Fold upregulation (p-value) ABCG2 91.0 0.007 SLC22A3 65.3 0.02 ABCC2 43.0 0.002 SLCO2B1 30.3 0.01 SLC7A7 6.8 0.0003 ABCF1 2.2 0.05
[0653] The volcano plot of NCI-N87 wildtype versus NCI-N87-PBDR is shown in
TABLE-US-00012 Gene Fold upregulation (p-value) ABCG2 114.5 0.00001 SLC22A3 61.1 0.002 SLCO2B1 54.4 0.0006 ABCC2 35.5 0.0003 SLC7A7 7.8 0.006 AQP7 6.4 0.03 SLC5A1 4.9 0.01 SLC28A3 2.9 0.01 ABCF1 2.3 0.0008 ATP7A 2.2 0.007 ABCB6 2.1 0.002
Example 6: Human Drug Transporter Expression—RT-PCR
Karpas-R Drug Transporter TaqMan RT-PCR
[0654] To confirm the upregulation of drug transporter genes potentially implicated in the acquired resistance phenotype, mRNA from Karpas wt, ADCR and PBDR cells were collected and cDNA synthesis performed with SuperScript III RT kit (LifeTech). Rt-PCR reaction mixes were prepared using TaqMan gene expression probes (LifeTech) for the drug transporter of choice or the housekeeping gene ABL-1 and fold change/regulation calculated using ΔΔCT method as described above. The change in expression levels for ABCG2, ABCC2 and SLCO2B1 in Karpas-ADCR and PBDR cells versus wildtype are shown in
[0655] The mean fold change in expression is shown in the table below.
TABLE-US-00013 Karpas ADC-R fold Karpas PBD-R fold Gene upregulation upregulation ABCG2 134 1.9 ABCC2 6.5 0.8 SLCO2B1 9.5 6
NCI-N87-R Drug Transporters TaqMan RT-PCR
[0656] To confirm the upregulation of drug transporter genes potentially implicated in the acquired resistance phenotype, mRNA from NCI-N87 wt, ADCR and PBDR cells were collected and cDNA synthesis performed with SuperScript III RT kit (LifeTech). Rt-PCR reaction mixes were prepared using TaqMan gene expression probes (LifeTech) for the drug transporter of choice or the housekeeping gene ABL-1 and fold change/regulation calculated using ΔΔCT method as described above. The change in expression levels for ABCG2, ABCC2 and SLCO2B1, SLC7A7 and SLC22A3 in NCI-N87-ADCR and PBDR cells versus wildtype are shown in
[0657] The mean fold change in expression is shown in the table below.
TABLE-US-00014 NCI-N87 ADC-R NCI-N87 PBD-R Gene fold upregulation fold upregulation ABCG2 111 150 ABCC2 55 56 SLCO2B1 49 71 SLC7A7 11 10 SLC22A3 220 201
DNA Damage Signalling Pathway
[0658] In contrast to the results obtained with the human drug transporter gene array, an RT.sup.2 PCR array for DNA damage signalling pathway showed no significantly upregulated genes in any of the acquired resistant lines. Overall, the data are consistent with the mechanism of resistance being upstream of the DNA damage produced by the PBD dimer.
Example 7: Human Drug Transporter Expression—Western Blot
Karpas-R ABC Transporter Western Blot
[0659] To confirm the drug transporter genes shown to be upregulated by PCR also gave rise to an increase in protein expression, 30-40 μg protein lysate from Karpas resistant and wt cells was loaded into a Mini-Proean TGX 4-15% Tris-Gly gel (BioRad) and run for 120v for 1 hour. The gel was then transferred onto a Trans-Blot Turbo NCL pack (BioRad). The nitrocellulose was then blocked with 5% milk for 30 mins, and incubated with rabbit monoclonal anti-ABCG2 (Abcam) or rabbit monoclonal anti-ABCC2 (Cell Signalling) overnight at 4° C. The membrane was washed with TBST and incubated with HRP-conjugated secondary antibody before developing the signal with the Amersham ECL Western blotting detection reagents and analysis system (GE Healthcare). A western blot of Karpas wt and resistant cell lines probed for ABCC2 and ABCG2 is shown in
NCI-N87-R ABC Transporter Western Blot
[0660] To confirm the drug transporter genes shown to be upregulated also gave rise to an increase in protein expression, 30-40 μg protein lysate from NCI-N87 resistant and wt cells was loaded into a Mini-Proean TGX 4-15% Tris-Gly gel (BioRad) and run for 120v for 1 hour.
[0661] The gel was then transferred onto a Trans-Blot Turbo NCL pack (BioRad). The nitrocellulose was then blocked with 5% milk for 30 mins, and incubated with rabbit monoclonal anti-ABCG2 (Abcam) or rabbit monoclonal anti-ABCC2 (Cell Signalling) overnight at 4° C. The membrane was washed with TBST and incubated with HRP-conjugated secondary antibody before developing the signal with the Amersham ECL Western blotting detection reagents and analysis system (GE Healthcare). A western blot of NCI-N87 wt and resistant cell lines probed for ABCC2 and ABCG2 is shown in
Example 8: Antibody Internalisation
Methodology
[0662] To investigate any difference in antibody internalisation in the Karpas acquired resistant cell lines compared to wt, cells were seeded in a poly-L-ornithine coated 96-well plate and allowed to attach at 37° C. HuMax-TAC was incubated with a 3× molar excess of FabFluor pH red antibody internalisation reagent (Essen Bioscience) for 15 mins at room temperature, then a 3× serial dilution set up with cell culture medium. The labelled antibody dilutions were added to the Karpas cells and the plate was immediately transferred to the IncuCyte Zoom, where images were taken at 10× magnification every 2 hours with phase contrast and red fluorescence filters. Mean red object area per well was calculated using the IncuCyte Zoom software.
[0663] Similarly, NCI-N87 wt and acquired resistant cells were seeded in a 96-well plate and allowed to attach at 37° C. Trastuzumab was incubated with a 3× molar excess of FabFluor pH red antibody internalisation reagent (Essen Bioscience) for 15 mins at room temperature, then a 3× serial dilution set up with cell culture medium. The labelled antibody dilutions were added to the cells and the plate was immediately transferred to the IncuCyte Zoom, where images were taken at 10× magnification every 2 hours with phase contrast and red fluorescence filters. Mean red object area per well was calculated using the IncuCyte Zoom software.
Results
[0664] A plot of Karpas wt and resistant cell line CD25 antibody internalisation is shown in
[0665] A plot of NCI-N87 wt and resistant cell line HER2 antibody internalisation is shown in
[0666] In both cases, similar levels and kinetics of internalisation were observed in the resistant lines compared to wildtype.
Example 9.1: Karpas Resistant Line ABC Inhibitor Combination (MK-571)
Methodology
[0667] To confirm the involvement of ABCC transporters in the acquired resistance to ADCT-301, Karpas ADC and PBD resistant cells were incubated with a non-toxic dose (50 μM) of MK-571 before seeding in a 96-well plate and 96-hour continuous exposure to ADCT-301. The in vitro cytotoxicity assay (MTS) was carried out as previously described.
Results
[0668] A plot of 96-hour continuous exposure MK-571 and ADCT-301 cytotoxicity of Karpas wt and Karpas ADCR cells is shown in
[0669] A plot of 96-hour continuous exposure MK-571 and ADCT-301 cytotoxicity of Karpas wt and Karpas PBDR cells is shown in
[0670] In both acquired resistant cell lines, addition of the inhibitor was able to partially reverse the resistance.
Example 9.2: Karpas Resistant Line ABC Inhibitor Combination (Multi-Inhibitor)
Methodology
[0671] Cells were seeded at 10,000 cells per well in a flat bottom 96-well plate, and NCI-N87 cell were incubated overnight to allow for cell attachment.
[0672] For ADC growth inhibition assays, cells were then incubated with serial dilutions of ADCT-301 or ADCT-502 in triplicate. For the SG3199 growth inhibition assays, cells were mixed with a serial dilution of SG3199 prepared in DMSO before seeding to ensure the DMSO concentration was the same in all wells and had no effect on cell growth.
[0673] Growth inhibition was measured after 96 hours in Karpas-299 cells and 144 hours in NCI-N87 cells using the CellTiter 96 AQueous One MTS Solution (Promega) and absorbance measured on a Multiskan Ascent plate reader (ThermoFisher) at 492 nm.
[0674] Growth inhibition was calculated as a percentage of absorbance compared to an untreated control, and IC50 values were calculated using the sigmoidal, 4PL, X is log(concentration) equation in GraphPad Prism.
[0675] For drug transporter inhibitor combination cytotox, the resistant cell lines were incubated overnight with either 5 μM MK-571 (ABCC2 inhibitor), 10 μM FTC (ABCG2 inhibitor), 5 μM Reversin-121 (ABCB1 inhibitor) or 10 nM Lovastatin (SLCO2B1 inhibitor) overnight before carrying out the ADC or PBD growth inhibition assay as previously described.
Results
[0676] In order to investigate further the contribution of ABC drug transporter upregulation in the acquired resistance to the PBD dimer and PBD dimer-based ADC resistant cell lines, inhibitors of ABCG2 (Fumitremorgin C, FTC) and ABCC2 (MK-571) were used in combination with the ADC or PBD dimer to assess the effect on growth inhibition using the MTS assay.
[0677] Karpas and NCI-N87 resistant cells were treated with a non-toxic dose of MK-571 (5 μM) or FTC (10 μM) for 24 hours before the addition of the ADC or PBD dimer. Both MK-571 and FTC showed a dramatic re-sensitisation of Karpas-299 ADCr and PBDr cells to ADCT-301 treatment (
[0678] Similar restoration of sensitivity was observed when the ABC transporter inhibitors were combined with SG3199 in Karpas ADCr and PBDr cells (
[0679] In order to correlate the inhibition of the ABC transporters with the reversal of ADC and PBD dimer resistance mechanistically, the comet assay was used to measure ICL formation in the resistant cell lines by the ADC or PBD dimer pre-treated with either 5 μM MK-571 or 10 μM FTC.
[0680] In all the resistant cell lines, treatment with transporter inhibitor resulted in increased formation of ADC or PBD-induced DNA interstrand cross-linking (
[0681] In the accompanying figures, each data point represents the average of at least 3 biological repeats with +/−SD error bars. p-values obtained using two-tailed, unpaired t-tests.
Example 10: Karpas-R Cross-Resistance with SG2000
[0682] To measure the cross-resistance of the Karpas ADCR and PBDR cells to the PBD dimer warhead SG2000, the in vitro cytotoxicity (MTS) assay was carried out as previously described in Example 2. The mean IC50 values and fold resistance compared to the wildtype (wt) cell line are shown in the table below.
TABLE-US-00015 Cell line SG2000 IC50 nM (fold resistance) Karpas wt 0.9 Karpas ADCr 2.8 (3.1) Karpas PBDr 4.7 (5.2)
Example 11: NCI-N87-R Cross-Resistance with Other PBD Dimers
SG3560
[0683] To investigate the cross-resistance of the NCI-N87 ADCR and PBDR cells to other PBD dimer containing ADCs, in vitro cytotoxicity was compared to the wt cells treated with the C2-linked PBD-ADC trastuzumab-SG3560, using the same MTS assay protocol previously described.
TABLE-US-00016 Trastuzumab-SG3560 IC50 ng/mL Cell line (fold resistance) NCI-N87 wt 1 NCI-N87 ADCr 95 (95) NCI-N87 PBDr 54 (54)
SG2000
[0684] To measure the cross-resistance of the NCI-N87 ADCR and PBDR cells to the PBD dimer warhead SG2000, the in vitro cytotoxicity (MTS) assay was carried out as previously described. The mean IC50 values and fold resistance compared to the wt cell line are shown in the table below.
TABLE-US-00017 Cell line SG2000 IC50 pM (fold resistance) NCI-N87 wt 64 NCI-N87 ADCr 483 (7.5) NCI-N87 PBDr 493 (7.7)
Example 12: NCI-N87 Resistant Line HER2 Antibody Binding
Methodology
[0685] NCI-N87 wt and resistant cells were blocked at 4° C. for 30 mins before being incubated with a serial dilution series of trastuzumab for 1 hour on ice. The cells were then washed and incubated with a F(ab′)2-Goat anti-Human IgG Fc secondary antibody conjugated to Alexa Fluor 488. After incubation for 1 hour on ice in the dark the cells were washed and run on a Fortessa X20 flow cytometer, and the MFI using the B530/30 laser/filter.
Results
[0686] The resulting curves are shown in
[0687] Receptor affinity was similar and a small decrease in peak MFI was observed in the resistant cell lines compared to wt cells, the effect being greatest in the PBDr cell line.
Example 13: Protein Analysis of ABC Transporters and siRNA Knockout of ABCC2
Methodology
[0688] Silencer® Select siRNA oligonucleotides targeting ABCC2 and nontargeting siRNAs were purchased from Ambion/ThermoFisher. For reverse transfection, Opti-MEM medium was mixed with siRNA to give a final concentration of 25 pmol/L, this was then combined with diluted Lipofectamine® RNAiMAX (Thermo Scientific). After 20-minute incubation at room temperature, the transfection mixture was aliquoted into 6-well plates. Cells were added to each well containing siRNA and RNAiMAX complex. 48 hours after transfection, cells were harvested and used for immunoblotting or growth inhibition assays as previously described.
Results
[0689] The two drug transporters with the most consistent mRNA upregulation across the resistant cell lines, which also responded to appropriate transporter inhibition to restore drug sensitivity were ABCG2 and ABCC2. Immunoblotting showed the ABCG2 protein to be upregulated in the Karpas-299 ADCr cells compared to the parental cell line, while any upregulation in PBDr cells could not be detected (
[0690] NCI-N87 cells were transfected with siRNA against ABCC2 and ABCG2. ABCG2 was not able to be effectively knocked out due to the very long half-life of the protein (data not shown), but ABCC2 was successfully depleted in both NCI-N87 ADCr and PBDr cells compared with a non-target siRNA control (
TABLE-US-00018 SEQUENCES SEQ ID NO. 1 (AB12 VH): QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRYIINWVRQAPGQGLEWMGRIIPILGVENYA QKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKDWFDYWGQGTLVTVSSASTKGP SVFPLA SEQ ID NO. 2 (AB12 VL): EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFP SEQ ID NO. 3 (AB12 VH CDR1): RYIIN SEQ ID NO. 4 (AB12 VH CDR2): RIIPILGVENYAQKFQG SEQ ID NO. 5 (AB12 VH CDR3): KDWFDY SEQ ID NO. 6 (AB12 VL CDR1): RASQSVSSYLA SEQ ID NO. 7 (AB12 VL CDR2): GASSRAT SEQ ID NO. 8 (AB12 VL CDR3): QQYGSSPLT SEQ ID NO. 11 (RB4v1.0 VH): QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTN YNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTV S SEQ ID NO. 12 (RB4v1.2 VH): QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQAPGQGLEWIGEIDPSDSYTN YNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTV S SEQ ID NO.13 (RB4v1.0 VK): EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPAR FSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK SEQ ID NO. 14 (RB4v1.2 VK): EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPAR FSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK SEQ ID NO. 15 (Epratuzumab VH): QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGYINPRNDYTE YNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDITTFYWGQG SEQ ID NO. 16 (Epratuzumab VL): DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYWASTRE SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSVVTFGQG SEQ ID NO. 17 (EMabC220?HC): QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGYINPRNDYTEY NQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDITTFYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTVPPVPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG SEQ ID NO. 18 (EMabC220-LC): DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYWASTRE SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES SEQ ID NO. 21 (J591 VH): EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYN QKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS SEQ ID NO. 22 (J591 VL): DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPD RFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK SEQ ID NO. 23 (J591 VH Delm): EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYN QKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS SEQ ID NO. 24 (J591 VK Delm): DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSR FSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK SEQ ID NO. 25 (J591BJ Delm heavy chain): EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYN QKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTVPPVPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG SEQ ID NO. 26 (J591BJ DeIm light chain): DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSR FSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGES SEQ ID NO.31 [1H12 VH, CDR underline] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISSGGSYTY YPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHPIYYTYDDTMDYWGQGTTVT VSS SEQ ID NO.32 [1H12 VL, CDR underline] EIVLTQSPGTLSLSPGERATLSCSASSSVSSGNFHWYQQKPGLAPRLLIYRTSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQWSGYPWTFGGGTKLEIK SEQ ID NO.33 [1H12 Heavy Chain] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISSGGSYTY YPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHPIYYTYDDTMDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297 (numbering according to Kabat) SEQ ID NO.34 [1H12 Light Chain] EIVLTQSPGTLSLSPGERATLSCSASSSVSSGNFHWYQQKPGLAPRLLIYRTSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQWSGYPWTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.35 [1H12 VH CDR] SYGMS SEQ ID NO.36 [1H12 VH CDR2] TISSGGSYTYYPDSVKG SEQ ID NO.37 [1H12 VH CDR3] HPIYYTYDDTMDY SEQ ID NO.38 [1H12 VL CDR] SASSSVSSGNFH SEQ ID NO.39 [1H12 VL CDR2] RTSNLAS SEQ ID NO.40 [1H12 VL CDR3] QQWSGYPWT SEQ ID NO.41 [HuBa-1-3d VH, CDR underline] QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGNTN YNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQGTMVT VSS SEQ ID NO.42 [HuBa-1-3d VL, CDR underline] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSSNQKNYLAWYQQKPGQPPKLLVYFASTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPPTFGQGTKLEIK SEQ ID NO.43 [HuBa-1-3d Heavy Chain] QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGNTN YNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297 SEQ ID NO.44 [HuBa-1-3d Light Chain] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSSNQKNYLAWYQQKPGQPPKLLVYFASTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPPTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.45 [HuBa-1-3d VH CDR1] DYAMH SEQ ID NO.46 [HuBa-1-3d VH CDR2] VISTYYGNTNYNQKFKG SEQ ID NO.47 [HuBa-1-3d VH CDR3] GGLREYYYAMDY SEQ ID NO.48 [HuBa-1-3d VL CDR] KSSQSLLNSSNQKNYLA SEQ ID NO.49 [HuBa-1-3d VL CDR2] FASTRES SEQ ID NO.50 [HuBa-1-3d VL CDR3] QQHYSTPPT SEQ ID NO.51 [HuBa-1-3d Heavy Chain, terminal K] QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGNTN YNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297 SEQ ID NO.61 [3A4 VH, CDR underline] QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNY NQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSS SEQ ID NO.62 [3A4 VL, CDR underline] DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK SEQ ID NO.63 [3A4 Heavy Chain] QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNY NQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG N* indicates Asn297 SEQ ID NO.64 [3A4 Light Chain] DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.65 [3A4 VH CDR] GYTFTDDYMS SEQ ID NO.66 [3A4 VH CDR2] DINPYNGDTN SEQ ID NO.67 [3A4 VH CDR3] DPGAMDY SEQ ID NO.68 [3A4 VL CDR1] RSSQSLLHSNGNTYLE SEQ ID NO.69 [3A4 VL CDR2] TVSN RFS SEQ ID NO.70 [3A4 VL CDR3] FQGSHVPLT SEQ ID NO.71 [3A4 Heavy Chain, terminal K] QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNY NQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK N* indicates Asn297 SEQ ID NO.72 [Human KAAG1] MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLSQR PHRTQGAGSPPETNEKLTNPQVKEK SEQ ID NO.73 [3A4-L2 VL, CDR underline] DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK SEQ ID NO.74 [3A4-L2 Light Chain] DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.75 [3A4-K4 VL, CDR underline] DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLEWYQQKPGQPPKLLIYTVSNRFS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPLTFGQGTKVEIK SEQ ID NO.76 [3A4-K4 Light Chain] DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLEWYQQKPGQPPKLLIYTVSNRFS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPLTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.81 [XA4 VH, CDR underline] QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVT VSS SEQ ID NO.82 [XA4 VL, CDR underline] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK SEQ ID NO.83 [XA4 Heavy Chain] QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297 SEQ ID NO.84 [XA4 Light Chain] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.91 [XA4 Heavy Chain, terminal K] QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297 SEQ ID NO.92 [XFT VH, CDR underline] QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYS PSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS SEQ ID NO.93 [XFT VL, CDR underline] DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVS NRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIK SEQ ID NO.94 [XFT Heavy Chain] QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYS PSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297 SEQ ID NO.95 [XFT Light Chain] DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.102 [XFT Heavy Chain, terminal K] QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYS PSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297 SEQ ID NO.103 [X09 VH, CDR underline] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSY NQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS SEQ ID NO.104 [X09 VL, CDR underline] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGR FSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK SEQ ID NO.105 [X09 Heavy Chain] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSY NQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297 SEQ ID NO.106 [X09 Light Chain] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGR FSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.113 [X09 Heavy Chain, terminal K] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSY NQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297 SEQ ID NO.114 [X09.2 VH, CDR underline] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSY NQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSS SEQ ID NO.115 [X09.2 VL, CDR underline] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGR FSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIK SEQ ID NO.116 [X09.2 Heavy Chain] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSY NQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297 SEQ ID NO.117 [X09.2 Light Chain] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGR FSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.118 [X09.2 Heavy Chain, terminal K] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSY NQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297