WO2020094834A1 - Anticorps anti-il-2r bêta/chaîne gamma commune - Google Patents

Anticorps anti-il-2r bêta/chaîne gamma commune Download PDF

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Publication number
WO2020094834A1
WO2020094834A1 PCT/EP2019/080658 EP2019080658W WO2020094834A1 WO 2020094834 A1 WO2020094834 A1 WO 2020094834A1 EP 2019080658 W EP2019080658 W EP 2019080658W WO 2020094834 A1 WO2020094834 A1 WO 2020094834A1
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Prior art keywords
amino acid
seq
acid sequence
region
cdr1
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PCT/EP2019/080658
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English (en)
Inventor
Cheng-I Wang
Jianrong Lionel LOW
Cheah Chen SEH
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Euchloe Bio Pte. Ltd.
CLEGG, Richard Ian
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Publication of WO2020094834A1 publication Critical patent/WO2020094834A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the fields of molecular biology and methods of medical treatment and prophylaxis.
  • the present invention relates to antigen-binding molecules capable of binding to interleukin 2 receptor b (IL-2R ; CD122) and common y chain (yc; CD132).
  • IL-2R interleukin 2 receptor b
  • yc common y chain
  • IL-2 is an essential cytokine that plays a central role in maintaining T cell homeostasis and mediating proper immune responses. Its high potency as an immune stimulator has led to clinical uses to treat a range of conditions, including cancers and AIDS; it is also widely used as an adjuvant for vaccination to stimulate activation and proliferation of various effector cells.
  • VLS vascular leak syndrome
  • IL-2 exerts its pleiotropic functions by binding to different combinations of receptor components expressed on different cell types: the alpha chain (IL-2Ra, also known as CD25), the beta chain (IL-2R , or CD122), and the common cytokine receptor gamma chain (IL-2Ry, yc, or CD132).
  • IL-2Ra also known as CD25
  • IL-2R beta chain
  • IL-2Ry common cytokine receptor gamma chain
  • CD132 common cytokine receptor gamma chain
  • Isolated IL-2Ra has been termed the“low affinity” IL-2 receptor (binding affinity KD ⁇ 10 nM) and is not involved in signal transduction.
  • a complex of IL-2R and yc binds IL-2 with intermediate affinity (KD ⁇ 1 nM), although IL-2R alone has very low affinity (KD ⁇ 100 nM) and yc alone has virtually no detectable binding affinity for IL-2.
  • a complex with all three subunits, IL-2Ra, IL-2R , and yc binds IL-2 with high affinity (KD ⁇ 10 pM).
  • IL-2Ra plays no role in signal transduction.
  • High-affinity a-b-yc IL-2Rs are typically found on CD4+ T regulatory cells (Tregs) as well as recently- activated T cells.
  • Intermediate-affinity b-yc IL-2Rs are present at a low level on naive CD8+ cells, but are prominent on antigen-experienced (memory) and memory-phenotype (MP) CD8+ T cells as well as natural killer (NK) cells. Both MPCD8+ T cells and NK cells express very high levels of I ⁇ -2Rb and readily respond to IL-2. Previous studies have indicated that VLS is caused by the release of proinflammatory cytokines from IL- 2-activated NK cells.
  • IL-2-induced pulmonary edema may result from direct binding of IL-2 to lung endothelial cells, which express functional high affinity a-b-yc IL-2Rs.
  • mAb monoclonal antibody
  • IL-2Ra -deficient host mice or by the use of an IL- 2/anti-IL-2 mAb (IL-2/mAb) complex in which the antibody prevents IL-2/ IL-2Ra interaction, thus preventing VLS.
  • the present invention provides an antigen-binding molecule, optionally isolated, comprising: a polypeptide comprising a moiety capable of binding to CD122, a moiety capable of binding to CD 132, a CH2 region and a CH3 region.
  • the moiety capable of binding to CD122 and the moiety capable of binding to CD132 are provided in tandem.
  • the moiety capable of binding to CD122 comprises or consists of a single chain variable fragment (scFv) capable of binding to CD122.
  • scFv single chain variable fragment
  • the moiety capable of binding to CD132 comprises or consists of a scFv capable of binding to CD132.
  • the antigen-binding molecule comprises, or consists of, two of said polypeptides.
  • an antigen-binding molecule comprising: (a) a polypeptide comprising a moiety capable of binding to CD122, a CH2 region and a CH3 region, (b) a polypeptide comprising the heavy chain variable (VH) region of an antigen-binding moiety capable of binding to CD132, a CH1 region, a CH2 region and a CH3 region, and (c) a polypeptide comprising the light chain variable (VL) region of an antigen-binding moiety capable of binding to CD132, and CL region.
  • VH heavy chain variable
  • VL light chain variable
  • the antigen binding molecule comprises an Fc region comprising the knob-into- hole (KiH) modification.
  • the CH3 region of the polypeptide of (a) comprises W at the position
  • the CH3 region of the polypeptide of (b) comprises S at the position corresponding to position 366, A at the position corresponding to position 368, Y at the position corresponding to position 407 and C at the position corresponding to position 349.
  • the moiety capable of binding to CD122 comprises or consists of a single chain variable fragment (scFv) capable of binding to CD122.
  • the antigen-binding moiety capable of binding to CD122 comprises:
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of one of SEQ ID NOs: 103 to 1 15
  • HC-CDR2 having the amino acid sequence of one of SEQ ID NOs: 1 16 to 127
  • HC-CDR3 having the amino acid sequence of one of SEQ ID NOs: 128 to 144; and a light chain variable (VL) region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of one of SEQ ID NOs: 145 to 161
  • LC-CDR2 having the amino acid sequence of one of SEQ ID NOs: 162 to 176
  • LC-CDR3 having the amino acid sequence of one of SEQ ID NOs: 177 to 194;
  • HC-CDR1 HC- CDR2
  • HC-CDR3 LC-CDR1 , LC-CDR2 or LC-CDR3 are substituted with another amino acid.
  • the antigen-binding moiety capable of binding to CD132 comprises:
  • HC-CDR1 having the amino acid sequence of one of SEQ ID NOs: 106, 108, 1 12, or 195 to 201
  • HC-CDR2 having the amino acid sequence of one of SEQ ID NOs: 1 19, 120, 124, or 202 to 209
  • HC-CDR3 having the amino acid sequence of one of SEQ ID NOs:210 to 225; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of one of SEQ ID NOs: 151 , or 226 to 235
  • LC-CDR2 having the amino acid sequence of one of SEQ ID NOs: 174, or 236 to 245
  • LC-CDR3 having the amino acid sequence of one of SEQ ID NOs: 189, or 247 to 258; or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC- CDR2, HC-CDR3, LC-CDR1 , LC-CDR2 or LC-CDR3 are substituted with another amino acid.
  • the antigen-binding moiety capable of binding to CD122 comprises:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177; or
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16 HC-CDR3 having the amino acid sequence of SEQ ID NO:128; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:149
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177; or (P2C4_B1 ) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 166
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177
  • P2C4_B5 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 181
  • P2C4_C4 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 166
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_C7 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_D10 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_E6 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 149
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177
  • P2C4_E7 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_F8 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2H7 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 104
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 17
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 129
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 146
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 163
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:178; or (P2D12) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 105
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 18
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 130; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 147
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 164
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 179; or (P1 G1 1 ) 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: 1 19
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 131 ; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 148
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 165
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 180
  • P2C4_A9 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 132
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177
  • P2C4_B6 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 107
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_E9 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 107
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 168
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_B8 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_B12 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16 HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 150
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 167
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177
  • P2C4_C1 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 149
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177
  • P2C4_C12 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_E2 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181 ; or (P2C4_E3) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 107
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_E8 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181 ; or (P2C4_F11 ) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_G2 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181 ; or (P2C4_G11 ) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4JH1 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4JH2 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:116
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:128
  • VL region incorporating the following CDRs LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_H3 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181
  • P2C4_C1 D10 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 149
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:181 ; or (P2C4_FW2) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 162
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177
  • P1 E7 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: 133; 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: 169
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 182; or (P1 B10) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 109
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 121
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 134
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 152
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 164
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 183; or (P1 F3) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:105
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 122
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 135
  • fa VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 153
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 164
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 184; or (P1 D10) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 10
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 19
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 136; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 154
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 170
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 185; or (P1 E1 ) 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: 1 19
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 137; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 155
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 171
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 186; or (P2B1 1 ) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 1 1 HC-CDR2 having the amino acid sequence of SEQ ID NO: 123 HC-CDR3 having the amino acid sequence of SEQ ID NO: 138; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 156
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 172
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 187; or (P2C9) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 12
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 124
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 139; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 157
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 173
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 188
  • P2C10 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: 1 19
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:140; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 158
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 174
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:189
  • P2C1 1 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 13
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 125
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 141
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 159
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 175
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190; or (P2E6) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 14
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 126
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 142; and a VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 160
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 176
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:191 ; or (P2E1 1 ) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 109
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 121
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 134
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 159
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 164
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 192; or (P2F9) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 15
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 127
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:143
  • 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: 193; or (P2F10) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 15
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 127
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 144;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 194.
  • the antigen-binding moiety capable of binding to CD122 comprises:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 128;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 145
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 177.
  • the antigen-binding moiety capable of binding to CD122 comprises:
  • VH region comprising an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:1 to 34;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:35 to 65.
  • the antigen-binding moiety capable of binding to CD122 comprises:
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:21 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:52;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:35;
  • P2C4_A4 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:39;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:40;
  • P2C4_B5 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ; and a VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:41 ; or
  • P2C4_C4 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:44;
  • P2C4_C7 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:45;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:46;
  • P2C4_E7 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:47;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:49;
  • VH7 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:2;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:36;
  • (P2D12) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:37;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:38;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:5;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:35; or (P2C4_B6) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:6; and
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:6;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:48;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:7;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:43;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:39;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:11 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:12;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:13;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:14;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:50;
  • P2C4_G2 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:15; and a VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42; or
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH2 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:18;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • VH3 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:19;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42;
  • P2C4_C1 D10 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:20;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:51 ;
  • P1 E7 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:22;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:53;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:54;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:55;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:56;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:57; or (P2B1 1 ) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:27; and
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:58;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:28;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:59;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:29;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:60;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:61 ;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:31 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:62;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:63;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:33;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:64;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:34;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:65.
  • the antigen-binding moiety capable of binding to CD122 comprises:
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:21 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:52.
  • the antigen-binding moiety capable of binding to CD132 comprises:
  • 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
  • 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 (P1A3) 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: 1
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:210
  • 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:236
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P2B9) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 195
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:202
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:21 1
  • VL region incorporating the following CDRs:
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:226
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:237
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:247; or (P1 A3_B3) 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:203
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:210
  • 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:236
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1 A3_B4) 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:203
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:210
  • 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:236
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:189; or (P1 A3_E9) 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:203
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:210
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 151
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:236
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1 A3_E8) 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:203
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:210
  • 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:236
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1A3_FW2) 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: 1
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:210
  • 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:236
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 189; or (P1 B6) 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
  • 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 (P1 C10) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 1 12
  • 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 (P1 D7) 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
  • 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
  • P1 E8 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
  • 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: 1
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:218
  • 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 (P2D1 1 ) 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
  • 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
  • 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-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: 1 19
  • 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 (P1 G4) 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: 1 19
  • 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 (P1 B12) 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: 1 19
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:224; and a VL region incorporating the following CDRs:
  • 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 (P1 C7) 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: 1 19
  • 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-CDR3 having the amino acid sequence of SEQ ID NO: 189.
  • the antigen-binding moiety capable of binding to CD132 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;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:248; or
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 106
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 1 19
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:210;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 151
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 189.
  • the antigen-binding moiety capable of binding to CD132 comprises:
  • VH region comprising an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:66 to 84;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:85 to 102.
  • the antigen-binding moiety capable of binding to CD132 comprises:
  • (P1A10) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:71 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:89;
  • (P1 A3) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:66;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85;
  • (P1A3_AQ) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:465;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:67;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:86; or
  • (P1A3_B3) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:68; and a VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85; or
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:68;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:87;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:68;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:69;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85;
  • P1A3_FW2 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:70;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:88;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:90;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:91 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:92;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:93;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:94;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:77;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:95; or (P2D1 1 ) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:78; and
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:96;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:79;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:97;
  • VH4 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:80;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:98;
  • (P2D3) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:81 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:99;
  • P1 G4 a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:82;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 100;
  • (P1 B12) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:83;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 101 ;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:84;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:102.
  • the antigen-binding moiety capable of binding to CD132 comprises:
  • (P1A10) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:71 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:89;
  • (P1 A3) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:66;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85;
  • (P1A3_AQ) a VH region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:465;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:85.
  • an antigen-binding molecule comprising or consisting of: two polypeptides, each polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:461.
  • an antigen-binding molecule comprising or consisting of: two polypeptides, each polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:467.
  • an antigen-binding molecule comprising or consisting of: two polypeptides, each polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:462.
  • an antigen-binding molecule comprising or consisting of: two polypeptides, each polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:468.
  • an antigen-binding molecule comprising or consisting of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:457;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:458.
  • an antigen-binding molecule comprising or consisting of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:459;
  • the antigen-binding molecule further comprises a cell membrane anchor region.
  • the antigen-binding molecule is an IL-2 receptor agonist (e.g. an agonist of the IL-2 receptor comprising a complex of CD122 and CD132).
  • the present invention also provides a nucleic acid, optionally isolated, encoding an antigen-binding molecule according to the present invention.
  • the present invention also provides an expression vector comprising a nucleic acid according to the present invention.
  • the present invention also provides a cell comprising an antigen-binding molecule, nucleic acid, or an expression vector according to the present invention.
  • the present invention also provides a method for producing an antigen-binding molecule according to the present invention, the method comprising culturing a cell comprising a nucleic acid or an expression vector according to the present invention under conditions suitable for expression of the antigen-binding molecule from the nucleic acid or expression vector.
  • the present invention also provides a composition comprising an antigen-binding molecule, nucleic acid, an expression vector according or a cell according to the present invention.
  • the present invention also provides an antigen-binding molecule, nucleic acid, expression vector, cell or composition according to the present invention, for use in a method of medical treatment or prophylaxis.
  • the present invention also provides an antigen-binding molecule, nucleic acid, expression vector, cell or composition according to the present invention, for use in a method of treatment or prevention of a T cell dysfunctional disorder, a cancer or an infectious disease.
  • the present invention also provides the use of an antigen-binding molecule, nucleic acid, expression vector, cell or composition according to the present invention, in the manufacture of a medicament for use in a method of treatment or prevention of a T cell dysfunctional disorder, a cancer or an infectious disease.
  • the present invention also provides 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, nucleic acid, expression vector, cell or composition according to the present invention.
  • 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.
  • HNSCC head and neck squamous cell carcinoma
  • 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.
  • the immune checkpoint protein is PD-1 , CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA.
  • the present invention also provides a method for generating or expanding a population of immune cells, comprising contacting immune cells in vitro, in vivo or ex vivo with an antigen-binding molecule, nucleic acid, expression vector, cell or composition according to the present invention.
  • the present invention also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present invention.
  • CAR chimeric antigen receptor
  • the present invention also provides an in vitro complex, optionally isolated, comprising an antigenbinding molecule or CAR according to the present invention bound to CD122 and/or CD132.
  • the present invention also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present invention.
  • CAR chimeric antigen receptor
  • the present invention also provides a nucleic acid, optionally isolated, encoding a CAR according to the present invention.
  • the present invention also provides an expression vector comprising a nucleic acid according to the present invention.
  • the present invention also provides a cell comprising a CAR, a nucleic acid, or an expression vector according to the present invention.
  • the present invention also provides a composition comprising a CAR, a nucleic acid, an expression vector or a cell according to the present invention.
  • the present invention also provides a CAR, a nucleic acid, an expression vector, a cell or a composition according to the present invention for use in a method of medical treatment or prophylaxis.
  • Treatment with IL-2 is an approved immunotherapy for the treatment of cancer, and works by promoting proliferation and activity of effector immune cells such as T cells and NK cells (see e.g. Skorombolas and Frelinger, Expert Rev Clin Immunol. 2014; 10(2): 207-217).
  • IL-2 has a very short half-life in serum, and so large doses and regular administration is required to achieve stimulation of T cell and NK cell proliferation/activity. This is problematic because high doses of IL-2 cause increases in levels of proinflammatory cytokines sometimes referred to as“cytokine storm”, which is thought to be a result of the widespread stimulation of immune cells. The cytokine storm is in turn thought to be responsible for many of the unwanted side effects of IL-2 treatment, including vascular leak syndrome (VLS).
  • VLS vascular leak syndrome
  • IL-2 is able to act on Tregs (which express the high-affinity IL-2Ra/B/yc receptors), and so treatment with IL-2 induces expansion of this suppressor T cell subset which can downregulate effector immune cell activity.
  • the inventors have designed and produced agonist antibodies which selectively bind to and activate intermediate-affinity IL-2RB/yc receptors.
  • the antibodies are demonstrated to mimic the effect of IL-2 on cells expressing CD122 and CD132, causing expansion of effector immune cells.
  • the bispecific antibodies of the present invention preferentially stimulate proliferation of effector immune cells (which express intermediate-affinity IL-2RB/yc receptors) over regulatory T cells (which express high levels of the high-affinity IL-2Ra/B/yc receptors). Moreover, they have an increased serum half-life as compared to IL-2, and can therefore be administered less frequently and/or at a lower dose.
  • the inventors have furthermore identified particular formats for the bispecific IL-2RB- and yc-binding antibodies associated with favourable biological activity, thermostability and freeze-thaw stability.
  • IL-2RB CD122
  • Common gamma chain vc; CD132
  • Human IL-2RB (also known as CD122, IL15RB and P70-75) is the protein identified by UniProt P14784-1 , v1 (SEQ ID NO:434).
  • the N-terminal 26 amino acids of SEQ ID NO:434 constitute a signal peptide, and so the mature form (i.e. after processing to remove the signal peptide) of human CD122 protein has the amino acid sequence shown in SEQ ID NO:435.
  • Amino acids 27 to 240 of SEQ ID NO:434 constitute the extracellular domain of CD122, shown in SEQ ID NO:436.
  • IL-2RB or“CD122” refers to CD122 from any species and includes isoforms, fragments, variants or homologues of CD122 from any species.
  • a“fragment”,“variant” or“homologue” of a protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein.
  • Fragments, variants, isoforms and homologues of a reference protein may be may be characterised by ability to perform a function performed by the reference protein.
  • A“fragment” generally refers to a fraction of the reference protein.
  • A“variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein.
  • An“isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein.
  • A“homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein.
  • human CD122 P14784-1 , v1 ; SEQ ID NO:434
  • cynomolgus macaque CD122 UniProt: Q38J85-1 , v1 ) are homologues of one another.
  • A“fragment” of a reference protein may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.
  • a fragment of CD122 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350,
  • the CD122 is mammalian CD122 (e.g. cynomolgous, human and/or rodent (e.g. rat and/or murine) CD122).
  • Isoforms, fragments, variants or homologues of CD122 may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of immature or mature CD122 from a given species, e.g. human CD122.
  • Isoforms, fragments, variants or homologues of CD122 may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference CD122 (e.g. full-length human CD122), as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of CD122 may display one or more of: association with one or more of CD132, IL-2Ra (CD25) or IL-15Ra (CD215), or binding to IL-2 or IL-15.
  • the CD122 has at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs:434 to 436.
  • Human common gamma chain (yc; also known as CD132, IL-2RG and CIDX) is the protein identified by UniProt P31785-1 , v1 (SEQ ID NO:437).
  • the N-terminal 23 amino acids of SEQ ID NO:437 constitute a signal peptide, and so the mature form (i.e. after processing to remove the signal peptide) of human CD132 protein has the amino acid sequence shown in SEQ ID NO:438.
  • Amino acids 23 to 262 of SEQ ID NO:437 constitute the extracellular domain of CD132, shown in SEQ ID NO:439.
  • CD132 refers to CD132 from any species and includes isoforms, fragments, variants or homologues of CD132 from any species.
  • the CD132 is mammalian CD132 (e.g. cynomolgous, human and/or rodent (e.g. rat and/or murine) CD132).
  • Isoforms, fragments, variants or homologues of CD132 may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of immature or mature CD132 from a given species, e.g. human CD132.
  • Isoforms, fragments, variants or homologues of CD132 may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference CD132 (e.g. full-length human CD132), as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of CD132 may display one or more of: association with one or more of CD122, IL-2Ra, L- 15Ra, IL-4R (CD124), IL-9R (CD129), IL-21 R (CD360) or IL7R (CD127), or binding to one or more of IL- 2, IL-15, IL-4, IL-9, IL-21 or IL-7.
  • a fragment of CD132 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 amino acids.
  • the CD132 has at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs:438 to 440.
  • CD122 and CD132 participate in the formation of receptors for IL-2.
  • CD122 and CD132 associate with IL- 2Ra (CD25) to form the trimeric, high-affinity IL-2 receptor (sometimes designated“IL-2Ra/ /yc” or “CD25/CD 122/CD 132”), which binds to IL-2 with a Kd of ⁇ 10 pM.
  • CD 122 and CD 132 are also capable of associating to form a functional intermediate-affinity IL-2 receptor (sometimes designated“IL-2R /yc” or “CD122/CD132”), which binds to IL-2 with a Kd of ⁇ 1 nM.
  • composition of the receptors, the number, and likely signalling capacity can vary with the cell type and activation stage.
  • IL-2 receptors are expressed at relatively low levels on resting naive T cells.
  • CD25 is expressed at higher amounts (8-10 fold) compared to CD122 and CD132.
  • CD25 is thought to bind IL-2 initially, effectively increasing its concentration at the cell surface and inducing a conformational change in IL-2 which then subsequently binds to the CD122 and CD132 (Liao et al., Immunity (2013) 38(1 ): 13-25).
  • NK cells and memory phenotype CD8 cells express high levels of CD122 and CD132 compared to naive cells and some NK cells can also express CD25 after stimulation with IL-2.
  • Tregs CD4 regulatory T cells constitutively express high levels of CD25.
  • Tregs act in multiple ways to down regulate many immune responses, including anti-tumor responses (see e.g.
  • the present invention provides antigen-binding molecules.
  • the antigen-binding molecules are capable of binding to CD122 and CD132.
  • the antigen-binding molecules are capable of binding to CD122 and CD132, and comprise an antigen-binding moiety capable of binding to CD122 and an antigen-binding moiety capable of binding to CD132.
  • an“antigen-binding molecule” as used herein refers to a polypeptide or polypeptide complex which is capable of binding to a target antigen or antigens, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they display binding to the relevant target antigen(s).
  • the antigen-binding molecule of the present invention comprises a moiety or moieties capable of binding to the target antigen(s).
  • the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen.
  • the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3): 181-202).
  • the moiety capable of binding to a target antigen comprises or consists of a antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a single-domain antibody (sdAb)) affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem.
  • a antigen-binding peptide/polypeptide e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a single-domain
  • the antigen-binding molecules of the present invention generally comprise antigen-binding moieties comprising a VH and a VL of an antibody capable of specific binding to the target antigen.
  • the antigenbinding moiety formed by a VH and a VL may also be referred to herein as an Fv region.
  • An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex.
  • An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain.
  • the polypeptides may associate covalently or non- covalently.
  • the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).
  • An antigen-binding molecule may comprise or consist of one or more polypeptides.
  • an antigen-binding molecule comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 polypeptides.
  • an antigen-binding molecule is a covalent or non-covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, 8, 10 or more polypeptides).
  • an antigenbinding molecule comprises two heavy chain polypeptides and two light chain polypeptides.
  • the antigen-binding molecules described herein preferably display specific binding to the relevant target (e.g. CD122 and/or CD132).
  • relevant target e.g. CD122 and/or CD132.
  • specific binding refers to binding which is selective for the antigen, and which can be discriminated from non-specific binding to non-target antigen.
  • An antigenbinding molecule that specifically binds to a target molecule preferably binds the target with greater affinity, and/or with greater duration than it binds to other, non-target molecules.
  • An antigen-binding molecule described herein may be capable of binding to CD122 as described herein.
  • An antigen-binding molecule described herein may be capable of binding to CD132 as described herein.
  • An antigen-binding molecule described herein may be capable of binding to CD122 as described herein and CD 132 as described herein.
  • the ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:41 1-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015)
  • the extent of binding of the antigen-binding molecule to an non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA.
  • binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with a dissociation constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1 x 10 n , where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule.
  • KD dissociation constant
  • This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, or 2.0.
  • the antigen-binding molecule binds to the target molecule with a KD of ⁇ 10 mM
  • ⁇ 1 mM ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM as determined by analysis according to SPR, Bio-Layer Interferometry or by RIA.
  • the antigen-binding molecule binds to the same or an overlapping epitope of the target molecule as a reference antigen-binding molecule which is capable of binding to the target molecule (i.e. CD122 or CD132).
  • the antigen-binding molecule displays competitive binding with a reference antigen-binding molecule which is capable of binding to the target molecule. Whether a given antigen-binding molecule displays such competitive binding can be determined by various methods known to the skilled person, including competition ELISA.
  • the antigen-binding molecule comprises the complementarity-determining regions (CDRs) of an antigen-binding molecule which is capable of binding to the target molecule (i.e. CD122 or CD132).
  • Antibodies generally comprise six CDRs; three in the light chain variable region (VL): LC-CDR1 , LC-CDR2, LC-CDR3, and three in the heavy chain variable region (VH): HC-CDR1 , HC-CDR2 and HC- CDR3.
  • the six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target molecule.
  • There are several different conventions for defining antibody CDRs such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 ), Chothia et al., J. Mol. Biol.
  • CDRs of the antigen-binding molecules described herein are defined according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 ).
  • the antigen-binding molecule may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to CD122, and mAbs capable binding to CD132 described herein.
  • mAbs monoclonal antibodies
  • Antigen-binding regions of antibodies such as single chain variable fragment (scFv), Fab and Fab ⁇ fragments may also be used/provided.
  • An‘antigen-binding region’ is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.
  • the antigen-binding molecule of the present invention is a CD122-binding molecule.
  • the antigen-binding molecule comprises or consists of a CD122-binding molecule.
  • the antigen-binding molecule comprises a heavy chain variable (VH) region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 of a CD122-binding antibody clone described herein, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC- CDR2, HC-CDR3 are substituted with another amino acid.
  • VH heavy chain variable
  • the antigen-binding molecule comprises a light chain variable (VL) region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 of a CD122-binding antibody clone described herein, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC-CDR2, LC-CDR3 are substituted with another amino acid.
  • VL light chain variable
  • the antigen-binding molecule comprises a VH region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 and a VL region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 of a CD 122-binding antibody clone described herein, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, HC-CDR3, LC-CDR1 , LC-CDR2 or LC-CDR3 are substituted with another amino acid.
  • the antigen-binding molecule comprises a VH region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VH region of a CD122-binding antibody clone described herein.
  • the antigenbinding molecule comprises a VL region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VL region of a CD122-binding antibody clone described herein.
  • the antigen-binding molecule comprises a VH region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VH region of a CD122-binding antibody clone described herein and a VL region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,
  • a CD122-binding antibody clone is selected from: P2C4, P2C4_A4, P2C4_B1 , P2C4_B5, P2C4_C1 , P2C4_C4, P2C4_C7, P2C4_D10, P2C4_E6, P2C4_E7, P2C4_F8, P2C4_C1 D10, P2C4_FW2, P2H7, P2D12, P1 G1 1 , P2C4_A9, P2C4_B6, P2C4_E9, P2C4_B8, P2C4_B12, P2C4_C12, P2C4_E2, P2C4_E3, P2C4_E8, P2C4_F1 1 , P2C4_G2, P2C4J31 1 , P2C4_H1 , P2C4_H2, P2C4JH3, P1 E7
  • the CD122- binding antibody clone is P2C4, P2C4_FW2, P2E6, P1 D10, P1 E7 or P1 G1 1 . In some embodiments the CD122-binding antibody clone is P2C4 or P2C4_FW2.
  • the antigen-binding molecule of the present invention is a CD132-binding molecule.
  • the antigen-binding molecule comprises or consists of a CD132-binding molecule.
  • the antigen-binding molecule comprises a heavy chain variable (VH) region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 of a CD132-binding antibody clone described herein, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC- CDR2, HC-CDR3 are substituted with another amino acid.
  • VH heavy chain variable
  • the antigen-binding molecule comprises a light chain variable (VL) region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 of a CD132-binding antibody clone described herein, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC-CDR2, LC-CDR3 are substituted with another amino acid.
  • VL light chain variable
  • the antigen-binding molecule comprises a VH region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 and a VL region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 of a CD 132-binding antibody clone described herein, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, HC-CDR3, LC-CDR1 , LC-CDR2 or LC-CDR3 are substituted with another amino acid.
  • the antigen-binding molecule comprises a VH region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VH region of a CD132-binding antibody clone described herein.
  • the antigenbinding molecule comprises a VL region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VL region of a CD132-binding antibody clone described herein.
  • the antigen-binding molecule comprises a VH region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VH region of a CD132-binding antibody clone described herein and a VL region which comprises or consists of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to VL region of a CD132-binding antibody clone described herein.
  • a CD132-binding antibody clone is selected from: P1A3, P1A3_B3, P1A3_E8, P1A3_E9, P2B9, P1A3_B4, P1A3_FW2, P1A10, P1 B6, P1 C10, P1 D7, P1 E8, P2B2, P2B7, P2D1 1 , P2F10, P2H4, P2D3, P1 G4, P1 B12 and P1 C7.
  • a CD132-binding antibody clone is selected from: P1A10, P1 B6, P1 C10, P1 D7, P1 E8, P2B2, P2B7, P2D1 1 , P2F10, P2H4, P2D3, P1 G4, P1 B12 and P1 C7.
  • the CD132-binding antibody clone is P1A10.
  • the CD132-binding antibody clone is P1A3 or P1A3_FW2.
  • substitutions are conservative substitutions, for example according to the following Table.
  • amino acids in the same block in the middle column are substituted.
  • amino acids in the same line in the rightmost column are substituted:
  • the antigen-binding molecule of the present invention does not comprise a combination of CDRs or VL/VH domains disclosed in WO 2017/021540 A1 (hereby incorporated by reference in its entirety.
  • the CD122-binding antigen-binding molecule according to the invention comprises or consist of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOs:265 to 308.
  • the CD122-binding antigenbinding molecule according to the invention comprises or consist of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOs:296 to 308.
  • the CD132-binding antigen-binding molecule according to the invention comprises or consist of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOs:309 to 329.
  • the CD132-binding antigenbinding molecule according to the invention comprises or consist of an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOs:316 to 329.
  • the CD122-binding antigen-binding molecule lacks HC-CDR1 , HC-CDR2, HC-CDR3, LC-CDR1 , LC-CDR2, and LC-CDR3 of one or more of the following clones: P2C4, P2C4_A4, P2C4_B1 , P2C4_B5, P2C4_C1 , P2C4_C4, P2C4_C7, P2C4_D10, P2C4_E6, P2C4_E7, P2C4_F8, P2C4_C1 D10, P2C4_FW2, P2H7, P2D12, P1 G1 1 , P2C4_A9, P2C4_B6, P2C4_E9, P2C4_B8, P2C4_B12, P2C4_C12, P2C4_E2, P2C4_E3, P2C
  • the CD122-binding antigenbinding molecule according to the present invention lacks the VL domain sequence and/or the VH domain sequence of one or more of said clones. In some embodiments the CD122-binding antigen-binding molecule according to the present invention lacks the VL domain sequence and/or the VH domain sequence of one or more of said clones.
  • the CD132-binding antigen-binding molecule according to the present invention lacks HC-CDR1 , HC-CDR2, HC-CDR3, LC-CDR1 , LC-CDR2, and LC-CDR3 of one or more of the following clones: P1A3, P1A3_B3, P1A3_E8, P1A3_E9, P1A3_B4, P1A3_FW2 and P2B9.
  • the CD132-binding antigen-binding molecule according to the present invention lacks the VL domain sequence and/or the VH domain sequence of one or more of said clones.
  • Antigen-binding molecules may be produced by a process of affinity maturation in which a modified antibody is generated that has an improvement in the affinity of the antibody for antigen, compared to an unmodified parent antibody.
  • Affinity-matured antigen-binding molecules may be produced by procedures known in the art, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994- 2004 (1995); Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al, J. Mol. Biol.
  • the antigen-binding molecule according to the present invention comprises, or consists of, an Fv region which binds to CD122. In some embodiments, the antigen-binding molecule comprises, or consists of, an Fv region which binds to CD132.
  • the antigen-binding molecule of the antigen-binding molecule described herein comprises, or consists of, a Fab region which binds to CD122. In some embodiments, the antigen-binding molecule comprises, or consists of, a Fab region which binds to CD132.
  • the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to CD122. In some embodiments, the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to a CD132.
  • “whole antibody” refers to an antibody having a structure which is substantially similar to the structure of an
  • immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.
  • Immunoglobulins of type G are -150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chain comprise a VL followed by a CL.
  • immunoglobulins may be classed as IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM.
  • the light chain may be kappa (K) or lambda (l).
  • the immunoglobulin heavy chain constant sequence is human immunoglobulin G 1 constant (IGHG1 ; UniProt: P01857-1 , v1 ; SEQ ID NO:440). Positions 1 to 98 of SEQ ID NO:440 form the CH1 region (SEQ ID NO:441 ). Positions 99 to 110 of SEQ ID NO:440 form a hinge region between CH1 and CH2 regions (SEQ ID NO:442). Positions 111 to 223 of SEQ ID NO:440 form the CH2 region (SEQ ID NO:443). Positions 224 to 330 of SEQ ID NO:440 form the CH3 region (SEQ ID NO:444).
  • a CH1 region comprises or consists of the sequence of SEQ ID NO:441 , or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%,
  • a CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID NO:442, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:442.
  • a CH2 region comprises or consists of the sequence of SEQ ID NO:443, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:443.
  • a CH3 region comprises or consists of the sequence of SEQ ID NO:444, or a sequence having at least 60%, preferably one of 70%, 75%, 80%,
  • CH3 regions may be provided with further substitutions in accordance with modification to an Fc region of the antigen-binding molecule as described herein.
  • a CH3 region comprises or consists of the sequence of SEQ ID NO:447, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:447.
  • a CH3 region comprises or consists of the sequence of SEQ ID NO:448, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:448.
  • the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin light chain constant sequence.
  • the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK; UniProt: P01834-1 , v2; SEQ ID NO:445).
  • the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.
  • a CL region comprises or consists of the sequence of SEQ ID NO:445, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%,
  • the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to CD122.
  • the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to CD132.
  • the antigen-binding molecules according to the present invention may be provided in any suitable format.
  • the antigen-binding molecule of the present invention comprises an antigenbinding moiety capable of binding to CD122 and an antigen-binding moiety capable of binding to CD132 may be provided in the same polypeptide chain.
  • the antigen-binding moiety capable of binding to CD122 and antigen-binding moiety capable of binding to CD132 are provided in tandem. That is, in some embodiments the antigenbinding moiety capable of binding to CD122 and antigen-binding moiety capable of binding to CD132 are adjacent to one another in the amino acid sequence of a polypeptide.
  • the antigen-binding moieties may be provided in a particular order relative to the N- and C-termini of the amino acid sequence of the polypeptide.
  • the moiety capable of binding to CD122 is N-terminal to the moiety capable of binding to CD132.
  • the moiety capable of binding to CD132 is N-terminal to the moiety capable of binding to CD122.
  • the polypeptide further comprises a linker sequence (e.g. a linker sequence as described herein) between the CD122-binding moiety and the CD132-binding moiety.
  • a linker sequence e.g. a linker sequence as described herein
  • multispecific antigen-binding molecules By“multispecific” it is meant that the antigen-binding molecule displays specific binding to more than one target. In particular, the antigen-binding molecule is binding to CD122 and CD132, and so is at least bispecific.
  • bispecific means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.
  • Multispecific antigen-binding molecules described herein display at least monovalent binding with respect to CD122, and also displays at least monovalent binding with respect to CD132.
  • Binding valency refers to the number of binding sites in an antigen-binding molecule for a given antigenic determinant.
  • bispecific antigen-binding molecules in scFv-KiHs-s-Fc, CrossMab and Duobody formats are provided herein, which are monovalent with respect to binding to CD122, and monovalent with respect to binding to CD132.
  • the antigen-binding molecule comprises one binding site for CD122, and one binding site for CD132. In some embodiments the antigen-binding molecule comprises more than one binding site (e.g. two, three) for CD122. In some embodiments the antigen-binding molecule comprises more than one binding site (e.g. two, three) for CD132. In some embodiments the antigen-binding molecule comprises more than one binding site (e.g. two, three) for CD122, and more than one binding site (e.g. two, three) for CD132.
  • the antigen-binding molecule is multivalent (e.g. bivalent, trivalent) for CD122. In some embodiments the antigen-binding molecule is multivalent (e.g. bivalent, trivalent) for CD132. In some embodiments the antigen-binding molecule is multivalent (e.g. bivalent, trivalent) for CD122, and multivalent (e.g. bivalent, trivalent) for CD132.
  • the antigen-binding molecule comprises two binding sites for CD122. In some embodiments the antigen-binding molecule comprises two binding sites for CD132. In some
  • the antigen-binding molecule comprises two binding sites for CD122, and two binding sites for CD 132.
  • Multispecific antigen-binding molecules may be provided in any suitable format, such as those formats described in Kontermann MAbs 2012, 4(2): 182-197, which is hereby incorporated by reference in its entirety.
  • an antigen-binding molecule may be a bispecific antibody conjugate (e.g. an lgG2, F(ab’)2 or CovX-Body), a bispecific IgG or IgG-like molecule (e.g.
  • bispecific antigen-binding molecules The skilled person is able to design and prepare bispecific antigen-binding molecules.
  • Methods for producing bispecific antigen-binding molecules include chemically crosslinking of antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules.
  • SPDP A/-succinimidyl-3-(-2-pyridyldithio)-propionate
  • SPDP can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.
  • bispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1— 2.13.16.
  • Bispecific antigen-binding molecules according to the present invention can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigenbinding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules:
  • a DNA construct encoding the light and heavy chain variable domains for the two antigenbinding fragments i.e. the light and heavy chain variable domains for the antigen-binding fragment capable of binding CD122 or CD132, and the light and heavy chain variable domains for the antigenbinding fragment capable of binding to another target protein
  • sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques.
  • Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific antibody can then optionally be purified.
  • the antigen-binding molecule comprises an Fv fragment, scFv or Fab fragment specific for CD122 and an Fv, scFv or Fab fragment specific for CD132.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • polypeptide comprising a VH, a CH2 domain and a CH3 domain
  • polypeptide comprising a VL and a CL domain
  • a CD132-binding region comprising:
  • polypeptide comprising a VH, a CH2 domain and a CH3 domain
  • polypeptide comprising a VL and a CL domain.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • polypeptide comprising a VH, a CH1 domain, a CH2 domain and a CH3 domain a polypeptide comprising a VL and CL domain;
  • a CD132-binding region comprising:
  • polypeptide comprising a VH, a CH1 domain, a CH2 domain and a CH3 domain a polypeptide comprising a VL and CL domain.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • a polypeptide comprising a VL, a VH, a CH2 domain and a CH3 domain; and a CD132-binding region comprising:
  • polypeptide comprising a VL, VH, a CH2 domain and a CH3 domain.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • a polypeptide comprising a VH, a VL, a CH2 domain and a CH3 domain; and a CD132-binding region comprising:
  • polypeptide comprising a VH, VL, a CH2 domain and a CH3 domain.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • polypeptide comprising a VL, a VH, a CH1 domain, a CH2 domain and a CH3 domain;
  • a CD132-binding region comprising:
  • polypeptide comprising a VL, VH, a CH1 domain, a CH2 domain and a CH3 domain.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • polypeptide comprising a VH, a VL, a CH1 domain, a CH2 domain and a CH3 domain;
  • a CD132-binding region comprising:
  • polypeptide comprising a VH, VL, a CH1 domain, a CH2 domain and a CH3 domain.
  • Fab fragments formed by association of polypeptides comprising such structure are referred to as“cross-Fab” or“crossover Fab” fragments.
  • the antigen-binding molecule comprises or consists of, a cross-Fab region which binds to CD122.
  • the antigen-binding molecule comprises, or consists of, a cross-Fab region which binds to CD132.
  • the antigen-binding molecule comprises a cross-Fab fragment specific for CD122 and/or a cross-Fab fragment specific for CD132.
  • the antigen-binding molecule according to the present invention comprises: a CD122-binding region comprising:
  • polypeptide comprising a VH, a CL domain, a CH2 domain and a CH3 domain a polypeptide comprising a VL and a CH1 domain;
  • a CD132-binding region comprising:
  • polypeptide comprising a VH, a CL domain, a CH2 domain and a CH3 domain a polypeptide comprising a VL and a CH1 domain.
  • aspects of the present invention relate to antigen-binding molecules comprising an Fc region.
  • Fc regions are composed of CH2 and CH3 regions from one polypeptide, and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.
  • Fc regions In IgM and IgE isotypes the Fc regions contain three constant domains (CH2, CH3 and CH4), and CH2 to CH4 from the two polypeptides together form the Fc region.
  • Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects.
  • IgG Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about through Fc- mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, NK cells and T cells) through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade.
  • immune cells e.g. macrophages, dendritic cells, NK cells and T cells
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:444. In some embodiments, the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:446.
  • the antigen-binding molecule comprises more than one polypeptide (e.g. 2 polypeptides), each comprising a an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:444.
  • the antigenbinding molecule comprises more than one polypeptide (e.g.
  • polypeptides each comprising a an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:446.
  • the antigen-binding molecule of the present invention comprises an Fc region comprising modification to reduce antibody effector function.
  • Fc-mediated effector functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • MAC membrane attack complex
  • cell degranulation cytokine and/or chemokine production
  • antigen processing and presentation Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1 ):63-73, which is hereby incorporated by reference in its entirety.
  • substitutions“L234A/L235A” and corresponding substitutions (such as e.g.
  • F234A/L235A in human lgG4 are known to disrupt binding of Fc to Fey receptors and inhibit ADCC, ADCP, and also to reduce C1q binding and thus CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, hereby incorporated by reference in entirety).
  • the antigen-binding molecule of the present invention comprises an Fc region comprising modification corresponding to the combination of substitutions L234A/L235A.
  • the antigen-binding molecule of the present invention comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region.
  • Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations.
  • the antigen antigen-binding molecule of the present invention comprises an Fc region comprising paired substitutions in the CH3 regions of the Fc region according to one of the following formats, as shown in Table 1 of Ha et al., Front. Immnol (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1 , 7.8.60, DD-KK, EW-RVT, EW-RVT s -s, SEED or A107.
  • the bispecific antigen-binding molecule of the present invention is provided with an Fc region comprising the“knob-into-hole” or“KiH” modification, e.g. as described e.g. in US 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001 ).
  • one of the CH3 regions of the Fc region comprises a“knob” modification
  • the other CH3 region comprises a“hole” modification.
  • the “knob” and“hole” modifications are positioned within the respective CH3 regions so that the“knob” can be positioned in the“hole” in order to promote heterodimerisation (and inhibit homodimerisation) of the polypeptides and/or stabilise heterodimers.
  • Knobs are constructed by substituting amino acids having small chains with those having larger side chains (e.g. tyrosine or tryptophan). Holes are created by substituting amino acids having large side chains with those having smaller side chains (e.g. alanine or threonine).
  • one of the CH3 regions of the Fc region of the antigen-binding molecule of the present invention comprises the substitution (numbering of positions/substitutions in the Fc region herein is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 ) T366W, and the other CH3 region of the Fc region comprises the substitution Y407V.
  • one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W
  • the other CH3 region of the Fc region comprises the substitutions T366S and L368A.
  • one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S and L368A.
  • one of the CH3 regions comprises the substitution S354C
  • the other CH3 region of the Fc region comprises the substitution Y349C.
  • Introduction of these cysteine residues results in formation of a disulfide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001 ), J Immunol Methods 248, 7-15).
  • the Fc region comprises the“KiHs-s” modification.
  • one of the CH3 regions comprises the substitutions T366W and S354C, and the other CH3 region of the Fc region comprises the substitutions T366S, L368A, Y407V and Y349C.
  • one of the CH3 regions comprises the substitutions K392D and K409D
  • the other CH3 region of the Fc region comprises the substitutions E356K and D399K.“DDKK” knob-into-hole technology is described e.g. in WO 2014/131694 A1 , and promotes assembly of the heavy chains providing the complementary amino acid residues.
  • the antigen-binding molecule of the present invention comprises an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci U S A. (2013) 1 10(13):5145-50, referred to as ‘Duobody’ format.
  • one of the CH3 regions comprises the substitution K409R
  • the other CH3 region of the Fc region comprises the substitution K405L.
  • the antigen-binding molecule of the present invention comprises an Fc region modified as described in Strop et al., J Mol Biol. (2012) 420(3):204-19, so-called ⁇ EE-RRR’ format.
  • one of the CH3 regions comprises the substitutions D221 E, P228E and L368E
  • the other CH3 region of the Fc region comprises the substitutions D221 R, P228R and K409R.
  • the antigen-binding molecule comprises an Fc region comprising the“EW-RVT” modification described in Choi et al., Mol Cancer Ther (2013) 12(12):2748-59.
  • one of the CH3 regions comprises the substitutions K360E and K409W
  • the other CH3 region of the Fc region comprises the substitutions Q347R, D399V and F405T.
  • the antigen-binding molecule of the present invention comprises an Fc region comprising the“SEED” modification as described in Davis et al., Protein Eng Des Sel (2010) 23(4): 195- 202, in which b-strand segments of human lgG1 CH3 and IgA CH3 are exchanged.
  • one of the CH3 regions comprises the substitutions S364H and F405A
  • the other CH3 region of the Fc region comprises the substitutions Y349T and T394F (see e.g. Moore et al., MAbs (201 1 ) 3(6):546-57).
  • one of the CH3 regions comprises the substitutions T350V, L351 Y, F405A and Y407V
  • the other CH3 region of the Fc region comprises the substitutions T350V, T366L, K392L and T394W (see e.g. Von Kreudenstein et al., MAbs (2013) 5(5):646-54).
  • one of the CH3 regions comprises the substitutions K360D, D399M and Y407A
  • the other CH3 region of the Fc region comprises the substitutions E345R, Q347R, T366V and K409V (see e.g. Leaver-Fay et al., Structure (2016) 24(4):641-51 ).
  • one of the CH3 regions comprises the substitutions K370E and K409W
  • the other CH3 region of the Fc region comprises the substitutions E357N, D399V and F405T (see e.g. Choi et al., PLoS One (2015) 10(12):e0145349).
  • the antigen-binding molecule comprises a KiH Fc region. In particular embodiments, the antigen-binding molecule comprises a KiHs-s Fc region.
  • the antigen-binding molecule comprises a polypeptide comprising a CH3 region comprising W at the position corresponding to position 366. In some embodiments the antigen-binding molecule comprises a polypeptide comprising a CH3 region comprising W at the position corresponding to position 366 and C at the position corresponding to position 354.
  • the antigen-binding molecule comprises a polypeptide comprising a CH3 region comprising S at the position corresponding to position 366, and A at the position corresponding to position 368. In some embodiments the antigen-binding molecule comprises a polypeptide comprising a CH3 region comprising S at the position corresponding to position 366, A at the position corresponding to position 368, and Y at the position corresponding to position 407. In some embodiments the antigenbinding molecule comprises a polypeptide comprising a CH3 region comprising S at the position corresponding to position 366, A at the position corresponding to position 368, Y at the position corresponding to position 407, and C at the position corresponding to position 349.
  • the antigen-binding molecule comprises: (a) a polypeptide comprising a CH3 region comprising W at the position corresponding to position 366, and C at the position corresponding to position 354; and (b) a polypeptide comprising a CH3 region comprising S at the position corresponding to position 366, A at the position corresponding to position 368, Y at the position corresponding to position 407, and C at the position corresponding to position 349.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:259.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:451.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:448.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:447, and a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:448.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:450.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:449, and a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:450.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:453.
  • the antigen-binding molecule comprises a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:452, and a polypeptide comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:453.
  • the present invention also provides polypeptide constituents of the antigen-binding molecules described herein.
  • the polypeptides may be provided in isolated or substantially purified form.
  • the antigen-binding molecule of the present invention may be, or may comprise, a complex of polypeptides.
  • a polypeptide comprises more than one domain or region
  • the plural domains/regions are preferably present in the same polypeptide chain. That is, the polypeptide comprises more than one domain or region is a fusion polypeptide comprising the domains/regions.
  • a polypeptide according to the present invention comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present invention comprises, or consists of, a VL as described herein.
  • the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).
  • CH antibody heavy chain constant regions
  • CL antibody light chain constant regions
  • the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).
  • the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments the polypeptide comprises a CH 1 region as described herein. In some embodiments the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments the polypeptide comprises a CH2 region as described herein. In some embodiments the polypeptide comprises a CH3 region as described herein.
  • the polypeptide comprises a CH3 region comprising any one of the following amino acid substitutions/combinations of amino acid substitutions (shown e.g. in Table 1 of Ha et al., Front. Immnol (2016) 7:394, incorporated by reference hereinabove): T366W; T366S, L368A and Y407V; T366W and S354C; T366S, L368A, Y407V and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A and Y407V; T350V, T366L, K392L and T394W; K360D, D399M and Y407A; E345R, Q347R, T366V and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347
  • the CH2 and/or CH3 regions of the polypeptide comprise one or more amino acid substitutions for promoting association of the polypeptide with another polypeptide comprising a CH2 and/or CH3 region.
  • polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments the polypeptide comprises a CL region as described herein. In some embodiments, the polypeptide according to the present invention comprises a structure from N- to C-terminus according to one of the following:
  • VH(anti-CD122) refers to the VH of an antigen-binding molecule capable of binding to CD122 as described herein
  • VL(anti-CD122) refers to the VL of an antigen-binding molecule capable of binding to CD122 as described herein
  • VH(anti-CD132) refers to the VH of an antigen-binding molecule capable of binding to CD132 as described herein
  • VL(anti-CD132) refers to the VL of an antigen-binding molecule capable of binding to CD132 as described herein.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:455.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:456.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:457.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%,
  • polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:460.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:461.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:467.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:462.
  • the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:468.
  • antigen-binding molecules comprising a polypeptide according to the present invention.
  • the antigen-binding molecules and polypeptides of the present invention comprise one or more linker sequences between amino acid sequences.
  • a linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule/polypeptide.
  • Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety.
  • a linker sequence may be a flexible linker sequence.
  • Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence.
  • Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
  • the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-30, 1-40 or 1-50 amino acids. In some embodiments a linker sequence comprises, or consists of, one or more copies (e.g. 2, 3 or 4 copies) of the amino acid sequence of SEQ ID NO:330, 331 , 332, 333, 463 or 464.
  • the antigen-binding molecules and polypeptides of the present invention may additionally comprise further amino acids or sequences of amino acids.
  • the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide.
  • the antigen-binding molecule/polypeptide may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus of the antigen-binding
  • the antigen-binding molecule/polypeptide comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.
  • the antigen-binding molecules and polypeptides of the present invention may additionally comprise a signal peptide (also known as a leader sequence or signal sequence).
  • Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.
  • the signal peptide may be present at the N-terminus of the antigen-binding molecule/polypeptide, and may be present in the newly synthesised antigen-binding molecule/polypeptide.
  • the signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule/polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding
  • Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 201 1 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).
  • SignalP Protein et al., 201 1 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176.
  • the antigen-binding molecule comprises one of the following formats:
  • the antigen-binding molecule is comprised of (a) a polypeptide comprising scFv specific for CD122 connected to CH2-CH3, wherein the CH3 domain comprises W at the position corresponding to position 366 and C at the position corresponding to position 354, and (b) a polypeptide comprising scFv specific for CD132 connected to CH2-CH3, wherein the CH3 domain comprises S at the position corresponding to position 366, A at the position corresponding to position 368, Y at the position corresponding to position 407, and C at the position corresponding to position 349.
  • the antigen-binding molecule is comprised of (a) a polypeptide comprising scFv specific for CD122 connected to CH2-CH3, wherein the CH3 domain comprises W at the position corresponding to position 366 and C at the position corresponding to position 354, (b) a polypeptide comprising VH specific for CD132 connected to CH1-CH2-CH3, wherein the CH3 domain comprises S at the position corresponding to position 366, A at the position corresponding to position 368, Y at the position corresponding to position 407, and C at the position corresponding to position 349, and (c) a polypeptide comprising VL specific for CD132 connected to CL.
  • the antigen-binding molecule is comprised of two polypeptides, each polypeptide comprising scFv specific for CD122 connected to scFv specific for CD132, connected to CH2-CH3.
  • the antigen-binding molecule comprises, or consists of: (i) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454 and
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:455.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:456.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:457;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:458.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:454;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:459;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:460.
  • the antigen-binding molecule comprises, or consists of two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:461.
  • the antigen-binding molecule comprises, or consists of two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:467.
  • the antigen-binding molecule comprises, or consists of two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:462.
  • the antigen-binding molecule comprises, or consists of two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:468.
  • the antigen-binding molecule described herein may be characterised by reference to certain functional properties.
  • the antigen-binding molecule described herein may possess one or more of the following properties:
  • PD-1 immune checkpoint proteins
  • thermostability and/or freeze-thaw stability e.g. as compared to a reference antigen-binding molecule, e.g. an antigen-binding molecule described in WO
  • the extent of binding of an antigen-binding molecule to an non-target is less than about 10% of the binding of the antibody to the target as measured, e.g., by ELISA, SPR, Bio-Layer Interferometry (BLI), MicroScale Thermophoresis (MST), or by a radioimmunoassay (RIA).
  • the binding specificity may be reflected in terms of binding affinity, where the antigen-binding molecule described herein binds to CD122 and/or CD132 with an affinity that is at least 0.1 order of magnitude greater than the affinity towards a non-target molecule.
  • the antigen-binding molecule described herein binds to CD122 and/or CD132 with an affinity that is one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 .5, or 2.0 orders of magnitude greater than the affinity towards another, non-target molecule.
  • Binding affinity of an antigen-binding molecule for its target is often described in terms of its dissociation constant (KD). Binding affinity can be measured by methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:41 1-442; or Rich et al., Anal Biochem.
  • MST MicroScale Thermophoresis
  • the antigen-binding molecule described herein binds to CD122 with a KD of 10 mM or less, preferably one of ⁇ 5 pM, ⁇ 2 pM, ⁇ 1 pM, ⁇ 500 nM, ⁇ 100 nM, ⁇ 75 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 15 nM, ⁇ 12.5 nM, ⁇ 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM, ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 500 pM.
  • the antigen-binding molecule described herein binds to CD132 with a KD of 10 pM or less, preferably one of ⁇ 5 pM, ⁇ 2 pM, ⁇ 1 pM, ⁇ 500 nM, ⁇ 100 nM, ⁇ 75 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 15 nM, ⁇ 12.5 nM, ⁇ 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM, ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 500 pM.
  • Affinity of binding to CD122 and/or CD132 may be analysed in vitro by ELISA assay.
  • Suitable assays are well known in the art and can be performed by the skilled person, for example, as described in Antibody Engineering, vol. 1 (2 nd Edn), Springer Protocols, Springer (2010), Part V, pp657-665.
  • the binding affinity of the antigen-binding molecule of the present invention to CD122 is greater than the binding affinity of an antigen-binding molecule described in WO 2017/021540 A1.
  • the antigen-binding molecule described herein binds to CD122 with a KD which is less than 1 times, e.g.
  • the binding affinity of the antigen-binding molecule of the present invention to CD132 is greater than the binding affinity of an antigen-binding molecule described in WO 2017/021540 A1.
  • the antigen-binding molecule described herein binds to CD132 with a KD which is less than 1 times, e.g. ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times or ⁇ 0.1 times the KD of binding to CD132 for an antigen-binding molecule described in WO 2017/021540 A1.
  • the antigen-binding molecule described herein preferably binds to CD122 in a region of CD122 which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when CD122 is expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments the antigen-binding molecule described herein is capable of binding to CD122 when CD122 is expressed at the cell surface.
  • the antigen-binding molecule described herein preferably binds to CD132 in a region of CD132 which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when CD132 is expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments the antigen-binding molecule described herein is capable of binding to CD132 when CD132 is expressed at the cell surface.
  • the antigen-binding molecule may bind to CD122 and/or CD 132-expressing cells, such as cells expressing CD122 and CD132 at the cell surface, e.g. immune cells, lymphocytes, T cells (CD4+ T cells, CD8+ T cells) or NK cells.
  • CD122 and/or CD 132-expressing cells such as cells expressing CD122 and CD132 at the cell surface, e.g. immune cells, lymphocytes, T cells (CD4+ T cells, CD8+ T cells) or NK cells.
  • the ability of an antigen-binding molecule to bind to a given cell type can be analysed by contacting cells with the antigen-binding molecule, and detecting antigen-binding molecule bound to the cells, e.g. after a washing step to remove unbound antigen-binding molecule.
  • the ability of an antigen-binding molecule to bind to CD 132-expressing cells and/or CD 122-expressing cells can be analysed by methods such as flow cytometry and immunofluorescence microscopy, e.g. as described in the experimental examples of the present application.
  • the antigen-binding molecules of the present invention may stimulate signalling through the intermediate- affinity IL-2 receptor (i.e. the polypeptide complex comprising CD122 and CD132, and not comprising CD25).
  • the antigen-binding molecules are preferably agonists of signalling through the intermediate- affinity IL-2 receptor.
  • the ability of a given antigen-binding molecule to stimulate signalling through the intermediate-affinity IL-2 receptor can be evaluated e.g. in an in vitro assay, e.g. as described in Example 4 herein. Briefly, cells expressing CD122 and CD132 at the cell surface can be contacted with the antigen-binding molecule, and activation of intracellular signalling can be determined by detecting an increase in the level of phosphorylated STAT5 (i.e. pSTAT5). pSTAT5 can be detected e.g. using antibody- or reporter-based methods as described herein.
  • the antigen-binding molecule of the present invention is capable of increasing the amount of pSTAT5 to more than 1 times, e.g. >1.01 times, >1.02 times, >1.03 times, >1.04 times, >1.05 times, >1.1 times, >1.2 times, >1.3 times, >1.4 times, >1.5 times, >1.6 times, >1.7 times, >1.8 times, >1.9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times, >10 times, >20 times, >30 times, >40 times, >50 times, >60 times, >70 times, >80 times, >90 times, or >100 times the level of pSTAT5 detected following culture in the absence of the antigen-binding molecule, or in the presence of an appropriate control antigen-binding molecule (e.g. isotype-matched control antigenbinding molecule), in a comparable assay.
  • an appropriate control antigen-binding molecule e.g. isotype-matched
  • the antigen-binding molecule of the present invention activates signalling through the intermediate-affinity IL-2 receptor to a greater extent than an antigen-binding molecule described in WO 2017/021540 A1.
  • culture of cells expressing CD122 and CD132 in the presence of an antigen-binding molecule according to the present invention increases the amount of pSTAT5 to more than 1 times, e.g.
  • the antigen-binding molecule of the present invention is capable of increasing proliferation of cells expressing CD122 and CD132.
  • Cell proliferation can be determined by analysing cell division over a period of time.
  • Cell division can be analysed, for example, by in vitro analysis of incorporation of 3 H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.
  • Proliferating cells may also be identified by analysis of incorporation of 5-ethynyl-2'- deoxyuridine (EdU) by an appropriate assay, as described e.g. in Buck et al., Biotechniques.
  • EdU 5-ethynyl-2'- deoxyuridine
  • the antigen-binding molecule of the present invention is capable of increasing the number or proportion of proliferating cells to more than 1 times, e.g. >1.01 times, >1.02 times, >1.03 times, >1.04 times, >1.05 times, >1 .1 times, >1 .2 times, >1.3 times, >1.4 times, >1.5 times, >1.6 times,
  • the antigen-binding molecule of the present invention increases the number or proportion of proliferating cells to a greater extent than an antigen-binding molecule described in WO 2017/021540 A1.
  • culture of cells expressing CD122 and CD132 in the presence of an antigen-binding molecule of the present invention increases the number/proportion of proliferating cells to more than 1 times, e.g. >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1.05 times, >1.1 times,
  • culture in the presence of an antigen-binding molecule of the present invention causes expansion of cells expressing CD122 and CD132 to a number of cells which is more than 1 times, e.g. >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1.05 times, >1.1 times, >1.2 times, >1.3 times,
  • the antigen-binding molecule of the present invention causes expansion of cells expressing CD122 and CD132 to a greater extent than an antigen-binding molecule described in WO 2017/021540 A1.
  • culture of cells expressing CD122 and CD132 in the presence of an antigen-binding molecule of the present invention causes expansion of the cells to a number of cells which is more than 1 times, e.g.
  • the antigen-binding molecule of the present invention preferentially stimulates proliferation/expansion of one or more of the following cell types over (i.e. in preference to) regulatory T cells (e.g. CD4+CD25+FoxP3+ T cells): antigen-specific T cells (e.g. virus-specific T cells), antigen-specific CD4 T cells, antigen-specific CD8 T cells, effector memory CD4 T cells, effector memory CD8 T cells, central memory CD4 T cells, central memory CD8 T cells, cytotoxic CD8+ T cells (i.e. CTLs) NK cells or antigen-specific NK cells.
  • regulatory T cells e.g. CD4+CD25+FoxP3+ T cells
  • antigen-specific T cells e.g. virus-specific T cells
  • antigen-specific CD4 T cells e.g. virus-specific CD4 T cells
  • antigen-specific CD8 T cells e.g. virus-specific CD8 T cells
  • effector memory CD4 T cells
  • the antigen-binding molecule of the present invention is capable of reducing expression of one or more immune checkpoint proteins. In some embodiments the antigen-binding molecule is capable of reducing expression of one or more immune checkpoint proteins by immune cells, e.g. T cells.
  • Immune checkpoint proteins are well known to the skilled person, and include e.g. PD-1 , CTLA-4, LAG-3, TIM-3, VISTA, TIGIT and BTLA.
  • the antigen-binding molecule of the present invention is capable of reducing expression of PD-1.
  • the ability of an antigen-binding molecule to reduce the expression of an immune checkpoint protein can be analysed by contacting a population of immune cells with the antigen-binding molecule, and subsequently analysing the cells for expression of the immune checkpoint protein, e.g. by flow cytometry.
  • the cells may be contacted with the antigen-binding molecule in vivo, e.g. through administration of the antigen-binding molecule to a subject, or cells obtained from a subject may be contacted in vitro or ex vivo with the antigen-binding molecule.
  • the antigen-binding molecule of the present invention causes a reduction in the level expression of PD-1 by T cells to less than less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times, ⁇ 0.05 times, or ⁇ 0.01 times the level of expression by PD-1 by T cells observed in the absence of the antigenbinding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.
  • the antigen-binding molecule of the present invention enhances anticancer activity of cancer antigen-specific immune cells, e.g. in vivo.
  • the ability of an antigen-binding molecule to enhance anticancer immune response can be analysed e.g. as described in Example 9 herein.
  • the antigen-binding molecule of the present invention has increased stability as compared to a reference antigen-binding molecule, e.g. an antigen-binding molecule described in WO 2017/021540 A1.
  • “stability” may refer to resistance to degradation, aggregation and/or unfolding.
  • An antigen-binding molecule which has increased/improved stability as compared to a reference antigenbinding molecule may display reduced degradation/propensity to degrade, reduced
  • Antigen-binding molecule degradation/aggregation may be determined by detecting and optionally quantifying degraded/aggregated/unfolded species, e.g. in a sample containing the antigen-binding molecule.
  • Antigen-binding molecule degradation, aggregation and/or unfolding may be determined by detecting and optionally quantifying monomer species, e.g. in a sample containing the antigen-binding molecule.
  • a sample containing an antigen-binding molecule having increased/improved stability as compared to a reference antigen-binding molecule may contain a reduced proportion of
  • a sample containing an antigen-binding molecule having increased/improved stability as compared to a reference antigen-binding molecule may contain an increased proportion of monomer species as compared to a sample containing the reference antigen-binding molecule.
  • the antigen-binding molecule of the present invention has increased
  • thermostability e.g. as compared to a reference antigen-binding molecule, e.g. an antigen-binding molecule described in WO 2017/021540 A1.
  • the antigen-binding molecule of the present invention has increased freeze-thaw stability, e.g. as compared to a reference antigen-binding molecule, e.g. an antigen-binding molecule described in WO 2017/021540 A1 .
  • Antigen-binding molecule stability may be evaluated according to methods well known in the art of molecular biology. Such methods may involve evaluating the antigen-binding molecules to determine the level of degradation (fragmentation), aggregation, unfolding and/or the proportion of
  • Antigen-binding molecule stability may be evaluated according to the methods described e.g. in Thiagarajan et al., mAbs (2016) 8(6) 1088-1097 (incorporated by reference herein), including analysis by size-exclusion chromatography (SEC), analysis of onset-of- melting temperatures (Tonset), thermal unfolding temperatures (T m ), and apparent enthalpies associated with unfolding transitions, by differential scanning calorimetry (DSC); analysis of effective surface charge via zeta potential and diffusion interaction parameter (KD) analysis; and analysis of intrinsic tryptophan fluorescence by fluorescence spectroscopy.
  • SEC size-exclusion chromatography
  • Tonset onset-of- melting temperatures
  • T m thermal unfolding temperatures
  • DSC apparent enthalpies associated with unfolding transitions
  • stability may be evaluated by analysis by size-exclusion
  • An antigen-binding molecule having increased stability as compared to a reference antigen-binding molecule may be determined by SEC analysis to have a larger monomer fraction, and/or smaller a degraded fraction (e.g. as determined by detection of a smaller number/proportion of low molecular weight species), and/or smaller aggregate fraction (e.g. as determined by detection of a smaller number/proportion of high molecular weight species) in a sample containing the antigen-binding molecule as compared to a sample containing the reference antigen-binding molecule.
  • the methods may involve evaluating the level of degradation, aggregation and/or unfolding, and/or the proportion of degraded/aggregated/unfolded/monomer species in a sample containing the antigen-binding molecule. In some embodiments the methods may involve evaluating the level of degradation, aggregation and/or unfolding, and/or the proportion of
  • Purification may comprise affinity-purification of the antigen-binding molecule, e.g. from cell culture supernatant of cells expressing the constituent polypeptides of the antigen-binding molecule.
  • Example 10 herein describes analysis of antigen-binding molecule containing samples by high-performance size exclusion chromatography (HP-SEC) following protein G affinity purification from cell culture supernatant of cells expressing the antigen-binding molecules.
  • HP-SEC high-performance size exclusion chromatography
  • the methods may involve evaluating the level of degradation, aggregation and/or unfolding, and/or the proportion of degraded/aggregated/unfolded/monomer species in a sample containing the antigen-binding molecule after the sample has been subjected to chemical or physical stress (e.g. high temperature, freeze-thaw stress, low pH, agitation and/or long-term storage).
  • chemical or physical stress e.g. high temperature, freeze-thaw stress, low pH, agitation and/or long-term storage.
  • Example 12.1 herein describes analysis of antigen-binding molecule containing samples by HP-SEC following incubation at different temperatures, for different periods of time.
  • Example 12.2 herein describes analysis of antigen-binding molecule containing samples by HP-SEC following different numbers of cycles of rapid or slow freeze/thaw.
  • Analysis of stability may involve expressing the antigen-binding molecule, optionally purifying the antigenbinding molecule, and evaluating the level of degradation, aggregation and/or unfolding, and/or the proportion of degraded/aggregated/unfolded/monomer species in a sample containing the antigen-binding molecule, e.g. by SEC.
  • An antigen-binding molecule having increased stability as compared to a reference antigen-binding molecule may have a smaller number/proportion of degraded/aggregate species and/or a larger number/proportion of monomer species in a sample containing the antigenbinding molecule as compared to a sample containing the reference antigen-binding molecule.
  • Thermostability of antigen-binding molecules can be analysed by methods well known to the skilled person, including Differential Scanning Fluorimetry and Differential Scanning Calorimetry (DSC), which are described e.g. in He et al., J Pharm Sci. (2010) which is hereby incorporated by reference in its entirety.
  • DSC Differential Scanning Calorimetry
  • the antigen-binding molecule of the present invention may be determined in such an assay to have a T m 1 value which is more than 1 times, e.g. >1.01 times, >1.02 times, >1 .03 times,
  • thermostability may involve subjecting a sample containing the antigen-binding molecule to thermal stress for a period of time, and subsequently evaluating the level of degradation, aggregation and/or unfolding, and/or the proportion of degraded/aggregated/unfolded/monomer species in a sample containing the antigen-binding molecule, e.g. by SEC.
  • An antigen-binding molecule having increased thermostability as compared to a reference antigen-binding molecule may have a smaller
  • Analysis of freeze-thaw stability may involve subjecting a sample containing the antigen-binding molecule to one or more freeze-thaw cycles, and subsequently evaluating the level of degradation, aggregation and/or unfolding, and/or the proportion of degraded/aggregated/unfolded/monomer species in a sample containing the antigen-binding molecule, e.g. by SEC.
  • An antigen-binding molecule having increased freeze-thaw stability as compared to a reference antigen-binding molecule may have a smaller number/proportion of degraded/aggregate species and/or a larger number/proportion of monomer species in a sample containing the antigen-binding molecule as compared to a sample containing the reference antigen-binding molecule.
  • a sample containing the antigen-binding molecule may be determined in an assay of stability (e.g. thermostability, e.g. freeze-thaw stability) analysed by SEC to have a proportion of monomer species which is greater than 1 times, e.g. >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1.05 times, >1.1 times, >1 .2 times, >1.3 times, >1.4 times, >1.5 times, >1.6 times, >1.7 times, >1.8 times, >1.9 times, >2 times the proportion of monomer species determined for a sample containing a reference antigen-binding molecule (e.g. an antigen-binding molecule described in WO 2017/021540 A1 ) in said assay.
  • stability e.g. thermostability, e.g. freeze-thaw stability
  • a sample containing the antigen-binding molecule may be determined in an assay of stability (e.g. thermostability, e.g. freeze-thaw stability) analysed by SEC to have a level of
  • degradation/aggregation and/or a proportion of degraded/aggregate species which is less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times the level of degradation/aggregation/proportion of degraded/aggregate species determined for a sample containing a reference antigen-binding molecule (e.g. an antigenbinding molecule described in WO 2017/021540 A1 ) in said assay.
  • a reference antigen-binding molecule e.g. an antigenbinding molecule described in WO 2017/021540 A1
  • the antigen-binding molecule of the present invention further comprises a cell membrane anchor region.
  • a‘cell membrane anchor region’ is a region providing for anchoring of the antigen-binding molecule to the cell membrane of a cell expressing the antigen-binding molecule.‘Anchoring’ may be reversible or irreversible.
  • the cell membrane anchor region is a transmembrane domain.
  • a transmembrane domain refers to any three-dimensional structure formed by a seguence of amino acids which is thermodynamically stable in a biological membrane, e.g. a cell membrane.
  • the transmembrane domain may comprise or consist of a seguence of amino acids which forms a hydrophobic alpha helix or beta-barrel.
  • the amino acid seguence of the transmembrane domain may be, or may be derived from, the amino acid seguence of a transmembrane domain of a protein comprising a transmembrane domain.
  • Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid seguence analysis tools such as TMHMM (Krogh et al., 2001 J Mol Biol 305: 567-580).
  • the amino acid seguence of the transmembrane domain may be, or may be derived from, the amino acid seguence of the transmembrane domain of a protein expressed at the cell surface.
  • the protein expressed at the cell surface is a receptor or ligand, e.g. an immune receptor or ligand.
  • the amino acid seguence of the transmembrane domain may be, or may be derived from, the amino acid seguence of the transmembrane domain of one of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I a, MHC class II a, MHC class II b, CD3 , CD35, CD3y, O ⁇ 3-z, TCRa TCR , CD4, CD8a, O ⁇ db, CD40, CD40L, PD-1 , PD-L1 , PD-L2, 4-1 BB, 4- 1 BBL, 0X40, OX40L, GITR, GITRL, TIM-3, Galectin 9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM- 1 , ICAM1 , LFA-1 , LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1 , CD2, CD48, 2B4, SL
  • the cell membrane anchor region may be a lipid anchor region.
  • a lipid anchor region comprises or consists of a lipid anchor (e.g. a GPI anchor).
  • A‘lipid anchor’ refers to a moiety capable of associating (e.g. covalently) with the lipid component of a biological membrane (e.g. cell membrane). Through such association, a protein having a lipid anchor attached thereto is‘anchored’ in the cell membrane.
  • a lipid anchor typically comprises a lipophilic group. Lipid anchors, lipophilic groups thereof and modification of proteins to attach lipid anchors is described for example in Resh 2013, Curr Biol. 23(10): R431-R435, which is hereby incorporated by reference in its entirety.
  • a lipid anchor may comprise or consist of an isoprenyl, myristoyl, palmitoyl, fatty acyl, diacylglycerol, steroyl, or phospholipid group, or a glycosylphosphatidyl inositol (GPI) anchor.
  • GPI glycosylphosphatidyl inositol
  • a lipid anchor region comprises or consists of a lipid anchor signal sequence.
  • a ‘lipid anchor signal sequence’ refers to an amino acid sequence directing processing of a protein to attach a lipid anchor. Following such processing the antigen-binding molecule comprises a lipid anchor.
  • the present invention also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present invention.
  • CAR chimeric antigen receptor
  • CARs Chimeric Antigen Receptors
  • CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signalling region.
  • An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker.
  • the cell membrane anchor region is provided between the antigen-binding region and the signalling region of the CAR.
  • the cell membrane anchor region provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell.
  • the CAR of the present invention comprises a cell membrane anchor region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the transmembrane region amino acid sequence for one of CDS-z, CD4, CD8 or CD28.
  • a region which is‘derived from’ a reference amino acid sequence comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence.
  • the signalling region of a CAR allows for activation of the T cell.
  • the CAR signalling regions may comprise the amino acid sequence of the intracellular domain of CDS-z, which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing T cell.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Signalling regions comprising sequences of other ITAM-containing proteins have also been employed in CARs, such as domains comprising the ITAM containing region of FcyRI (Haynes et al., 2001 J Immunol 166(1 ): 182-187).
  • CARs comprising a signalling region derived from the intracellular domain of CDS-z are often referred to as first generation CARs.
  • Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR- expressing T cells upon binding to the target protein.
  • Suitable co-stimulatory molecules include CD28, 0X40, 4-1 BB, ICOS and CD27.
  • CARs having a signalling region including additional co-stimulatory sequences are often referred to as second generation CARs.
  • CARs are engineered to provide for co-stimulation of different intracellular signalling pathways.
  • signalling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas the 4-1 BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins.
  • TNF TNF receptor associated factor
  • Signalling regions of CARs therefore sometimes contain co-stimulatory sequences derived from signalling regions of more than one co-stimulatory molecule.
  • CARs comprising a signalling region with multiple co-stimulatory sequences are often referred to as third generation CARs.
  • the CAR of the present invention comprises one or more co-stimulatory sequences comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the intracellular domain of one or more of CD28, 0X40, 4-1 BB, ICOS and CD27.
  • an optional hinge region may provide separation between the antigen-binding region and the transmembrane domain, and may act as a flexible linker. Hinge regions may be flexible domains allowing the binding moiety to orient in different directions. Hinge regions may be derived from lgG1 or the CH2CH3 region of immunoglobulin.
  • the CAR of the present invention comprises a hinge region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of IgG 1 or the CH2CH3 region of immunoglobulin.
  • CARs may be combined with costimulatory ligands, chimeric costimulatory receptors or cytokines to further enhance T cell potency, specificity and safety (Sadelain et al., The basic principles of chimeric antigen receptor (CAR) design. Cancer Discov. 2013 April; 3(4): 388-398. doi: 10.1 158/2159- 8290. CD-12-0548, specifically incorporated herein by reference).
  • a cell comprising a CAR according to the invention.
  • the CAR according to the present invention may be used to generate CAR-expressing immune cells, e.g. CAR-T or CAR-NK cells.
  • Engineering of CARs into immune cells may be performed during culture, in vitro, for transduction and expansion, such as happens during expansion of T cells for adoptive T cell therapy.
  • the present invention provides a nucleic acid encoding an antigen-binding molecule or CAR according to the present invention.
  • the nucleic acid is purified or isolated, e.g. from other nucleic acid, or naturally-occurring biological material.
  • the present invention also provides a vector comprising nucleic acid encoding an antigen-binding molecule or CAR according to the present invention.
  • the nucleic acid and/or vector according to the present invention may be provided for introduction into a cell, e.g. a primary human immune cell.
  • Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g.
  • the viral vector may be a lentiviral, retroviral, adenoviral, or Herpes Simplex Virus vector.
  • the lentiviral vector may be pELNS, or may be derived from pELNS.
  • the vector may be a vector encoding CRISPR/Cas9.
  • the nucleic acid according to the present invention comprises, or consists of, a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NO: 1
  • nucleic acid sequence encoding the same amino acid sequence as one of SEQ ID NOs:335 to 433 as a result of codon degeneracy.
  • the present invention also provides a cell comprising or expressing an antigen-binding molecule or CAR according to the present invention. Also provided is a cell comprising or expressing a nucleic acid or expression vector according to the invention.
  • the cell may be a eukaryotic cell, e.g. a mammalian cell.
  • the mammal may be a human, or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).
  • the cell may be from, or may have been obtained from, a human subject.
  • the cell may be an immune cell.
  • the cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte.
  • the lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof.
  • the cell may express e.g. CD3 polypeptides (e.g. CD3y CD3 O ⁇ 3z or CD35), TCR polypeptides (TCRa or TCR ), CD27, CD28, CD4 or CD8.
  • the cell is a T cell.
  • the T cell is a CD3+ T cell.
  • the T cell is a CD3+, CD8+ T cell.
  • the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
  • CTL cytotoxic T lymphocyte
  • the cell is an antigen-specific T cell.
  • an“antigen-specific” T cell is a cell which displays certain functional properties of a T cell in response to the antigen for which the T cell is specific, or a cell expressing said antigen.
  • the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.
  • an antigen-specific T cell may display one or more of the following properties: cytotoxicity, e.g.
  • the antigen for which the T cell is specific may be a peptide or polypeptide of a virus, e.g.
  • Epstein-Barr virus (EBV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplex virus (HSV) or human papilloma virus (HPV).
  • the present invention also provides a method for producing a cell comprising a nucleic acid or expression vector according to the present invention, comprising introducing a nucleic acid or expression vector according to the present invention into a cell.
  • the present invention also provides a method for producing a cell expressing an antigen-binding molecule or CAR according to the present invention, comprising introducing a nucleic acid or expression vector according to the present invention in a cell.
  • the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid or expression vector by the cell.
  • the methods are performed in vitro.
  • introducing an isolated nucleic acid or expression vector according to the invention into a cell comprises transduction, e.g. retroviral transduction.
  • the isolated nucleic acid or expression vector is comprised in a viral vector, or the vector is a viral vector.
  • the method comprises introducing a nucleic acid or expression vector according to the invention by electroporation, e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety.
  • the present invention also provides cells obtained or obtainable by the methods according to the present invention.
  • Antigen-binding molecules and CARs according to the invention may be prepared according to methods for the production of polypeptides known to the skilled person.
  • polypeptide(s) of interest may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis.
  • peptides/polypeptides can by synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.
  • antigen-binding molecules and CARs according the invention may be produced by recombinant expression.
  • Molecular biology techniques suitable for recombinant production are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, which is hereby incorporated by reference in its entirety.
  • Expression may be from a nucleotide sequence.
  • the nucleotide sequence may be contained in a vector.
  • A“vector” as used herein is an oligonucleotide molecule (DNA or RNA) used as a vehicle to transfer foreign genetic material into a cell.
  • the vector may be an expression vector for expression of the foreign genetic material in the cell.
  • Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed.
  • a vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the invention.
  • the vector may be a plasmid, MAC, virus, etc.
  • the vector may be a eukaryotic expression vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell.
  • the vector may be a mammalian expression vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
  • CMV cytomegalovirus
  • operably linked may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette).
  • a regulatory sequence is operably linked to the selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of the nucleotide sequence.
  • the resulting transcript may then be translated into a desired peptide or polypeptide.
  • the antigen-binding molecules according to the present invention are comprised of more than one polypeptide chain.
  • production of the antigen-binding molecules may comprise transcription and translation of more than one polypeptide chain, and subsequent association of the polypeptide chains to form the antigen-binding molecule.
  • any cell suitable for the expression of polypeptides may be used.
  • the cell may be a prokaryote or eukaryote.
  • the cell is a prokaryotic cell, such as a cell of archaea or bacteria.
  • the bacteria may be Gramnegative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli.
  • the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK, HeLa or COS cells.
  • a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK, HeLa or COS cells.
  • the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells.
  • very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags.
  • Specific plasmids may also be utilised which enhance secretion of the protein into the media.
  • Production may involve culture or fermentation of a eukaryotic cell modified to express the peptide or polypeptide.
  • the culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors.
  • Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted peptide or polypeptide. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).
  • Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches.
  • the bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.
  • the polypeptide of interest is preferably isolated. Any suitable method for separating proteins from cell culture known in the art may be used. In order to isolate the polypeptide from a culture, it may be necessary to first separate the cultured cells from media containing the polypeptide of interest. If the polypeptide of interest is secreted from the cells, the cells may be separated from the culture media that contains the secreted polypeptide of interest by centrifugation. If the polypeptide of interest collects within the cell it will be necessary to disrupt the cells prior to centrifugation, for example using sonification, rapid freeze-thaw or osmotic lysis.
  • Centrifugation will produce a pellet containing the cultured cells, or cell debris of the cultured cells, and a supernatant containing culture medium and the polypeptide of interest.
  • polypeptide of interest may be isolated from the supernatant or culture medium, which may contain other protein and non-protein components.
  • a common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.
  • precipitating agent such as ammonium sulfate
  • polypeptide of interest may be desired or necessary to concentrate the peptide or polypeptide.
  • a number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.
  • Antigen-binding molecules according to the present invention also find use in methods for
  • the antigen-binding molecules according to the present invention find use in generating/expanding populations of cell types expressing CD122 and CD132 (e.g. at the cell surface).
  • the cells may be e.g. T cells, antigen-specific T cells (e.g. virus-specific T cells), antigen-specific CD4 T cells, antigen-specific CD8 T cells, effector memory CD4 T cells, effector memory CD8 T cells, central memory CD4 T cells, central memory CD8 T cells, cytotoxic CD8+ T cells (i.e. CTLs) NK cells or antigen- specific NK cells.
  • T cells antigen-specific T cells (e.g. virus-specific T cells), antigen-specific CD4 T cells, antigen-specific CD8 T cells, effector memory CD4 T cells, effector memory CD8 T cells, central memory CD4 T cells, central memory CD8 T cells, cytotoxic CD8+ T cells (i.e. CTLs) NK cells or antigen- specific NK cells.
  • the cells may be antigen-specific immune cells, e.g. antigen-specific T cells.
  • the cells may be specific for a peptide/polypeptide of a virus, e.g. adenovirus, Epstein-Barr virus (EBV), cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).
  • a virus e.g. adenovirus, Epstein-Barr virus (EBV), cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocy
  • Virus-specific immune cells may be an adenovirus-specific T cells (AdVSTs), Epstein-Barr virus-specific T cells (EBVSTs), cytomegalovius-specific T cells (CMVSTs), human papilloma virus-specific T cells (HPVSTs), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B virus-specific T cells (HBVSTs), hepatitis C virus-specific T cells (HCVSTs), human immunodeficiency virus-specific T cells (HIVSTs), lymphocytic choriomeningitis virus-specific T cells (LCMVSTs), or herpes simplex virus-specific T cells (HSVSTs).
  • AdVSTs adenovirus-specific T cells
  • EBVSTs Epstein-Barr virus-specific T cells
  • CMVSTs cytomegalovius-specific T cells
  • HPVSTs human papilloma virus-specific T cells
  • the cells may comprise/express a chimeric antigen receptor (CAR) or nucleic acid encoding a CAR.
  • CAR chimeric antigen receptor
  • the cells may comprise/express a TGF decoy receptor, or nucleic acid encoding a TGF decoy receptor.
  • the methods comprise contacting cells expressing CD 122 and CD 132 in the presence of an antigenbinding molecule according to the present invention.
  • the cells expressing CD122 and CD132 are stimulated by the antigen-binding molecule to undergo cell division (i.e. proliferate), resulting in an increase in the number of cells.
  • the methods comprise generating/expanding cells in vitro. In some embodiments, the methods comprise generating/expanding cells ex vivo. In some embodiments the methods comprise culturing cells in vitro in the presence of an antigen-binding molecule according to the present invention.
  • Culture of cells may be performed using suitable medium and under suitable environmental conditions (e.g. temperature, pH, humidity, atmospheric conditions, agitation etc.) for the in vitro culture of immune cells, which are well known to the person skilled in the art of cell culture.
  • suitable environmental conditions e.g. temperature, pH, humidity, atmospheric conditions, agitation etc.
  • cultures of cells may be maintained at 37°C in a humidified atmosphere containing 5% CO2.
  • Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc.
  • the cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person.
  • cells are cultured in a bioreactor.
  • cells are cultured in a bioreactor described in Somerville and Dudley,
  • cells are cultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100 bioreactor.
  • immune cells expressing CD122 and CD132 may be generated or expanded from within population of immune cells. It will be appreciated that the population of immune cells comprises the immune cells expressing CD122 and CD132.
  • the population of immune cells from which the population of immune cells expressing CD122 and CD132 are generated/expanded according to the methods of the present invention comprise at least one immune cell expressing CD122 and CD132.
  • immune cells expressing CD122 and CD132 may be generated or expanded from within population of PBMCs.
  • the methods may involve expansion of T cells (e.g. antigen-specific T cells) from within a population of immune cells (e.g. PBMCs, PBLs).
  • T cells e.g. antigen-specific T cells
  • the immune cells (e.g. PBMCs, PBLs) used in the methods of the invention may be freshly obtained, or may be thawed from a sample of immune cells which has previously been obtained and frozen.
  • generation or expansion of a population of immune cells may involve culture of a population of PBMCs.
  • a population of immune cells may be generated/expanded from within a population of T cells (e.g. a population of T cells of heterogeneous type and/or specificity), which may have been obtained from a blood sample or a population of PBMCs.
  • Culture of the population of immune cells from which the cells expressing CD122 and CD132 are generated/expanded may result in an increase of the number of cells expressing CD122 and CD132, and/or result in an increased proportion of such cells in the cell population at the end of the culture.
  • the methods comprise treating cells to increase expression (e.g. surface expression) of CD122 and/or CD132, and expansion of cells expressing CD122 and CD132.
  • T cell activation e.g. by stimulation using anti-CD3 (e.g. clone OKT3) and anti-CD28
  • anti-CD3 e.g. clone OKT3
  • anti-CD28 induces upregulation of CD122 and CD132.
  • Antigen-specific T cells also have upregulated expression of CD122 and CD132.
  • the methods comprise contacting immune cells with antigen or cells presenting antigen.
  • the population of cells is generated/expanded in vivo following administration of an antigen-binding molecule according to the present invention (or administration of cells expressing the antigen-binding molecule) to a subject.
  • compositions and formulations are provided.
  • compositions comprising the antigen-binding molecules, nucleic acids, expression vectors and cells described herein.
  • the antigen-binding molecules, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration which may include injection or infusion.
  • Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium.
  • Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.
  • the antigen-binding molecule, nucleic acid, expression vector, CAR, composition or cells according to the present invention are formulated for injection or infusion, e.g. into a blood vessel or tumor.
  • methods are also provided for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: isolating an antigen-binding molecule, nucleic acid, expression vector or cells described herein; and/or mixing an antigen-binding molecule, nucleic acid, expression vector or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • a further aspect the invention described herein relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a cancer, the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, nucleic acid, expression vector or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • antigen-binding molecules CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.
  • the invention described herein provides antigen-binding molecules, nucleic acids, expression vectors, cells and compositions described herein for use in a method of medical treatment or prophylaxis.
  • the invention described herein also provides the use of antigen-binding molecules, nucleic acids, expression vectors, cells and compositions described herein in the manufacture of medicaments for treating or preventing a disease or condition.
  • the invention described herein also provides methods of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or
  • an antigen-binding molecule prophylactically effective amount of an antigen-binding molecule, nucleic acid, expression vector, cell or composition described herein.
  • Treatment may, for example, be reduction in the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition.
  • Treatment or alleviation of a disease/condition may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of the condition or to slow the rate of development.
  • treatment or alleviation may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition.
  • Prevention of a disease/condition may refer to prevention of a worsening of the condition or prevention of the development of the disease/condition, e.g.
  • the articles of the present invention find use in the treatment of any disease/condition which would benefit from an increase in the number/frequency of immune cells such as T cells (in particular effector T cells) and/or NK cells.
  • T cells in particular effector T cells
  • NK cells in particular effector T cells
  • the antigen-binding molecules and pharmaceutical compositions described herein find use to treat or prevent T cell dysfunctional disorders, cancers and infectious disease.
  • the therapeutic and prophylactic utility of the present invention extends to the treatment of any subject that would benefit in an increase in the number of (i.e. expansion of a population of) cells expressing CD122 and CD132 (e.g. effector T cells and/or NK cells).
  • a T cell dysfunctional disorder may be a disease or condition in which normal T cell function is impaired causing downregulation of the subject’s immune response to pathogenic antigens, e.g. generated by infection by exogenous agents such as microorganisms, bacteria and viruses, or generated by the host in some disease states such as in some forms of cancer (e.g. in the form of tumor-associated antigens).
  • pathogenic antigens e.g. generated by infection by exogenous agents such as microorganisms, bacteria and viruses, or generated by the host in some disease states such as in some forms of cancer (e.g. in the form of tumor-associated antigens).
  • the T cell dysfunctional disorder may comprise T cell exhaustion or T cell anergy.
  • T cell exhaustion comprises a state in which CD8+ T cells fail to proliferate or exert T cell effector functions such as cytotoxicity and cytokine (e.g. IFNy) secretion in response to antigen stimulation.
  • Exhausted T cells may also be characterised by sustained expression of one or more markers of T cell exhaustion, e.g. PD-1 , CTLA-4, LAG-3, TIM-3.
  • the antigen-binding molecules and pharmaceutical compositions described herein find use to treat or prevent T cell dysfunctional disorders, cancers and infectious disease, wherein treatment with the antigen-binding molecules and pharmaceutical compositions results in reduced expression by T cells of one or more markers of T cell exhaustion. In some cases, the treatment results in reduced expression by T cells of PD-1.
  • the T cell dysfunctional disorder may be manifest as an infection, or inability to mount an effective immune response against an infection.
  • the infection may be chronic, persistent, latent or slow, and may be the result of bacterial, viral, fungal or parasitic infection.
  • treatment may be provided to patients having a bacterial, viral or fungal infection.
  • bacterial infections include infection with Helicobacter pylori.
  • viral infections include infection with HIV, hepatitis B or hepatitis C.
  • the T-cell dysfunctional disorder may be associated with a cancer, such as tumor immune escape.
  • a cancer such as tumor immune escape.
  • Many human tumors express tumor-associated antigens recognised by T cells and capable of inducing an immune response.
  • Cancers may also be treated where there is no indication of a T-cell dysfunctional disorder, but the use of an antigen-binding molecule, cell or composition according to the present invention stimulates proliferation and expansion of T cells (particularly effector T cells) and allows the subject to and mount an effective immune response.
  • the cancer to be treated/prevented in accordance with the invention described herein may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor.
  • the cancer may be benign or malignant and may be primary or secondary (metastatic).
  • a neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue.
  • the cancer may be of tissues/cells derived from e.g.
  • adrenal gland adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney oesophagus
  • glial cells heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.
  • the cancer to be treated may be a cancer of a tissue selected from the group consisting of colon, rectum, nasopharynx, cervix, oropharynx, stomach, liver, head and neck, oral cavity, oesophagus, lip, mouth, tongue, tonsil, nose, throat, salivary gland, sinus, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovary or mesothelium.
  • Tumors to be treated may be nervous or non-nervous system tumors.
  • Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma.
  • Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer , colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancer and sarcoma.
  • NHL Non-Hodgkin’s lymphoma
  • CML chronic myelogenous leukemia
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the cancer to be treated may be 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.
  • HNSCC head and neck squamous cell carcinoma
  • the cancer to be treated/prevented in accordance with the present invention is a virus-associated cancer, e.g. an EBV-associated cancer or a HPV-associated cancer.
  • “EBV associated” and“HPV associated” cancers may be a cancers which are caused or exacerbated by infection with the respective viruses, cancers for which infection is a risk factor and/or cancers for which infection is positively associated with onset, development, progression, severity or metastasis.
  • EBV-associated cancers which may be treated with cells produced by methods of the disclosure include nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC).
  • HPV-associated medical conditions that may be treated with cells produced by methods of the disclosure include at least dysplasias of the genital area(s), cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, anal intraepithelial neoplasia, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, genital cancers, oral papillomas, oropharyngeal cancer.
  • the cancer to be treated in accordance with various aspects of the present disclosure is one or more of nasopharyngeal carcinoma (NPC; e.g. Epstein-Barr Virus (EBV)-positive NPC), cervical carcinoma (CC; e.g. human papillomavirus (HPV)-positive CC), oropharyngeal carcinoma (OPC; e.g. HPV-positive OPC), gastric carcinoma (GC; e.g. EBV-positive GC), hepatocellular carcinoma (HCC; e.g. Hepatitis B Virus (HBV)-positive HCC), lung cancer (e.g. non-small cell lung cancer (NSCLC)) and head and neck cancer (e.g. cancer originating from tissues of the lip, mouth, nose, sinuses, pharynx or larynx, e.g. head and neck squamous cell carcinoma (HNSCC)).
  • NPC nasopharyngeal carcinoma
  • EBV Epstein-Bar
  • the treatment may be aimed at reducing the number of cells of the cancer, and/or reducing the size of a tumour, and/or inhibiting signalling mediated by an immune checkpoint protein (e.g. PD-1 ).
  • an immune checkpoint protein e.g. PD-1
  • Administration of the antigen-binding molecules and compositions described herein may delay or prevent the onset of symptoms of the cancer.
  • Administration of the antigen-binding molecules and compositions described herein may reduce the severity of symptoms of the cancer.
  • Administration of the antigenbinding molecules and compositions described herein may delay or prevent the onset of invasion and/or metastasis.
  • Administration of the antigen-binding molecules and compositions described herein reduce invasion and/or metastasis.
  • An infection may be any infection or infectious disease, e.g. bacterial, viral, fungal, or parasitic infection.
  • T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections (including viral, bacterial and parasitic), as well as in cancer (Wherry Nature Immunology Vol.12, No.6, p492-499, June 2011 ).
  • bacterial infections examples include infection by 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.
  • the bacterial infection may be sepsis or tuberculosis.
  • viral infections examples include infection 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).
  • influenza virus measles virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HMV human immunodeficiency virus
  • LCMV lymphocytic choriomeningitis virus
  • HPV Herpes simplex virus
  • HPV human papilloma virus
  • fungal infections examples include infection by Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp.
  • the fungal infection may be fungal sepsis or histoplasmosis.
  • parasitic infections examples include infection by Plasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi).
  • Plasmodium species e.g. Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi.
  • the parasitic infection may be a disease such as malaria, leishmaniasis and toxoplasmosis.
  • Methods of medical treatment may also involve in vivo, ex vivo, and adoptive immunotherapies, including those using autologous and/or heterologous cells or immortalised cell lines.
  • the antigen-binding molecules of the present invention are also useful in connection with methods comprising adoptive cell transfer (ACT).
  • ACT adoptive cell transfer
  • the antigen-binding molecules of the present invention are useful in methods for generating/expanding populations of immune cells in vitro or ex vivo, which may then be administered to subject. Methods for generating/expanding populations of immune cells may also be performed in vivo.
  • the present invention provides a method of treatment or prophylaxis comprising adoptive transfer of immune cells (e.g. T cells, effector T cells, virus-specific T cells, NK cells) produced (i.e. generated or expanded) according to the methods of the present invention.
  • adoptive cell transfer generally refers to a process by which immune cells are obtained from a subject, typically by drawing a blood sample from which the immune cells are isolated. The immune cells are then typically treated or altered in some way, optionally expanded, and then administered either to the same subject or to a different subject.
  • the treatment is typically aimed at providing an immune cell population with certain desired characteristics to a subject, or increasing the frequency of immune cells with such characteristics in that subject.
  • the immune cells may be e.g. T cells, antigen-specific T cells (e.g. virus-specific T cells), antigen-specific CD4 T cells, antigen-specific CD8 T cells, effector memory CD4 T cells, effector memory CD8 T cells, central memory CD4 T cells, central memory CD8 T cells, cytotoxic CD8+ T cells (i.e. CTLs) NK cells or antigen-specific NK cells.
  • the immune cells preferably express CD122 and CD132.
  • the immune cells are derived from the patient that they are introduced to (autologous cell therapy). That is, cells may have been obtained from the patient, generated according to methods described herein, and then returned to the same patient. Methods disclosed herein may also be used in allogeneic cell therapy, in which cells obtained from a different individual are introduced into the patient. Adoptive T cell transfer is described, for example, in Chia WK et al., Molecular Therapy (2014), 22(1 ): 132-139, Kalos and June 2013, Immunity 39(1 ): 49-60 and Cobbold et al., (2005) J. Exp. Med. 202: 379- 386, which are hereby incorporated by reference in their entirety.
  • adoptive transfer is performed with the aim of introducing, or increasing the frequency of, immune cells in a subject.
  • the present invention provides a method of treating or preventing a disease or condition in a subject, comprising:
  • the subject from which the PBMCs are isolated is the subject administered with the generated/expanded cells (i.e., adoptive transfer is of autologous cells). In some embodiments, the subject from which the PBMCs are isolated is a different subject to the subject to which the
  • generated/expanded cells are administered (i.e., adoptive transfer is of allogenic cells).
  • the method may comprise one or more of the following steps: taking a blood sample from a subject; isolating PBMCs from the blood sample; generating or expanding a population of immune cells by culture in the presence of an antigen-binding molecule according to the present invention; collecting the generated or expanded population of immune cells; mixing the generated or expanded population of immune cells with an adjuvant, diluent, or carrier; administering the generated or expanded population of immune cells or composition to a subject.
  • the method may additionally comprise administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule according to the present invention.
  • an antigen-binding molecule or composition according to the invention is preferably in a "therapeutically effective” or“prophylactically effective” amount, this being sufficient to show benefit to the subject.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease or disorder. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub.
  • the articles of the present invention are administered in an amount sufficient to cause an increase in the number of (i.e. cause expansion a population of) cells expressing CD122 and CD132 (e.g. effector T cells and/or NK cells).
  • Administration of an antigen-binding molecule encompasses administration of a cell
  • Administration may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the disease/condition to be treated/prevented.
  • the antigen-binding molecule or composition described herein and an additional agent, e.g. a therapeutic agent, may be administered simultaneously or sequentially.
  • the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment/prevention of a cancer.
  • the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and/or hormone therapy.
  • Simultaneous administration refers to administration of the antigen-binding molecule, nucleic acid, vector, cell or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.
  • administration refers to administration of one of the antigen-binding molecule/composition or therapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval.
  • the antigen-binding molecule is administered in combination with a chimeric antigen receptor (CAR) or a CAR-expressing cell (e.g. a CAR-T cell).
  • CAR chimeric antigen receptor
  • the antigenbinding molecule is administered in combination with a population of immune cells, e.g. a population of immune cells generated/expanded according to the methods described herein.
  • the antigen-binding molecule is administered in a method comprising adoptive cell transfer (ACT), as described herein.
  • ACT adoptive cell transfer
  • the antigen-binding molecule is administered in combination with an agent capable of inhibiting signalling mediated by an immune checkpoint protein.
  • Immune checkpoint proteins are sometimes referred to as immune checkpoint inhibitors.
  • Immune checkpoint proteins help keep immune responses in check and thereby protect against autoimmunity, but can also act to inhibit desirable effector immune cell activity such as killing of cancer cells by T cells, or killing of cells infected with a pathogen (particularly in instances of chronic infection). Inhibition of signalling mediated by immune checkpoint proteins is desirable in such settings as it releases effector immune cells from immune checkpoint protein-mediated inhibition, thereby promoting effector immune responses.
  • Agents capable of inhibiting signalling mediated by a given immune checkpoint protein include, e.g. agents capable of binding to the immune checkpoint protein or a ligand for the immune checkpoint protein and inhibiting signalling mediated by the immune checkpoint protein; agents capable of reducing gene/protein expression of the immune checkpoint protein or a ligand for the immune checkpoint protein (e.g.
  • RNA encoding the immune checkpoint protein/ligand through inhibiting transcription of the gene(s) encoding the immune checkpoint protein/ligand, inhibiting post-transcriptional processing of RNA encoding the immune checkpoint protein/ligand, reducing stability of RNA encoding the immune checkpoint protein/ligand, promoting degradation of RNA encoding the immune checkpoint protein/ligand, inhibiting post-translational processing of the immune checkpoint protein/ligand, reducing stability the immune checkpoint protein/ligand, or promoting degradation of the immune checkpoint protein/ligand), and small molecule inhibitors.
  • the immune checkpoint protein is PD-1 , CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA. In some embodiments the immune checkpoint protein is selected from PD-1 , CTLA-4, LAG-3 and TIM-3.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by PD-1.
  • the agent capable of inhibiting signalling mediated by PD-1 may be a PD-1-targeted agent, or an agent targeted against a ligand for PD-1 such as PD-L1 or PD-L2.
  • the agent capable of inhibiting signalling mediated by PD-1 may e.g. be an antibody capable of binding to PD-1 , PD-L1 or PD-L2 and inhibiting PD-1-mediated signalling.
  • the agent capable of inhibiting signalling mediated by PD-1 is an antibody/fragment described in W02016/068801 or WO2016/1 1 1645, both of which are hereby incorporated by reference in their entirety.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by CTLA-4.
  • the agent capable of inhibiting signalling mediated by CTLA-4 may be a CTLA-4-targeted agent, or an agent targeted against a ligand for CTLA-4 such as CD80 or CD86.
  • the agent capable of inhibiting signalling mediated by CTLA-4 may e.g. be an antibody capable of binding to CTLA-4, CD80 or CD86 and inhibiting CTLA-4-mediated signalling.
  • the agent capable of inhibiting signalling mediated by CTLA-4 is an antibody/fragment described in WO2017/194265, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by LAG-3.
  • the agent capable of inhibiting signalling mediated by LAG-3 may be a LAG-3-targeted agent, or an agent targeted against a ligand for LAG-3 such as MHC class II.
  • the agent capable of inhibiting signalling mediated by PD-1 may e.g. be an antibody capable of binding to LAG-3 or MHC Class II and inhibiting LAG-3-mediated signalling.
  • the agent capable of inhibiting signalling mediated by LAG-3 is an antibody/fragment described in WO2017/149143, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by TIM-3.
  • the agent capable of inhibiting signalling mediated by TIM-3 may be a TIM-3-targeted agent, or an agent targeted against a ligand for TIM-3 such as Galectin 9.
  • the agent capable of inhibiting signalling mediated by TIM-3 may e.g. be an antibody capable of binding to TIM-3 or Galectin 9 and inhibiting TIM- 3-mediated signalling.
  • the agent capable of inhibiting signalling mediated by PD-1 is an antibody/fragment described in WO2016/068802 or WO2016/068803, both of which are hereby incorporated by reference in their entirety.
  • Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or g-rays).
  • the drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
  • the drug may be formulated as a pharmaceutical composition or medicament.
  • the formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.
  • a treatment may involve administration of more than one drug.
  • a drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the chemotherapy may be a co-therapy involving administration of two drugs, one or more of which may be intended to treat the cancer.
  • the chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
  • routes of administration e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
  • the chemotherapy may be administered according to a treatment regime.
  • the treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment.
  • the treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc.
  • a single treatment regime may be provided which indicates how each drug is to be administered.
  • Chemotherapeutic drugs and biologies may be selected from: alkylating agents such as cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; purine or pyrimidine antimetabolites such as azathiopurine or mercaptopurine; alkaloids and terpenoids, such as vinca alkaloids (e.g.
  • paclitaxel TaxolTM
  • topoisomerase inhibitors such as the type I topoisomerase inhibitors camptothecins irinotecan and topotecan, or the type II topoisomerase inhibitors amsacrine, etoposide, etoposide phosphate, teniposide
  • antitumor antibiotics e.g. anthracyline antibiotics
  • dactinomycin doxorubicin (AdriamycinTM), epirubicin, bleomycin, rapamycin
  • antibody based agents such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-CTLA-4, anti-4-1 BB, anti- GITR, anti-CD27, anti-BLTA, anti-OX43, anti-VEGF, anti-TNFa, anti-IL-2, antiGpllb/llla, anti-CD-52, anti- CD20, anti-RSV, anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFR antibodies, monoclonal antibodies or antibody fragments, examples include: cetuximab, panitumumab, infliximab, basiliximab, bevacizumab (Avastin®), abciximab, daclizumab, gemtuzumab, alemtuzumab, rituximab (Mabthera
  • chemotherapeutic drugs may be selected from: 13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine, 5- Azacitidine 5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine, Abraxane, Accutane®, Actinomycin-D Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®, All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole, Arabinosylcytosine, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, Arsenic Trioxide, Asparaginase, ATRA Avastin®,
  • Camptothecin-1 1 Capecitabine, CaracTM, Carboplatin, Carmustine, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-1 1 , Cyclophosphamide, Cytadren®, Cytarabine Cytosar-U®, Cytoxan®, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin
  • TESPA Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan, Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin, TrexallTM, Trisenox®, TSPA, TYKERB®, VCR, VectibixTM, Velban®, Velcade®, VePesid®, Vesanoid®, ViadurTM, Vidaza®, Vinblastine, Vinblastine Sulfate, Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, ZevalinTM, Zinecard®,
  • Multiple doses of the antigen-binding molecule or composition may be provided.
  • One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.
  • Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.
  • doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
  • the antigen binding molecule may be conjugated to a drug moiety or a detectable moiety.
  • the antigen-binding molecules described herein may be used in methods that involve detection of CD122 and/or CD132, or cells expressing CD122 and/or CD132 (e.g. at the cell surface).
  • the methods may be in vitro or in vivo methods. Such methods may involve detection of the bound complex of the antigenbinding molecule and CD122 and/or CD132, or cells expressing CD122 and/or CD132.
  • a method comprising contacting a sample containing, or suspected to contain, CD122 and/or CD132 or cells expressing CD122 and/or CD132, and detecting the formation of a complex of the antigen-binding molecule and CD122 and/or CD132 or cells expressing CD122 and/or CD132.
  • Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA.
  • the methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a detectable moiety as described hereinabove.
  • the detectable moiety is a fluorescent label, a luminescent label, an immuno-detectable label or a radio-label.
  • the detectable moiety may be selected from: a radio-nucleotide, positron-emitting radionuclide (e.g. for positron emission tomography (PET)), MRI contrast agent or fluorescent label.
  • PET positron emission tomography
  • MRI contrast agent or fluorescent label.
  • Methods of this kind may provide the basis of methods for the diagnostic and/or prognostic evaluation of a disease or condition. Such methods may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body.
  • the methods may involve detecting or quantifying CD122 and/or CD132, or cells expressing CD122 and/or CD132, e.g. in a patient sample.
  • the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation.
  • Other diagnostic/prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.
  • a sample may be taken from any tissue or bodily fluid.
  • the sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual.
  • CSF cerebrospinal fluid
  • the sample may be obtained or derived from a tissue or tissues which are affected by the disease/condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition).
  • a tissue or tissues which are affected by the disease/condition e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition.
  • the subject to be treated in accordance with aspects the invention described herein may be any animal or human.
  • the subject is preferably mammalian, more preferably human.
  • the subject may be a nonhuman mammal, but is more preferably human.
  • the subject may be male or female.
  • the subject may be a patient.
  • a subject may have been diagnosed with a disease or condition requiring treatment (e.g. a cancer), may be suspected of having such a disease/condition, or may be at risk of
  • the subject is preferably a human subject.
  • the subject to be treated according to a therapeutic or prophylactic method of the invention herein is a subject having, or at risk of developing, a cancer.
  • a subject may be selected for treatment according to the methods based on characterisation for 5 certain markers of such disease/condition.
  • the present invention also provides a kit of parts.
  • the kit may have at least one container having a predetermined quantity of an antigen-binding molecule, nucleic acid, expression 10 vector, CAR, composition or cells described herein.
  • the kit may provide the antigen-binding molecule, nucleic acid, expression vector, CAR, composition or cells together with instructions for administration to a patient in order to treat a specified
  • the kit may comprise materials for producing antigen-binding molecule or composition described herein.
  • the kit may additionally instructions for administration to a patient in order to treat a specified
  • the kit may comprise materials and/or instructions for producing an antigen-binding molecule, nucleic acid, expression vector, CAR, cell or composition described herein.
  • the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. anti-infective agent or chemotherapy agent).
  • another therapeutic agent e.g. anti-infective agent or chemotherapy agent.
  • the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition.
  • Pairwise and multiple sequence alignment for the purposes of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J.
  • the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
  • Figures 1A and 1B Graphs showing binding of different formats of bispecific anti-IL2R /yc antibodies to (1A) yc-Fc and (1B) IL2R -Fc as determined by ELISA.
  • FIGS. 2A to 2C Graphs and bar chart showing binding of bispecific and monospecific IL2R - and/or yc-binding antibodies to cells expressing human IL2R , yc or IL-2Ra at the cell surface, as determined by flow cytometry.
  • (2A) Graphs showing analysis of binding of P2C4/P1A3, P2C4/P1A10, a monospecific anti-yc (‘neg/alL2Ry’), a monospecific anti-IL-2R ((‘alL2R /neg’) to cells transfected with constructs encoding human IL-2R and yc. Negative unstained, secondary antibody only and isotype control conditions are indicated.
  • Figures 3A and 3B Bar charts showing binding of bispecific IL-2R - and yc-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.
  • FIGS 4A and 4B Graphs and bar chart showing binding of bispecific and monospecific IL-2R - and/or yc-binding antibodies to cells expressing rhesus IL-2R and yc at the cell surface, as determined by flow cytometry.
  • (4A) Graphs showing analysis of binding of P2C4/P1A3, P2C4/P1A10, a monospecific anti-yc (‘neg/alL2Ry’), a monospecific anti-IL-2R ((‘alL2R /neg’) to cells transfected with constructs encoding rhesus IL-2R and yc. Negative unstained, secondary antibody only and isotype control conditions are indicated.
  • (4B) Bar chart summarising normalised MFIs for binding of the indicated antibodies to cells transfected with constructs encoding rhesus IL-2R and yc.
  • Figure 5 Bar chart showing binding of bispecific IL-2R - and yc-binding antibodies to primary cynomolgus macaque T cell subsets, as determined by flow cytometry.
  • Figures 6A and 6B Graphs showing analysis of proliferation of NK92 cells in response to treatment with bispecific IL-2R - and yc-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 yc-binding antibodies.
  • 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 yc-binding antibodies or IL-2. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated.
  • 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 yc-binding antibodies or the indicated cytokines.
  • Figures 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 yc-binding antibodies or IL-2.
  • 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 yc-binding antibodies or the indicated cytokines. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated.
  • (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 CD8+ EBV-specific T cells as determined by CellTrace Violet staining.
  • 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-2R - and yc-binding antibodies or IL-2. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated.
  • 11D Absolute numbers of naive CD4+ T cells.
  • FIG. 12A to 12M Bar charts showing analysis of proliferation of pre-activated cynomolgus T cell subsets in response to treatment of cynomolgus PBMCs with bispecific IL-2R - and yc-binding antibodies or IL-2. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated.
  • (12D Absolute numbers of naive CD4+ T cells.
  • (12F Absolute numbers of central memory CD4+ T cells.
  • FIG. 13 Graph showing analysis of induction of STAT5 phosphorylation in NK92 cells in response to treatment with bispecific IL-2R - and yc-binding antibodies or IL-2. EC50 values for induction of STAT5 phosphorylation are shown.
  • 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-2R - and yc- binding antibodies or IL-2. EC50 values for induction of STAT5 phosphorylation are shown. (14A)
  • 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- 2R - and yc-binding antibodies or IL-2. EC50 values for induction of STAT5 phosphorylation are shown.
  • FIGS 17A to 17E Graphs showing kinetics of induction of STAT5 phosphorylation in human immune cell subsets following treatment of PBMCs with bispecific IL-2R - and yc-binding antibodies or IL-2.
  • (17D Percentage pSTAT5-positive monocytes.
  • 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 yc-binding antibodies or IL-2.
  • FIG. 19 Western blot showing induction of STAT6 phosphorylation by IL-4 in THP-1 cells following treatment with bispecific IL-2R - and yc-binding antibodies, isotype control antibody, or IL-2. Total STAT6 and actin were included as controls.
  • Figures 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 yc-binding antibodies or IL-2. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated.
  • (20D Absolute numbers of NK cells.
  • 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 yc-binding antibodies or IL-2. Unstimulated cells (media) and anti-CD3/CD28 bead-stimulated controls (beads) are indicated.
  • FIG 22 Graph showing levels of bispecific IL-2R /yc antibody (P2C4:P1 A3) in the serum of cynomolgus macaques at the indicated time point, following administration of the indicated amount of antibody, as determined by ELISA.
  • Figures 23A and 23B Graphs showing expression of (23A) IL-2R and (23B) go 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-2R and yc as determined by flow cytometry.
  • nMFI median fluorescence Intensity
  • FIGS 24A and 24B Graphs showing expression of (24A) IL-2R and (24B) yc on EBV-specific immune cell subsets. The graphs show normalized median fluorescence Intensity (nMFI) of antibody staining for IL-2R and yc as determined by flow cytometry.
  • nMFI median fluorescence Intensity
  • Figure 25 Schedule of administration of VSTs with or without bispecific IL-2R - and Yc-binding antibodies (BiAb), isotype control antibody or IL-2 to murine EBV-LCL tumour model.
  • BiAb bispecific IL-2R - and Yc-binding antibodies
  • 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 yc- 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.
  • Figures 27A to 27C Schematic representations of the (27A) anti-CD122 scFv / anti-CD132 scFv- Fc(KiHs-s), (27B) anti-CD122 scFv / anti-CD132 Fab-Fc(KiHs-s) and (27C) anti-CD122 scFv-anti-CD132 scFv-Fc format molecules.
  • FIGS 28A to 28D Graphs showing results of SEC-HPLC analysis of protein G-purified bispecific IL- 2R - and yc-binding antibodies (28A) P2C4FW2(scFv)/P1A3(Fab)-Fc(KiHs-s), (27B)
  • FIGS 29A and 29B Graphs showing analysis of proliferation of NK92 cells in response to treatment with the indicated antibodies or IL-2. EC50 values for induction of NK92 cell proliferation are shown. 29A shows the results obtained using P2C4FW2(scFv)/P1A3(Fab)-Fc(KiHs-s) (i.e. P2C4FW2/P1A3 Fab-scFv) and P2C4FW2(scFv)/P1A10(Fab)-Fc(KiHs-s) (i.e.
  • P2C4FW2/P1A10 Fab-scFv shows the results obtained using P2C4FW2(scFv)-P1A3_AQ(scFv)-Fc (i.e. P2C4FW2/P1 A3 Tan scFv) and
  • FIG. 30A to 30D Graphs showing the results of analysis of proliferation of pre-activated, primary human T cell subsets in response to treatment with different amounts of IL-2, isotype control antibody, P2C4FW2(scFv)/P 1 A3_AQ(scFv)-Fc(KiHs-s) (i.e. P2C4/P1A3), P2C4FW2(scFv)/P1A10(scFv)-Fc(KiHs-s) (i.e.
  • (30A) shows the total number of CD4 cells
  • (30B) shows the total number of CD8 cells
  • (30C) shows the number of CD4 Treg cells
  • (30D) shows the ratio of CD8 cells to CD4 Treg cells determined at the end of the experiment.
  • Figures 31 A to 31 D Graphs showing the results of analysis of proliferation of pre-activated, primary human T cell subsets in response to treatment with different amounts of IL-2, isotype control antibody, P2C4FW2(scFv)/P 1 A3_AQ(scFv)-Fc(KiHs-s) (i.e. P2C4/P1A3), P2C4FW2(scFv)/P1A10(scFv)-Fc(KiHs-s) (i.e. P2C4/P1A10), P2C4FW2(scFv)/P1A10(Fab)-Fc(KiHs-s) (i.e.
  • P2C4FW2/P1A10 Fab-scFv) or P2C4FW2(scFv)-P1A10(scFv)-Fc i.e. P2C4FW2/P1A10 Tan scFv.
  • 31A shows the percentage of dividing CD4 cells, and
  • 31 B the percentage of dividing CD8 cells at the end of the experiment, as determined by reduction in CellTrace signal.
  • (31 C) shows the percentage of CD4 cells, and (31 D) the percentage of CD8 cells, staining positive for the cell proliferation marker Ki-67 at the end of the experiment.
  • FIGS 32A to 32I Graphs showing results of SEC-HPLC analysis of bispecific IL-2R - and yc- binding antibodies following incubation at different temperatures for 7 days.
  • (32A) to (32C) show the results obtained after incubation of P2C4FW2(scFv)/P1A3(Fab)-Fc(KiHs-s) for 7 Days at (32A) 4°C, (32B) 25°C and (32C) 37°C.
  • (32D) to (32F) show the results obtained after incubation of
  • (32G) to (32I) show the results obtained after incubation of P2C4FW2(scFv)-P1A10(scFv)-Fc for 7 Days at (32G) 4°C, (32H) 25°C and (32I) 37°C.
  • FIGS 33A to 33F Graphs showing results of SEC-HPLC analysis of bispecific IL-2R - and yc- binding antibodies following incubation at different temperatures for 28 days.
  • (33A) to (33C) show the results obtained after incubation of P2C4FW2(scFv)/P1A10(Fab)-Fc(KiHs-s) for 28 Days at (33A) 4°C, (33B) 25°C and (33C) 37°C.
  • (33D) to (33F) show the results obtained after incubation of
  • FIGS 34A to 34C Graphs showing analysis of proliferation of NK92 cells in response to treatment with IL-2, isotype control antibody, or the indicated bispecific IL-2R - and yc-binding antibodies after incubation at different temperatures (4°C, 25°C or 37°C) for 7 Days.
  • (34A) shows the results obtained for P2C4FW2(scFv)/P1A3(Fab)-Fc(KiHs-s)
  • (34B) shows the results obtained for
  • FIGS 35A and 35B Graphs showing analysis of proliferation of NK92 cells in response to treatment with IL-2, isotype control antibody, or the indicated bispecific IL-2R - and yc-binding antibodies after incubation at different temperatures (4°C, 25°C or 37°C) for 28 Days.
  • (35A) shows the results obtained for P2C4FW2(scFv)/P1A10(Fab)-Fc(KiHs-s)
  • (35B) shows the results obtained for P2C4FW2(scFv)- P1A10(scFv)-Fc.
  • FIGS 36A to 36F Graphs showing results of SEC-HPLC analysis of P2C4FW2(scFv)- P1A10(scFv)-Fc following 1 , 2 or 3 rapid or slow freeze-thaw cycles.
  • (36A) to (36C) show the results obtained after (36A) 1 , (36B) 2 or (36C) 3 rapid freeze-thaw cycles.
  • (36D) to (36F) show the results obtained after (36D) 1 , (36E) 2 or (36F) 3 slow freeze-thaw cycles.
  • Figures 37A and 37B Graphs showing analysis of proliferation of NK92 cells in response to treatment with P2C4FW2(scFv)-P1A10(scFv)-Fc following 1 , 2 or 3 rapid or slow freeze-thaw cycles.
  • (37A) shows the results obtained after 1 , 2 or 3 rapid freeze-thaw cycles.
  • (37B) shows the results obtained after 1 , 2 or 3 slow freeze-thaw cycles.
  • Figure 38 Schematic representation of tandem scFv format molecules P1 A3(scFv)- P2C4FW2(scFv)-Fc and P1A10(scFv)-P2C4FW2(scFv)-Fc.
  • Figures 39A and 39B Graphs showing results of SEC-HPLC analysis of protein G-purified bispecific IL- 2Rb- and yc-binding antibodies P1A3(scFv)-P2C4FW2(scFv)-Fc (39A) and P1A10(scFv)- P2C4FW2(scFv)-Fc (39B).
  • Figure 40 Graph showing analysis of proliferation of NK92 cells in response to treatment with bispecific IL-2R - and yc-binding antibodies or IL-2. EC50 values for induction of NK92 cell proliferation are shown.
  • Figures 41 A to 41 F Graphs showing results of SEC-HPLC analysis of bispecific IL-2R - and yc- binding antibodies P1A3(scFv)-P2C4FW2(scFv)-Fc (41A, 41 B and 41C) and P1A10(scFv)- P2C4FW2(scFv)-Fc (41 D, 41 E and 41 F) after incubation at 4°C for 7 days (41 A and 41 D), 25°C for 7 days (41 B and 41 E), or 37°C for 7 days (41 C and 41 F).
  • Figures 42A to 42E Graphs showing results of SEC-HPLC analysis of bispecific IL-2R - and yc- binding antibodies P1A3(scFv)-P2C4FW2(scFv)-Fc (42A and 42B) and P1A10(scFv)-P2C4FW2(scFv)-Fc (42C, 42D and 42E) after incubation at 4°C for 28 days (42A and 42C), 25°C for 28 days (42B and 42D), or 37°C for 28 days (42E).
  • FIGS 43A and 43B Graphs showing analysis of proliferation of NK92 cells in response to treatment with bispecific IL-2R - and yc-binding antibodies or IL-2 following incubation at different temperatures (4°C, 25°C or 37°C) for 7 Days, or in the absence of an incubation step. EC50 values for induction of NK92 cell proliferation are shown. 43A shows the results for P1 A3(scFv)-P2C4FW2(scFv)-Fc, and 43B shows the results for P1A10(scFv)-P2C4FW2(scFv)-Fc. Figures 44A and 44B.
  • the inventors design, produce and characterise antibodies capable of binding to IL-2R and yc.
  • Example 1 IL-2R8 and vc binding antibodies
  • Anti-IL-2R antibody clones and anti-yc antibody clones were isolated from a human antibody phage display library via in vitro selection.
  • bispecific antibodies were constructed using IL-2R -binding clone P2C4 in combination with one of the yc-binding antibody clones P1 A3 or P1 A10.
  • the bispecific antibodies were designated ‘P2C4/P1A3’ and‘P2C4/P1A10’, respectively.
  • the closest matching antibody germline genes for clone P2C4 are IGHV1-46*01 and IGLV2-14*01.
  • the closest matching antibody germline genes for clone P1 A3 are IGHV4-34*01 and IGKV2-28*01.
  • the closest matching antibody germline genes for clone P1A10 are IGHV1-24*01 and IGKV2-28*01.
  • bispecific antibody formats were prepared: scFv-KiH-Fc, CrossMab and Duobody formats.
  • bispecific antibodies were expressed by transient transfection of HEK 293 cells, and yields were as follows:
  • P2C4/P1 A3 4-14 mg/L
  • P2C4/P1A10 28-40 mg/L
  • P2C4/P1A3 14-160mg/L
  • P2C4/P1A10 63 mg/L
  • P2C4/P1A10 (P2C4) 77 mg/L; (P1A10) 1 10 mg/L
  • P2C4/P1A3and 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
  • Binding of P2C4/P1 A3to IL-2R or go was measured by ELISA analysis, using recombinant IL-2R -Fc and coated on maxisorp plates.
  • 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.
  • the bispecific antibodies analysed in this assay were:
  • scFv P2C4: scFv (P1A3) - KiHs-s-Fc - designated‘P2C4/P1A3’ in the Figures.
  • scFv P2C4_FW2: scFv (P1A3_FW2) - KiHs-s-Fc - designated‘P2C4_FW2/P1A3_FW2’ in the
  • Fab (P2C4) Fab (P1 A3) in CrossMab format - designated‘P2C4/P1 A3 Crossmab’ in the Figures.
  • P2C4/P1A3 or P2C4/P1A10 were captured on anti-human Fc biosensor tips, and 5 different
  • P2C4/P1A10 Whilst the affinity of binding to YC 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.
  • HEK293-6E cells were transfected with plasmids encoding human IL-2Ra-GFP, or IL- 2R -OFP and YC-GFP.
  • 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.
  • nMFI Normalized Median Fluorescence Intensity
  • PBMCs peripheral blood mononuclear cells
  • T cell markers CD3, CD4, CD8, CD45RA, CCR7, Foxp3 and CD25 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+CCR7+), T central memory (CD45RA-CCR7+), T effector memory (CD45RA-CCR7-), T effector memory re-expressing CD45RA (TEMRA; CD45RA+CCR7-) and Treg (CD4+CD25+Foxp3+).
  • nMFI Normalized Median Fluorescence Intensity
  • P2C4/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.
  • 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 cell markers CD3, CD28 and CD95 to delineate the following T cell subsets: Naive (CD28+CD95-), Effector (CD28-CD95+) and Memory (CD28+CD95+).
  • nMFI Normalized Median Fluorescence Intensity

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Abstract

La présente invention concerne des anticorps anti-CD122 et/ou γc et des fragments de ceux-ci. L'invention concerne également des compositions comprenant ces anticorps, ainsi que des utilisations et des procédés les utilisant.
PCT/EP2019/080658 2018-11-09 2019-11-08 Anticorps anti-il-2r bêta/chaîne gamma commune WO2020094834A1 (fr)

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WO2022212848A1 (fr) * 2021-04-02 2022-10-06 Teneobio, Inc. Anticorps agonistes anti-il-2r et procédés d'utilisation
WO2023150735A3 (fr) * 2022-02-04 2023-10-05 The Board Of Trustees Of The Leland Stanford Junior University Polypeptides de cytokines de substitution

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