IL2RBETA/COMMON GAMMA CHAIN ANTIBODIES

Abstract

Antigen binding molecules capable of binding to CDI22 and/or common y chain (CDI32) are disclosed herein. Also disclosed are compositions comprising such antigen binding molecules, and uses and methods using the same.

Claims

1. An antigen-binding molecule, optionally isolated, which is capable of binding to common ? chain (CD132), wherein the antigen-binding molecule comprises: (P1A10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 196 HC-CDR2 having the amino acid sequence of SEQ ID NO: 204 HC-CDR3 having the amino acid sequence of SEQ ID NO: 212; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 227 LC-CDR2 having the amino acid sequence of SEQ ID NO: 238 LC-CDR3 having the amino acid sequence of SEQ ID NO: 248; or (P1B6) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 213; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 239 LC-CDR3 having the amino acid sequence of SEQ ID NO: 249; or (P1C10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 112 HC-CDR2 having the amino acid sequence of SEQ ID NO: 124 HC-CDR3 having the amino acid sequence of SEQ ID NO: 214; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 228 LC-CDR2 having the amino acid sequence of SEQ ID NO: 240 LC-CDR3 having the amino acid sequence of SEQ ID NO: 250; or (P1D7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 197 HC-CDR2 having the amino acid sequence of SEQ ID NO: 206 HC-CDR3 having the amino acid sequence of SEQ ID NO: 215; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 229 LC-CDR2 having the amino acid sequence of SEQ ID NO: 241 LC-CDR3 having the amino acid sequence of SEQ ID NO: 251; or (P1E8) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 198 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 216; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 230 LC-CDR2 having the amino acid sequence of SEQ ID NO: 242 LC-CDR3 having the amino acid sequence of SEQ ID NO: 252; or (P2B2) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 207 HC-CDR3 having the amino acid sequence of SEQ ID NO: 217; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 253; or (P2B7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 218; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 231 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 254; or (P2D11) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 199 HC-CDR2 having the amino acid sequence of SEQ ID NO: 208 HC-CDR3 having the amino acid sequence of SEQ ID NO: 219; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 232 LC-CDR2 having the amino acid sequence of SEQ ID NO: 243 LC-CDR3 having the amino acid sequence of SEQ ID NO: 255; or (P2F10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 200 HC-CDR2 having the amino acid sequence of SEQ ID NO: 209 HC-CDR3 having the amino acid sequence of SEQ ID NO: 220; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 233 LC-CDR2 having the amino acid sequence of SEQ ID NO: 244 LC-CDR3 having the amino acid sequence of SEQ ID NO: 256; or (P2H4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 221; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 234 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 257; or (P2D3) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 201 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 222; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1G4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 223; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 258; or (P1B12) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 224; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 235 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1C7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 225; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 245 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189.

2. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule comprises: (P1A10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 71; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 89; or (P1B6) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 72; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 90; or (P1C10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 73; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 91; or (P1D7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 74; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 92; or (P1E8) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 75; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 93; or (P2B2) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 76; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 94; or (P2B7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 77; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 95; or (P2D11) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 78; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 96; or (P2F10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 79; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 97; or (P2H4) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 80; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 98; or (P2D3) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 81; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 99; or (P1G4) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 82; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 100; or (P1B12) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 83; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 101; or (P1C7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 84; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 102.

3. The antigen-binding molecule according to claim 1, wherein the antigen binding molecule is capable of binding to CD122 and CD132.

4. The antigen-binding molecule according to claim 1, which is an IL-2 receptor agonist.

5. The antigen-binding molecule according to claim 1, which is capable of reducing expression of PD-1 by T cells.

6. A nucleic acid encoding an antigen-binding molecule, optionally isolated, which is capable of binding to common ? chain (CD132), wherein the antigen-binding molecule comprises: (P1A10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 196 HC-CDR2 having the amino acid sequence of SEQ ID NO: 204 HC-CDR3 having the amino acid sequence of SEQ ID NO: 212; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 227 LC-CDR2 having the amino acid sequence of SEQ ID NO: 238 LC-CDR3 having the amino acid sequence of SEQ ID NO: 248; or (P1B6) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 213; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 239 LC-CDR3 having the amino acid sequence of SEQ ID NO: 249; or (P1C10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 112 HC-CDR2 having the amino acid sequence of SEQ ID NO: 124 HC-CDR3 having the amino acid sequence of SEQ ID NO: 214; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 228 LC-CDR2 having the amino acid sequence of SEQ ID NO: 240 LC-CDR3 having the amino acid sequence of SEQ ID NO: 250; or (P1D7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 197 HC-CDR2 having the amino acid sequence of SEQ ID NO: 206 HC-CDR3 having the amino acid sequence of SEQ ID NO: 215; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 229 LC-CDR2 having the amino acid sequence of SEQ ID NO: 241 LC-CDR3 having the amino acid sequence of SEQ ID NO: 251; or (P1E8) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 198 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 216; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 230 LC-CDR2 having the amino acid sequence of SEQ ID NO: 242 LC-CDR3 having the amino acid sequence of SEQ ID NO: 252; or (P2B2) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 207 HC-CDR3 having the amino acid sequence of SEQ ID NO: 217; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 253; or (P2B7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 218; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 231 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 254; or (P2D11) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 199 HC-CDR2 having the amino acid sequence of SEQ ID NO: 208 HC-CDR3 having the amino acid sequence of SEQ ID NO: 219; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 232 LC-CDR2 having the amino acid sequence of SEQ ID NO: 243 LC-CDR3 having the amino acid sequence of SEQ ID NO: 255; or (P2F10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 200 HC-CDR2 having the amino acid sequence of SEQ ID NO: 209 HC-CDR3 having the amino acid sequence of SEQ ID NO: 220; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 233 LC-CDR2 having the amino acid sequence of SEQ ID NO: 244 LC-CDR3 having the amino acid sequence of SEQ ID NO: 256; or (P2H4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 221; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 234 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 257; or (P2D3) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 201 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 222; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1G4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 223; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 258; or (P1B12) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 224; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 235 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1C7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 225; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 245 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189.

7. The nucleic acid encoding an antigen-binding molecule according to claim 6, wherein the antigen-binding molecule comprises: (P1A10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 71; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 89; or (P1B6) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 72; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 90; or (P1C10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 73; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 91; or (P1D7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 74; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 92; or (P1E8) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 75; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 93; or (P2B2) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 76; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 94; or (P2B7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 77; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 95; or (P2D11) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 78; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 96; or (P2F10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 79; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 97; or (P2H4) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 80; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 98; or (P2D3) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 81; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 99; or (P1G4) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 82; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 100; or (P1B12) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 83; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 101; or (P1C7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 84; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 102.

8. The nucleic acid encoding an antigen-binding molecule according to claim 6, wherein the antigen binding molecule is capable of binding to CD122 and CD132.

9. The nucleic acid encoding an antigen-binding molecule according to claim 6, which is an IL-2 receptor agonist.

10. The nucleic acid encoding an antigen-binding molecule according to claim 6, which is capable of reducing expression of PD-1 by T cells.

11. A method of treating or preventing a T cell dysfunctional disorder, a cancer, or an infectious disease, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, wherein the antigen-binding molecule comprises: (P1A10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 196 HC-CDR2 having the amino acid sequence of SEQ ID NO: 204 HC-CDR3 having the amino acid sequence of SEQ ID NO: 212; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 227 LC-CDR2 having the amino acid sequence of SEQ ID NO: 238 LC-CDR3 having the amino acid sequence of SEQ ID NO: 248; or (P1B6) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 213; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 239 LC-CDR3 having the amino acid sequence of SEQ ID NO: 249; or (P1C10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 112 HC-CDR2 having the amino acid sequence of SEQ ID NO: 124 HC-CDR3 having the amino acid sequence of SEQ ID NO: 214; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 228 LC-CDR2 having the amino acid sequence of SEQ ID NO: 240 LC-CDR3 having the amino acid sequence of SEQ ID NO: 250; or (P1D7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 197 HC-CDR2 having the amino acid sequence of SEQ ID NO: 206 HC-CDR3 having the amino acid sequence of SEQ ID NO: 215; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 229 LC-CDR2 having the amino acid sequence of SEQ ID NO: 241 LC-CDR3 having the amino acid sequence of SEQ ID NO: 251; or (P1E8) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 198 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 216; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 230 LC-CDR2 having the amino acid sequence of SEQ ID NO: 242 LC-CDR3 having the amino acid sequence of SEQ ID NO: 252; or (P2B2) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 207 HC-CDR3 having the amino acid sequence of SEQ ID NO: 217; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 253; or (P2B7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 218; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 231 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 254; or (P2D11) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 199 HC-CDR2 having the amino acid sequence of SEQ ID NO: 208 HC-CDR3 having the amino acid sequence of SEQ ID NO: 219; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 232 LC-CDR2 having the amino acid sequence of SEQ ID NO: 243 LC-CDR3 having the amino acid sequence of SEQ ID NO: 255; or (P2F10) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 200 HC-CDR2 having the amino acid sequence of SEQ ID NO: 209 HC-CDR3 having the amino acid sequence of SEQ ID NO: 220; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 233 LC-CDR2 having the amino acid sequence of SEQ ID NO: 244 LC-CDR3 having the amino acid sequence of SEQ ID NO: 256; or (P2H4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 108 HC-CDR2 having the amino acid sequence of SEQ ID NO: 120 HC-CDR3 having the amino acid sequence of SEQ ID NO: 221; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 234 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 257; or (P2D3) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 201 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 222; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1G4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 223; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 258; or (P1B12) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 224; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 235 LC-CDR2 having the amino acid sequence of SEQ ID NO: 174 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1C7) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 106 HC-CDR2 having the amino acid sequence of SEQ ID NO: 119 HC-CDR3 having the amino acid sequence of SEQ ID NO: 225; and a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 245 LC-CDR3 having the amino acid sequence of SEQ ID NO: 189.

12. The method according to claim 11, wherein the antigen-binding molecule comprises: (P1A10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 71; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 89; or (P1B6) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 72; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 90; or (P1C10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 73; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 91; or (P1D7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 74; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 92; or (P1E8) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 75; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 93; or (P2B2) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 76; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 94; or (P2B7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 77; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 95; or (P2D11) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 78; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 96; or (P2F10) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 79; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 97; or (P2H4) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 80; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 98; or (P2D3) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 81; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 99; or (P1G4) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 82; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 100; or (P1B12) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 83; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 101; or (P1C7) a VH region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 84; and a VL region comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 102.

13. The method according to claim 11, wherein the antigen binding molecule is capable of binding to CD122 and CD132.

14. The method according to claim 11, wherein the cancer is selected from the group consisting of: colon cancer, colon carcinoma, colorectal cancer, nasopharyngeal carcinoma, cervical carcinoma, oropharyngeal carcinoma, gastric carcinoma, hepatocellular carcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urothelial carcinoma, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or mesothelioma.

15. The method according to claim 11, wherein the antigen binding molecule is administered in combination with a therapeutically effective amount of an agent capable of inhibiting signalling mediated by an immune checkpoint protein.

16. The method according to claim 15, wherein the immune checkpoint protein is PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA.

17. The method according to claim 11, wherein the infectious disease causes an inability to mount an effective immune response against an infection, and wherein the infection is a chronic and/or latent infection.

18. The method according to claim 11, wherein the infectious disease is caused by a bacterial, fungal, parasitic, or viral infection.

19. The method according to claim 11, wherein the infectious disease caused by a bacterial infection is selected from: Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) and Pseudomonas aeruginosa; wherein the infectious disease caused by a fungal infection is selected from: Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp; wherein the infectious disease caused by a parasitic infection is selected from: diseases caused by Plasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi), malaria, leishmaniasis, and toxoplasmosis; or wherein the infectious disease caused by a viral infection is selected from by influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplex virus and human papilloma virus (HPV).

20. The method according to claim 11, wherein the T cell dysfunctional disorder comprises T cell exhaustion or T cell anergy.

Description

BRIEF DESCRIPTION OF THE FIGURES

[1728] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[1729] FIGS. 1A and 18. Graphs showing binding of different formats of bispecific anti-IL2RB/?c antibodies to (1A) ?c-Fc and (1B) IL2RG-Fc as determined by ELISA.

[1730] FIGS. 2A to 2C. Graphs and bar chart showing binding of bispecific and monospecific IL2R?-add/or ?c-binding antibodies to cells expressing human IL2R?, ?c or IL-2R? at the cell surface, as determined by flow cytometry, (2A) Graphs showing analysis of binding of P2C4/P1A3, P2C4/P1A10, a monospecific anti-?c (neg/?IL2R?), a monospecific anti-IL-2RB (?IL2R?/neg) to cells transfected with constructs encoding human IL-2R? and ?c. Negative unstained, secondary antibody only and isotype control conditions are indicated (28) Graphs showing analysis of binding of P2C4/P1A3, P2C4/P1A10, a monospecific anti-?c (neg/?IL2R?), a monospecific anti-IL-2R? (?IL2R?/neg) to cells transfected with construct encoding IL-2R?. Negative unstained, secondary antibody only and isotype control conditions, and positive ?IL2R? control conditions are indicated. (2C) Bar chart summarising normalised median fluorescence intensity (nMFI) for binding of the indicated antibodies to cells transfected with constructs encoding IL-2R? and ?c.

[1731] FIGS. 3A and 38. Bar charts showing binding of bispecific IL-2R?- and ?c-binding antibodies to primary human T cell subsets, as determined by flow cytometry. (3A and 3B) Bar chart summarising normalised MFIs for binding of the indicated antibodies to the indicated CD4+(3A) and CD8+(3B) human T cell subsets.

[1732] FIGS. 4A and 48. Graphs and bar chart showing binding of bispecific and monospecific IL-2R?- and/or ?c-binding antibodies to cells expressing rhesus IL-2R? and ?c at the cell surface, as determined by flow cytometry. (4A) Graphs showing analysis of binding of P2C4/P1A3, P2C4/P1A10, a monospecific anti-?c (neg/?IL2R?), a monospecific anti-IL-2R? ((?IL2RB/neg) to cells transfected with constructs encoding rhesus IL-2R? and ?c. Negative unstained, secondary antibody only and isotype control conditions are indicated. (48) Bar chart summarising normalised MFIs for binding of the indicated antibodies to cells transfected with constructs encoding rhesus IL-2R? and ?c.

[1733] FIG. 5. Bar chart showing binding of bispecific IL-2R?- and ?c-binding antibodies to primary cynomolgus macaque T cell subsets, as determined by flow cytometry.

[1734] FIGS. 6A and 68. Graphs showing analysis of proliferation of NK92 cells in response to treatment with bispecific IL-2R?- and ?c-binding antibodies or the indicated cytokines. EC50 values for induction of NK92 cell proliferation are shown. 6A and 6B show the results from different experiments investigating different bispecific IL-2R?- and ?c-binding antibodies.

[1735] FIGS. 7A to 7L. Bar charts and graphs showing analysis of proliferation of pre-activated, primary human T cell subsets in response to treatment with bispecific IL-2R?- and ?c-binding antibodies or IL-2 Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated. (7A)

[1736] Absolute numbers of CD4+ T cells. (7B) Absolute numbers of CD8+ T cells. (7C) Absolute numbers of Tregs. (7D) Graphs showing CD4+CD25+FoxP3+ regulatory T cell compartment following stimulation with the indicated agents. (7E) Absolute numbers of naive CD8+ T cells. (7F) Absolute numbers of naive CD4+ T cells. (7G) Absolute numbers of central memory CD8+ T cells. (7H) Absolute numbers of central memory CD4+ T cells. (7I) Absolute numbers of effector memory CD8+ T cells. (7J) Absolute numbers of effector memory CD4+ T cells. (7K) Graphs showing dividing effector memory CD8+ T cells as determined by CellTrace Violet staining. (7L) Percentage of CD8+ effector memory cells which are dividing.

[1737] FIGS. 8A to 8H. Graphs showing analysis of proliferation of pre-activated, primary human T cell subsets in response to treatment with different amounts of bispecific IL-2R?- and ?c-binding antibodies of the indicated cytokines. (8A) Absolute numbers of CD4+ T cells. (8B) Absolute numbers of CD8+ T cells. (8C) Absolute numbers of Tregs. (8D) Ratio of the absolute number of CD8+ T cells to the absolute number of Tregs. (8E) Absolute numbers of effector memory CD4+ T cells. (8F) Absolute numbers of effector memory CD8+ T cells. (8G) Percentage of CD8+ effector memory cells which are dividing. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated. (8H) Absolute numbers of pre-expanded Tregs after treatment with bispecific IL-2R?- and ?c-binding antibodies or the indicated cytokines.

[1738] FIGS. 9A to 9I. Bar charts showing analysis of proliferation of pre-activated, T cell subsets in response to treatment of human PBMCs with bispecific IL-2R?- and ?c-binding antibodies or IL-2 Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated. (9A) Absolute numbers of CD4+ T cells (9B) Absolute numbers of CD8+ T cells. (9C) Absolute numbers of Tregs. (9D) Absolute numbers of na?ve CD8+ T cells. (9E) Absolute numbers of na?ve CD4+ T cells. (9F) Absolute numbers of central memory CD8+ T cells. (9G) Absolute numbers of central memory CD4+ T cells. (9H) Absolute numbers of effector memory CD8+ T cells. (9I) Absolute numbers of effector memory CD4+ T cells.

[1739] FIGS. 10A to 10G. Bar charts and graphs showing analysis of proliferation of antigen-specific T cells in response to treatment with bispecific IL-2R?- and ?c-binding antibodies or the indicated cytokines. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated. (10A and 10D) Absolute numbers of CD4+EBV-specific T cells. (10B and 10E) Absolute numbers of CD8+EBV-specific T cells. (10C) Absolute numbers of CD56+EBV-specific T cells. (10F) Percentage of CD8+EBV-specific T cells which are dividing. (10G) Graphs showing dividing C08+EBV-specific T cells as determined by CellTrace Violet staining.

[1740] FIG. 11A to 11L. Bar charts showing analysis of proliferation of cynomolgus T cell subsets in response to treatment of cynomolgus PBMCs with bispecific IL-2Rj3- and ye-binding antibodies or IL-2. Unstimulated cells (media) and anti-C03/C02B bead-stimulated controls (beads) are indicated. (11A) Absolute numbers of C04+ T cells. (11B) Absolute numbers of COB+ T cells (11C) Absolute numbers of Tregs. (110) Absolute numbers of naive C04+ T cells. (11E) Absolute numbers of effector memory C04+ T cells. (11F) Absolute numbers of central memory C04+ T cells. (11G) Absolute numbers of naYve COB+ T cells. (11H) Absolute numbers of effector memory C08+ T cells (111) Absolute numbers of central memory C08+ T cells. (11J) Absolute numbers of NK cells. (11K) Absolute numbers of B cells. (11L) Ratio of the absolute number of COB+ T cells to the absolute number of C04+ T cells.

[1741] FIG. 12A to 12N. Bar charts showing analysis of proliferation of pre-activated cynomolgus T cell subsets in response to treatment of cynomolgus PBMCs with bispecific IL-2Rj3- and ye-binding antibodies or IL-2. Unstimulated cells (media) and anti-C03/C028 bead-stimulated controls (beads) are indicated. (12A) Absolute numbers of C04+ T cells. (12B) Absolute numbers of C08+ T cells. (12C) Absolute numbers of Tregs. (120) Absolute numbers of naive C04+ T cells. (12E) Absolute numbers of effector memory C04+ T cells. (12F) Absolute numbers of central memory C04+ T cells. (12G) Absolute numbers of naYve C08+ T cells. (12H) Absolute numbers of effector memory C08+ T cells. (121) Absolute numbers of central memory C08+ T cells (12J) Ratio of the absolute number of C08+ T cells to the absolute number of C04+ T cells. (12K) Percentage of effector memory C04+ T cells which are dividing. (12L) Percentage of central memory C04+ T cells which are dividing. (12M) Percentage of effector memory COB+ T cells which are dividing. (12N) Percentage of central memory COB+ T cells which are dividing.

[1742] FIG. 13. Graph showing analysis of induction of STAT5 phosphorylation in NK92 cells in response to treatment with bispecific IL-2RI3- and ye-binding antibodies or IL-2. EC50 values for induction of STAT5 phosphorylation are shown.

[1743] FIGS. 14A to 14H. Graphs showing analysis of induction of STAT5 phosphorylation in human immune cell subsets following treatment of PBMCs with different amounts of bispecific IL-2Rj3- and ye-binding antibodies or IL-2. EC50 values for induction of STAT5 phosphorylation are shown. (14A) Percentage pSTAT5-positive naYve C04+ T cells. (14B) Percentage pSTAT5-positive memory C04+ T cells. (14C) Percentage pSTAT6-positive Tregs (140) Percentage pSTAT5-positive 8 cells. (14E) Percentage pSTAT5-positive na ive C08+ T cells. (14F) Percentage pSTAT5-positive memory C08+ T cells. (14G) Percentage pSTAT5-positive NK cells. (14H) Percentage pSTAT5-positive monocytes.

[1744] FIGS. 15A to 15C. Graphs showing analysis of induction of STAT5 phosphorylation in human Immune cell subsets following treatment of pre-activated PBMCs with different amounts of bispecific IL-2Rj3- and ye-binding antibodies or IL-2. EC50 values for induction of STAT5 phosphorylation are shown. (15A) Percentage pSTAT5-positive C04+ T cells. (15B) Percentage pSTAT5-positive COB+ T cells (15C) Percentage pSTAT5-positive NK cells.

[1745] FIG. 16. Western blot showing kinetics of induction of STATE phosphorylation in NK92 cells following treatment with bispecific IL-2R?- and ?c-binding antibodies or IL-2. Total STAT6 and actin were included as controls.

[1746] FIGS. 17A to 17E. Graphs showing kinetics of induction of STAT5 phosphorylation in human immune call subsets following treatment of PBMCs with bispecific IL-2R?- and ?c-binding antibodies or IL-2. (17A) Percentage pSTAT5-positive T cells. (17B) Percentage pSTAT5-positive CD8+ T cells (17C) Percentage pSTAT5-positive CD4+ T cells. (17D) Percentage pSTAT5-positive monocytes. (17E) Percentage pSTAT5-positive B cells.

[1747] FIGS. 18A to 18C. Graphs showing kinetics of induction of STAT5 phosphorylation in antigen-specific T cells following treatment of with bispecific IL-2R?- and ?c-binding antibodies or IL-2. (18A) Percentage pSTAT5-positive EBV-specific T cells. (18B) Percentage pSTAT5-positive CD8+EBV-specific T cells. (18C) Percentage pSTAT5-positive CD4+ EBV-specific T cells.

[1748] FIG. 19. Western blot showing induction of STAT6 phosphorylation by IL-4 in THP-1 cells following treatment with bispecific IL-2R?- and ?c-binding antibodies, isotype control antibody, or IL-2. Total STAT6 and actin were included as controls.

[1749] FIGS. 20A to 20K. Bar charts showing analysis of proliferation of immune cell subsets in response to treatment of freshly-obtained, non-activated human PBMCs with bispecific IL-2R?- and ?c-binding antibodies or IL-2. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated. (20A) Absolute numbers of CD4+ T cells. (20B) Absolute numbers of CD8+ T cells. (20C) Absolute numbers of Tregs. (20D) Absolute numbers of NK cells. (20E) Absolute numbers of B cells. (20F) Absolute numbers of na?ve CD4+ T cells. (20G) Absolute numbers of na?ve CD8+ T cells. (20H) Absolute numbers of central memory CD4+ T cells. (20I) Absolute numbers of central memory CD8+ T cells. (20J) Absolute numbers of effector memory CD4+ T cells. (20K) Absolute numbers of effector memory CD8+ T cells.

[1750] FIGS. 21A to 21C. Bar charts showing analysis of proliferation of immune cell subsets in response to treatment of non-activated human T cells with bispecific IL-2R?- and ?c-binding antibodies or IL-2 Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated. (21A) Absolute numbers of CD4+ T cells. (21B) Absolute numbers of CD8+ T cells. (21C) Absolute numbers of Tregs.

[1751] FIG. 22. Graph showing levels of bispecific IL-2R?/?c antibody (P2C4:P1A3) in the serum of cynomolgus macaques at the indicated time point, following administration of the indicated amount of antibody, as determined by ELISA.

[1752] FIGS. 23A and 23B. Graphs showing expression of (23A) IL-2R? and (23B) ?c on human immune cell subsets with or without activation using anti-CD3/CD28. The graphs show normalized median fluorescence Intensity (nMFI) of antibody staining for IL-2AB and ?c as determined by flow cytometry.

[1753] FIGS. 24A and 24B. Graphs showing expression of (24A) IL-2R? and (24B) ?c on EBV-specific immune cell subsets. The graphs show normalized median fluorescence intensity (nMFI) of antibody staining for IL-2R? and ?c as determined by flow cytometry.

[1754] FIG. 25. Schedule of administration of VSTs with or without bispecific IL-2R?- and ?c-binding antibodies (BiAb), isotype control antibody or IL-2 to murine EBV-LCL tumour model.

[1755] FIGS. 26A to 26I. Graphs showing analysis of proliferation of T cell subsets and PD-1 expression in an in vivo murine EBV-LCL tumour model following treatment with VSTs and bispecific IL-2R?- and ?c-binding antibodies, isotype control antibody, or IL-2. (26A) Absolute numbers of CD3+ T cells at 8 days post-VST treatment. (26B) Absolute numbers of CD3-CD4+ T cells at 8 days post-VST treatment. (26C) Absolute numbers of CD3+CD8+ T cells at 8 days post-VST treatment. (26D) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells, and CD3 T cell PD-1 expression analysed by MFI, from blood at 22 days post-VST treatment. (26E) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells, and CD3 T cell PD-1 expression analysed by MFI, from the spleen at 22 days post-VST treatment. (26F) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells, and CD3 T cell PD-1 expression analysed by MFI, from the liver at 22 days post-VST treatment. (26G) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells, and CD3 T cell PD-1 expression analysed by MFI, from tumour-draining lymph nodes at 22 days post-VST treatment. (26H) Absolute numbers of CD3+. CD3+CD4+ and CD3+CD8+ T cells, and CD3 T cell PD-1 expression analysed by MFI, from tumour at 22 days post-VST treatment. (26I) Total organ tumour load shown by the absolute total number of CD19+ cells in spleen, liver, tumour-draining lymph node and tumour.

[1756] FIGS. 27A to 27D. Graphs showing analysis of proliferation of pre-activated human NK cells following treatment with different amounts of bispecific IL-2R?- and ?c-binding antibodies or the indicated cytokines. Isotype antibody was used as a control. (27A) Absolute numbers of CD16+CD56+ NK cells. EC50 values are shown. (27B) Absolute numbers of CD16-CDS6+ NK cells. (27C) Percentage of dividing cells that are CD16+CD66+, determined by CellTrace Violet (CTV). EC50 values are shown. (27D) Percentage of dividing cells that are CD16-CD56+, determined by CTV. EC50 values are shown.

[1757] FIGS. 28A to 28D. Graphs showing analysis of proliferation of CAR-T cells following treatment with different amounts of bispecific IL-2R?- and ?c-binding antibodies or IL-2. Isotype antibody was used as a control. EC50 values are shown for 28A-D. (28A) Absolute numbers of CD4+ CAR-T cells. (28B) Absolute numbers of CD4+ CAR-T cells. (28C) Percentage of dividing CAR-T cells that are CD4+, determined by CellTrace? Violet (CTV). (28D) Percentage of dividing CAR-T cells that are CD8+, determined by CTV.

[1758] FIG. 29. Schedule of administration of VSTs and Tregs with or without bispecific IL-2R?- and ?c-binding antibodies (BiAb), isotype control antibody (iso) or IL-2 to murine EBV-BLCL tumour model.

[1759] FIGS. 30A to 30K. Graphs showing analysis of proliferation of T cell subsets in an in vivo murine EBV-BLCL tumour model following treatment with VSTs and Tregs plus bispecific IL-2R?- and ?c-binding antibodies, isotype control antibody (Iso), or IL-2. (30A) Absolute numbers of CD3+ T cells at 7, 14 and 21 days post-treatment. (30B) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells from the spleen at 22 days post-treatment. (30C) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells from the liver at 22 days post-treatment. (30D) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells from tumour-draining lymph node at 22 days post-treatment. (30E) Absolute numbers of CD3+, CD3+CD4+ and CD3+CD8+ T cells from injection site at 22 days post-treatment. (30F) Ratio of the absolute number of CD8+ T cells to the absolute number of Tregs in spleen, liver and tumour-draining lymph node at 22 days post-treatment. (30G) Total number of CD3+, CD3+CD4+ and CD3+CD8+ T cells pooled from spleen, liver, tumour-draining lymph node and injection site. (30H) Ratio of the total number of CD8+ T cells to the total number of Tregs pooled from spleen, liver, tumour-draining lymph node, and injection site at 22 days post-treatment. (30I) Absolute numbers of EBV-BLCL tumour cells from spleen, liver and lymph node(s) at 22 days post-treatment. (30J) Total organ tumour load shown by the absolute total number of CD19+ tumour cells in spleen, liver, and tumour-draining lymph node. (30K) Total number of CD107a+. IFN-?+ and perforin+ CD8 T cells pooled from spleen, liver, tumour-draining lymph node, and injection site at 22 days post-treatment.

EXAMPLES

[1760] In the following Examples, the inventors design, produce and characterise antibodies capable of binding to IL-2R? and ?c.

Example 1: IL-2R? and ?c Binding Antibodies

[1761] Anti-IL-2R? antibody clones and anti-?c antibody clones were isolated from a human antibody phage display library via in vitro selection.

[1762] Illustrative bispecific antibodies were constructed using IL-2R?-binding clone P2C4 in combination with one of the ?c-binding antibody clones P1A3 or P1A10. The bispecific antibodies were designated P2C4/P1A3 and P2C4/P1A10, respectively.

[1763] The closest matching antibody germline genes for clone P2C4 are IGHV1-46*01 and IGLV2-14*01.

[1764] The closest matching antibody germline genes for clone P1A3 are IGHV4-34*01 and IGKV2-28*01.

[1765] The closest matching antibody germline genes for clone P1A10 are IGHV1-24*01 and IGKV2-28*01.

[1766] Three bispecific antibody formats were prepared: scFv-KIH-Fc, CrossMab and Duobody formats. The bispecific antibodies were expressed by transient transfection of HEK 293 cells, and yields were as follows: [1767] ScFv-KiH-Fc: [1768] P2C4/P1A3: 4-14 mg/L; P2C4/P1A10: 28-40 mg/L [1769] CrossMab: [1770] P2C4/P1A3: 14-160 mg/L; P2C4/PIA 10: 63 mg/L [1771] Duobody: [1772] P2C4/P1A10: (P2C4) 77 mg/L; (P1A10) 110 mg/L

[1773] Except where otherwise indicated, in the following examples P2C4/P1A3 and P2C4/P1A10 were investigated in the scFv-KiH-Fc format, in which scFv comprising VH and VL domains for P2C4 are fused via a linker to Fc comprising the knob modification is expressed with scFv comprising VH and VL domains for P1A3 (P2C4/P1A3) or P1A10 (P2C4/P1A10) fused via a linker to Fc comprising the hole modification.

Example 2: Analysis of Binding to IL-2 Receptors

[1774] 2.1 Analysis of Binding Affinity by ELISA

[1775] Binding of P2C4/P1A3 to IL-2R? or ?c was measured by ELISA analysis, using recombinant IL-2R?-Fc and ?c-Fc coated on maxisorp plates.

[1776] Biotinylated P2C4/P1A3 was added at various concentrations. Detection of binding was performed using a colorimetric assay using HRP-conjugated streptavidin which converts TMB substrate to a blue solution. The reaction was stopped using hydrochloric acid, and absorbance was measured at 450 nm and 670 nm.

[1777] The results are shown in FIGS. 1A and 1B. P2C4/P1A3 was shown to bind to both IL-2R? and ?c. EC50s for binding were calculated and are shown in the Figures.

[1778] The bispecific antibodies analysed in this assay were: [1779] scFv (P2CA) scFv (P1A3)KiH-Fcdesignated P2C4/P1A3 in the Figures. [1780] scFv (P2C4_FW2); scFv (P1A3_FW2)KIH-Fcdesignated P2C4_FW2/P1A3_ FW2 in the Figures. [1781] Fab (P2C4): Fab (P1A3) in CrossMab formatdesignated P2C4/P1A3 Crossmab in the Figures.

[1782] 2.2 Analysis of Binding Affinity by Bio-Layer Interferometry

[1783] The affinity of binding of P2C4/P1A3 and P2C4/P1A10 to IL-2R? and ?c was measured by Bio-Layer Interferometry (BLI).

[1784] P2C4/P1A3 or P2C4/P1A10 were captured on anti-human Fc biosensor tips, and 5 different concentrations of monomeric IL-2R? or ?c were allowed to bind to the captured antibodies. Dissociation of the antigen from the immobilised antibodies was performed for 5 min Binding affinity was calculated by fitting binding curves using the 1:1 Langmuir model.

[1785] The affinity data are summarised in the table below.

TABLE-US-00003 IL -2R? ?c P2C4/P1A3 k.sub.on = 2.21 ? 10.sup.5 M.sup.1s.sup.1 k.sub.off = 6.62 ? 10.sup.3 s.sup.1 k.sub.on = 6.22 ? 10.sup.4 M.sup.1s.sup.1 k.sub.off = 4.42 ? 10.sup.3 s.sup.1 Ko = 3.00 ? 10.sup.8 M Ko = 8.47 ? 10.sup.8 M P2C4/P1A10 k.sub.on = 1.66 ? 10.sup.5 M.sup.1s.sup.1 k.sub.off = 4.40 ? 10.sup.3 s.sup.1 k.sub.off = 1.56 ? 10.sup.5 M.sup.1s.sup.1 k.sub.off = 9.61 ? 10.sup.3 s.sup.1 Ko = 2.82 ? 10.sup.8 M Ko = 6.18 ? 10.sup.8 M

[1786] Similar binding to IL-2R? for P2C4/P1A3 and P2C4/P1A10 was observed (30 nM vs 28.2 nM). This was to be expected because the bispecific antibodies have the same IL-2R?-binding clone P2C4.

[1787] Whilst the affinity of binding to ?c was similar for P2C4/P1A3 and P2C4/P1A10 (84.7 nM vs 61.8 nM). P2C4/P1A10 was found to have a faster on-rate and a faster off-rate than P2C4/P1A3.

[1788] 2.3 Analysis of Binding to IL-2RD and Yc Expressed at the Cell Surface

[1789] To determine whether P2C4/P1A3 and P2C4/P1A10 are able to bind to IL-2 receptors expressed on the surface of cells, HEK293-6E cells were transfected with plasmids encoding human IL-2R?-GFP, or IL-2R?-OFP and ?c-GFP.

[1790] Transfected cells were stained with P2C4/P1A3, P2C4/P1A10 or an isotype control antibody, followed by detection with a fluorochrome-conjugated secondary antibody for analysis by flow cytometry.

[1791] Normalized Median Fluorescence Intensity (nMFI) was calculated in the GFP+ cell population (for calls transfected with constructs encoding IL-2R?-GFP) or the GFP+/OFP+ cell population (for cells transfected with constructs encoding IL-2R?-OFP and ?c-GFP) by subtracting the MFI obtained when secondary antibody only was added to the cells (negative control condition).

[1792] The results of the analysis are shown in FIGS. 2A to 2C. Both P2C4/P1A3 and P2C4/P1A10 showed specific binding to cells expressing human IL-2R? and ?c, but did not bind to cells expressing IL-2Ra.

[1793] 2.4 Analysis of Binding to Human T Cell Subsets

[1794] To identify the subsets of human T cells that P2C4/P1A3 and P2C4/P1A10 bind to, human peripheral blood mononuclear cells (PBMCs) were isolated and stained with P2C4/P1A3, P2C4/P1A10 or isotype control antibody, followed by detection with a fluorochrome-conjugated secondary antibody. Cells were then stained with antibodies for the T cell markers CD3, CD4, CD8, CD45RA, CCR7, Foxp3 and CD25 to enable the delineation of the following T cell subsets: Naive (CD45RA+CCR?+). T central memory (CD45RA-CCR7+), T effector memory (CD45RA-CDR7?), T effector memory re-expressing CD45RA (TEMRA: CD45RA+CCR7?) and Treg (CD4+CD25+Foxp3+).

[1795] Samples were analysed by flow cytometry. Normalized Median Fluorescence Intensity (nMFI) was calculated by subtracting the MFI of the secondary antibody control.

[1796] The results are shown in FIGS. 3A and 3B. P2CA/P1A3 and P2C4/P1A10 were found to bind to all of the different human T cell subsets tested. P2C4/P1A10 displayed reduced level of binding as compared to P2C4/P1A3.

[1797] 2.5 Analysis of Binding to Rhesus IL-2R? and ?c Expressed at the Cell Surface

[1798] Cross-reactivity of P2C4/P1A3 and P2C4/P1A10 for rhesus IL-2R? and ?c was analysed essentially as described in Example 2.3 above, using HEK293-6E cells transfected with plasmids encoding rhesus IL-2R?-OFP and ?c-GFP.

[1799] The results of the analysis are shown in FIGS. 4A and 4B. Both P2C4/P1A3 and P2C4/P1A10 showed specific binding to cells expressing rhesus IL-2R? and ?c.

[1800] 2.6 Analysis of Binding to Cynomolgus Macaque T Cells

[1801] Cynomolgus macaque PBMCs were isolated and stained with P2C4/P1A3, P2C4/P1A10 or isotype control antibody, followed by a fluorochrome-conjugated secondary antibody. Cells were then stained with T call markers CD3, CD28 and CD95 to delineate the following T cell subsets: Naive (CD28+CD95?). Effector (CD28-CD95+) and Memory (CD28+CD95+).

[1802] Samples were analysed by flow cytometry. Normalized Median Fluorescence Intensity (nMFI) was calculated by subtracting the MFI of the secondary antibody control.

[1803] The results are shown in FIG. 5. P2C4/P1A3 and P2C4/P1A10 were found to bind to na?ve, effector and memory subsets of cynomolgus T calls P2C4/P1A10 displayed reduced level of binding as compared to P2C4/P1A3.

Example 3: Analysis of Induction of Cell Proliferation by IL-2R6- and ?c-Binding Bispecific Antibodies

[1804] 3.1 Analysis of the Effect on NK Cells

[1805] To analyse the functional activity of the IL-2R?- and ?c-binding bispecific antibodies, a stimulation assay was performed using the NK92 cell line which expresses both IL-2R? and ?c.

[1806] Anti-IL-2R? antibody clones and anti-?c antibody clones identified from human antibody phage display library were paired to form various bispecific antibody combinations, based on a single chain variable fragment (scFv) linked to a IgG1 knob or hole Fc. These antibodies were then used in a NK92 cell stimulation assay.

[1807] Briefly, cells were washed and stimulated with antibodies or cytokines for 3 days. Trastuzumab was used as a negative control. To quantify cell proliferation, alamarBlue reagent was added and fluorescence signal was measured at fluorescence excitation wavelength 544 nm and fluorescence emission wavelength 590 nm.

[1808] The results are shown in FIG. 6A. Several combinations anti-IL2RB and anti-?c clones are capable of Inducing NK92 cell proliferation.

[1809] In a separate assay, the following bispecific antibodies were analysed. [1810] scFv (P2C4): scFv (P1A3)KiH-Fcdesignated P2C4/P1A3 in the Figure. [1811] scFv (P2C4) scFv (P1A10)KIH-Fcdesignated P2C4/P1A10 in the Figure. [1812] Fab (P2C4): Fab (P1A3) in CrossMab formatdesignated P2C4/P1A3 Crossmab in the Figure. [1813] Fab (P2C4): Fab (P1A10) in CrossMab formatdesignated P2C4/P1A10 Crossmab in the Figure. [1814] Fab (P2C4): Fab (P1A10) in Duobody formatdesignated P2C4/P1A10 Duobody in the Figure.

[1815] The results are shown in FIG. 68. P2C4/P1A3 and P2C4/P1A10 induced NK92 proliferation in a dose-dependent manner, with an average EC50 of 0.43 nM and 0.16 nM respectively from four independent experiments.

[1816] 3.2 Analysis of the Effect on Primary Human T Cells

[1817] To analyse the effects of P2C4/P1A3 and P2C4/P1A10 on primary human T cells, T cells were isolated from human PBMCs and pre-activated for three days with anti-CD3-coated plates (2 ?g/ml) plus soluble anti-CD28 (1 ?g/ml). Cells were then rested in fresh media for a day before being labelled with CellTrace Violet, Cells were seeded at 100,000 per well and treated with P2C4/P1A3, P2C4/P1A10 (200 nM, 40 nM, 8 nM and 1.6 nM), IL-2 (20 nM, 4 nM, 0.8 nM, 0.16 nM) or anti-CD3/CD28 beads. Isotype antibody and untreated cells were included as negative controls. After four days, cells were stained with T cell markers CD3, CD4, CD8, CD45RO, CCR7, Foxp3 and CD25 to delineate T cell subsets: [1818] CD4+na?ve T cells: CD3+CD4+FoxP3?CCR7+CD45RO? [1819] CD8+na?ve T cells: CD3+CD8+CCR7+CD45RO? [1820] CD4+ central memory T cells: CD3+CD4+FoxP3?CCR7+CD45RO+ [1821] CD8+ central memory T cells: CD3+CD8+CCR7+CD45RO+ [1822] CD4+ effector memory T cells: CD3+CD4+FoxP3?CCR7?CD45RO+ [1823] CD8+ effector memory T cells: CD3+CD8+CCR7?CD45RO+ [1824] CD4+ Tregs: CD3+CD4+CD25+FoxP3+

[1825] Counting beads were included to allow absolute cell numbers to be determined by flow cytometry.

[1826] The results are shown in FIG. 7A to 7L. Treatment of pre-activated T cells with P2C4/P1A3 and P2C4/P1A10 was found to induce expansion of CD8+ T cells whilst inducing only minimal expansion of CD4+FoxP3+ regulatory T cells (Treg)see FIGS. 7B and 7C. Absolute numbers of Trags were ?10-fold lower following treatment with P2C4/P1A3 or P2C4/P1A10 as compared to treatment with IL-2 (FIG. 70).

[1827] With respect to the individual T cell subsets, CD8+ T effector memory subset responded the most to stimulation with P2C4/P1A3 and P2C4/P1A10 (FIG. 71). Proliferation of CD4+ T effector memory cells was also observed in P2C4/P1A10-treated cells. Based on CellTrace Violet staining, a high percentage of dividing CD8+ T effector memory cells were observed following P2C4/P1A3 and P2C4/P1A10 treatment (FIGS. 7K and 7L).

[1828] In a separate experiment, pre-activated T cells were stimulated with 8 different concentrations of P2C4/P1A3, P2C4/P1A10, isotype control antibody. IL-2 or IL-15. The ratio of CD8 to Treg cells was determined by dividing the absolute number of COB T cells with the absolute number of Tregs.

[1829] The results are shown in FIGS. 8A to 8G. Both P2C4/P1A3 and P2C4/P1A10 induced dose-dependent proliferation of pre-activated (i.e. anti-CDS/CD28 stimulated) T cells. The effect on CD8+ T cells was more pronounced than the effect on CD4+ T cells, P2C4/P1A10 was a more potent stimulator of proliferation than P2C4/P1A3. Both P2C4/P1A3 and P2C4/P1A10 did not induce significant proliferation of Tregs, with numbers similar to that of the isotype control-treated cells (see e.g. FIG. 8C). The ratio of CD8 to Treg cells indicated that both P2C4/P1A3 and P2C4/P1A10 preferentially expand CD8 T cells over Tregs, and to a greater extent than IL-2 or IL-15 (FIG. 8D). Stimulation of the CD4+ and CD8+ T effector memory T cell subsets was also dose-dependent (FIGS. 8E and 8F). A high percentage of dividing CD8+ T effector memory cells was detected following stimulation with P2CA/P1A10 or P2C4/P1A3 (FIG. 8G).

[1830] Pre-expanded human Treg cells were stimulated with P2C4/P1A3, P2C4/P1A10, isotype control antibody IL-2 or IL-15. After four days, cells were stained with CD3, CD4, CD8, Foxp3 and CD25 and assessed by flow cytometry to determine absolute counts using counting beads. Treg cells were defined as CD3+CD4+CD25+Foxp3+ cells.

[1831] The results are shown in FIG. 8H. A dose-dependent trend in the number of Treg cells was detected following stimulation with IL-2 and IL-15, but not following treatment with P2C4/P1A3 or P2C4/P1A10, indicating that neither antibody sustains nor expands Treg numbers in vitro.

[1832] 3.3 Analysis of the Effect on Primary Human PBMCs

[1833] To determine whether the same stimulatory effect for P2C4/P1A3 and Gigkaine could be observed in stimulated human PBMCs. PBMCs were isolated and pre-activated with anti-CD3/CD28 beads for three days. Cells were then rested in fresh media for a day before labelling with CellTrace Violet. Cells were seeded at 400 000 per well and treated with P2C4/P1A3, P2C4/P1A10 (200 nM, 4D nM, 8 nM and 1.6 nM), IL-2 (20 nM, 4 nM, 0.8 nM, 0.16 nM) or anti-CD3/CD28 beads. Isotype antibody and untreated control conditions were included as negative controls. After four days, cells were stained with T cell markers CD3, CD4, CD8, CD45RO, CCR7, Foxp3 and CD25 to delineate T cell subsets (see Example 3.2). Counting beads were included to allow absolute cell numbers to be determined by flow cytometry.

[1834] The results are shown in FIGS. 9A to 9I. In agreement with the data obtained for treatment of pre-activated primary human T cells, P2C4/P1A3 and P2C4/P1A10 also were found to induce preferential expansion of CD8+ T cells over Tregs, and CD4+ T cell proliferation was additionally observed with 2C4/P1A10-treated cells.

[1835] 3.4 Analysis of the Effect on Antigen-Specific T Cells

[1836] To determine the effects of P2C4/P1A3 and P2C4/P1A10 stimulation on antigen-specific T cells (e.g. virus-specific T cells), EBV-specific T cells (EBVSTs) were thawed and rested for a day in fresh media, and subsequently treated with P2C4/P1A3, P2C4/P1A10, IL-2 or anti-CD3/CD28 beads. Isotype antibody and untreated control conditions were included as negative controls. After four days, cells were stained with T cell markers CD3, CD4, CD8, CD45RA, CDR7, Foxp3 and CD25 to delineate T call subsets, and CD56 to enable detection of NK cells.

[1837] Counting beads were included to allow absolute cell numbers to be determined by flow cytometry.

[1838] The results are shown in FIGS. 10A to 10G. P2C4/P1A3 and P2C4/P1A10 were found to induce the expansion of both CD4+ and CD8+ virus-specific T cells (FIGS. 10A and 10B). In addition, both antibodies were able to induce the expansion of CD56+ NK cells within the virus-specific T cell population (FIG. 10C). P2C4/P1A3 and P2C4/P1A10 were found to induce proliferation of CD4+ and CD8+ virus-specific T cells in a dose-dependent manner (FIGS. 10D and 10E). A high percentage of dividing CD8+ virus-specific T cells was also detected in response to P2C4/P1A10 and P2C4/P1A3 treatment (FIGS. 10F and 10G).

[1839] 3.5 Analysis of the Effect on Cynomolgus PBMCs

[1840] Frozen cynomolgus PBMCs were thawed and rested overnight in complete media before labelling with Cell Trace Violet and seeded at 200 000 cells per well. Cells were then treated with P2C4/P1A3, P2C4/P1A10, isotype antibody (200 nM, 40 nM, 8 nM, 1.6 nM) or human recombinant IL-2 (20 nM, 4 nM, 0.8 nM, 0.16 nM). Media and anti-CD3/CD28 beads were included as controls. After four days, cells were stained with T cell markers CD3, CD4, CD8, CD2B, CD95. Foxp3 and CD25 to delineate cynomolgus T cell subsets: [1841] CD4+ Na?ve T cells: CD3+CD4+CD28+CD95? [1842] CD4+ effector memory T cells: CD3+CD4+CD28-CD95+ [1843] CD4+ central memory T cells: CD3+CD4+CD28+CD95+ [1844] CD8+ Na?ve T cells: CD3+CD8+CD28+CD95? [1845] CD8+ effector memory T cells: CD3+CD8+CD28-CD95+ [1846] CD8+ central memory T cells: CD3+CD8+CD28+CD95+

[1847] Cells were also stained with CD16 and CD2D to respectively identify NK and B cells. Counting beads were included to allow absolute cell numbers to be determined by flow cytometry.

[1848] The results are shown in FIGS. 11A to 11K. The effect of proliferation was most pronounced with IL-2 treatment. P2C4/P1A10 induced slight proliferation of CD4+, CD8+ T cells and NK cells in comparison to P2C4/P1A3 and isotype antibody control. Dose-dependent proliferation by P2C4/P1A10 was observed for CD4+ effector memory T cells. CD8+Na?ve T cells. CD8+ effector memory T cells and NK cells. Treg proliferation was observed in cells treated with IL-2 but not P2C4/P1A3 or P204/P1A10.

[1849] FIG. 11L shows the ratio of the absolute number of CD8+ T cells to the absolute number of CD4+ T cells from directly stimulated cynomolgus PBMCs treated with P2C4/P1A3, P2C4/P1A10, isotype antibody or IL-2. The ratio of CD8 to CD4 T cells indicated that P2C4/P1A10 and IL-2 preferentially expand CD8 over CD4 T cells to a greater extent than P2C4/P1A3 and isotype antibody control.

[1850] 3.6 Analysis of the Effect on Pre-Activated Cynomolgus PBMCs

[1851] Frozen cynomolgus PBMCs were thawed end rested overnight in complete media before pre-activation for three days with CD3/CD2/CD28 non-human primate T cell activating beads at a beads:cells ratio of 1:2. Cells were then rested in fresh media for a day before labelling with Cell Trace Violet. Cells were seeded at 180 000 per well and treated with P204/P1A3, P2C4/P1A10, isotype antibody (200 nM, 40 nM, 8 nM, 1.6 (M) or human recombinant IL-2 (20 nM, 4 nM, 0.8 nM, 0.15 nM). Media and anti-CD3/CD28 beads were included as controls. After four days, cells were stained with T cell markers CD3, CD4, CD8, CD28, CD95, Foxp3 and CD25 to delineate T cell subsets, as above.

[1852] Counting beads were included to allow absolute cell numbers to be determined by flow cytometry.

[1853] The results are shown in FIGS. 12A to 12I. P2C4/P1A3 and P2C4/P1A10 induced the proliferation of both CD4+ and CD8+ T cells but not Treg. P2C4/P1A10 induced the proliferation of all CD4+ and CD8+ T cell subsets. Dose dependent proliferation was observed in both CD4+ and CD8+ effector memory and central memory T cells under P2C4/P1A3 treatment.

[1854] FIG. 12J shows the ratio of the absolute number of CD8+ T calls to the absolute number of CD4+ T cells from pre-activated cynomolgus PBMCs treated with P2C4/P1A3, P2C4/P1A10, isotype antibody or IL-2. The ratio of CD8 to CD4 T cells indicate that P2C4/P1A3 and P2C4/P1A10 preferentially expand CD8 over CD4 T cells to a greater extent than IL-2 and isotype antibody control.

[1855] FIGS. 12K to 12N show analysis of proliferation of CD8+ and CD4+ T cells. A high percentage of dividing CD8+ T effector memory and CD8+ T central memory cells were detected following stimulation with P2C4/P1A10 or P2C4/P1A3. In addition, P2C4/P1A10 also induced a high percentage of dividing CD4+ T effector memory and CD4+ T central memory cells.

[1856] 3.7 Analysis of the Effect on Pre-Activated Human NK Cells

[1857] To determine the effects of IL-2R?- and ?c-binding bispecific antibodies on human NK calls, primary NK cells were isolated from human PBMCs and pre-activated for three days with irradiated K562-4-1BBL-CD64-CD86 cell line. Cells were labelled with CellTrace? Violet for tracking of cell proliferation, then treated with P2C4/P1A3, P2C4/P1A10, IL-2 and IL-15. Isotype antibody and no treatment wells were included as negative controls. After five days, cells were stained for CD56 and CD16 NK cell markers. Counting beads were also added to determine the absolute cell numbers, and samples were analysed by now cytometry.

[1858] The results are shown in FIGS. 27A to 27D. P2C4/P1A3 and P2C4/P1A10 were both found to induce proliferation of activated NK cells. Both the CD56+CD16+ and CD56+CD16? NK subsets were found to proliferate in response to P2C4/P1A3 and P2C4/P1A10 treatment in a dose-dependent manner, as shown by the dose-dependent increase in absolute counts of both types of NK cells (27A, 27B) and the percentages of the corresponding proliferating NK cell subsets Illustrated by CellTrace? Violet (CTV)-diluted cells (27C, 27D).

[1859] 3.8 Analysis of the Effect on CAR-T Cells

[1860] To determine the effects of IL-2R?- and ?c-binding bispecific antibodies on CAR-T calls, primary T cells were isolated from human PBMCs and then transduced with the CAR construct so that the CAR is expressed. Alter 24 days of cell expansion, cells were labelled with CellTrace? M Violet for tracking of cell proliferation, then treated with P2CA/P1A3, P204/P1A10, isotype antibody or IL-2. After five days, cells were stained for CAR expression and T cell markers to identify the T cell subsets Counting beads were also added to determine the absolute cell numbers, and samples were analysed by flow cytometry.

[1861] The results are shown in FIGS. 28A to 28D. P2C4/P1A3 and P2C4/P1A10 both induce proliferation of CAR-T cells. The antibodies were found to induce expansion of the CD4+ and CD8+ CAR-T cells in a dose-dependent manner, as shown by the dose-dependent increase in absolute counts of CD4+(28A) and CD8+(28B) CAR-T call subsets A greater percentage of CD8+ CAR-T cells were found to be proliferating (28D) compared to CD4+ CAR-T cells (28C), determined by CellTrace? Violet (CTV), suggesting that CD8+ CAR-T cells are more responsive to P2C4/P1A3 and P2C4/P1A10 treatment then CD4+ CAR-T cells.

Example 4: Analysis of Induction of Intracellular Signalling by IL-2RB- and ?c-Binding Bispecific Antibodies

[1862] 4.1 Analysis of Induction of STAT5 Phosphorylation in NK Cells

[1863] NK92 cells were washed and rested in IL-2-free media for 1 h prior to stimulation with various concentrations of P2C4/P1A3, P2CA/P1A10, Isotype control antibody or IL-2 for 30 min. Cells were subsequently fixed, permeabilised and stained for phosphorylated STAT5 using a fluorescently-labelled antibody, and the samples were then analysed by flow cytometry.

[1864] The results are shown in FIG. 13. Both P2C4/P1A3 and P2C4/P1A10 were found to stimulate phosphorylation of STAT5 in NK92 cells in a dose-dependent manner. Activation by P2C4/P1A10 achieved a higher level of STAT5 phosphorylation as compared to activation by P2C4/P1A3.

[1865] 4.2 Analysis of Induction of STAT5 Phosphorylation in Primary Human Immune Cell Subsets

[1866] PBMCs were thawed and rested overnight prior to being seeded at 400.000 cells per well in serum-free media. Cells were rested for two hours and subsequently stimulated with 8 different concentrations of P2C4/P1A3, P2C4/P1A10, isotype control antibody or IL-2. After 30 minutes, cells were analysed by flow cytometry for phosphorylation of STAT5 as well as for immune cell markers CD3, CD4, CD8, CD45RA, CD45RO, Foxp3, CD25, CD56, CD19 and CD14 to delineate T subsets, B, NK cells and monocytes.

[1867] The results are shown in FIGS. 14A to 14H. P2C4/P1A10 induced phosphorylation of STAT5 in several T cell subsets, as well as in NK cells, in a dose-dependent manner. P2C4/P1A3 induced minimal phosphorylation of STAT5. Minimal phosphorylation of STAT5 was also observed in B cells and monocytes.

[1868] 4.3 Analysis of Induction of STAT5 Phosphorylation in Pre-Activated Primary Human Immune Cell Subsets

[1869] PBMCs were thawed and rested overnight before pre-activation with anti-CD3/CD28 beads for three days. Cells were then rested in fresh media for a day before seeding at 200 000 par well in serum-free media. Cells were rested for two hours and subsequently stimulated with 8 different concentrations of P2C4/P1A3, P2C4/P1A10, isotype control antibody or IL-2. After 30 minutes, cells were analysed by flow cytometry for phosphorylation of STAT5 as well as for immune cell markers CD3, CD4, CD8, and CD56 to delineate T subsets and NK cells.

[1870] The results are shown in FIGS. 15A to 15C. Both P2C4/P1A10 and P2C4/P1A3 induced the phosphorylation of STAT5 within pre-activated CD4+, CD8+ T cell subsets and NK cells in a dose-dependent manner, and to a greater extent than within non-activated cells.

[1871] 4.4 Analysis of the Kinetics of Induction of STAT5 Phosphorylation in NK Cells

[1872] NK92 calls were rested in serum-free media and treated with P2C4/P1A3, P2C4/P1A10. Isotype control antibody at 100 nM or IL-2 at 20 nM for 5, 10, 20, 30, 60 and 120 min. Cells were harvested at the indicated time points for assessment of STAT5 phosphorylation (Y694) via western blotting. Total STAT5 and actin were included as controls.

[1873] P2C4/P1A3 and P2C4/P1A10 were able to induce pSTAT5 in a time-dependent manner in comparison to Isotype antibody treatment (FIG. 16).

[1874] 4.5 Analysis of the Kinetics of Induction of STAT5 Phosphorylation in Primary Human Immune Cell Subsets

[1875] Freshly isolated human PBMCs were stimulated with 50 nM P2C4/P1A3, P2C4/P1A10, Isotype control antibody or 2 nM IL-2 in a reverse lime course of 0, 5, 10, 20, 40, 60 and 120 min. Cells were then fixed, permeabilised and stained with CD3, CD4, CD8, CD14, CD19 and pSTAT5 (Y694) for the identification of immune cell subsets. The data are presented as mean percentage of pSTAT5-positive cells of the PBMC subsets from 3 donors.

[1876] The results are shown in FIGS. 17A to 17E. Both P2C4/P1A3 and P2G4/P1A1D induced STAT5 phosphorylation in T cells. Maximal stimulation was achieved by both antibodies at the 5 min time point P2C4/P1A10 also stimulated a higher percentage of pSTAT5-positive cells than P204/P1A3 over the time course of 2 h. Stimulation of PBMCs with P2C4/P1A3 and P204/P1A10 did not result in a significantly greater percentage of pSTAT5-positive monocytes and B cells as compared to the isotype control antibody.

[1877] 4.6 Analysis of the Kinetics of Induction of STAT5 Phosphorylation in Antigen-Specific T Cells

[1878] EBV-specific T cells were thawed and rested in fresh media, and subsequently stimulated with 50 nM P2C4/P1A3, P2C4/P1A10, isotype control antibody or 2 nM IL-2 in a reverse time course of 0, 5, 10, 20, 40, 60 and 120 min. Cells were then fixed, permeabilised and stained with CD3, CD4, CD8 and pSTAT5 (Y694) for the identification of EBV-specific T cell subsets. The data are presented as mean percentage of pSTAT5-positive cells of the virus-specific T cell subsets from 3 donors.

[1879] The results are shown in FIGS. 18A to 18C. Similar to the data obtained using human PBMCs (Example 4.5), both P2C4/P1A3 and P2C4/P1A10 stimulated STAT5 phosphorylation in EBV-specific T cells, and P2C4/P1A10 induced higher percentage of STAT5-positive cells than P2C4/P1A3 over the time course of 2 h.

[1880] 4.7 Effects on Other Cytokine Receptors

[1881] To assess whether P2C4/P1A3 or P2C4/P1A10 binding of IL-2Ry prevents IL-4 signalling through the IL-4 receptor, THP-1 cells were treated with P2C4/P1A3, P2C4/P1A10. Isotype Control antibody (100 nM) or IL-2 (20 nM), with or without IL-4 (200 ng/ml) for 30 minutes. Cell lysates were assessed by western blotting to determine phosphorylation of STATE (Y641). Total STAT6 and actin were included as controls.

[1882] IL-4 induced pSTAT6 to a similar extent between different conditions, even in the presence of P2C4/P1A3/P2C4/P1A10 (FIG. 19). This suggests that despite binding to IL-2R?, P2C4/P1A3 and P2C4/P1A10 do not affect IL-4-mediated signalling.

Example 5: Analysis of ToxicityInduction of Proliferation of Non-Activated Immune Cells

[1883] 5.1 Analysis of Stimulation of Proliferation by Non-Activated PBMCs

[1884] To measure the effects of P2C4/P1A3 and P2C4/P1A10 on non-activated, freshly-obtained PBMCs. PBMCs were isolated and directly treated with P2C4/P1A3, P2C4/P1A10 (200 nM, 40 nM, 8 nM and 1.6 nM), IL-2 (20 nM, 4 nM, 0.8 nM and 0.16 nM) or anti-CD3/CD28 beads as positive control. Isotype antibody and untreated control conditions were included as negative controls. After four days, cells were stained with T cell markers CD3, CD4, CD8, CD45RO, CCR7, Foxp3 and CD25 to for the delineation of T cell subsets, and with CD19 and CD56 for the identification of 8 cells and NK cells, respectively. Counting beads were included to enable absolute cell numbers to be determined by flow cytometry.

[1885] The results of the analysis are shown in FIGS. 20A to 20K. P2C4/P1A3 and P2C4/P1A10 did not induce significant proliferation of non-activated PBMCs as compared to isotype control antibody. This was observed for all T cell subsets including CD4, CD8, Treg, na?ve, T central memory (Tcm) and T effector memory cells (Tem), as well as NK cells. This contrasts with IL-2, which stimulated expansion of T and NK calls even at lower doses. Minimal proliferation was also observed for B cells in response to treatment with IL-2.

[1886] T cell activation requires three signals (1) TCR-(CD3)/MHC interaction, (2) co-stimulation i.e. CD28 and (3) cytokine signalling i.e. IL-2, As P2C4/P1A3 and P2C4/P1A10 do not induce proliferation of T cells under direct stimulation, this indicated that signals (1) and (2) are required before T cells become responsive to the antibodies, in agreement with the results obtained with preactivated calls.

[1887] These data suggest that P2C4/P1A3 and P2C4/P1A10 preferentially expand activated T cells, and may be associated with reduced toxicity as compared to treatment with IL-2 (which expands both activated and non-activated cells).

[1888] 5.2 Analysis of Stimulation of Proliferation by Non-Activated T Cells

[1889] Human T cells were isolated and directly treated with P2C4/P1A3, P2C4/P1A10. IL-2 or anti-CD3/CD28 beads. Isotype antibody and untreated control conditions were included as negative controls. After four days, cells were stained with T cell markers CD3, CD4, CD5, CD45RA, CCR7, Foxp3 and CD25 to delineate T cell subsets. Counting beads were included to enable absolute cell numbers to be determined by flow cytometry.

[1890] The results are shown in FIGS. 21A to 21C. Similar to the observation with direct stimulation of human PBMCs, P2C4/P1A3 and P2C4/P1A10 did not induce proliferation of T cells without pre-activation, indicating that antigen recognition/CD3 activation and co-stimulation signals are required before T cells become responsive to these antibodies. This contrasts with IL-2, which indiscriminately expanded T cells even at low doses.

Example 6: Analysis of Pharmacokinetics in non-Human Primates

[1891] A simple pharmacokinetics (PK) study was performed to measure the clearance of P2C4/P1A3 in non-human primates.

[1892] 3 cynomolgus macaques were injected with a single dose of 1 mg/kg, 5 mg/kg and 10 mg/kg P2C4/P1A3 and blood collection was done at pre-dose, 1 h, 24 h, 72 h and 120 h post-antibody injection time-points. Plasma is obtained from the collected blood and a sandwich ELISA were performed to measure the levels of P2C4/P1A3.

[1893] Sandwich ELISA was performed using coated anti-human CH2 antibody, and detection of P2C4/P1A3 was by using anti-human Fc-HRP. An ELISA standard curve was derived using purified P2C4/P1A3 for calculation of the absolute concentration of antibody in blood.

[1894] The results are shown in FIG. 22. Maximum blood antibody levels were detected at 1 h post antibody dosing, and remained in the system up to 120 h.

[1895] IL-2 is known to have a much shorter serum half-lifesee e.g. Skrombolas and Frelinger, Expert Rev Clin Immunol. (2014)10(2): 207-217, which reports that a study of the serum half-life of IL-2 introduced intravenously found a bi-phasic event with phase I (biodistribution throughout the body) resulting in t.sub.1/2 of approximately 7 min and phase II (extravasation from plasma into tissue) at approximately 60 min.

Example 7: Analysis of IL-2RS and Yc Expression on Human PBMCs and Antigen-Specific T Cells

[1896] Human PBMCs were thawed and rested overnight in cell culture media. The cells were then activated using anti-CD3/CD28 beads.

[1897] After three days, cells were rested in media for a day before staining with commercially available anti-IL-2R? or ?c antibodies plus antibodies for markers of human immune subsets. Cells were then analysed by flow cytometry to determine the expression of IL-2R? and ?c before (?) and after (+) pre-activation. Normalized Median Fluorescence Intensity (nMFI) was calculated by subtracting MFI values for the fluorescence-minus one (FMO) control.

[1898] The results are shown in FIGS. 23A and 23B. Activation of human PBMCs with anti-CD3/CD28 was shown to upregulate surface expression of both IL-2R? and ye across three different donor samples, particularly on T cell subsets.

[1899] In a separate experiment, EBV-specific immune cells were thawed and rested in fresh media overnight prior to being stained with commercially available anti-IL-2R? or ?c antibodies plus antibodies for markers of human T cell subsets and NK cells. Cells were then analysed by flow cytometry to determine the expression of IL-2R? and ?c. Normalized Median Fluorescence Intensity (nMFI) was calculated by subtracting MFI values for the fluorescence-minus one (FMO) control.

[1900] The results are shown in FIGS. 24A and 24B. Expression of IL-2R? and ?c was detected on different immune cell subsets within the EBV-specific T cells derived from three different donors.

Example B: Production of Anti-IL-2R?/?c Antibody P2C4IP1A10 in Duobody Format

[1901] P2C4/P1A10 was made in the Duobody format. Briefly, monospecific anti-IL-2R? P2C4 IgG1-K409R and anti-?c P1A10 IgG1-F405L antibodies were produced and purified, mixed, then subjected to reduction with 75 mM 2-MEA at pH 8.5, 31? C. for 5 h, 2-MEA was removed by dialysis, and the antibodies were left to re-oxidise at 4? C. The fully formed bispecific Duobody were purified by anion exchange chromatography.

Example 9: Analysis of the Effect of Anti-IL-2R?/?c Antibodies on Anti-Cancer Immune Responses

[1902] Example 8.4 of WO 2017/021540 A1 reports the ability of CD8+ T cells expanded by treatment with bispecific agonist anti-IL-2R? and -?c antibodies to kill cancer cells. Specifically, T cells expanded from PBMCs obtained from EBV seropositive donors by culture in presence of P2C4:P1A3 are shown to kill LCLs.

[1903] Example 12 and FIGS. 41 and 42 of WO 2017/021540 A1 demonstrate the ability of bispecific agonist anti-HL-2R? and -?c antibodies to stimulate proliferation of T cells and NK cells in vivo in cynomolgus macaques.

[1904] In the present Example, bispecific agonist anti-IL-2R? and -?c antibodies are shown to promote an anti-cancer immune response in vivo.

[1905] Tumours are established by subcutaneous injection of mice with LOLs. Specifically. EBV-transformed lymphoblastoid B-cell line (LCLs) was mixed with Matrigel and injected subcutaneously to the right flank of NSG mice.

[1906] Mice were subsequently administered with autologous EBV-specific CTLs (VSTs), with or without P2C4/P1A3, P2C4/P1A10, isotype control antibody, or IL-2, at 19 days post-tumour inoculation, IL-2 treatments were given at 40 000 U/kg, intra-peritoneally (i.p.) consecutively for 5 days for a total of 5 doses. Antibody treatments were given at 5 mg/kg, i.p. every 14 days, for a total of 2 doses. The administration schedule is shown in FIG. 25.

[1907] Mouse blood was collected at 8 days post-VST treatment and flow cytometric analysis showed elevated numbers of total human CD3, CD4 and CD8 T cells in mice treated with P2C4/P1A3 and P2CA/P1A10 as compared to mica treated with isotype control antibody or IL-2. The results are shown in FIGS. 26A to 26C.

[1908] At the end of the experiment, mice were euthanised at 22 days post-VST treatment and blood, spleen, liver, tumour-draining lymph node and flank tumour were harvested for flow cytometric analysis.

[1909] The results are shown in FIGS. 26D to 26H. Similar to results at 8 days post-VST treatment, mice treated with P2C4/P1A3 and P2C4/P1A10 had elevated numbers of total human CD3, CD4 and COB T cells in blood and organs. CD3 T cells from mice treated with P2C4/P1A3 and P2C4/P1A10 also had lower expression of PD-1 as compared to cells from mice treated with IL-2 and isotype control antibody.

[1910] Total organ tumour load in mice was calculated from the total numbers of GD 19+ cells in spleen, liver, flank tumour and tumour-draining lymph node (FIG. 261). Mice treated with P2CA/P1A3 and P2C4/P1A10 had lower total organ tumour burden as compared to mice treated with isotype control antibody, IL-2 or no VSTs.

Example 10: Analysis of the Effect of anti-IL-2R?/?c Antibodies on Anti-Cancer Immune Responses in the Presence of Tregs

[1911] In the present Example, bispecific agonist anti-IL-2R? and -?c antibodies are shown to promote an anti-cancer immune response in vivo, without accompanying increases in immunosuppressive regulatory T cells (Tregs) in a mouse model of EBV-BLCL metastatic lymphoma.

[1912] Tumours are established by subcutaneous injection of mice with LCLs. Specifically. EBV-transformed B lymphoblastoid cell lines (LCLs) was mixed with Matrigel and injected subcutaneously to the right flank of NSG mice.

[1913] Mice were subsequently administered with autologous EBV-specific CTLs (VSTs) and Tregs, with or without P2C4/P1A3, P2C4/P1A10, isotype control antibody, or IL-2, at 20 days post-tumour inoculation.

[1914] IL-2 treatments were given at 40 000 U/kg, intra-peritoneally (i.p.) consecutively for 5 days for a total of 5 doses Antibody treatments were given at 5 mg/kg, i.p. every 14 days, for a total of 2 doses. The administration schedule is shown in FIG. 29.

[1915] Analysis of peripheral blood collected from mice at 7, 14 and 21 days post-treatment showed that slightly higher numbers of circulating VSTs were detected in mics which received bispecific anti-IL-2R? and -?c antibodies compared to isotype (Iso) or IL-2, as shown in FIG. 30A.

[1916] At the end of the experiment, mice were euthanised at 22 days post-treatment and spleen, liver, tumour-draining lymph node and injection site were harvested for flow cytometric analysis. The results are shown in FIGS. 30B to 30E. Mice Treated with P2C4/P1A3 and P2C4/P1A10 were found to have elevated numbers of total human CD3, CD4 and CD8 T cells in spleen (30B), liver (30C), tumour-draining lymph node(s) (30D) and injection site (30E). **, p<0.01: *, p<0.05.

[1917] The ratio of CD8 cells to Tregs has been shown to be predictive of a favourable outcome in multiple tumour types (de Leeuw R J et al. Clin Cancer Res 2012, 18:3022-9). FIG. 30F shows that the spleen, liver and lymph node(s) of mice treated with P2GA/P1A3 or P2C4/P1A10 were found to demonstrate higher CD8/Treg ratios compared to the same organs from mice treated with IL-2. This shows that P2C4/P1A3 and P2C4/P1A10 preferentially expand CD& T cells over Tregs, compared to the effect seen with IL-2.

[1918] The numbers of CD3, CD4 and CD8 T cells from spleen, liver, tumour-draining lymph node and injection site were pooled to provide total numbers of cells. The results are shown in FIG. 30G. The total pooled CD8/Treg ratio is shown in FIG. 30H. The total CD8/Treg ratio of mice treated with P2C4/P1A3 or P2C4/P1A10 was found to be higher than the total CD8/Treg ratio from mice treated with isotype (Iso) or IL-2; *, p<0.05.

[1919] The numbers of EBV-BLCLs present in the spleen, liver and lymph node(s) at the end of the experiment were also analysed. The results are shown in FIG. 30I. Mice treated with P2C4/P1A3 and P2C4/P1A10 were found to have reduced numbers of EBV-BLCL tumour cells compared to mica treated with IL-2**, p<0.01; *, p<0.05.

[1920] Total organ tumour load in mice was calculated from the total numbers of CD19+ tumour cells found in spleen, liver, and tumour-draining lymph node. The results are shown in FIG. 30J. Mice treated with P2CA/P1A3 and P2C4/P1A10 were found to have lower total organ tumour burden as compared to mice treated with isotype control antibody. IL-2 or no VSTs; **, p<0.01.

[1921] Next, the cytolytic activity of the expanded CD8 T cells was assessed by identifying the total number of CD8 T cells from spleen, liver, tumour-draining lymph node and injection site secreting the effector molecules interferon-? (IFN-?), CD107a and perforin. The results are shown in FIG. 30K. Higher numbers of effector molecule-secreting CD8 T cells were found to be present following treatment with P2C4/P1A3 and P2C4/P1A10 compared to treatment with IL-2. *, P<0.05.

[1922] In conclusion, bispecific anti-IL-2R and -ye antibodies were shown to provide sustained expansion of COB T cells in vivo without accompanying increases in Tregs, leading to improved tumour control.

Example 11: Analysis of the Effect of Anti-IL-2R?/?c Antibodies on Survival

[1923] A murine model of metastatic lymphoma is generated by intravenous injection of EBV-BLCLs to investigate the effect of bispecific agonist anti-IL-2R and -ye antibodies versus IL-2 on survival. Mice treated with anti-IL-2R and -ye antibodies are found to have improved survival as compared to mice not treated with anti-IL-2R and -ye antibodies.