COMBINATION THERAPY

Abstract

The present disclosure relates to combination therapies for the treatment of pathological conditions, such as cancer. In particular, the present disclosure relates to combination therapies comprising treatment with Antibody Drug Conjugates (ADCs) ADCs which bind to CD25 (CD25-ADCs) and radiotherapy.

Claims

1. A method of inducing or enhancing an immune response against a cancer in a subject, the method comprising administering to the subject an effective amount of an anti-CD25 antibody drug conjugate (ADC) in combination with radiotherapy, wherein the treatment comprises administering the anti-CD25 ADC before the radiotherapy.

2-4. (canceled)

5. A method for treating a cancer in a subject, the method comprising administering to the subject an effective amount of an anti-CD25 antibody drug conjugate (ADC) in combination with radiotherapy, wherein the treatment comprises administering the anti-CD25 ADC before the radiotherapy.

6-8. (canceled)

9. The method of claim 5, wherein the CD25-ADC and radiotherapy are administered on the same day.

10-30. (canceled)

31. The method of claim 5, wherein: (i) the immune-suppressive activity of a population of regulatory immune cells in the subject is reduced by at least 90% before the radiotherapy is administered; and/or (ii) the size of a population of regulatory immune cells in the subject is reduced by at least 90% before the radiotherapy is administered.

32. The method of claim 5, wherein the regulatory immune cells are Treg cells.

33. The method of claim 5, wherein the subject: a) has a disorder or has been determined to have a disorder; b) has been determined to have a cancer which expresses CD25 or CD25+ tumour-associated non-tumour cells, such as CD25+ infiltrating cells; or c) is radiosensitive.

34-35. (canceled)

35. The method of claim 5, wherein: a) the radiotherapy is focal radiotherapy; b) the radiotherapy is tumour targeted; or c) the radiotherapy is selected from the group consisting of: external beam radiotherapy, stereotactic radiation therapy, Intensity-Modulated Radiation Therapy, particle therapy, brachytherapy, delivery of radioisotopes, intraoperative radiotherapy, Auger therapy, Volumetric modulated arc therapy, Virtual simulation, 3-dimensional conformal radiation therapy, and intensity-modulated radiation therapy.

36-38. (canceled)

39. The method of claim 5, wherein the radiotherapy is sub-therapeutic dose for treatment of the disorder with radiotherapy alone.

40. The method of claim 5, wherein the total radiotherapy dose is no greater than 40 Gy.

41-48. (canceled)

49. The method of claim 5, wherein the radiotherapy is administered as a single dose.

50. The method of claim 5, wherein the radiotherapy is administered as fractionated doses.

51. The method of claim 50, wherein each fractionated dose is no greater than 20 Gy.

52-60. (canceled)

61. The method of claim 50, wherein the radiotherapy is administered in two, three, four, five, six, eight, or ten fractionated doses.

62-67. (canceled)

68. The method of claim 50, wherein the fractionated doses are administered once daily (QD), once every other day (Q2D), once every third day (Q3D) or once weekly (QW).

69-73. (canceled)

74. The method of claim 5, wherein the cancer comprises a solid tumor, and wherein the treatment induces or enhances an immune response against the at least one of the solid tumors.

75-80. (canceled)

81. The method of claim 74, wherein the solid tumour comprises CD25−ve neoplastic cells, or is associated with CD25+ve infiltrating cells.

82. (canceled)

83. The method of claim 74, wherein the solid tumour is selected from the group consisting of pancreatic cancer, 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.

84-86. (canceled)

87. The method of claim 73, wherein the cancer is 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).

88. The method of claim 73, wherein the cancer is associated with elevated levels of regulatory immune cells.

89. The method of claim 5, wherein the CD25-ADC is administered in combination with a checkpoint inhibitor or other immunostimulatory agent.

90. (canceled)

91. The method of claim 89, wherein the checkpoint inhibitor is: a) a PD1 antagonist selected from pembrolizumab, nivolumab, MEDI0680, PDR001 (spartalizumab), Camrelizumab, AUNP12, Pidilizumab Cemiplimab (REGN-2810), AMP 224, BGB-A317 (Tislelizumab), and BGB-108; b) a PD-L1 antagonist selected from atezolizumab (Tecentriq), BMS-936559/MDX-1105, durvalumab/MEDI4736, and MSB0010718C (Avelumab); c) a Glucocorticoid-Induced TNFR-Related (GITR) protein agonist selected from MEDI1873, TRX518, GWN323, MK-1248, MK 4166, BMS-986156 and INCAGN1876 d) an OX40 agonist selected from MEDI0562, MEDI6383, MOXR0916, RG7888, OX40mAb24, INCAGN1949, GSK3174998, and PF-0451860; or e) a CTLA-4 antagonist selected from ipilimumab and Tremelimumab.

92-101. (canceled)

102. The method of claim 5, wherein the CD25-ADC comprises a PBD drug moiety, optionally wherein the CD25-ADC is a conjugate of formula:
L-(D.sup.L).sub.p, where D.sup.L is of formula I or II: ##STR00041## wherein: L is an antibody (Ab) which is an antibody that binds to CD25; 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; ##STR00042## 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; ##STR00043## 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 ##STR00044## 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 ##STR00045## 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; wherein, if D.sup.L is of formula I: R.sup.L1′ is a linker for connection to the antibody (Ab); 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; ##STR00046## 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; ##STR00047## 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 ##STR00048## 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 ##STR00049## 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; wherein, if D.sup.L is of formula I: R.sup.22 is of formula IIIa formula IIIb or formula IIIc: ##STR00050## 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; ##STR00051## where; R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and unsubstituted C.sub.1-2 alkyl; ##STR00052## 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′, ##STR00053## 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 antibody (Ab); 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.

103-104. (canceled)

105. The method of claim 5, wherein the anti-CD25-ADC is Camidanlumab Tesirine.

106-109. (canceled)

110. The method of claim 5, wherein the anti-CD25 ADC is administered 1 to 21 days before the radiotherapy.

111. The method of claim 5, wherein the anti-CD25 ADC is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days before the radiotherapy.

112. The method of claim 5, wherein the anti-CD25 ADC is administered 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours before the radiotherapy.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0654] Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:

[0655] FIG. 1. Sequences

[0656] FIG. 2. In vivo MC38 tumour volume following mono-treatment with surrogate ADCx25, anti-PD1 treatment, or control ADC (as per Example 1)

[0657] FIG. 3. In vivo MC38 tumour volume showing synergy between low-dose surrogate ADCx25 and anti-PD1 treatment (as per Example 1)

[0658] FIG. 4. Re-challenge of tumour-free survivors from MC38 study (per Example 2)

[0659] FIG. 5. In vivo CT26 tumour volume following mono-treatment with surrogate ADCx25, anti-PD1 treatment, or control ADC (as per Example 3)

[0660] FIG. 6. In vivo CT26 tumour volume showing synergy between low-dose surrogate ADCx25 and anti-PD1 treatment (as per Example 3)

[0661] FIG. 7. Re-challenge of tumour-free survivors from CT26 efficacy study (as per Example 4)

[0662] FIG. 8. ADCx25 anti-tumour activity is dependent on CD8+ T-cells

[0663] FIG. 9. ADCx25 plus PD-1 anti-tumour activity is dependent on CD8+ T-cells

[0664] FIG. 10. Spleen T-cell immuno-profiling after SurADCx25 dosing in healthy immuno-competent mice

[0665] FIG. 11. Lymph node T-cell immuno-profiling after SurADCx25 dosing in healthy immuno-competent mice

[0666] FIG. 12. Blood T-cell immuno-profiling after SurADCx25 dosing in healthy immuno-competent mice

[0667] FIG. 13. Thymus T-cell immuno-profiling after SurADCx25 dosing in healthy immuno-competent mice

[0668] FIG. 14. Tumour T-cell immuno-profiling after SurADCx25 dosing in CT26 tumor-bearing immuno-competent mice

[0669] FIG. 15. Spleen T-cell immuno-profiling after SurADCx25 dosing in CT26 tumor-bearing immuno-competent mice

[0670] FIG. 16. Blood T-cell immuno-profiling after SurADCx25 dosing in CT26 tumor-bearing immuno-competent mice

[0671] FIG. 17. surADCx25 and radiotherapy combination in vivo study (main)

[0672] FIG. 18. surADCx25 and radiotherapy combination in vivo study (rechallenge)

[0673] FIG. 19. surADCx25 and radiotherapy combination in vivo study (bilateral) [0674] Each line represents the volume of a single tumour (right OR left flank) from a single animal [0675] The panel title indicates if the panel data relates to tumours from the LEFT (non-irradiated) or RIGHT (irradiated) flank [0676] The panel title also indicates if the animal from which the tumour originated that received: vehicle (control), RadioTx (Radiotherapy only), CD25-ADC (surADCx25 only), or CD25-ADC+RadioTx (combination of radiotherapy & surADCx25)

[0677] FIG. 20. surADCx25 and radiotherapy combination in vivo study (sequencing) [0678] Each line represents the volume of a single tumour (right OR left flank) from a single animal [0679] The panel title indicates if the panel data relates to tumours from the LEFT (non-irradiated) or RIGHT (irradiated) flank [0680] The panel title also indicates the order in which the Radiotherapy and surADCx25 were administered: [0681] for “CD25-ADC+RadioTx” the ADC was administered first, on Day 0, with the radiotherapy being administered second, on Day1; [0682] for “RadioTx+CD25-ADC” the radiotherapy was administered first, on Day 0, with the ADC being administered second, on Day1.

[0683] FIG. 21. surADCx25 and radiotherapy combination in vivo study (sequencing) [0684] Kaplan-Meier analysis of survival

STATEMENTS OF INVENTION

[0685] 1. A method of inducing or enhancing an immune response against a disorder in a subject, the method comprising administering to the subject an effective amount of an anti-CD25 ADC in combination with radiotherapy. [0686] 2. The method of statement 1, wherein the immune response is a CD8+ T cell response, a CD4+ T cell response, or an antibody response. [0687] 3. The method of any one of statements 1 to 2, wherein the immune response is a CD8+ T cell response. [0688] 4. The method of statement 1, wherein the immune response is a memory cell response. [0689] 5. A method for treating a disorder in an subject, the method comprising administering to the subject an effective amount of an anti-CD25 ADC in combination with radiotherapy. [0690] 6. The method according to statement 5, wherein the method further comprises a step of selecting the subject for treatment and a subject is selected for treatment with the anti-CD25 ADC if: [0691] (i) the subject has been treated with radiotherapy; [0692] (ii) the subject is being treated with radiotherapy; and/or [0693] (iii) the subject is radiosensitive. [0694] 7. The method according to any previous statement, wherein the treatment comprises administering the anti-CD25 ADC before the radiotherapy, simultaneous with the radiotherapy, or after the radiotherapy. [0695] 8. The method of statement 7, wherein the CD25-ADC and radiotherapy are administered concomitantly. [0696] 9. The method of statement 7, wherein the CD25-ADC and radiotherapy are administered on the same day. [0697] 10. The method of statement 7, wherein the radiotherapy is administered no longer than 3 weeks before the CD25-ADC. [0698] 11. The method of statement 10, wherein the radiotherapy is administered no longer than 1 week before the CD25-ADC. [0699] 12. The method of statement 10, wherein the radiotherapy is administered no longer than 1 day before the CD25-ADC. [0700] 13. The method of any one of statements 1 to 7, wherein the CD25-ADC is administered before the radiotherapy. [0701] 14. The method of statement 13, wherein the CD25-ADC is administered at least 24 hours before the radiotherapy. [0702] 15. The method of statement 14, wherein the CD25-ADC is administered at least 2 days before the radiotherapy. [0703] 16. The method of any one of statgvfements 13 to 15, wherein the CD25-ADC is administered no longer than 21 days before the radiotherapy. [0704] 17. The method of statement 16, wherein the CD25-ADC is administered no longer than 14 days before the radiotherapy. [0705] 18. The method of statement 16, wherein the CD25-ADC is administered no longer than 7 days before the radiotherapy. [0706] 19. A method of inducing or enhancing an immune response against a disorder in a subject, the method comprising: [0707] (a) selecting for treatment a subject who has, is, or will be treated with radiotherapy; and [0708] (b) administering to the subject an effective amount of an anti-CD25 ADC. [0709] 20. A method for treating a disorder in a subject, the method comprising: [0710] (a) selecting for treatment a subject who has, is, or will be treated with radiotherapy; and [0711] (b) administering to the subject an effective amount of an anti-CD25 ADC. [0712] 21. The method of either one of statements 19 or 20, wherein the subjects are selected for treatment by a method comprising (i) identifying a subject who has, is, or will be treated with radiotherapy, then (ii) selecting the subject for treatment if they have, are, or will be treated with radiotherapy. [0713] 22. The method of any one of statements 19 to 21, wherein the subject is selected for treatment with the CD25-ADC if they have received, or are expected to receive, radiotherapy on the same day as the ADC administration. [0714] 23. The method of any one of statements 19 to 21, wherein the subject is selected for treatment with the CD25-ADC if they have received radiotherapy no longer than 3 weeks before administration of the ADC. [0715] 24. The method of statement 23, wherein the subject is selected for treatment with the CD25-ADC if they have received radiotherapy no longer than 1 week before administration of the ADC. [0716] 25. The method of statement 24, wherein the subject is selected for treatment with the CD25-ADC if they have received radiotherapy no longer than 1 day before administration of the ADC. [0717] 26. The method of any one of statements 19 to 21, wherein the subject is selected for treatment with the CD25-ADC if they are expected to receive radiotherapy at least 24 hours after the ADC administration. [0718] 27. The method of statement 26, wherein the subject is selected for treatment with the CD25-ADC if they are expected to receive radiotherapy at least 2 days after the ADC administration. [0719] 28. The method of either one of statements 26 or 27, wherein the subject is selected for treatment with the CD25-ADC if they are expected to receive radiotherapy no longer than 21 days after the ADC administration. [0720] 29. The method of statements 28, wherein the subject is selected for treatment with the CD25-ADC if they are expected to receive radiotherapy no longer than 14 days after the ADC administration. [0721] 30. The method of statements 28, wherein the subject is selected for treatment with the CD25-ADC if they are expected to receive radiotherapy no longer than 7 days after the ADC administration. [0722] 31. The method of any one of statements 1 to 8, wherein: [0723] (i) the immune-suppressive activity of a population of regulatory immune cells in the subject is reduced by at least 90% before the radiotherapy is administered; and/or [0724] (ii) the size of a population of regulatory immune cells in the subject is reduced by at least 90% before the radiotherapy is administered. [0725] 32. The method of statement 9, wherein the regulatory immune cells are Treg cells. [0726] 33. The method according to any preceding statement, wherein the subject has a disorder or has been determined to have a disorder. [0727] 34. The method according to statement 11, wherein the subject has, or has been has been determined to have, a cancer which expresses CD25 or CD25+ tumour-associated non-tumour cells, such as CD25+ infiltrating cells. [0728] 34. The method according to any previous statement, wherein the subject is radiosensitive. [0729] 35. The method according to any previous statement, wherein the radiotherapy is focal radiotherapy. [0730] 36. The method according to any previous statement wherein the radiotherapy is tumour targeted. [0731] 37. The method of any one of statements 1 to 37, wherein the radiotherapy is selected from the group consisting of: external beam radiotherapy, stereotactic radiation therapy, Intensity-Modulated Radiation Therapy, particle therapy, brachytherapy, delivery of radioisotopes, intraoperative radiotherapy, Auger therapy, Volumetric modulated arc therapy, Virtual simulation, 3-dimensional conformal radiation therapy, and intensity-modulated radiation therapy. [0732] 38. The method of any one of statements 1 to 38, wherein the radiotherapy is optimized to minimize immunosuppressive effects on immune cells and/or maximise the cytotoxic effect on the targeted tissue. [0733] 39. The method of any one of statements 1 to 38, wherein the radiotherapy is sub-therapeutic dose for treatment of the disorder with radiotherapy alone. [0734] 40. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 40 Gy. [0735] 41. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 30 Gy. [0736] 42. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 24 Gy. [0737] 43. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 20 Gy. [0738] 44. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 18 Gy. [0739] 45. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 16 Gy. [0740] 46. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 15 Gy. [0741] 47. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 12 Gy. [0742] 48. The method of any one of statements 1 to 38, wherein the total radiotherapy dose is no greater than 10 Gy. [0743] 49. The method of any one of statements 1 to 48, wherein the radiotherapy is administered as a single dose. [0744] 50. The method of any one of statements 1 to 48, wherein the radiotherapy is administered as fractionated doses. [0745] 51. The method of statement 50, wherein each fractionated dose is, or is no greater than, 20 Gy. [0746] 52. The method of statement 50, wherein each fractionated dose is, or is no greater than, 15 Gy. [0747] 53. The method of statement 50, wherein each fractionated dose is, or is no greater than, 12 Gy. [0748] 54. The method of statement 50, wherein each fractionated dose is, or is no greater than, 10 Gy. [0749] 55. The method of statement 50, wherein each fractionated dose is, or is no greater than, 8 Gy. [0750] 56. The method of statement 50, wherein each fractionated dose is, or is no greater than, 6 Gy. [0751] 57. The method of statement 50, wherein each fractionated dose is, or is no greater than, 5 Gy. [0752] 58. The method of statement 50, wherein each fractionated dose is, or is no greater than, 4 Gy. [0753] 59. The method of statement 50, wherein each fractionated dose is, or is no greater than, 3 Gy. [0754] 60. The method of statement 50, wherein each fractionated dose is, or is no greater than, 2 Gy. [0755] 61. The method of any one of statements 50 to 60, wherein the radiotherapy is administered in two fractionated doses. [0756] 62. The method of any one of statements 50 to 60, wherein the radiotherapy is administered in three fractionated doses. [0757] 63. The method of any one of statements 50 to 60, wherein the radiotherapy is administered in four fractionated doses. [0758] 64. The method of any one of statements 50 to 60, wherein the radiotherapy is administered in five fractionated doses. [0759] 65 The method of any one of statements 50 to 60, wherein the radiotherapy is administered in six fractionated doses. [0760] 66. The method of any one of statements 50 to 60, wherein the radiotherapy is administered in eight fractionated doses. [0761] 67. The method of any one of statements 50 to 60, wherein the radiotherapy is administered in ten fractionated doses. [0762] 68. The method of any one of statements 50 to 67, wherein a fractionated dose is administered once daily (QD). [0763] 69. The method of any one of statements 50 to 67, wherein a fractionated dose is administered once every other day (Q2D). [0764] 70. The method of any one of statements 50 to 67, wherein a fractionated dose is administered once every third day (Q3D). [0765] 71. The method of any one of statements 50 to 67, wherein a fractionated dose is administered once weekly (QW). [0766] 72. The method according to any previous statement, wherein the disorder is a proliferative disease. [0767] 73. The method of statement 72, wherein the proliferative disorder is cancer. [0768] 74. The method of either one of statements 72 or 73, wherein the proliferative disorder or cancer is, or is characterized by, one or more solid tumours. [0769] 75. The method of statement 74, wherein the treatment induces or enhances an immune response against a solid tumour. [0770] 76. The method of either one of statements 74 or 75, wherein the solid tumour is remote from the radiotherapy administration site. [0771] 77. The method of any one of statements 74 to 76, wherein the solid tumour is an established tumour. [0772] 78. The method of statement 77, wherein the established tumour is a tumour diagnosed or identified in a naïve subject. [0773] 79. The method of statement 77, wherein the established tumour is a relapsed tumour. [0774] 80. The method of any one of statements 74 to 79, wherein the solid tumour is a metastatic tumour. [0775] 81. The method of any one of statements 74 to 80, wherein the solid tumour comprises or consists of CD25−ve neoplastic cells. [0776] 82. The method of any one of statements 74 to 81, wherein the solid tumour is associated with CD25+ve infiltrating cells; [0777] optionally wherein the solid tumour is associated with high levels of CD25+ve infiltrating cells. [0778] 83. The method of any one of statements 74 to 82, 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. [0779] 84. The method any one of statements 74 to 81, wherein the solid tumour is associated with low levels of CD25+ve infiltrating cells. [0780] 85. The method of any one of statements 74 to 81, wherein the solid tumour is not associated with CD25+ve infiltrating cells. [0781] 86. The method of either one of statements 72 or 73, wherein the proliferative disorder or cancer is lymphoma or leukaemia. [0782] 87. The method of statement 86, wherein the proliferative disorder or cancer is selected from: [0783] Hodgkin's Lymphoma; [0784] 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 [0785] 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). [0786] 88. The method of any one of statements 72 to 82, 86 or 87, wherein the proliferative disorder or cancer is associated with elevated levels of regulatory immune cells, such as Treg cells. [0787] 89. The method of any one of statements 1 to 88, wherein the CD25-ADC is administered in combination with a checkpoint inhibitor or other immunostimulatory agent. [0788] 90. The method of statement 89, wherein the CD25-ADC may be administered before the checkpoint inhibitor or other immunostimulatory agent, simultaneous with the checkpoint inhibitor or other immunostimulatory agent, or after the checkpoint inhibitor or other immunostimulatory agent. [0789] 91. The method of either one of statements 89 or 90, wherein the checkpoint inhibitor is a PD1 antagonist. [0790] 92. The method of statement 91, wherein the PD1 antagonist is selected from pembrolizumab, nivolumab, MEDI0680, PDR001 (spartalizumab), Camrelizumab, AUNP12, Pidilizumab Cemiplimab (REGN-2810), AMP-224, BGB-A317 (Tislelizumab), and BGB-108. [0791] 93. The method of either one of statements 89 or 90, wherein the checkpoint inhibitor is a PD-L1 antagonist. [0792] 94. The method of statement 93, wherein the PD-L1 antagonist is selected from atezolizumab (Tecentriq), BMS-936559/MDX-1105, durvalumab/MEDI4736, and MSB0010718C (Avelumab). [0793] 95. The method of either one of statements 89 or 90, wherein the checkpoint inhibitor is a GITR (Glucocorticoid-Induced TNFR-Related protein) agonist. [0794] 96. The method of statement 95, wherein the GITR (Glucocorticoid-Induced TNFR-Related protein) agonist is selected from MEDI1873, TRX518, GWN323, MK-1248, MK 4166, BMS-986156 and INCAGN1876. [0795] 97. The method of either one of statements 89 or 90, wherein the checkpoint inhibitor is an OX40 agonist. [0796] 98. The method of statement 97, wherein the OX40 agonist is selected from MEDI0562, MEDI6383, MOXR0916, RG7888, OX40mAb24, INCAGN1949, GSK3174998, and PF-04518600. [0797] 99. The method of either one of statements 89 or 90, wherein the checkpoint inhibitor is a CTLA-4 antagonist. [0798] 100. The method of statement 99, wherein the CTLA-4 antagonist is selected from ipilimumab and Tremelimumab. [0799] 101. The method according to any previous statement, wherein the treatment further comprises administering a chemotherapeutic agent. [0800] 102 The method of any one of statements 1 to 101, wherein the CD25-ADC comprises a PBD drug moiety, optionally wherein the CD25-ADC is as defined herein in statements 1-110 of the section herein entitled “CD25-ADCs”. [0801] 103. The method of any one of statements 1 to 102, wherein the CD25-ADC is ADCx25. [0802] 104. The method of any one of statements 1 to 102, wherein the CD25-ADC is ADCT-301. [0803] 105. The method of any one of statements 1 to 102, wherein the CD25-ADC is Camidanlumab Tesirine. [0804] 106. The method according to any previous statement, wherein the subject is human. [0805] 107. An antibody-drug conjugate compound as defined in any one of statements 1 to 106 for use in a method of any one of statements 1 to 106. [0806] 108. A composition or pharmaceutical composition comprising an antibody-drug conjugate compound as defined in any one of statements 1 to 106 for use in a method of any one of statements 1 to 106. [0807] 109. Use of an antibody-drug conjugate compound as defined in any one of statements 1 to 106 in the preparation of a medicament for use in a method of any one of statements 1 to 106.

EXAMPLES

Example 1

[0808] In vivo efficacy study of surrogate-ADCx25 in an immuno-competent syngeneic mouse model using mouse colon cancer MC38 cells.

INTRODUCTION

[0809] MC38 is a CD25−ve mouse colon cancer-derived model used pre-clinically in immunotherapy-type studies which is known to have infiltration of Treg and Teff cells.

[0810] In Arce Vargas et al., 2017, Immunity 46, 1-10, Apr. 18, 2017 (http://dx.doi.org/10.1016/j.immuni.2017.03.013) selective depletion of tumor infiltrating Treg cells in the MC38 model was shown using an Fc enhanced version of PC61, a rat antibody directed against mouse CD25 and synergy with PD1 was described. The wild-type PC61 was conjugated to the PBD dimer drug-linker SG3249 (the PBD drug-linker used in ADCx25/ADCT-301/Camidanlumab Tesirine) and designated as Surrogate-ADCx25 (or SurADCx25). The efficacy of Surrogate-ADCx25 was studied as monotherapy or in combination with anti-PD1 (Anti-PD1, clone RPM1-14, BioXcell cat #BE0146) in the MC38 syngeneic mouse model.

[0811] Study Design

[0812] Female C57BL/6 mice (C57BL/6NCrl, Charles River) were nine weeks old on Day 1 of the study and had a body weight (BW) range of 17.8 to 24.2 g. At the completion of the initial study described in this example, tumor-free survivors were transferred to a secondary rechallenge study, described in Example 2.

[0813] On the day of implant 5×10.sup.5 MC38 cells (0.1 mL suspension) were subcutaneously implanted into the right flank of each test animal. Tumors were monitored as their volumes approached the target range of 80-120 mm.sup.3. Fifteen days after tumor cell implantation, on Day 1 of the study, animals were sorted into ten groups (n=10/group) with individual tumor volumes of 63 to 172 mm3, and group mean tumor volumes of 103-172 mm3.

[0814] All doses were administered intraperitoneally (i.p.) on Day 1 except for anti-PD-1 which was administered once on Days 2, 5, 8. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal. Tumors were measured twice per week until the study was ended on Day 59. Each animal was euthanized when its tumor attained the endpoint tumor volume of 1000 mm3 or on the final day, whichever came first.

[0815] Surrogate-ADCx25 was administered intraperitoneally (i.p.) as single dose (0.1, 0.5 and 1 mg/kg) on day 1 either alone or in combination with anti-PD1 antibody (given at standard dosing regime, i.e. 5 mg/kg at day 2, 5 and 8). As a control, Isotype Control ADC (B12-SG3249) was administered as single dose (1 mg/kg) on day 1 either alone or in combination with anti-PD1 antibody (given at standard dosing regime), while anti-PD1 antibody was administered alone at standard dosing regimen.

[0816] Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:

Tumor Volume (mm.sup.3)=w.sup.2×l/2, where w=width and l=length, in mm, of the tumor.

[0817] Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.

[0818] Results

[0819] Surrogate-ADCx25 had strong and dose-dependent anti-tumor activity per se in the MC38 syngeneic model. The isotype control ADO had significant lower activity than surrogate-ADCx25 at 1 mg/kg. (FIG. 2). A strong synergy was observed when combining a low single dose of surrogate-ADCx25 with anti-PD1 antibody (FIG. 3). High efficacy of higher doses of surrogate-ADCx25 in the present model prevented assessment of synergy at higher doses.

[0820] In vivo, a single dose of sur-ADCx25 at 0.5 or 1 mg/kg induced strong and durable anti-tumor activity against established CD25-negative solid tumors with infiltrating Treg cells (MC38 syngeneic model).

TABLE-US-00010 Response summary PR CR TFS Vehicle 0 0 0 Sur ADCX25, 0.1 mg/kg 0 1 1 Sur ADCX25, 0.5 mg/kg 2 8 8 Sur ADCX25, 1 mg/kg 2 8 8 Anti-PD1, 5 mg/kg 0 3 3 B12-SG3249, 1 mg/kg 2 2 2 Sur ADCX25, 0.1 mg/kg + 1 6 6 anti-PD1 Sur ADCX25, 0.5 mg/kg + 1 9 9 anti-PD1 Sur ADCX25, 1 mg/kg + 0 10  10  anti-PD1 B12-SG3249, 1 mg/kg + 2 5 5 anti-PD1

TABLE-US-00011 Coefficient of Drug Interaction (CDI) Sur ADCX25, 0.1 mg/kg; anti-PD1,5 mg/kg; Sur ADCX25, 0.1 mg/kg + anti-PD1 Day 21 0.268 (Synergism)

[0821] Response Table Criteria (Also Applicable to Example 3 and Example 5):

[0822] Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal.

[0823] In a PR response, the tumor volume is 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm.sup.3 for one or more of these three measurements.

[0824] In a CR response, the tumor volume is less than 13.5 mm.sup.3 for three consecutive measurements during the study.

[0825] Any animal with a CR response at the end of the study was additionally classified as a tumor-free survivor (TFS).

[0826] Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied.

[0827] CDI Methodology Also Applicable to Example 3 and Example 5):

[0828] The Coefficient of Drug Interaction (CDI) (11) in were assessed for subadditive, additive, or supra-additive (synergism) properties on Day 21, the last day all evaluable animals remained on study.

[0829] The CDI was determined according to the equation below:

CDI=AB/AxB [0830] Where, [0831] x=mean tumor volume [0832] AB=xAB/xC [0833] A=xA/xC [0834] B=xB/xC

[0835] CDI<1 is supra-additive (i.e. synergism); CDI=1 is additive; CDI>1 is subadditive

Example 2

[0836] Re-Challenge of Tumor-Free Survivors from Example 1 MC38 Efficacy Study

[0837] The complete responders from Example 1 and 10 naïve control female C57BL/6 mice were or 17-18 weeks old on Day 1 of this study and had a BW range of 20.9 to 39.0 g.

[0838] On Day 1 of the re-challenge study, 5×105 MC38 cells (0.1 mL suspension) were subcutaneously implanted into the left flank (contralateral to the original cell implant) and tumor growth was monitored. No ADC or anti-PD-1 treatment was administered in the rechallenge study.

[0839] Animal handling and tumour measurement was as in Example 1 unless otherwise stated.

[0840] Results: Re-challenged animals did not develop new tumors indicating ADCx25 was able to induce tumor-specific protective immunity (see FIG. 4).

Example 3

[0841] In vivo efficacy study of surrogate-ADCx25 in an immuno-competent syngeneic mouse model using mouse colon cancer CT26 cells

[0842] Introduction

[0843] CT26 is a CD25−ve mouse colon cancer-derived model used pre-clinically in immunotherapy-type studies which is known to have infiltration of Treg and Teff cells.

[0844] Study Design

[0845] Female BALB/c mice (BALB/cNCrl, Charles River) were nine weeks old on Day 1 of the study and had a body weight (BW) range of 17.2 to 23.3 g. At the completion of the study, tumor-free survivors were transferred to they re-challenge study described in Example 4.

[0846] On the day of implant 3×10.sup.5 CT26 cells (0.1 mL suspension) were subcutaneously implanted into the right flank of each test animal. Tumors were monitored as their volumes approached the target range of 80-120 mm.sup.3. Ten days after tumor cell implantation, on Day 1 of the study, animals were sorted into ten groups (n=10/group) with individual tumor volumes of 75 to 162 mm.sup.3, and group mean tumor volumes of 110-111 mm.sup.3. All doses were administered intraperitoneally (i.p.) on Day 1 except for anti-PD-1 which was administered once on Days 2, 5, 8. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal. Tumors were measured twice per week until the study was ended on Day 48. Each animal was euthanized when its tumor attained the endpoint tumor volume of 2000 mm.sup.3 or on the final day, whichever came first.

[0847] Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:

Tumor Volume (mm.sup.3)=w2×l/2, where w=width and l=length, in mm, of the tumor.

[0848] Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.

[0849] Results: In vivo, a single dose of sur-ADCx25 at 0.5 or 1 mg/kg induced strong and durable anti-tumor activity against established CD25-negative solid tumors with infiltrating Treg cells (CT26 syngeneic model); see FIG. 5 and FIG. 6.

TABLE-US-00012 Response summary PR CR TFS Vehicle 0 0 0 Sur ADCX25, 0.1 mg/kg 0 0 0 Sur ADCX25, 0.5 mg/kg 1 2 2 Sur ADCX25, 1 mg/kg 1 3 3 Anti-PD1, 5 mg/kg 0 0 0 Isotype-ADC, 1 mg/kg 0 0 0 Sur ADCX25, 0.1 mg/kg + 1 1 1 anti-PD1 Sur ADCx25, 0.5 mg/kg + 0 7 7 anti-PD1 Sur ADCX25, 1 mg/kg + 0 8 8 anti-PD1 Isotype-ADC, 1 mg/kg + 0 1 1 anti-PD1

TABLE-US-00013 Coefficient of Druq Interaction (CDI) SurADCX25, 0.1 mg/kg; anti-PD1,5 mg/kg; Sur ADCX25, 0.1 mg/kg + anti-PD1 Day 20 0.285 (Synergism)

Example 4

[0850] Re-Challenge of Tumor-Free Survivors from Example 3 CT26 Efficacy Study

[0851] The complete responders from Example 3 and 10 naïve control female BALB/c mice were or 16-17 weeks old on Day 1 of the study and had a BW range of 18.2 to 24.4 g.

[0852] On Day 1 of the re-challenge study 3×10.sup.5 CT26 cells (0.1 mL suspension) were subcutaneously implanted into the left flank (contralateral to the cell implant of example 3) and tumor growth was monitored and measured as described in example 3.

[0853] No treatment was administered in the re-challenge study.

[0854] Results: Re-challenged animals did not develop new tumors indicating ADCx25 was able to induce tumor-specific protective immunity (see FIG. 7).

Example 5

[0855] surADCx25 Anti-Tumour Activity Against CD25-Ve Tumours is Dependent on C8+ T-Cells

[0856] Study Design

[0857] Female C57BL/6 mice (C57BL/6NCrl, Charles River) were eleven weeks old on Day 1 of the study and had a body weight (BW) range of 18.6 to 28.2 g.

[0858] On the day of implant 5×10.sup.5 MC38 cells (0.1 mL suspension) were subcutaneously implanted into the right flank of each test animal. Tumors were monitored as their volumes approached the target range of 80-120 mm.sup.3.

[0859] Fifteen days after tumor cell implantation, on Day 0 of the study, animals were sorted into groups (n=10/group) with individual tumor volumes of 75 to 126 mm3, and with group mean tumor volumes of 86-89 mm.sup.3. All doses were administered intraperitoneally (i.p.).

[0860] Anti-CD8-2.43 murine monoclonal antibody was administered once daily on Days 0, 5, 8 and 13 in order to deplete and suppress levels of CD8+ T-cells.

[0861] SurADCx25 dose was administered on Day 1.

[0862] Anti-PD-1 was administered once daily on Days 2, 5, 8.

[0863] The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.

[0864] Tumors were measured twice per week until the study was ended (day 29 for anti-CD8+surADCx25 and anti-CD8+anti-PD1+surADCx25 groups; day 44 for surADCx25 alone and surADCx25+anti-CD8).

[0865] Each animal was euthanized when its tumor attained the endpoint tumor volume of 1000 mm.sup.3 or on the final day, whichever came first.

[0866] Results

[0867] Sur-ADCx25 anti-tumor activity, either alone (FIG. 8) or combined with an anti-PD1 antibody (FIG. 9), was significantly reduced when CD8+ T-cells are depleted, indicating that surADCx25 activity is CD8+ T-cell-dependent and that overall effector T-cell responses were not negatively impacted by sur-ADCx25.

TABLE-US-00014 Response summary PR CR TFS Vehicle 0 0 0 Anti-PD1 2 0 0 Sur ADCX25 0 1 1 Sur ADCX25 + anti-CD8 0 0 0 Sur ADCX25 + anti-PD1 2 5 5 Sur ADCX25 + anti-PD1 + 0 0 0 anti-CD8

TABLE-US-00015 Coefficient of Drug Interaction (CDI) Sur ADCX25, 0.5 mg/kg; anti-PD1,5 mg/kg; Sur ADCX25, 0.5 mg/kg + anti-PD1 Day 22 0.471 (Synergism)

Example 6

[0868] T-Cell Immuno-Profiling after SurADCx25 Dosing in Healthy Immuno-Competent Mice

[0869] Study Design

[0870] Eight to twelve week old, female C57BL/6 mice were dosed intravenously on day 1 with either surADCx25 (0.5 mg/kg) or an isotype control ADC (0.5 mg/kg). A non-dosed group acted as control (each group contained 24 mice).

[0871] Terminal samples (blood, spleen, lymph node and thymus) were obtained on days 1 (4 hrs post-dose), 7, 14 and 21 from 6 animals per group at each time-point. Additional non-terminal blood samples (mandibular bleeds) were obtained on days 4, 11 and 18 from 6 animals per group at each time-point.

[0872] Samples (tissue and blood) were processed for flow cytometry assessment and CD4+ T-cells (CD45.sup.+ CD3.sup.+ CD4.sup.+ CD8.sup.−), CD8.sup.+ T-cells (CD45.sup.+ CD3.sup.+ CD4.sup.− CD8.sup.+) and T.sub.reg cell (CD45.sup.+ CD3.sup.+ CD4.sup.+ CD25.sup.+ FoxP3.sup.+) content determined. Data represent the mean±SEM of T.sub.reg cell population in the assayed tissue as a percentage of CD45.sup.+.

[0873] Results

[0874] Spleen: [0875] .fwdarw. a clear depletion of spleen Tregs was observed at 1 day & 7 days post surADCx25 administration, with Treg levels mostly recovering by day 14 (see FIG. 10A; % shown indicates the % reduction compared to vehicle) [0876] .fwdarw. there was no observed impact on the level of spleen CD8+ Teff cells (see FIG. 10B)

[0877] Lymph Nodes: [0878] .fwdarw. a clear depletion of lymph node Tregs was observed at 1 day & 7 days post surADCx25 administration, with Treg levels mostly recovering by day 14 (see FIG. 11A; % shown indicates the % reduction compared to vehicle) [0879] .fwdarw. there was no observed impact on the level of lymph node CD8+ Teff cells (see FIG. 11B)

[0880] Blood: [0881] .fwdarw. Increased variability in vehicle and isotype-control values due to low levels of events; measured with additional non-terminal blood samples (days 4, 11 and 18). [0882] .fwdarw. a clear depletion of blood Tregs was observed at 1 day, 7 days, and 11 days post surADCx25 administration, with Treg levels recovering by day 14 (see FIG. 12A; % shown indicates the % reduction compared to vehicle) [0883] .fwdarw. there was no observed impact on the level of blood CD8+ Teff cells (see FIG. 12B)

[0884] Thymus: [0885] .fwdarw. a clear increase of thymus Tregs & CD8+ Teffs was observed at 7 days post surADCx25 administration, with Treg & CD8+ Teff levels mostly recovering by day 14 (see FIGS. 13A & B)

SUMMARY

[0886] A single dose of SurADCx25 caused significant depletion of Tregs in spleen, lymph nodes and blood (>95%).

[0887] There was a clear increase in the thymus in the amount of Tregs at 7 days post-dose.

[0888] There was no depletion of Teff cells from spleen, lymph nodes and blood caused by SurADCx25, however, an increase in thymus Teff cells was also observed 7 days post dosing of SurADCx25.

[0889] Around Day 15, Tregs levels in blood, spleen, thymus and lymph nodes are restored to normal (vehicle control).

Example 7

[0890] T-Cells Immuno-Profiling after surADCx25 Dosing in CT26 Tumor-Bearing Immune-Competent Mice

[0891] Study Design

[0892] Female BALB/c mice were ten weeks old on Day 1 of the study.

[0893] Cultured CT26 cells were harvested during log phase growth and resuspended in phosphate buffered saline (PBS) at a concentration of 3×10.sup.6 cells/mL. Tumors were initiated by subcutaneously implanting 3×10.sup.5 CT26 cells into the right flank of each test animal. Fourteen days after tumor cell implantation, on Day 1 of the studies, animals were sorted into groups (n=24 or 18) with mean tumor volumes of 115-116 mm.sup.3.

[0894] SurADCx25 was administered intraperitoneally (i.p.) on Day 1. Anti-PD-1 was administered i.p. once daily on Days 2, 5, 8. Group 1 received the PBS vehicle and served as the control. Group 2 received anti-PD-1 at 5 mg/kg. Groups 3 received surADCx25 at 1 mg/kg. Group 4 received surADCx25 at 1 mg/kg in combination with anti-PD-1 at 5 mg/kg.

[0895] Samples were collected for analysis by flow cytometry on Days 1 (pre-dose; Group 1 only, n=6), 3 (Groups 1-4, n=6) and 9 (Groups 1-4, n=6). Full blood volume was collected from each animal via terminal cardiac puncture and was processed for flow cytometry.

[0896] Immediately following blood collection, the tumor and the spleen were harvested from each animal and processed for flow cytometry and CD4+ T-cells (CD45.sup.+ CD3.sup.+ CD4.sup.+ CD8.sup.−), CD8+ T-cells (CD45.sup.+ CD3.sup.+ CD4.sup.− CD8.sup.+) and Treg cell (CD45.sup.+ CD3.sup.+ CD4.sup.+ CD25.sup.+ FoxP3.sup.+) content determined.

[0897] Results

[0898] Tumour: [0899] .fwdarw. a significant and sustained depletion of tumour Tregs was observed from 2 days through 11 days post surADCx25 administration (see FIG. 14A) [0900] .fwdarw. an increased tumour CD8+ Teff/Tregs ratio was observed from 2 days through 11 days post surADCx25 administration (see FIG. 14B).

[0901] Spleen: [0902] .fwdarw. a significant and sustained depletion of spleen Tregs was observed from 2 days through 11 days post surADCx25 administration (see FIG. 15A) [0903] .fwdarw. an increased spleen CD8+ Teff/Tregs ratio was observed from 2 days through 11 days post surADCx25 administration (see FIG. 15B).

[0904] Blood: [0905] .fwdarw. a significant and sustained depletion of blood Tregs was observed from 2 days through 11 days post surADCx25 administration (see FIG. 16A) [0906] .fwdarw. an increased blood CD8+ Teff/Tregs ratio was observed from 2 days through 11 days post surADCx25 administration (see FIG. 16B).

[0907] Summary and Conclusions

[0908] A single dose of surADCx25 to immuno-competent mice bearing established CT26 tumors caused significant and sustained depletion of Tregs in tumors, blood, and spleen.

[0909] The simultaneous increase observed in CD8+ Teff/Tregs ratio in tumors, blood, and spleen indicates that surADCx25 did not negatively impact the overall Teff cell response.

[0910] Together, the data set out in Examples 5 to 7 indicate that SurADCx25 depletion of Tregs together with activation of the CD8+ Teff response is an important mode of action in surADCx25's anti-tumour activity.

Example 8

[0911] In Vivo Studies of Combined Treatment with surADCx25 and Radiotherapy

[0912] Methodology

[0913] Female BALB/c mice (BALB/cAnNHsd, Envigo) were 6-7 weeks old (main study) at day of implant and had a body weight (BW) range of 17.8 to 18.7 g.

[0914] On the day of implant of the main study, 5×10.sup.5 CT26 cells (0.2 mL suspension) were subcutaneously implanted into the right high axilla of each test animal. Tumors were monitored as their volumes approached the target range. Seven days after tumor cell implantation, on Day 1 of the study, animals were sorted into six groups (n=10/group) with group mean tumor volumes of 95 mm.sup.3. surADCx25 (also described herein as “sur301”) was administered intravenously (i.v.) on Day 1, while tumor-targeted radiation treatment was administered once on Day 2 at 5 Gy. Tumors were measured thrice per week until the study was ended on Day 62. Each animal was euthanized when its tumor attained the endpoint tumor volume of 2000 mm.sup.3 or on the final day, whichever came first.

[0915] At the completion of the main study, tumor-free survivors were transferred to a secondary rechallenge study. The complete responders and 10 age matched, naïve control female BALB/c mice were used in this re-challenge study.

[0916] On Day 1 of the re-challenge study, 5×10.sup.5 CT26 cells (0.2 mL suspension) were subcutaneously implanted into the left high axilla (contralateral to the original cell implant) and tumor growth was monitored. No treatment was administered in the re-challenge study.

[0917] In both the main and re-challenge studies, tumors were measured in two dimensions using calipers, and volume was calculated using the formula:

Tumor Volume (mm.sup.3)=w.sup.2×l/2, where w=width and l=length, in mm, of the tumor.

[0918] Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.

[0919] Results: Main Study

[0920] See FIG. 17 for full results.

TABLE-US-00016 Response summary PR CR TFS Vehicle 0 0 0 Sur301, 0.25 mg/kg 0 0 0 Sur301, 0.5 mg/kg 0 1 1 Radiotherapy (Rx), 5 Gy 0 3 0 Sur301 ,0.25 mg/kg + Rx, 5 Gy 0 6 6 Sur301, 0.5 mg/kg + Rx, 5 Gy 0 8 8

TABLE-US-00017 Coefficient of Drug Interaction (CDI) Sur301, 0.25 mg/kg; Rx, 5 Gy; Sur301, 0.25 mg/kg + Rx, 5 Gy Day 15 0.88 (Synergism)

TABLE-US-00018 Coefficient of Drug Interaction (CDI) Sur301,0.5 mg/kg; Rx, 5 Gy; Sur301, 0.5 mg/kg + Rx, 5 Gy Day 15 0.47 (Synergism)

[0921] [CDI<1 indicates a synergistic effect; CDI=1 indicates an additive effect; CDI>1 indicates an antagonistic effect.]

[0922] Results: Re-Challenge Study

[0923] See FIG. 18 for full results.

[0924] Conclusions

[0925] Synergy between sur301 and radiotherapy is observed at both of the analysed dose levels.

[0926] In the re-challenge study, no tumour growth was observed on re-inoculation of Tumour Free Survivors from either the groups treated with the sur301+Rx combination (14 individuals in total), or the sole TFS from the sur301, 0.5 mg/kg group. This is consistent with an immune-based therapeutic mechanism such as the abscopal effect.

Example 9

[0927] In Vivo Studies of Combined Treatment with surADCx25 and Radiotherapy; Bilateral Testing for an Abscopal Effect

[0928] Methodology

[0929] Female BALB/c mice (BALB/cAnNHsd, Envigo) were 7-8 weeks old at day of implant.

[0930] On the day of implant, 5×10.sup.5 CT26 cells (0.2 mL suspension) were subcutaneously implanted into the right and left high axilla of each test animal. Tumors were monitored as their volumes approached the target range. Ten days after tumor cell implantation, on Day 0 of the study, animals were sorted into groups (n=10/group) with group mean tumor volumes of 105 mm.sup.3 (right tumors) and 99 mm.sup.3 (left tumors).

[0931] Two treatment schedules were tested: [0932] 1) CD25-ADC was administered intravenously (i.v.) on Day 0, and tumor-targeted radiation was administered once to the tumors on the right flanks on Day 1 at 5 Gy. Tumours on the left flank were not irradiated. [0933] 2) Tumor-targeted radiation was administered once to the tumors on the right flanks on Day 0 at 5 Gy and CD25-ADC was administered intravenously (i.v.) on Day 1. Tumours on the left flank were not irradiated.

[0934] Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:

Tumor Volume (mm.sup.3)=w.sup.2×l/2, where w=width and l=length, in mm, of the tumor.

[0935] Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.

[0936] Results

TABLE-US-00019 Coefficient of IRRADIATED TUMORS Day 20 Drug interaction CD2S-ADC 0.94 Radiotherapy (synergism) CD25-ADC + Radiotherapy

TABLE-US-00020 Coefficient of NON-IRRADIATED TUMORS Day 20 Drug interaction CD25-ADC 0.91 Radiotherapy (synergism) CD25-ADC + Radiotherapy

[0937] Conclusions

[0938] In the bilateral CT26 tumor model, combination of CD25-ADC with focal radiotherapy resulted in synergistic anti-tumor activity in both the irradiated and non-irradiated distal tumor (abscopal effect) and the combination significantly increased survival compared to the single treatments.

Example 10

[0939] In vivo studies of combined treatment with surADCx25 and radiotherapy; testing the effect of order of administration

[0940] Methodology

[0941] The base methodology is as described for Example 9.

[0942] In this part of study, and as noted above, there were two parallel groups of animals: [0943] A) Animals where a single 0.5 mg/kg dose of surADCx25 (also described herein as “sur301”) was administered intravenously (i.v.) on Day 0, while tumor-targeted radiation treatment was administered only to the tumour on the right flank once on Day 1 at 5 Gy; and [0944] B) Animals where a tumor-targeted radiation treatment was administered only to the tumour on the right flank once on Day 0 at 5 Gy, while a single 0.5 mg/kg dose of surADCx25 (also described herein as “sur301”) was administered intravenously (i.v.) on Day 1.

[0945] Results

[0946] See FIGS. 20 & 21 for full results.

CONCLUSIONS

[0947] Sequential administration of CD25-ADC followed by radiotherapy resulted in superior anti-tumor activity compared to the reverse order of administration (radiotherapy first, followed by CD25-ADC), suggesting prior Treg depletion allows for optimal anti-tumor activity mediated by radiotherapy.